Si-RNA composition for inducing cardiac fibroblasts to transdifferentiate into endothelial cells and application of si-RNA composition

Abstract

The invention relates to the field of biological medicine, in particular to a si-RNA composition for inducing cardiac fibroblasts to transdifferentiate into endothelial cells and application of the si-RNA composition. The si-RNA composition comprises a si-RNA (Ribonucleic Acid) aiming at a gene Huwe1, a si-RNA aiming at a gene Ltbp3, a si-RNA aiming at a gene Dysf and a si-RNA aiming at a gene Hivep3. The HLDH (Huwe1, Ltbp3, Dysf and Hive3) combined knockout can be used for successfully inducing the transformation of fibroblasts into endothelial cells in an in-vitro ischemia and hypoxia state. A carrier containing shRNA capable of expressing and generating the si-RNA composition is delivered into the heart, so that cardiac fibroblasts undergo transdifferentiation in vivo and participate in angiogenesis, and therefore, increase of fibrosis area and adverse ventricular remodeling after myocardial ischemia are limited through ways such as fibrosis remodeling, the heart function is retained, and myocardial ischemia is improved and repaired.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and in particular to a si-RNA composition for inducing the transdifferentiation of cardiac fibroblasts into endothelial cells and its application. Background Technology

[0002] Myocardial ischemia, especially myocardial infarction (MI), is a major cause of heart failure (HF) and cardiovascular death. As myocardial ischemia worsens, the heart structure gradually changes, including left ventricular dilation, thinning of the ventricular wall, and increased myocardial fibrosis, leading to a gradual decline in cardiac function. When blood flow to the myocardium is blocked, part of the heart is in a state of ischemia and hypoxia, causing excessive activation of cardiac fibroblasts (CFs), which transform into myofibroblasts, promoting myocardial fibrosis and further affecting normal cardiac function, potentially leading to heart failure. While timely interventional treatment can restore coronary blood flow after coronary artery occlusion, the loss of coronary blood flow reserve is difficult to correct. Furthermore, coronary microvascular dysfunction exists throughout the heart, not just in the myocardium supplied by the narrowed vessel; therefore, coronary microvascular sparseness is significantly correlated with patient prognosis. Therefore, how to effectively improve vascular density after myocardial ischemia and promote cardiac repair has become one of the current hot topics in cardiovascular disease (CVD) research.

[0003] Angiogenesis is closely related to endothelial cell regeneration. Traditionally, it was believed that angiogenesis primarily depended on the proliferation and migration of existing endothelial cells (ECs). However, increasing evidence suggests that cardiac fibroblasts, after myocardial ischemia-injury, not only play a role in fibrosis but also participate in cardiac repair processes by partially transdifferentiating into endothelial cells. Therefore, the transdifferentiation of cardiac fibroblasts into endothelial cells to promote angiogenesis has emerged as a potential cardiac repair strategy.

[0004] Recent research has advanced the study of different methods and mechanisms of reprogramming to improve the generation of endothelial cells and the acquisition of vascular tissue function, and to strive to create methods that can be translated into clinical applications. These include the transdifferentiation of iPSCs (induced pluripotent stem cells) or mesenchymal stem cells (MSCs), including adipose-derived mesenchymal stem cells, into endothelial cells. While iPSCs or MSCs have higher transdifferentiation efficiency, stem cell-derived endothelial cells are prone to excessive proliferation in vivo, forming mutant tumors. The potential risk of tumor formation hinders their further development and in vivo application. Therefore, directly reprogramming somatic cells into endothelial cells is, to some extent, safer. In methods for reprogramming fibroblasts into endothelial cells, the SOX17 / ETV2 combination or ER71 / ETV2 can directly reprogram dermal fibroblasts into reprogrammed endothelial cells. Regarding angiogenesis, delivering anti-miR-200b to the hind limb injury site in mice increased the contribution of fibroblasts to hind limb perfusion vessels in a FLI1-dependent manner, enhancing tissue perfusion, wound closure, and angiogenesis. Mouse tail tip fibroblasts were simultaneously reprogrammed into cardiomyocyte-like, endothelial, or smooth muscle cell-like structures via microRNA-mimic, miR-208b-3p, ascorbic acid, and bone morphogenetic protein 4, as well as directly reprogrammed cells to form vascular tissue-like structures. These methods primarily involve in vitro cell transdifferentiation followed by transplantation into vivo, but this easily leads to in vivo rejection. This method is difficult to implement in the early stages after myocardial ischemia, and the transdifferentiated endothelial cells are unlikely to participate in angiogenesis, thus failing to achieve the ultimate effect of improving cardiac function. Therefore, directly transdifferentiating fibroblasts into endothelial cells in vivo has better clinical translation prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides an si-RNA composition for inducing the transdifferentiation of cardiac fibroblasts into endothelial cells and its application.

