Compositions and methods for the diagnosis and treatment of microvasculature dysfunction and related diseases

Abstract

The proinflammatory and vasoconstrictor agent endothelin-1 (ET-1) is a pathogenic molecule involved in an array of cardiovascular diseases. Different from ET-1 signaling for vasoconstriction in large arteries, we demonstrate herein that the coronary microvasculature exhibits unique signaling mechanisms (independent of PKC, CPI-17, and intracellular calcium storage) for constriction. The pathophysiological levels of ET-1 preferentially act on the microvasculature to exert long-lasting vasoconstriction through the activation of Rho kinase after binding to the ET-1 receptor ETAR. The ETAR antagonist BQ123 only blocks but is not able to reverse the vasoconstriction to ET-1. In contrast, ROCK inhibitors (e.g., H-1152) effectively reverse arteriolar constriction to ET-1. Therefore, ROCK inhibition is a potent and specific choice for treating coronary ischemic diseases (abnormal vasoconstriction) associated with microvascular dysfunction with overproduced ET-1. ROCK inhibitors may also be used to treat microvascular diseases related to diabetic retinopathy and microvascular dysfunction from cancer therapy or immunosuppressive medications. Since H-1152 can also reduce the basal tone of the microvasculature, ROCK inhibitors are also good diagnostic agent(s) when used during angiogram or by noninvasive methods.

