Application of MMP10 inhibitor in preparation of product for preventing and treating vascular calcification

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of an MMP10 inhibitor in preparation of a product for preventing and treating vascular calcification. The invention provides an action mechanism of MMP10 secretion mediating bypass artery calcification, and vascular calcification aggravated by endothelium TRIM35 deletion can be reversed by inhibiting MMP10 activity. The invention also provides an MMP10 inhibitor which can reduce calcium salt deposition and phenotypic transformation of smooth muscle cells, so that TRIM35 endothelial cell specific knockout mouse bypass artery blood vessel calcification is reversed. The method deepens the understanding of the vascular calcification pathological process, and promotes the development of related subjects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of an MMP10 inhibitor in the preparation of a product for preventing and treating vascular calcification. Background Art

[0002] Vascular calcification refers to the phenomenon of calcium deposition on the blood vessel wall, resulting in an increase in the stiffness of the blood vessel wall and a decrease in compliance. Vascular calcification is a common pathological manifestation, usually associated with factors such as atherosclerosis, hypertension, diabetic angiopathy, vascular injury, chronic kidney disease, and aging. The progression of vascular calcification is often accompanied by endothelial dysfunction, which can increase the release of inflammatory factors in the blood vessel wall and promote calcium salt deposition in the blood vessel wall. Currently, the research on vascular calcification mainly focuses on predicting the prognosis of atherosclerotic patients and the cardiovascular risk of chronic kidney disease patients, with less attention paid to patients after arterial bypass surgery.

[0003] Vascular calcification is an active, adjustable, treatable, and preventable process, which provides a theoretical basis for the research and development of drug targets. Research has found that short-chain fatty acids (such as propionate) play an important role in vascular calcification. They can inhibit the inflammatory response and "apply the brakes" to the process of vascular calcification by maintaining the homeostasis of intestinal microbiota, reducing the production of lipopolysaccharide, and protecting the integrity of the mucosal barrier. In particular, propionate has been found to significantly inhibit vascular calcium salt deposition in experimental rats, reduce macrophage infiltration in the blood vessel wall, and lower the levels of various substances such as tumor necrosis factor and interleukin in the plasma. Therefore, short-chain fatty acids and their related metabolic pathways may become important targets for drugs against vascular calcification. In addition, there is a close relationship between the intestinal microbiota and vascular calcification. Research has found that remodeling the intestinal microbiota through short-chain fatty acids (such as propionate) is expected to improve intestinal barrier function, reduce the inflammatory response, and prevent further vascular calcification. In addition, fecal microbiota transplantation studies have also found that specific intestinal microbiota (such as Akkermansia muciniphila) can weaken vascular calcification and reduce the inflammatory response in the body. Therefore, the research and development of drugs targeting the intestinal microbiota may become a new approach for the treatment of vascular calcification.

[0004] Currently, the research on drug targets for vascular calcification mainly focuses on short-chain fatty acids and the intestinal microbiota. These research results provide strong theoretical support and practical guidance for the development of new therapeutic drugs. However, the mechanism of vascular calcification is complex, involving the interaction of multiple biological macromolecules such as genes, receptors, and enzymes. Therefore, further in-depth research is needed to discover more drug targets and provide more effective means for clinical treatment.

[0005] MMP10, also known as matrix metalloproteinase 10 or matrilysin-2, is a protease encoded by the human MMP10 gene. MMP10 is mainly involved in the degradation of the extracellular matrix, especially the degradation of proteoglycans and fibronectin. It plays an important role in various physiological and pathological processes, including tissue remodeling and repair, cell migration, inflammatory responses, and tumor progression. MMP10 may promote cell migration and tissue repair during the inflammatory process. It is related to the viability and metastasis of stem cells. In addition, MMP10 is strongly expressed in osteoclasts and most monocytes in the bone marrow. It is also induced by cytokines in human keratinocytes and is also expressed in human head and neck and lung cells. The gene expression of MMP10 is regulated by multiple factors, including cell signal transduction, growth factors, inflammatory mediators, etc. The prior art does not clearly show a potential relationship between MMP10 and vascular calcification. The present invention further studies the mechanism of action of MMP10 in vascular calcification and explores an intervention strategy based on MMP10, which is of great significance for the prevention and treatment of vascular calcification-related diseases. Summary of the Invention

[0006] To solve the above problems, the present invention provides the mechanism of action of MMP10 in vascular calcification, discovers new intervention targets, and provides a theoretical basis for the development of new methods and drugs for the treatment of vascular calcification.

[0007] On the one hand, the present invention provides the use of an MMP10 inhibitor in the preparation of a product for preventing and treating vascular calcification and diseases caused by vascular calcification.

[0008] Specifically, the vascular calcification has the characteristics of up-regulated expression of osteoblast markers and down-regulated expression of contractile markers. The osteoblast markers include: Runx2, BMP2, SP7, MSX2, osteopontin, osteocalcin, and alkaline phosphatase; the contractile markers include: α-SMA, SMM-HC, VIMENTIN, and SM22α.

[0009] Specifically, the diseases caused by vascular calcification include atherosclerosis, cardiovascular diseases, hypertension, chronic kidney disease, or diabetic angiopathy.

[0010] More specifically, the mechanism of action of the product includes: inhibiting the up-regulation of osteoblast markers Runx2, BMP2, and alkaline phosphatase; improving calcium salt deposition and collagen deposition.

[0011] Specifically, the inhibitor is used to inhibit the biological activity of MMP10, or to inhibit the expression of MMP10.

