Use of PCSK5 molecule in the treatment and / or prevention of ischemic diseases

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the application of PCSK5 molecule in the treatment and / or prevention of ischemic diseases. Specifically, the experiments of the present invention have demonstrated that PCSK5 is a molecular target for ischemic diseases, thereby providing applications such as treatment and / or prevention for ischemic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of PCSK5 molecule in the treatment and / or prevention of ischemic diseases. Background Art

[0002] Ischemic diseases are major diseases with a wide range of affected sites (such as cardiovascular, brain, limbs, etc.) that seriously threaten human life and health. Some treatment methods for various ischemic diseases have been disclosed in the prior art, such as:

[0003] Patent document (CN116251115A) discloses the application of miR-342-5p in the preparation of drugs for preventing and treating lower limb ischemic diseases;

[0004] Patent document (CN113662930A) discloses the application of a dianthrone compound in the preparation of drugs for preventing and / or treating myocardial ischemic diseases and related disorders;

[0005] Patent document (CN116019802A) discloses the use of crocetin in the preparation of drugs for treating and preventing cerebral ischemic diseases.

[0006] Coronary heart disease, as a common ischemic disease, seriously affects the quality of life of patients and even endangers their lives, bringing a heavy burden to society. As an ischemic disease with diverse etiologies and clinical manifestations, the pathogenesis of coronary heart disease is very complex. The curative effect of some patients is not good and the recurrence rate is relatively high, which restricts the clinical treatment progress of coronary heart disease. Therefore, the research on the pathogenesis and treatment methods of coronary heart disease is still one of the difficult problems that the medical community needs to solve, and there is an urgent need to find new methods and approaches for the prevention and treatment of coronary heart disease.

[0007] Vascular and myocardial functions are very important for the heart to maintain normal contraction. The role of vascular and myocardial regeneration in coronary heart disease has become a research hotspot in recent years. Some studies have shown that in patients with coronary heart disease, the heart vascular function is abnormal, the blood vessels are blocked and ischemic, myocardial cells undergo apoptosis, and the severity of their condition and the recurrence probability are closely related to the severity of blood vessel blockage and myocardial cell death, suggesting that promoting heart and blood vessel regeneration is an important new direction for the treatment of coronary heart disease. The secretion and maturation of growth factors are the basis for promoting heart and blood vessel regeneration. Among them, proprotein convertase subtilisin / kexin type 5 (PCSK5) plays a key regulatory role in the secretion and maturation of growth factors. Epigenetic modification is one of the important mechanisms for external environmental factors to regulate the expression of PCSK5 in the body. However, the role and mechanism of PCSK5 in regulating the occurrence and development of ischemic diseases remain to be further studied. Summary of the Invention

[0008] In a first aspect, the present invention provides a biomarker for ischemic diseases, wherein the biomarker is a proprotein convertase subtilisin / kexin type 5 (PCSK5) molecule.

[0009] Preferably, the PCSK5 molecule includes PCSK5 protein and / or pcsk5 gene.

[0010] Preferably, the pcsk5 gene includes pcsk5 mRNA.

[0011] Preferably, the biomarker detection sample is derived from blood and / or ischemic tissue, more preferably, the ischemic tissue includes heart, blood vessels, brain tissue or limbs. Further preferably, the detection sample is derived from endothelial and / or myocardial cells of cardiac tissue, or ischemic limbs.

[0012] Preferably, the ischemic diseases include cardiovascular ischemic diseases, cerebral ischemic diseases, limb ischemic diseases, ischemic bowel disease, ischemic retinopathy, ischemic nephropathy, ischemic muscle contracture, and ischemic gastritis.

[0013] Further preferably, the cardiovascular ischemic disease includes but is not limited to one or more of coronary heart disease, myocardial infarction, angina pectoris, atherosclerosis or hypertensive ischemic cardiomyopathy.

[0014] Further preferably, the cerebral ischemic disease includes, but is not limited to, one or more of transient ischemic attack, cerebral ischemia, cerebral infarction, cerebral embolism, ischemic stroke, vertebrobasilar insufficiency or cerebral artery steal syndrome.

[0015] Further preferably, the ischemic diseases of the limbs include but are not limited to one or more of lower limb arteriosclerosis obliterans, varicose veins of the lower limbs, deep vein thrombosis of the lower limbs, arterial embolism, popliteal artery aneurysm, valvular insufficiency of the lower limb communicating veins, diabetic foot, lower limb arterial ischemic disease, diabetic acromial ischemic disease, thromboangiitis obliterans, obliterative arteriosclerosis or distal limb ischemic disease.

[0016] Preferably, the ischemic bowel disease includes, but is not limited to, one or more of ischemic enteritis, mesenteric artery occlusion, mesenteric artery stenosis, and mesenteric vein thrombosis.

[0017] Preferably, the ischemic retinopathy includes, but is not limited to, one or more of retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, hypertensive retinopathy, and arteriosclerotic retinopathy.

[0018] Preferably, the ischemic kidney diseases include one or more of, but are not limited to, renal artery stenosis, renal artery occlusion, renal infarction, ischemic nephritis, ischemic nephrotic syndrome, and ischemic renal failure.

[0019] Preferably, the ischemic gastric diseases include one or more of, but are not limited to, ischemic gastritis, ischemic gastric ulcer, ischemic gastric spasm, and ischemic gastroparesis.

[0020] Preferably, the ischemic liver diseases include one or more of, but are not limited to, ischemic liver injury, ischemic hepatitis, ischemic hepatic infarction, and ischemic hepatic fibrosis.

[0021] Preferably, the ischemic lung diseases include one or more of, but are not limited to, pulmonary embolism, pulmonary hypertension, ischemic pneumonia, pulmonary hemangioma, and ischemic pulmonary edema.

[0022] Preferably, the ischemic muscle diseases include one or more of, but are not limited to, ischemic muscle spasm, ischemic myopathy, ischemic myositis, and ischemic muscular atrophy.

[0023] Preferably, the ischemic bone diseases include one or more of, but are not limited to, ischemic osteonecrosis, ischemic osteitis, ischemic osteoarthritis with osteonecrosis, and ischemic bone defect.

[0024] In a second aspect of the present invention, there is provided a regulator of the biomarker according to the first aspect above, wherein the regulator increases the expression level of PCSK5 protein and / or increases the expression level of pcsk5 gene.

[0025] According to the requirements of the specific embodiments, the regulator can be any reagent in the prior art, as long as it can regulate the mRNA expression level of pcsk5 gene and / or the expression level of PCSK5 protein.

[0026] Preferably, the regulator includes any one or more of the following groups:

[0027] 1) PCSK5 protein or its mutant;

[0028] 2) pcsk5 mRNA;

[0029] 3) a vector containing the pcsk5 gene;

[0030] 4) a regulatory factor that promotes the expression of pcsk5 gene;

[0031] 5) a regulatory factor that increases the activity of PCSK5 protein,

[0032] Exemplarily, the amino acid sequence of the PCSK5 protein can be as shown in NP_001156616.1 or NP_006191.2; the nucleotide sequence of the pcsk5 gene can be as shown in positions 17409678 to 17815076 of NC_000085.7, or positions 75889809 to 76362975 of NC_000009.12, or positions 88048502 to 88519580 of NC_060933.1; the nucleotide sequence of the pcsk5 mRNA can be as shown in NM_001163144.1 or NM_006200.6.

[0033] Preferably, the expression or overexpression vector includes a viral vector or a non-viral vector;

[0034] Preferably, the viral vector includes a lentiviral vector, a pseudovirus vector, an adenoviral vector, a herpes simplex virus vector, a cytomegalovirus vector or an adeno-associated virus vector;

[0035] Preferably, the non-viral vector includes a lipid nanoparticle (LNP), a liposome complex, a cationic polymer, a chitosan polymer or an inorganic nanoparticle.