Description

[0001] Attorney Docket No.11164-015WO1 Compositions and Methods for the Diagnosis and Treatment of Microvasculature Dysfunction and Related Diseases STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government Support under Grant Nos. R01EY1018420, R01EY023335, and R01EY024624 awarded by the National Institutes of Health. The Government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority of U.S. Provisional Application No. 63 / 501,944, filed May 12, 2023, which is hereby incorporated by reference in its entirety. BACKGROUND Heart tissue displays a high density of coronary microvessels. The abnormal function of these small blood vessels can cause symptoms such as angina pectoris (chest pain), with severe consequences, such as myocardial infarction or sudden cardiac death. The American College of Cardiology - National Cardiovascular Data Registry and the National Heart, Lung and Blood Institute database - indicates that at least 3-4 million women and men have signs and symptoms of myocardial Ischemia with No Obstructive Coronary Artery (INOCA) disease, which is known as Myocardial Infarction and Non-Obstructive Coronary Arteries (MINOCA) or Cardiac Syndrome X (CSX). In other cases, “obstructive” coronary artery disease (CAD) occurs either with >70% narrowing in diameter (stenosis) of the large coronary arteries or with 50% to 70% stenosis when associated with inducible ischemia and other measurable parameters based on recent evidence-based guidelines of the European Society of Cardiology and American College of Cardiology / American Heart Association (ACC / AHA). Coronary microvascular dysfunction / disease (CMD) with increased coronary resistance and / or reduced vasodilator function in the coronary microcirculation appears to be the underlying cause of inadequate Attorney Docket No.11164-015WO1 blood flow or ischemia in as much as 60% to 90% of INOCA patients, which elevates the risk of undergoing a major adverse cardiovascular event. Moreover, microvascular angina can co- exist and often be responsible for the persistent symptoms among patients with obstructive CAD and individuals with angina after coronary revascularization because CMD contributes to myocardial ischemia in many such patients. Vasculopathy and dysfunction of coronary microvessels have also been confirmed in heart transplant recipients. Unfortunately, despite the identified pathophysiological and prognostic role of CMD in several conditions, to date, there is no specific treatment for CMD. Moreover, CMD is also responsible for angina in individuals with cardiomyopathy and heart valve disease as well as acute coronary syndrome cases such as Takotsubo syndrome. Microvascular dysfunctions are also associated with various diseases, including aging, hypertension, stroke, cognitive impairment, dementia, depression, Alzheimer’s disease, and the development / progression of diabetic retinopathy, systemic lupus erythematosus (SLE) and preeclampsia or eclampsia. Microcirculatory dysfunction is also inducible among patients receiving anticancer therapies, immunosuppressive drugs or abusing substances such as cocaine. As noted above, microvascular disease is a main culprit that plays a role as a common denominator in different clinical phenotypes. However, currently, there are no medications that can effectively treat and prevent microvascular diseases. Some of the available antianginal drugs are only marginally helpful and the treatment of CMD remains a major challenge. Despite the recent advancements in the diagnosis and treatment of heart diseases, there is no sufficient demonstration of improved clinical outcomes in CMD with currently available therapies. Before the latest diagnostic devices were available, CMD was diagnosed or identified in patients complaining of chest pain after exclusion or ruling out the contribution of coronary obstructions from physical blockage (e.g., atherosclerotic plaque) and / or structure alterations (arterial narrowing) in large conduit arteries. These large vessel occlusions can be diagnosed by coronary angiography under X-rays and contrast dye to view the vascular wall; coronary blood flow can be assessed through intravascular ultrasound catheters. Even as of today, the most advanced technology can only assess / visualize the coronary arteries with a resolution limited to ~200 µm in diameter. The signal-to-noise ratio is reduced with decreasing vessel size. These limitations have restricted the application of coronary angiography in diagnosing populations with CMD because this disease occurs in vessels smaller than 100 µm in diameter. Directly Attorney Docket No.11164-015WO1 measuring arteriolar size change in the µm range is not a practical method. Therefore, indirectly assessing the microvascular function (vasodilation capacity), i.e., index of microcirculatory resistance (IMR) and coronary flow reserve (CFR), is used as the quantitative methodology to evaluate the functional reactivity and capacity of the microvessels for the proper diagnosis of CMD. Coronary blood flow velocity can be measured through invasive or noninvasive methods, before and after administering a pharmacological vasodilator such as acetylcholine (testing endothelium function) or adenosine / adenosine analog (testing endothelium- independent function with a maximum dose). Currently, different angiographic wires (e.g., thermodilution vs. Doppler-based velocity wires) are used during invasive angiogram procedures and the cutoff values can be a little different. In general, a CFR less than 2.0 or IMR more than 25 leads to a diagnosis of CMD. Unfortunately, the clinical utility of these diagnostic methods is hampered by serious adverse reactions reported in patients receiving intracoronary acetylcholine infusion because it causes vasoconstriction if endothelial function is impaired by disease. Reports also indicate that intracoronary infusion of adenosine increases the risk of advanced atrioventricular blocks and rates of adenosine-triggered ventricular arrhythmias because these vasodilators are receptor- dependent and consequently activate pathways unrelated to CFR. An agent without receptor- dependent properties is preferred to be a diagnostic agent for CFR or IMR assessment. Repeated failure of developing effective therapy for treating CMD urges better understanding of the root cause. From a physiological perspective, proper function of the tissue / organ depends upon an adequate supply of blood flow (oxygen / nutrients) to and the removal of waste products from the tissue / organ via the microcirculation (arterioles, capillaries, and venules). Therefore, regulation of the activity / reactivity of these microvessels is critically important, and their dysfunction could contribute to the development and progression of ischemic diseases. Under physiological circumstances, the large arteries contribute only about 5-7% to the overall coronary resistance, while coronary arterioles (around 100 µm or smaller in diameter) control more than 70% of overall coronary resistance and are considered the primary regulation site of flow to the heart. The ability of these small blood vessels to constrict or dilate in response to the changing metabolic demands of specific tissues is of paramount importance for cardiovascular (CV) homeostasis and it is dependent upon signaling that occurs within endothelial cells and smooth muscle cells. Any perturbation in these signaling processes in Attorney Docket No.11164-015WO1 resistance arterioles can shift blood flow away from homeostasis. Therefore, the function of arterioles dictates the supply of blood flow, as well as blood pressure, and tissue survival. Despite widely observed microvascular dysfunction, as discussed above, and unmet needs on the clinical side, the basic research side has not identified key root problem(s). Proceedings in the microcirculation field have not led to any approved medications that are specific for treating diseases originating from arteriolar or venular dysfunction. Further, despite marketed vasodilation and / or antianginal drugs being empirically used for medical conditions such as coronary microvascular angina and hypertensive retinopathy, treatment modalities to restore tissue blood flow and microvascular function are relatively ineffective. Thus, there is a critical need to diagnose microvascular dysfunction and uncover / identify potential therapeutic targets and agents that can effectively reverse abnormal microvascular function (i.e., alleviating vasospasm) and restore tissue flow (i.e., improving vasodilation) at the microvascular domain during tissue ischemia. SUMMARY The dysregulation of microvascular blood flow is directly related to the development and progression of many cardiovascular diseases, especially the increased microvascular constriction that can limit blood flow (ischemia) to support cell / tissue survival. The over- production of the potent vasoconstrictor and proinflammatory factor endothelin-1 (ET-1) has been considered as a possible cause for severe vasoconstriction and ischemia in various types of tissues. However, studies of vasoconstriction elicited by ET-1 have rarely been conducted in microvessels, including coronary arterioles. Even though so-called “coronary arterioles” are used in some studies, confounding factors deserve critical discussion to understand the limitations (see below). The results described herein demonstrate that Rho kinase (ROCK) inhibitors, such as H- 1152, can effectively reduce coronary and / or peripheral microvascular constriction and can potentially treat coronary microvascular diseases and angina, including those that are insensitive to nitrate and calcium channel blockers. The results described herein also suggest that ROCK inhibitors, such as H-1152, may be used to treat diabetic retinopathy related to retinal microvascular complications. Overall, the results described herein suggest that ROCK inhibitors, such as H-1152, can be used to treat a range of microvascular diseases related to ET- 1-mediated ROCK activation. The results described herein further suggest that ROCK Attorney Docket No.11164-015WO1 inhibitors, such as the receptor-independent vasodilator H-1152, can be used as a diagnostic agent during coronary angiography. Accordingly, provided herein are methods of treating or preventing microvascular dysfunction in a subject in need thereof that comprise administering to the subject an effective amount of a ROCK inhibitor. In some embodiments, the microvascular dysfunction can be associated with elevated levels of ET-1, for example, as measured using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). Also provided herein are methods of inducing microvascular vasodilation in a subject that comprise administering to the subject an effective amount of a ROCK inhibitor. Also provided herein are methods of reversing ET-1-induced vasoconstriction in a subject in need thereof that comprise administering to the subject an effective amount of a ROCK inhibitor. Also provided herein are methods of treating or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject that comprise administering to the subject an effective amount of a ROCK inhibitor. In some embodiments, disease, disorder or condition is associated with elevated levels of ET-1, for example, as measured using ELISA or RIA. In some embodiments, disease, disorder or condition fails to respond to a calcium channel blocker or vasodilators such as nitrates. In some embodiments, disease, disorder or condition can comprise microvascular angina (also known as cardiac syndrome X; CSX). In some embodiments, disease, disorder or condition can comprise coronary microvascular dysfunction / disease (CMD). In some embodiments, disease, disorder or condition can comprise microvascular vasculopathy or dysfunction among heart transplant recipients. These patients do not complain of chest pain despite severe microvascular findings, because all the innervation to the heart was surgically interrupted. In some embodiments, disease, disorder or condition can comprise drug-resistant hypertension. In some embodiments, disease, disorder or condition can comprise drug-induced hypertension, such as hypertension, coronary spasm, and / or CMD, induced by the Attorney Docket No.11164-015WO1 administration of an anticancer agent (e.g., chemotherapeutic agents such as 5-fluorouracil, anthracycline and bevacizumab). In some embodiments, disease, disorder or condition can comprise coronary artery spasm, percutaneous coronary intervention (PCI)-related refractory myocardial ischemia, cerebral vasospasm following subarachnoid hemorrhage, drug-resistant systemic hypertension, drug-induced hypertension (e.g., bevacizumab-induced hypertension), drug-induced cardiotoxicity (e.g., cardiotoxicity induced by a chemotherapeutic agents such as 5-fluorouracil, anthracycline or an immunosuppressive agent such as cyclosporin A, tacrolimus, etc.), pulmonary arterial hypertension, diabetes-induced microvascular dysfunction, microangiopathy, silent stroke, substance abuse (e.g., cocaine) related myocardial infarction, or any combination thereof. In some embodiments described herein, the methods can further comprise measuring circulating levels of ET-1 in the subject to determine an appropriate dosing regimen of the ROCK inhibitor. In some of the embodiments described herein, the effective amount of the ROCK inhibitor comprises an effective amount to treat or prevent a coronary microvascular dysfunction or vasculopathy among heart transplant recipients who are taking immunosuppressive medications such as tacrolimus. Also provided herein are methods of treating cancer in a subject in need thereof that comprise administering to the subject an effective amount of a cancer therapy in combination with an effective amount of a ROCK inhibitor. Also provided herein are methods of improving the efficacy of a cancer therapy that comprise co-administering an effective amount of a ROCK inhibitor in combination with the cancer therapy. In some of these embodiments, administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor simultaneously with the cancer therapy. In other embodiments, administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor after cancer therapy. In other embodiments, administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor before cancer therapy. In certain embodiments, the effective amount of the ROCK inhibitor comprises an effective amount to treat or prevent a cardiovascular disease caused by the cancer therapy, such as drug-induced hypertension or drug-induced cardiotoxicity. Attorney Docket No.11164-015WO1 In some of these embodiments, cancer therapy can comprise administration of radiotherapy. In other embodiments, the cancer therapy can comprise administration of a chemotherapeutic agent, such as a DNA-alkylating agent, an antitumor antibiotic agent, an antimetabolic agent, a tubulin stabilizing agent, a tubulin destabilizing agent, a hormone antagonist agent, a topoisomerase inhibitor, a protein kinase inhibitor, a HMG-CoA inhibitor, a cyclin-dependent kinase (CDK) inhibitor, tyrosine kinase inhibitors (TKI), hypoxia-inducible factor 2 (HIF2α) inhibitor, angiogenesis antagonists, immune checkpoint inhibitors (ICI), monoclonal and bispecific antibodies, antibody drug conjugates (ADCs), a cyclin inhibitor, a caspase inhibitor, a metalloproteinase inhibitor, an antisense nucleic acid, a triple-helix DNA, a nucleic acid aptamer, a molecularly-modified viral, bacterial or exotoxic agent, or any combination thereof. In some examples, the chemotherapeutic agent can comprise cytidine arabinoside, cytarabine, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin- D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, taxol and derivatives thereof, tamoxifen, dacarbazine, rituximab, daunorubicin, 1- β-D-arabinofuranosylcytosine, fludarabine, docetaxel, FOLFOX4, etc. In some examples, TKIs can comprise imatinib, dasatinib, nilotinib, bosutinib, ponatinib, pazopanib, sunitinib, sorafenib, regorafenib, cabozantinib, lenvatinib, axitinib, tivozanib, etc. Antiangiogenic agents including antibodies can comprise aflibercept, fruquintinib, ramucirumab, bevacizumab, etc. ICIs can comprise pembrolizumab, nivolumab, and cemiplimab as anti-PD-1 antibodies, ipilimumab as an anti-CTLA-4 antibody, as well as atezolizumab, avelumab, and durvalumab as anti-PD-L1 antibodies. Antibody and ADCs can comprise, for example, rituximab, trastuzumab, denosumab, pertuzumab, glofitamab, tisotumab vedotin, sacituzumab govitecan, enfortumab vedotin, gemtuzumab ozogamicin, etc. Proteasome inhibitors can comprise bortezomib, carfilzomib, ixazomib, or any combination thereof. Also provided are methods of treating or preventing cancer treatment-induced hypertension in a subject undergoing cancer treatment that comprise administering to the subject an effective amount of a ROCK inhibitor. Also provided are methods of treating or preventing substance abuse-related cardiovascular disease in a subject that comprise administering to the subject an effective amount of a ROCK inhibitor. Attorney Docket No.11164-015WO1 In some embodiments, the substance abuse-related cardiovascular disease comprises cocaine-induced cardiotoxicity. In certain embodiments, the substance abuse-related cardiovascular disease comprises cocaine-induced myocardial infarction. Also provided are diagnostic methods for assessing microvascular function (e.g., by calculating CFR and IMR) during coronary angiogram in a subject. These methods can be used alone, or as a test performed as part of a conventional coronary angiogram protocol, which administers adenosine (intravenous or intracoronary) to create hyperemia. These methods can comprise measuring coronary blood flow velocity in the subject before and after administration of a ROCK inhibitor (e.g., H-1152) at single or multiple doses; and calculating the subject’s CFR (i.e., a ratio of flow increase by a ROCK inhibitor to the resting flow without inhibitor during diastole) and / or IMR as the indexes of coronary vasodilator function. The CFR value based on adenosine (CFRad) in a healthy normal subject is generally above 2.5; while CFRad value less than 2.0 indicates CMD. A CFRad value between 2.0-2.5 is a gray zone and more clinical information will be considered for making a diagnosis. By intracoronary administration of ROCK inhibitors such as H-1152, a set of CFR based on H- 1152 (CFRh) can be established to match current CFRad-based standard. At a fixed dose, patients’ CFRh is anticipated to show a similar trend as CFRad. The use of ROCK inhibitors in these diagnostic methods can offer several advantages, including (1) replacing adenosine in patients unable to tolerate adenosine administration; (2) providing additional interpretation for CFRad scores, especially CFRad scores between 2.0 and 2.5; (3) providing additional guidance regarding possible therapeutic strategies for treating CMD in patients with CFRad scores between 2.0 and 2.5; and / or (4) for subjects receiving a coronary angiogram due to clear evidence of ischemia but show no signs of obstructive disease, a bolus ROCK inhibitor may quickly improve the ischemia condition when the time is an essence. Therefore, intracoronary administration can serve both diagnostic and therapeutic purposes. In some embodiments, the coronary blood flow velocity in the subject can be measured invasively, for example, during a coronary angiography or angioplasty procedure. In certain embodiments, the coronary blood flow velocity in the subject is measured invasively, for example, using a surgically placed flow probe, a Doppler velocity catheter or guide wire, or a coronary sinus thermodilution catheter. Attorney Docket No.11164-015WO1 In other embodiments, the coronary blood flow velocity in the subject can be measured noninvasively, for example, using magnetic resonance imaging (MRI) or positron emission tomography (PET). The subject’s coronary flow reserve can be a ratio of peak hyperemic flow velocity (i.e., in response to an intracoronary vasodilator) to resting peak flow velocity (i.e., without a vasodilator). In some embodiments, when the subject exhibits a less than anticipated change in the subject’s CFR assessed by administration of the ROCK inhibitor at a fixed dose, the method further comprises administering to the subject an effective amount of a ROCK inhibitor to achieve better blood flow. A subject who requires a high intracoronary ROCK inhibitor dose to achieve better CFR can be classified as a candidate for treatment with the ROCK inhibitor. In some of the embodiments described herein, the ROCK inhibitor can selectively inhibit ROCK2 activity over ROCK1 activity. In other embodiments described herein, the ROCK inhibitor can selectively inhibit ROCK1 activity over ROCK2 activity. In some embodiments of the methods described herein, the ROCK inhibitor can be defined by Formula I wherein L is O or SO2; R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R3is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; Attorney Docket No.11164-015WO1 R5is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; R7is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R8is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; n is 1, 2, 3 or 4; m is, independently for each occurrence, 1, 2, 3, or 4; R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; and R″ is (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts. In some embodiments of the methods described herein, the ROCK inhibitor can comprise an isoquinoline sulfonamide. In some embodiments of the methods described herein, the ROCK inhibitor can be defined by Formula IA wherein R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; Attorney Docket No.11164-015WO1 R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; and R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts. In some embodiments of the methods described herein, the ROCK inhibitor can comprise belumosudil, dimethyl fasudil, fasudil, hydroxyfasudil, netarsudil, ripasudil, AR- 12286, AT-13148, BA-1049 (R), FCN-016, GSK429286A, H-1152, PHP-201, RKI-1447, SAR407899, Y-27632, Y-30141, Y-32885, Y-39983, a prodrug thereof, a salt thereof, a hybrid compound thereof, or any combination thereof. In some embodiments of the methods described herein, the ROCK inhibitor can comprise fasudil (HA-1077), the structure of which is shown below. In some embodiments of the ROCK inhibitor can comprise glycyl-H 1152, the structure of which is shown below. It is also commonly synthesized as Glycyl-H 1152 dihydrochloride (right). Attorney Docket No.11164-015WO1 In some embodiments of the methods described herein, the ROCK inhibitor can comprise H-1152 (also referred to H-1152P), the structure of which is shown below. It is also commonly synthesized as H-1152 dihydrochloride (right) In some embodiments of the methods described herein, the ROCK inhibitor can comprise ripasudil (K-115), the structure of which is shown below. In some embodiments of the ROCK inhibitor can comprise netarsudil (AR-13503), the structure of which is shown below. In some can comprise belumosudil (KD025, SLx-2119), the structure of which is shown below. Attorney Docket No.11164-015WO1 In some embodiments of ROCK inhibitor can comprise OPL-0401 (SAR407899), the structure of which is shown below. Also provided herein are pharmaceutical compositions and kits comprising an effective amount of a ROCK inhibitor to treat or prevent microvascular dysfunction in a subject in need thereof, an effective amount of a ROCK inhibitor to induce microvascular vasodilation in a subject, an effective amount of a ROCK inhibitor to reverse ET-1-induced vasoconstriction in a subject, an effective amount of a ROCK inhibitor to treat or prevent a disease, disorder, or condition associated with microvascular dysfunction in a subject, and / or an effective amount of a ROCK inhibitor to treat or prevent substance abuse-related cardiovascular disease in a subject. Also provided herein are pharmaceutical compositions and kits comprising an effective amount of a ROCK inhibitor and an effective amount of a chemotherapeutic agent. DESCRIPTION OF DRAWINGS Figures 1A-1C show the videomicroscopic method used to evaluate microvascular function in the Examples. Figure 1A is a schematic diagram illustrating the videomicroscopic techniques used to assess microvascular function (i.e., dilation and constriction) with basal tone. Figures 1B and 1C show cannulated and pressurized human retinal arteriole both at a stable basal tone (Figure 1B) and maximally dilated (Figure 1C) in calcium-free solution. Attorney Docket No.11164-015WO1 Figures 2A-2D are plots characterizing coronary arteriolar constriction in response to ET-1. Arteriolar constrictions to high (10 nM) and low (0.1 nM) ET-1 concentrations are directly compared. Roles of ET-1 receptor antagonists, extracellular and intracellular calcium are studied. Figures 3A-3D are plots characterizing the impact of differential extracellular Ca2+entry, through L- or T-type calcium channels, in coronary arteriolar constriction in response to ET-1 and protein kinase C (PKC) activator phorbol 12, 13-dibutyrate (PDBu). Figures 4A-4C are plots characterizing the mechanisms of coronary arteriolar constriction to ET-1, specifically the role of phospholipase C and myosin light chain kinase (MLCK). Figures 5A-5B are plots showing the role of PKC and MLCK in coronary arteriolar constriction to PKC activation by PDBu. Figures 6A-6D are plots characterizing the mechanisms of coronary arteriolar constriction to ET-1, specifically the role of PKC and ROCK. Figure 7 is a plot characterizing the mechanisms of coronary arteriolar constriction to ET-1, specifically the role of myosin light chain phosphatase versus ROCK. Figures 8A-8D show molecular evidence of MYPT1 and MLC phosphorylation by ET- 1 via ROCK activation. Figures 9A-9B demonstrate the phosphorylation of MLC in cultured coronary arteriolar smooth muscle cells by ET-1 and inversely regulated by H-1152. Figures 10A-10F show the characteristics of ROCK isoform expression in different species; their expressions in porcine coronary arterioles in comparison to cardiomyocytes and left anterior descending (LAD) artery. Figures 11A-11D demonstrate the differential expression of CPI-17 in the coronary arterial network, specifically illustrating vessel size-dependent CPI-17 expression. Figures 12A-12B characterize the effect of ROCK inhibitor on resting vascular tone independent of endothelial nitric oxide (NO). Figures 13A-13B are plots showing the vasomotor heterogeneity between coronary and retinal arterioles. Figure 14 is a schematic illustration of the subcellular mechanisms by which ET-1 and PKC activator PDBu evoked coronary arteriolar constrictions. Attorney Docket No.11164-015WO1 Figure 15 is a plot demonstrating that dasatinib, an anticancer tyrosine kinase inhibitor, causes constriction of small ophthalmic resistance arteries, and that this response can be reversed by H-1152. DETAILED DESCRIPTION Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. At various places in the present specification, divalent linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. The term “n-membered” where n is an integer typically describes the number of ring- forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group. As used herein, the phrase “optionally substituted” means unsubstituted or substituted. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. It is to be understood that substitution at a given atom is limited by valency. Throughout the definitions, the term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C1-4, C1-6, and the like. As used herein, the term “Cn-malkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs Attorney Docket No.11164-015WO1 such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms. As used herein, “Cn-m alkenyl” refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. As used herein, “Cn-m alkynyl” refers to an alkyl group having one or more triple carbon-carbon bonds and having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. As used herein, the term “Cn-malkylene”, employed alone or in combination with other terms, refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethan-1,2-diyl, propan-1,3-diyl, propan-1,2-diyl, butan- 1,4-diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms. As used herein, the term “Cn-m alkoxy”, employed alone or in combination with other terms, refers to a group of formula -O-alkyl, wherein the alkyl group has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-m alkylamino” refers to a group of formula -NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-malkoxycarbonyl” refers to a group of formula -C(O)O- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-malkylcarbonyl” refers to a group of formula -C(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Attorney Docket No.11164-015WO1 As used herein, the term “Cn-m alkylcarbonylamino” refers to a group of formula -NHC(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-m alkylsulfonylamino” refers to a group of formula -NHS(O)2-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “aminosulfonyl” refers to a group of formula -S(O)2NH2. As used herein, the term “Cn-m alkylaminosulfonyl” refers to a group of formula -S(O)2NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “di(Cn-m alkyl)aminosulfonyl” refers to a group of formula -S(O)2N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “aminosulfonylamino” refers to a group of formula - NHS(O)2NH2. As used herein, the term “Cn-malkylaminosulfonylamino” refers to a group of formula - NHS(O)2NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “di(Cn-malkyl)aminosulfonylamino” refers to a group of formula -NHS(O)2N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “aminocarbonylamino”, employed alone or in combination with other terms, refers to a group of formula -NHC(O)NH2. As used herein, the term “Cn-malkylaminocarbonylamino” refers to a group of formula - NHC(O)NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “di(Cn-malkyl)aminocarbonylamino” refers to a group of formula -NHC(O)N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Attorney Docket No.11164-015WO1 As used herein, the term “Cn-m alkylcarbamyl” refers to a group of formula -C(O)- NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “thio” refers to a group of formula -SH. As used herein, the term “Cn-malkylsulfinyl” refers to a group of formula -S(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-m alkylsulfonyl” refers to a group of formula -S(O)2-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “amino” refers to a group of formula –NH2. As used herein, the term "aryl," employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term "Cn-m aryl" refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g., phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, the aryl group is a substituted or unsubstituted phenyl. As used herein, the term “carbamyl” to a group of formula –C(O)NH2. As used herein, the term “carbonyl”, employed alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O). As used herein, the term “di(Cn-m-alkyl)amino” refers to a group of formula -N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “di(Cn-m-alkyl)carbamyl” refers to a group of formula – C(O)N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “halo” refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br. In some embodiments, a halo is F or Cl. As used herein, “Cn-m haloalkoxy” refers to a group of formula –O-haloalkyl having n to m carbon atoms. An example haloalkoxy group is OCF3. In some embodiments, the Attorney Docket No.11164-015WO1 haloalkoxy group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term “Cn-mhaloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+1 halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbons including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (C3-10). Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O) or C(S)). Cycloalkyl groups also include cycloalkylidenes. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, and the like. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, the cycloalkyl has 6-10 ring-forming carbon atoms. In some embodiments, cycloalkyl is adamantyl. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring- forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-10 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. A five- Attorney Docket No.11164-015WO1 membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five- membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4- thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl. As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Example heterocycloalkyl groups include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)2, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members. At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an Attorney Docket No.11164-015WO1 azetidine ring may be attached at any position of the ring, whereas a pyridin-3-yl ring is attached at the 3-position. The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified. Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone – enol pairs, amide - imidic acid pairs, lactam – lactim pairs, enamine – imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In some embodiments, the compounds described herein can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, enantiomerically enriched mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures (e.g., including (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+) (dextrorotatory) forms, (-) (levorotatory) forms, the racemic mixtures thereof, and other mixtures thereof). Additional asymmetric carbon atoms can be present in a substituent, such as an alkyl group. All such isomeric forms, as well as mixtures thereof, of these compounds are expressly included in the present description. The compounds described herein can also or further contain linkages wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring or double bond (e.g., carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds). Accordingly, all cis / trans and E / Z isomers and rotational isomers are expressly included in the present description. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms of that compound. Optical isomers can be obtained in pure form by standard procedures known to those skilled in the art, and include, but are not limited to, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Attorney Docket No.11164-015WO1 Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972), each of which is incorporated herein by reference in their entireties. It is also understood that the compounds described herein include all possible regioisomers, and mixtures thereof, which can be obtained in pure form by standard separation procedures known to those skilled in the art, and include, but are not limited to, column chromatography, thin-layer chromatography, and high-performance liquid chromatography. Unless specifically defined, compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. Unless otherwise stated, when an atom is designated as an isotope or radioisotope (e.g., deuterium, [11C], [18F]), the atom is understood to comprise the isotope or radioisotope in an amount at least greater than the natural abundance of the isotope or radioisotope. For example, when an atom is designated as “D” or “deuterium”, the position is understood to have deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium). All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated. In some embodiments, preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts. Example acids can be inorganic or organic acids and include, but are not limited to, strong and weak acids. Some example acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid. Example bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some Attorney Docket No.11164-015WO1 example strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides and arylamines, wherein; alkoxides include lithium, sodium and potassium salts of methyl, ethyl and t-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl and cyclohexyl substituted amides. In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non- toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an Attorney Docket No.11164-015WO1 organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002. As used herein, the term “subject,” refers to any animal, including mammals. For example, the term “subject” includes, but is not limited to, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiment, the subject can comprise “a subject at risk thereof,” meaning a subject that is at a higher risk of developing a disease or disorder associated with microvascular dysfunction as compared to the general population. Such a higher risk may be due to another condition from which the subject is suffering, or due to family history of the subject. Methods of Use Provided herein are methods of diagnosing, treating, and / or preventing microvascular dysfunction in a subject in need thereof that comprise administering to the subject an effective amount of a ROCK inhibitor. Also provided herein are methods of diagnosing, treating, and / or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject that comprise administering to the subject an effective amount of a ROCK inhibitor. The term “local blood flow” as used herein refers to the blood flow perfused via a few arterioles to a regional tissue, rather than the global or systemic perfusion, to support the local or regional needs of oxygen and nutrients. The term “microvascular dysfunction”, which is used interchangeably with “microvascular disease” in publications, as used herein refers to a heterogeneous set of conditions affecting the function of the microvasculature, especially the arterioles, or resulting from the dysfunction of the microvessels ranging from reduced local blood flow or tissue maximal perfusion due to undesired vasodilation ability, especially via arterioles less than 100 micrometers (µm) in diameter. Microvascular dysfunction is meant to also encompass undesired vasoconstriction of blood vessels such as vascular spasm, which restricts perfusion of blood flow to the local tissue. Attorney Docket No.11164-015WO1 The term “coronary flow reserve” (CFR) reflects the maximal capacity of the microvessels in the heart that dilate from the resting conditions after being subjected to the stimulation by a pharmacological vasodilator or under metabolic stress. With current available technology, a number of studies in patients without microvascular disease have found that a normal CFR is more than 2.5. The term “index of microcirculatory resistance” (IMR) is a guidewire-based measurement that allows quantitative assessment of the minimum microcirculatory resistance in a target coronary artery territory. It is obtained following an invasive procedure to measure both coronary pressure and flow. With current available technology, a number of studies in patients without microvascular disease have found that a normal IMR is less than 25. In some embodiments, the microvascular dysfunction can be associated with elevated levels of the potent vasoconstrictor and proinflammatory agent endothelin-1 (ET-1), for example, as measured using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). ET-1, a 21-amino acid peptide, is a proinflammatory and pathogenic agent and a potent endogenous vasoconstrictor. The synthesis and release of ET-1 are augmented and participate in the pathogenesis across a broad spectrum of CV and non-CV diseases, including refractory hypertension, neurological inflammation, obesity, preeclampsia, autoimmune diseases, cancers, sickle cell disease, diabetes-related retinopathy, SLE, and nephropathy. In patients with hypertension or ischemic heart disease, the plasma level of ET-1 rises 2- to 4-fold from a normal value of 1-3 pg / ml. An increased circulating level of ET-1 is generally associated with poor clinical outcomes and survival rates in patients with myocardial infarction and is regarded as an independent predictor of myocardial no-flow, reduced left ventricular function, and long-term mortality. Small arterioles, compared with their upstream arteries, can preferentially respond to ET-1 and this response can be augmented under disease states, which is capable of impairing vasodilator function. Therefore, the microvasculature is more vulnerable to ET-1 attack and preferentially suffers from accentuated vasoconstriction and compromised vasodilation, consequently leading to microvascular dysregulation and tissue ischemia, which aggravate disease progression. In some embodiments, the blood vessel affected by the microvascular dysfunction, or the blood vessel in which vasodilation is induced, is an arteriole or a venule. In some embodiments, unless specifically mentioned, the microvessel is referred to the arteriolar network. In some embodiments, the blood vessel is a coronary arteriole. In some embodiments, Attorney Docket No.11164-015WO1 the arteriole is less than about 100 µm in diameter. In some embodiments, the blood vessel in which vasodilation is induced displays basal tone. In some embodiments, the term “basal tone” of a microvessel as used herein refers to an arteriole that exhibits a tonic (i.e., partially constricted) state of constriction without using exogenous pharmacological constrictors, such as preconstrictors norepinephrine, thromboxane A2, prostaglandin E2, KCl, and ET-1 etc. The arterial blood vessel (> 300 µm in diameter), described herein as the large arterial vessel, does not display a significant basal tone. These vessels used for the in vitro vasodilation study may have been preconstricted with pharmacological vasoconstrictors, which confound the vasoconstriction / vasodilation signaling pathways. A number of conditions are closely associated with microvascular dysfunction, including aging, amyloidosis, chronic thromboembolic pulmonary hypertension, dementia, diabetes mellitus, substance abuse (such as tobacco or cocaine abuse), heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), systemic hypertension, hypertrophic obstructive cardiomyopathy, idiopathic cardiomyopathy, inflammatory disease, ischemic cardiomyopathy, no-reflow phenomenon, obesity, obstructive sleep apnea, peripheral neuropathy, schizophrenia, stress related cardiomyopathy, systemic lupus erythematosus, systemic sclerosis, tumor angiogenesis, and vasospasm. Additional conditions associated with abnormal / enhanced vasoconstriction include angina, congestive heart failure, transplanted heart, erectile dysfunction, preeclampsia, migraine, stroke, and Raynaud phenomenon. In some embodiments, disease, disorder or condition is associated with elevated levels of ET-1 as measured using ELISA or RIA. In some embodiments, disease, disorder or condition can comprise coronary artery spasm, percutaneous coronary intervention (PCI)-related refractory myocardial ischemia, cerebral vasospasm following subarachnoid hemorrhage, drug-resistant systemic hypertension, drug-induced hypertension (e.g., bevacizumab-induced hypertension), drug-induced cardiotoxicity (e.g., cardiotoxicity induced by a chemotherapeutic agents such as 5-fluorouracil and doxorubicin or an immunosuppressive agent such as cyclosporin A, tacrolimus, etc.), pulmonary arterial hypertension (PAH), drug-induced PAH (e.g., dasatinib), diabetes-induced microvascular dysfunction, microangiopathy, silent stroke, or any combination thereof. In certain embodiments, disease, disorder or condition can comprise microvascular angina (also known as cardiac syndrome X; CSX). Attorney Docket No.11164-015WO1 In certain embodiments, disease, disorder or condition can comprise coronary microvascular dysfunction / disease (CMD). In some embodiments, disease, disorder or condition can comprise drug-resistant hypertension. In some embodiments, disease, disorder or condition can comprise drug-induced hypertension, such as hypertension induced by the administration of an anticancer agent. In some embodiments, the disease, disorder or condition fails to respond to at least one existing medication for the treatment of hypertension. In some embodiments, disease, disorder or condition fails to respond to a calcium channel blocker, or vasodilators such as nitrates. The terms “fails to respond,” “non-responsive,” “refractory,” or “resistant” to treatment, as used herein interchangeably, mean that existing medications have no effect or a low effect, such that they do not achieve a satisfactory outcome with respect to treatment of the microvascular dysfunction. The terms “partially responsive” and “insufficiently responsive” mean that there is some effect of the existing medications, but the effect is only partial and a more effective treatment is still desired. In some embodiments described herein, the methods can further comprise measuring circulating levels ET-1 in the subject to determine an appropriate dosing regimen of the ROCK inhibitor. Inducing vasodilation against the elevated vascular tone of the microvasculature may be clinically desirable in various circumstances, including treating certain diseases or disorders, e.g., diseases or disorders that involve microvascular dysfunction caused by ET-1, as detailed above. Additional circumstances may be preventing or reducing the risk of developing certain diseases (e.g., cardiac diseases), or ameliorating certain conditions. An example of such use is the need to maintain a sufficient local blood flow in the heart to reduce the risk of developing cardiac problems. Accordingly, also provided herein are methods of inducing microvascular vasodilation in a subject that comprise administering to the subject an effective amount of a ROCK inhibitor to improve local blood flow. The term "microvascular vasodilation" as used herein refers to the widening of blood vessels of the microvasculature and is the opposite of vasoconstriction, which is the narrowing of a blood vessel. Microvascular vasodilation results from the relaxation of smooth muscle cells within the vessel walls, in particular in smaller arterioles. Because the Attorney Docket No.11164-015WO1 arterioles contribute to most of the vascular resistance that regulates local blood flow within organs and tissues, dilation of these microvessels improves local blood flow. Also provided herein are methods of reversing ET-1-induced vasoconstriction in a subject in need thereof that comprise administering to the subject an effective amount of a ROCK inhibitor. The term “vasoconstriction” as used herein relates to the narrowing of blood vessels resulting from the contraction of the muscular wall of the vessels, in particular the small arterioles, which reduces tissue blood flow. In some of the embodiments described above, the ROCK inhibitor can be administered in combination with an additional active agent, such as an agent for treating hypertension or for controlling blood pressure. Examples of such additional active agents include, for example, diuretics (e.g., thiazides), beta-blockers (e.g., atenolol), calcium channel antagonists (e.g., dihydropyridines, phenylalkylamines, and benzothiazepines), angiotensin converting enzyme (ACE) inhibitors (e.g., benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril, and ramipril), and angiotensin II receptor blockers (e.g., valsartan, telmisartan, losartan, irbesartan, azilsartan, and olmesartan). In some of the embodiments described above, administration of the ROCK inhibitor, as described herein to increase blood flow to ischemic tissue, in combination with the additional active agent can allow the additional active agent to be administered at a lower dose, even at a sub-therapeutic dose, thereby preventing or ameliorating side effects. The term "sub-therapeutic dose" as used herein means less than an amount of the additional active agent needed to produce a therapeutic effect when the additional active agent is administered alone. Many cancer therapies induce systemic hypertension with tissue ischemia and promote coronary microvascular complications. Without wishing to be bound by theory, it is believed that these cancer therapies cause arteriolar constriction by promoting ET-1-dependent activation of ROCK or by directly activating ROCK signaling. Accordingly, ROCK inhibitors can effectively reverse the adverse effect of anticancer drugs. Thus, ROCK inhibitors can be administered both to remedy hypertension and tissue ischemia, and to boost the efficacy of chemotherapy and radiotherapy that are less effective under hypoxia / ischemia. Accordingly, also provided herein are methods of treating cancer in a subject in need thereof that comprise administering to the subject an effective amount of cancer therapy in combination with an effective amount of a ROCK inhibitor. Attorney Docket No.11164-015WO1 Also provided herein are methods of improving the efficacy of a cancer therapy that comprise co-administering an effective amount of a ROCK inhibitor in combination with the cancer therapy. In some of these embodiments, administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor simultaneously with the cancer therapy. In other embodiments, administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor after administration of the cancer therapy. In other embodiments, administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor before administration of the cancer therapy. In certain embodiments, the effective amount of the ROCK inhibitor comprises an effective amount to treat or prevent a cardiovascular disease caused by the cancer therapy, such as drug-induced hypertension or drug-induced cardiotoxicity. In some of these embodiments, the cancer therapy can comprise administration of radiotherapy. As used herein, radiation therapy refers to the use of high-energy radiation for the treatment of cancer. High-energy radiation for use in radiation therapy may be provided by X- rays, gamma rays and neutrons, among others. Different methods of radiation therapy are well known in the art and are suitable for use with the methods of the invention. These methods include, but are not limited to, external beam radiation, brachytherapy, intensity-modulated radiotherapy (IMRT), implant radiation, systemic radiation and stereotactic radiotherapy. In other embodiments, the cancer therapy can comprise administration of a chemotherapeutic agent (i.e., chemotherapy). As used herein, chemotherapy refers to the administration of one or more chemical substances or drugs for the treatment of cancer. A suitable chemotherapeutic agent for use in embodiments of methods described herein can be any chemical substance known to be useful for treating cancer, for example, DNA- alkylating agents, anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial or exotoxic agents. Examples of particularly suitable agents for use in methods of the present invention include, but are not limited to, cytidine arabinoside, cytarabine, methotrexate, vincristine, Attorney Docket No.11164-015WO1 etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, taxol and derivatives thereof, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, fludarabine, docetaxel, bevacizumab, trastuzumab, FOLFOX4, bortezomib, carfilzomib, ixazomib, or any combination thereof. Also provided are methods of treating or preventing cancer treatment-induced hypertension in a subject undergoing cancer treatment that comprise administering to the subject an effective amount of a ROCK inhibitor. Also provided are methods of treating or preventing substance abuse-related cardiovascular disease in a subject that comprise administering to the subject an effective amount of a ROCK inhibitor. In some embodiments, the substance abuse-related cardiovascular disease comprises cocaine-induced cardiotoxicity. In certain embodiments, the substance abuse-related cardiovascular disease comprises cocaine-induced myocardial infarction. In some embodiments, substance abuse (e.g., cocaine) can cause coronary artery spasm and / or CMD, with evidence suggesting that ET-1 expression was elevated. Also provided are methods of treating or preventing coronary microvascular dysfunction and / or vasculopathy in a subject who has received or will receive an organ or tissue transplant that comprise administering to the subject an effective amount of a ROCK inhibitor. In some embodiments, the method can comprise administering to the subject an effective amount of a ROCK inhibitor and an effective amount of an immunosuppressive agent (e.g., tacrolimus and / or cyclosporine A). In some embodiments, the organ or tissue transplant can comprise a heart transplant. Also provided are diagnostic methods for assessing microvascular function during coronary angiogram as an add-on test to the current coronary angiogram protocol, in which adenosine (intravenous or intracoronary) or other vasodilators are administered to create hyperemia. These methods can comprise measuring coronary blood flow velocity in the subject before and after administration of a ROCK inhibitor (e.g., H-1152) (multiple doses may be used if desired); and calculating the subject’s CFR (i.e., a ratio of flow increase by a ROCK inhibitor to the resting flow without inhibitor) and IMR as the indexes of coronary vasodilatory Attorney Docket No.11164-015WO1 function. The CFR value based on intracoronary adenosine (CFRad) in a healthy normal subject is generally above 2.5; while CFRad value less than 2.0 indicates CMD. CFRad value between 2.0-2.5 is a gray zone and more clinical information will be considered for making a diagnosis. By intracoronary administration of ROCK inhibitors such as H-1152, a set of CFR based on H-1152 (CFRh) will be established to match current CFRad-based standard. At a fixed dose, subjects’ CFRh is anticipated to show a similar trend as CFRad. In some embodiments, when the subject exhibits a less-than-anticipated change in the subject’s CFR induced by administration of the ROCK inhibitor, the method further comprises administering to the subject an effective amount of a ROCK inhibitor to cause a desirable increase of blood flow. In certain examples, the less-than-anticipated changes include CFR and IMR scores corresponding to the CFRad less than 2.0 or IMRad more than 25 by using current criteria generated based on administering adenosine. Cut-off values of CFRh or IMRh under a hyperemia condition after administering H-1152 or other ROCK inhibitor will be set through clinical trials. In some embodiments, the coronary blood flow velocity in the subject can be measured invasively, for example, during a coronary angiography or angioplasty procedure. In certain embodiments, the coronary blood flow velocity in the subject is measured invasively, for example, using a surgically placed flow probe, a Doppler velocity catheter or guide wire, or a coronary sinus thermodilution catheter. In other embodiments, the coronary blood flow velocity in the subject can be measured noninvasively, for example, using MRI or PET. The subject’s coronary flow reserve is calculated as a ratio of peak diastolic flow velocity induced by a pharmacological vasodilator to resting peak diastolic flow velocity without a pharmacological vasodilator. As used herein, the phrase “effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. Effective doses will also vary depending on the route of administration, as well as the possibility of co-usage with other agents. Methods of administration include, but are not limited to, oral, parenteral, e.g., intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, mucosal (e.g., Attorney Docket No.11164-015WO1 intranasal, buccal, vaginal, rectal, intraocular, sublingual), intrathecal, intravitreal, topical, intradermal routes and through feeding tubes. The mode of administration may be systemic or local. In some embodiments, the composition is formulated for administration by an injection. In some embodiments, the composition is formulated for administration by an intravenous injection. In some embodiments, the composition is formulated for administration by an intracoronary route. In some embodiments, the composition is formulated for administration through catheters (IV catheter, peripherally inserted central catheter, central line etc.) and ports (implantable port or port-a-cath). In some embodiments, the composition is administered by an intramuscular injection. The following exemplification of carriers, modes of administration, dosage forms, etc., are listed as known possibilities from which the carriers, modes of administration, dosage forms, etc., may be selected for use with the present invention. Those of ordinary skill in the art will understand, however, that any given formulation and mode of administration selected should first be tested to determine that it achieves the desired results. The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. The determination of the doses of the active ingredient to be used for human use is based on commonly used practices in the art and will be finally determined by physicians in clinical trials. An expected approximate equivalent dose for administration to a human can be calculated based on the in vivo experimental evidence disclosed herein below, using known formulas (e.g., Reagan-Show et al. (2008) Dose translation from animal to human studies revisited. The FASEB Journal 22:659-661). According to this paradigm, the adult human Attorney Docket No.11164-015WO1 equivalent dose (mg / kg body weight) equals a dose given to a mouse (mg / kg body weight) multiplied with 0.081. In some embodiments, the composition is administered in a single dose, hereinbelow referred to as “acute administration”. In some embodiments, the composition is administered in more than a single dose such as in multiple doses, for example once a day, more than once a day such as two or three times a day, or less than once a day such as once in every two days, three days, or a week. These modes of administration are referred to hereinbelow as “chronic administration.” Pharmaceutical Compositions and Kits Also provided herein are pharmaceutical compositions and kits comprising an effective amount of a ROCK inhibitor to treat or prevent microvascular dysfunction in a subject in need thereof, an effective amount of a ROCK inhibitor to induce microvascular vasodilation in a subject, an effective amount of a ROCK inhibitor to reverse ET-1-induced vasoconstriction in a subject, and / or an effective amount of a ROCK inhibitor to treat or prevent a disease, disorder, or condition associated with microvascular dysfunction in a subject. Also provided herein are pharmaceutical compositions and kits comprising an effective amount of a ROCK inhibitor and an effective amount of a chemotherapeutic agent. Pharmaceutical compositions can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), retinal (intravitreal), oral including through feeding tubes, or parenteral. Parenteral administration includes intravenous, intraarterial, intracoronary, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, (e.g., intrathecal or intraventricular administration). Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. In some embodiments, the compounds provided herein, or a pharmaceutically acceptable salt thereof, are suitable for parenteral administration. In some embodiments, the compounds provided herein are suitable for intravenous administration. In some embodiments, the compounds Attorney Docket No.11164-015WO1 provided herein are suitable for oral administration. In some embodiments, the compounds provided herein are suitable for topical administration. Pharmaceutical compositions and formulations for topical administration may include, but are not limited to, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. In some embodiments, the pharmaceutical compositions provided herein are suitable for parenteral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for intravenous administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for oral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for topical administration. Also provided are pharmaceutical compositions which contain, as the active ingredient, a compound provided herein in combination with one or more pharmaceutically acceptable carriers (e.g., excipients). In making the pharmaceutical compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be, for example, in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. Some examples of suitable excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include, without limitation, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy- benzoates; sweetening agents; flavoring agents, or combinations thereof. The active compound can be effective over a wide dosage range and is generally administered in an effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of Attorney Docket No.11164-015WO1 administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject’s symptoms, and the like. The compositions provided herein can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound described herein can include a single treatment or a series of treatments. Dosage, toxicity and therapeutic efficacy of the compounds provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds exhibiting high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects. Rho Kinase (ROCK) Inhibitors As used herein the term "Rho-associated protein kinase", “Rho kinase” or "ROCK" has its general meaning in the art. ROCK is a member of the serine-threonine protein kinase family. ROCK has been identified as an effector molecule of RhoA, a small GTP-binding protein (G protein) that plays a key role in multiple cellular signaling pathways. ROCK exists in two isoforms, ROCK1 and ROCK2. ROCK1 and ROCK2 are highly homologous with an overall amino acid sequence identity of 65%. The methods described herein can involve administration of a ROCK inhibitor. Likewise, the compositions and kits described herein can include a ROCK inhibitor. As used herein, the term "ROCK inhibitor" refers to a natural or synthetic compound which inhibits ROCK1, and / or ROCK2 activity. In some embodiments, the ROCK inhibitor can be selective. Attorney Docket No.11164-015WO1 Selective ROCK inhibitors can display increased inhibitory activity towards one ROCK isoform as compared to another ROCK isoform. For example, a “selective” Rho kinase 1 (ROCK1) inhibitor refers to a ROCK1 inhibitor that has at least 2, 5, 10, 20, 50, 100, or 200- fold greater inhibitory activity (for example, as determined by calculation of IC50, Ki, or other measure of affinity or effect) for a particular isozyme of ROCK1 compared to other members of the ROCK family. Similarly, a “selective” Rho kinase 2 (ROCK2) inhibitor refers to a ROCK2 inhibitor that has at least 2, 5, 10, 20, 50, 100, or 200-fold greater inhibitory activity (for example, as determined by calculation of IC50, Ki, or other measure of affinity or effect) for a particular isozyme of ROCK2 compared to other members of the ROCK family. For example, in some embodiments, the ROCK inhibitor can selectively inhibit ROCK1 activity over ROCK2 activity. In other embodiments, the ROCK inhibitor can selectively inhibit ROCK2 activity over ROCK1 activity. In some embodiments, the ROCK inhibitor can inhibit both ROCK1 activity and ROCK2 activity with similar capability. ROCK inhibitors are well known in the art. For example, isoquinoline derivatives, especially fasudil, are typical ROCK inhibitors. Fasudil (hexahydro-l-(5- isoquinolylsulfonyl)- lH-l,4-di-azepime), also named as HA-1077, is an isoquinoline sulfonamide derivative and the only clinically available ROCK inhibitor for treating cerebral vasospasm, which is often due to subarachnoid hemorrhage. However, this drug is not approved in the USA or the EU. It is co- developed by Asahi Kasei of Japan and Department of Pharmacology of Nagoya University. Hydroxyfasudil is an active metabolite of fasudil in vivo, which has a higher affinity to ROCK than fasudil. Another isoquinoline derivative, H-1152 (aka H-1152P), is optimized based on fasudil. Through competitively binding to the ATP binding pocket, Y-27632, another type of ROCK inhibitor, inhibits both ROCK1 and ROCK2. Optimization of these compounds leads to a more potent ROCK inhibitor, Y-39983, which is a benefit for the treatment of the glaucoma (Kubo T, Yamaguchi A, Iwata N, The therapeutic effects of Rho-ROCK inhibitors on CNS disorders. Ther Clin Risk Manag 2008;4(3):605-15). SLx-2119, a ROCK2-specific inhibitor, has recently been developed (Boerma M, Fu Q, Wang J, Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin. Blood Coagul Fibrinolysis 2008;19(7):709-18). A series of fasudil analogs were synthesized and their selectivity and inhibitory activity against ROCK were evaluated (Satoh N, Toyohira Y, Itoh H, Stimulation of norepinephrine transporter function by fasudil, a Rho kinase inhibitor, in cultured bovine adrenal medullary cells. Naunyn Schmiedebergs Arch Attorney Docket No.11164-015WO1 Pharmacol 2012;385(9):921-31; Nakabayashi S, Nagaoka T, Tani T, Retinal arteriolar responses to acute severe elevation in systemic blood pressure in cats: role of endothelium-derived factors. Exp Eye Res 2012; 103:63-70; Sun X, Minohara M, Kikuchi H, The selective Rho- kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune encephalomyelitis. J Neuroimmunol 2006;180(l-2): 126-34; Yu JZ, Ding J, Ma CG, Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil, a Rho kinase inhibitor. J Neurosci Res 2010;88(8): 1664-72; Hou SW, Liu CY, Li YH, Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through anti-inflammatory effect. CNS Neurosci Ther 2012;18(11):909-17; LoGrasso PV, Feng Y. Rho kinase (ROCK) inhibitors and their application to inflammatory disorders. Curr Top Med Chem 2009;9(8):704-23; Engel J Jr. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classification and Terminology. Epilepsia 2001;42(6):796-803; Fisher RS, van Emde Boas W, Blume W, Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 2005;46(4): 470-2. Inan S, Buyukafsar K. Antiepileptic effects of two Rho-kinase inhibitors, Y-27632 and fasudil, in mice. Br J Pharmacol 2008; 155(1):44-51; Meihui Chen, Anmin Liu, Ying Ouyang, Yingjuan Huang, Xiaojuan Chao, Rongbiao Pi Fasudil and its analogs: a new powerful weapon in the long war against central nervous system disorders? Expert Opin. Investig. Drugs 2013; 22: 537-550.). Two ROCK inhibitors (ripasudil and netasudil) were approved as eyedrops to treat glaucoma and ocular hypertension in Japan and the USA, respectively. The fourth marketed ROCK inhibitor, belumosudil was approved for treating chronic graft versus host disease. None of these medications are approved for treating microvascular diseases. Other examples of ROCK inhibitors include those described in the international patent publications WO98 / 06433, WO00 / 09162, WO00 / 78351, WO01 / 17562, WO02 / 076976, EP1256574, WO02 / 100833, WO03 / 082808, WO2004 / 009555, WO2004 / 024717, WO2004 / 108724, WO2005 / 003101, WO20Q5 / 035501, WO2005 / 035503, WO2005 / 035506, WO2005 / 058891 , WO2005 / 074642, WO2005 / 074643, WO2005 / Q80934, WO2005 / 082367, WO2005 / 082890, WO2005 / 097790, WO2005 / 100342, WO2005 / 103050, WO2005 / 105780, WO2005 / 108397, WO2006 / 044753, WO2006 / 051311, WO2006 / 057270, WO2006 / 058120 , WO2006 / 072792WO2011107608A1, and WO2007026920A2, each of which is hereby incorporated by reference. Attorney Docket No.11164-015WO1 Other examples of ROCK inhibitors are described in Feng, Y. et al. Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential. J Med Chem.2016;59(6):2269-2300. In some embodiments the ROCK inhibitor can be an isoquinoline / isoquinolinone-based ROCK inhibitor. In some embodiments, the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethyl fasudil, a fasudil-based hybrid compound, H- 1152, H-1152P, H-1152 dihydrochloride, ripasudil, netarsudil, belumosudil, SAR407899, a prodrug thereof, a salt thereof, or a combination thereof. In some embodiments, the ROCK inhibitor can be selected from the group consisting of AR-12286, AT-13148, BA-1049 (R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, a prodrug thereof, a salt thereof, or a combination thereof. In some embodiments, the ROCK inhibitor can be defined by Formula I wherein L is O or SO2; R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R3is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R5is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— Attorney Docket No.11164-015WO1 NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; R7is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R8is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; n is 1, 2, 3 or 4; m is, independently for each occurrence, 1, 2, 3, or 4; R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; and R″ is (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts. In some embodiments, the ROCK inhibitor can comprise an isoquinoline sulfonamide. In some embodiments, the ROCK inhibitor can be defined by Formula IA wherein R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; and Attorney Docket No.11164-015WO1 R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts. In some embodiments, the ROCK inhibitor can comprise fasudil, hydroxyfasudil, dimethyl fasudil, H-1152, Y-27632, Y-30141, Y-32885, Y-39983, ripasudil, netarsudil, RKI- 1447, GSK429286A, belumosudil, AT-13148, BA-1049 (R), or any combination thereof. In some embodiments, the ROCK inhibitor can comprise fasudil, H-1152, ripasudil, RKI-1447, Y-27632, GSK429286A, Y-30141, or any combination thereof. In some embodiments, the ROCK inhibitor can comprise H-1152, fasudil, dimethyl fasudil, Y-27632, or any combination thereof. In some embodiments, the ROCK inhibitor can comprise fasudil (HA-1077), the structure of which is shown below. In some embodiments, the glycyl-H 1152, the structure of which is shown below. In some embodiments, H 1152 or a salt thereof, such as H-1152 dihydrochloride, the structure of which is shown below. Attorney Docket No.11164-015WO1 In some embodiments, the ROCK inhibitor can comprise ripasudil, the structure of which is shown below. In some embodiments, the ROCK inhibitor can comprise belumosudil, the structure of which is shown below. In some netarsudil, the structure of which is shown below. In some the structure of which is shown below. In some embodiments, BA-1049, the structure of which is shown below. Attorney Docket No.11164-015WO1 In some SAR407899, the structure of which is shown below. In some embodiments, the ROCK inhibitor can comprise hydroxyfasudil, the structure of which is shown below. HN N In some embodiments, the dimethyl fasudil, the structure of which is shown below. N In some embodiments, the Y-27632, the structure of which is shown below. Attorney Docket No.11164-015WO1 In some embodiments, the Y-30141, the structure of which is shown below. In some embodiments, the comprise Y-32885, the structure of which is shown below. In some embodiments, the can comprise Y-39983, the structure of which is shown below.