[0012] More specifically, the MMP10 inhibitor includes at least one of the following: (1) Chemically synthesized inhibitors; (2) Plant extracts; (3) Antibiotics.

[0013] Preferably, the chemically synthesized inhibitors include, but are not limited to: GM6001, BB-94, or compounds designed and optimized for specific domains of MMP10; The plant extracts include, but are not limited to: one or more of artemisinin, curcumin, tea polyphenols, grape seed extract, and Platycodi Radix extract; The antibiotics include, but are not limited to: doxycycline.

[0014] Specifically, the product is a drug.

[0015] More specifically, the drug further includes pharmaceutically acceptable excipients.

[0016] Even more specifically, the pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0017] Preferably, the pharmaceutically acceptable excipients are at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0018] Specifically, the diseases caused by vascular calcification include atherosclerosis, cardiovascular diseases, hypertension, chronic kidney disease, or diabetic angiopathy.

[0019] In one aspect, the present invention provides the use of a genetic engineering product that inhibits the expression of MMP10 in the preparation of a product for preventing and treating vascular calcification and diseases caused by vascular calcification.

[0020] Specifically, the genetic engineering product includes a vector carrying siRNA and / or miRNA that inhibits the expression of MMP10.

[0021] In another aspect, the present invention provides a drug for preventing and treating vascular calcification and diseases caused by vascular calcification. The drug includes an active ingredient and excipients. The active ingredient is selected from any one or more of the following: (1) MMP10 inhibitor; (2) Genetic engineering product that inhibits the expression of MMP10.

[0022] Specifically, the MMP10 inhibitor is one or more of a chemically synthesized inhibitor, a neutralizing antibody, a plant extract, and an antibiotic.

[0023] More specifically, the chemically synthesized inhibitor includes GM6001, BB-94, or a compound designed and optimized for a specific domain of MMP10; The plant extract includes one or more of artemisinin, curcumin, tea polyphenols, grape seed extract, and Platycodi Radix extract; The antibiotic includes doxycycline.

[0024] Specifically, the drug further includes a pharmaceutically acceptable excipient.

[0025] Even more specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a sweetening agent, and a flavoring agent.

[0026] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl paraben, propyl paraben, magnesium stearate, and mineral oil.

[0027] Specifically, the diseases caused by vascular calcification include atherosclerosis, cardiovascular disease, hypertension, chronic kidney disease, or diabetic angiopathy.

[0028] In another aspect, the present invention provides the use of an MMP10 inhibitor in the preparation of a therapeutic drug after arterial bypass surgery.

[0029] Specifically, the MMP10 inhibitor is one or more of a chemically synthesized inhibitor, a plant extract, and an antibiotic.

[0030] More specifically, the chemically synthesized inhibitor includes GM6001, BB-94, or a compound designed and optimized for a specific domain of MMP10; The plant extract includes one or more of artemisinin, curcumin, tea polyphenols, grape seed extract, and Platycodi Radix extract; The antibiotic includes doxycycline.

[0031] Specifically, the drug further includes a pharmaceutically acceptable excipient.

[0032] More specifically, the pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifying agents, preservatives, antioxidants, buffering agents, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, suspending aids, solubilizing agents, thickening agents, stabilizers, sweeteners, and fragrances.

[0033] Preferably, the pharmaceutically acceptable excipients are at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0034] Specifically, the pharmaceutical dosage forms are tablets, pills, powders, granules, capsules, solutions, emulsions, suspensions, injections, or intravenous drip agents.

[0035] Technical effects achieved by the present invention: Over-secretion of MMP10 by endothelial cells promotes smooth muscle ossification deposition and bypass artery calcification: Proteomics has found that high plasma MMP10 levels are highly correlated with vascular calcification after arterial bypass surgery. After extracting RNA from primary endothelial cells of calcified bypass arteries and performing high-throughput RNA sequencing, it was found through differential gene analysis that the expression of MMP10 increased in endothelial cells; qPCR, Western blot, and ELISA confirmed an increase in the synthesis and secretion of MMP10 by endothelial cells in calcified arteries; in vitro stimulation experiments by directly adding recombinant MMP10 protein to stimulate smooth muscle cell lines found an increase in calcium salt deposition. The above results indicate that endothelial cells may mediate bypass artery calcification by increasing MMP10 secretion.

[0036] Inhibiting the activity of MMP10 can reverse the vascular calcification exacerbated by endothelial secretion of MMP10: In vitro experiments found that reducing the activity of MMP10 can reduce calcium salt deposition and phenotypic transformation of smooth muscle cells; in vivo experiments found that the vascular calcification of bypass arteries was reversed after applying the MMP10 inhibitor GM6001 and MMP10 neutralizing antibody in situ in mouse bypass arteries. Description of the Drawings

[0037] Figure 1Results graphs showing plasma MMP10 predicting poor prognosis in coronary artery bypass graft patients in the UK Biobank and Framingham Offspring Study public databases. Among them, A is the overall analysis flowchart; B is a heatmap showing that MMP10 is highly correlated with risk factors for poor outcomes in CABG patients; C is the association between CVD death risk, MACE events and MMP10 in the crude model or adjusted model; D and E are ROC curves for MMP10 predicting 10-year death risk and MACE risk; F is the verification of the correlation between plasma MMP10 and vascular calcification events in the Framingham Offspring Study database, and multivariate analysis of MMP10 level, gender, BMI, smoking status and blood pressure level was performed using logistic regression.