[0006] To achieve the above objectives, the technical solution of this application is as follows: a si-RNA composition for inducing the transdifferentiation of cardiac fibroblasts into endothelial cells, comprising: siRNA targeting the Huwe1 gene; siRNA targeting the Ltbp3 gene; siRNA targeting the Dysf gene; siRNA targeting the Hivep3 gene.

[0007] Preferably, the si-RNA targeting the gene Huwe1 has a sense strand with a sequence as shown in SEQ ID NO: 1 and an antisense strand with a sequence as shown in SEQ ID NO: 2.

[0008] Preferably, the si-RNA targeting gene Ltbp3 has a sense strand with a sequence as shown in SEQ ID NO:3 and an antisense strand with a sequence as shown in SEQ ID NO:4.

[0009] Preferably, the si-RNA targeting the gene Dysf has a sense strand with a sequence as shown in SEQ ID NO: 5 and an antisense strand with a sequence as shown in SEQ ID NO: 6.

[0010] Preferably, the si-RNA targeting gene Ltbp3 has a sense strand with a sequence as shown in SEQ ID NO: 7 and an antisense strand with a sequence as shown in SEQ ID NO: 8.

[0011] The RNA composition described above, which induces cardiac fibroblasts to transdifferentiate into endothelial cells, is used in the preparation of drugs for treating myocardial ischemia.

[0012] A vector comprising shRNA capable of expressing shRNA that generates the si-RNA composition described above for inducing the transdifferentiation of cardiac fibroblasts into endothelial cells.

[0013] Preferably, the vector is an adenovirus carrying a fibroblast-specific promoter.

[0014] Preferably, the fibroblast-specific promoter is Tcf21.

[0015] The carriers described above are used in the preparation of drugs for treating myocardial ischemia.

[0016] This application reveals that HLDH (Huwe1, Ltbp3, Dysf, Hivep3) knockout can successfully induce the transformation of fibroblasts into endothelial cells under in vitro ischemic and hypoxic conditions. Immunofluorescence and Western blotting experiments confirmed that fibroblasts expressed widespread endothelial cell markers such as CD31 and VE-CAD, and exhibited the endothelial cell functional marker eNOS. High-throughput transcriptome sequencing revealed that transcriptome analysis of induced endothelial cells showed enrichment of endothelial cell-related focal adhesion, tight junction, and gap junction functional pathways.

