Application of MFAP5 and adeno-associated virus vector carrying MFAP5-siRNA in myocardial injury repair treatment medicine

Abstract

The invention discloses an application of MFAP5 and an adeno-associated virus vector carrying MFAP5-siRNA in myocardial injury repair treatment drugs, and belongs to the field of medical biology, the repair process after myocardial injury can be remarkably promoted by knocking down expression of an MFAP5 gene, after the MFAP5 gene is knocked down, the cardiac function after myocardial infarction can be improved, fibroblast proliferation and migration can be inhibited, and the myocardial injury repair treatment effect can be improved. Further, the myocardial injury repair is realized. Regulation of MFAP5 gene expression is expected to become an effective strategy for relieving heart failure progression after ischemic myocardial injury.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and specifically relates to the application of MFAP5 and adeno-associated virus vectors carrying MFAP5-siRNA in myocardial injury repair and treatment drugs. Background Art

[0002] After myocardial infarction, fibroblasts are the main collagen-producing cells in the damaged myocardium. Fibroblasts exhibit different cell phenotypes in three overlapping phases after myocardial infarction: the inflammatory phase, the proliferative phase, and the repair phase. In the proliferative phase, the number of fibroblasts in the infarct zone increases through the proliferation and migration of fibroblasts from the non-infarcted area, transforming into myofibroblasts expressing α-smooth muscle actin (α-SMA) and synthesizing a large amount of extracellular matrix (ECM) proteins. In the mature stage of repair, myofibroblasts degrade stress fibers containing α-SMA and transform into matrix fibroblasts that maintain scars, which highly express cartilage oligomeric matrix protein and cartilage mucin. Studies have shown that activated fibroblasts play a dual role in the infarcted myocardium. In the proliferative phase, activated myofibroblasts prevent heart rupture and slow down harmful dilatation remodeling by forming an ordered matrix network, increasing the tensile strength of the infarct zone. However, excessive or uncontrolled fibroblast activation can lead to fibrotic remodeling of the infarct edge and distal myocardium, thereby inducing dysfunction and chronic heart failure. Therefore, fibroblasts are regarded as potential targets for the treatment of myocardial infarction. In-depth research on the molecular signals and regulatory mechanisms of their phenotypic transformation is of great significance for the treatment of myocardial infarction or heart failure.

[0003] Currently, there is no specific drug or treatment for fibroblast phenotypic transformation in the clinic that can directly slow down the progression of cardiac fibrosis after myocardial injury. Therefore, there is an urgent need to find a new treatment method to promote the repair of myocardial injury and improve the fibrosis process.

[0004] MFAP5 (Microfibril-associated protein 5), also known as MAGP-2 (Microfibril-associated Glycoprotein 2), belongs to the microfibril-associated protein family (MFAPs). The molecules in this family are a class of small molecule glycoproteins located in the microfibril bead region. They belong to the extracellular matrix proteins and are found to specifically bind to tropoelastin through their carboxyl termini. MFAPs include five subfamily members, namely MFAP1, MFAP2 (also known as MAGP or MAGP1), MFAP3, MFAP4 and MFAP5. They are small in size and located in microfibrils, so they are classified into the same family; as the main component of extracellular matrix glycoproteins, MFAPs regulate microfibril assembly, elastin production and tissue homeostasis; they are expressed in different tissues and have unique structures, thereby mediating unique roles under physiological and pathological conditions, such as participating in osteopenia, neutropenia, lung function, neointimal hyperplasia, fat metabolism and cancer development, revealing the unique roles of different members of MFAP proteins in tissue development.

[0005] MFAP5 is a microfibril-associated protein that binds to fibrillin to affect microfibril function, interacts with the microfibril / elastin network, and mediates cell adhesion through its N-terminal RGD (Arg-Gly-Asp) sequence. MFAP5 can also function as a matricellular protein by reacting with the Notch signaling cascade and can bind to αvβ3 integrin. This suggests that MFAP5 has the potential to alter cell phenotypes by regulating major cell signaling pathways. Many studies have found that it is highly expressed in stromal fibroblasts of various human cancers, and higher expression is associated with enhanced cancer fibrosis, angiogenesis, and chemotherapy resistance. And MFAP5 expression is also increased in fibroblasts under fibrotic conditions, such as idiopathic pulmonary fibrosis and systemic sclerosis-related skin and lung fibrosis. These previous studies suggest that MFAP5 may promote scarring and fibrosis broadly, but the role of MFAP5 in cardiac fibrosis is still unknown. Summary of the invention

[0006] In order to promote the repair of myocardial injury and improve the process of fibrosis, the present invention proposes the use of MFAP5 and an adeno-associated virus vector carrying MFAP5-siRNA in myocardial injury repair therapeutic drugs.