[0036] In a specific embodiment of the present invention, the expression or overexpression vector is an adenoviral vector or an adeno-associated virus vector.

[0037] Preferably, the regulatory reagent can promote cardiac regeneration and repair after myocardial infarction, promote angiogenesis in ischemic diseases, promote the angiogenic response of endothelial cells, and promote cardiomyocyte proliferation.

[0038] Preferably, the regulatory reagent can improve the cardiac systolic function after myocardial infarction, such as increasing the ejection fraction and / or the fractional shortening of the short axis, reducing the infarct area after myocardial infarction, and increasing the proportion of cardiomyocytes with PH3 + and / or Aurora B + in cardiomyocytes, increasing the vascular density and arterial density after ischemic injury, reducing the percentage of apoptotic cardiomyocytes, promoting blood flow recovery in the blood vessels after ischemia, endothelial cell proliferation, endothelial cell migration, and lumen formation of endothelial cells.

[0039] Preferably, the regulatory reagent can promote the expression of IGF1, FGF2, VEGFA, PCNA and / or CCND1, activate the IGF / AKT / ERK signaling pathway, and promote the phosphorylation of IGF-1R and AKT / ERK.

[0040] In the third aspect of the present invention, there is provided an application of the regulatory reagent of the second aspect above in the preparation of a product for treating and / or preventing ischemic diseases.

[0041] According to the needs of specific implementation, the application further includes treating complications of ischemic diseases.

[0042] Preferably, the regulatory reagent treats ischemic diseases through cardiomyocyte proliferation, endothelial cell proliferation, heart regeneration, and / or angiogenesis.

[0043] Preferably, the regulatory reagent treats and / or prevents ischemic diseases by promoting cardiac regeneration and repair after myocardial infarction, promoting angiogenesis in ischemic diseases, promoting the angiogenic response of endothelial cells, and promoting cardiomyocyte proliferation.

[0044] Preferably, the regulatory reagent promotes the expression of growth factors (VEGFA, IGF-1, and FGF2) and proliferation-related genes (PCNA and CCND1), activates the IGF-1 / AKT / ERK signaling pathway, and then promotes the proliferation of cardiomyocytes and endothelial cells and / or the regeneration of blood vessels (especially cardiac regeneration and repair after myocardial infarction, angiogenesis in ischemic diseases, the angiogenic response of endothelial cells, and / or cardiomyocyte proliferation), thereby treating ischemic diseases.

[0045] The definition of ischemic diseases is the same as that in the first aspect of the present invention.

[0046] In the fourth aspect of the present invention, there is provided an application of the regulatory reagent described in the second aspect above in the preparation of products for promoting cardiac regeneration and repair (preferably cardiac regeneration and repair after myocardial infarction), angiogenesis (preferably angiogenesis in ischemic diseases), the angiogenic response of endothelial cells, and / or cardiomyocyte proliferation.

[0047] According to the needs of specific embodiments, the regulatory reagent treats ischemic diseases by promoting cardiac regeneration and repair after myocardial infarction, angiogenesis in ischemic diseases, the angiogenic response of endothelial cells, and cardiomyocyte proliferation.

[0048] The definition of ischemic diseases is the same as that in the first aspect of the present invention.

[0049] In the fifth aspect of the present invention, there is provided a drug or a pharmaceutical composition, and the drug or the pharmaceutical composition includes the regulatory reagent described in the second aspect above.

[0050] Preferably, the drug or pharmaceutical composition may comprise pharmaceutically acceptable carriers, excipients or salts commonly used in the prior art. The drug can be administered by any suitable route, such as gastrointestinal administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, infusion, intracerebral, intrathecal, transdermal, rectal, etc.). The drug can be in any suitable dosage form, such as a gastrointestinal dosage form or a parenteral dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder aerosols, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, etc. The various dosage forms of the drug can be prepared according to the conventional production methods in the pharmaceutical field.

[0051] In a specific embodiment, the administration includes intravenous or intra-organ administration, such as intra-organ administration during imaging.

[0052] In a sixth aspect of the present invention, an overexpression vector of the pcsk5 gene is provided.

[0053] Preferably, the overexpression vector is an adeno-associated virus or an adenovirus vector.

[0054] Preferably, the overexpression vector contains all or part of the cDNA sequence, all or part of the CDS sequence, or the genomic sequence of the pcsk5 gene.

[0055] Exemplarily, the amino acid sequence of the PCSK5 protein can be as shown in NP_001156616.1 or NP_006191.2; the nucleotide sequence of the pcsk5 gene can be as shown in positions 17409678 to 17815076 of NC_000085.7, or positions 75889809 to 76362975 of NC_000009.12, or positions 88048502 to 88519580 of NC_060933.1; the nucleotide sequence of the pcsk5 mRNA can be as shown in NM_001163144.1 or NM_006200.6.

[0056] In a seventh aspect of the present invention, a method for promoting cardiac regeneration and repair after myocardial infarction, angiogenesis in ischemic diseases, angiogenic response of endothelial cells, and / or proliferation of cardiomyocytes is provided.

[0057] Preferably, the method includes administering an effective amount of the above-mentioned regulatory reagent, the above-mentioned overexpression vector, and / or the above-mentioned drug or pharmaceutical composition to a diseased individual.

[0058] In an eighth aspect of the present invention, the present invention provides a method for treating and / or preventing ischemic diseases.

[0059] Preferably, the method includes administering an effective amount of the above-mentioned regulatory reagent, the above-mentioned overexpression vector, and / or the above-mentioned drug or pharmaceutical composition to an affected individual.

[0060] The definition of ischemic diseases is the same as that in the first aspect of the present invention.

[0061] Preferably, the affected individual includes a human or a non-human animal, such as a non-human mammal.

[0062] In a ninth aspect of the present invention, a method for screening candidate drugs for treating and / or preventing ischemic diseases is provided.

[0063] Preferably, the method includes administering a candidate drug to an animal model of ischemic diseases and screening candidate drugs that can increase the expression of the pcsk5 gene and / or the expression level of the PCSK5 protein.

[0064] This method can be for therapeutic purposes or non-therapeutic purposes. This method only screens which drugs can be used to treat coronary heart disease and / or ischemic diseases, that is, the therapeutic effect is not inevitable. The screening of drugs is only a possibility.

[0065] In a tenth aspect of the present invention, an application of the biomarker in the first aspect above as a target in screening products for treating and / or preventing ischemic diseases is provided.

[0066] Preferably, the screening includes screening regulatory reagents that increase the level or activity of the PCSK5 protein and / or increase the expression level of the pcsk5 gene as products for treating and / or preventing ischemic diseases and / or promoting cardiac regeneration and repair, angiogenesis, angiogenic response of endothelial cells, and / or cardiomyocyte proliferation.

[0067] Exemplarily, the amino acid sequence of the PCSK5 protein can be as shown in NP_001156616.1 or NP_006191.2; the nucleotide sequence of the pcsk5 gene can be as shown in positions 17409678 to 17815076 of NC_000085.7, or positions 75889809 to 76362975 of NC_000009.12, or positions 88048502 to 88519580 of NC_060933.1; the nucleotide sequence of the pcsk5 mRNA can be as shown in NM_001163144.1 or NM_006200.6.

[0068] The definition of ischemic diseases is the same as that in the first aspect of the present invention.

[0069] This application only screens which products can be used for the treatment and / or prevention of coronary heart disease, that is, the therapeutic effect is not inevitable, but only a possibility. The above application does not belong to the method for the diagnosis and / or treatment of diseases.

[0070] In the eleventh aspect of the present invention, there is provided an application of the regulatory reagent described in the fifth aspect above in promoting the expression of growth factors and / or proliferation-related genes.

[0071] Preferably, the growth factors include one or more of VEGFA, IGF-1 or FGF2.

[0072] Preferably, the proliferation-related genes include PCNA or CCND1.