[0038] Figure 2 Results graphs showing osteogenic transformation changes in the smooth muscle layer of calcified bypass arteries. Among them, A is Van Kossa staining of calcified bypass arteries in mice; B is immunofluorescence of BMP2 (red), an indicator of osteogenic transformation of SMCs in calcified bypass arteries in mice; C is immunofluorescence of RUNX2 (red), an indicator of osteogenic transformation of SMCs in calcified bypass arteries in mice.

[0039] Figure 3 Results graphs showing calcification of smooth muscle cells driven by primary endothelial cells of bypass arteries. Primary endothelial cells of calcified bypass arteries (Isograft pEC) were isolated, co-cultured with primary smooth muscle cells, and primary endothelial cells from non-operated patients (Ctrl pEC) were extracted for control of smooth muscle co-culture. A is alizarin red staining to detect the formation of calcium nodules in pVSMCs indirectly co-cultured with Ctrl pEC or calcified artery pEC; B is direct microscopic observation of the formation of calcium nodules in pVSMCs indirectly co-cultured with Ctrl pEC or calcified artery pEC; C is statistical analysis of calcified nodules (Figure A) by alizarin red staining; D is quantitative analysis of alkaline phosphatase activity in pVSMCs indirectly co-cultured with Ctrl pEC or calcified artery pEC; E and F are the RNA level and protein level of pVSMCs indirectly co-cultured with Ctrl pEC or calcified artery pEC, respectively. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0040] Figure 4Results graph of MMP10 secreted by endothelial cells inducing smooth muscle cell calcification. Primary endothelial cells of calcified bypass arteries were isolated and co-cultured with primary smooth muscle cells, and primary endothelial cells without surgery were extracted for control of smooth muscle co-culture. Among them, A is a heat map of RNA sequencing of primary endothelial cells, showing the six cytokine-related genes with the highest up-regulated and down-regulated levels between the control group and pECs of calcified bypass arteries; B is a volcano plot showing the gene characteristics of pECs in the control group and calcified bypass arteries; C is immunoblotting to detect the expression levels of MMP10 in pECs of the control group and calcified bypass arteries; D is qPCR to detect the RNA levels of MMP10 in pECs of the control group and calcified bypass arteries; E is enzyme-linked immunosorbent assay (ELISA) to detect the secretion levels of MMP10 in the conditioned media of pECs in the control group and calcified bypass arteries; F is immunofluorescence staining of MMP10 (green) and CD31 (red) in control blood vessels and calcified bypass arteries; G and F are respectively quantitative analysis of the positive cell count in the intimal layer of each section; H is an in vitro stimulation experiment of MMP10, and alizarin red staining shows calcium nodules in pVSMCs directly stimulated by MMP10; I is quantitative analysis of calcified nodules. *P<0.05, ***P<0.001, ****P<0.0001.

[0041] Figure 5 Results graph of the effect of inhibiting the activity function of MMP10 on smooth muscle calcification. Primary endothelial cells of calcified bypass arteries (Isograft pEC) were isolated and co-cultured with primary smooth muscle cells, and primary endothelial cells without surgery (Ctrl pEC) were extracted for control of smooth muscle co-culture. Among them, A is alizarin red staining to detect calcium nodules in pVSMCs treated with DMSO or GM6001 (10 uM); B is direct microscopic observation of the formation of calcium nodules in pVSMCs treated with DMSO or GM6001 (10 uM); C is statistical analysis of alizarin red staining of calcified nodules (Figure A); D-G are Von Kossa staining (D), Masson staining (E), and hematoxylin-eosin staining (F and G) of 8W bypass arteries locally applied with DMSO or MMP10 neutralizing antibody; H is immunofluorescence of the expression of p52 (green) and CD31 (red) in 8W bypass arteries locally applied with DMSO or MMP10 neutralizing antibody; I is immunofluorescence of the expression of MMP10 (green) and CD31 (red) in 8W bypass arteries locally applied with DMSO or MMP10 neutralizing antibody. Specific implementation mode

[0042] The present invention will be further elaborated in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are those without specific conditions, and usually follow conventional conditions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0043] Basic Embodiment 1. Experimental Instruments, Equipment and Reagents The main experimental instruments and equipment are shown in Table 1.

[0044] Table 1

[0045] The main antibodies are shown in Table 2.

[0046] Table 2

[0047] The main reagents are shown in Table 3.

[0048] Table 3

[0049] Configuration of the main reagents: (1) 10×PBS buffer stock solution (pH 7.4): Add 80 g of NaCl, 2 g of KCl, 35.8 g of NaH2PO4·12H2O and 2.7 g of KH2PO4 to 800 mL of deionized water in sequence and stir to dissolve. After preliminary dissolution, adjust the pH value to 7.4, then use a magnetic vortex mixer to stir until completely dissolved, and finally add deionized water to make up the volume to 1 L. It needs to be sterilized when used in cell experiments, and is diluted with deionized water at a ratio of 1:10 to PBS working solution before use.

[0050] (2) PBST: Add 500 μl of Tween-20 to the prepared PBS working solution to obtain a PBST working solution with 0.05% Tween-20.

[0051] (3) Calcification medium: First prepare DMEM complete medium (500 mL of DMEM medium plus 50 mL of fetal bovine serum plus 5.5 mL of triple antibody), and then add 1 M calcium chloride to a final concentration of 3 mM and 100 mM β-glycerophosphate disodium pentahydrate to a final concentration of 10 mM to the DMEM complete medium to form the calcification medium.

[0052] (4) 5×SDS-PAGE Loading Buffer Stock Solution: Add the components in Table 4 into a 50 mL centrifuge tube, dissolve them, make up to 50 mL with deionized water, dispense and store in a -20 °C refrigerator. When using, dilute it with deionized water at a ratio of 1:4 to prepare the 5×SDS-PAGE Loading Buffer Working Solution.