[0017] Furthermore, this application provides a vector coated with 4in1 shRNA (sh-Huwe1, sh-Ltbp3, sh-Dysf, sh-Hivep3), which is an adenovirus vector (AAV9-shHLDH) carrying the fibroblast-specific promoter Tcf21. This vector is delivered into the heart, allowing cardiac fibroblasts to undergo transdifferentiation and participate in angiogenesis in vivo. This limits the increase in fibrotic area and adverse ventricular remodeling after myocardial ischemia through fibrotic remodeling and other pathways, while preserving cardiac function. Experimental results show that HLDH knockout significantly increases blood perfusion in the peri-infarct area of ​​mice, indicating that it can promote vascular support for surviving myocardium, limit scar formation and cardiac fibrosis in the late repair stage after myocardial ischemia, and slow the progression of heart failure. In animal experiments, the drug was administered immediately after myocardial ischemia surgery, and in small animals, it was confirmed that AAV9-shHLDH delivered into the heart after myocardial ischemia can improve myocardial ischemia repair through angiogenesis and fibrotic remodeling. This method may be applicable to the simultaneous administration of AAV9-shHLDH after interventional procedures following myocardial infarction, providing possibilities for further clinical translation. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0019] Figure 1 shows the experimental results of Huwe1, Ltbp3, Dysf, and Hivep3 in si-RNA knockdown mice: (A) Flowchart of the induction of cardiac fibroblast transdifferentiation into endothelial cells; (B) qRT-PCR detection of the mRNA expression of Huwe1, Ltbp3, Dysf, and Hivep3 in the knockdown group relative to the CON group, n=4, t test; CON: fibroblast transfected with negative control si-RNA group; si-HLDH: transfected with si-Huwe1, si-Ltbp3, si-Dysf, and si-Hivep3 group; Figure 2 shows the results of endothelial cell marker expression on the cell membrane of cardiac fibroblasts after induction: (A) Immunofluorescence representative image showing the expression of endothelial cell markers CD31 and VE-CAD after fibroblast induction into endothelial cells, scale bar = 50 µm; (B) Proportion of cells expressing endothelial cell markers, n = 6, t test, CON: fibroblasts transfected with negative control si-RNA group; si-HLDH: transfected with si-Huwe1, si-Ltbp3, si-Dysf, si-Hivep3 group; Figure 3 shows the results of increased expression of endothelial markers and decreased expression of fibroblast markers in induced endothelial cells: (A) Representative immunoblot images of endothelial cell marker expression in CF and iEC: induced endothelial cell markers VE-CAD and CD31 expression increased, while fibroblast marker VIMENTIN expression decreased; (B) Quantitative analysis of Western blot results using ImageJ. n=3, data are presented as mean ± standard deviation, t-test; (C) Increased expression of endothelial cell functional marker eNOS after induction, n=3; (D) Quantitative analysis of Western blot results using ImageJ, n=3, t-test, VE-CAD: Ve-cadherin; Figure 4 shows the transcript analysis results of induced endothelial cells: (A) Volcano plot of 6001 differentially expressed genes with P<0.05 and log2FC>1; (B) KEGG pathway analysis showing the enrichment of differentially expressed genes upregulated in iEC samples compared to CF samples. Figure 5 shows the construction and validation results of fibroblast lineage tracer mice: (A) Schematic diagram showing the strategy for constructing the Col1a2DreERT:H11-RSRtdTomato allele; (B) Genotyping of the tail of Col1a2DreERT:H11-RSRtdTomato mice; (C) Flowchart of the induction protocol and validation process for Col1a2DreERT:H11-RSRtdTomato mice using tomoxifen; (D) Fluorescence microscopy image showing Tdtomato in a