Compositions and methods for treating ischemic conditions

Abstract

Pharmaceutical compositions and methods for treating an ischemic condition in a subject are provided. [Solution] A composition for treating an ischemic condition in a subject can include the hexosamine D-mannosamine (ManN). The method can include administering an effective amount of ManN to a subject in need thereof. The administration can be effective to promote endothelial cell proliferation and angiogenesis in the subject. The subject can be in need of induction of angiogenesis due to an ischemic condition caused by disease or trauma. A composition for inhibiting protein glycosylation in a cell can include ManN. The method for inhibiting protein glycosylation in a cell can include administering an effective amount of ManN to the cell.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 094,032, filed on October 20, 2020, which is incorporated herein by reference in its entirety.

[0002] This disclosure generally relates to compositions and methods for treating ischemic conditions. [Background technology]

[0003] Angiogenesis is a complex process involving the growth of new blood vessels from the existing vascular system and occurs in both physiological and pathological contexts. In tumors, angiogenesis facilitates rapid growth and metastasis through the delivery of nutrients and oxygen, as well as the removal of metabolic waste products.[1] Vascular development requires the coordinated activation of multiple signaling pathways, including VEGF / VEGFR, angiopoietin (Ang) / Tie2, Notch, ephrin / Eph, and PDGF / PDGFR.[2,3] Stimulating angiogenesis may facilitate the treatment of several conditions characterized by reduced perfusion, including diabetic ulcers, myocardial, and limb ischemia.[4,5] Conversely, blocking angiogenesis is a clinically validated strategy for treating malignancies and intraocular neovascularization.[1,6]

[0004] Endothelial cell (EC) metabolism is hypothesized to play a crucial role in regulating angiogenesis in both normal and pathological settings. Metabolic switches of ECs, such as fatty acid, glucose, and glutamine metabolism, have been reported to induce angiogenesis [7, 8]. Tumor vascular ECs are known to depend on glycolysis for ATP production, for example, through enhanced expression of the glucose transporter GLUT1. Reduced glycolysis in tumor ECs inhibits their proliferation [9]. Furthermore, abnormal glycosylation patterns have been described during oncogenic transformation and cancer progression, and it has been suggested that inhibition of glycosylation may lead to suppression of major angiogenic pathways, including VEGF / VEGFR2 and Notch

[10] . Recent evidence points to glycans as novel angiogenesis regulators through alterations in protein glycosylation

[11] . For example, it has been reported that the glycan-binding protein galectin 1 interacts with VEGFR2, leading to ligand-independent receptor activation, which may contribute to tumor resistance to anti-VEGF therapy

[11] . Therefore, EC metabolism has been identified as a novel target for anti-angiogenic therapies, particularly through the inhibition of energy metabolism and glycosylation. [Overview of the project]

[0005] This disclosure provides pharmaceutical compositions and methods for treating ischemic conditions in subjects. In embodiments, the composition for treating ischemic conditions in subjects comprises hexosamine D-mannosamine (ManN). In embodiments, the method for treating ischemic conditions in subjects comprises administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need.

[0006] In embodiments, the administration is effective in promoting endothelial cell proliferation and angiogenesis in the subject. In embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject that requires it. In embodiments, the method further comprises administering an effective amount of VEGF to a subject that requires it.

[0007] In embodiments, the ischemic state is caused by disease or trauma. In embodiments, administration is intravenous, intraperitoneal, or intravitreous.

[0008] In embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, which includes administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need thereof.

[0009] In the embodiments, the administration is effective in reducing ischemia in the subject. In the embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need. In the embodiments, the method further comprises administering an effective amount of VEGF to a subject in need.

[0010] In the embodiment, the subject needs to induce angiogenesis through an ischemic state, which is caused by disease or trauma.

[0011] In embodiments, administration is intravenous, intraperitoneal, or intravitreous.

[0012] In embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting protein glycosylation in cells, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to cells.

[0013] In the embodiments, the administration is in vivo. In the embodiments, the administration is ex vivo. In the embodiments, the administration is effective in stimulating EC proliferation and angiogenesis. In the embodiments, the administration is effective in activating unfolded protein responses induced by JNK and ER stress. In the embodiments, the administration is effective in inducing changes in N-glycan and O-glycan profiles. [Brief explanation of the drawing]

[0014] [Figure 1A-1F]This document presents an example demonstrating the effect of mannosamine (ManN) on bovine choroidal microvascular endothelial cell (BCEC) proliferation. Figure 1A shows images of crystal violet-stained BCEC samples treated with ManN, with or without VEGF. BCECs were treated with various concentrations of ManN ranging from 0.5 μM to 1 mM for 5–6 days, with or without 5 ng / ml VEGF. At the end of the experiment, the cells were fixed and stained with crystal violet. The cell coverage area of ​​each treatment group was quantified using ImageJ software. Figure 1B is a chart showing the effect of ManN on cell number, with or without VEGF. Cell number was quantified by adding AlamarBlue, and fluorescence was measured at 530 nm / 590 nm. Three independent samples were used. Figure 1C is a chart showing the effect of ManN on bovine retinal microvascular endothelial cell (BREC) proliferation. Three biologically independent samples were used. Figure 1D is an image showing the effect of hexosamines other than ManN on BCEC proliferation in samples. Each treatment group was tested in pairs. Figure 1E is a chart and image showing the effect of ManN on wound BCEC samples. BCEC confluent monolayers were scratched with a 1 ml pipette tip, washed, and then incubated for 40 hours in low-glucose DMEM containing 1% FBS. Three independent samples were used (n=3). Scale bar = 400 μm. Images were taken, and the gap between wound fronts at the tip was quantified using AxioVision LE Rel.4.4 software. Representative images from crystal violet staining are shown. Figure 1F is a chart showing the effect of ManN in the BCEC transwell migration assay. Four independent samples were used (n=4). Asterisks indicate significant differences compared to controls. Lines were used between specific groups when statistical analysis was performed using different controls. Representative experiments from two independent studies are shown. Data are mean + / - SD, and statistical analysis was performed by a two-sided two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figure 2A-2C]These are Western blot images showing activation of ERK, AKT, mTOR, AMPKα, CREB, ACC, and eNOS, and are not specific to ManN. Enhanced activation of ERK (Thr202 / Tyr204), AKT (Ser473), and CREB (Ser133) in BCEC after treatment with ManN with VEGF for various time periods (Figure 2A), or after pretreatment with ManN for 8 hours followed by 15 minutes of VEGF stimulation (Figure 2B). For the samples shown in Figure 2C, BCEC was treated with 40 μM ManN, ManNAc, or mannose for various time periods. Western blot analysis examined the phosphorylation of total mTOR, ACC, eNOS, AMPKα, ERK, AKT, CREB, and mTOR (Ser2448), ACC (Ser79), eNOS (Ser1177), AMPKα (Thr172), ERK (Thr202 / Tyr204), AKT (Ser473), and CREB (Ser133). β-actin served as a loading control. Molecular weight (KDa) is labeled on the right. Representative experiments from two independent studies are shown. [Figure 3A-3E]This example demonstrates that ManN specifically activates the JNK pathway in BCEC. Figure 3A is a Western blot image. BCEC cells grown in growth medium (GM: low glucose DMEM containing 10% bovine calf serum (BCS), 10 ng / ml VEGF, and 5 ng / ml bFGF) were switched to a growth factor-free medium and subsequently treated with 4 μM–4 mM ManN or mannose. After 4 hours, cell lysates were collected and subjected to Western blot analysis for phosphorylated JNK (Thr183 / Tyr185), p38 (Thr180 / Tyr182), and ERK (Thr202 / Tyr204), as well as total JNK, p38, and ERK. Figure 3B is a Western blot image showing that ManN can activate JNK and its downstream c-Jun, but mannose cannot. β-actin served as a loading control. For each study, representative experiments from 2-3 independent studies are shown. Figure 3C is a chart showing the effect of ManN on pre-treated samples. BCECs plated in a 96-well plate were pre-treated with the specific JNK inhibitor SP600125 (5 μM) for 2 hours, followed by treatment with either 40 μM or 2 mM ManN, with or without 5 ng / ml VEGF. After 6 days, cell proliferation was quantified using AlamarBlue. Three independent samples were used (n=3). Figure 3D is a Western blot image showing siRNA screening against JNK1 and JNK2. 24 hours after siRNA transfection, BCECs were lysed and proteins were subjected to Western blot analysis. β-actin served as a loading control. Quantification of targeted knockdown is shown. Figure 3E is a chart showing exemplary results indicating that approximately 80% knockdown of JNK1 and / or JNK2 by two independent siRNAs was associated with a significant reduction in the stimulating effect of ManN on BCEC proliferation. Three independent samples (n=3) were used. Data are expressed as mean + / - SD, and asterisks indicate statistically significant differences compared to the control. Lines were used between specific groups when performing statistical analysis using different controls. Statistical analysis was performed using a two-sided, two-sample unequal variance t-test.*p<0.05, **p<0.01. [Figure 4A-4G] This document presents an example demonstrating the effect of ManN on protein glycosylation. Figure 4A is a Western blot image showing the reduction in VEGFR2 molecular weight after ManN treatment. BCEC cells were treated with 40 μM of various hexosamines, their derivatives, and monosaccharides, or with 5 ng / ml of VEGF for 24 hours. VEGFR2 Western blot analysis was performed. Figure 4B is a Western blot image showing the dose-dependent effect of ManN on VEGFR2 molecular weight in BCEC cells. Figure 4C is a Western blot image showing that mannose can dose-dependently reverse the effect of 2 mM ManN on VEGFR2 molecular weight change, although mannose alone had no effect even at 10 mM. Figure 4D is a chart showing that 5 mM mannose can completely reverse the bell effect of ManN on BCEC proliferation, with or without 5 ng / ml of VEGF. Plated BCEC cells were attached to 96 wells, followed by the addition of ManN. After 2 hours, the cells were treated with different concentrations of mannose, with or without VEGF. Six days later, cell proliferation was quantified using AlamarBlue. Three independent samples (n=3) were used. Figure 4E is a Western blot image showing the reversible effect of ManN. After treatment with 40 μM ManN for 24 hours, BCECs were washed three times with low-glucose DMEM. Cells were held in low-glucose DMEM for a further 8 or 24 hours. VEGFR2 Western blot analysis was performed. Figure 4F is a Western blot image showing the reduction in molecular weight of VEGFR2, neuropilin-1, CD31, and c-met in HUVECs after treatment with various concentrations of ManN. Figure 4G is a Western blot image showing the reduction in molecular weight of VEGFR2, β1 integrin, and bFGFR1 in hDMVECs with various concentrations of ManN. β-actin served as a loading control. Data are mean + / - SD, and asterisks indicate significant differences compared to the control. For each study, representative experiments from 2–5 independent studies are shown. Statistical analysis was performed using a two-sided 2-sample unequal variance t-test. *p<0.05, **p<0.01. [Figures 5A-5D] This example demonstrates that ManN specifically induces the expression of unfolded protein response (UPR) responsive proteins. Figure 5A shows Western blot images. BCECs were grown in growth medium (GM) to a concentration of approximately 80%. The medium was changed to growth factor-free medium containing 10% BCS in the presence or absence of 40 or 400 μM ManN or mannose over various time periods. At the end of each incubation, cell lysates were collected and proteins were separated on 4–12% Bis-Tris gels for Western blot analysis. Figure 5B shows Western blot images of cells treated for 24 hours with various concentrations of ManN, mannose, 5 ng / ml VEGF, or a combination of ManN and VEGF. For Western blot analysis, cell lysates were separated on NuPAGE 3–8% Tris-Acetate gels. Figure 5C is a Western blot image showing that 4-PBA effectively blocks CHOP induction in BCEC, accompanied by recovery of ATF-6 transcription factor expression upon 400 μM ManN treatment, whereas TUDCA does not. BCEC cells were pretreated with two chemical chaperones: 2 mM 4-PBA or 500 μM TUDCA. After 16 hours, cells were switched to growth factor-free medium for 4 hours in the presence of ManN. GM: Growth medium. Figure 5(d) 4-PBA significantly blocked the bell-shaped effect of ManN on BCEC proliferation. Pretreatment of cells with 1 mM 4-PBA for 8 hours suppressed the additive effect of 40 μM ManN and 5 ng / ml VEGF and protected cells from the toxic effects induced by 2 mM ManN. Three independent samples were used. For each study, representative experiments from two to three independent studies are shown. Data are in mean + / - SD, and asterisks indicate statistically significant differences compared to the control. Statistical analysis was performed by a two-sided, two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figure 6A-6J]This chart shows the effects of ManN on non-endothelial cells derived from bovine, mouse, or human. ManN did not promote the growth of Calu6 (Figure 6A), A673 (Figure 6B), U87MG (Figure 6C), and 4T1 (Figure 6D) tumor cells. 10% FBS was used as a positive control for Calu6 and A673, while 10 ng / ml bFGF and 1 μg / ml human apo-transferrin were used as positive controls for U87MG and 4T1, respectively. Similarly, ManN, alone or in combination with growth factors, did not induce increased proliferation on AML12 (Figure 6E), bovine pituitary cells (Figure 6F), NIH3T3 cells (Figure 6G), human RPE (Figure 6H), human dermal fibroblasts (Figure 6I), and human keratinocytes (Figure 6J). Proliferation was quantified using AlamarBlue or MTS (for 4T1 cells). Three independent samples (n=3) were used. Charts in Figures 6A–6J show representative Western blot analyses demonstrating the dose-dependent effects of 400 μM (2, 4) and 2 mM (3, 5) ManN and mannose on bFGFR1 or β1 integrin (for 4T1, AML12, NIH3T3 cells, human skeletal muscle cells, human dermal fibroblasts, and human keratinocytes) compared to an untreated control (1). β-actin served as a loading control. GM: Growth medium. For Western blot analysis, proteins were isolated on NuPAGE 3–8% Tris-Acetate gels. For each study, representative experiments from two independent studies are shown. Asterisks indicate statistically significant differences compared to the control. Parentheses were used between specific groups when statistical analysis was performed using different controls. Data were mean or mean + / - SD for n=2. Statistical analysis was performed by a two-sided, two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figures 7A-7F]Examples of the effects of protein glycosylation inhibitors on BCEC proliferation are shown. Figure 7A includes images of samples showing dose-dependent stimulation of BCEC proliferation by various glycosylation inhibitors. Inhibitors were added for 3 days at concentrations ranging from 0.01 to 100 μM, with or without 5 ng / ml of VEGF. At the end of the experiment, cells were fixed and stained with crystal violet. Representative experiments are shown: kyfunesin (Kif), an ERα-1,2-mannosidase I and Golgi α-mannosidase I inhibitor, and castanospermine (Cas), an α-glucosidase inhibitor. The cell coverage region of various treatment groups was quantified using ImageJ software. Figure 7B is a chart showing the dose-dependent effects of Kif and Cas on promoting BCEC proliferation, with or without 5 ng / ml of VEGF. Three independent samples were used (n=3). Figure 7C includes a Western blot image showing that both inhibitors reduced VEGFR2 molecular weight and induced Bip expression in a dose-dependent manner, as assessed by Western blot analysis. Proteins from whole cell lysates were separated using a 3–8% Tris-Acetate gel. BCEC was treated with the various inhibitors for 24 hours. Western blot quantification was performed by densitometry. β-actin was the loading control. Figure 7D is a chart showing the acceleration of monolayer gap closure by Kif (H2O) and Cas (DMSO) in a BCEC scratch assay, using Kif (H2O) and Cas (DMSO) controls. The gap was quantified using AxioVision LE Rel.4.4 software. Three independent samples were used (n=3). Scale bar = 400 μm. Figure 7E is a Western blot image showing the activation of AKT and JNK in BCEC with 40 μM glycosylation inhibitor and 10 ng / ml VEGF. However, CAS did not activate ERK. Quantification of phosphorylated AKT, JNK, and ERK was performed by densitometry analysis of total protein. Figure 7F is a chart showing that 2 hours of pretreatment of BCEC with 5 μM SP600125 significantly blocked the effects of both glycosylation inhibitors on BCEC proliferation. Three independent samples were used. Representative experiments are shown from 2–4 independent studies.The data shown are mean + / - SD. Statistical analysis was performed by a two-sided 2-sample unequal variance t-test. *p<0.05, **p<0.01. [Figures 8A-8D] This study demonstrates how topical application of ManN and VEGF stimulates angiogenesis and accelerates wound healing in mice. Figure 8A is a chart showing the effect of ManN on wounds. Wounds were created in the dorsal skin of mice using a 6 mm punch. VEGF and ManN were administered daily at a dose of 20 μg per wound in 25 μl of PBS for the first four days, with PBS serving as the control. A 10-day wound healing study was conducted with 5 mice in each group. Wound closure rate (%) was quantified using ImageJ software in two independent studies. Asterisks indicate significant differences compared to the control at each time point. Figure 8B includes images obtained from the 4-day wound healing study using images of the wound healing process on days 1, 2, and 4. n=5 animals / treatment group was used. Figure 8c includes representative images of CD31 immunohistochemical staining in the PBS control group and the VEGF and ManN combination group (scale bar = 200 μm). Figure 8D is a chart showing the quantification of CD31-positive vascular density (red dotted circle) around the wound area by the eye under a microscope (20X magnification). Data are mean + / - SD. Statistical significance was further confirmed using the Wilcoxon rank-sum test between the treatment groups of interest. Asterisks indicate significant differences compared to the PBS control. For each study, a representative experiment is shown. n=3 animals / treatment group was used. Lines were used between specific groups when statistical analysis was performed using different controls. Statistical analysis was performed by a two-sided 2-sample unequal variance t-test. *p<0.05, **p<0.01. [Figures 9A-9D]This example demonstrates that ManN accelerates hemoperfusion recovery in a mouse ischemic hindlimb model. Figure 9A includes images obtained from serial laser Doppler analysis of hemoperfusion in the hindlimbs of ManN-treated, Kif-treated, and control mice. Different colors are used to show hemoperfusion in the ischemic limb (ligated; left side) versus the non-ischemic limb (pseudo; right side). Representative images at week 0 and week 1 are shown. Figure 9B is a chart showing the quantification of the hemoperfusion ratio between region 2 (ischemic; left limb) and region 1 (non-ischemic; right limb), with n=8 animals / treatment group. Figure 9C includes images of sample tissue. Skeletal muscle tissue was harvested and fixed three weeks after surgery. CD31 immunostaining was performed on these tissue sections to label the vascular system. H&E staining was also performed. Representative CD31-stained and H&E histological images of the ischemic hindlimb 21 days after surgery are shown. Scale bar = 50 μm. Figure 9D is a chart showing the quantification of vascular density by CD31 immunostaining performed using ImageJ software, n=8 animals / treatment group, 3 independent experiments; data are mean + / - SEM. Statistical analysis was performed by a two-sided, two-sample unequal variance t-test. *p<0.05, **p<0.01. [Figure 10A-10B] An example showing ManN promoting retinal neovascularization in mice is presented. Figure 10A includes tissue images. Intravitreal injection of ManN increases vascular density. Adult mice were given a single intravitreal injection of 500 ng ManN, Kif, or 200 ng bFGF. PBS was used as a vehicle control. Seven days after injection, PFA-fixed retinas were subjected to CD31 immunofluorescence. Representative images of CD31-positive vessels are shown. n=10 animals / treatment group, 3 independent experiments, scale bar=50 μm. Figure 10B is a chart showing vascular density determined by ImageJ software, n=10 animals / treatment group, 3 independent experiments. Data were mean + / - SEM. Statistical analysis was performed by a two-sided, two-sample unequal variance t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 11A-11D]This is an example showing that ManN, not structurally related molecules, stimulates the proliferation of endothelial cells. Figure 11A is a chart showing the additive effect of ManN and bFGF on BCEC proliferation. Bell-shaped effect of ManN on BCEC proliferation BCEC were treated with ManN ranging from 0.4 to 400 μM for 5 to 6 days, with or without 20 ng / ml of bFGF. At the end of the experiment, proliferation was quantified using AlamarBlue. Figure 11B is a chart showing that the additive effect of VEGF and ManN on BCEC proliferation depends on the glycolytic pathway. Proliferation assays were performed in low-glucose DMEM medium without growth factors, or DMEM medium without glucose and pyruvate. Asterisks indicate significant difference compared with the untreated control. Statistical analysis was also performed to compare the VEGF alone and VEGF + ManN treatment groups for cells grown in two different assay media. Figures 11C and 11D are charts showing the effect of various concentrations of agents from 0.04 μM to 5 mM on BCEC in the absence (Figure 11C) or presence (Figure 11D) of 5 ng / ml VEGF. Independent samples of n=3 were used. For each study, a representative experiment from two independent studies is shown. Data are presented as the mean + / - SD. Statistical analysis was performed by a two-tailed, two-sample t-test with unequal variances. *p < 0.05, **p < 0.01. Mode for Carrying Out the Invention