[0002] Attorney Docket No.11164-015WO1 In some embodiments, the comprise RKI-1447, the structure of which is shown below. In some the structure of which is shown In some embodiments, the ROCK inhibitor can comprise AT-13148, the structure of which is shown below.

[0003] Attorney Docket No.11164-015WO1 In some embodiments, the ROCK inhibitor can comprise PHP-201 (sovesudil), the structure of which is shown below. In some embodiments, the ROCK inhibitor can comprise a hybrid fasudil derivative defined by the formula below, where R is H or methyl, and n is 1 or 2. Such compounds are described, for example, in J. Med. Chem. 2022, 65, 3, 1867–1882. EXAMPLES The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results. Example 1: Rho Kinase (ROCK) Inhibition for Microvascular Disease Treatment All tissues need blood circulation for proper function by providing sufficient oxygen / nutrients and removing harmful wastes. The microvascular network exerts this important function through the regulation of blood flow to the tissue by arterioles, maintenance of fluid / substance exchange in capillaries, and drainage of blood from venules. The small arterioles (<100 µm in diameter) are the “gatekeeper” that control and regulate tissue blood flow by vasoconstriction and vasodilation. Excessive vasoconstriction and prolonged dysregulation, as seen in many cardiovascular diseases, can lead to tissue ischemia / hypoxia and subsequent organ damage. In the heart, the clinical presentation of this problem is chest pain Attorney Docket No.11164-015WO1 (angina pectoris) and heart failure. However, currently, there is no FDA-approved effective medication for treating microvascular dysfunction-associated ischemia. In many forms of cardiovascular disease (CVD) associated with ischemia, as well as inflammation, the ET-1 level either in local tissue or systemic plasma is elevated. In this Example, we demonstrate that ET-1 is the fundamental culprit causing preferential arteriolar constriction, which is not effectively relieved by currently available medications. Based on our data, some clinical observations are well explained. These observations include that calcium channel blocker (e.g., nifedipine), and direct nitric oxide releasing nitrates at clinically relevant concentrations do not always relieve microvascular angina; nor clinical development of ET-1 receptor antagonists shown clinical benefit in CMD filed. More importantly, we also demonstrate that the vasoconstrictor action of ET-1 is mediated by ROCK activation and that inhibition of ROCK (e.g., using a specific inhibitor such as H-1152) consistently produces vasodilation and abolishes vascular adverse effects of ET-1 on microvessels. Our data support that ROCK inhibitors, such as H-1152, can be the solution to this unmet medical need. Overview The activation of ROCK signaling and protein kinase C (PKC) pathways by ET-1 has been linked to the development of various cardiovascular diseases, including vascular pathophysiology. Although PKC activation by ET-1 at the level of the macrocirculation (i.e., vessels greater than 300 µm in diameter) has been reported, it remains undetermined whether PKC signaling is responsible for the vasoconstriction to ET-1 in the resistance arterioles (i.e., vessels less than 100 µm in diameter) of the microvascular domain where tissue blood flow is dominantly regulated. In this Example, we describe our methods of studying the coronary arteriolar microvessels, which naturally display resting basal tone without vasoconstrictor(s). We then present data establishing the signaling mechanisms of ET-1 through ROCK activation, independent of PKC signaling, in coronary arteriolar constriction. These data support the idea that ROCK inhibitors, such as H-1152, can be used to specifically treat ET-1-associated vascular dysfunction, especially at the microvascular level. Materials and Methods Videomicroscopic Techniques for Assessing Microvascular Function. To directly study the microvessel activity and reactivity without confounding influences from changes in systemic parameters such as pressure, flow, metabolic activity, and neurohumoral controls, the microvessels are isolated, cannulated, and pressurized for in vitro study. This technique also Attorney Docket No.11164-015WO1 allows the pressure and flow to be independently controlled, which is not feasible with in vivo preparations. The intravital microscopic technique that was used to assess the arteriolar function is shown in Figure 1A. The arterioles (30-100 µm in diameter) were isolated from organs of interest and then cannulated with a pair of glass micropipettes (i.e., inflow and outflow pipette). The arterioles were pressurized to 60 cm H2O (~44 mmHg) with a dual-reservoir system to mimic the pressure environment found in the microcirculation in vivo. At 36-37°C physiological salt solution (PSS) in the vessel chamber, the vessel gradually constricted to a stable level of 50-60% of its maximum diameter within 60 minutes. This state of vasoconstriction at the resting level is called “basal tone.” The development of spontaneous basal tone under PSS, without adding any pharmacological vasoconstrictors, is one of the essential / necessary criteria to verify the viability of the vessel in our preparations (see left inset-block of the diagram in Figure 1A). This characteristic is the intrinsic nature of the microvessels, as seen in alive animals, to allow vasodilation to occur when tissue needs to recruit blood flow or to further constrict when the tissue is over-perfused. This unique behavior is not seen in large conduit vessels, which exhibit little basal tone for blood flow regulation. Another viability criterion is that the isolated microvessels need to exhibit vasodilation to the endothelium-dependent vasodilators such as bradykinin or serotonin to ensure the functional integrity of the endothelium. The intact vascular smooth muscle function is verified by the dilation of the vessel to a smooth muscle- specific vasodilator sodium nitroprusside. Under our experimental conditions, the isolated microvessels display natural characteristics and behavior as observed in vivo. With this setup, the luminal pressure can be changed without flow alteration by simultaneously moving (elevating or lowering) both reservoirs in the same direction to the desired hydrostatic pressure. To elicit flow without changes in luminal pressure, the reservoirs are set at the desired pressure as described above and then the reservoirs are moved in an opposite direction to create a longitudinal pressure gradient across the vessel segment. The degree of flow (or shear stress) is determined by the amount of pressure gradient applied to the vessel. In some studies, the intraluminal pressure of the vessel was recorded using the servo- null micropressure technique to ensure a constant luminal pressure during flow induction (Figure 1A). The agonist (or antagonist) is prepared in the vehicle solution and administered at 20 µL in volume cumulatively or with a single application to the vessel chamber with 2 mL in Attorney Docket No.11164-015WO1 volume. The vessel chamber was flushed with the new solution every 20-30 minutes to ensure the osmolarity and chemical concentrations of the bath solution were not changed due to water evaporation with time. Animals. Domestic pigs (8-12 weeks old of either sex; 7-10 kg) purchased from Barfield Farms (Rogers, TX) were sedated with Telazol (4.4 mg / kg, i.m.) and Xylazine (2.2 mg / kg, i.m.), and anesthetized with 2-4% isoflurane. Heparin (1,000 units / kg) was administered into the marginal ear vein to prevent clotting. The heart and eyes were removed and immediately placed in a saline chamber on ice for further vessel dissection and isolation. It is well documented that the physiology, histology, and pathophysiology of the cardiovascular system, including coronary and ophthalmic circulations, resemble humans. The data obtained from the pig model are better representative of human vascular biology and applicable to translational medicine. Isolation and Cannulation of Microvessels. Single coronary or retinal arterioles (0.6 to 1.0 mm in length) were carefully dissected out in a tissue chamber containing physiological salt solution (PSS: in mmol / L NaCl 145.0, KCl 4.7, CaCl22.0, MgSO41.17, NaH2PO41.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS buffer 3.0) using a pair of Dumont microdissection forceps (Fine Science Tools, Foster City, CA) with the aid of a stereomicroscope (model SZX12, Olympus, Melville, NY). After careful removal of any remaining connective tissues, the arteriole was then transferred for cannulation to a Lucite vessel chamber containing PSS with 1% albumin (USB, Cleveland, OH). One end of the arteriole was cannulated using a glass micropipette filled with PSS-albumin solution, and the outside of the arteriole was securely tied to the pipette with 11-0 ophthalmic suture (Alcon, Fort Worth, TX). The other end of the vessel was cannulated with a second micropipette and secured with a suture. After cannulation, the vessel and pipettes were transferred to the stage of an inverted microscope (model CKX41, Olympus) coupled to a video camera (Sony DXC-190, Labtek, Campbell, CA), video micrometer (Cardiovascular Research Institute, Texas A&M Health Science Center, College Station, TX) and PowerLab data acquisition system (ADInstruments, Colorado Springs, CO) for continuous measurement and recording of the internal diameter throughout the experiment (Figure 1A). The cannulating pipettes were connected to independent pressure reservoirs. By adjusting the height of the reservoirs, the vessel was pressurized to 55-60 cmH2O (40-44 mmHg) intraluminal pressure without flow. This level of pressure was used based on pressure ranges that have been documented in Attorney Docket No.11164-015WO1 arterioles in vivo. Arterioles with side branches and leaks were excluded from further study and all arterioles used developed spontaneous basal tone. At the end of each functional experiment, the vessel was relaxed with 0.1 mmol / L sodium nitroprusside in EDTA (1 mmol / L)-Ca2+-free PSS to obtain its maximal diameter at 55-60 cmH2O intraluminal pressure. Immunoblots and Immunohistochemistry. The vascular proteins were isolated from the intact conduit coronary artery (left anterior descending branches) and downstream arterioles (<100 µm in diameter) from pigs unless stated otherwise. For the study of protein kinase phosphorylation, the microvessels were pressurized and pharmacological interventions were applied under the same protocols as studying vasomotor function described above. Then the vessels were snap-frozen for further protein / enzyme analysis. In some studies, the vascular cells in primary culture were obtained from freshly isolated vessels. The cultured cells were lysed into a radioimmunoprecipitation assay lysis buffer. Tissues were ground using pellet pestles (Kontes) with two grinding cycles. Lysate supernatants were collected after spinning at 12,000g rpm at 4°C for 20 minutes. For protein assay, the same amount of protein (5-10 μg / well) was loaded and further separated by 4-15% gradient SDS-PAGE gel (Bio-Rad, Hercules, CA), then transferred onto nitrocellulose membrane (Bio-Rad) for immunoblot. For pressurized vessel studies, 2-4 arterioles were collected and quickly snap-frozen in liquid nitrogen before being lysed for protein isolation. The protein samples were loaded to run SDS- PAGE gel, and protein signal strength was normalized by smooth muscle actin (SMA; Sigma- Aldrich, St. Louis, MO), unless stated otherwise. The following antibodies were purchased from Santa Cruz Biotechnology, Dallas, TX: ROCK1, ROCK2, pCPI-17, CPI-17, GAPDH, tropomyosin, HRP labeled anti-rabbit and anti-mouse secondary antibodies. The pMYPT1- Thr850 antibody was purchased from Millipore, Billerica, MA. The antibodies pMLC Ser19 and MLC were purchased from Cell Signaling Technology, Danvers, MA. SuperSignal West Pico Chemiluminescent Substrate was purchased from Thermo Scientific (Waltham, MA). For immunohistochemistry analysis, the isolated vessels and cardiac tissues were subjected to frozen sections (10-µm thickness) and immunostaining. Fluorescence-labeled secondary antibodies were purchased from Jackson Laboratory (Bar Harbor, Maine). Analysis. The vasomotor response was analyzed as the ratio of diameter changes (in response to agonist) vs. maximum dilation capacity (exposure of the vessel to 0.1 mmol / L sodium nitroprusside in the absence of extravascular calcium) as “% Maximum Dilation,” or the vessel diameter was normalized with the resting diameter and presented as “% Resting Attorney Docket No.11164-015WO1 Diameter.” Data are presented as mean±SEM. The one-way or two-way ANOVA with Tukey post hoc analysis was used to determine the statistical significance of the result of the intervention, when appropriate. The significance of changes in protein expression was analyzed with Student's t-test. P < 0.05 is considered statistical significance. Technical Note: Due to technical challenges in studying microvessels, e.g., visualizing / imaging / isolating, network complexity, and the limited available tissue for molecular characterization, the advance in microvascular research has been either linear or stagnating over the past decade. Consequently, the knowledge on the mechanism of microvascular dysregulation was mainly extrapolated from studies in large conduit vessels or cultured vascular / non-vascular cells. Furthermore, the microvessels are susceptible to physical manipulations, and great care during isolation preparation is necessary to preserve viability and vasomotor function such as the development of spontaneous basal tone. Notably, these studies are performed in microvessels less than 100 µm in diameter where blood flow is dominantly regulated. Thus, the data presented in this document were from microvessels that exhibited spontaneous basal tone (i.e., without adding any pharmacological preconstrictors). Other laboratories have commonly used pharmacological vasoconstrictors such as a thromboxane analog (U46619), prostaglandin F2α, KCl, and ET-1, to induce an “artificial basal tone” in vessels that fail to develop a spontaneous basal tone. These preconstrictors can lead to inaccurate information on vasoreactivity and responsiveness and also evoke confounding signaling molecules because of triggering / mingling / superimposing of additional pathways that mask or alter the original vascular behavior and signal transduction. This concern is especially true in the study of large arteries because they do not display a significant level of basal tone or in microvessels that fail to develop basal tone. Therefore, preconstrictors are consequently added to study the vasodilation mechanisms. Here, in our preparations, the arterioles (< 100 µm in diameter) isolated from the heart, brain, retina, skeletal muscle, or mesentery develop spontaneous basal tone without using any pharmacological constrictors, and these vessels exhibit normal dilation to endothelium-dependent agonists (bradykinin or serotonin) mimicking vasomotor behavior observed in vivo. These criteria are prerequisites for every study we have performed. As discussed above, the information obtained from the large vessels might not be appropriate to extrapolate to the microvessels because of the inherent differences in vascular Attorney Docket No.11164-015WO1 behavior and function at cellular and molecular levels. These differences may highlight the reason for the failure of treatment of microvascular diseases by using marketed medications that are not intended to be used for this purpose. Understanding the molecular mechanism, corresponding to exerted microvascular function, is important for designing effective therapy for these unmet clinical needs. Characterization of Coronary Arteriolar Constriction in Response to ET-1: Role of Calcium and ET-1 Receptors ET-1’s vasomotor effect is through binding its receptors (ETAand ETBsubtypes). Many studies have shown that arterial constriction, including of the coronary arteries, is mediated by the release of intracellular calcium (Ca2+) from the sarcoplasmic reticulum following ETAreceptor activation. Moreover, ET-1 can also elicit endothelial nitric oxide (NO) or prostacyclin release to exert vasodilation via endothelial ETB receptor activation. However, these findings are derived from large conduit vessels without spontaneous basal tone, which required preconstriction with pharmacological agonists (e.g., high concentration of KCl, U46619, PDBu, etc.). It is unclear whether the same vasomotor mechanisms observed in large conduit vessels can be extrapolated or adopted to the small coronary arterioles. To address these questions, the pig coronary arterioles (< 100 µm in diameter) were isolated and pressurized for functional study in the presence of spontaneous basal tone. The studies were designed to determine the role of extracellular versus intracellular Ca2+in vasomotor responses to ET-1 and to identify the responsible receptor subtypes for this response. As shown in Figure 2A, we first characterized the coronary arteriolar response to ET-1. This is the first investigation on the constriction of such small isolated coronary arterioles (<100 µm) to ET-1 with spontaneous resting basal tone (R). Control arterioles isolated from pigs developed stable basal tone (spontaneously constricted from the maximum diameter 73±3 µm to 40±4 µm) in the presence of PSS-albumin (containing normal 2.0 mmol / L Ca2+at 60 cmH2O luminal pressure) solution. ET-1 caused coronary arteriolar constriction in a concentration-dependent manner with a threshold concentration of 0.1 pmol / L. The coronary arterioles almost shut down their lumens by constricting to about 10%-15% of resting diameter in response to the highest concentration tested (10 nmol / L). Another group of coronary arterioles developed a similar level of basal tone (42±5 µm resting diameter) in the presence of PSS-albumin with Ca2+solution and then dilated from resting diameter to a maximum diameter 84±5 µm after changing the vessel bath to a Ca2+-free solution (containing 1 mmol / L EDTA in Attorney Docket No.11164-015WO1 PSS). These dilated arterioles (in the absence of extracellular Ca2+) did not constrict to ET-1, except at the highest concentration (10 nmol / L). The data indicate that the extracellular Ca2+entry is necessary for the coronary arterioles to develop spontaneous basal tone and for their response to ET-1 at the tested concentrations. It should be noted that the studies on coronary arterial constriction from other labs showed a threshold constriction response greater than 1 nmol / L ET-1. Our coronary arterioles are at least 100-fold more sensitive and responsive to ET-1 than those previously recorded. The normal plasma ET-1 concentration in vivo is about 1-3 pmol / L and blocking this endogenous ET-1 in vivo with its specific receptor blocker causes a decrease in coronary resistance and an increase in coronary blood flow, indicating that this endogenous level of ET-1 (1-3 pmol / L) causes coronary arteriolar constriction. Our in vitro data demonstrated that 1 pmol / L ET-1 is sufficient to evoke a small degree of coronary arteriolar constriction (Figure 2A). This result also indicates that the arterioles in our studies preserve the viability and responsiveness to ET-1 as observed in the in vivo intact heart. Most importantly, previous studies of ET-1 with concentrations greater than or equal to 1 nmol / L are un-physiological and even supra- pathophysiological because reported plasma ET-1 in patients with coronary or other diseases hardly exceeds 50 pmol / L, taking into consideration that the estimated interstitial ET-1 concentration could be 5-fold higher than that in the plasma. It should be noted that the diameter changes were recorded at the end of 5 minutes after exposure to each concentration of ET-1 because the solution in the vessel chamber needs to be refreshed every 30-40 minutes to avoid the rising osmolarity of the bath solution, which can cause a confounding effect on the vasomotor activity. Therefore a 5-minute time exposure for each concentration of ET-1 was chosen for establishing this concentration-dependent vasoconstrictor response. Figure 2B is a plot showing time-courses of coronary arteriolar constriction in response to ET-1 (0.1 and 10 nmol / L) from resting (R) diameters (0.1 nmol / L ET-1: 45±1 µm; 10 nmol / L ET-1: 51±3 µm). It takes about 10-20 minutes to fully develop vasoconstriction, and the constriction lasts at least 1 hour. The data indicate that ET-1 elicits slow but sustained coronary arteriolar constriction. For all the rest of the studies, arteriolar diameters were documented at 20 minutes with ET-1 exposure unless specified. As shown in Figure 2C, the role of extracellular and intracellular Ca2+in vasoconstriction to ET-1 was determined by using 2-aminoethoxydiphenyl borate (2-APB), an Attorney Docket No.11164-015WO1 inhibitor of intracellular Ca2+release from the sarcoplasmic reticulum via IP3 signaling and the store-operated Ca2+channels. First, the coronary arterioles were exposed to the Ca2+-free solution (containing 1 mmol / L EDTA in PSS) to eliminate the contribution of extracellular Ca2+to vasoconstriction. The vessels lost their basal tone (45±5 µm resting diameter; n=5) and dilated maximally in the absence of bath Ca2+.*P < 0.05 versus percent resting diameter at R. Administration of 0.1 nmol / L ET-1 (at “zero” time) failed to evoke vasoconstriction. In contrast, in the second group of vessels (44±6 µm resting diameter; n=8), administration of 10 nmol / L ET-1 evoked gradual vasoconstriction, stabilizing within 15-20 minutes and yielding about 40% reduction in diameter in the absence of extracellular Ca2+.#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 In the third group of vessels (40±5 µm resting diameter; n=7), administration of 2-APB (100 µmol / L) in the presence of Ca2+-free PSS abolished vasoconstriction to 10 nmol / L ET-1. This result indicates that the high concentration of ET-1 (10 nmol / L) evokes an additional vasocontraction mechanism by activating the release of intracellular Ca2+. These supra-high concentrations of ET-1 (> 1 nmol / L) are commonly used for the study of ET-1 signaling for vasoconstriction or other bioactivities either in large conduit vessels or in so-called “small resistance arterioles” in vivo or in vitro, including cell cultures. Therefore, these activated “extra” pharmacological activities render the interpretation of physiological / pathological action of ET-1 difficult because the activated release of intracellular Ca2+can trigger and participate in numerous signaling events and bioactivities unrelated to the initial extracellular Ca2+-dependent vasoconstriction. To precisely determine the impact of ET-1 on the microcirculation (< 100 µm in diameter) and to delineate its underlying mechanism, we emphasize the importance of utilization of the concentration of ET-1 at ≤ 0.1 nmol / L for physiological and pathophysiological interpretations. As shown in Figure 2D, the involved ET-1 receptors in coronary arteriolar constriction to ET-1 were assessed using the specific ETAand ETBreceptor blockers BQ123 and BQ788, respectively. In the absence of ET-1 receptor blockers, ET-1 (0.1 nmol / L) caused a gradual constriction of control vessels (39±3 µm), stabilizing within 15-20 minutes to about 60% of resting (R) diameter.