[0073] The beneficial technical effects of the present invention:

[0074] The present invention finds that overexpression of PCSK5 in the hearts of mice with ischemic disease models can promote the proliferation of cardiomyocytes and endothelial cells after myocardial infarction in mice, promote the expression of growth factors (VEGFA, IGF-1 and FGF2) and proliferation-related genes (PCNA and CCND1), activate the IGF-1 / AKT / ERK signaling pathway, and thus promote the proliferation of cardiomyocytes and endothelial cells and / or the regeneration of blood vessels (especially the cardiac regeneration and repair after myocardial infarction, the angiogenesis of ischemic diseases, the angiogenic response of endothelial cells and / or the proliferation of cardiomyocytes), and promote the recovery of cardiac function in mice. Overexpression of PCSK5 in the lower limb ischemia model promotes the recovery of blood flow in the ischemic lower limbs of mice and promotes angiogenesis.

[0075] The expression of PCSK5 is up-regulated in the infarct area of coronary heart disease patients, and PCSK5 in serum is highly expressed in coronary heart disease patients with good prognosis. Targeting the expression of the PCSK5 gene and related signaling pathway molecules can help in the treatment and / or prevention of ischemic diseases.

[0076] The above only summarizes some aspects of the present invention and should not be considered as limiting the present invention in any aspect.

[0077] All patents and publications mentioned in this specification are incorporated into the present invention by reference as a whole. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention.

[0078] The following examples further illustrate the present invention in detail and should not be considered as limiting the present invention or the scope of the specific methods described in the present invention.

[0079] The "products" described in the present invention include reagents for detecting biomarkers and / or reagents for inhibiting biomarkers. Including but not limited to drugs, kits, devices, etc.

[0080] As used in this invention, the term "diagnosis" means ascertaining whether a patient has had, has, or will have a disease or disorder, or ascertaining the progression or likely future progression of a disease.

[0081] As used in this invention, "treatment" means slowing, interrupting, halting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after it has begun to develop, but does not necessarily involve complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0082] As used in this invention, "effective amount" means the amount or dose of a drug of this invention that, when administered to an individual or organ in a single or multiple doses, provides the desired treatment or prophylaxis.

[0083] As used in this invention, the term "comprising" or "including" is an open-ended description that includes the specified components or steps described, as well as other specified components or steps that do not materially affect.

[0084] As used in this invention, "and / or" includes all combinations of the items connected by this term and should be considered as if each combination had been separately listed herein. For example, "A and / or B" includes "A", "A and B", and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0085] As used in this invention, "individual" can be a human or non-human animal, and the non-human animal can be a non-human mammal such as a mouse, cow, sheep, rabbit, pig, monkey, etc.

[0086] The Chinese and English counterparts of some terms appearing in this invention are shown in Table 1 below:

[0087] Table 1

[0088]

[0089] BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The statistical P values in each drawing are as shown in the figures, specifically as follows:

[0091] Figure 1: A, Spatial feature map of pcsk5 gene expression in the border area between controls (healthy individuals) and myocardial infarction patients in spatial transcriptomics data. B, Western blot analysis and statistical chart of relative expression levels of PCSK5 and GAPDH proteins in mouse heart tissues in the border area at 7 days (D7), 14 days (D14), 21 days (D21), and 28 days (D28) after myocardial infarction, n = 6. C, Representative peak map of H3K27ac at the Pcsk5 locus in mouse hearts at postnatal day 0 (P0), day 7 (P7), and day 21 (P21). D, Detection of Pcsk5 mRNA expression in mouse hearts at postnatal day 1 (P1), day 14 (P14), and day 56 (P56), n = 4.

[0092] Figure 2 : A, Detection of PCSK5 expression levels in the sera of coronary heart disease patients and non - coronary heart disease patients by ELISA; B, Correlation between PCSK5 expression levels in the sera of coronary heart disease patients and ejection fraction (%); C, Correlation between PCSK5 expression levels in the sera of coronary heart disease patients and high - sensitivity troponin.

[0093] Figure 3 : Effect of pcsk5 gene on cardiac repair in chronic myocardial infarction in mice; among them, A, Ejection fraction (%) and fractional shortening (%) of cardiac ultrasound analysis in the control group (AAV - Con) and PCSK5 - overexpressing mice (AAV - Pcsk5); B, Masson staining of cardiac sections in the control group (AAV - Con) and PCSK5 - overexpressing mice (AAV - Pcsk5), n = 6.

[0094] Figure 4 : Effect of pcsk5 gene on cardiac and vascular regeneration in chronic myocardial infarction in mice; among them, A - B, Immunofluorescence detection of PH3 + and Aurora B + in cardiomyocytes at 28 days after myocardial infarction in the control group (AAV - Con) and PCSK5 - overexpressing mice (AAV - Pcsk5). C, Immunofluorescence detection of the number of blood vessels of IB4 + and SMA + at 28 days after myocardial infarction in the control group (AAV - Con) and PCSK5 - overexpressing mice (AAV - Pcsk5), n = 6. D, Detection of myocardial apoptosis by Tunel staining at 28 days after myocardial infarction in the control group (AAV - Con) and PCSK5 - overexpressing mice (AAV - Pcsk5), n = 6.

[0095] Figure 5: Effects of the pcsk5 gene on the VEGF / IGF1 signaling pathway in chronic myocardial infarction in mice. A, C-D, WB was used to detect the expression of cell proliferation-related genes (PCNA and CCND1) and growth factor (VEGFA), as well as the activation of the IGF / AKT / signal pathway (P-IGF1R / IGF1R, P-AKT / AKT, and P-ERK / ERK) in the control group (AAV-Con) and PCSK5-overexpressing mice (AAV-Pcsk5). B, qPCR was used to detect the expression of proliferation-related genes (Pcna and Ccnd1) and growth factors (Igf1, Vegf, and Fgf2) in the control group (AAV-Con) and PCSK5-overexpressing mice (AAV-Pcsk5), n = 6.

[0096] Figure 6 : Effects of the pcsk5 gene on cardiac repair in neonatal mouse myocardial infarction; among them, A, ejection fraction (%) and fractional shortening (%) of echocardiographic analysis of the hearts of mice in the control group (Ad-Con) and PCSK5-overexpressing group (Ad-PCSK5); B, Masson staining of heart sections and infarct area (%) of mice in the control group (Ad-Con) and PCSK5-overexpressing group (Ad-PCSK5), n = 6.

[0097] Figure 7 : Effects of the pcsk5 gene on cardiac and vascular regeneration in neonatal mouse myocardial infarction; among them, A-C, immunofluorescence was used to detect PH3 + , Aurora B + , and EdU + of cardiomyocyte numbers at 28 days after myocardial infarction in the control group (Ad-Con) and PCSK5-overexpressing group (Ad-PCSK5). n = 6.

[0098] Figure 8 : Effects of the pcsk5 gene on cardiac and vascular regeneration in neonatal mouse myocardial infarction; among them, A, immunofluorescence was used to detect the number of IB4 + and SMA + blood vessels at 28 days after myocardial infarction in the control group (Ad-Con) and PCSK5-overexpressing mice. B, immunofluorescence was used to detect the number of apoptotic cardiomyocytes at 28 days after myocardial infarction in the control group (Ad-Con) and PCSK5-overexpressing group (Ad-PCSK5), n = 6.

[0099] Figure 9: Effects of the PCSK5 gene on the VEGF / IGF1 signaling pathway in neonatal mice with chronic myocardial infarction. A, Western blot was used to detect the expression of genes related to cell proliferation and growth factors, as well as the activation of the IGF1 / AKT / ERK signaling pathway in mice of the control group (Ad-Con) and the PCSK5 overexpression group (Ad-PCSK5). B, Quantitative PCR was used to detect the expression of genes related to proliferation and growth factors in mice of the control group (Ad-Con) and the PCSK5 overexpression group (Ad-PCSK5), n = 6.