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0016] Unless otherwise defined, all technical and scientific terms, and any acronyms, used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, exemplary methods, devices, and materials are described herein.

[0017] The present disclosure provides pharmaceutical compositions and methods for treating an ischemic condition in a subject, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need thereof. Since ManN is converted in vivo to ManN-6-phosphate (ManN-6p), the present disclosure provides the use of such metabolic precursors and derivatives.

[0018] ManN is a hexosamine that has the ability to inhibit protein post-translational modification, activate stress pathways, and exhibit additivity with VEGF in promoting endothelial cell (EC) proliferation and angiogenesis. The effects of ManN on ECs and angiogenesis have not been previously reported. Without being bound by theory, the use of known glycosylation inhibitors in combination with ManN may establish a correlation between changes in glycosylation patterns and angiogenesis in mammalian ECs. The effect of ManN on endothelial cells may be independent of VEGFR2 activation.

[0019] ManN was discovered as a component of bacterial cell walls in the 1960s

[15] , and accounts for 5 to 10% of capsular polysaccharides

[45] . Related N-acetylmannosamine is considered an intermediate in the biosynthesis of sialic acid

[46] . Over the years, multiple effects of ManN on enzymes, growth factor-mediated signaling, protein stability, and cell viability have been described[47~50]. Most of these effects are not specific to ManN, and may be induced by other hexosamines. Furthermore, they required high concentrations[45, 48]. ManN has been reported to have anti-tumor properties

[47] , stimulate osteogenic differentiation[48, 51], and protect articular cartilage

[49] . More recently, ManN has been used as an intermediate to facilitate high-throughput screening in the modification of various molecules / nanoparticles

[52] and in the synthesis of unnatural ManNAc analogs for expression of thiols on cell surface sialic acid

[53] . However, to date, the effects of ManN on ECs have not been described.

[0020] ManN has previously been reported to affect lipid-linked oligosaccharide (LLO) formation in MDCK cells, possibly by inhibiting α-1,2-mannosyltransferase

[24] . Upon ManN treatment, the major oligosaccharides associated with dolichol were Man5GlcNAc2 and Man6GlcNAc2, rather than Glc3Man9GlcNAc2, which is normally found in MDCK cells. Furthermore, ManN has been reported to alter protein GPI biosynthesis and hybrid glycan production in the ER [54-56]. However, none of the angiogenesis-related proteins previously examined are GPI anchors. Without being constrained by theory, a decrease in Man-9 may be a direct result of inhibiting LLO donor synthesis, i.e., Glc3Man9GlcNAc20PP-Dol formation, and subsequently transposing the dolichol donor to polypeptides. In embodiments, Man-5 can be significantly increased over 24 hours by treating cells with 40 μM ManN. ManN may not affect α-mannosidase in the ER.

[0021] Activation of PI3K-AKT, PLCγ-ERK, and p38 is associated with the survival, proliferation, and migration of VEGFR2-mediated ECs. Other cellular metabolic stress sensors, such as AMPK (AMP-activated protein kinase), may also confer stress adaptation and promote EC survival via eNOS

[57] . Without being constrained by theory, activation of ERK, AKT, mTOR, AMPKα, eNOS, and ACC is a common phenomenon for hexosamine and mannose. However, activation of the JNK / c-Jun and UPR pathways in BCECs is specific to ManN and glycosylation inhibitors. Glycosylation is required for correct protein folding in the ER

[26] . A link between LLO inhibition and UPR activation has been reported

[58] . Indeed, despite the complexity of ManN action, LLO inhibition followed by UPR activation seems to be a plausible explanation for the reported ManN effects.

[0022] EC can cope with acute / mild ER stress caused by glycosylation inhibition by activating the UPR pathway. UPR detects misfolded proteins accumulated in the ER and initiates a response to maintain cellular homeostasis via the induction of Bip, a major ER chaperone protein

[29] . BiP often binds to hydrophobic patches exposed on nascent or incompletely folded proteins that are not glycosylated. ManN shows a stronger induction of Bip expression compared to hexosamine. Similar effects on stress pathway activation can result from the glycosylation inhibitors Kif and Cas.

[0023] Glycosylation inhibition is considered a novel pharmacological strategy targeting metabolic pathways essential for excessive angiogenesis in various pathological conditions, and glycosylation inhibitors are expected to possess anti-angiogenic and anti-metastatic properties [10, 59, 60]. Glycosylation has been shown to be involved in cellular stress responses and compensatory angiogenesis in response to VEGF-VEGFR2 signaling blockade

[61] . Stress-induced O-GlcNAcylation has previously been reported to promote survival in various cell types in response to DNA damage, ER stress, glucose deficiency, and hypoxia

[62] . Without being constrained by theory, glycosylation inhibition may be associated with promoting angiogenesis, and inhibiting glycosylation within the tumor microenvironment may result in stimulation rather than suppression of tumor angiogenesis.