*P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 minutes). Pretreatment of coronary arterioles with BQ123 (1 µmol / L) for 20 minutes did not alter the resting diameter (43±3 µm) but abolished the vasoconstriction to ET-1 (0.1 nmol / L). Administration of BQ788 (0.1 µmol / L) to the vessels did not affect the resting Attorney Docket No.11164-015WO1 diameter (41±4 µm) or ET-1-induced vasoconstriction. The efficacy of BQ788 was verified by blocking the vasoconstriction in response to sarafotoxin S6c (ETBreceptor agonist) in a separate study. These results indicate that the coronary arteriolar constriction evoked by ET-1 is mediated by the activation of ETA receptors and the role of ETB receptors is minimal, if any, in these small coronary arterioles. On the other hand, the role of endothelial ETBreceptors is more apparent in the upstream large arteries. These results show the unique vasomotor signaling in small coronary arterioles different from large upstream parent vessels. In sum, the Ca2+influx from the extracellular space is responsible for the vasoconstriction evoked by a physiological / pathophysiological concentration of ET-1 (0.1 nmol / L). The supra pharmacological concentrations of ET-1 (> 0.1 nmol / L) appear to also trigger Ca2+release from the internal store (i.e., sarcoplasmic reticulum) to elicit vasoconstriction. Therefore, different ET-1 concentrations elicit different signal pathways for vasoconstriction. The physiological / pathophysiological concentrations of ET-1 (≤ 0.1 nmol / L) do not evoke Ca2+release from internal stores. The vasoconstriction elicited by ET-1 is mediated by the activation of ETAreceptors solely. Unlike large conduit arteries, the activation of endothelial ETBreceptors for vasodilation is not apparent in coronary arterioles. Because ET-1 concentrations in vivo (low pmol / L range) are much lower than what has been used in numerous previous studies (> 1 nmol / L) in large or small arteries, the vasoconstriction mechanisms identified in those studies are not applicable to the microvessels for both physiological and pathophysiological considerations. Differential Extracellular Ca2+Entry in Coronary Arteriolar Constriction to ET-1 and PKC Activation Studies have shown that large arteries constrict to PDBu, a direct PKC activator and partially depend on extracellular Ca2+. The relative contribution of extracellular Ca2+to PDBu- induced constriction of coronary arterioles remains unknown. It is also unclear whether ET-1 and PDBu can activate the same Ca2+-entry channels for coronary arteriolar constriction. These questions are important in terms of helping the understanding of the mechanism of ET-1 versus PKC activation for coronary arteriolar constriction, since activation of these signaling pathways contributes to the development of various cardiovascular diseases. The L-type and T-type voltage-operated Ca2+channels were the focus because their inhibitors are commonly used for treating patients with systemic hypertension. Attorney Docket No.11164-015WO1 As shown in Figure 3A, isolated coronary arterioles developed spontaneous basal tone and were exposed to different inhibitors (Ca2+-free solution, L-type Ca2+channel blocker nifedipine, or T-type Ca2+channel blocker NNC 55-0396) for 20 minutes before ET-1 (0.1 nmol / L) administration (at “zero” time). This approach was used to examine whether pretreatment of these inhibitors could prevent ET-1-induced vasoconstriction. The data showed that coronary arterioles (45±5 µm resting diameter) lost their resting basal tone (R) and failed to respond to ET-1 under Ca2+-free conditions, indicating the essential role of extracellular Ca2+in maintaining basal tone and the continuous Ca2+entry into the vessel to exert vasoconstriction in response to ET-1. In the normal PSS, the resting basal tone (44±4 µm diameter) was inhibited by 1 µmol / L nifedipine and by 1 µmol / L NNC 55-0396, indicating that Ca2+entry from L- and T-type Ca2+channels in maintaining basal tone. However, unlike in the Ca2+-free solution, the ET-1-induced vasoconstriction was still developed in the presence of Ca2+channel blockers with a moderately greater inhibition by NNC 55-0396. These data indicate that Ca2+entry via voltage-operated channels plays a small role in the initiation of ET-1-induced vasoconstriction if any. In Figure 3A,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at time of addition of ET-1 (0 minutes). As shown in Figure 3B, isolated coronary arterioles developed spontaneous basal tone and were exposed to ET-1 to evoke vasoconstriction, then different inhibitors (Ca2+-free, nifedipine, NNC 55-0396) were applied to the vessels to test whether this vasoconstriction can be reversed. This approach was used to examine whether these inhibitors can reverse the vasoconstriction that has already fully developed to mimic the prolonged vasoconstriction in disease states. The result showed that ET-1 (0.1 nmol / L) evoked coronary arteriolar constriction by reducing about 40% of their resting (R) diameter. This vasoconstriction (from 42±2 µm resting diameter) was fully reserved, and reached maximal diameter, by exposing the vessels to the Ca2+-free solution. In contrast, both nifedipine (with 46±3 µm resting diameter) and NNC 55-0396 (with 42±2 µm resting diameter) at 1 µmol / L only partially reversed the vasoconstriction, with a better inhibition by NNC 55-0396. These data indicate that a continuous Ca2+entry is essential to maintain ET-1-evoked vasoconstriction and that T-type Ca2+channels may play some role in this regard. On the other hand, the L-type Ca2+channels do not appear to play a significant role in maintaining vasoconstriction, because the effect of nifedipine on vasoconstriction is minimal besides its apparent inhibition of resting vessel tone. Attorney Docket No.11164-015WO1*P < 0.05 versus percent resting diameter at R. In Figure 3B,#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 minutes). As shown in Figure 3C, the roles of Ca2+entry and L- and T-type Ca2+channels in PDBu-induced vasoconstriction were investigated and compared with the ET-1 response. Isolated coronary arterioles developed spontaneous basal tone and then were exposed to different inhibitors (Ca2+-free, L- nifedipine, NNC 55-0396) for 20 minutes before PDBu (0.1 µmol / L) administration (at “zero” time). In the Ca2+-free bathing solution, the arterioles (47±2 µm resting diameter) dilated to their maximum diameter and failed to constrict to PDBu. This result indicates that the vasoconstriction in response to PKC activation, similar to ET-1, requires extracellular Ca2+entry. Similarly, pretreatment with nifedipine (1 µmol / L; 39±4 µm resting diameter) or NCC 55-0396 (1 µmol / L; 40±5 µm resting diameter) under normal level of Ca2+in PSS caused the vessels to lose basal tone and abolished vasoconstriction to PDBu (0.1 µmol / L). These data not only demonstrate the efficacy of Ca2+channel inhibitors nifedipine and NNC 55-0396 but also indicate the opening of L-type and T-type Ca2+channels in mediating the development of basal tone and the initiation of vasoconstriction in response to PDBu. This vasoconstriction mechanism appears to be different from that induced by ET-1. In Figure 3C,*P < 0.05 vs. percent resting diameter at R. To investigate the role of Ca2+entry and L- and T-type Ca2+channels in maintaining PDBu-induced vasoconstriction, coronary arterioles with basal tone were constricted with PDBu (0.1 µmol / L) and then different inhibitors (Ca2+-free, nifedipine, NNC 55-0396) were applied to the vessels to examine whether the constriction can be reversed. As shown in Figure 3D, the PDBu caused vasoconstriction by reducing the resting basal diameter (R; 40±3 µm) by about 50%. This vasoconstriction was reversed by a Ca2+-free solution, 1 µmol / L nifedipine, and 1 µmol / L NNC 55-0396. These findings indicate that continuous Ca2+entry via both L- and T-type Ca2+channels contributes significantly to the maintenance of sustained vasoconstriction evoked by PKC activation, in a manner different from that evoked by ET-1. These data indicate the differential activation of plasma membrane Ca2+channels for vasoconstriction to ET-1 vs. PDBu. In Figure 3D,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of PDBu (0 minutes). In sum, ET-1 evoked coronary arteriolar constriction requires extracellular Ca2+entry, and the opening of T-type Ca2+channels may, in part, mediate this process. On the other hand, L-type Ca2+channels play little role in coronary arteriolar constriction to ET-1, but they solely Attorney Docket No.11164-015WO1 mediate the vasoconstriction evoked by PKC activation. It is interesting to note that L-type Ca2+channels contribute little to vasoconstriction induced by PKC in large conduit arteries harvested from pigs. This highlights the heterogeneity in vasomotor mechanisms between large vs. small vessels. Although both ET-1 and PKC activations can lead to various cardiovascular diseases, the difference in their vasoconstriction mechanisms in response to ET-1 and PKC stimulation stresses the importance of using targeted therapeutic strategies. To achieve a high efficacy in disease treatment, the identification of involved pathogenic factors and the determination of involved vessel size are necessary. Mechanisms of Coronary Arteriolar Constriction to ET-1: Role of Phospholipase C and Myosin Light Chain Kinase (MLCK) The signaling transduction mechanism responsible for coronary arteriolar constriction to ET-1 has not been established, despite the activations of phospholipase C (PLC) and myosin light-chain (MLC) kinase (MLCK) identified in large vessel studies. The activation of PLC following receptor activation and the subsequent phosphorylation (activation) of MLC after the rise of intracellular Ca2+and formation of the Ca2+-calmodulin complex for MLCK activation is a central dogma pathway contributing to vasoconstriction to various agonists, including ET-1, in large conduit vessels. To determine whether this classical vasoconstriction pathway applies to coronary arteriolar constriction in response to ET-1, the pig coronary arterioles (< 100 µm) were isolated for functional studies as described above. After the development of resting basal tone, the vessels were subject to treatment with inhibitors of PLC and MLCK, and the vasomotor response to a physiological concentration of ET-1 was examined. As shown in Figure 4A, the role of phospholipase C (PLC) in mediating ET-1-induced coronary arteriolar constriction was examined in the presence of selective PLC inhibitor U- 73122 (5 µmol / L). In the absence of U-73122, the control coronary arterioles constricted to ET- 1 (0.1 nmol / L) by reducing their resting (R) diameter (46±3 µm) by about 40%. In another group of vessels (46±2 µm resting diameter), pretreatment with U-73133 caused some loss of resting basal tone and reduced vasoconstriction in response to ET-1 (0.1 nmol / L). This result indicates a role of PLC in coronary arteriolar constriction to ET-1. In coronary smooth muscle, PLC can activate Ca2+entry via non-selective cation channels. This notion is consistent with the observations presented in Figures 2A and 2C that extracellular Ca2+entry is necessary for developing resting tone and constricting to ET-1. In Figure 4A,*P < 0.05 versus percent resting Attorney Docket No.11164-015WO1 diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 minutes). As shown in Figure 4B, the role of MLCK in coronary arteriolar constriction to ET-1 (0.1 nmol / L) was examined using the selective MLCK inhibitor ML-9. In the absence of ML-9, ET-1 (0.1 nmol / L) produced constriction of control coronary arterioles by reducing their resting (R) diameter (43±3 µm) by about 40%. In the presence of ML-9 (10 µmol / L), the vessels (resting diameter: 43±2 µm) lost their basal tone but the vasoconstriction evoked by ET-1 (0.1 nmol / L) remained. This result indicates that the MLCK activity is essential for the development of basal tone but not for the initiation of vasoconstriction to ET-1. Therefore, inhibition of MLCK does not prevent coronary arteriolar constriction to ET-1. In Figure 4B,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 minutes). It is important to determine whether ET-1-induced constriction can be reversed by MLCK inhibition. Accordingly, we performed a further study to determine whether MLCK activation contributes to the sustained constriction of ET-1. As shown in Figure 4C, the vasoconstriction induced by ET-1 is initially reversed by ML-9 (with 44±3 µm resting diameter), but the vasoconstriction gradually developed with time in the presence of ML-9. This indicates that the basal vascular tone portion is sensitive to MLCK inhibition as suggested by the data shown in Figure 4B and that the vasoconstriction to ET-1 is insensitive to MLCK inhibition. Therefore, ML-9 fails to reverse ET-1-induced vasoconstriction. In contrast, the ROCK inhibitor H-1152 effectively reverses the vasoconstriction to ET-1 (with 44±3 µm resting diameter) and maintains its dilation status. These microvascular data appear to not agree with established knowledge that the molecular signaling associated with smooth muscle contraction is centered around MLC phosphorylation by MLCK. The data presented in Figures 4B and 4C demonstrate that the resting microvascular tone is controlled by the MLCK activity but the vasoconstriction to ET-1 is likely mediated by a pathway independent of MLCK. Because ML-9 does not reverse vasoconstriction to ET-1, but H-1152 does, it is believed that ET-1 activates a signaling pathway mediated by ROCK that increases MLC phosphorylation by inhibiting MLC phosphatase (MLCP) activity because the level of MLC phosphorylation is determined by the balance of the activity of MLCK and MLCP. In Figure 4C,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 . Attorney Docket No.11164-015WO1 In sum, the coronary arteriolar constriction evoked by ET-1 is mediated by PLC without the involvement of MLCK. This vasoconstriction mechanism is different from what has been reported in large conduit vessels. The MLCK-independent mechanism in coronary arterioles may be explained by the potential activation of ROCK, which can modulate vasoconstriction through the inhibition of MLCP. To the best of our knowledge, this is the first finding on the MLCK-independent vasoconstriction to ET-1 in small coronary arterioles. The role of ROCK in mediating coronary arteriolar constriction to ET-1 is further supported by additional studies presented below. Role of MLCK in Coronary Arteriolar Constriction to PKC Activation by PDBu Because excessive activation of ET-1 and PKC contributes to many microvascular diseases, it is important to understand their similarities and differences in the mechanism of vasoconstriction at the level of microcirculation. As shown in Figures 4A-4C above, coronary arterioles exhibit a unique MLCK-independent pathway for vasoconstriction in response to ET- 1. However, it is undetermined whether vasoconstriction to PKC activation is also MLCK- independent since both vasoconstrictors rely on the extracellular Ca2+entry (see Figures 3A- 3D). To address this question, the MLCK inhibitor ML-9 was used to test whether this drug can prevent and / or reverse PDBu-induced vasoconstriction. The effects of ML-9 and ROCK inhibitor H-1152 were also compared. As shown in Figure 5A, PDBu evoked vasoconstriction by reducing diameter to about 40% of resting (R) diameter (50±2 µm), which was about 60% vasoconstriction. Adding ML-9 to this constricted vessel produced transient vasodilation (i.e., 178% of resting diameter) with the diameter gradually returning to the control level (i.e., ~125% of resting diameter) 20 minutes later. This diameter change corresponded to about 70% of the relaxed diameter, which is about 30% vasoconstriction. This magnitude of vasoconstriction in the presence of ML-9 is 50% lower than the initial vasoconstriction in the absence of ML-9 at time zero. This indicates that the PDBu-evoked vasoconstriction is partially (i.e., 50%) reversed by the MLCK inhibition. Adding the broad-spectrum PKC inhibitor Gö6983 to the vessels (38±2 µm resting diameter) completely reversed the PDBu-induced vasoconstriction. In contrast, H-1152 did not affect vasoconstriction to PDBu (49±4 µm resting diameter). These data demonstrated that PDBu activates PKC-dependent vasoconstriction and that MLCK plays a significant role in the development of resting tone. On the other hand, MLCK has about 50% contribution to vasoconstriction in response to PKC activation. Moreover, this vasoconstriction is independent Attorney Docket No.11164-015WO1 of ROCK signaling. In Figure 5A,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of PDBu (at 0 minutes) As shown in Figure 5B, pre-exposure of the vessels to Gö6983 did not alter the resting (R) vascular tone (55±5 µm) but prevented the development of vasoconstriction in response to PDBu. These results indicate that PKC does not contribute to the development of resting tone. However, PDBu evokes PKC-mediated vasoconstriction. These results echo the findings presented in Figure 5A. In the presence of ML-9 (48±3 µm) or H-1152 (40±3 µm), coronary arterioles lost the resting tone by doubling (i.e., ~200%) their diameters. These data indicate that MLCK and ROCK contribute to the development of resting vascular tone. Adding PDBu to the vessel bath, containing either ML-9 or H-1152, produced gradual vasoconstrictions with time, and the final diameters were slightly below the resting level (R), i.e., ~75-90% of resting diameter. The magnitudes of these vasoconstrictions corresponded to about 55-63% vasoconstriction from dilated vessels (i.e., 200% resting diameter). These magnitudes of vasoconstriction are within the range of initial PDBu-induced constriction without any inhibitors as shown in Figure 5A (i.e., constricting from the level of 100% resting diameter to 40% of resting diameter: ~60%). These data indicated that MLCK and ROCK signalings have limited (or no) contributions to the initiation of PKC-mediated vasoconstriction. In Figure 5B,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of PDBu (0 minutes). In sum, PDBu-evoked coronary arteriolar constriction is mediated by the activation of PKC because this vasomotor response is blocked by the broad-spectrum PKC inhibitor Gö6983. The initiation of PKC-mediated vasoconstriction appears to be independent of Ca2+ / calmodulin / MLCK signaling because pre-inhibition of MLCK by ML-9 does not prevent PDBu-induced vasoconstriction (Figure 5B). This is the first study to document the MLCK- independent vasoconstriction elicited by PKC activation in microvessels. It is likely that PKC activation that directly phosphorylates MLC or causes Ca2+influx through PKC-coupled Ca2+channels explains the vasoconstriction we observed. On the other hand, MLCK activation contributes to about 50% of sustained vasoconstriction evoked by PKC (Figure 5A). The other 50% of vasoconstriction, insensitive to MLCK inhibition, is likely derived from the direct activation of MLC by PKC, or possibly PKC-coupled Ca2+influx, as suggested in Figure 5B. It appears that the coronary arteriolar constriction to PKC activation can be initiated even if MLCK activity is compromised. Moreover, once vasoconstriction occurs, the MLCK Attorney Docket No.11164-015WO1 participates in sustaining this vasomotor activity. This appears to add another layer of complexity to using MLCK inhibitors for treating PKC-activated vascular diseases effectively because of the activation of multiple pathways by PKC at the level of smooth muscle contractile elements. However, importantly, ROCK signaling plays no role in PKC-mediated vasoconstriction. Mechanisms of Coronary Arteriolar Constriction to ET-1: Role of PKC and ROCK It has been suggested that vasoconstriction in response to ET-1 stimulation is mediated by PKC activation. However, this hypothesis was based on data obtained either from large conduit arteries or from microvessels that did not exhibit spontaneous basal tone. Thus, the contribution of PKC signaling to ET-1-induced coronary arteriolar constriction remains to be determined. Moreover, PKC activation is known to mediate many cardiovascular diseases via a myriad of signaling mechanisms, so determining whether PKC, as a broad kinase family, is involved in the vasoconstriction of ET-1 has clinical implications. To address this issue, the pig coronary arterioles were isolated and pressurized as described above. The vessels were subjected to pharmacological interventions to elucidate the involvement of PKC and ROCK in vasoconstriction to ET-1 and to determine whether inhibition of these two signaling molecules can prevent and / or reverse the vasoconstriction. To test whether PKC activation is involved in the ET-1-induced coronary arteriolar constriction, the vasoconstrictions in response to ET-1 and PKC activator PDBu were compared in the absence or presence of a broad spectrum, cell-permeable PKC inhibitor bisindolylmaleimide XI (BIM XI, 1 µmol / L). As shown in Figure 6A, both ET-1 (0.1 nmol / L) and PDBu (0.1 µmol / L) constricted coronary arterioles comparably from their resting (R) basal diameter (ET-1 group: 46±6 µm; PDBu group: 43±2 µm). Pretreatment of vessels with BIM XI caused vasodilation and abolished the vasoconstriction induced by PDBu. However, in another group of vessels with BIM XI treatment, the vasoconstriction evoked by ET-1 remained. This result indicates that the vasoconstriction evoked by ET-1 is not mediated by the activation of PKC, which is different from the findings at upstream large coronary arteries. In Figure 6A,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 or PDBu (0 minutes). To investigate whether ET-1- and PDBu-induced vasoconstrictions can be prevented by the inhibition of Rho-kinase (ROCK) activation, vasoconstrictions to ET-1 and PDBu were Attorney Docket No.11164-015WO1 examined using ROCK inhibitor H-1152. As shown in Figure 6B, coronary arterioles constricted to ET-1 (0.1 nmol / L; 44±4 µm resting diameter) and PDBu (0.1 µmol / L; 47±4 µm resting diameter) comparably. Pretreating the coronary arterioles (40±3 µm resting diameter) with H-1152 (3 µmol / L) abolished the resting (R) basal tone but the vasoconstriction in response to PDBu was potentiated. However, in another group of vessels (44±5 µm resting diameter), the vasoconstriction in response to ET-1 was prevented by H-1152. These results indicate that ROCK activation mediates vasoconstriction induced by ET-1, but not by PDBu, likely through the inhibition of phosphatase activity (see Figure 7 and Figures 8A-8D below). Therefore, the ROCK inhibitor H-1152 can be used specifically to prevent vasomotor activity induced by ET-1 in coronary arterioles. These data also suggest that H-1152 can potentially be used to prevent the induction of microvascular constriction by ET-1 during disease development. In Figure 6B,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 or PDBu (0 minutes). To determine whether vasoconstriction to ET-1 can be reversed by ROCK and / or PKC inhibition, the vessels were first constricted with ET-1 (0.1 nmol / L) to reach a stable diameter (i.e., reduced resting (R) diameter by about 40-50%). The status of vasoconstrictions was then examined after the addition of the ROCK inhibitor (H-1152, 3 µmol / L) or a broad-spectrum PKC inhibitor BIM XI (1 µmol / L). As shown in Figure 6C, H-1152, but not BIM XI, effectively reversed the ET-1-induced vasoconstriction within 30 seconds after drug administration, and the vessels remained dilated throughout the course of the study. The reversibility by BIM XI is rather insignificant compared with H-1152. This study demonstrated that ET-1-induced vasoconstriction is not mediated by PKC activation. These results indicate that H-1152 is a potent agent to reverse vasoconstriction evoked by ET-1 and suggest that H- 1152 can be used for treating microvascular spasms evoked by ET-1. In Figure 6C,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of ET-1 (0 minutes). The same approach was used to address whether vasoconstriction evoked by PDBu (0.1 µmol / L) can be reserved by H-1152 (3 µmol / L) and / or by BIM XI (1 µmol / L). As shown in Figure 6D, PDBu-induced vasoconstriction was reversed by BIM XI, which is consistent with the result in Figure 5A when a different PKC inhibitor Gö6983 was used. However, H-1152 only slightly attenuated the vasoconstriction to PDBu in the first minute of drug administration, and the prolonged vasoconstriction was observed throughout the course of the study. This study Attorney Docket No.11164-015WO1 indicates that H-1152 is not an effective agent to reverse vasoconstriction evoked by PKC activation. In Figure 6D,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of PDBu (0 minutes) In sum, the finding of PKC-independent vasoconstriction to ET-1 is unique to the resistance arterioles because PKC activation is known to be responsible for the constriction of large arterial vessels in response to ET-1 stimulation. Therefore, the findings in large upstream vessels cannot be extrapolated to the downstream microvessels in terms of the mechanistic action of ET-1. The involvement of ROCK activation in microvascular constriction to ET-1 is supported by the present study. Most importantly, H-1152 can selectively and effectively prevent, as well as reverse, the ET-1-evoked vasoconstriction in coronary arterioles, supporting the clinical application of this inhibitor for the prevention and treatment of ET-1-induced coronary microvascular dysfunction. Mechanisms of Coronary Arteriolar Constriction to ET-1: Role of Myosin Light- Chain Phosphatase (MLCP) versus ROCK Our studies demonstrate the involvement of ROCK activation in coronary arteriolar constriction to ET-1 (Figure 6). The interpretation of these data relies on the specificity of H- 1152 for ROCK inhibition. It should be noted that while the inhibition of ROCK activity by H- 1152 has been characterized using biochemical enzyme assays, cultured smooth muscle cell preparations, or vascular strip contraction studies, there are no data to support whether this is the case in vasomotor regulation of small arterioles. Most importantly, this concern needs to be addressed at the level of intact vessels that exhibit spontaneous basal tone as seen in vivo without the participation of confounding activation or deactivation of other signaling pathways by pharmacological agents. Therefore, a mechanistic understanding of the action of H-1152 in the intact microvessel is essential if H-1152 is to be a candidate for treating microvascular dysfunction related to ROCK activation. Along the same line, because ROCK activation leads to the inhibition of MLCP and consequently promotes vasoconstriction, we expect that H-1152 would increase MLCP activity and cause vasodilation. In this case, the effect of H-1152 should be reversed by the MLCP inhibitor. In other words, the inhibitory effect of H-1152 on ET-1- induced vasoconstriction should be abolished in the presence of an MLCP inhibitor. The experiments below support this context. The role of MLCP was examined in isolated coronary arterioles using a potent and selective cell-permeable MLCP inhibitor calyculin A (CLA). CLA directly inhibits protein Attorney Docket No.11164-015WO1 phosphatase type 1 catalytic subunit (PP1cδ), one of the 3 subunits of the MLCP. The other two components of MLCP are a regulatory complex of MYPT1 (aka MBS, M110) as the downstream target of ROCK and PKC and a 21-kDa accessory subunit with an unknown function. CLA promotes vasoconstriction by directly inhibiting phosphatase activity. It is unclear whether the inhibitory action of H-1152 on ET-1-evoked coronary vasoconstriction is mediated by the direct activation of phosphatase or the inhibition of the ROCK molecule upstream of the phosphatase. Since CLA affects Ca2+channel activity in the plasma membrane, we addressed these issues by exposing coronary arterioles to the Ca2+-free solution and to H- 1152 pretreatment and then comparing the vasomotor reaction to ET-1 (0.1 nmol / L) in the absence and presence of CLA (0.1 µmol / L). As shown in Figure 2A and Figures 3A and 3B, the ET-1-induced vasoconstriction was absent in the Ca2+-free solution (45±5 µm resting diameter, n=5, Figure 7) and the vasoconstriction to ET-1 (0.1 nmol / L) was sensitive to H-1152 (44±5 µm resting diameter, n=7, Figure 7; see also Figure 4C and Figures 6B and 6C). In contrast, as shown in Figure 7, the vasoconstriction induced by CLA (0.1 µmol / L) was independent of extracellular Ca2+(50±5 µm resting diameter; n=6). Although the vasoconstriction to ET-1 was abolished by H-1152 (49±2 µm resting diameter; n=4), this vasoconstriction was preserved by adding CLA. These data indicate that, first, unlike ET-1, CLA triggers vasoconstriction signaling without extracellular Ca2+entry, suggesting the action of CLA downstream from plasma membrane Ca2+channels. This context is consistent with the outcome of phosphatase inhibition independent of extracellular Ca2+entry. Second, H-1152 did not affect the vasoconstriction induced by CLA, suggesting the target of CLA downstream from ROCK. These results also indicate that H-1152 blocks the ET-1-induced vasoconstriction by increasing phosphatase activity indirectly, e.g., through ROCK inhibition, rather than acting on the phosphatase directly. These studies not only demonstrate the specificity of H-1152 as a ROCK inhibitor but also indicate the action of CLA downstream from ROCK as a phosphatase inhibitor. In Figure 7,*P < 0.05 versus percent resting diameter at R, and#P < 0.05 versus percent resting diameter observed at the time of addition of CLA (0 minutes). In sum, the possibility that H-1152 inhibits ET-1-induced vasoconstriction through a direct action on MLCP activity is excluded from this study. Our study demonstrates the specificity of H-1152 as a potent inhibitor of ROCK in intact microvessels. Moreover, the Attorney Docket No.11164-015WO1 inhibitory action of H-1152 on ROCK is supported by a series of molecular studies described below. Molecular Evidence of MYPT1 and MLC Phosphorylation by ET-1 via ROCK Activation Myosin light chain (MLC) activation through the phosphorylation process is important for smooth muscle contraction and the level of MLC phosphorylation (pMLC) can be modulated by MLCP, which is negatively controlled by the phosphorylation of myosin phosphatase target subunit 1 (pMYPT1) in the phosphatase. Thus, the level of pMLC at serine 19 (S19), corresponding to the contractile activity of the smooth muscle, can be increased by the inhibition of phosphatase activity via MYPT1 phosphorylation at threonine 850 (T850). However, it is unclear whether ET-1 directly activates the MLC or indirectly via inhibition of phosphatase activity by MYPT1 for coronary arteriolar constriction. It is also unclear whether ROCK is involved in the phosphorylation of MLC and MYPT1 for vasoconstriction. To address these questions, pig coronary arterioles (< 100 µm in diameter) were isolated, pressurized, and allowed to develop basal tone as described for the functional studies. After pharmacological treatments (ET-1, PDBu, or H-1152), 2-3 vessels in each group from the same animal were snap frozen in liquid nitrogen for biochemical analysis. The concentration of pharmacological agents used for molecular studies was the same as that used for functional studies in intact vessels. The expressions of coronary arteriolar pMLC and pMYPT1 in response to ET-1 were analyzed, and the results were compared with that obtained from PKC activator PDBu. Coronary arterioles were subjected to immunoblotting of pMYPT1 and pMLC using smooth muscle actin (SMA) expression as the reference of equal protein loading. As shown in Figure 8A, pMYPT1 and pMLC are elevated in vessels treated with ET-1 (0.1 nmol / L) compared with the vessels treated with vehicle (Ctl). In contrast, PDBu (0.1 µmol / L) only causes phosphorylation of MLC (pMLC) with no elevated pMYPT1. These data indicate the involvement of MYPT1 phosphorylation in ET-1-evoked signaling for vasoconstriction, a pathway different from that triggered by PKC activation. Phosphorylation of MYPT1 is known to inhibit phosphatase activity, thus it is concluded that ET-1-induced coronary arteriolar constriction is mediated by the inhibition of phosphatase via MYPT1 phosphorylation. These molecular data agree with the functional data that ET-1 and PDBu activate different pathways for coronary arteriolar constriction (Figures 3A-3D and Figures 5A-5B), i.e., phosphatase- Attorney Docket No.11164-015WO1 dependent vs. phosphatase-independent signaling, respectively. The representative data were analyzed from 4 independent experiments. As shown in Figure 8B, when arterioles under 3 treatment conditions (control, ET-1 and PDBu) are collected during the same day, treatments with both ET-1 and PDBu cause strong activation of MLC (pMLC) at the S19 site, only the treatment with ET-1 cause the phosphorylation of MYPT1 at the T850 site. Because pMYPT1 is elevated by ET-1, but not by PDBu, ET-1 and PDBu appear to trigger different vasoconstriction pathways. The data indicate that PDBu elicits Ca2+-calmodulin-MLCK pathway for vasoconstriction. On the other hand, ET-1 elicits a Ca2+-sensitization mechanism for vasoconstriction through the inhibition of MLCP activity upon MYPT1 phosphorylation. The representative data were analyzed from 4 independent experiments. Ctl = Control. As shown in Figure 8C, the quantitative analysis of pMYPT1 with smooth muscle actin (SMA) normalization from 4 independent experiments is presented. The data showed that ET-1 significantly elevates pMYPT1 in coronary arterioles. *P < 0.05 versus Control. As shown in Figure 8D, ET-1 consistently induces MYPT1 and MLC phosphorylation. It should be noted that MLC phosphorylation induced by ET-1 was abolished by H-1152, indicating the activation of the Ca2+-calmodulin-MLCK axis is not responsible for the pMLC- dependent vasoconstriction, instead, ROCK activation is the key signaling pathway mediating vasoconstriction to ET-1. Because H-1152 did not affect the basal level of pMYPT1 but abolished the elevated pMYPT1, it is concluded that activation of ROCK by ET-1 is responsible for the MYPT1 phosphorylation. Overall, these data demonstrate the involvement of ROCK activation upstream of myosin phosphatase in coronary arteriolar constriction to ET- 1. The quantitative analysis was derived from the data after normalization with MLC from 4 independent experiments. In Figure 8D, *P < 0.05 versus other groups. In sum, ET-1 evokes coronary arteriolar constriction by increasing pMLC, a result of myosin phosphatase inhibition by phosphorylated (activated) its MYPT1 subunit following ROCK activation. In contrast, PDBu / PKC elicits direct MLC phosphorylation and / or indirect activation of Ca2+-calmodulin-MLCK axis for vasoconstriction independent of ROCK / pMYPT1 signaling. Our molecular data support the role of ROCK in ET-1-induced coronary arteriolar constriction. The ROCK inhibitor H-1152 blocks this signaling pathway and inhibits the vasoconstriction elicited by ET-1. Moreover, H-1152 is ineffective in inhibiting contractile signaling, and thus vasoconstriction, elicited by PKC activation. However, calcium Attorney Docket No.11164-015WO1 channel blockers can be utilized if it is clear PKC activation is involved based on our data. This conclusion is consistent and reflected by the results of the functional studies in intact arterioles presented in Figures 5A-5B and Figures 6A-6D. Phosphorylation of MLC in Isolated Coronary Arteriolar Smooth Muscle Cells by ET-1 and Inversely Regulated by H-1152 Although ET-1 evokes constriction of intact coronary arterioles in a concentration- dependent manner, it is unclear whether the activation of contractile signaling (pMLC) in smooth muscle cells corresponds to the increased level of ET-1 or just a simple increase in the Ca2+sensitivity of the contractile filaments within the cell. This question is important in terms of addressing the signal transduction mechanism of ET-1 activation. Moreover, it is unclear whether the change of pMLC level in the smooth muscle is a function of ET-1 concentration and whether H-1152 can inversely regulate the ET-1-evoked MLC phosphorylation level in a manner consistent with that observed in the functional study of intact vessels. This information is important for the interpretation of molecular data corresponding to the functional behavior of the vessel and for supporting the idea of antagonizing ET-1 by inhibiting MLC phosphorylation through ROCK inhibitor H-1152. As shown in Figure 9A, ET-1 increased MLC phosphorylation (pMLC) of primary culture of smooth muscle cells isolated from pig coronary arterioles (< 100 µm in diameter) in a concentration-dependent manner. In the absence of ET-1 (C), the cells expressed a basal level of pMLC, indicating the viability and functional integrity of MLC of these cells consistent with the development of spontaneous basal tone of the intact vessel. These data indicate that ET-1 is capable of inducing pMLC and supports the role of pMLC in coronary arteriolar constriction to ET-1. The quantitative data were derived from 3 independent experiments. The intensity of immunoblotting bands was normalized to the level obtained from 10 nmol / L ET-1 and expressed as relative intensity. In Figure 9A, M = mol / L. To address the impact of H-1152 on ET-1-induced pMLC, smooth muscle cells isolated from coronary arterioles (< 100 µm in diameter) were treated with 0.1 nmol / L ET-1 (same as used for functional study) in primary culture in the absence (C) and presence of different concentrations of H-1152. As shown in Figure 9B, ET-1 activates MLC (pMLC) in the absence of H-1152, and the expression level of pMLC is reduced by H-1152 in a concentration- dependent manner. These data indicate that the level of MLC phosphorylation is dependent upon ROCK activity and support the concept that coronary arteriolar constriction to ET-1 is Attorney Docket No.11164-015WO1 mediated by pMLC through ROCK activation. Moreover, the ET-1-activated pMLC is almost abolished by a micromolar concentration of H-1152, in a manner consistent with the abolished basal tone and constriction to ET-1 (0.1 mmol / L) by the same level of H-1152 in coronary arterioles (Figures 5A-5B and Figures 6A-6D). The quantitative data were derived from 3 independent experiments. The intensity of immunoblotting bands was normalized to the level obtained under control (C) conditions without H-1152 and expressed as relative intensity. In Figure 9B, M = mol / L. In sum, the molecular data on the elevation of ET-1-induced MLC phosphorylation agree well with the degree of vasoconstriction elicited by ET-1 in a concentration-dependent manner (Figures 2A-2D). The data on the concentration-dependent inhibition of MLC phosphorylation by H-1152 can help to select the effective H-1152 concentration for antagonizing the vasoconstrictor activity of ET-1 or basal tone. Therefore, H-1152 can potentially be used to treat ET-1-associated microvascular spasms and angina in cardiovascular events related to ROCK activation. Characteristics of ROCK Isoform Expression in Cardiomyocytes, Left Anterior Descending Artery and Coronary Arterioles Although two ROCK isoforms, ROCK1 and ROCK2, have been identified, there is no information regarding their distribution and relative expression in the coronary microvasculature. Herein, we address these important issues because our data indicate the vital roles of ROCK in mediating coronary arteriolar constriction to ET-1. Coronary arteries and arterioles were carefully isolated from pig hearts, and all vessels were pooled, homogenized, and lysed for immunoprecipitation (IP). The lysates were centrifuged to obtain supernatant (S) and precipitated (P) samples. These samples were loaded with input (I) in the SDS-PAGE gel and immunoblotted with antibodies against ROCK1 and ROCK2. As shown in Figure 10A, coronary vessels express both ROCK1 and ROCK2 isoforms and can be precipitated and subsequently detected with their specific antibodies without cross-reaction. As shown in Figure 10B, the ROCK isoforms were expressed in aortas from different species, including mice, pigs, and different strains of rats (SD: Sprague Dawley; F: Fischer; LE: Long-Evans). The human samples were retinal tissues obtained from patients subjected to enucleation due to ocular tumors (conducted after informed consent with approval from the Baylor Scott & White Health Institutional Review Board and followed the tenets of the Attorney Docket No.11164-015WO1 Declaration of Helsinki). The retinal samples were isolated from the area without containing tumor tissue for this study. As shown in Figure 10B, ROCKs are ubiquitously expressed in vasculatures across different species. A truncated ROCK1 (130KD, arrow) was detected in the aortic tissue except for the mouse and human samples. ROCK1 is a direct cleavage substrate of activated caspase-3 involved in myocardial apoptosis and cardiomyopathy. The cleavage of ROCK1 results in a 130-KD subspecies that can activate caspase-3 through a positive feedforward loop, which may consequently promote apoptotic signals in myocardial hypertrophy and / or heart failure. However, the physiological and pathophysiological roles of truncated ROCK1 in conduit arteries are unknown. Interestingly, all tested rat strains mainly express the 130 KD truncated ROCK1 isoform with trace 160 KD ROCK1 being barely visible. It is unclear whether ROCK isoforms are also expressed in cardiomyocytes and whether their expression levels are different from coronary arterioles. This question is important in terms of addressing whether the target of the ROCK inhibitor is mainly in the myocardium or coronary vasculature. As shown in Figure 10C, both cardiomyocytes and arterioles express ROCK1 and ROCK2 with 4- and 2-fold higher expression levels, respectively, in coronary arterioles. The cardiomyocyte samples contained tropomyosin but not α-actin, indicating the samples were deprived of the contamination of vascular tissues. The vascular samples contained α-actin with little or no tropomyosin, indicating the high purity of vascular samples. The expression intensity of the immunoblots was normalized with GAPDH and expressed as relative intensity (n = 3 independent experiments). In Figure 10C, *P < 0.05 versus Cardiomyocyte. Since there is no information concerning the relative expression of ROCK1 versus ROCK2 in the coronary vasculature and it is unclear whether their expression levels are different between large conduit arteries and small resistance arterioles, we addressed these questions to help the future design of isoform-specific ROCK inhibitors. The left anterior descending (LAD) arteries (the main trunk of the coronary artery in the heart; ~3-4 mm in diameter) and their small downstream arterioles (< 100 µm in diameter) were isolated for immunoblotting. As shown in Figure 10D, both ROCK isoforms were expressed in the coronary arterial system with no apparent difference in the expression levels between large versus small vessels after normalization with their own α-actin expression (n = 3 independent Attorney Docket No.11164-015WO1 experiments). It is worth noting that the truncated ROCK1 (130KD) is readily detected in LAD arteries but not in small coronary arterioles. Because ROCK2 appears to be the major isoform expressed in the coronary arterioles, we performed immunohistochemical studies of ROCK2 to localize its expression in heart tissues. As shown in Figure 10E, the arterioles were identified with the expression of smooth muscle actin (green), which colocalized with the vascular expression of ROCK2 (merged image). It appears that both cardiomyocytes and arterioles expressed ROCK2 in agreement with the western blot data (Figure 10C). The immunohistochemical staining in arterioles is much stronger than that of cardiomyocytes, suggesting the greater expression of ROCK2 in arterioles. These immunohistochemical data agree with the western blot of ROCK2 expression. The expression of ROCK2, smooth muscle vs. endothelial cells, has not been determined in coronary arterioles. This issue is important because their differential expressions might influence the arteriolar response to ET-1 and its vasomotor regulation. To address this question, coronary arterioles (< 100 µm in diameter) were isolated for immunohistochemical staining with ROCK2 after cryosection. The arteriolar sections were subjected to staining with antibodies conjugated with red-fluorescent dye against eNOS and smooth muscle actin to localize endothelial cells and smooth muscle cells, respectively. ROCK2 was stained with an antibody conjugated with green-fluorescent dye. As shown in Figure 10F, smooth muscle expression of ROCK2 is evident. This is expected because vasoconstriction evoked by ET-1 is endothelium-independent and is mediated by ROCK. Noticeably, endothelial cells also express ROCK2. In sum, coronary arterioles and cardiomyocytes express both ROCK1 and ROCK2 isoforms, with the ROCK2 isoform predominantly expressed in coronary arterioles. Coronary arteriolar endothelial cells also express the ROCK2 isoform. However, its role in the regulation of the vasomotor function of coronary arterioles remains to be determined. The expression of truncated ROCK1 (130KD) in conduit arteries, including coronary arteries, is intriguing. It is speculated that this expression may increase the susceptibility of the vessel to disease development. Nevertheless, this study also demonstrates another characteristic of coronary arterioles different from their upstream conduit vessels. Differential Expression of CPI-17 in the Coronary Arterial Network: Vessel Size- Dependent CPI-17 Expression Attorney Docket No.11164-015WO1 PKC-potentiated myosin phosphatase inhibitor of 17 kDa (CPI-17) is a phosphorylation-dependent inhibitory protein for the MLCP. Phosphorylation of CPI-17 at Thr38, reported in studies of large conduit arteries in vitro, by PKC or ROCK can enhance its inhibitory potency toward MLCP to promote vasoconstriction. It is unclear whether ET-1, at a physiological / pathophysiological concentration, can activate CPI-17 downstream from ROCK to exert MLCP inhibition and elicit coronary arteriolar constriction. Moreover, there is no study to demonstrate whether CPI-17 activation is necessary for coronary arteriolar constriction to ET-1. To address these questions, aortas, cerebral arteries, coronary arteries (1 mm in diameter) and arterioles (~100 µm in diameter) were isolated from pigs for biochemical and immunohistochemical studies of CPI-17 phosphorylation (pCPI-17). As shown in Figure 11A, in large conduit arteries (pig cerebral arteries and aortas), both CPI-17 and pCPI-17 were readily detected, but their expressions were not detectable in pig coronary arterioles (< 100 µm in diameter). Such a unique CPI-17 expression leads to a hypothesis that its expression is dependent upon the size of blood vessels. To further study the differential expression of CPI-17 in different sizes of blood vessels in the same vascular bed, the coronary arteries (~ 1000 µm in diameter) and coronary arterioles (100 µm in diameter) were isolated and subjected to western blot analysis of CPI-17. As shown in Figure 11B, CPI-17 is expressed abundantly in large coronary arteries but is barely detected in coronary arterioles with the same amount of protein load (10 µg). Moreover, the CPI-17 expression remained very low by increasing the protein load 5 times (50 µg). It should be noted that these small-vessel-size protein samples might be “contaminated” by some larger vessels (100-150 µm in diameter) during microvessel isolation. Nevertheless, these data indicate that CPI-17 might not be of functional importance in terms of regulating myosin phosphatase activity in coronary arterioles because of its sparse expression. The quantitative study of CPI-17 and ROCK2 protein expression in different sizes of coronary arteries was further investigated to support the concept of vascular size-dependent CPI-17 expression. As shown in Figure 11C, CPI-17 was barely detectable in small coronary arterioles (30-100 µm in diameter), but its expression levels were higher in arteries with increasing vessel size. It should be noted that the ROCK2 protein expression is independent of vessel size. The amount of protein 10 µg was loaded into each lane. The expression levels, after normalization with smooth muscle actin (SMA), were presented as the Relative Ratio in Attorney Docket No.11164-015WO1 reference to the data obtained from the 1000-µm vessel size from 6 independent experiments. In Figure 11C, *P < 0.05 versus other groups. To strengthen the results from the western blot analysis above, the immunostaining of CPI-17 in large conduit vessels and small coronary arterioles was performed in cryosections. The DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus of the cells. As shown in Figure 11D, CPI-17 is barely detected in small coronary arterioles (~20-50 µm in the left- middle panel), but its expression in a large coronary artery (~550 µm in the right panel) is apparent. This result is consistent with the differential CPI-17 protein expression from the western blot analysis (Figure 11C). In sum, these findings demonstrate that coronary arterioles (< 100 µm in diameter) express little, if any, of CPI-17. However, the CPI-17 expression is increased with increasing vessel size, suggesting the regulation of myosin phosphatase activity by CPI-17 is increasingly important in large conduit vessels. This finding supports the biochemical and mechanical contractile study of isolated vascular smooth muscle cells, vascular strips, or vascular rings harvested from large conduit vessels that CPI-17 plays a critical role in smooth muscle contraction by inhibiting myosin phosphatase activity, and thus increasing Ca2+sensitivity of contractile filaments for sustained vasocontraction. However, this contractile signaling mechanism might not be important in small coronary arterioles since CPI-17 is barely detected, almost absent, in these microvessels. An artery-size based CPI-17 expression along the same vascular tree and low or no expression at the arteriole level is demonstrated herein for the first time. In contrast, the ROCK signaling toward myosin phosphatase regulation is predominant in small coronary arterioles for vasoconstriction. Our studies show the unique biochemical characteristics of coronary arterioles in vasomotor regulation. These findings may highlight the reason for the failure of treatment of microvascular diseases based on the large-vessel data and emphasize the importance of targeting microvessels for effective therapy of microvascular disease. Effect of ROCK Inhibitor on Resting Basal Tone Independent of Endothelial Nitric Oxide (NO) As demonstrated in a series of studies presented above, the coronary arteriolar constriction evoked by ET-1 is primarily mediated by the sequential events of ETA receptor activation, extracellular Ca2+entry, ROCK activation, MYPT1 phosphorylation, myosin phosphatase inhibition, and consequently increase in MLC phosphorylation. However, there is Attorney Docket No.11164-015WO1 no information available regarding the effect of ROCK inhibition on the basal vasomotor tone of coronary arterioles. This concern is important because the level of resting blood flow is mainly influenced by the level of arteriolar basal tone in tissues and organ systems, including the heart. Moreover, it is unclear whether the vasodilation evoked by the ROCK inhibitor is mediated by the release of NO from the endothelium. These questions are also important because the coronary arteriolar basal tone is generally elevated in patients with myocardial ischemia and / or hypertension in association with microvascular dysfunction. Therefore, determining the sensitivity and potency of the ROCK inhibitor in antagonizing the basal tone is essential for blood flow improvement and hypertension management by reducing resting arteriolar resistance. Furthermore, if the vasodilation induced by ROCK inhibitors is mediated by the release of endothelial NO, then the efficacy of reversing vascular resistance would be compromised due to endothelial dysfunction that is known to associate with coronary diseases. Therefore, the consideration of the potential limitation of using ROCK inhibitors for medical applications needs to be addressed. The current clinical approach is also focused on improving endothelial function such as treating patients with statin drugs. To answer these critical questions, coronary arterioles (< 100 µm in diameter) were isolated and pressurized for functional study by constructing the concentration-response curve of ROCK inhibitors. We compared the efficacy of reversing basal tone by H-1152 and by Y-27632, one of the most commonly used ROCK inhibitors for biomedical research. The ROCK-induced vasodilation was examined in the absence and presence of NO synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor to eliminate NO production. As shown in Figure 12A, isolated coronary arterioles developed resting basal tone (R) and dilated concentration-dependently to H-1152 (51±3 µm resting diameter) and Y-27632 (50±5 µm resting diameter). The IC50 value of 30 nmol / L for H-1152 in dilating coronary arterioles was 100 times more potent than that for Y-27632 at 3 µmol / L. The response to H- 1152 showed a linear concentration-dependent relationship up to 300 nmol / L when the arterioles were close to their maximum dilations. This indicates a high specificity of H-1152 to its molecular target(s). The arterioles dilated maximally at 1 µmol / L of H-1152 and all the vessels regained resting tone after washing out H-1152. There were no signs of weakened H- 1152 responsiveness with repeated experiments and no immediate negative impact of H-1152 on vasomotor function. Attorney Docket No.11164-015WO1 As shown in Figure 12B, in the presence of L-NAME (10 µmol / L), the coronary arteriolar vasodilation in response to H-1150 was not altered, indicating that the observed vasodilation is independent of the release of endothelial NO. These results indicate that H-1152, compared to Y-27632, is a more specific, sensitive