[0100] Figure 10 : PCSK5 promotes the activation of the VEGF signaling pathway in ischemic lower limbs. Western blot was used to detect the expression of the signaling pathway and genes related to proliferation in mice of the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con), n = 6.

[0101] Figure 11 : PCSK5 promotes blood flow recovery in ischemic lower limbs. A, Color Doppler was used to detect the blood flow recovery in mice of the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con); B, Limb perfusion ratio; C, Immunofluorescence was used to detect the blood vessel density in mice of the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con), n = 6.

[0102] Figure 12 : Effects of PCSK5 on the angiogenesis ability of endothelial cells. A, EdU staining was used to detect the proliferation of endothelial cells in the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con), n = 9. B, Cell counting was used to detect the changes in the number of endothelial cells in the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con), n = 5. C, Transwell was used to detect the migration of endothelial cells in the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con). Tube formation assay was used to detect the tube formation ability of endothelial cells in the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con), n = 5.

[0103] Figure 13 : Effects of PCSK5 on the angiogenesis ability of endothelial cells. A, Western blot was used to detect the expression of growth factors and genes related to proliferation in the PCSK5 overexpression group (Ad-PCSK5) and the control group (Ad-Con), n = 3. B, Quantitative PCR was used to detect the expression of growth factors and genes related to proliferation in the PCSK overexpression group (Ad-PCSK5) and the control group (Ad-Con). n = 6.

[0104] Figure 14: Immunofluorescence assay was used to detect the proliferation of primary cardiomyocytes from neonatal rats in the overexpression group of PCSK5 (Ad-PCSK5) and the control group (Ad-Con), n = 6.

[0105] Figure 15 : Effects of PCSK5 on the growth factors and the expression of genes related to proliferation in primary cardiomyocytes from neonatal rats. A, Western blot was used to detect the expression of signaling pathways and genes related to proliferation in the overexpression group of PCSK5 (Ad-PCSK5) and the control group (Ad-Con). B, Quantitative real-time PCR was used to detect the expression of growth factors and genes related to proliferation in the overexpression group of PCSK5 (Ad-PCSK5) and the control group (Ad-Con), n = 6. Specific embodiments

[0106] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention. In the methods of the following embodiments, unless otherwise specified, the biochemical reagents used are commercially available reagents; the methods without detailed steps described are all conventional methods.

[0107] Sources of some reagents involved in the present invention:

[0108] PCSK5 antibody pairs for ELISA reagents: Purchased from Thermo Fisher Scientific, product number PA5-98039; purchased from Abclonal, product number A5450; purchased from Huamei Bio, product number CSB-PA017644LA01HU;

[0109] PCSK5 protein antibody: Purchased from Thermo Fisher Scientific, product number PA5-98039, purchased from Proteintech, product number 16470-1-AP;

[0110] ELISA kit: Purchased from Abclonal, product number: RK04587;

[0111] Reverse transcription kit: Purchased from TOYOBO, product number: FSQ-101;

[0112] Real-time quantitative PCR kit: Purchased from Norway Zan, product number: Q341.

[0113] Some experimental methods involved in the present invention:

[0114] I. The specific implementation steps of the chronic left anterior descending coronary artery ligation myocardial infarction model are as follows (this model is a classic coronary heart disease model, and the cited literature has been marked in Example 1):

[0115] In the myocardial infarction model of neonatal mice (P7), the mice were anesthetized on an ice bed for about 4 minutes, and then the chest was opened to fully expose the heart. In the myocardial infarction model of adult mice (8 - 10 weeks old), they were anesthetized by intraperitoneal injection with 100 mg / kg body weight of ketamine and 10 mg / kg body weight of xylazine, then intubated with a small animal ventilator and ventilated with air (supplemented with oxygen), and the heart was manually exposed. The left anterior descending coronary artery (LAD) was permanently ligated with absorbable silk thread (7 - 0 for adults, 8 - 0 for neonates). The suture was passed through about 2 mm below the left auricle of the heart. The mice in the sham - operated group were subjected to the same operation without ligating the LAD. The mice were placed in a warm cage with the temperature maintained at 37 °C until they were fully awake. Cardiac function was measured by echocardiography (FUJIFILM Visual Sonics Co., Ltd., Toronto, Canada, Vevo 3100, 30 MHz, 450 probe). Cardiac function was measured on the M - mode image through the long axis of the left ventricle. The mice were euthanized by carbon dioxide asphyxiation and tissues were collected.

[0116] II. The specific implementation steps of the lumen formation experiment are as follows:

[0117] (1) One day before the experiment, Matrigel and the pipette tips used in the experiment were placed in the 4 °C refrigerator overnight. Before the experiment, 250 μl of melted Matrigel was added to a 24 - well plate and placed in an incubator at 37 °C for 30 min;

[0118] (2) The cells were digested and counted. 2×10 4 / 500 μl of cell suspension was added to the 24 - well plate, with 3 replicates for each group;

[0119] (3) It was placed in an incubator at 37 °C and photographed and observed after 12 h.

[0120] III. The specific implementation steps of the Tranwells cell migration experiment are as follows:

[0121] (1) The endothelial cells were digested with trypsin, counted, and seeded into the upper chamber of the Transwell, with 6×10 4 cells per well and 200 μl of culture medium per well.

[0122] (2) 800 μl of culture medium was added to each well in the lower chamber, with 3 replicates for each group, and incubated in an incubator at 37 °C for 12 h.

[0123] (3) The culture medium was aspirated, the cells on the upper layer of the chamber were gently wiped off with a cotton swab, and fixed with 4% paraformaldehyde at room temperature for 10 min.

[0124] (4) Staining: Stain with 0.5% crystal violet for 30 min at room temperature.

[0125] (5) Wash once with ddH2O, air dry, observe and photograph under a microscope, and count the number of migrated cells.

[0126] IV. The specific implementation steps of the EdU cell proliferation experiment are as follows:

[0127] 1) Place cell slides in a 12-well plate, digest the cells and seed them into each well of the 12-well plate at a density of 3×10 4 Let the cells incubate overnight until they are fully spread and restored to a normal state.

[0128] 2) Prepare a 2X EdU working solution by diluting EdU 1:500 with the culture medium in advance.

[0129] 3) Add 0.5 ml of the pre-warmed 2X EdU working solution (20 μM) at 37 °C to the cells cultured in the 12-well plate, so that the final concentration of EdU in the 12-well plate becomes 10 μM.

[0130] 4) Continue to incubate the cells for 4 hours. After EdU labels human aortic smooth muscle cells, remove the cell culture medium, wash once with PBS, and add 1 ml of 4% paraformaldehyde to fix at room temperature for 15 minutes.

[0131] 5) Remove the 4% paraformaldehyde, wash the cells in each well 3 times with 1 ml of PBS, 5 minutes each time.

[0132] 6) Permeabilize the cells in each well with 1 ml of PBS containing 0.3% Triton X-100 at room temperature for 10 minutes. Remove the permeabilization solution and wash the cells in each well with 1 ml of PBS.

[0133] 7) Prepare the Click reaction solution for 2 wells of the 12-well plate according to Table 2 below.

[0134] Table 2: Click reaction solution

[0135] Reagent Volume Click Reaction Buffer 430 μl CuSO4 20 μl Azide 488 1 μl Click Additive Solution 50 μl Total Volume 501 μl

[0136] Add 0.25 ml of the Click reaction solution to each well and incubate at room temperature for 30 minutes in the dark.

[0137] 8) Aspirate the Click reaction solution and wash 3 times with PBST, 5 minutes each time.

[0138] 9) Dilute Hoechst 33342 with PBS at a ratio of 1:1000 in advance, add 0.25 ml of the diluted Hoechst 33342 to each well, and incubate at room temperature for 10 minutes in the dark. Wash 3 times with PBST, 5 minutes each time.

[0139] 10) Mount the slides with an anti-fluorescence quenching agent, take images with a fluorescence microscope, and count the percentage of EdU-positive cells.