[0053] Table 4

[0054] (5) RIPA Cell Lysis Buffer: Add the components in Table 5 into 1 L of deionized water in sequence to reach the target concentration and content, then adjust the pH value to 7.6 with concentrated hydrochloric acid, store 50 mL each in a 4 °C refrigerator. When using, add protease inhibitor and phosphatase inhibitor to each tube according to the purpose, then dispense into 1 mL each and store in a -20 °C refrigerator.

[0055] Table 5

[0056] (6) 4×Tris-HCl / SDS: pH 8.8: Add 182 g of Tris and 4 g of SDS into 800 mL of deionized water in sequence, adjust the pH to 8.8 with concentrated hydrochloric acid, and then make up to 1 L with deionized water.

[0057] pH 6.8: Add 60.5 g of Tris and 4 g of SDS into 800 mL of deionized water in sequence, adjust the pH to 6.8 with concentrated hydrochloric acid, and then make up to 1 L with deionized water.

[0058] (7) SDS-PAGE Gel: The components are shown in Tables 6 - 7.

[0059] Table 6

[0060] Total volume: 15 mL.

[0061] Table 7

[0062] (8) 10×SDS-PAGE Electrophoresis Buffer Stock Solution: Add the components in Table 8 into 800 mL of deionized water in sequence and make up to 1 L, dissolve with a magnetic vortex mixer and store at room temperature. When using, dilute it with deionized water at a volume ratio of 1:9 to prepare 1×SDS-PAGE electrophoresis buffer.

[0063] Table 8

[0064] (9) 10× Transfer Buffer Stock Solution: Add each component in Table 9 to 800 mL of deionized water in sequence and then make up to 1 L. Dissolve with a magnetic vortex mixer and store at room temperature. For PVDF membranes, dilute the 10× Transfer Buffer Stock Solution, methanol, and deionized water in a volume ratio of 1:1.5:7.5 to prepare the working solution of SDS-PAGE transfer buffer.

[0065] Table 9

[0066] (10) 5% Skim Milk: Weigh 10 g of skim milk powder for experiments and add it to 200 mL of PBS. Stir well to dissolve and use immediately.

[0067] (11) Trizol: First, prepare Solution I by adding 125 g of guanidine isothiocyanate to 146.5 mL of deionized water. Heat and stir at 65 °C on a magnetic vortex mixer until dissolved. After complete dissolution, take 10 mL of Solution I and add and mix the components in Table 10 in sequence (note to store in the dark at 4 °C).

[0068] Table 10

[0069] (12) GM6001 Solution (10 uM): Take 3.89 μg of GM6001 powder and dissolve it in 1 mL of DMSO. Shake well on a vortex mixer until completely dissolved. Wrap it with aluminum foil and sterilize under ultraviolet light, then store at 4 °C for later use.

[0070] (13) GM6001 Applied Gel: ① Prepare the gel: Take 2 g of Pluronic F-127 powder and pour it into pre-cooled sterilized PBS (10 mL). Rotate at low temperature overnight (since Pluronic F-127 powder is difficult to dissolve and will solidify into a colloid when the temperature exceeds 30 °C, it needs to be stored at low temperature). After complete dissolution, perform high-temperature sterilization. ② Add GM6001: Prepare the in-situ application gel one day before arterial bypass surgery. Take 77.8 μg of GM6001 powder and add it to 1 mL of Pluronic F-127 solution. The dissolution process is the same as in step ①, and the final concentration is 200 uM.

[0071] 2. RNA Extraction and qPCR.

[0072] (1)RNA Extraction: The operation needs to be carried out in a laminar flow hood, wearing a mask and gloves, and using nuclease-free tips and EP tubes. First, aspirate the cell culture medium and wash 2 - 3 times with PBS. Then, add 1 mL of Trizol to the culture dish and lyse at room temperature for 5 minutes. Pipette the lysate repeatedly and transfer it to a 1.5 mL nuclease-free EP tube. Next, add 200 μL of chloroform, shake vigorously for 15 seconds, and let it stand at room temperature for 5 minutes. Place the tube in a pre-cooled ultracentrifuge and centrifuge at 14,000 rpm at 4°C for 15 minutes. After taking out the centrifuge tube, it can be seen that the lysate is divided into three layers. The upper layer is the RNA phase, the middle layer is the protein phase, and the lower layer is the DNA phase. Tilt the EP tube at a 45° angle and aspirate 400 μL of the upper liquid into a new EP tube, taking care not to let the tip touch the white protein precipitate in the middle layer. Add 400 μL of isopropanol and mix well, then let it stand at room temperature for 5 minutes, and then centrifuge at 14,000 rpm at 4°C for 15 minutes. A white RNA precipitate can be seen at the bottom of the tube. Discard the supernatant, add 1 mL of 75% ethanol, gently pipette to wash and make the RNA precipitate float, centrifuge at 7,500 rpm at 4°C for 5 minutes, and discard the supernatant. Try to aspirate the residual liquid using a 10 μL pipette tip, and place the EP tube flat on the workbench to open and air-dry the tube wall. Finally, add 50 μL of DEPC water to dissolve the RNA and measure the concentration. Take 1 μg according to the concentration to synthesize cDNA and carry out subsequent experiments. The remaining RNA is stored frozen at -80°C in the refrigerator.