and potent ROCK inhibitor in reversing basal tone without apparent toxic effects. This vasodilator action is not mediated by the release of endothelial NO. Therefore, the clinical application of this ROCK inhibitor is not expected to rely on the functional status of the endothelium. This unique property is advantageous for clinical implications because the endothelial function related to NO bioavailability is generally compromised in many cardiovascular diseases. Therefore, the endothelial NO-independent action of H-1152 is expected to have a broad application for disease treatment; H-1152 may be combined with statin medications as a maintenance therapy. Overall, our data indicate that ROCK activation, without going through CPI-17 signaling, is the primary pathway mediating coronary arteriolar constriction in response to ET-1. Our studies also demonstrate that H-1152 is a selective and effective inhibitor for ROCK in both preventing and reversing vasoconstriction evoked by ET-1 in coronary arterioles. Our data support the inventive technology that ROCK inhibitor H-1152 is an excellent candidate to treat ET-1-associated cardiovascular diseases, including microvascular dysfunction. Tissue Heterogeneity in Vasomotor Regulation and Utilization of H-1152 Studies of the fundamental mechanisms of vasomotor regulation in the microcirculation have provided a framework for characterizing the development of microvascular dysregulation in animal models of various diseases, i.e., hypertension, diabetes / hyperglycemia, ischemia / reperfusion injury, atherosclerosis, hyperlipidemia, lipopolysaccharide (LPS) shock, pathological angiogenesis, and C-reactive protein with inflammatory insults. Microvessels from different vascular beds and associated diseases exhibit their own unique features and mechanisms leading to microvascular dysfunction. We have investigated healthy ophthalmic / retinal microcirculation and compared its vasomotor regulation with the coronary microvasculature. Although both coronary and retinal circulations exhibit the same phenomenon of metabolic-dependent blood flow control, we found that they display some different signaling pathways for vasomotor regulation and disease susceptibility. For example, we found that acetylcholine is a potent coronary arteriolar vasoconstrictor in pigs when given extraluminally but it is a vasodilator in pig retinal arterioles. Attorney Docket No.11164-015WO1 Moreover, thrombin is a vasodilator in pig coronary arterioles but a potent vasoconstrictor in pig retinal arterioles. Serotonin is a potent endothelium-dependent vasodilator in coronary arterioles, but retinal arterioles fail to respond to serotonin. Specifically, as shown in Figure 13A, isolated porcine coronary and retinal arterioles were pressurized to 60 cmH2O. The coronary arterioles developed basal tone (45±4 µm, n=5) under 36-37°C bath temperature and constricted concentration-dependently to acetylcholine. However, acetylcholine produced weak dilations in retinal arterioles (43±3 µm resting diameter, n=5). Likewise, as shown in Figure 13B, under the same preparation as described in Figure 13A, thrombin evoked dilation in coronary arterioles (47±4 µm resting diameter, n=5) but constriction in retinal arterioles (44±3 µm resting diameter, n=5). Coronary and retinal arterioles also display different signaling pathways for vasomotor regulation. For example, coronary arteriolar constriction elicited by PKC activation is independent of Rho kinase signaling (i.e., insensitive to H-1152), but the latter plays an important role in mediating PKC-induced retinal arteriolar constriction (i.e., sensitive to H- 1152). In addition, dilation of coronary arterioles to bradykinin is mediated in part via endothelium-derived hyperpolarizing factor, whereas retinal arteriolar dilation to bradykinin relies entirely on the release of endothelial NO. Porcine coronary arterioles express arginase-I for vasomotor regulation, but retinal arterioles mainly express the arginase-II isoform, which contributes differentially to the development of vascular dysfunction. Under experimental pathological conditions, we found that 90 minutes of ischemia impairs retinal arteriolar dilation to endothelium-dependent agonists, but this duration of ischemia is not sufficient to elicit coronary arteriolar dysfunction unless subsequent reperfusion is imposed. We also found that activation of proinflammatory c-Jun N-terminal kinase (JNK) signaling can exert abnormal vasoconstriction in the pig retinal microvasculature. However, in pig coronary arterioles, JNK signaling does not contribute to vascular injury. Despite the main function of blood flow regulation in the microcirculation being the same across different tissues, it appears that the retinal and coronary microvasculature can exhibit different susceptibilities and signaling pathways for vasomotor regulation and dysregulation. Because multiple pathogenic factors, in addition to ET-1, can activate ROCK, the application of ROCK inhibitor H-1152 would benefit the blood flow recruitment and tissue survival in general. It should be noted that our findings do not exclude the possibility of the activation of identical pathways in different tissues / organs for some diseases. On the other hand, our Attorney Docket No.11164-015WO1 findings suggest that systemic intervention might not always be helpful or effective for treating tissue or organ-specific microvascular disease. Instead, the target (or tissue)-selective approach should be considered due to the heterogeneity of vasomotor regulation / dysregulation and the possible involvement of different signaling mechanisms in a specific organ / tissue. In this document, we demonstrate the novel signaling mechanism of coronary arteriolar constriction to ET-1 and the cardinal role of ROCK activation in mediating this vasoconstriction (or spasm). Our data suggest the unique benefit of using H-1152 in neutralizing the adverse effect, specifically in microvascular disease-associated ROCK activation in general. Since ET-1 is one of the major pathogenic factors and its biological actions are mediated by ROCK, the utilization of H-1152 is deemed desirable in this regard. This concept may be applied to other tissue / organs such as the retinal and other microvascular beds. Mechanisms of Coronary Arteriolar Constrictions Evoked by ET-1 and PKC Activator PDBu Studies in conduit arteries show that the vasoconstriction evoked by ET-1 is mediated by the activation of PKC signaling. Because ET-1 and PKC activation can contribute to pathogeneses of many coronary diseases, it is valuable to elucidate their molecular signaling underlying the vasoconstriction mechanism in coronary arterioles where the myocardial blood flow is dominantly regulated. Based on the data presented in this Example, a model diagram depicting subcellular mechanisms of coronary arteriolar constrictions evoked by ET-1 and PKC activator PDBu can be generated. This diagram is shown in Figure 14. ET-1 (0.1 nmol / L) binds and activates the ETA receptor, coupling to phospholipase C (PLC), and triggers extracellular calcium (Ca2+) entry, likely via T-type voltage-gated Ca2+channels (T-VGCC) and / or Ca2+-permeable nonselective cation channels (NSCC). The increased cytosolic Ca2+stimulates an active GTP-bound form of RhoA for ROCK activation.37The activated ROCK phosphorylates the regulatory subunit of myosin phosphatase target subunit 1 (MYPT1) of myosin light-chain phosphatase (MLCP) and consequently inactivates the phosphatase activity. The vasoconstriction occurs upon the elevation of phosphorylation (P) of the 20 kDa regulatory myosin light chain (MLC20) following the inactivation of MLCP. Both the development / maintenance of resting basal tone and vasoconstriction evoked by ET-1 require extracellular Ca2+entry for initiating ROCK-dependent regulation of MLC phosphorylation. Although the direct participation of ROCK in MLC phosphorylation cannot be excluded, our studies demonstrate the requisite role of ROCK in mediating coronary Attorney Docket No.11164-015WO1 arteriolar constriction to ET-1. It is worth noting that a supra pharmacological concentration of ET-1 (10 nmol / L), which is not seen under physiological and / or pathophysiological conditions in vivo, triggers Ca2+release from the internal store, i.e., sarcoplasmic reticulum (SR) via IP3 and / or store-operated Ca2+channels. This superimposed Ca2+elevation likely triggers other biological events in addition to vasomotor activation. It should be noted that the resting microvascular tone is maintained by the extracellular Ca2+entry and the Ca2+ / calmodulin (CaM)-dependent phosphorylation of MLC20 by myosin light-chain kinase (MLCK). The activity of ROCK also contributes to the maintenance of resting tone by regulating MLC20 phosphorylation. Therefore, both MLCK inhibitor (ML-9) and ROCK inhibitor (H-1152) abolish the resting vascular tone. Activation of PKC signaling by PDBu (0.1 µmol / L) elicits extracellular Ca2+entry by opening L-type voltage-gated Ca2+channels (L-VGCC), and the subsequent activation of the CaM / MLCK axis contributes to about 50% of sustained vasoconstriction. However, Ca2+ / calmodulin / MLCK axis appears to not be involved in the initiation of PKC-mediated vasoconstriction since ML-9 fails to prevent the contractile reaction to PDBu. This suggests the possibility of direct phosphorylation / activation of MLC20by PKC triggered chain reactions. Although the activated PKC has been shown to phosphorylate MYPT1 to promote vasoconstriction in conduit vessels (dashed line in the diagram), the level of pMYPT1 is not increased by PDBu in coronary arterioles, indicating that the vasoconstriction to PKC activation is independent of MLCP inhibition. Phosphorylation of CPI-17 by either PKC or ROCK (brown octagon in the diagram) also causes vasoconstriction by deactivating MLCP. However, despite its abundance in conduit arteries, CPI-17 is barely detected in coronary arterioles, suggesting its limited role in modulating microvascular constriction through PKC or ROCK activation. Because coronary arteriolar constriction to PKC activation is insensitive to H-1152, this vasomotor activity appears to be dissociated from the ROCK. Thus, coronary arteriolar constrictions evoked by ET-1 and PKC are mediated by different signaling pathways. The sequential events responsible for ET-1-induced vasoconstriction are highlighted with thick lines in the schematic diagram and the inhibitors used for probing the involved pathways are also shown. Example 2: Rho Kinase (ROCK) Inhibition in Conjunction with Cancer Therapy, Immunosuppressive Medications, and Substance Abuse (e.g., Drug or Alcohol Abuse) Attorney Docket No.11164-015WO1 In this Example, we extend our findings above to cancer therapy because solid evidence is accumulated for some chemotherapy drugs of causing coronary microvascular complications. New evidence is emerging that many anti-cancer therapies (e.g., antibody, kinase inhibitors) lead to systemic hypertension and contribute to adverse coronary microvascular dysfunction. Such conditions lead to a new subspecialty called onco-cardiology or cardio-oncology. However, there are no specific medications to address these problems due to insufficient research in studying microvessels, the origin of this clinical problem. Herein, we propose that anticancer drugs (doxorubicin, 5-FU, bevacizumab, and tyrosine kinase inhibitors such as dasatinib, nilotinib), with hypertension, microvascular diseases, and / or cardiotoxicity as part of well-established side effects. In this Example, we extend our findings above to immunosuppressive therapy involving tacrolimus, cyclosporine A or other drugs in the same category because clinical evidence suggests their contribution to the coronary microvascular dysfunction and vasculopathy. A challenging part for heart transplant recipients is that they are not able to sense chest pain, because the innervations from the donated heart are not connected to the recipient’s nervous system. Evidence has suggested that coronary adverse events from drugs like doxorubicin or tacrolimus are associated with elevated ET-1. In addition, drug-induced artery constriction, with underlying cause from abnormal ET-1 expression, was reported but these drugs have not been systemically explored in experiments using coronary arterioles. We propose that some offending drugs activate ROCK signaling, directly or indirectly (e.g. increasing ET-1 expression) and that H-1152 can effectively reverse the adverse effect of anticancer drugs. Thus, H-1152 can be the first drug that not only remedies hypertension and tissue ischemia but also improves quality of life and may reduce chances of death from cardiovascular side effects due to anticancer therapies. When co-administrating with anticancer therapies, H-1152 may also boost the efficacy of chemotherapy and radiotherapy that are less effective under hypoxia. In this Example, we also extend our findings above to substance abuse (e.g. cocaine, nicotine and alcohol etc.) related to coronary spasm and / or microvascular dysfunction. Background Microvascular dysfunction contributes to a myriad of cardiovascular diseases associated with diabetes mellitus, hypertension, hyperlipidemia, obesity, hyperglycemia, and many other medical complications. Despite microvascular dysfunction being known to be highly prevalent Attorney Docket No.11164-015WO1 and associated with adverse clinical outcomes, effective medical treatment remains unavailable due to a limited understanding of the pathophysiology of microvascular dysfunction. The microvascular disease links to blood flow dysregulation and can ultimately lead to irreversible tissue damage and organ failure. Due to technical challenges in studying microvessels, e.g., visualizing / imaging / isolating, network complexity, and the limited available tissue for molecular characterization, the advance in microvascular research has been either linear or stagnating over the past decade. Consequently, the knowledge concerning the mechanism of microvascular dysregulation was mainly extrapolated from studies in large conduit vessels or cultured vascular / non-vascular cells. Using the methods we have developed for studying microvascular function, we have identified that the overproduction of endothelin-1 (ET-1), the most potent vasoconstrictor known in the human cardiovascular system with proinflammatory activity, contributes to microvascular dysfunction in the diseased heart and retina through activation of Rho kinase (ROCK). Moreover, ROCK inhibitor H-1152 both prevents and reverses the vasoconstriction of ET-1 and consequently restores microvascular function. In our hands, H-1152 shows the best potency / specificity to reverse ET-1’s action, and we have completed proof-of-concept studies at the translational level in animal models of cardiovascular disease. In this Example, we propose to extend the utilization of H-1152 for treating cancer therapy-, immunosuppressive therapy-, and substance-abuse related CVD that has not been investigated in the microcirculation by using isolated pressurized arterioles. Certain anticancer drugs with known cardiovascular side effects exhibit vasoconstrictor activity, which is believed to be elicited directly by the activation of ROCK or indirectly by the overproduction of ET-1. Evidence also suggests that cocaine can induce ET-1 expression from vascular endothelial cells, which can lead to contraction of underlying smooth muscle cells. Likewise, the deterioration of microvascular endothelial function among heart transplant recipients was associated with an enhanced coronary ET-1 concentration during follow-up. We propose that these adverse effects can be reversed by H-1152. For example, H-1152 can be applied in conjunction with cancer therapy not only to mitigate hypertension and cardiovascular complications but also to improve flow / oxygen delivery and thus enhance the responsiveness of chemotherapy and radiotherapy that are less effective under tissue hypoxia / ischemia. When co- administered with narcotic medications, H-1152 may relieve tissue ischemia. When co- Attorney Docket No.11164-015WO1 administered with immunosuppressive medications, H-1152 may reduce or slow down the progress of coronary microvascular dysfunction / disease. Summary of Proposed Treatments Microvascular dysfunction has a myriad of clinical presentations, ranging from ischemic heart disease to diabetic complications, nephropathy, systemic / pulmonary hypertension, stroke, blindness, preeclampsia, and dementia with cognitive impairment. However, there is no approved medication specifically for treating microvascular diseases. A key factor in failing to identify effective drugs is the lack of a mechanistic approach to understanding microvascular dysfunction and targeting the underlying pathophysiology. The isolated and pressurized microvessel technology described above has allowed us to mechanistically address microvascular diseases. We have demonstrated that the microvascular arterioles exhibit unique properties different from the upstream conduit vessels in physiology and pathophysiology and that the overproduction of ET-1 contributes to the microvascular dysfunction in a pig model with heart and retinal diseases. The pathophysiology (i.e., excessive arteriolar constriction) derived by ET-1 is mediated by ROCK activation in the microcirculation, which can be functionally restored by its specific inhibitor H-1152. Therefore, H-1152 has the potential to become the first effective drug to address the unmet need in treating microvascular diseases. A recent review on the comprehensive meta-analysis of global clinical trials in chemotherapy of 6,241 patients indicated that cancer treatment-induced hypertension is the most frequent case of cardiovascular toxicities, especially the combination of cancer drug treatment. Hypertension has been recognized as the most common comorbidity among various types of cancers, which is one of the high-risk factors for cancer survivors suffering from the comorbidity of heart diseases. Furthermore, anticancer drugs including doxorubicin, 5-FU, bevacizumab, dasatinib and nilotinib; immunosuppressive including tacrolimus can promote coronary artery diseases with microvascular dysfunctions. The potential mechanisms such as vascular rarefaction, endothelial dysfunction, impaired vasodilation, oxidative stress, and potentiated vasoconstrictor effects have been suggested for hypertension and microvascular disorder in association with anticancer therapy. Animal data suggest the role of reduced vasodilator nitric oxide and vascular rarefaction in hypertension, particularly with the use of angiogenesis-inhibiting drugs such as tyrosine kinase inhibitors by blocking access to vascular endothelial growth factor receptor 2 (VEGFR2) signaling and antibody drugs by directly Attorney Docket No.11164-015WO1 binding to vascular endothelial growth factor. However, our understanding of the mechanism of hypertension associated with anticancer therapy in humans is very limited and the treatment is largely empirical rather than based on solid scientific evidence. Currently, there is no available or sufficient clinical trial data to define specific guidelines for antihypertensive therapies in patients with cancer; therefore, a pragmatic approach is generally adopted. Medications that are used for treating chemotherapy-related hypertension include angiotensin-converting enzyme and angiotensin II receptor inhibitors, calcium channel blockers and other antihypertensive agents, which are not directly related to the etiology of hypertension and microvascular disorder associated with anticancer drugs. Moreover, these antihypertensive drugs are for the management of chronic hypertension, and evidence supporting their use for managing hypertension induced by cancer therapy is still insufficient. It should be noted that various complications, including organ damage, hypotension, angioedema, hyperkalemia, diarrhea, hyperglycemia, renal injury, dizziness, urinary urgency, fatigue, weight gain, bradycardia, and several others related to the use of antihypertensive therapeutics are well recognized. Therefore, the unfavorable side effects, inconsistent efficacy, and empirical use of these conventional antihypertensive drugs in conjunction with anticancer therapies remain of concern. It is worth noting that we have reported the adverse effects of ET-1 on endothelial function, vasodilation, oxidative stress, and promoting vasoconstriction, which all are actions that can contribute to the development of hypertension and tissue ischemia. Interestingly, tissue ischemia / hypoxia, a common phenomenon in most malignant tumors, can lead to angiogenesis, dysfunctional vascularization, promoting cell mobility / metastasis, and resistance to chemotherapy and radiotherapy by inducing cell quiescence and reducing chemosensitivity. Therefore, we propose that hypertension and vascular complications induced by some common anticancer drugs (e.g., doxorubicin, 5-FU, bevacizumab, nilotinib, etc.); immunosuppressive medications (e.g., tacrolimus, cyclosporine A, etc.), and substance abuse (e.g., cocaine) are mediated by ET-1 via microvascular ROCK activation, or directly activate ROCK signaling. We have observed the vasoconstrictor effect of dasatinib in three pilot experiments, two in pig retinal arterioles and one in mouse ophthalmic arteries. Dasatinib is one of the second- generation tyrosine kinase inhibitors (TKIs), along with nilotinib and bosutinib, used as frontline therapy in chronic myeloid leukemia. Although these new TKIs show superior clinical outcomes, they also exhibit increased vascular events and toxicity. For example, growing Attorney Docket No.11164-015WO1 clinical evidence suggests that dasatinib causes drug-induced PAH. At its effective clinical concentrations, we found that dasatinib evoked 15-20% constriction of arterioles, which is estimated, based on the diameter and flow relationship, to reduce blood flow by more than 50%. This is consistent with the hypothesis that the observed cardiovascular complications of some anticancer drugs may be due to arteriolar constriction. Herein, we also propose that ROCK inhibitor H-1152 can effectively reverse the vasoconstrictor action of anticancer drugs and improve the effectiveness of anticancer therapy. This H-1152 technology can extend the scope of treating microvascular diseases to anticancer therapy. For initial study, four different anticancer drugs were chosen to represent four different categories of anticancer mechanism: doxorubicin as an anthracycline antibiotic / topoisomerase inhibitor, 5-FU as a thymidylate synthase inhibitor, bevacizumab as a VEGF / angiogenesis inhibitor, and dasatinib as a TKI. These examples cover a broad spectrum of anticancer therapeutics. One immunosuppressive drug tacrolimus and addictive substance cocaine or its metabolites will also be studied for their vasoconstriction effects in vitro. We will investigate the vasomotor activity of coronary and retinal arterioles isolated from pigs because this large animal model resembles humans in cardiovascular physiology / pathophysiology and is applicable to translational medicine. It is worth noting that the heart is a major organ that suffers from hypertensive impacts with the development of coronary microvascular dysfunction by anticancer drugs. Moreover, changes in the retinal microvasculature of healthy individuals are independently associated with future risk of type 2 diabetes, congestive heart failure, and cardiovascular mortality. Notably, the development of retinal microvascular disorder precedes the clinical onset of diabetic retinopathy in type 1 and type 2 diabetes. Therefore, studying the vasomotor activity of arterioles in these two organ systems has important clinical value for the understanding of CVD development as well as treatment. The coronary and retinal arterioles, 40-80 µm in diameter, were isolated and pressurized in a dual-reservoir cannulation system and studied using videomicroscopic techniques as described above. It should be noted that the microvessels studied in our lab exhibit spontaneous basal tone and present vasomotor activity as seen in vivo. Therefore, the collected data reflect the microvascular behavior in the intact microcirculation. We evaluated both the impact of Attorney Docket No.11164-015WO1 these chemotherapeutic agents on the microvasculature and the ability of ROCK inhibitors, such as H-1152, to reverse any negative effects on the microvasculature. Figure 15 is a plot demonstrating that dasatinib, an anticancer tyrosine kinase inhibitor, causes constriction of small ophthalmic resistance arteries, and that this response can be reversed by H-1152. Briefly, small ophthalmic resistance arteries were isolated from mice for vasomotor study using videomicroscopic techniques. The vessels developed basal tone (resting diameter <100 µm) after pressurizing to 75 cmH2O (55 mmHg) with a dual-reservoir system. Dasatinib, an anticancer tyrosine kinase inhibitor, was added to the physiological saline solution surrounding the vessels. Dasatinib (5 µM) caused significant vasoconstriction (i.e., decrease in resting diameter), which was reversed by the subsequent administration of Rho kinase inhibitor H-1152 (3 µM). Maximum diameter was obtained by replacing the vessel bath with a Ca2+-free physiological saline solution. The compounds, compositions, and methods of the appended claims are not limited in scope by the specific compounds, compositions, and methods described herein, which are intended as illustrations of a few aspects of the claims. Any compounds, compositions, and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compounds, compositions, and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth Attorney Docket No.11164-015WO1 used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