[0140] V. The specific implementation steps for RNA extraction are as follows:

[0141] 1) Add 500 μl of Trizol to the cells or tissue fragments, transfer them to a new 1.5 ml EP tube, lyse for 10 minutes, or directly store at -80 °C.

[0142] 2) Add 100 μl of chloroform to the EP tube, shake it up and down to make it pink, centrifuge at 12,000 rpm at 4 °C for 15 minutes to separate the liquid layers.

[0143] 3) Carefully aspirate the upper layer of liquid (RNA) and transfer it to a new 1.5 ml RNase-free EP tube, being careful not to touch the middle layer. Add an equal volume of isopropanol and mix well by shaking. Precipitate overnight at 4 °C.

[0144] 4) Centrifuge at 12,000 rpm at 4 °C for 15 minutes in a pre-cooled centrifuge. Aspirate and discard the supernatant, being careful not to aspirate the precipitate. Add 75% alcohol prepared with DEPC water, invert the tube up and down to wash without dispersing the precipitate, centrifuge at 7,500 rpm for 5 minutes, discard the supernatant, open the lid and let it dry, but do not over-dry.

[0145] 5) Dissolve the RNA with 10 - 20 μl of appropriate DEPC water according to the amount of precipitate, and measure the concentration and purity.

[0146] VI. Reverse transcription and real-time quantitative PCR

[0147] Reverse transcription reaction:

[0148] 1) Take 1 μg of RNA and make up to 7 μl with nuclease-free water, pre-denature at 65 °C for 5 minutes in a PCR instrument.

[0149] 2) Add 2 μl of 5× reverse transcription buffer, 0.5 μl of Primer Mix, and 0.5 μl of reverse transcriptase to the pre-denatured RNA above, and mix well by pipetting.

[0150] 3) Perform the reaction with the parameters shown in Table 3 below in a PCR instrument

[0151] Table 3: PCR reaction parameters

[0152] Temperature Time 37℃ 15 min 95℃ 5 min 4℃ Hold

[0153] 4) After the reaction is completed, add 190 μl of ddH 2 O to dilute the cDNA 20-fold, store at -20 °C or perform real-time quantitative PCR operation.

[0154] Real-time quantitative PCR:

[0155] The real-time quantitative PCR system is shown in Table 4 below (10 μl):

[0156] Table 4: Real-time quantitative PCR system

[0157] cDNA 1 μl 10 μM Forward Primer 0.5 μl 10 μM Reverse Primer 0.5 μl SYBR Green supermix 5 μl <![CDATA[ddH 2 O]]> 3 μl

[0158] The RT-PCR cycling parameters are set as shown in Table 5 below:

[0159] Table 5: RT-PCR cycling parameters

[0160]

[0161] After the program ends, the primer specificity is judged according to the melting curve. Using β-actin as an internal reference, according to the 2 -ΔΔCt method to calculate the relative mRNA abundance of the gene: ΔCt = Ct 目的基因 -Ct 内参基因 , ΔΔCt = ΔCt 实验组 -ΔCt 对照组 , relative mRNA abundance = 2 -ΔΔCt .

[0162] Among them,

[0163] Forward primer: 5’-CCAACCACTGGGCAGTCAAA-3’ (SEQ ID NO: 1);

[0164] Reverse primer: 5’-CTGACCTTTTAATCGTCCTGCT-3’ (SEQ ID NO: 2).

[0165] VII. The specific implementation steps of chromatin immunoprecipitation are as follows:

[0166] Prepare chromatin as shown in Table 6 below:

[0167] Table 6: Chromatin preparation operation method

[0168]

[0169]

[0170] 1) Estimate the cell content in the lysis buffer based on the DNA content recovered in the above steps, and calculate according to 7000 ng of DNA per million cells. According to the calculated cell amount in the lysis buffer, take chromatin containing one million cell amounts (make up the volume to 300 μl with low-salt lysis buffer), add an appropriate amount of antibody (2 μg of H3K27ac), and supplement PMSF and PIC. Incubate with rotation at 4°C for 6 - 8 h to allow the antibody to fully bind to the proteins in the lysis buffer.

[0171] 2) Add 30 μl of Protein A / G agarose beads to the above system and incubate on a rotary shaker at 4°C for 2 - 4 h to allow the antibodies to bind fully to the agarose beads.

[0172] 3) After incubation, wash away the chromosomes non-specifically bound to the agarose beads. Centrifuge a 1.5 ml EP tube at 3000 rpm for 1 minute in a 4°C centrifuge to precipitate the agarose beads at the bottom, and gently discard the supernatant; wash twice with pre-cooled 150 mM Lysis Buffer, three times with Wash Buffer, and finally once with TE Buffer, taking care to avoid aspirating the agarose beads during the process of discarding the supernatant.

[0173] 4) Add 200 μl of Elution Buffer to the agarose beads, place in a shaking metal bath at 65°C for 4 - 6 h to elute and de-crosslink the DNA fully, and add 200 μl of TE Buffer, 4 U of DNase-free RNase, and 1 μg / ml proteinase K to it, incubate in a metal bath at 55°C for 2 - 4 h to degrade the proteins and RNA fully; and recover the DNA in the above system according to the steps of the DNA recovery kit, and elute the recovery column with pre-warmed 10 μM Tris-HCl at pH = 8.0 to 55°C. The recovered DNA is subsequently used for q-PCR experiments or library construction for high-throughput sequencing.

[0174] VIII. The steps of the mouse hindlimb ischemia model are as follows:

[0175] (1) In this invention, 12-week-old mice are selected for the experiment. Anesthetize by intraperitoneal injection of 2% sodium pentobarbital. Fix the mice on the operating board and depilate.

[0176] (2) Make a vertical incision at the center of the groin and cut along the direction of the femoral artery. Use micro forceps to separate the subcutaneous fascia and adipose tissue to expose the femoral artery and femoral vein. Slightly posterior to the bifurcation of the common femoral artery, separate the femoral artery and femoral vein, and ligate the femoral artery. Continue to separate downward by 5 mm, ligate the femoral artery, cut the blood vessel between the two ligation points with fiber scissors, and strip and remove the blood vessel. Suture the skin.

[0177] (3) Place the bilateral hindlimbs of the mice under the probe of a laser Doppler blood flow detector to scan the blood flow condition of the hindlimbs. Analyze the blood flow ratio of the two hindlimbs through the software mlDTmainV53. A ratio of the blood flow in the affected side to the blood flow in the contralateral normal limb less than 0.25 indicates successful model establishment.

[0178] (4) After successful model establishment, detect the blood flow condition of the hindlimbs on days 0, 7, and 14 respectively.

[0179] IX. The specific implementation steps of PCSK5 ELISA determination are as follows:

[0180] (1) ELISA coating and detection were performed according to the ELISA kit instructions;

[0181] (2) The blood sample was at room temperature, centrifuged at 3000 g for 10 minutes, and the serum was taken for subsequent detection

[0182] (3) The kit was restored to room temperature 30 minutes before the experiment;

[0183] (4) 100 μL of the standard product and the sample were added to the enzyme-labeled wells pre-coated with the PCSK5 antibody;

[0184] (5) After incubation at 37 °C for 90 minutes, the liquid was discarded;

[0185] (6) 100 μL of the biotin-labeled antibody working solution was added and incubated at 37 °C for 180 minutes;

[0186] (7) Washing was repeated, the liquid was discarded, and it was spun dry. 100 μL of streptavidin labeled with horseradish peroxidase was added to each reaction well and incubated at 37 °C for 30 minutes;

[0187] (8) Washing was repeated, 100 μL of the chromogenic agent was added, and it was incubated at 37 °C in the dark for 15 minutes;

[0188] (9) 100 μL of the stop solution was added, and the absorbance value was immediately measured at 450 nm. With the standard product concentration as the abscissa and the absorbance value as the ordinate, a standard curve was plotted to calculate the PCSK5 concentration, and the PCSK5 concentration of the sample was calculated.