[0073] (2)Reverse Transcription: ① Remove residual DNA, the system is shown in Table 11: Table 11

[0074] The PCR program is shown in Table 12: Table 12

[0075] ② cDNA reverse transcription, the system is shown in Table 13: Table 13

[0076] The reverse transcription program is shown in Table 14: Table 14

[0077] After the program ends, centrifuge at low speed and add 80 μL of DEPC water for dilution. Aspirate the part for subsequent qPCR, and the remaining is aliquoted and stored long-term at -80°C or short-term at -20°C.

[0078] (3)qPCR: The reaction system is shown in Table 15: Table 15

[0079] Primer information is shown in Table 16: Table 16

[0080] 3. Western blot (1)Gel preparation: ① Prepare the SDS-PAGE separating gel according to the formula (prepare separating gels with different concentrations according to the target molecular weight), check the airtightness of the gel preparation rack and then pour it into the gel preparation rack; ② Add 1 mL of isopropanol to remove air bubbles and flatten the liquid surface to avoid uneven separating gel, and then leave it at room temperature for 20 minutes until it solidifies; ③ After the separating gel solidifies, pour out the isopropanol for pressing the gel and rinse it, then prepare the SDS-PAGE stacking gel and fill the glass plate, insert the comb, and let it stand until it solidifies.

[0081] (2)Electrophoresis: ① Carefully remove the comb, firmly clamp the gel plate in the electrophoresis clip and place it in the electrophoresis tank; ② Prepare 1× electrophoresis buffer and pour it into the electrophoresis tank, add the protein sample to be tested and protein marker into the comb holes, and start electrophoresis. Compress the protein sample at a constant voltage of 60 V. When the bromophenol blue loading runs out of the stacking gel plane, adjust the voltage to 120 - 150 V and continue to run through the separating gel.

[0082] (3)Transfer: During the waiting time of electrophoresis, prepare 1× transfer buffer. After electrophoresis is completed, use the wet transfer method to transfer the proteins in the gel to the PVDF membrane pre-activated with methanol at a constant current of 300 mA.

[0083] (4)Blocking: After the transfer is completed, take out the transferred PVDF membrane, mark the position of the marker, soak it in an incubation box containing 5% milk, and block it on a shaker at room temperature for 1 hour.

[0084] (5)Incubate with primary antibody: Dilute the primary antibody according to the appropriate ratio and incubate it overnight in an incubation box at 4℃.

[0085] (6)Incubate with secondary antibody: After the incubation with the primary antibody is completed, wash the membrane on a PBS shaker for 5 minutes, wash it 3 times in total; Dilute the secondary antibody in 5% milk and incubate it at room temperature for 1 hour.

[0086] (7)Developing: After washing the membrane 3 times again, place the membrane in the developing instrument, drop the prepared developing solution (prepare it immediately before use), develop the image, and record the data and take pictures.

[0087] 4. ELISA (1)Sample collection: Replace the primary endothelial cell culture medium with serum-free DMEM. After 48 h, centrifuge at 300 g for 10 min to remove the precipitate, then detect or aliquot immediately, and store the remaining part in a -20℃ refrigerator.

[0088] (2)Reagent Preparation: All reagents are included in the MMP10 Elisa Kit (ab277454, Abcam). ① 1× Wash Buffer: Pipette 50 mL of 20× concentrated wash buffer into a 1 L graduated cylinder, add distilled water to 1000 mL, mix gently to avoid foaming. Transfer to a clean bottle. Store at 4°C in the refrigerator. The 1× wash buffer can be stably stored for 30 days. ② 1× Detection Buffer: Pipette 5 mL of 10× concentrated detection buffer into a 100 mL graduated cylinder, add distilled water to 50 mL, mix gently to avoid foaming. Store at 4°C in the refrigerator. The 1× detection buffer can be stably stored for 30 days. ③ Detection Antibody Working Solution: Mix well before dilution. Dilute the concentrated detection antibody at a ratio of 1:100 with 1× detection buffer according to the number of standards and samples to be tested. ④ Streptavidin Working Solution: Mix well before dilution. Dilute the concentrated streptavidin at a ratio of 1:100 with 1× detection buffer according to the number of standards and samples to be tested.

[0089] (3)Sample Dilution: Dilute the cell culture supernatant with cell culture medium according to the results of the preliminary experiment.

[0090] (4)Preparation of Standard Curve: Centrifuge briefly before opening the cap. Resuspend the standard with distilled water. The resuspension volume is marked on the label of the standard. Vortex gently to ensure thorough mixing. The concentration of the standard after resuspension is 4000 pg / ml. Let it stand for 10 - 30 minutes after resuspension. Mix well before dilution.

[0091] (5)Preparation of Standard Curve for Cell Culture Supernatant Samples: Take 230 μL of the concentrated standard and add 230 μL of cell culture medium to serve as the highest concentration (2,000 pg / mL) of the standard curve. Add 230 μL of cell culture medium to each tube. Perform a 1:1 serial dilution using the high - concentration standard. Ensure thorough mixing each time when pipetting. Use cell culture medium as the zero concentration of the standard curve.