[0024] In embodiments, ManN may be used to promote angiogenesis in a mouse skin injury model, accompanied by accelerated wound closure. In embodiments, ManN may be used to stimulate angiogenesis and blood flow restoration in ischemic hind limbs of mice. Combinations of VEGF-A with ManN, or other glycosylation inhibitors, may offer advantages over monotherapy for the treatment of ischemic injury. The lack of a direct permeability-enhancing effect of ManN may result in less edematous tissue. In this context, damage to the pulmonary endothelium is a central pathogenic event of respiratory failure associated with various infections, including SARS-CoV-2

[65] . Endothelial mitogens like ManN, which lack permeability effects, may help protect and stabilize blood vessels and thus limit tissue damage.

[0025] In embodiments, intravitreal administration of ManN may be used, for example, in therapeutic applications in ocular diseases to enhance retinal neovascularization. While 10–15% of patients with moderate AMD progress to the neovascular type, the remaining patients may develop geographic atrophy (GA) [1]. Previous studies have shown that choroidal capillary loss is frequently detected in GA, and that choroidal capillary regeneration / protection may be a strategy for treating GA

[66] .

[0026] In embodiments, the administration is effective in promoting endothelial cell proliferation and angiogenesis in the subject. In embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject that requires it. In embodiments, the method further comprises administering an effective amount of VEGF to a subject that requires it.

[0027] In embodiments, the ischemic state is caused by disease or trauma. This disclosure provides treatments for several conditions characterized by reduced perfusion, including but not limited to diabetic ulcers, macular degeneration, peripheral arterial disease (PAD), limb ischemia, cerebral or cerebral ischemia, and coronary artery ischemia.

[0028] In embodiments, administration is intravenous, intraperitoneal, or intravitreous.

[0029] In embodiments, the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, which includes administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need thereof.

[0030] In embodiments, the administration is effective in reducing ischemia in a subject. In embodiments, ischemia may include cerebral ischemia. The administration may be effective in preventing, reducing, or treating conditions associated with cerebral ischemia, such as edema, ischemic stroke, or infarction. In embodiments, the method further comprises administering an effective amount of an N-glycosylation inhibitor to a subject in need. In embodiments, the method further comprises administering an effective amount of VEGF to a subject in need.

[0031] In the embodiment, the subject needs to induce angiogenesis through an ischemic state, which is caused by disease or trauma.

[0032] In embodiments, administration is intravenous, intraperitoneal, or intravitreous.

[0033] In embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting protein glycosylation in cells, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to cells.

[0034] In the embodiments, the administration is in vivo. In the embodiments, the administration is ex vivo. In the embodiments, the administration is effective in stimulating EC proliferation and angiogenesis. In the embodiments, the administration is effective in activating unfolded protein responses induced by JNK and ER stress.

[0035] In the embodiments, administration is effective in inducing changes in the N-glycan and O-glycan profiles. In the embodiments, administration is effective in inducing a reduction in Man6GlcNAc2 (Man-6), Man-8, and Man-9, an accumulation of Man-5 and Man-7, and a reduction in O-glycosylation after treatment with ManN, compared to an untreated control.

[0036] Conversely, in embodiments, the present disclosure provides pharmaceutical compositions and methods for inhibiting angiogenesis, and methods for treating malignancies and intraocular neovascularization in subjects, including administering an effective amount of hexosamine D-mannosamine (ManN) inhibitor to a subject in need, or reducing the amount of ManN available to the subject.

[0037] The implementation of this invention will utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, within the scope of the art, unless otherwise specified. Such techniques are described in A Laboratory Manual, 2. nd ed. (Sambrook et al., 1989), Oligonucleotide Synthesis (MJGait, ed., 1984), Animal Cell Culture (RIFreshney, ed., 1987), Methods in Enzymology (Academic Press, Inc.), Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987, and regular updates), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22 thThis is fully explained in publications such as (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0038] As used herein, “comprise,” “comprising,” “include,” “including,” “has,” “having,” “contain,” “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion of the enumerated components, subject to any limitations expressly indicated elsewhere. For example, a pharmaceutical composition and / or method “including” a list of elements (e.g., components, features, or steps) may include other elements (or components or steps) that are not expressly enumerated or specific to the pharmaceutical composition and / or method, but are not necessarily limited to those elements (or components or steps).

[0039] The aspects and embodiments of the disclosure described herein are understood to include the terms "consists of" and / or "essentially consists of." As used herein, the transitional phrases "consists of" and "consisting of" exclude any elements, steps, or components not specified. For example, as used in the claims, "consists of" or "consisting of" limits the claims to the components, materials, or steps specifically enumerated in the claims, with the exception of impurities that are normally associated (i.e., impurities in a given component). If the phrase "consists of" or "consisting of" appears in a clause of the claims rather than immediately following the preamble, the phrase "consists of" or "consisting of" limits only the elements (or components or steps) described in that clause, and does not exclude other elements (or components) as a whole from the claims.

[0040] As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a pharmaceutical composition and / or method that includes materials, steps, features, components, or elements in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not substantially affect the basic and novel properties of the claimed subject matter. The term “essentially consisting of” occupies a compromise between “equipped with” and “consisting of.”

[0041] When introducing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that one or more of the elements exist. The terms “comprising,” “including,” and “having” are intended to mean comprehensive and that additional elements other than those listed may exist.

[0042] When the term "and / or" is used to list two or more items, it means that any one of the listed items may be used on its own or in combination with one or more of the listed items. For example, the expression "A and / or B" is intended to mean either A or B, or both, i.e., A only, B only, or a combination of A and B. The expression "A, B and / or C" is intended to mean A only, B only, C only, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0043] It should be understood that descriptions in range form are merely for convenience and conciseness and should not be interpreted as a firm limitation on the scope of this disclosure. Therefore, a range description should be considered to specifically disclose all possible subranges within that range, as well as individual numerical values. For example, a range description such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as individual digits within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. Furthermore, in this specification, values ​​or ranges may be expressed as “approximately,” “approximately” one particular value, and / or “approximately” another particular value. Where such values ​​or ranges are expressed, other embodiments disclosed will include the listed specific values, from one particular value to and / or other specific values. Similarly, where values ​​are expressed as approximations, it will be understood that by using the antecedent “approximately,” a particular value forms another embodiment. There are several values ​​disclosed herein, and it will be further understood that each value is disclosed herein not only as the value itself, but also “about” that particular value. In embodiments, “about” may be used to mean, for example, within 10% of the listed value, within 5% of the listed value, or within 2% of the listed value.

[0044] As used herein, “patient” or “subject” means a human or other mammal subject being treated.

[0045] As used herein, the term “pharmaceutical composition” means a pharmaceutically acceptable composition, which comprises a pharmaceutically active agent and, in some embodiments, further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of a pharmaceutically active agent and a carrier.

[0046] The term “combination” refers to either a fixed combination in a single drug dosing unit or a parts kit for combination administration, in which one or more active compounds and combination partners (e.g., another drug described below, also referred to as “therapeutic agent” or “adjunct agent”) may be administered simultaneously, independently, or separately within a time interval. In some situations, combination partners exhibit a synergistic effect. As used herein, terms such as “combination administration” or “combination administration” are intended to encompass the administration of selected combination partners to a single subject (e.g., a patient) that requires them, and to include treatment regimens in which the drugs are not necessarily administered via the same route or at the same time.

[0047] As used herein, the term “combination of pharmaceuticals” means a product obtained by mixing or combining two or more active ingredients, including products in which the combination of active ingredients is fixed or unfixed. The term “fixed combination” means that both the active ingredients, e.g., a compound and a combination partner, are administered to the patient simultaneously in the form of a single entity or dose. The term “unfixed combination” means that both the active ingredients, e.g., a compound and a combination partner, are administered to the patient simultaneously, concurrently, or sequentially as separate entities without any particular time constraints, thereby providing the patient with therapeutically effective concentrations of the two compounds. The latter also applies to cocktail therapies, e.g., the administration of three or more active ingredients.

[0048] As used herein, the term “pharmaceutically acceptable” means that, in addition to other formulations that are safe for use in animals, more specifically in humans and / or non-human mammals, it is approved by a federal or state regulatory authority or listed in the United States Pharmacopeia or any other commonly accepted pharmacopoeia.

[0049] As used herein, the term “pharmaceutically acceptable carrier” refers to excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or vehicles administered with a demethylene compound. Such carriers may be sterile liquids such as water and oil, and oils may include oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose can also be carriers. Methods for preparing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the term “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, isotonic agent, and absorption retarder, etc., suitable for pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Such use in this composition is intended unless conventional media or agents are incompatible with the active compound.

[0050] As used herein, “therapeutic effective dose” refers to the amount of a pharmaceutically active compound that is sufficient to treat, improve, or reduce in any way the symptoms associated with a disease or condition. When used in relation to a method, the method is sufficient to treat, improve, or reduce in any way the symptoms associated with a disease or condition. For example, in relation to a disease, an effective dose is sufficient to prevent or inhibit its onset, or, if the disease symptoms have already begun, to mitigate, improve, stabilize, recover from, or delay the progression of the disease, or to reduce the pathological consequences of the disease. In either case, the effective dose may be administered as a single dose or in divided doses.

[0051] As used herein, the terms “to treat,” “to cure,” or “to treat” encompass at least improvement of the symptoms associated with a disease in a patient, in which case improvement is used in a broad sense to mean at least a reduction in the degree of a parameter such as the symptoms associated with the disease or condition being treated. Thus, “treatment” includes situations in which a disease, disorder, or pathological condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) so that the patient will no longer suffer from that condition, or at least the symptoms that characterize that condition.

[0052] As used herein, unless otherwise specified, the terms “prevent,” “prevent,” and “prevention” refer to the prevention of the onset, recurrence, or spread of a disease or disorder, or one or more of its symptoms. In certain embodiments, these terms refer to treatment or administration of a compound or dosage form provided herein, with or without one or more additional activators, to a subject at risk of developing a disease or disorder provided herein, particularly before the onset of symptoms. These terms encompass the inhibition or reduction of symptoms of a particular disease. In certain embodiments, a subject with a family history of the disease is a potential candidate for a preventive regimen. In certain embodiments, a subject with a history of recurrent symptoms is also a potential candidate for prevention. In this regard, the term “prevention” may be used interchangeably with the term “preventive treatment.”

[0053] As used herein, unless otherwise specified, the “prophylactic effective dose” of a compound is an amount sufficient to prevent a disease or disorder or to prevent its recurrence. A prophylactic effective dose of a compound means the amount of a therapeutic agent, either alone or in combination with one or more other agents, that provides a prophylactic benefit in the prevention of a disease. The term “prophylactic effective dose” may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0054] When used herein, unless otherwise specified, the compounds described herein are intended to encompass all possible stereoisomers unless a specific stereochemistry is specified. Where structural isomers of a compound are interchangeable across low energy barriers, the compound may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism, or so-called valence tautomerism in the compound, e.g., involving an aromatic moiety. The term “derivative” refers to a chemical substance that is structurally related to another substance, or a chemical substance that can be produced from another substance (i.e., the substance from which it originates), for example, by chemical or enzymatic modification.

[0055] As used herein, the term “pharmaceutically acceptable salt” refers to an acid- or base-addition salt of a compound, such as a multidrug conjugate, as described herein. A pharmaceutically acceptable salt is any salt that preserves the activity of the parent agent or compound and does not impart any harmful or undesirable effect to the subject to which it is administered, or in the circumstances under which it is administered. A pharmaceutically acceptable salt may be derived from amino acids, including but not limited to cysteine. Methods for producing compounds as salts are known to those skilled in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J Pharm. Sci. 66:1, 1977). In some embodiments, “pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base of a drug or compound described herein that is non-toxic, bioacceptable, or otherwise biosuitable for administration to a subject. For general information, see Berge, et al., J. Pharm. Sci., 1977, 66, 1-19. A preferred pharmaceutically acceptable salt is one that is pharmacologically effective and suitable for contact with the target tissue without excessive toxicity, irritation, or allergic response. The agents or compounds described herein may have a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or two or more of each type, and may therefore react with several inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

[0056] Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monophosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propioates, oxalates, malons, succinates, suberates, sebacinates, fumarates, maleates, butin-1,4-diones, and hexin-1,6-diones. This includes phosphates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besilates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutylates, citrates, lactates, [gamma]-hydroxybutylates, glycolates, tartrates, and mandelates. [Examples]

[0057] As disclosed in the following examples, hexosamine mannosamine (hereinafter, 2-amino-2-deoxy-D-mannose or ManN) inhibits protein glycosylation but stimulates EC proliferation in vitro. The biological effects of ManN in other in vitro and in vivo models and their possible mechanisms of action were investigated. ManN is an EC mitogen and survival factor in bovine and human microvascular ECs and is additive with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and unfolded protein responses induced by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, accelerating limb blood flow recovery compared to controls. Furthermore, intraocular injection of ManN induces retinal angiogenesis. Therefore, the activation of stress pathways following the inhibition of protein glycosylation can promote EC proliferation and angiogenesis, representing a potential therapeutic strategy for treating ischemic injuries.