[0189] X. The specific implementation steps of Western Bot are as follows:

[0190] 1) According to the steps for measuring protein concentration in the protein concentration kit, the sample was added to measure the standard curve and the protein concentration of the sample;

[0191] 2) Incubate in an oven at 37 °C for 25 - 30 min;

[0192] 3) The absorbance of the sample to be measured at OD562 nm was measured with an enzyme-labeled instrument;

[0193] 4) According to the measured data, a protein standard curve was plotted to calculate the protein concentration of the sample to be measured.

[0194] 5) Calculate the volume required for 20 μg of the loaded protein, add 6×Loading buffer, pipette and mix well; Centrifuge briefly

[0195] 6) Place the EP tube on a metal bath at 100 °C for 10 min to denature the protein and fully open the secondary structure;

[0196] 7) Select a PAGE gel with an appropriate concentration according to the protein size. Add the sample to be tested or protein Marker into the sample loading wells of the pre-prepared PAGE gel;

[0197] 8) First, run the stacking gel at a constant voltage of 60 V to align the bands and make the bands into a single line;

[0198] 9) After the sample enters the separating gel, run the gel at a constant voltage of 120 V;

[0199] 10) Activate the PVDF membrane with methanol in advance and soak the filter paper and sponge in the transfer buffer;

[0200] 11) After electrophoresis, transfer the membrane. Place the sponge, filter paper, gel, PVDF membrane, and filter paper and sponge in sequence from black to white, and pay attention to carefully removing the air bubbles between the gel and the membrane;

[0201] 12) Transfer the membrane at a constant current of 300 mA on ice, and select an appropriate transfer time according to the size of the target protein;

[0202] 13) After membrane transfer, the contact surface between the PVDF membrane and the gel is the front side;

[0203] 14) Prepare 5% milk in advance and block at room temperature for 1 h;

[0204] 15) Dilute the primary antibody according to the ratio in the instruction manual and incubate the primary antibody overnight at 4°C;

[0205] 16) Take out the membrane incubated overnight at 4°C and warm it to room temperature, and wash it three times with TBST for 15 min each time;

[0206] 17) Incubate the secondary antibody at room temperature for 1 h;

[0207] 18) Wash it three times with TBST for 15 min each time;

[0208] 19) Use the ECL developing solution to develop the film, photograph and save the results under different exposure conditions, and analyze and calculate the gray value with the Image J software.

[0209] Example 1 PCSK5 expression increases after myocardial infarction

[0210] We found through spatial transcriptome data analysis that compared with healthy people, PCSK5 expression was upregulated in the infarct border zone of myocardial infarction patients ( Figure 1A). Ligation of the left anterior descending coronary artery is a classic animal model of coronary heart disease. The disease occurrence and manifestations caused by ligation of the left anterior descending coronary artery can better simulate the symptoms of human coronary heart disease and the biochemical changes it causes (References: Du J, et al. A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration. Cell Stem Cell. 2022 Apr 7; 29(4): 545-558.e13; Mohamed TMA, et al. Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration. Cell. 2018 Mar 22; 173(1): 104-116.e12. doi: 10.1016 / j.cell.2018.02.014; Reboll MR, et al. Meteorin-like promotes heart repair through endothelial KIT receptor tyrosine kinase. Science. 2022 Jun 17; 376(6599): 1343-1347). We constructed an animal model of coronary heart disease by ligating the left anterior descending coronary artery of mice in the manner described in the literature. Compared with the sham operation group (relative expression level of PCSK5: 1.00), after myocardial infarction in mice, with the increase of myocardial infarction time, the expression of PCSK5 protein was up-regulated (7 days after myocardial infarction: relative expression level of PCSK5: 1.50, P = 2.32E-07; 14 days after myocardial infarction: relative expression level of PCSK5: 1.46, P = 9.12E-07; 21 days after myocardial infarction: relative expression level of PCSK5: 1.52, P = 1.19E-07; 28 days after myocardial infarction: relative expression level of PCSK5: 1.56, P = 2.99E-08) (see Figure 1 B, in which Figure 1The bar graphs on the right side of B correspond to the sham operation group, myocardial infarction model group - D7, myocardial infarction model group - D14, myocardial infarction model group - D21, and myocardial infarction model group - D28 from left to right). It shows that the PCSK5 molecule may be upregulated in the infarct border zone of the heart in coronary heart disease. After mammals are born, the cardiac regeneration ability decreases, and many genes related to cardiac regeneration have a decrease in the active histone modification H3K27ac and a down - regulation in expression after mouse birth (References: Eldad Tzahor, et al. Cardiac regeneration strategies: Staying young at heart. Science. 2017 Jun 9; 356(6342): 1035 - 1039; Mei Xin, et al. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Rev Mol Cell Biol. 2013 Aug; 14(8): 529 - 41; Ge Tao, et al. Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury. Nature. 2016 Jun 2; 534(7605): 119 - 23.). Further analysis of ChIP - seq of the active histone modification H3K27ac in the hearts of mice at different developmental stages found that with the development of mice, the enrichment of the active histone modification H3K27ac in the Pcsk5 promoter region decreased ( Figure 1 C), and the expression of Pcsk5 mRNA was down - regulated ( Figure 1 D). This suggests that Pcsk5 may be related to cardiac regeneration.

[0211] Example 2: The expression of PCSK5 in the serum of myocardial infarction patients increases

[0212] We collected the serum of coronary heart disease patients (MI group) and control serum of non - coronary heart disease patients (Healthy group), and detected the expression level of PCSK5 in the serum by ELISA. We found that compared with non - coronary heart disease patients, the level of PCSK5 in the serum of coronary heart disease patients was significantly increased (non - coronary heart disease patients = 14.55 ng / ml, coronary heart disease patients: 23.52 ng / ml, P = 8.2E - 12) ( Figure 2 A). Further, we collected the ejection fraction and high - sensitivity cTnT data of coronary heart disease patients and found that the level of PCSK5 in the serum of coronary heart disease patients was positively correlated with the ejection fraction (r = 0.40, P = 0.0022) (Figure 2 B), which was negatively correlated with the myocardial injury marker high-sensitivity cTnT (r=-0.39, P=0.0038)( Figure 2 C). The above results indicate that PCSK5 is highly expressed in the serum of coronary heart disease patients, and the expression level of PCSK5 in the serum is positively correlated with cardiac function and negatively correlated with myocardial injury.

[0213] Example 3 Tail vein injection of a pcsk5 gene overexpression vector targeting the heart can promote cardiac regeneration and repair after myocardial infarction in mice