cross-section of the heart, scale bar = 10 μm, ip: intraperitoneal injection. Figure 6 shows the results of lineage tracing verification of HLDH promoting the transdifferentiation of cardiac fibroblasts into endothelial cells in mice: (A) Flowchart of animal experiments; (B) Immunofluorescence results of heart sections of fibroblast lineage tracing mice 8 weeks after I / R and injection of AAV9-shHLDHh or AAV-blank, white arrows show the co-localization of fibroblasts and endothelial cell marker vWF in the periphery of myocardial infarction, and CD31 and tdTomato double-positive cells appear yellow, scale bar = 10 μm; (C) Representative image of tdTomato++ + vWF+ cells magnified; (D) Representative image of tdTomato++ + CD31+ cells magnified, labeled as above; (E) Proportion of tdTomato++ + vWF+ double-positive cells and tdTomato++ + CD31+ double-positive cells in random fields of view of the two groups, quantified using ImageJ software, n=6, t test; Figure 7 shows the results of lineage tracing verification of HLDH knockdown promoting angiogenesis in mice: (A) Immunohistochemical results of VE-CAD in the heart section of fibroblast lineage tracing mice 8 weeks after I / R, scale bar = 20 μm; (B) Quantitative analysis results of VE-CAD positive cells using ImageJ software, n = 6, t test; (C) Example image of short-axis M-mode ultrasound of mouse heart; (D) Statistical graph of cardiac ultrasound of fibroblast lineage tracing mice 8 weeks after I / R, n = 6, t test; EjectionFraction: ejection fraction; Fractional Shortening: shortening fraction; Baseline: baseline echocardiographic data one day before I / R surgery; Figure 8 shows the cardiac function results in mice after AAV9-shHLDH delivery to preserve myocardial ischemia: (A) Schematic diagram of the animal experiment process; (B) Line graphs of ejection fraction, a cardiac function index in mice 1 day before I / R surgery, and 1 day, 1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after surgery, n=10; (C) Line graphs of cardiac short-axis shortening rate in mice 1 day before I / R surgery, and 1 day, 1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after surgery, n=10, Two-way ANOVA test. This indicates that P < 0.05. P < 0.01 indicates a statistical effect; (D) I / R: heart weight / body weight ratio and heart weight / tibia length ratio in mice 12 weeks post-surgery, n = 10, t-test, HW: heart weight; TL: tibia length; BW: body weight; Figure 9 shows the long-term blood perfusion results of HLDH knockdown promoting myocardial ischemia in mice: (A) Laser microcirculation blood flow imaging shows the representative map of the average blood perfusion in the precordial region before myocardial ischemia surgery and 12 weeks after myocardial ischemia 30 min reperfusion. The color bar shows the degree of blood perfusion, with red representing vigorous blood perfusion and blue representing poor blood perfusion; (B) Statistical graph of the difference in the ratio of average blood perfusion in the ROI region to the baseline; (C) Representative map and statistical results of VE-CAD immunohistochemistry of the heart section of C57BL / 6J mice 12 weeks after I / R, scale bar = 20 μm, n = 10, t test; Figure 10 shows the results of HLDH knockdown reducing myocardial fibrosis in mice: (A) Representative images of the heart under HE staining at 1x and 40x magnification 10 weeks after myocardial ischemia using the HLDH knockdown strategy and the control group, scale bar = 20 μm; (B) Representative image of heart cross-section stained with Masson's trichrome, red for myocardium and blue for fibrous tissue in the infarct area; (C) Statistical analysis of myocardial infarction area 10 weeks after myocardial ischemia using the HLDH knockdown strategy and the control group, n = 10, t test. Detailed Implementation