[0007] In the first aspect, the present invention demonstrates through examples that MFAP5 is highly expressed in the peri-infarction region of myocardial infarction with ischemic myocardial injury, indicating that MFAP5 can be used in myocardial injury repair therapeutic drugs. By knocking down the expression of the MFAP5 gene, cardiac function after myocardial infarction can be improved, fibroblast proliferation and migration can be inhibited, and myocardial injury can be repaired.

[0008] In the second aspect, the present invention demonstrates through specific examples that the expression of the MFAP5 gene can be knocked down by an adeno-associated virus vector carrying MFAP5-siRNA, which can be used for the preparation of a drug for the repair and treatment of myocardial injury. Preferably, the adeno-associated virus vector is AAV9 or AAV6. The siRNA is a double-stranded RNA, consisting of two nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0009] Beneficial effects: The present invention can significantly promote the repair process after myocardial injury by knocking down the expression of the MFAP5 gene. After knocking down the MFAP5 gene, the cardiac function after myocardial infarction can be improved, the proliferation and migration of fibroblasts can be inhibited, and the repair of myocardial injury can be achieved. Regulating the expression of the MFAP5 gene is expected to become an effective strategy to alleviate the progression of heart failure after ischemic myocardial injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The expression of MFAP5 in mice one week after myocardial infarction in Example 1; wherein A is the expression of MFAP5 in heart tissue collected from mice one week after myocardial infarction modeling, and B is the RT-PCR result of mRNA in different regions of heart tissue from mice 14 days after myocardial infarction modeling.

[0011] Figure 2 These are the expression data of MFAP5, ɑ-SMA, Col1a1, and Col1a3 during the fibroblast activation process in Example 2.

[0012] Figure 3 These are the expression data of MFAP5, ɑ-SMA, Col1a1, and Col1a3 during fibroblast activation after MFAP5 knockdown in Example 2.

[0013] Figure 4 Schematic diagram of the effect of MFAP5 knockdown on fibroblast proliferation and migration in Example 2; wherein A is the result of fibroblast proliferation after MFAP5 knockdown, and B is the result of fibroblast migration after MFAP5 knockdown.

[0014] Figure 5 Schematic diagram of the results of myocardial damage repair after myocardial infarction in mice after MFAP5 knockdown in Example 3; wherein A is a schematic diagram of the area of ​​repair after myocardial infarction in mice promoted by knocking out MFAP5 in fibroblasts, Figure 5 B in the figure is the percentage of fibrosis area in A to the left ventricular area; Figure 5 C in the figure shows the result of Col1 staining.

[0015] Figure 6These are the cardiac ultrasound results 28 days after surgery in Example 3, wherein A is the left ventricular ejection fraction, and B is the left ventricular fractional shortening. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0017] Example 1

[0018] In this example, myocardial infarction was induced in mice, and the expression of MFAP5 in myocardial cells in the peri-infarction area was measured. The results showed that the expression of MFAP5 in myocardial cells in the peri-infarction area increased.

[0019] The specific experimental process and results are as follows:

[0020] The mice were C57BL / 6JSlac mice purchased from the Slack Experimental Animal Center and were raised in the animal room of the Central Laboratory of Wannan Medical College.

[0021] Surgery group (MI): The mice were subjected to ligation of the left anterior descending coronary artery to induce ischemic myocardial injury (myocardial infarction).

[0022] Sham operation group (sham): mice were not subjected to left anterior descending coronary artery ligation.

[0023] One week after the myocardial infarction model was established in mice, heart tissues were collected (n=3, the number of samples was 3) and proteins were extracted. The results are shown in Figure 1 As shown in A, the expression of MFAP5 in cardiac tissue increased significantly after myocardial infarction. GAPDH in A represents glyceraldehyde-3-phosphate dehydrogenase (as a protein internal reference).