[0214] To clarify the regulatory effect of the pcsk5 gene on coronary heart disease, the coding region sequence of the pcsk5 gene (NM_001163144.1) was inserted into an adeno-associated virus vector (element order: pAAV9-CMV-PCSK5-Flag) to construct an overexpression vector of the pcsk5 gene, which was injected into the tail vein of mice using a tail vein injector, with an injection volume of 150 μl. Since adeno-associated virus requires 1 week to stably express and the mice need 1 week to recover after surgery before modeling, tail vein virus injection was performed 7 days before myocardial infarction modeling (references: Cui M, et al. Nrf1 promotes heart regeneration and repair by regulating proteostasis and redox balance. Nat Commun. 2021 Sep 6;12(1):5270; Li Y, et al. gp130 Controls Cardiomyocyte Proliferation and Heart Regeneration. Circulation. 2020 Sep 8;142(10):967-982). After myocardial infarction modeling, echocardiography showed that overexpression of PCSK5 could improve the systolic function of the heart after myocardial infarction compared with the modeling group (ejection fraction (EF%) could reach 48%, while the modeling group was only 37%, P=0.0042; fractional shortening (FS%) could reach 23%, while the modeling group was only 17%, P=0.0123) (see Figure 3 A). Samples were taken 28 days after surgery (chronic phase), and Masson staining showed that overexpression of PCSK5 could reduce the infarct area (only 12.6% in the overexpression group, while 22.0% in the control group, P=0.0322) and fibrosis level after myocardial infarction compared with the modeling group (see Figure 3 B). We performed immunofluorescence staining on tissue sections of myocardial infarction to observe myocardial proliferation-related indicators and found that overexpression of PCSK5 (AAV-Pcsk5) could increase PH3 compared with the control group (empty vector, without pcsk5 gene, AAV-Con) +The proportion of cardiomyocytes (AAV-Pcsk5: 1.6%, AAV-Con: 0.8%, P = 1.5E-7) and Aurora B + The proportion of cardiomyocytes (AAV-Pcsk5: 2.8%, AAV-Con: 0.5%, P = 0.0001) (see Figure 4 Figure A-4B), indicating that overexpression of PCSK5 can promote the proliferation of cardiomyocytes after cardiac injury in mice. At the same time, IB4 and α-SMA staining showed that overexpression of PCSK5 can increase the vascular density (IB4: AAV-Pcsk5: 3142, AAV-Con: 1831, P = 8.8E-6) and arterial density (α-SMA: AAV-Pcsk5: 78, AAV-Con: 35, P = 8.8E-5) after cardiac injury in mice (see Figure 4 Figure C), and Tunel staining showed that overexpression of PCSK5 can reduce the apoptosis of cardiomyocytes after myocardial infarction (AAV-Pcsk5: 31%, AAV-Con: 44%, P = 0.006) (see Figure 4 Figure D). Further, we extracted RNA and proteins from the left ventricular tissue. qPCR showed that overexpression of PCSK5 can promote the expression of IGF1, VEGFA, and proliferation-related genes PCNA and CCND1 ( Figure 5 Figure A), and WB showed that overexpression of PCSK5 can promote the activation of IGF1 and VEGFA signaling pathways and the expression of proliferation-related genes PCNA and CCND1 (Table 7, Figure 5 Figures B-D, where, Figure 5 In each bar graph of Figures B-D, from left to right are the control group (AAV-Con) and the overexpression group (AAV-Pcsk5). It shows that injecting the pcsk5 gene overexpression vector can effectively promote cardiac repair after myocardial infarction.

[0215] Table 7 Expression results of gene mRNA or protein in tissue sections of acute myocardial infarction in different treatment groups

[0216] Gene / Protein AAV-Con AAV-Pcsk5 P Value Igf1 mRNA 1.022 1.758 p＝0.042 Fgf2 mRNA 1.080 1.891 p＝0.024 Vegfa mRNA 1.358 3.274 p＝0.004 Pcna mRNA 1.017 1.523 p＝0.015 Ccnd1 mRNA 1.059 1.862 p＝0.022 Pcsk5 mRNA 1.251 7.830 p＝0.01 VEGFA Protein 1.000 1.708 p＝0.0003 PCNA Protein 1.000 2.460 p = 2.07E-05 CCND1 Protein 1.000 2.109 p = 2.75E-05 PCSK5 Protein 1.000 1.794 p＝0.0001 P-IGF1R / IGF1R 1.000 1.688 p＝0.0001 P-AKT / AKT 1.000 2.048 p＝0.0022 P-ERK / ERK 1.000 1.792 p＝0.0022

[0217] Example 4 Local injection of pcsk5 gene overexpression vector into the myocardium can promote cardiac regeneration and repair after myocardial infarction in neonatal mice

[0218] To determine whether PCSK5 promotes neonatal mouse cardiomyocyte proliferation and heart regeneration, we performed myocardial infarction surgery in 7-day-old neonatal mice (reference: Li Y, et al. gp130 Controls Cardiomyocyte Proliferation and Heart Regeneration. Circulation. 2020 Sep 8;142(10):967-982), and injected an adenovirus vector overexpressing the pcsk5 gene (insert sequence NM_006200.6, as the PCSK5 overexpression group, Ad-PCSK5) and a control vector (empty vector, without the Pcsk5 gene, as the control group, Ad-Con) into the infarcted area respectively. Four weeks after myocardial infarction, echocardiography showed that compared with the Ad-Con group, overexpression of PCSK5 restored its cardiac function (ejection fraction could reach 57%, while that of the Ad-Con group was only 35%, p = 0.0019; fractional shortening could reach 30%, while that of the Ad-Con group was only 17%, p = 0.0021)( Figure 6 A). In addition, Masson staining showed that overexpression of PCSK5 reduced the infarct area (13.4% in the Ad-Con group and 7.6% in the Ad-PCSK5 group, p = 0.0022) and fibrosis level( Figure 6 B). Immunostaining showed that the percentages of PH3 + and Aurora B + cardiomyocytes in the Ad-PCSK5 group of mice were significantly higher than those in the Ad-Con group of mice (PH3 + cardiomyocytes: 4% in the Ad-PCSK5 group and 1% in the Ad-Con group, p = 0.0006; Aurora B + cardiomyocytes: 4.0% in the Ad-PCSK5 group and 1.5% in the Ad-Con group, p = 0.0001)( Figure 7 upper and lower panels of A and upper and lower panels of 7B). EdU staining further confirmed that the percentage of proliferating cardiomyocytes in the Ad-PCSK5 group of mice increased compared with that in Ad-Con mice (EdU + cardiomyocytes: 1.9% in the Ad-PCSK5 group and 0.8% in the Ad-Con group, p = 0.0013)( Figure 7 upper and lower panels of C). Immunofluorescence assessment showed that overexpression of PCSK5 enhanced the vascular density in the infarct border zone (number of IB4 + vessels: 2776 in the Ad-PCSK5 group and 2130 in the Ad-Con group, p = 0.0027; see Figure 8 upper panel and lower left panel of A) and arterial density (α-SMA +Number of blood vessels: 37 in the Ad-PCSK5 group and 12 in the Ad-Con group, p = 0.0007, see Figure 8 A (upper and middle lower figures). Compared with the control mice, the percentage of apoptotic cardiomyocytes in the infarct border zone of mice overexpressing PCSK5 was significantly reduced (TUNEL + Cardiomyocytes: 16.6% in the Ad-PCSK5 group and 29.7% in the Ad-Con group, p = 8.78E-05, see Figure 8 B (upper and lower right figures). In addition, we observed that the expression of growth factor and proliferation-related genes and the activation of the IGF / AKT / ERK signaling pathway in the heart tissue of the infarct peripheral region in the group overexpressing PCSK5 (Ad-PCSK5 group) were higher than those in the control group (Ad-Con group) (Table 8, Figure 9 A (left figure), Figure 9 A (upper right figure), Figure 9 A (lower right figure), 9B, where Figure 9 in each bar graph, from left to right are the control group (Ad-Con) and the overexpression group (Ad-PCSK5). Our results show that PCSK5 can promote blood vessel and heart regeneration after heart injury in neonatal mice and improve heart function.