[0020] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application. The terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0022] The antibody reagents used in the following examples are sourced from the sources shown in Table 1.

[0023] Table 1 List of antibody reagents used in the experiment The culture medium preparation process used in the following examples is as follows: Fibroblast culture medium: Add 50 ml of FBS to every 450 ml of basal DMEM medium to prepare DMEM complete medium containing 10% FBS. Shake thoroughly until air bubbles disappear, then aliquot into 50 ml centrifuge tubes. Seal with sealing film and store at 4°C. Before use, preheat in a 37°C water bath for 30 min. Add GlutaMAX™ supplement to a final concentration of 2 mM two weeks after opening.

[0024] Endothelial cell culture medium: Aliquot the complete culture medium into appropriate containers and store at -80°C. Before each use, add 2.5 ml FBS and 500 µl endothelial cell growth supplement (ECGS) to every 47 ml of basal ECM medium to prepare a complete ECM medium containing 5% FBS + 1% ECGS. Seal the container with sealing film and store at 4°C. Preheat the container to 37°C in a water bath 30 min before use. Add GlutaMAX™ supplement two weeks after opening the medium to a final concentration of 2 mM.

[0025] Low serum medium: Aliquot the complete medium components appropriately and store at -80°C. For each use, add 1 ml FBS and 500 µl ECGS to every 48.5 ml of basal ECM medium to prepare a complete ECM medium containing 2% FBS + 1% ECGS. Seal with sealing film and store at 4°C. Preheat in a 37°C water bath for 30 min before use. Add GlutaMAX™ supplement to a final concentration of 2 mM two weeks after opening.

[0026] The experimental animals used in the following examples were obtained from the following sources: a) C57BL / 6J wild-type mice (1 day / 8 weeks old, SPF grade) were purchased from Huafukang Biotechnology Co., Ltd.

[0027] b) H11-CAG-RSR-tdTomato gene mice (6 weeks old, SPF grade) were purchased from Shanghai Southern Model Biotechnology Co., Ltd. (Beijing).

[0028] c) Col1a2-2A-DreERT2 gene-infected mice (6 weeks old, SPF grade) were purchased from Shanghai Southern Model Biotechnology Co., Ltd. (Beijing).

[0029] The process of creating the animal model of myocardial ischemia-reperfusion used in the following examples is as follows: Ischemia / Reperfusion (I / R) model: Surgery was performed under isoflurane inhalation anesthesia. Male C57BL / 6J mice were anesthetized. After ensuring complete anesthesia, the hair on the chest and underarms was shaved to expose the surgical area. The ventilator was started and relevant parameters were adjusted. The mouse's tongue was gently pulled out using ophthalmic forceps, and the glottis, a bright spot changing with respiration, was located and observed under cold light illumination. At the moment the glottis opened, a straight 26G intravenous catheter was inserted into the trachea and connected to the ventilator. The frequency of chest wall movement was observed to match the ventilator's set frequency. Next, the skin was cut approximately 0.5 cm from the left forelimb, and the thoracic cavity was incised between the third and fourth ribs approximately at the anterior axillary line to expose the heart and locate the left anterior descending (LAD) coronary artery. The LAD is located approximately 5 mm to the left of the pulmonary conus, appearing as a thin, superficial pink line running downwards and disappearing into the lower middle part of the left anterior wall of the heart. After accurately locating the left atrial appendage (LAD), a needle was inserted 1 mm below the lower border of the left atrial appendage to a depth of approximately 0.5 mm, passing through the left LAD and entering the vicinity of the pulmonary conus. Following puncture, the left anterior descending artery (LAD) was ligated. The color change of the left ventricular anterior wall myocardium after ligation was observed, changing from red to pale. Additionally, ischemic pallor of the skin in the upper and lower limbs could be used as a supplementary indicator. After 30 minutes of ischemic treatment, the ligation was loosened, and the thoracic cavity was closed layer by layer. Once the mice regained spontaneous breathing, the endotracheal tube was removed. The sham-operated group received the same anesthesia and surgical procedures, along with the same nursing care, but without ligation of the left anterior descending coronary artery.

[0030] Example 1: In vitro induction of mouse cardiac fibroblasts to transdifferentiate into endothelial cells (1) Using siRNA combination to knock down HLDH gene in vitro Small interfering RNAs (si-RNAs) of four genes, Huwe1, Ltbp3, Dysf, and Hivep3 (HLDH), were constructed: si-Huwe1, si-Ltbp3, si-Dysf, and si-Hivep3, as well as a negative control (sequences are shown in Table 2).