[0058] Example 1 The effect of ManN on EC proliferation was evaluated. A library of 619 highly purified metabolites encompassing a wide range of chemicals was screened for their ability to influence bovine choroidal microvascular EC (BCEC) growth, in the presence or absence of VEGF. This assay and similar assays have been previously used to identify and characterize angiogenic stimulants and inhibitors [12-14]. Under the conditions tested, proliferation was little to no detection in the absence of VEGF.

[0059] Initial screening was performed by testing each compound at concentrations of approximately 1 and 10 μM (assuming a molecular weight of 100 Da for each compound) with and without 5 ng / ml VEGF, which can induce an approximately 4-5-fold increase in cell proliferation. Six compounds with varying chemical properties showed some inhibitory or stimulating activity. Analysis focused on one of these, ManN (hexosamine originally identified as a component of bacterial cell walls

[15] ), as it showed the most potent and consistent effect. ManN had a significant stimulating effect in the dose range of 5–500 μM and was additive with VEGF in promoting BCEC proliferation. The dose-dependent effect of ManN on BCEC proliferation in the absence or presence of VEGF is shown in Figures 1A and 1B. When cells were treated with 50 μM ManN and 5 ng / ml VEGF, compared to VEGF alone, 50 μM ManN alone resulted in a maximum increase of approximately 6.5 times on the EC coating surface (Figure 1A), or a maximum increase of approximately 2.5–3 times in fluorescence units with the addition of AlamarBlue (Figure 1B). AlamarBlue detects mitochondrial activity as an indicator of cell viability that correlates with a specific range of cell numbers

[16] . The effect of ManN showed a bell-shaped dose-response curve with inhibition at higher concentrations (Figures 1A and 1B). Additive effects of ManN in promoting BCEC proliferation were also observed with bFGF (Figure 11A) and bovine retinal EC (BREC) (Figures 11C and 11D).

[0060] Various hexosamines (galactosamine, glucosamine, and their N-acetyl derivatives) were tested with ManN in a BCEC proliferation assay. However, none of these hexosamines showed any significant stimulating effect (Figure 1D). Several structurally related molecules, including D-isoglucosamine (fructosamine), meglumine, muramic acid, N-acetylneuraminic acid (sialic acid present in all mammalian cells), glucose, and mannose, were also tested. None of these molecules stimulated BCEC proliferation, with or without VEGF (Figures 11C and D).

[0061] ManN entered and accumulated in cells in a concentration-dependent manner. When BCEC was treated with 400 uM ManN for 2 hours, 0.66 nmol of ManN was detected in 1 mg of cell lysate. Upon entering cells, ManN is immediately converted to ManN-6-phosphate (ManN-6p), but not to mannose

[17] . No ManN incorporation was detected in N-glycans. Efficient uptake of ManNAc and mannose has been reported [18, 19].

[0062] The effect of ManN on BCEC proliferation was dependent on cellular glycolysis. Additive relationship between ManN and VEGF was lost when glucose-free medium was used. On the other hand, VEGF activity was independent of the glycolytic pathway (Figure 11B). However, even in the presence of VEGF, significant cytotoxicity was observed with ManN at a concentration of around 4 μM in glucose-free medium.

[0063] To further characterize the effects of ManN on EC survival, proliferation, and migration, confluent BCEC monolayers were mechanically scratched. Figure 1E shows that after 48 hours, compared to the control group, 40 μM ManN or 50 ng / ml VEGF significantly accelerated BCEC migration and / or proliferation, as reflected by more complete closure of the "scratched" areas. Additiveness was observed when cells were treated with both ManN and VEGF, as in the proliferation assay (Figure 1E). In addition, 40 μM ManN showed significant additiveity with VEGF in promoting BCEC migration (Figure 1F).

[0064] The observations were extended to human retinal microvascular ECs (hRMECs), HUVECs, and human dermal microvascular endothelial cells (hDMVECs). ManN itself stimulated the growth of HUVECs and hDMVECs. Furthermore, in all EC types tested, there was a dose-dependent additive relationship with VEGF, and toxicity was minimal even at 5 mM. Similarly, stimulation of migration and wound closure was observed in HUVECs treated with 40 μM ManN alone (migration assay) and / or in combination with 50 ng / ml VEGF (scratch assay).

[0065] Example 2 Activation of ERK, AKT, mTOR, CREB, AMPK, ACC, and eNOS is not specific to ManN. Crosstalk between signaling and metabolic pathways in the vascular system, such as insulin signaling and glucose metabolism in ECs, has been reported to involve activation of AKT and STAT 3. Together, they affect glycolysis, EC sprouting, proliferation, and migration

[20] . We evaluated the effects of ManN and / or VEGF on the activation of major signaling pathways known to promote proliferation in BCECs, e.g., ERK, AKT, mTOR, and CREB (cAMP response element-binding protein). ManN activated ERK, AKT, mTOR, and CREB at 40 μM. Stimulation of ERK, AKT, and CREB was rapid, occurring within 10–30 minutes after ManN addition (Figure 2A–2C). Furthermore, enhanced activation of ERK, AKT, and CREB was observed in the presence of both ManN and VEGF compared to ManN or VEGF alone (Figure 2A and 2B). The effect of ManN on the activation of the ACC (Acetyl-CoA carboxylase) / eNOS (endothelial nitric oxide synthase 3) pathway was evaluated. Activation of the energy sensor AMPK (AMP-activated protein kinase) leads to eNOS activation and NO (nitric oxide) production, the latter having a bell-like effect on EC proliferation

[21] . Both eNOS and ACC were significantly activated by 40 μM ManN within 10–30 minutes (Figure 2D). However, the activation of ERK, AKT, mTOR, CREB, ACC, and eNOS was not specific to ManN. In fact, other hexosamines such as ManNAc and mannose induced similar activation of these signaling pathways (Figure 2C). While the activation of these common proliferation pathways likely contributed without being constrained by theory, several unique mechanisms may be involved in the EC fission-promoting effect of ManN.

[0066] Example 3 Using a series of specific pharmacological inhibitors, we identified JNK / c-jun as a unique signaling pathway activated by ManN among hexosamines. Western blot analysis revealed that JNK, one of the three MAPK family members (ERK, p38, and JNK), was specifically activated by ManN. When growing BCECs were switched to a growth assay medium free of growth factors, JNK and its downstream c-Jun were markedly activated by ManN in a dose-dependent manner, but not by mannose (Figures 3A and 3B). ManN activated the JNK pathway, but the other hexosamines tested were not. Treatment of BCECs with the JNK-specific inhibitor SP600125 (5 μM) abolished the effect of ManN on BCEC growth (Figure 3C).

[0067] The effect of ManN on BCEC was evaluated after transfection with siRNA against JNK (i.e., JNK1 and JNK2, since JNK3 is not expressed in BCEC). Knockdown of approximately 80% of either JNK1 and / or JNK2 by two independent siRNAs against JNK1 or JNK2 eliminated the mitotic effect of ManN on BCEC at uM concentrations (Figures 3D and 3E), indicating that both JNK1 and JNK2 are important for stress signaling.

[0068] Example 4 ManN affects protein glycosylation in endothelial cells. Additive properties of ManN with VEGF may potentially occur at the transcriptional and / or translational level, or through the VEGF-VEGFR2-mediated signaling pathway. However, when cells were treated with various concentrations of ManN for 4 hours (for gene expression levels) or 24 hours (for protein expression levels), neither the transcription of VEGF, VEGFR2, and GLUT1 and 4, nor the total VEGFR2 protein expression, were significantly altered in BCECs (Figures 4A-4C; Figures 5A and 5B; Figures 7C and 7E). The same was true for BRECs and hRMVECs. Biotinylation studies showed no change in the amount of VEGFR2 on the cell surface. However, VEGF-responsive VEGFR2 phosphorylation was decreased in ManN-pretreated cells, suggesting that VEGFR2 activation was inhibited rather than enhanced in BCECs. Ligand-independent VEGFR2 activation did not occur after ManN addition in BCECs. The same was true for HUVEC (SFIG.10a) and hDMVEC.

[0069] The apparent molecular weight of VEGFR2 shifted significantly from 40 μM after ManN treatment in both BCEC (Figures 4A-4C and 4E) and BREC. A new low molecular weight band (approximately 170–200 kDa) appeared in a dose-dependent manner in ManN-treated BCEC compared to the control (major band of approximately 230 kDa and minor band of approximately 210 kDa) (Figures 4B, C, and E; Figures 5A and 5B; and Figure 7C). This shift was specific to ManN among hexosamine and its derivatives (Figure 4A). VEGF alone did not affect molecular weight. Adding VEGF to ManN did not cause any additional shift (Figure 4A). The low molecular weight VEGFR2 band is unlikely to be a degradation product, as removal of ManN completely reversed its effect on molecular weight after 24 hours (Figure 4E). However, based on PNGase F treatment, it appears that not all glycosylation on VEGFR2 was abolished by ManN, at least at uM concentrations. Experiments with the potent VEGFR2 inhibitor, the small molecule tyrosine kinase inhibitor axitinib [6], show that the reduction in VEGFR2 molecular weight and the stimulation of BCEC proliferation by ManN are independent of VEGFR2 signaling.

[0070] Significant changes in VEGFR2 protein mass were also observed in hRMVEC, HUVEC (Figure 4F), and hDMVEC (Figure 4G) with 40 μM ManN, but the additive effect of ManN and VEGF on the proliferation of these cells occurred at the mM level.

[0071] To better understand how ManN may affect post-translational modification of VEGFR2, cells were treated with ManN in the presence of one of four monosaccharides (mannose, glucose, galactose, or fucose) in a maximum molar ratio of 1:10. These monosaccharides are known to be important in protein N-glycosylation. Our results suggest that mannose can dose-dependently block the effects of ManN on VEGFR2 molecular weight and BCEC proliferation (Figures 4C and 4D). The effect of mannose may not be limited to preventing ManN entry into cells via the same transporter, as the effect was observed when BCECs were initially treated with ManN for 2 hours to ensure good cellular uptake. Glucose had a similar effect to mannose, but galactose or fucose did not.

[0072] The decrease in protein levels after ManN administration in BCEC was not limited to VEGFR2. Other N-glycosylated growth factor receptors / coreceptors or adhesion molecules, including αv integrin, neuropilin-1, VE-cadherin, and bFGFR1, were similarly affected.

[0073] Example 5 The effect of ManN on the general protein glycosylation profile was evaluated. N-glycosylation is a complex process that relies on multiple enzymes that successively act on glycoproteins to generate hybrid and high-mannose glycan structures as they pass through the secretory pathway from the ER to the Golgi apparatus

[22] . It plays a crucial role in determining the fate of newly synthesized glycoproteins in the ER, their correct folding, cellular destination, and appropriate function.

[0074] Several key enzymes involved in protein N-glycosylation in both the ER and Golgi apparatus were evaluated. α-mannosidase from Canavalia cordata is a broadly specific exoglycosidase that catalyzes the hydrolysis of terminal non-reducing α1-2, α1-3, and α1-6 binding mannose residues from oligosaccharides in both organelles, controlling the final hydrolysis step in the N-glycan maturation pathway: the conversion of high-mannose to complex N-glycans. This enzyme has been used to screen for potential N-glycosylation inhibitors

[23] . ManN showed inhibitory activity at 400 μM, considerably higher than the effective mitotic-promoting concentration in BCEC, whereas other hexosamines or their derivatives did not. No effect of up to 2 mM of ManN on α- or β-glucosidases was detected.

[0075] N-linked glycans from BCEC were isolated by enzymatic cleavage, subsequently purified, and characterized using MALDI-TOF-MS. Treatment with 40 μM ManN resulted in a significant time-dependent reduction of Man6GlcNAc2 (Man-6), Man-8, and Man-9 in total oligomannose N-glycan content compared to untreated controls, although significant early-stage accumulation of Man-5 and Man-7 was observed after ManN treatment. ManN has previously been shown to inhibit lipid-bound oligosaccharide (LLO) synthesis, alter protein GPI biosynthesis and hybrid glycan production, and incorporate into glycans in MDCK cells

[24] . The time-dependent accumulation of Man-5 suggested that inhibition of mannosidase is unlikely to be the mechanism of pro-angiogenic activity in BCEC.

[0076] The complex N-glycan composition was profiled by measuring monosaccharide content. Significant decreases in fucose (8 hours), mannose (12 hours), galactose (24 hours), and Neu5Ac (8 and 24 hours) were found in ManN-treated cells compared to untreated control cells, consistent with the inhibitory activity of ManN on overall protein N-glycosylation.

[0077] O-glycan modification is another form of post-translational modification of proteins, in which a serine or threonine residue is covalently bonded to a GalNAc residue

[22] . GalNAc residues can be further modified by several glycosyltransferases that act sequentially to extend the glycan chain, either in a branched or linear manner, depending on substrate specificity. ppGalNAcT (polypeptidyl GalNAc transferase) catalyzes the transfer of α-GalNAc from UDP-GalNAc to a Ser or Thr residue of glycoproteins, producing the Tn antigen. When the Tn antigen is produced, it can have three different fates: (i) it can be sialized on C6 by the enzyme ST6GalNAcT; (ii) it can be substituted on C3 or C6 by β-GlcNAc, giving rise to core-3 or core-6, respectively; or (iii) it can be galactosylated on C3 by C1GalT1 to form core-1, which can also be sialized to produce mono- or di-sialyl core-1O-glycan

[22] .