[0024] 14 days after the infarction model was established in mice, samples were taken from different infarction areas (remote zone, RZ; border zone, BZ) to extract mRNA. The results of RT-PCR (reverse transcription polymerase chain reaction) were as follows: Figure 1 As shown in B, the expression of MFAP5 in the RZ and BZ of the surgical group was higher than that in the sham group, and the expression of MFAP5 in the BZ of the surgical group was higher than that in the RZ. The changes in fibroblast phenotypic transformation mainly occurred at the junction of damaged and normal myocardium, that is, the peri-MI area, indicating that the high expression of MFAP5 in the peri-MI area during ischemic myocardial injury may play a role in the repair of myocardial injury.

[0025] Example 2

[0026] This example measured the expression of MFAP5 during the activation process of cardiac fibroblasts in vitro, the activation of fibroblasts after MFAP5 knockdown, and the effect of MFAP5 knockdown on the proliferation and migration of fibroblasts.

[0027] The results showed that the expression of MFAP5 increased during the activation process of cardiac fibroblasts in vitro. MFAP5 could promote fibroblast activation, and knocking down MFAP5 inhibited the proliferation and migration of fibroblasts.

[0028] The specific experimental process and results are as follows:

[0029] 1. Mouse cardiac fibroblast cell lines (MCFSs, purchased from ScienCells) were cultured in vitro and stimulated with TGFβ (transforming growth factor-β, 10ug / ML, HZ-1011, Proteintech) for 0h, 24h, and 48h. RNA was extracted and the expression of MFAP5, myofibroblast marker (ɑ-SMA), and fibrosis-related genes (Col1a1, Col1a3) was detected by RT-PCR. The results are shown in Figure 2 As shown, the research results show that the expression of ɑ-SMA, Col1a1, Col1a3 and MFAP5 is upregulated after TGFβ stimulation, and the expression of MFAP5 is more obvious 48 hours after TGFβ stimulation, which is consistent with the in vivo experimental results (the in vivo experimental results refer to the results of increased expression of MFAP5 after myocardial infarction in Example 1).

[0030] 2. Analysis of transient transfection of MFAP5 siRNA in MCFs After continuous stimulation with TGFβ for 0h, 24h, and 48h, RNA was extracted and the expression of MFAP5, myofibroblast marker (ɑ-SMA), and fibrosis-related genes (Col1a1, Col1a3) was detected by RT-PCR. Figure 3 As shown, the results showed that the expression of ɑ-SMA and Col1a3 was downregulated after MFAP5 siRNA, suggesting that MFAP5 accelerated the activation of fibroblasts.

[0031] 3. Determine the effect of MFAP5 knockdown on fibroblast proliferation and migration.

[0032] MFAP5 siRNA (purchased from Jiman Bio, siRNA consists of two nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2), MCFs were transiently transfected with MFAP5 siRNA, and stimulated with TGFβ for 0h, 24h, and 48h. The results of MTT (tetrazolium colorimetry, a commonly used cell survival rate and proliferation ability detection technology, suitable for various adherent cells and some suspension cells) were as follows Figure 4 As shown in A ( Figure 4 Scramble means reference control group). After knocking down MFAP5, the proliferation of activated MCFs (mouse cardiac fibroblasts) decreased. At the same time, Transwell experiment was performed after TGFβ stimulation for 48 hours. The results are as follows Figure 4 As shown in B, knockdown of MFAP5 reduced the migration of MCFs. The above data indicate that knockdown of MFAP5 inhibits the proliferation and migration of fibroblasts.

[0033] SEQ ID NO.1: 5'-GGUGAACGAUCCCUACA-3';

[0034] SEQ ID NO. 2: 5'-UGUAGAGGGAUCGUUCACC-3'.

[0035] Example 3

[0036] In this example, mice were infected with the MFAP5 knockdown AAV9 vector driven by the Tcf21 promoter, and its repair effect on myocardial injury was determined. The results showed that it can promote the repair of myocardial injury.

[0037] The specific experimental process and results are as follows:

[0038] In this example, mice were C57BL / 6JSlac mice purchased from Slack Experimental Animal Center and raised in the animal room of the Central Laboratory of Wannan Medical College.

[0039] 1. Set up MFAP5 knockdown group and control group

[0040] MFAP5 knockdown group (MFAP5 FBKO ): The Tcf21 promoter-driven MFAP5 knockdown AAV9 vector (AAV9-Tcf21p-mMFAP5-EGFP, the concentration of this expression vector was 1×10 13 μg / mL) (purchased from Jiman Biotechnology) was injected into the mouse through the tail vein, with an injection volume of 500uL, and one injection.