[0092] (6)Detection steps: ① Prepare all required reagents and working concentration standards. ② Remove the unnecessary strips, put them back into the aluminum foil bag with desiccant, and reseal the bag. ③ Immerse the ELISA plate: Add 300 μL of 1× wash buffer and let it stand for 30 seconds. After discarding the wash buffer, pat the microplate dry on absorbent paper. Immediately use the microplate after washing to avoid drying it. ④ Add standards: Add 100 μL of standards diluted 2-fold serially to the standard wells. Add 100 μL of medium to the blank well. ⑤ Cell culture supernatant: Add 100 μL of cell culture supernatant to the sample wells. ⑥ Add detection antibody: Add 50 μL of "detection antibody working solution" to each well (see reagent preparation). Ensure continuous sample addition in steps 4, 5, and 6 without interruption. Complete the sample addition process within 15 minutes. ⑦ Incubation: Seal the plate with a sealing film. Incubate at room temperature for 2 hours with shaking at 100 - 300 rpm / min. ⑧ Washing: Discard the liquid, add 300 μL of wash buffer to each well and wash 6 times. Pat dry on absorbent paper each time. To obtain ideal experimental performance, residual liquid must be completely removed. ⑨ Add enzyme and incubate: Add 100 μL of "streptavidin working solution" to each well (see reagent preparation). ⑩ Incubation: Seal the plate with a new sealing film. Incubate at room temperature for 45 minutes with shaking at 100 - 300 rpm / min. ⑪ Washing: Repeat step ⑧. ⑫ Add substrate for color development: Add 100 μL of chromogenic substrate to each well, protect from light, and incubate at room temperature for 30 minutes. ⑬ Add stop solution: Add 100 μL of stop solution to each well. The color changes from blue to yellow. ⑭ Detection and reading: Within 30 minutes, use an ELISA reader for dual-wavelength detection to measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm. The calibrated OD value is the measured value at 450 nm minus the measured value at 570 nm.

[0093] (7)Result analysis: Calculate the average OD values of the standards and samples, and then subtract the OD value of the zero-concentration standard. Use the standard concentration as the abscissa and the OD value as the ordinate, and perform regression fitting with computer software to generate a standard curve. Regression analysis determines the best-fitting curve.

[0094] 5. Immunohistochemical staining and immunofluorescent staining (1)Thawing of frozen sections: Take the sections and let them return to room temperature for about 20 minutes, gently rinse with tap water for about 3 - 5 minutes to remove OCT compound. This step is not required for cell fluorescence staining.

[0095] (2)Fixation + membrane permeabilization: Fix with 4% paraformaldehyde for 15 minutes, then wash 3 times with PBS, 5 minutes each time. Permeabilize with 0.2% Triton X-100 for 10 minutes, then wash 2 times with PBS and 1 time with PBST, 5 minutes each time.

[0096] (3)Blocking: Block with 5% BSA for 30 min. For tissue staining, circle the tissue with a histochemical pen in advance, then add 5% BSA to cover all the tissue. For cell fluorescence, directly add 1 mL of 5% BSA to each well of the 24-well plate.

[0097] (4)Primary antibody incubation: Wash off the BSA, add the appropriate concentration of primary antibody and incubate overnight (prepared with 5% BSA). The next day, wash off the primary antibody and then wash twice with PBS and once with PBST, 5 min each time.

[0098] (5)Secondary antibody incubation: Prepare the secondary antibody with the appropriate concentration for the corresponding species and incubate for 1 h (prepared with 5% BSA). If it is a fluorescent secondary antibody, it needs to be incubated in the dark.

[0099] (6)Subsequent steps of immunohistochemistry: This step is for immunohistochemistry. Prepare the DAB working solution in advance, add it dropwise and observe the color change under the microscope. When the color turns significantly yellow, add tap water to terminate the reaction, and then wash three times with PBS. Stain with hematoxylin for 5 - 20 s, and adjust according to the tissue condition. Then continuously wash with tap water until the hematoxylin stain is washed off. If the staining is too deep, use hydrochloric acid ethanol for decolorization. Dehydrate with absolute ethanol for 5 min and then dry in an oven at 37°C for 5 - 10 min. Then, dropwise add the mounting medium and cover the slide.

[0100] (7)Subsequent steps of immunofluorescence: Add DAPI (1:2000, diluted with 5% BSA) to stain the cell nuclei for 10 min. After air drying, add 25 - 30 μL of anti-fluorescence quenching agent and cover the slide.

[0101] 6. Co-culture calcium deposition experiment and alizarin red staining with endothelial cell conditioned medium (1)Collection of conditioned medium: When the primary endothelial cells reach 80% confluence, replace the endothelial cell medium with serum-free DMEM medium and continue to culture for 48 h before collection.

[0102] (2)Centrifugation: Centrifuge at 1000 g for 10 minutes, collect the supernatant of the conditioned medium and discard the precipitate. Store it routinely in a -80°C refrigerator.

[0103] (3)Co-culture with conditioned medium: Mix the conditioned medium and the calcification medium in a volume ratio of 1:1 and use it to culture primary smooth muscle cells. Replace the mixed medium every two days.

[0104] (4)Alizarin red staining: At 5 - 7 days of co-culture, remove the mixed medium, wash three times with distilled water (do not use PBS), then fix with 4% paraformaldehyde for 15 min and wash three times with distilled water. Finally, stain each well with 1 mL of alizarin red staining solution for 3 - 5 min and then wash with distilled water.

[0105] 7. Von Kossa staining (1) Rewarming: Take out the sections from the refrigerator, place them at room temperature for 5 min, and then wash off the OCT compound under running water.

[0106] (2) Von Kossa staining: Circle the tissue with an immunohistochemistry pen, then drop the Von Kossa silver solution and irradiate it under strong light for 60 min. Rinse with tap water for 1 min.

[0107] (3) Washing with hypo solution: Drop the hypo solution on the rinsed sections for 2 min.

[0108] (4) Staining the nucleus with nuclear fast red: Drop the nuclear fast red staining solution on the sections and stain for 5 min, then rinse under tap water for 3 min.