Attorney Docket No.11164-015WO1 WHAT IS CLAIMED IS:

1. A method of treating or preventing microvascular dysfunction in a subject in need thereof comprising administering to the subject an effective amount of a Rho Kinase (ROCK) inhibitor.

2. The method of claim 1, wherein the microvascular dysfunction is associated with elevated levels of endothelin-1 (ET-1).

3. A method of inducing microvascular vasodilation in a subject comprising administering to the subject an effective amount of a Rho Kinase (ROCK) inhibitor.

4. A method of reversing endothelin-1-induced vasoconstriction in a subject in need thereof, the method comprising administering to the subject an effective amount of a Rho Kinase (ROCK) inhibitor.

5. A method of treating or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject, the method comprising administering to the subject an effective amount of a Rho Kinase (ROCK) inhibitor.

6. The method of claim 5, wherein the disease, disorder or condition is associated with elevated levels of endothelin-1 (ET-1).

7. The method of any of claims 5-6, wherein the disease, disorder or condition fails to respond to a calcium channel blocker.

8. The method of any of claims 5-7, wherein the disease, disorder or condition fails to respond to a vasodilator, such as a calcium channel blocker or nitrate.

9. The method of any of claims 5-8, wherein the disease, disorder or condition fails to respond to a statin.Attorney Docket No.11164-015WO1 10. The method of any of claims 5-9, wherein the disease, disorder or condition fails to respond to an anti-anginal agent, such as ranolazine.

11. The method of any of claims 5-10, wherein the disease, disorder or condition comprises microvascular angina (also known as cardiac syndrome X; CSX).

12. The method of any of claims 5-11, wherein the disease, disorder or condition comprises Coronary microvascular dysfunction / disease (CMD) as a stand-alone diagnosis, or from other conditions such as systemic lupus erythematosus (SLE) or substance abuse.

13. The method of any of claims 5-12, wherein the disease, disorder or condition comprises drug-resistant hypertension.

14. The method of any of claims 5-13, wherein the disease, disorder or condition comprises drug-induced hypertension, such as hypertension induced by the administration of an anticancer agent.

15. The method of any of claims 5-14, wherein the disease, disorder or condition comprises coronary artery spasm, percutaneous coronary intervention (PCI)-related refractory myocardial ischemia, cerebral vasospasm following subarachnoid hemorrhage, drug-resistant systemic hypertension, drug-induced hypertension (e.g., bevacizumab-induced hypertension), drug- induced cardiotoxicity (e.g., cardiotoxicity induced by a chemotherapeutic agent such as 5- fluorouracil or an immunosuppressive agent such as cyclosporin A, tacrolimus, etc.), pulmonary arterial hypertension, substance abuse (e.g., cocaine) related myocardial infarction, diabetes-induced microvascular dysfunction, microangiopathy, silent stroke, or any combination thereof.

16. The method of any of claims, wherein the disease, disorder or condition comprises coronary microvascular dysfunction or vasculopathy from an immunosuppressive medication, such as tacrolimus or cyclosporine A.Attorney Docket No.11164-015WO1 17. The method of claim 16, wherein the subject comprises an organ transplant recipient.

18. The method of claim 17, wherein the subject comprises a heart transplant recipient.

19. The method of claim 17, wherein the subject comprises a recipient of an organ transplant other than a heart transplant.

20. The method of any of claims 1-19, wherein the ROCK inhibitor selectively inhibits ROCK2 activity over ROCK1 activity.

21. The method of any of claims 1-19, wherein the ROCK inhibitor selectively inhibits ROCK1 activity over ROCK2 activity.

22. The method of any of claims 1-21, wherein the ROCK inhibitor is defined by Formula Iwherein L is O or SO2; R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R3is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R5is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN;Attorney Docket No.11164-015WO1 R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; R7is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R8is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; n is 1, 2, 3 or 4; m is, independently for each occurrence, 1, 2, 3, or 4; R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; and R″ is (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.

23. The method of any of claims 1-22, wherein the ROCK inhibitor comprises an isoquinoline sulfonamide.

24. The method of any of claims 1-23, wherein the ROCK inhibitor is defined by Formula IA whereinR1is H, hydroxy, NH2, NHR′; or NR′R′;Attorney Docket No.11164-015WO1 R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; and R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.

25. The method of any of claims 1-24, wherein the ROCK inhibitor comprises fasudil (HA- 1077).

26. The method of any of claims 1-24, wherein the ROCK inhibitor comprises Glycyl-H 1152.

27. The method of any of claims 1-24, wherein the ROCK inhibitor comprises H-1152.

28. The method of any of claims 1-24, wherein the ROCK inhibitor comprises ripasudil.

29. The method of any of claims 1-24, wherein the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethyl fasudil, H-1152, H-1152P, H-1152 dihydrochloride, ripasudil, netarsudil, belumosudil, SAR407899, a prodrug thereof, a salt thereof, or a combination thereof.

30. The method of any of claims 1-21, wherein the ROCK inhibitor is selected from the group consisting of AR-12286, AT-13148, BA-1049 (R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, a prodrug thereof, a salt thereof, or a combination thereof.Attorney Docket No.11164-015WO1 31. The method of any of claims 1-30, further comprising measuring circulating levels of endothelin-1 (ET-1) in the subject to determine an appropriate dosing regimen of the ROCK inhibitor.

32. The method of any of claims 1-31, wherein the subject exhibits elevated circulating levels of endothelin-1 (ET-1).

33. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a cancer therapy in combination with an effective amount of a ROCK inhibitor.

34. A method of improving the efficacy of a cancer therapy, the method comprising co- administering an effective amount of a ROCK inhibitor in combination with the cancer therapy 35. The method of any of claims 33-34, wherein administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor simultaneously with the cancer therapy.

36. The method of any of claims 33-34, wherein administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor after administration of the cancer therapy.

37. The method of any of claims 33-34, wherein administering the effective amount of a ROCK inhibitor in combination with the cancer therapy comprises administering the ROCK inhibitor before administration of the cancer therapy.

38. The method of any of claims 33-37, wherein the effective amount of a ROCK inhibitor comprises an effective amount to treat or prevent a cardiovascular disease caused by the cancer therapy, such as drug-induced hypertension or drug-induced cardiotoxicity.Attorney Docket No.11164-015WO1 39. The method of any of claims 33-38, wherein the cancer therapy comprises administration of a chemotherapeutic agent.

40. The method of claim 39, wherein the chemotherapeutic agent comprises a DNA- alkylating agent, an anti-tumor antibiotic agent, an anti-metabolic agent, a tubulin stabilizing agent, a tubulin destabilizing agent, a hormone antagonist agent, a topoisomerase inhibitor, a protein kinase inhibitor, a HMG-CoA inhibitor, a CDK inhibitor, a cyclin inhibitor, a caspase inhibitor, a proteasome inhibitor, a metalloproteinase inhibitor, an antisense nucleic acid, a triple-helix DNA, a nucleic acid aptamer, a molecularly-modified viral, bacterial or exotoxic agent, or any combination thereof.

41. The method of any of claims 39-40, wherein the chemotherapeutic agent comprises cytidine arabinoside, cytarabine, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin- D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, taxol and derivatives thereof, tamoxifen, dacarbazine, rituximab, daunorubicin, 1- β-D-arabinofuranosylcytosine, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, fludarabine, docetaxel, bevacizumab, trastuzumab FOLFOX4, bortezomib, carfilzomib, ixazomib, or any combination thereof.

42. The method of any of claims 33-41, wherein the cancer therapy comprises administration of radiotherapy.

43. A method of treating or preventing cancer treatment-induced hypertension in a subject undergoing cancer treatment, the method comprising administering to the subject an effective amount of a Rho Kinase (ROCK) inhibitor.

44. A method of treating or preventing substance abuse-related cardiovascular disease in a subject, the method comprising administering to the subject an effective amount of a ROCK inhibitor.Attorney Docket No.11164-015WO1 45. The method of claim 44, wherein the substance abuse-related cardiovascular disease comprises cocaine-induced cardiotoxicity.

46. The method of any of claims 44-45, wherein the substance abuse-related cardiovascular disease comprises cocaine-induced myocardial infarction.

47. The method of any of claims 33-46, wherein the ROCK inhibitor selectively inhibits ROCK2 activity over ROCK1 activity.

48. The method of any of claims 33-46, wherein the ROCK inhibitor selectively inhibits ROCK1 activity over ROCK2 activity.

49. The method of any of claims 33-48, wherein the ROCK inhibitor is defined by Formula Iwherein L is O or SO2; R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R3is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R5is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN;Attorney Docket No.11164-015WO1 R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; R7is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R8is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; n is 1, 2, 3 or 4; m is, independently for each occurrence, 1, 2, 3, or 4; R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; and R″ is (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.

50. The method of any of claims 33-49, wherein the ROCK inhibitor comprises an isoquinoline sulfonamide.

51. The method of any of claims 33-50, wherein the ROCK inhibitor is defined by Formula IA whereinR1is H, hydroxy, NH2, NHR′; or NR′R′;Attorney Docket No.11164-015WO1 R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; and R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.

52. The method of any of claims 33-51, wherein the ROCK inhibitor comprises fasudil (HA-1077).

53. The method of any of claims 33-51, wherein the ROCK inhibitor comprises Glycyl-H 1152.

54. The method of any of claims 33-51, wherein the ROCK inhibitor comprises H-1152.

55. The method of any of claims 33-51, wherein the ROCK inhibitor comprises ripasudil.

56. The method of any of claims 33-51, wherein the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethyl fasudil, H-1152, H-1152P, H-1152 dihydrochloride, ripasudil, netarsudil, belumosudil, SAR407899, a prodrug thereof, a salt thereof, or a combination thereof.

57. The method of any of claims 33-48, wherein the ROCK inhibitor is selected from the group consisting of AR-12286, AT-13148, BA-1049 (R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, a prodrug thereof, a salt thereof, or a combination thereof.Attorney Docket No.11164-015WO1 58. A method for assessing microvascular function in a subject, the method comprising: measuring coronary blood flow velocity in the subject before and after administration of a ROCK inhibitor; and determining the subject’s coronary flow reserve before and after administration of a ROCK inhibitor; and calculating a change in the subject’s coronary flow reserve assessed by administration of the ROCK inhibitor; wherein a less than anticipated change of coronary flow reserve in the subject assessed by administration of the ROCK inhibitor indicates that the subject exhibits coronary microvascular dysfunction.

59. The method of claim 58, wherein the coronary blood flow velocity in the subject is measured invasively, for example, during a coronary angiography or angioplasty procedure.

60. The method of claim 58, wherein the coronary blood flow velocity in the subject is measured non-invasively, for example, using MRI or PET.

61. The method of any of claims 58-60, wherein the subject’s coronary flow reserve is a ratio of peak diastolic flow velocity to resting peak diastolic flow velocity.

62. The method of any of claims 58-61, wherein a change in CFR at a fixed intracoronary dose of less than about 2.5, such as a change in CFR at a fixed intracoronary dose of less than about 2.3, or a change in CFR at a fixed intracoronary dose of less than about 2 indicates that the subject exhibits coronary microvascular dysfunction.

63. The method of any of claims 58-62, wherein when the subject exhibits a less than anticipated change in the subject’s coronary flow reserve induced by administration of the ROCK inhibitor, the method further comprises administering to the subject an effective amount of a ROCK inhibitor to achieve better blood flow.Attorney Docket No.11164-015WO1 64. The method of claim 63, further comprising selecting the patient for prescription of a ROCK inhibitor to treat underlying coronary microvascular dysfunction.

65. The method of claim 63, wherein when the effective amount of a ROCK inhibitor fails to improve CFR or provides for only a minor improvement in CFR, and wherein the method further comprises selecting an alternative therapy to treat the subject.

66. The method of any of claims 58-61, wherein a change in CFR at a fixed intracoronary dose of greater than about 2.5, such as a change in CFR at a fixed intracoronary dose of greater than about 2.7, or a change in CFR at a fixed intracoronary dose of greater than about 3 indicates that the subject does not exhibits coronary microvascular dysfunction.

67. The method of any of claims 58-66, wherein the method also applies to to cerebral or peripheral angiogram procedure when a ROCK inhibitor is used for calculating blood flow reserve or an index of microcirculatory resistance (IMR).

68. The method of any of claims 58-66, wherein the method also applies to a noninvasive diagnostic method (e.g., CT, ultrasound, MRI, PET etc.) based on changed blood supply before and after administering ROCK inhibitors.

69. The method of any of claims 58-68, wherein the ROCK inhibitor selectively inhibits ROCK2 activity over ROCK1 activity.

70. The method of any of claims 58-68, wherein the ROCK inhibitor selectively inhibits ROCK1 activity over ROCK2 activity.

71. The method of any of claims 58-70, wherein the ROCK inhibitor is defined by Formula IAttorney Docket No.11164-015WO1wherein L is O or SO2; R1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R3is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R5is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; R7is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R8is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; n is 1, 2, 3 or 4; m is, independently for each occurrence, 1, 2, 3, or 4; R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; and R″ is (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.Attorney Docket No.11164-015WO1 72. The method of any of claims 58-71, wherein the ROCK inhibitor comprises an isoquinoline sulfonamide.

73. The method of any of claims 58-72, wherein the ROCK inhibitor is defined by Formula IA whereinR1is H, hydroxy, NH2, NHR′; or NR′R′; R2is H, halogen, hydroxy, (C1-C6)alkyl, or (C1-C6)alkoxy; R4is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR′, NR′R′, or CN; R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R″, (C1-C6)alkylene-C(O)—R′, (C1-C6)alkylene- C(O)O—R′, (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR′, (C1-C6)alkylene- C(O)NR′R′, C(O)OR′, C(O)R′, C(O)NH2, C(O)NHR′, C(O)NR′R′, C(O)—(C1-C6)alkylene— NH2, C(O)—(C1-C6)alkylene—NHR′, C(O)—(C1-C6)alkylene—NR′R′, C(O)O—(C1- C6)alkylene—NH2, C(O)O—(C1-C6)alkylene—NHR′, or C(O)O—(C1-C6)alkylene—NR′R′; and R′ is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C10)heterocyclyl, or (C6-C10)aryl; or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.

74. The method of any of claims 58-73, wherein the ROCK inhibitor comprises fasudil (HA-1077).Attorney Docket No.11164-015WO1 75. The method of any of claims 58-73, wherein the ROCK inhibitor comprises Glycyl-H 1152.

76. The method of any of claims 58-73, wherein the ROCK inhibitor comprises H-1152.

77. The method of any of claims 58-73, wherein the ROCK inhibitor comprises ripasudil.

78. The method of any of claims 58-73, wherein the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethyl fasudil, H-1152, H-1152P, H-1152 dihydrochloride, ripasudil, netarsudil, belumosudil, SAR407899, a prodrug thereof, a salt thereof, or a combination thereof.

79. The method of any of claims 58-70, wherein the ROCK inhibitor is selected from the group consisting of AR-12286, AT-13148, BA-1049 (R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, a prodrug thereof, a salt thereof, or a combination thereof.

80. A pharmaceutical compositions or kit comprising an effective amount of a ROCK inhibitor to treat or prevent microvascular dysfunction in a subject in need thereof, an effective amount of a ROCK inhibitor to induce microvascular vasodilation in a subject, an effective amount of a ROCK inhibitor to reverse ET-1-induced vasoconstriction in a subject, an effective amount of a ROCK inhibitor to treat or prevent a disease, disorder, or condition associated with microvascular dysfunction in a subject, and / or an effective amount of a ROCK inhibitor to treat or prevent substance abuse-related cardiovascular disease in a subject.

81. A pharmaceutical composition or kit comprising an effective amount of a ROCK inhibitor and an effective amount of a chemotherapeutic agent.

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