[0041] WT (control group): 500uL of blank group virus solution was injected into the mouse through the tail vein once.

[0042] 2. AAV9-Tcf21p-mMFAP5-EGFP was injected into adult mice through the tail vein to achieve targeted fibroblast MFAP5 knockout. After the administration was completed, the left anterior descending coronary artery was ligated (myocardial infarction model) one week after absorption. The cardiac ultrasound results 28 days after the operation were as follows Figure 6 As shown in the results, it was found that knockdown of MFAP5 in fibroblasts improved cardiac function (EF, FS) after myocardial infarction, EF refers to left ventricular ejection fraction, and FS refers to left ventricular fractional shortening.

[0043] 3. Collect heart specimens 28 days after surgery, make paraffin sections, and use Masson staining to observe the size of myocardial infarction. The results are as follows: Figure 5As shown in Figure A, Masson staining revealed that in the MFAP5 knockdown group, the area of ​​injury after myocardial infarction was reduced and the degree of cardiac fibrosis was alleviated, indicating that knocking out MFAP5 in fibroblasts can promote the repair of mouse heart injury. The vertical axis of Figure B represents the percentage of fibrosis area to left ventricular area.

[0044] 4. Col1 staining (green immunofluorescence staining) was used to observe the collagen morphology and determine the maturity of collagen. The results are as follows: Figure 5 As shown in B, MFAP5 FBKO The collagen morphology of group mice was round. Example 3 showed that MFAP5 promoted cardiac repair by regulating fibroblast phenotypic transformation (fibroblast to myofibroblast transformation) and collagen maturation after cardiac injury.

[0045] Adeno-associated virus (AAV) is an ideal vector for targeting cardiac fibroblasts because of its non-pathogenicity, ability to infect a variety of cells (including dividing and non-dividing cells) and good gene delivery efficiency. The present invention utilizes AAV vectors to carry gene silencing molecules (small interfering RNA, siRNA) to reduce the expression level of fibrosis-related genes (MFAP5). AAV vectors can also be used to deliver anti-fibrotic factors or specific proteins that stimulate fibroblasts to regenerate myocardium to improve the repair effect after myocardial injury. In order to improve the targeting of AAV in fibroblasts, the present invention selects a specific AAV serotype (AAV9), and can also select AAV6, AAV6 and AAV9 to enhance its distribution and infection efficiency in cardiac tissue, thereby increasing the gene therapeutic effect. Through Example 3, it is also illustrated that AAV vectors provide a new approach for the treatment of myocardial fibrosis, which can improve cardiac function after myocardial injury and reduce the risk of heart failure.

[0046] Heart failure is a syndrome caused by myocardial diastolic or systolic dysfunction. Adult myocardial cells are particularly susceptible to dysfunction and cell death due to hypoxia after being damaged (such as myocardial infarction), which eventually leads to the loss of a large number of myocardial cells and causes heart failure. Heart failure is almost the final outcome of all heart diseases in the late stage, which greatly affects the quality of life and health of patients. The present invention shows that knocking down the expression of the MFAP5 gene can significantly promote the repair process after myocardial injury, bringing new hope for the treatment of heart failure. In the present invention, heart failure and related diseases associated with ischemic myocardial injury are not limited to specific types, and cover various known diseases caused by ischemic myocardial injury. Typical examples of diseases include (but are not limited to) stable angina pectoris, unstable angina pectoris, ischemic cardiomyopathy, and asymptomatic coronary heart disease. These diseases may progress to heart failure during ischemic injury. Therefore, regulating the expression of the MFAP5 gene is expected to be an effective strategy to alleviate the progression of heart failure after ischemic myocardial injury.

[0047] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. Application of MFAP5 in therapeutic drugs for myocardial injury repair.

2. The use according to claim 1, characterized in that: By knocking down the expression of the MFAP5 gene, cardiac function after myocardial infarction can be improved, fibroblast proliferation and migration can be inhibited, and myocardial damage can be repaired.

3. Use of an adeno-associated virus vector carrying MFAP5-siRNA in a drug for the treatment of myocardial injury repair, characterized in that: The expression of MFAP5 gene was knocked down by adeno-associated virus vector carrying MFAP5-siRNA.

4. The use according to claim 3, characterized in that: The adeno-associated virus vector is AAV9 or AAV6.

5. The use according to claim 3, characterized in that: The siRNA consists of two nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

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