[0078] O-glycan analysis was performed on BCEC lysates by MALDI-Tof mass spectrometry. Since no unique enzymes were available to cleave all different forms of O-glycans, reductive beta-elimination was performed to understand the O-glycan skeleton

[25] . Permethylation was performed before MALDI-Tof / Tof mass spectrometry to protect against desialylation during mass spectral data acquisition

[25] . Overall reduction in O-glycosylation after treatment with 40 μM ManN. In particular, we observed decreasing trends in ionic intensity at m / z for 895 (sialyl-core 1, Galβ1-3GalNAc-), 1256 (diciallylated core 1), 983 (core 2, GlcNAcβ1-6(Galβ1-3)-GalNAc-), and 1187 (di-galactosylated core 2).

[0079] Example 6 The effect of ManN on UPR activation by increasing Bip and CHOP expression was evaluated. Asparagine-linked N-glycosylation is one of the most common modification reactions in eukaryotic cells and occurs in proteins that are cotranslatically translocated across the ER during biosynthesis or incorporated into the ER

[22] . After N-linked oligosaccharides are transferred to nascent proteins by OST (oligosaccharyltransferase), ER-resident glucosidases and mannosidases produce a series of glycan trimming intermediates that are specifically recognized by ER-localized lectins to fold, degrade, or direct the nascent proteins into export pathways. One consequence of inhibiting protein glycosylation is impaired protein folding, leading to ER stress [26, 27]. Physiological responses to UPR are mediated by altered gene expression, including the regulation of the ER Hsp70 chaperone BiP (also known as glucose regulatory protein 78, which binds to immunoglobulin proteins) and another multifunctional transcription factor CHOP (CCAAT-enhancer-binding protein homolog) [28, 29]. For example, UPR dysfunction during aging creates an environment that tolerates protein aggregation, unresolved ER stress, and chronic inflammation

[30] .

[0080] To investigate potential ER stress mediated by ManN, we studied Bip and CHOP expression in ManN or mannose-treated cells by Western blot analysis. Our data showed that ManN could significantly turn on Bip expression in a concentration-dependent manner when growing cells were deprived of growth factor supply, whereas mannose or VEGF could not, with Bip accumulation evident at 24 hours (Figures 5A and 5B) and 48 hours (Figure 5A). CHOP induction appeared faster at approximately 6 hours in a dose-dependent manner (Figure 5A). No synergistic effect between ManN and VEGF in promoting Bip or CHOP expression was mentioned (Figure 5B).

[0081] We tested two well-known chemical chaperones, 4-PBA (4-phenylbutyric acid)

[31] and TUDCA (tauroursodeoxycholic acid)

[32] , to alleviate ER stress in ManN-treated BCEC. Both have previously been shown to reduce the tunicamycin-induced eIF2α-ATF4-CHOP arms of UPR and Bip expression. We found that 2 mM 4-PBA prevented ManN-induced CHOP expression at 400 μM and 5 mM and could restore ManN-induced ATF-6 (activating transcription factor-6) expression at 400 μM, but 500 μM TUDCA could not (Figure 5C). Similarly, the restoration of ATF-6 expression by TUDCA was much weaker compared to 4-PBA. As a transmembrane ER glycoprotein, ATF-6 is cleaved, releasing a 50 kDa amino-terminal fragment that translocates to the nucleus, activating ER chaperone transcription and ER-associated degradation components such as Bip and CHOP upon accumulation of improperly folded proteins in the ER

[28] . Pretreatment of cells with 1 mM 4-PBA for 4 hours effectively reverses the bell-shaped activity of ManN in BCEC proliferation in the absence or presence of VEGF. Additive activity between ManN and VEGF was almost completely eliminated (Figure 5D).

[0082] Example 7 The effects of ManN on non-endothelial cells were evaluated. To extend observations in ECs, we examined various non-EC types from different species. These included NIH3T3 fibroblasts and AML12 hepatocytes (mouse), ARPE-19 RPE cells (human), and newly isolated bovine pituitary cells. We also tested several human cell types associated with in vivo models, such as cutaneous fibroblasts and keratinocytes. Furthermore, we screened four human or mouse cancer cell lines (A673, U87MG, Calu6, and 4T1) (Figure 6). To investigate post-translational modifications of proteins in non-ECs, we monitored molecular weight changes using bFGFR1 or β1 integrins. Similar to BCECs, ManN could induce molecular weight changes in all of these non-ECs, but mannose could not (Figure 6 inset). However, unlike BCEC (Figure 1B), BREC (Figure 1C), hRMVEC, HUVEC, and hDMVEC, no proliferative effect of ManN was observed at μM-mM concentrations, either alone or in combination with other growth stimulants (Figure 6). Efficient ManN uptake and equivalent levels of free ManN were detected in all cell types. Cytotoxicity varied among different cell types, with AML12 being the most sensitive to a 5 mM ManN level, while human RPE cells and human keratinocytes were the least sensitive (Figures 6E and 6H). Growth inhibition by 25 mM mannose in vitro has been reported in several tumor lines with low levels of PMI (phosphomannose isomerase)

[19] . At 5 mM, ManN showed significant toxicity on 4T1 cells, likely due to higher PMI levels in 4T1 compared to all reported sensitive tumor lines, while mannose did not (Figure 6D).

[0083] Example 8 Similar to ManN, inhibitors of protein N-glycosylation stimulate EC growth. To determine whether broad alterations of protein glycosylation promote cell proliferation, two well-characterized inhibitors, kifunensine (KIF) and castanospermine (Cas), were tested [33-37]. BCEC growth was stimulated in a dose-dependent manner in the absence or presence of 5 ng / ml VEGF (Figures 7A and 7B). A reduction in VEGFR2 molecular weight on SDS-PAGE was evident after 24 hours of treatment with Kif or Cas (Figure 7C).

[0084] At 40 μM, Kif significantly activated ERK and AKT in BCEC (Figure 7E), HUVEC, and hDMVEC. Activation of ERK by Cas was less evident in both BCEC and hDMVEC. However, both inhibitors were able to activate the JNK pathway in BCEC (Figure 7E). Blocking JNK activation with 5 μM SP600125 significantly reduced the effect of both glycosylation inhibitors on BCEC proliferation (Figure 7F). Figure 7C shows the dose-dependent induction of Bip expression when growing BCECs were switched to growth factor-free medium for 24 hours in the presence of Kif or Cas at concentrations that promote cell proliferation.

[0085] Both Kif and Cas exhibited significant activity in the BCEC "scratch" assay, and the gap was closed more rapidly by each molecule over 48 hours compared to the control (Figure 7D). The insert panel in Figure 7D shows representative images from assays using Kif or Cas. Quantitative analysis showed a significant acceleration of gap closure in a dose-dependent manner compared to the control.

[0086] Example 9 The relationship between the in vitro effect of ManN on endothelial cells and in vivo angiogenesis was investigated through its effects in a mouse splint wound model. In this model, the repair process is entirely dependent on epithelialization, cell proliferation, and angiogenesis, which closely reflect the biological processes of human wound healing

[38] . The effects of ManN and VEGF were tested individually or in combination. Topical application of 20 μg of VEGF or 20 μg of ManN was performed daily for the first three days after wounding. When VEGF and ManN were combined, a significant acceleration of wound closure was observed during the early stages of healing (Figure 8A). Compared to VEGF or ManN monotherapy, the combination resulted in significantly faster wound closure from day 2 (Figure 8B). On day 4, the mean wound closure rates were 81.5%, 75.6%, 66.9%, and 29.8% in the PBS-, ManN-, VEGF-, and combination treatment groups, respectively. On day 4, the number of small vessels surrounding the wound area was quantified. A significant increase in CD31-positive vessels was observed in the combination group compared to PBS control, VEGF, or ManN alone (Figures 8C and 8D).

[0087] Therefore, ManN, when combined with VEGF, promotes angiogenesis in a skin injury model. In this acute model, wound closure occurs rapidly without treatment.

[0088] The stability of ManN was evaluated in wound fluid contaminated with bacteria, a common characteristic of wounds. ManN was added to freshly collected wound fluid from a mouse model of skin infection by Staphylococcus aureus, a common cause of skin and soft tissue infections in humans

[39] . After incubation at 37°C for up to 24 hours in such wound fluid, no significant loss of free ManN was detected. Therefore, ManN may be useful in treating infected wounds, possibly in combination with antimicrobial agents or other drugs.

[0089] One of the known properties of VEGF is the rapid induction of vascular permeability after injection into the skin of guinea pigs [1]. The effect of ManN on the induction of vascular permeability was evaluated using the same assay. However, when tested at 1 ng to 5 μg, ManN did not induce a permeability-enhancing effect, while 25 ng of VEGF did induce vascular permeability.

[0090] Example 10 The angiogenic effects of ManN and Kif in a mouse hindlimb ischemia model were evaluated. The activity of ManN in a chronic ischemia model, which may more specifically reflect its effects as an endothelial mitogen and pro-angiogenic factor, was evaluated, and a mouse hindlimb ischemia model was considered. Several variants have been described depending on which vessels are occluded [40, 41]. The selected variant was femoral artery ligation and excision, which resulted in more severe ischemia compared to simple femoral artery ligation

[40] . Occlusion of two vessels resulted in more severe ischemia, but had the disadvantage of inducing not only severe pain and suffering in mice but also frequent ulceration and necrosis

[40] .

[0091] Oral administration of ManN was tested in this femoral artery ligation-resection model. Kif was used because it had been previously administered intraperitoneally for in vivo studies

[42] . Laser Doppler perfusion imaging (LDPI) was used as a non-invasive method to monitor the time and extent of blood flow recovery in the ischemic limb

[43] . Serial blood flow monitoring was performed using LDPI, and blood flow recovery was indicated by the increase in the perfusion ratio of the ischemic (ligated; left) versus non-ischemic (pseudo; right) hind limb after ligation. Immediately after surgery, mice were orally fed 20% ManN or 1 mg / ml Kif ip every other day, as described in the methods. One week after surgery, the perfusion ratio in the H2O-fed group showed approximately 25% blood flow recovery, which is in good agreement with published data in the same mouse strain with the same type of lesion [40, 44]. However, the blood flow recovery rates in the ManN and Kif-treated groups were approximately 40% and 47%, respectively, indicating an accelerated rate of blood flow recovery compared to mice treated with H2O (Figures 9A and 9B). The blood perfusion ratio continued to increase to approximately 50% of the sham-treated limb three weeks after ManN and Kif treatment, which was significantly higher than the control group (Figures 9A and 9B).

[0092] Consistent with improved blood flow, ischemic hind limbs in the ManN-treated and Kif-treated groups showed increased vascular density compared to the control group, as assessed by CD31 immunostaining of surrounding muscle tissue 3 weeks after ligation. Compared to the H2O-treated control group, vascular density was 2.3 and 1.8 times higher in the ManN-treated and Kif-treated groups, respectively (Figures 9C and 9D).

[0093] Following oral administration, there was a relatively rapid decrease in ManN plasma levels. Plasma free ManN levels reached a peak level of approximately 100 nmol / ml in plasma after 1 hour. After 3 hours, only about half of that amount was detectable. Two hours after oral feeding of 20% ManN, muscle samples were taken from the ischemic leg. A considerable amount of ManN reached the ischemic leg, containing 0.17+ / -0.18 nmol / mg of free ManN protein and 0.91+ / -0.24 nmol / mg of ManN-6p protein. At least in BCEC, the effect of ManN on protein mass persisted for at least 8 hours in the absence of exogenous ManN (Figure 4E), indicating that even relatively short exposure may be sufficient to induce pharmacological effects.

[0094] Example 11 The effects of ManN and Kif on the induction of retinal neovascularization were evaluated. Findings from cultured eye-derived ECs were extended to a suitable in vivo model system. Mouse retinas have been widely used for the past several decades to study both physiological and pathological neovascularization

[41] . To obtain a detailed description of the retinal vascular system, images from retinal flat mounts were processed for vascular area fractions (the ratio of the area covered by blood vessels to the total retinal area). Using this model, the effect of ManN on retinal neovascularization was evaluated. Kif was also tested in this model because it is a water-soluble inhibitor and its mechanism of glycosylation inhibition is well established

[35] . Furthermore, it shares with ManN the ability to activate ERK, AKT, and stress pathways in BCECs (Figure 7E).

[0095] 500 nanograms of ManN or Kif were injected intravitreously, and the retinal vascular system was examined 7 days later. In this model, intravitreous administration of 200 ng of bFGF was used as a positive control. In the bFGF, ManN, and Kif treatment groups, retinal vascular density increased by approximately 35%, 30%, and 20%, respectively, compared to the PBS group (Figures 10A and 10B).