[0219] Table 8: Expression results of gene mRNA or protein in the heart tissue of the infarct peripheral region in different treatment groups

[0220] Gene / Protein Ad-Con Ad-Pcsk5 P Value Igf1 mRNA 1.039 2.010 p＝0.042 Fgf2 mRNA 1.010 1.664 p＝0.0008 Vegfa mRNA 1.073 2.026 p＝0.0269 Pcna mRNA 1.051 2.090 p＝0.035 Ccnd1 mRNA 1.049 1.460 p＝0.0297 VEGFA Protein 1.000 1.778 p = 5.26E-06 PCNA Protein 1.000 1.257 p＝0.0101 CCND1 Protein 1.000 1.344 p＝0.0043 PCSK5 Protein 1.000 1.805 p＝0.0043 P-IGF1R / IGF1R 1.000 1.753 p = 2.18E-05 P-AKT / AKT 1.000 1.538 p＝0.0051 P-ERK / ERK 1.000 1.260 p＝0.0013

[0221] Example 5 Local injection of pcsk5 gene overexpression vector can promote angiogenesis in ischemic lower limbs

[0222] To further verify the effect of PCSK5 on angiogenesis, we performed lower limb ischemia surgery on adult C57 mice and locally injected an adenovirus vector (prepared as in Example 4) overexpressing PCSK5 into the ischemic lower limbs of the mice. At 0 days (Day0), 3 days (Day 3), 7 days (Day 7), and 14 days (Day 14) after surgery, the changes in blood flow of the mice were dynamically monitored by color Doppler ([[]] Figure 11 A). We found that overexpression of PCSK5 could promote blood flow recovery after lower limb ischemia in mice. At day 7, there was a significant difference in the limb perfusion ratio between the Ad-Con and Ad-PCSK5 groups. Specifically: Day 7: Ad-Con: 0.43, Ad-PCSK5: 0.58, p = 0.0031; Day 14: Ad-Con: 0.61, Ad-PCSK5: 0.78, p = 0.0004 ( Figure 11 B. Starting from day 7, the broken line above the broken line graph represents the Ad-PCSK5 group). Immunofluorescence staining showed capillary density (using CD31 +Cell characterization, Ad-Con: 164, Ad-PCSK5: 259, p = 0.0041) and artery density (using α-SMA + Cell characterization, Ad-Con: 23, Ad-PCSK5: 46, p = 0.0083) increased ( Figure 11 C). The above results further indicate that PCSK5 can promote angiogenesis after lower limb ischemia, thereby promoting blood flow recovery. Further, we extracted tissues from the ischemic lower limbs for WB detection of the expression of angiogenesis-related genes and found that overexpression of PCSK5 can promote the expression of angiogenesis-related gene VEGFA (Ad-Con: 1.000, Ad-PCSK5: 1.523, p = 3.00E-04), as well as proliferation-related genes PCNA (Ad-Con: 1.000, Ad-PCSK5: 2.557, p = 7.98E-06) and CCND1 (Ad-Con: 1.000, Ad-PCSK5: 1.622, p = 0.0030)( Figure 10 ). The above results suggest that PCSK5 may promote angiogenesis and arteriogenesis by promoting the expression of angiogenesis-related genes.

[0223] Example 6 PCSK5 can promote the angiogenic response of endothelial cells

[0224] To further explore the effect of PCSK5 on the angiogenic response of endothelial cells. PCSK5 was overexpressed in cultured human umbilical vein endothelial cells. After 48 hours, cell counts were performed every 24 hours to observe cell proliferation. The number of umbilical vein endothelial cells increased after overexpression of PCSK5 ( Figure 12 B). Further, we used the EdU method to detect cell proliferation and found that the number of EdU-positive cells in human umbilical vein endothelial cells increased after overexpression of PCSK5 (Ad-Con was 28.7%, Ad-PCSK5 was 39.7%, P = 0.036, Figure 12 A). The above results indicate that overexpression of PCSK5 can promote the proliferation of endothelial cells. We used the Transwell assay to detect the migration ability of cells and found that overexpression of PCSK5 promoted the migration ability of endothelial cells (Ad-Con was 21, Ad-PCSK5 was 44, p = 0.0021, Figure 12 C, left and middle panels), and promoted the lumen formation of endothelial cells (Ad-Con was 1.00, Ad-PCSK5 was 1.58, p = 2.00E-04, Figure 12C (right figure)). Further, we overexpressed PCSK5 in human umbilical vein endothelial cells and found that overexpression of PCSK5 could promote the expression of cell proliferation-related genes (PCNA: Ad-Con was 1.000, Ad-PCSK5 was 1.269, p = 0.0152; CCND1: Ad-Con was 1.000, Ad-PCSK5 was 1.561, p = 0.0467)( Figure 13 A) and growth factors (FGF2 mRNA: Ad-Con was 1.107, Ad-PCSK5 was 3.012, p = 1.55E-5, and, VEGFA mRNA: Ad-Con was 1.004, Ad-PCSK5 was 2.205, p = 4.78E-5), etc.( Figure 13 B). In summary, our study found that PCSK5 promoted the angiogenic response of endothelial cells.

[0225] Example 7 PCSK5 can promote cardiomyocyte proliferation

[0226] We further wanted to verify the effect of PCSK5 on cardiomyocyte proliferation in vitro experiments. We isolated primary neonatal rat cardiomyocytes and overexpressed PCSK5 in primary neonatal rat cardiomyocytes. Through immunofluorescence staining, we found that overexpression of PCSK5 increased the proportion of PH3-positive cardiomyocytes (Ad-Con was 2.068%, Ad-PCSK5 was 4.822%, p = 0.046) and Aurora B-positive (Ad-Con was 2.35%, Ad-PCSK5 was 5.187%, p = 0.085) cardiomyocytes. Further EdU detection confirmed that overexpression of PCSK5 could promote the proliferation of primary neonatal rat cardiomyocytes (Ad-Con was 4.662%, Ad-PCSK5 was 7.029%, p = 0.0376)( Figure 14 ). We detected the activation of the PI3K / AKT signaling pathway by WB and found that overexpression of PCSK5 could promote the phosphorylation of IGF1R and AKT / ERK, thereby promoting the expression of proliferation-related genes (PCNA, CCND1, P-IGF1R / T-IGF1R, P-AKT / T-AKT, P-ERK / T-ERK, Table 9, Figure 15 A), and qPCR detection found that overexpression of PCSK5 could promote the expression of IGF-1 and proliferation-related genes (Igf1, Fgf2, Vegfa, Pcna, Ccnd1, Table 9, Figure 15 B). The above results indicate that PCSK5 can promote cardiomyocyte proliferation by activating the IGF-1 / AKT signaling pathway.

[0227] Table 9 Expression results of genes mRNA or protein in primary neonatal rat cardiomyocytes of different treatment groups

[0228] Gene / Protein Ad-Con Ad-Pcsk5 P Value PCNA Protein 1.000 1.437 p＝0.0002 CCND1 Protein 1.085 1.365 p＝0.0445 P-IGF1R / T-IGF1R 1.000 2.966 p＝0.0099 P-AKT / T-AKT 1.167 1.618 p＝0.006 P-ERK / T-ERK 1.026 1.302 p＝0.038 Igf1 mRNA 1.104 2.867 p＝0.030 Fgf2 mRNA 1.197 3.728 p＝0.034 Vegfa mRNA 1.377 4.055 p＝0.015 Pcna mRNA 1.120 2.947 p＝0.002 Ccnd1 mRNA 1.236 2.210 p＝0.054

[0229] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0230] In addition, it should be noted that, in the case of no conflict, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

[0231] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for increasing the level or activity of PCSK5 protein and / or pcsk5 Use of a gene expression level regulating agent in the preparation of a product for treating and / or preventing lower limb ischemic diseases; the regulating agent comprises pcsk5 Gene vector.

2. The use according to claim 1, characterized in that: Include pcsk5 Gene vectors include viral vectors and non-viral vectors.

3. The use according to claim 2, characterized in that: The viral vector includes a lentiviral vector, a pseudoviral vector, an adenoviral vector, a herpes simplex virus vector, a cytomegalovirus vector or an adeno-associated virus vector.

4. The use according to claim 2, characterized in that: The non-viral vector includes lipid nanoparticles (LNP), liposome complexes, cationic polymers, chitosan polymers or inorganic nanoparticles.

5. The use according to claim 1, characterized in that: Treat and / or prevent lower limb ischemic diseases by promoting angiogenesis.

6. The use according to claim 1, characterized in that: The lower limb ischemic diseases include lower limb ischemic diseases caused by one or more of arteriosclerosis obliterans, arterial embolism, popliteal artery aneurysm, diabetic foot, diabetic acroischemic disease and thromboangiitis obliterans.

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