[0109] (5) Dehydration and mounting: Dry in an oven at 37 °C (5 - 10 min), then drop neutral resin and mount the sections.

[0110] 8. Experimental animals and breeding All C57BL / 6J mice were purchased and bred in the Experimental Animal Department of Central South University and approved by the Experimental Animal Ethics Committee of Central South University. TRIM35 endothelial-specific knockout mice were provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0111] Primary endothelial cells and primary smooth muscle cells were both extracted from the aortic tissue of the arterial bypass mouse model.

[0112] 9. Statistics Statistical analysis and making statistical charts were performed using R language (version R version 4.1.1). All measurement data were expressed as mean ± standard error (mean ± SEM). When comparing, log-rank test, two-tailed t-test or one-way ANOVA was used, and Bonferroni post hoc test was used. When the p-value was less than 0.05, it was considered to have a statistically significant difference.

[0113] Example 1 MMP10 in plasma can be used to predict the calcification risk and poor prognosis of coronary artery bypass grafting patients. The specific experimental data are shown in Figure 1 .

[0114] The present invention first found through the UK Biobank and the Framingham cohort that plasma MMP10 can predict the calcification risk and poor prognosis of coronary artery bypass grafting patients. The experimental procedure of Example 1 is shown in Figure 1 A. As Figure 1As shown in Figure B, the plasma MMP10 concentration is strongly correlated with the risk factors of poor outcomes in CABG patients. In addition, according to the crude model and the fully adjusted model, MMP10 is positively correlated with CVD mortality and new major adverse cardiovascular events (MACE) ( Figure 1 C), suggesting that higher MMP10 levels tend to predict the occurrence of MACE and CVD death. In addition, the present invention uses receiver operating characteristic (ROC) curve analysis to further evaluate the ability of MMP10 to predict the risk of death, and the results further support our hypothesis ( Figure 1 D-E). All evidence indicates that human plasma MMP10 is related to the incidence of MACE and CVD mortality in CABG patients and can be a powerful predictor of poor prognosis.

[0115] The present invention verified the correlation between plasma MMP10 level and vascular calcification through the Framingham Offspring Study. Thoracic aortic calcification score (TAC score) is considered the most common form of extracoronary artery calcification. In the present invention, data from 7 examinations from 1998 to 2001 were used. Among the participants, those without TAC measurement at follow-up were excluded, and finally 126 participants were included. Multivariate analysis of MMP10 level, gender, BMI, smoking status and blood pressure level was performed using logistic regression. Model 1 included MMP10 level; Model 2 included MMP10 level, gender and BMI; Model 3 included MMP10 level, gender, BMI, current smoking status and blood pressure level. In the analysis, we detected the change in MMP10 level per unit corresponding to each 1 / 1000 unit of TAC. All participants provided informed consent before data collection. Logistic regression analysis showed that high levels of MMP10 were associated with higher TAC scores in CABG patients ( Figure 1 F).

[0116] Example 2 Osteogenic transformation occurs in smooth muscle in calcified bypass arteries. The specific experimental results are shown in Figure 2 .

[0117] The groups were: a control group 2 weeks after bypass without calcification and an experimental group 8 weeks after bypass with calcification. Immunofluorescence staining of the marker proteins BMP2 (red, primary antibody: A14708, ABclonal; secondary antibody: A-21207, Invitrogen) and RUNX2 (red, primary antibody: A11753, ABclonal; secondary antibody: A-21207, Invitrogen) for smooth muscle osteogenic transformation.

[0118] As can be seen from the figure, in the calcified arteries 8 weeks after bypass surgery, the expressions of BMP2 and RUNX2 in the calcified smooth muscle were significantly upregulated.

[0119] This part reveals that after arterial bypass surgery, osteogenic transformation of smooth muscle occurs in the stenotic arteries, as Figure 2 shown in A, the lesion area of the calcified artery after bypass mainly aggregates in the smooth muscle layer. Figure 2 B and Figure 2 C show osteogenic transformation of smooth muscle in the calcified bypass artery through immunofluorescence staining.

[0120] Example 3 Endothelial cells in the calcified bypass artery drive smooth muscle cell calcification, and the experimental results are shown in Figure 3 .

[0121] The grouping was as follows: endothelial cells of the non-calcified bypass artery were used as the control group, and the calcified endothelial cells were used as the experimental group. The control group and experimental group cells were co-cultured in indirect contact with smooth muscle cells, and were respectively induced to calcify with normal DMEM medium and calcification induction medium supplemented with calcium chloride (3 mM calcium chloride and 10 mM glycerophosphate were added to the DMEM medium).

[0122] Figure 3 A and 3B are respectively the direct view and microscopic field view of calcified nodules stained with alizarin red, Figure 3 C is Figure 3 the statistical chart of A. Figure 3 D detected the ALP activity after stimulating SMCs with the calcification medium and control medium using an alkaline phosphatase (ALP) kit (Beyotime). The level of ALP activity represents the ability of smooth muscle cells to undergo osteogenic transformation. The steps for ALP activity determination include sample preparation, preparation of reaction mixture, setting of reaction conditions, enzymatic reaction, measurement of absorbance, and finally calculation of ALP activity. In this experiment, the ALP enzyme in the sample converts the substrate into a product, and the ALP activity is quantified by its absorbance. The experimental steps were carried out strictly according to the instructions of the kit. At the same time, the transcription ( Figure 3 E) and protein expression levels ( Figure 3 F) of the markers BMP2 and RUNX2 of the smooth muscle cells undergoing osteogenic transformation were also detected. The experimental results illustrate that the endothelial cells of the calcified artery are the key to inducing osteogenic transformation of smooth muscle cells.