[0096] Materials and methods Small Molecule Library MSMLS (Mass Spectrometry Metabolite Library of Standards) IROA TECHNOLOGIES, Bolton, MA (currently Sigma) is a collection of 619 high-quality small molecules (purity >95%) spanning a wide range of primary metabolites, including carboxylic acids, amino acids, biogenic amines, polyamines, nucleotides, coenzymes, vitamins, and lipids. Plates were rotated at 300g after reconstitution according to the manufacturer's instructions.

[0097] chemical compound D-mannosamine hydrochloride was obtained from Sigma (M4670) or Spectrum Chemical MFG Corp (M3220). 1-Amino-1-deoxy-D-fructose hydrochloride (D-isoglucosamine) (803278), D-(+)-galactosamine (1287722), D-(+)-glucosamine (1294207), N-acetyl-mannosamine (A8176), N-acetyl-galactosamine (A2795), N-acetyl-glucosamine (A8625), meglumine (M9179), muramic acid (M2503), N-acetylnoraminic acid (A2388), D-(+)-glucose (D9434), D-(+)-mannose (1375182), meglumine (M9179), Streptomyces Tunicamycin (T7765) and SP600125 (S5567) derived from sp. were obtained from Sigma. Hypure cell culture grade water, used to dissolve the compounds (endotoxin <0.005 EU / ml), was obtained from Hyclone. Axitinib was obtained from Santa Cruz (SC-217679). Tauroursodeoxycholic acid (TUDCA) was from Calbiochem (1180-95-6), and 4-phenylbutyric acid (4-PBA) (P21005), castanospermine (Cas, C3784), kifunensin (K1140), and DMSO (D2650) were from Sigma. DMSO (D2650) was used as a solvent for Cas.

[0098] antibody Unless otherwise specified, the antibodies used in this study were from Cell Signaling Technology Inc (Danvers, MA). Total: VEGFR2 (2479), ERK (4695), p38 (9212), JNK (9252), mTOR (2983), AKT (4691), CREB (9104), CHOP (2895), ACC (3676), ATF-6 (65880), Bip (3183), AMPKα (5832), FGFR1 (9740), eNOS (9586), VE-cadherin (2500), c-Met (3127 or 3148), neuropilin (3725), CD31 (3528), c-Jun (9165). Phosphor-Antibody: VEGFR2 (Tyr1175, 2478, or 3770), ERK1 / 2 (Thr202 / Tyr204, 4376), p38 (Thr180 / Tyr182, 4511), JNK (Thr183 / Tyr185, 9251), mTOR (Ser2448, 5536), AKT (Ser473, 4060), CREB (Ser133, 9191), ACC (Ser79, 3661), eNOS (Ser1177, 9571), AMPKα (Thr172, 50081), c-Jun (Ser73, 9164), β1 Integrin (4706 & 34971), αv-integrin (4711), JNK1 (3708), JNK2 (4672), JNK3 (2305). Anti-β-actin was derived from sigma.

[0099] cell Primary human umbilical vein endothelial cells (HUVEC, passages 4-10) were obtained from Lonza (C2519AS, lot number 234871) and cultured on 0.1% gelatin-coated plates in endothelial cell growth medium (EGM) containing 2% FBS, BBE (Bovine Brain Extract), heparin, human EGF, hydrocortisone, ascorbic acid, GA-1000 (gentamicin, amphotericin B), and VEGF. Bovine retinal microvascular endothelial cells (BREC, #BRMVEC-3) and bovine choroidal microvascular endothelial cells (BCEC, #BCME-4) (both from VEC Technologies (Renssellaer, NY)) were cultured on fibronectin-coated plates (1 μg / cm³). 2 The growth medium consisted of 10% calf serum (BCS), 5 ng / ml bFGF, and 10 ng / ml human VEGF. 165 The cells were treated with low-glucose DMEM supplemented with bFGF(233-FB) and VEGF. 165Cell (293-VE) was purchased from R&D Systems. Human retinal microvascular endothelial cells (passage <15) were from Cell Systems Corporation (Kirkland, WA). They were grown on 0.1% gelatin-coated plates in medium 131 containing 5% fetal bovine serum, hydrocortisone (1 μg / ml), human fibroblast growth factor (3 ng / ml), heparin (10 μg / ml), human epidermal growth factor (1 ng / ml), and dibutyryl cyclic AMP (0.08 mM) (MVGS, S 005-25, Gibco Invitrogen). The human RPE cell line ARPE-19 was from ATCC. Cells were gently elevated with 0.025% trypsin and plated in RtEGM medium (Clonetics) containing 2% FBS, L-glutamine, human bFGF, and GA-1000. Once the cells had attached to the plates, the cultures were maintained using serum-free RtEGM medium to obtain the best results. ARPE-19 cells were obtained from ATCC (CRL-2302) and cultured according to the supplier's instructions. NIH3T3 cells were obtained from ATCC (CRL-1658). Human adult dermal MVECs (CC-2543) were cultured in EGM-2MV (CC-4147, Lonza). Keratinocytes (ATCC, PCS-200-011) were cultured in dermal cell base medium (PCS-200-030) and keratinocyte growth kit (PCS-200-040). Human primary dermal fibroblasts (ATCC PCS-201-012) were cultured in fibroblast base medium (ATCC, PCS-201-030) and growth kit (ATCC, PCS-201-040). Growth stimulants used in the assay included human EGF (R&D Systems, 236-EG), mouse TGFβ (R&D Systems, 410-MT), KGF (Sigma, K1757), or 10% FBS growth medium. 4T1 cells were obtained from ATCC (CRL-2539) and cultured in RPMI-1640 containing 10% FBS (Omega Scientific, Tarzana, CA) and antibiotics.A673 (CRL-1598), A549 (CCL-185), and U87MG (HTB-14) cells were derived from ATCC and cultured in high-glucose DMEM containing 10% FBS. FBS (S12550) was purchased from R&D Systems. BCS (SH30073.03) was obtained from Hyclone. All cell lines used in the study were negative for mycoplasma contamination from various suppliers.

[0100] Cell proliferation assay Growth assays were performed using BCEC and BREC

[13] . Logarithmic-phase growing BCEC or BREC (passage < 10) were trypsinized, resuspended, and seeded at a density of 1200–1500 cells per well in 200 μl volumes into 96-well plates (uncoated) of low-glucose DMEM supplemented with 10% calf serum, 2 mM glutamine, and antibiotics (growth medium). All reagents were added at the indicated final concentrations. After 3–6 days, cells were incubated with Alamar Blue for 4 hours. Fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The experiment was repeated at least three times. To create a hypoxic state, cells were placed in a hypoxic incubator containing a gas mixture of 1% O2, 5% CO2, and 94% N2. Each 96-well plate included untreated and VEGF-treated (10 / ng / ml) wells to monitor plate-to-plate variability. 20% methanol or 0.05% DMSO served as negative controls. When Cas was tested in these cells, 0.05% DMSO served as a negative control. Human RMVECs and human adult DMVECs were divided into 96 gelatin-coated wells (2000 cells per well) in low-glucose DMEM containing 10% FBS. 1200 cells / well were set up for the growth assay in low-glucose medium containing 0.5% FBS. Data were collected on day 4 or day 5. HUVECs (p7-10) were grown on gelatin-coated plates until a concentration density of 70-80% was reached.

[0101] On the day of the assay, cells were dissociated with 0.05% trypsin and neutralized with 0.5% FBS-containing EBM. Cells were briefly rotated and then resuspended in 0.5% FBS medium. Cells were counted and plated into 96 wells, 1000 cells / well. Three sets of wells were used for each treatment. Data were collected on day 3, and then cells were fixed with 4% paraformaldehyde for 15 minutes, followed by the addition of crystal violet. The cell coverage area was quantified after being imaged using ImageJ software.

[0102] Fibroblast growth assays were performed in low-glucose DMEM with or without 1% FBS, or with or without 10 ng / ml bFGF or 100 ng / ml human EGF, and the assay was completed on day 3. ARPE-19 cells were gently elevated with 0.025% trypsin and plated in RtEGM medium (Clonetics) containing 2% FBS, L-glutamine, human bFGF, and GA-1000. Once the cells adhered to the plate, the cultures were maintained using serum-free RtEGM medium. For the growth assay, 1500 human RPE cells were plated in 96-well plates of low-glucose DMEM containing 1% FBS. A673, U87MG, Calu6, and AML12 cells were grown to confluence, then harvested and resuspended in appropriate assay medium. For the proliferation assay, cells were plated at a density of 1000–2000 cells / well in low-glucose DMEM containing 5% FBS or as otherwise described. Bovine pituitary cells (pituitary follicular astrocytes) were isolated as previously described

[67] . For the proliferation assay using human epidermal keratinocytes, human DMVECs, and human dermal fibroblasts, 1000 cells / well were plated in low-glucose DMEM containing 1% FBS, with or without various growth factors. The assay was completed on day 3 for bovine pituitary cells and on day 4 for all other cell types. For 4T1, 1000 cells were plated in RPMI-1640 containing 2% BME and 2% FBS on 96 basement membrane extract (BME) coated wells and processed after 4 hours

[68] . Four days later, tumor cell growth was measured using the MTS assay (Promega, Madison, WI), a colorimetric assay that measures the metabolic activity of viable cells. Recombinant human transferrin was obtained from EMD Millipore (Temecula, CA). Recombinant mouse apo-transferrin was obtained from Sigma.

[0103] siRNA knockdown BCEC, 1.5 × 10 5Cells were plated into 6-well culture plates at a cell / well density and cultured overnight. Old medium was replaced with 2 ml of antibiotic-free culture medium. siRNAs including siNegative (Ambion, AM4611), siRNA against JNK1#2 (Invitrogen, NM_001192974.2_siRNA_266), JNK1#4 (Invitrogen, NM_001192974.2_siRNA_485), siRNA against JNK2#2 (Invitrogen, XM_005208371.4_siRNA_1240), and JNK2#4 (Invitrogen, XM_005208371.4_siRNA_696) were mixed with Lipofectamine RNAiMAX reagent (ThermoFisher Scientific, 13778150) in Opti-MEM I reduced serum medium (Gibco, 31985062). In short, cells in each well were transfected using a mix containing 25 pmol of siRNA, 7.5 μl of RNAiMAX reagent, and 125 μl of Opti-MEM medium, resulting in a final siRNA concentration of 12.5 nM. The RNAiMAX and Opti-MEM mixture was used as a non-siRNA control. Cells were incubated with siRNA. After 8 hours, the siRNA-containing medium was replaced with fresh medium. 24 hours and / or 48 hours after siRNA transfection, cells were used for growth assays and protein extraction.

[0104] PNGase F processing Glycerol-free PNGase F was obtained from New England Biolabs (Ipswich, MA). Briefly, BCEC was dissolved with an NP-40-containing protease inhibitor (Thermo Scientific, Waltham, MA). The lysate was clarified at 4°C and 5000g for 25 minutes. The total protein content was measured using the Pierce BCA protein assay kit (Thermo Scientific). 20 mg of protein was mixed with 10x denaturation buffer and H2O to a total volume of 10 ml. The glycoprotein was denatured at 100°C for 10 minutes, followed by the addition of Glycobuffer and PNGase F. The reaction was carried out at 37°C for 2 hours.

[0105] Western blot Cells were allowed to reach approximately 80% concentration in 12-well plates. Cells were pretreated with ManN, Kif, or Cas for varying durations, with or without subsequent VEGF addition, using H2O as a solvent control for ManN. At various time points, plates were removed from the incubator and kept on ice. Cell monolayers were initially washed once with ice-cold PBS, then lysed in 250 μl of Pierce RIPA buffer (ThermoFisher Scientific, Rockford, IL), or in a 100-fold protease / phosphatase inhibitor cocktail containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, and 1% NP-40 (Cell signaling, #5872). Lysates were collected and mixed with 4X Bolt LDS Sample Buffer (Novex, Carlsbad, CA) in the presence of a Halt protease inhibitor and phosphatase inhibitor cocktail (ThermoFisher Scientific, #NP0007). Samples were subjected to SDS-PAGE (Bolt 4-12% Bis-Tris Plus, Invitrogen) using Bolt MES SDS electrophoresis buffer (running buffer) or NuPAGE (3-8% Tris-Acetate gel) using Tris-Acetate SDS electrophoresis buffer (Novex). HUVEC (passages 6-8) were plated on EBM-2 basal medium (Lonza) containing 0.2% FBS. After overnight incubation, cells were serum-starved in EBM-2 medium for 4 hours, followed by 50 ng / ml VEGF at various lengths. 165Alternatively, the samples were treated with a vehicle control. Equal volumes of protein lysates were analyzed by SDS-PAGE and blotted with the indicated antibodies. Proteins were transferred using Tris-Glycine buffer containing 20% ​​methanol (proteonomics grade) (Apex BioResearch Products). The membranes were first incubated with 5% milk in TBST, pH 7.6 (TEKnova, Hollister, CA), followed by blotting with primary and secondary antibodies. ECL anti-rabbit IgG, a whole antibody conjugated to horseradish peroxidase from donkey or sheep anti-mouse, was obtained from GE Healthcare (UK Limited). The SuperSignal West Dura Extended Duration substrate was from ThermoFisher Scientific. In some cases, the same PVDF membranes were removed by incubation in Restore Plus Western Blot Stripping Buffer (ThermoFisher Scientific) for 8 minutes to show whole-specific protein expression, followed by a second removal for β-actin expression.

[0106] Migration assay As described in "Western Blot," HUVECs (passages 6-8) were cultured and serum-starved. Then, 10,000 cells in 150 μl of EBM-2 medium were added to the upper chamber of an 8 μm pore-size cell culture insert (Falcon) coated with 0.1% gelatin. The lower compartment was filled with 600 μl of EBM-2 medium containing various agents. The plate was incubated at 37°C to enable migration. After 4 hours, the cells were fixed with 4% PFA for 20 minutes, and then stained with crystal violet (Sigma-Aldrich) at room temperature for 20 minutes. Migrating cells at the bottom of the insert membrane were quantified by counting the entire area of ​​the insert at 40x magnification. The experiment was performed in sets of three and repeated three times. BCEC migration was similarly set up, except that the wells were coated with FN, the cells were suspended in 1% serum medium, and the migration time was 18-24 hours.

[0107] Scratch assay This assay used BCEC (passages 6-10) and HUVEC (passages 6-8). Cells were grown in 6-well plates to approximately 80% concentration, washed twice with PBS, then starved for 5 hours in serum-free DMEM (low glucose, Hyclone), after which a "scratch" was created using a 1 ml tip. The cell monolayer was briefly washed once with serum-free medium, followed by various treatments in medium containing 1% FBS. After 48 hours, the assay was stopped by adding 2 ml of 4% paraformaldehyde. After 20 minutes, the fixed cells were stained with 1 ml of crystal violet (Sigma). The plates were gently washed under running tap water, air-dried, and photographed. Images were acquired using a ZEISS Discovery V8 SteREO microscope equipped with a PixeLINK Megapixel FireWire camera. Wound closure was quantified using AxioVision LE Rel.4.4 software. Six images were taken for each sample, and six measurements (in pixels) were performed on each image using AxioVision LE Rel.4.4 software.

[0108] Analysis of N-glycans, monosaccharides, sialic acid, and O-glycans As soon as the BCECs reached a concentration of approximately 80%, they were collected by washing them twice with phosphate-buffered saline (PBS, Sigma) and scraping them off. The cells were pelletized by centrifugation at 300g for 3 minutes and washed once with cold PBS. The cells were homogenized and total protein was measured. All subsequent analyses were based on known protein amounts.

[0109] N-linked glycans were removed from glycoprotein samples using the PNGase-F kit (New England BioLabs, P0705S). Briefly, 300 μg of protein sample was reconstituted in 180 μl of UltraPure water. 20 μl of 10x denaturation buffer was added, and the mixture was boiled in a 100°C water bath for 14 minutes. The sample was cooled to room temperature and centrifuged at 2700 g for 1 minute. Then, 50 μl of 10x NP-40 was added, and the sample was kept at room temperature for 30 minutes with vortexing at 5-minute intervals, followed by the addition of 25 μL of 10x reaction buffer and thorough mixing. Next, 5 μl of PNGaseF (2500U) was added to the sample and gently mixed. The sample was incubated at 37°C for 16 hours. The released N-glycans were purified using solid-phase extraction. In short, N-glycans were purified by sequentially passing a reaction mixture through a pre-conditioned Sep-Pak C18 1cc cartridge (Waters) and a HyperSep PGC (polygraphitized charcoal) cartridge (25 mg, 1 mL Thermo Scientific). The cartridges were washed with 4 ml of water, and only the PGC was washed with an additional 1 ml of water. The N-glycans bound to the PGC were eluted using 30% acetonitrile containing 0.1% TFA in water. Finally, the purified N-glycans were lyophilized and labeled with 2-AB. In short, the sample was dissolved in a 10 μl solution of 0.44 M 2-AB (2-aminobenzamide) in 35% acetic acid in DMSO containing 1 M sodium cyanoborohydride. The sample was incubated at 65°C for 2.5 hours. 2-AB labeled glycans were purified using GlycoClean S cartridges (GLYKO) according to their glycan cleanup protocols. Excess reagent was removed from the samples using Glycoclean S cartridges (Prozyme), and the labeled glycans were dried using SpeedVac and stored at -20°C. Profiling of the 2-AB labeled glycans was obtained using a Dionex CarboPac PA1 (4×250mm) anion exchange column with a guard column (4×50mm) at a flow rate of 1 ml / min.Glycans were separated in 100 mM sodium hydroxide with a 0-250 mM sodium acetate gradient over 0-75 minutes. Data was acquired at sensitivity 7, λ. ex 330 nm, λ em Data were collected using a Dionex ICS-3000 HPLC system equipped with an Ultimate 3000 fluorescence detector (Dionex) set to 420 nm. Data were processed using Chromeleon software (Thermo Scientific).

[0110] Monosaccharide composition analysis was performed using HPAEC-PAD

[69] (Thermo-Dionex ICS3000), and the nmole amount of each monosaccharide present in 25 μg of protein was calculated. Samples were hydrolyzed using 2N trifluoroacetic acid (TFA) at 100°C for 4 hours. Subsequently, acid is removed using a dry nitrogen flush. To ensure complete removal of acid, samples were co-evaporated twice with 100 μl of 50% isopropyl alcohol (IPA). Finally, samples were dissolved in Milli-Q water and injected into HPAEC-PAD. Monosaccharide profiling was performed using a Dionex CarboPac® PA1 column (250 mm × 4 mm; with a 50 mm × 4 mm guard column). An isocratic solvent mixture of 19 mM sodium hydroxide and 0.95 mM sodium acetate was used for 25 minutes at a flow rate of 1 ml per minute. Data were acquired using a standard Quad waveform manufactured and supplied for carbohydrates. All neutral sugars and amino sugars were identified and quantified by comparison to an authentic monosaccharide standard mixture consisting of L-fucose, D-galactosamine, D-glucosamine, D-galactose, D-glucose, and D-mannose

[70] .

[0111] Sialic acid was released using mild acid hydrolysis. Briefly, the sample was treated with 2M acetic acid at 80°C for 3 hours, followed by removal of excess acid using a speed vacuum. Sialic acid was then tagged with DMB reagent and analyzed using the RP-UPLC-FL (Waters Acquity UPLC) system. The amount of sialic acid in the sample was quantified using a known amount of standard Neu5Ac.

[0112] For O-glycan analysis, homogenized cell samples were treated with 50 mM NaOH in the presence of 1 M NaBH4 at 45°C for 16 hours. The reaction mixture was slowly neutralized using ice-cold 30% acetic acid. Next, the neutralized reaction mixture was passed through a Dowex 50-X cation exchange resin to remove sodium ions and then lyophilized. Excess boric acid generated during neutralization was then removed by co-evaporation using acidified methanol and methanol, respectively. Finally, the O-glycans were purified by passing them through a C18 cartridge. The dried and purified O-glycans were then methylated and used for the analysis of permethylated O-glycans. The permethylated samples were then dissolved in absolute methanol and mixed with an SDHB (Super-DHB) MALDI matrix in a 1:1 v / v ratio and spotted onto a MALDI plate. Mass spectral data were acquired using a Bruker AutoFlex mass spectrometer in positive reflectron mode. Mass spectral data were analyzed and annotated using GlycoWork Bench software, with masses consistent with the proposed structure being noted. Single isotope ion intensities were obtained for calculation.

[0113] To measure the cellular uptake of ManN and its subsequent conversion to ManN-6P, BCEC was placed in a 60 mm dish, with approximately 6 × 10⁶ cells per dish. 5Cells were grown to a cell density. ManN was added to the culture at a final concentration of 400 μM. The cells were then incubated for 2 hours. The monolayer was washed three times with PBS at room temperature and lifted by a cell scraper on ice in 10 ml of PBS. The cell pellet was obtained by centrifugation at 400 g for 5 minutes and stored at -80°C for further use. The cell pellet was suspended in 200 μl of ultra-pure ice-cold water in the presence of 1 μl of protease inhibitor. The cells were sonicated for 1 minute with a 30-second pulse and vortexed to form a homogeneous solution. 2.5 μl of homogenate was used in triplicates for protein estimation using the BCA assay method. Standard curves of BSA at concentrations from 0 to 800 μg / ml were performed to quantify the total protein amount. The cell homogenate was filtered through a pre-washed 3K filter, and the filtrate was dried using a speed-vac. Dried samples were reconstituted in 100 μl of ultratrapure water, and samples containing 200 μg equivalents of protein were injected into HPAEC-PAD. The sugars present in the samples were quantified using known amounts (1 nmol) of ManN, glucose, mannose, and ManN-6P standards. All standards except ManNH2-6P were obtained from Sigma-Aldrich. ManNH2-6P was obtained from Omicron Biochemicals, Inc. (South Bend, IN). The amount of monosaccharides present in different cells is expressed as nmol / mg of total protein. All analyses were performed on a Thermo-Dionex ICS system using a CarboPac-PA-1 column with 100 mM NaOH and 250 mM NaOAc as HPLC buffers.

[0114] Biotinylation of surface proteins BCEC cells were plated into 10 cm cell culture dishes three days before cell surface protein isolation. Cells were washed three times with Dulbecco's PBS containing CaCl2 and MgCl2, followed by incubation on ice with EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA; 0.5 mg / ml in Dulbecco's) for 30 minutes. Cells were washed twice with Dulbecco's, and unreacted biotin was blocked with 20 mM glycine for 15 minutes. To prevent reduction of disulfide crosslinks in biotin molecules during the cell lysis process, 100 μM oxidized glutathione (Sigma-Aldrich, St. Louis, MO) was added to the final wash. For cell lysis, cells were added to 500 μl of lysis buffer in PBS (2% NP-40, 1% Triton X-100, 10% glycerol, 100 μM oxidized glutathione, and EDTA-free protease inhibitor tablets (Roche, Mannheim, Germany)). The lysed cell extract was scraped from the plate and transferred to an Eppendorf tube, then incubated on ice on a shaker for 30 minutes. The cell extract was incubated with 30 U of DNase (22°C, 50 minutes, Roche, Mannheim, Germany) and centrifuged for 20 minutes (20,800 × g, 4°C) to pellet the insoluble material. The protein concentration of the supernatant was determined. Equal amounts of protein (approximately 2 mg) from each extract were used for cell surface protein isolation. The supernatant was then treated with biotin agarose beads (Pierce ImmunoPure Immobilized D-biotin, Thermo The solution was pre-clarified using a solution from Scientific (20221), and the pre-clarified solution was used for cell surface protein isolation using streptavidin beads. The beads were washed four times with lysis buffer, four times with 300 mM NaCl in lysis buffer, and twice with 50 mM Tris-HCl, pH 7.8. The proteins were eluted twice at 30°C with elution buffer (50 mM DTT in 50 mM Tris-HCl, pH 7.8), and the eluates were then pooled. Three biological replicas and one non-biotinized control were used in the study.

[0115] Real-time Q PCR for gene expression analysis RNA was purified using the RNeasy mini-kit (Qiagen). 50 ng of total RNA per reaction was used for real-time PCR (Taqman) analysis. The reaction mixture was set up in a MicroAmp Fast Optical 96-well reaction plate, sealed with MicroAmp photoadhesion film, and run on a ViiA7 real-time PCR system (Applied Biosystems). Absolute quantification using standard curves was performed with Sequence Detection System (SDS) software. The expression levels of each gene were further quantified by comparison with the housekeeping gene RPL19 in the same sample. Taqman primers and probe mixes were obtained from Thermo Fisher Scientific. Bovine VEGF-A (Bt03213282), bovine RPL19 (Bt03229687), and bovine-specific VEGFR2 (Bt03258877), GLUT1 (Bt03215313), and GLUT4 (Bt03215316).

[0116] α-mannosidase, α- and α-glucosidase activity assays α-mannosidase activity was measured using the substrate p-nitrophenyl α-mannopyranoside (1 mM). Enzyme derived from Canavalia gladiata (M7257) (final concentration 0.077 U) was incubated at 37°C in 50 μl of 50 mM potassium phosphate buffer, pH 7.5. α-glucosidase was assayed with the substrate p-nitrophenyl α-glucoside (7 mM). Enzyme derived from Saccharomyces cerevisiae type 1 (Sigma, G5003) (final concentration 0.1 U) was incubated at 37°C in 50 μl of PBS, pH 7.5. β-glucosidase was assayed with the substrate 4-nitrophenyl β-D-glucopyranoside (Roche). Almond-derived enzyme (Sigma, G0395) (final concentration 0.002 U) was incubated at 37°C in 50 μl of PBS, pH 7.5, containing 1% SDS. Incubation was stopped by adding an equal volume of acid-based stop solution (R&D systems, 895032). Enzyme activity was measured at 405 nm. α-glucosidase derived from Saccharomyces cerevisiae type I (G5003) was analyzed using p-nitrophenyl α-D-glucopyranoside (Sigma, N1377) as the substrate.