[0123] Example 4 MMP10 secreted by endothelial cells induces smooth muscle cell calcification, and the experimental results are shown in Figure 4 .

[0124] To explore the mechanism by which endothelial cells drive smooth muscle cell calcification, we performed RNA sequencing (RNA-seq) on the endothelial cells of calcified arteries and conducted in-depth analysis. Figure 4 The grouping of A was as follows: Endothelial cells without calcification in the bypass artery were extracted as the control group, and endothelial cells with calcification in the bypass artery were used as the experimental group. Figure 4 A shows the cytokine-related genes with the highest and lowest differential expression. Figure 4 B shows the volcano plot of downregulated and upregulated genes in RNA sequencing. Figure 4 C - 4E respectively verified the transcription, expression, and secretion of MMP10 in endothelial cells with calcification in the bypass artery, and together they showed that the synthesis and secretion of MMP10 by endothelial cells in calcified bypass arteries were significantly upregulated. Figure 4 For F, by performing immunofluorescence staining of the bypass artery, the grouping was as follows: The bypass artery without calcification was used as the control group, and the bypass artery with calcification was used as the experimental group; the endothelial cell marker CD31 (primary antibody: red, 102502, Biolegend; secondary antibody: A - 21209, Invitrogen) and MMP10 (green, primary antibody: A3033, ABclonal; secondary antibody: A - 21206, Invitrogen) were stained respectively. Figure 4 G is Figure 4 The statistical chart of F. Figure 4 F and Figure 4 G verified the increased expression and secretion of MMP10 in calcified arteries through in vivo experiments. This part also verified the exact role of MMP10 protein in inducing smooth muscle cell calcification through in vitro experiments. Figure 4 The grouping of H was as follows: 0, 5, 10, 20 ng / mL of MMP10 active protein was directly added to smooth muscle cells respectively, and alizarin red staining was performed. Figure 4 I is Figure 4 The quantification of H. Figure 4 H and 4I together showed that smooth muscle cells could show calcium salt deposition through direct intervention of MMP10.

[0125] It shows that MMP10 secreted by endothelial cells is a key factor in smooth muscle cell calcification.

[0126] Example 5 Targeted inhibition of MMP10 activity can relieve smooth muscle calcification in bypass arteries, and the experimental results are shown in Figure 5 .

[0127] After determining that endothelial cells induce calcification of bypass artery smooth muscle cells through MMP10 secretion, the present invention uses GM6001 (an MMP10 protein inhibitor) and MMP10 neutralizing antibody to jointly verify the potential targeting strategy of MMP10. By treating the endothelial-smooth muscle cell co-culture model with GM6001, we observed that after inhibiting MMP10 activity, calcium salt deposition in smooth muscle cells was significantly reduced ( Figure 5 A-C). In the mouse bypass artery model, in-situ administration of MMP10 neutralizing antibody on the bypass artery also significantly reduced calcification and collagen deposition ( Figure 5 D-E). HE staining showed that after inhibiting MMP10, the thickened neointima was significantly reduced ( Figure 5 F-G). Finally, the activation of the non-canonical NF-κB pathway and the expression of MMP10 after stimulation with MMP10 neutralizing antibody were detected. There was no significant difference in the accumulation of p52 and MMP10 ( Figure 5 H-I), indicating that the alleviation of vascular calcification is indeed driven by blocking the biological function of MMP10 rather than its expression, or activation of the non-canonical NF-κB pathway.

[0128] The above results demonstrate that targeting MMP10 is a novel therapeutic method for protecting bypass artery blood vessels and preventing vascular calcification.

Claims

1. Application of MMP10 inhibitors in the preparation of products for preventing and treating vascular calcification and diseases caused by vascular calcification.

2. The use according to claim 1, characterized in that: The vascular calcification is characterized by up-regulated expression of osteoblast markers and down-regulated expression of contractile markers. The osteoblast markers include Runx2, BMP2, SP7, MSX2, osteopontin, osteocalcin and alkaline phosphatase; the contractile markers include α-SMA, SMM-HC, VIMENTIN and SM22α.

3. The use according to claim 1, characterized in that: The diseases caused by vascular calcification include atherosclerosis, cardiovascular disease, hypertension, chronic kidney disease or diabetic vascular disease.

4. The use according to any one of claims 1 to 3, characterized in that: The mechanism of action of the product includes: inhibiting the upregulation of osteoblast markers Runx2, BMP2 and alkaline phosphatase; improving calcium salt deposition and collagen deposition.

5. The use according to claim 1, characterized in that: The inhibitor is used to inhibit the biological activity of MMP10, or inhibit the expression of MMP10.

6. The use according to claim 5, characterized in that: The MMP10 inhibitor comprises at least one of the following: (1) Chemical synthesis inhibitors; (2) Plant extracts; (3) Antibiotics.

7. Use of genetic engineering products that inhibit the expression of MMP10 in the preparation of products for preventing and treating vascular calcification and diseases caused by vascular calcification.

8. The use according to claim 7, characterized in that: The genetic engineering product includes a vector carrying siRNA and / or miRNA that inhibits the expression of MMP10.

9. A drug for preventing and treating vascular calcification and diseases caused by vascular calcification, characterized in that: It comprises active ingredients and excipients, wherein the active ingredients are selected from any one or more of the following: (1) MMP10 inhibitors; (2) Genetically engineered products that inhibit the expression of MMP10.

10. Application of MMP10 inhibitors in the preparation of therapeutic drugs after arterial bypass surgery.

Images