[0117] Measurement of ManN in wound fluid from S. aureus-infected mice To test the stability of ManN in wound fluid collected from mice with skin infections, as described below, 1.5 μl of 5% ManN solution was added to each 200 μl of wound fluid, first diluted 1:1 (v / v) with PBS. At each time point, the samples were removed from a 37°C incubator and stored at -80°C. Plasma proteins were precipitated by adding ice-cold acetonitrile in a plasma:acetonitrile 1:3 (v / v) ratio. The samples were kept on ice for 1 hour, then centrifuged at 12000 g for 10 minutes at 7°C to form pellets. The supernatant was transferred to another tube, dried on a Speed ​​Vac, then reconstituted in UltraPure distilled water, and filtered through a pre-washed Nanosep 3K Omega filter (Pall Corporation). The filtrate was dried on a Speed ​​Vac. The dried samples were dissolved in 100 μl of water, and 2 μL of plasma or wound fluid samples were subjected to HPLC analysis. Neutral sugars and amino sugars were separated using a Dionex CarboPac registered trademark PA1 column (4 mm × 250 mm) with a 4 mm × 50 mm guard column. A uniform concentration gradient of 19 mM sodium hydroxide containing 0.95 mM sodium acetate was run for 20 minutes at a flow rate of 1 ml / min. Data were collected using a Dionex ICS-3000 HPLC system equipped with a pulsed current measurement detector using a standard Quad waveform. ManN was identified and quantified by comparison with monosaccharide standards using Thermo Scientific Chromeleon software. No ManN samples served as negative controls.

[0118] Skin wound healing model All animal experimentation procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego, and were conducted in an ethical manner and in accordance with the guidelines of the Animal Care Program (ACP).

[0119] The model has been previously described

[38] . Briefly, C57BL / 6 female mice (8-10 weeks old) were obtained from Jackson labs (Sacramento, CA). Using a punch (Acu Punch, Acuderm inc. Ft. Lauderdale, FL), a new full-thickness punch wound was created on the dorsal side of the animals in a Class II biological safety cabinet, splinted with a sterile neoprene ring (6 mm outer diameter, 4 mm inner diameter), and secured with 5-6 sutures (4-0 nylon) under the influence of isoflurane. Sterilization techniques were followed for all surgical procedures. Buprenorphine was administered subcutaneously before awakening from anesthesia due to anticipated pain. Mice were monitored until fully awake and housed individually to minimize injury / bite / fighting to the surgical site. Recombinant human VEGF was used. Roche-Genentech (Telbermin, recombinant human VEGF) 165 The treatment agent was donated by [organization name]. The treatment agent was prepared in PBS, sterile filtered, and 25 μl of the solution was applied directly to the wound bed daily for the first 4-5 days under the influence of isoflurane, followed by daily observation. Wound closure was monitored by standard imaging, and the wound area was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).

[0120] Four days post-injury, the wound was excised with a 2 mm rim of surrounding tissue and placed in 10% formalin for up to 24 hours. The wound was then bisected down the middle, and 5 μm paraffin sections were treated for hematoxylin and eosin (H&E) and Masson's trichrome staining. Epithelial gaps were histologically measured using AxioVision LE Rel.4.4 software. Skin tissue was fixed in 10% formalin for 24 hours. Paraffin embedding and sectioning were performed at the UCSD, Moores Cancer Center Histology Core. The 5 μm paraffin sections were deparaffinized, rehydrated, and then heat-induced antigen recovery was performed in 10 mM citrate buffer (pH 6.0). Immunostaining was performed as previously described

[20] . Anti-CD31 (SZ31, rat IgG2a) (Dianova, Warburgstrasse 45, 20354 Hamburg, Germany) was used at a concentration of 2 μg / ml. CD31-positive stained small blood vessels were microscopically counted on 10 fields (20x magnification) taken around the wound.

[0121] Vascular permeability assay Vascular permeability was assessed using a modified Mile assay

[14] . Hairless male guinea pigs (control: HA-Hrhr / IAF, 75 days old, 450-500 g, Charles River Laboratories) were anesthetized by intraperitoneal (ip) administration of xylazine (5 mg / kg) and ketamine (75 mg / kg). The animals were then intravenously injected (in the penile vein) with 1 ml of 1% Evans blue dye. Fifteen minutes later, different doses of ManN were administered intradermally (0.05 ml / site) into the trunk region behind the shoulder. All reagents were diluted with PBS for intradermal administration. 25 ng of VEGF per site. 165 A positive control was used. Thirty minutes after intradermal injection, the animals were euthanized by intravenous injection of pentobarbital (200 mg / kg). Skin tissue was cut from the connective tissue and photographed.

[0122] Mouse skin infection model A mouse skin infection model has been established

[39] . Briefly, this study used intermediate logarithmic phase Staphylococcus aureus subcultured from overnight cultures in Todd-Hewitt broth. Six- to eight-week-old C57BL / 6 mice were obtained from Charles River Laboratories. The mice were shaved with Nair cream to remove hair and then infected. 5 × 10 7 CFU of S. aureus was intradermally injected into the left groin of mice. Three days later, the abscess was surgically removed and homogenized on ice. The fluid was collected and rotated at 14,000 rpm. For further use, the clarified supernatant was diluted with PBS 1:1. The animals were housed in clean cages, and the experimental procedure was then performed under pathogen-free conditions. The presence of bacteria in the wound fluid was confirmed using a Todd Hewitt Broth (THB) plate.

[0123] Hindlimb ischemia model and blood flow evaluation C57BL / 6 male mice (6-8 weeks old) underwent unilateral hindlimb surgery under anesthesia with a ketamine / xylazine cocktail [41, 43]. Briefly, the left femoral artery was separated from the vein and nerve, proximal ligated, and resected. The right hindlimb served as a control. Blood flow was measured using a laser Doppler perfusion imaging device (PeriScan PSI; Perimed). Ischemic and non-ischemic limb perfusion was measured before and after surgery, and at 1, 2, and 3 weeks. Postoperatively, mice were randomly assigned to different groups (8 mice per group). 20% ManN in 200 μl was administered orally every other day starting 3 days postoperatively. 1 mg / ml Kif in 200 μl was administered by ip injection every other day. H2O was used as a vehicle control. Final blood flow values ​​were expressed as the ratio of ischemic hindlimb perfusion to non-ischemic hindlimb perfusion from the same animal. Vascular region quantification was performed as described [41, 43].

[0124] retinal neovascularization Retinal neovascularization after intravitreous administration was evaluated

[41] . Briefly, 6-8 week old C57BL / 6 male mice were randomly assigned to different groups and anesthetized with a ketamine / xylazine cocktail. The indicated amounts of ManN, Kif, or bFGF (R&D systems, AF-233-NA) in 1 μl of PBS and PBS vehicle control were intravitreously injected using a 33-gauge Hamilton syringe. Seven days after injection, the animals were euthanized. The eyes were then extracted and fixed with 4% paraformaldehyde (PFA) for 30 minutes. The retina was isolated and stained with anti-CD31 immunofluorescence (IF) to demonstrate the vascular system. Evaluation was performed by the principal investigator, who was blinded to the treatment. For CD31 IF, rat anti-mouse antibody (BD Biosciences, CAT#550274) was diluted 1:100 and incubated overnight at 4°C. After incubation with Alexa Fluor-488 conjugate anti-rat antibody (Life Technologies, A11006) for 4 hours, whole-mount images were imaged via the 488nm channel using an A1R confocal STORM super-resolution system (Nikon). Vascular density of the choroid and retina was quantified by ImageJ. Each experiment was repeated three times to obtain similar results, and each treatment group consisted of five individual samples.

[0125] statistics and fertility The legend in the figure shows statistical parameters, including n values. Sample size was determined to ensure sufficient power, as recommended by the Biostatistics and Bioinformatics Department, Moores Cancer Center. We used a two-sided two-sample unequal variance t-test. Since the method does not require any usual assumptions about the variables, we further confirmed statistical significance for several in vitro datasets using the Wilcoxon rank-sum test between the treatment groups in question. Statistical inference was based on the p-values ​​for each comparison using the R function "wilcox.test". We used a linear mixed effects (LME) model to investigate wound area (percentage) between three treatment groups (ManN, VEGF, VEGF+ManN) and the PBS group. Two LME models were fitted. In the first LME model, the control group was treated as the reference group. We included day effects (considering days as a categorical variable rather than a continuous variable) and their interaction with the treatment as fixed effects, and we also included subject ID as a random effect to capture the correlation between measurements of different days for the same subject. At baseline, there were no differences between the different groups. In the second LME model, we releveled the VEGF+ManN group as the reference group to investigate the comparison between a single treatment group and combination therapy. For each LME model, we investigated the different treatment effects and their corresponding p-values ​​on days 3, 5, and 8, respectively, relative to the reference group. Data were considered significant if p<0.05. Significant p-values ​​are shown in the figure below: ***p<0.001, **p<0.01, *p<0.05. For each experiment, representative experimental results from 2-5 independent studies are shown.

[0126] manner Embodiment 1. A method for treating an ischemic state in a subject, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need of treatment.

[0127] Embodiment 2. The method according to Embodiment 1, wherein the administration is effective in promoting endothelial cell proliferation and angiogenesis in the subject.

[0128] Embodiment 3. The method according to Embodiment 1 or 2, further comprising administering an effective amount of an N-glycosylation inhibitor to a subject requiring it.

[0129] Embodiment 4. The method according to any one of Embodiments 1 to 4, further comprising administering an effective amount of VEGF to a subject in need thereof.

[0130] Embodiment 5. The method according to any one of Embodiments 1 to 5, wherein the ischemic state is caused by a disease or trauma.

[0131] Embodiment 6. The method according to any one of Embodiments 1 to 6, wherein the administration is intravenous, intraperitoneal, or intravitreous.

[0132] Embodiment 7. A method for inducing angiogenesis in a subject, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to a subject in need thereof.

[0133] Embodiment 8. The method according to Embodiment 7, wherein the administration is effective in reducing ischemia in the subject.

[0134] Embodiment 9. The method according to Embodiment 7 or 8, further comprising administering an effective amount of an N-glycosylation inhibitor to a subject requiring it.

[0135] Embodiment 10. The method according to any one of Embodiments 7 to 9, further comprising administering an effective amount of VEGF to a subject in need thereof.

[0136] Embodiment 11. The method according to any one of Embodiments 7 to 10, wherein the subject needs to have angiogenesis induced due to an ischemic state caused by disease or trauma.

[0137] Embodiment 12. The method according to any one of Embodiments 7 to 11, wherein the administration is intravenous, intraperitoneal, or intravitreous.

[0138] Embodiment 13. A method for inhibiting protein glycosylation in cells, comprising administering an effective amount of hexosamine D-mannosamine (ManN) to the cells.

[0139] Embodiment 14. The method according to Embodiment 13, wherein the administration is in vivo.

[0140] Embodiment 15. The method according to Embodiment 13 or 14, wherein the administration is ex vivo.

[0141] Embodiment 16. The method according to any one of Embodiments 13 to 15, wherein the administration is effective in stimulating EC proliferation and angiogenesis.

[0142] Embodiment 17. The method according to any one of Embodiments 13 to 16, wherein the administration is effective in activating an unfolded protein response induced by JNK and ER stress.

[0143] Embodiment 18. The method according to any one of Embodiments 13 to 17, wherein the administration is effective in inducing changes in the N-glycan and O-glycan profiles.

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Claims

1. A pharmaceutical composition for treating an ischemic state in a subject and for promoting endothelial cell proliferation and angiogenesis, comprising an effective amount of D-mannosamine (ManN), the pharmaceutical composition for treating an ischemic state in a subject.

2. The pharmaceutical composition according to claim 1, further comprising an effective amount of VEGF.

3. The pharmaceutical composition according to claim 1 or 2, wherein the ischemic state is caused by a disease or injury.

4. A pharmaceutical composition according to any one of claims 1 to 3, for oral, intravenous, intraperitoneal, or intravitreal administration to a subject requiring the same.

5. A pharmaceutical composition containing an effective amount of D-mannosamine (ManN) for inducing angiogenesis in a subject.

6. The pharmaceutical composition according to claim 5, wherein the amount of D-mannosamine (ManN) is effective in reducing ischemia in the subject.

7. The pharmaceutical composition according to claim 5 or 6, further comprising an effective amount of VEGF.

8. The pharmaceutical composition according to any one of claims 5 to 7, wherein the subject needs to induce angiogenesis due to an ischemic state caused by disease or trauma.

9. A pharmaceutical composition according to any one of claims 5 to 8, for oral, intravenous, intraperitoneal, or intravitreal administration to a subject requiring the same.

10. A pharmaceutical composition for inhibiting protein glycosylation in a target, comprising an effective amount of D-mannosamine (ManN).

11. The pharmaceutical composition according to claim 10, further comprising an effective amount of VEGF.

12. The pharmaceutical composition according to claim 10 or 11, wherein the need to inhibit protein glycosylation of the target is due to an ischemic state caused by disease or injury.

13. The pharmaceutical composition according to any one of claims 10 to 12 for intravenous, intraperitoneal, or intravitreal administration to the subject requiring it.

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