[0031] Table 2 si-RNA sequences C57BL / 6J mice were used, and cardiac fibroblasts from suckling mice within one day of birth were isolated and cultured. When the P0 generation cardiac fibroblast density reached 90%, CD31 magnetic beads were used to remove contaminating endothelial cells to further purify the fibroblasts and eliminate the influence of existing endothelial cells on the experimental results. When the cell density reached approximately 60%, 25 nM si-Huwe1 and 25 nM si-Ltbp3 were simultaneously transfected into the fibroblasts via liposomes. Twelve h later, 25 nM si-Dysf and 25 nM si-Hivep3 were simultaneously transfected into the fibroblasts, and the culture medium was changed. Forty-eight h after transfection, a second transfection was performed following the same procedure, creating the experimental group (si-HLDH group). The control group (si-RNA negative control, CON group) was transfected into fibroblasts with 50 nM si-nc each time, and the culture medium was changed every other day. To verify the knockdown efficiency, RNA was extracted from cardiac fibroblasts in each group 4 days after the last transfection and reverse transcribed. The knockdown efficiency of the target genes was then detected using qRT-PCR. The results showed that compared to the CON group, the mRNA levels of four genes—Huwe1, Ltbp3, Dysf, and Hivep3—were significantly reduced in the si-HLDH group. Specifically, the relative expression levels of Huwe1 were 0.21 ± 0.03, Ltbp3 was 0.21 ± 0.05, Dysf was 0.35 ± 0.04, and Hivep3 was 0.24 ± 0.05 (Mean ± SEM). Figure 1 B).

[0032] The above results demonstrate that this siRNA knockdown strategy can effectively achieve the knockdown objective. Therefore, subsequent experiments will use the above-mentioned siRNA combination transfection method to transfect mouse fibroblasts. Following transfection, the culture medium will be replaced with low-serum ECM medium containing 2% FBS, and the cells will be cultured in an incubator containing 1% O2. Seven days after induction, successfully induced endothelial cells will be sorted using CD31 magnetic beads and expanded for culture. In vitro, fibroblasts will be induced to transdifferentiate into endothelial cells. Figure 1 A) (1) After inducing transdifferentiation of neonatal rat cardiac fibroblasts into endothelial cells in vitro, the success rate of transdifferentiation was verified using Western blot, immunofluorescence, and RNA-seq. CD31 and VE-Cadherin are extensive markers of endothelial cells, mediating their adhesion function. After expanding the culture of induced endothelial cells, the cells were fixed with 4% paraformaldehyde solution, and the expression and localization of endothelial cell markers in each group were detected by immunofluorescence assay. The expression of CD31 and E-CAD was observed in fibroblasts. Combined with the results of nuclear staining, clusters of induced endothelial cells were found after transdifferentiation, indicating that an active monoclonal population of induced endothelial cells may have emerged. Figure 2 A, B).

[0033] (2) To further investigate the differences between endothelial cells induced by fibroblasts and primitive endothelial cells, induced endothelial cells were sorted using CD31 magnetic beads after induction and expanded into 24-well plates pre-coated with 0.1% Gelatin using ECM medium containing 5% FBS. After the emergence of a cell clonal population, the cells were digested in situ with 0.25% trypsin and replanted into larger culture dishes. After 1-2 passages, the cells were collected for subsequent experiments. CFs that were passaged and had their medium changed but not induced were used as a control group. Cell proteins were collected 10 to 14 days after iEC expansion culture. Western blot showed that in vitro induced fibroblasts expressed the widely expressed endothelial cell marker proteins CD31 and Ve-Cadherin, while the expression of the fibroblast marker VIMENTIN was decreased. Figure 3 A, B). The functional endothelial cell marker eNOS was also detected, and the results showed that eNOS expression was increased in iECs compared to CFs, indicating that the induced endothelial cells possessed certain endothelial cell functions. Figure 3 C, D).

[0034] (3) RNA-seq was used to investigate the transcriptome expression of induced endothelial cells derived from fibroblast reprogramming. After obtaining read count data, DESeq2 was used to analyze the differential gene expression between the experimental group (iEC) and the control group (CF). The transcriptome expression patterns of iEC and CF were investigated. Volcano plots were used to show the distribution and upregulation of differentially expressed genes (DEGs) in the two groups. Using a p-value less than 0.05 and a log2 (foldchange) greater than 1 as the criteria for screening differentially expressed genes, iEC had 2095 upregulated genes and 3906 downregulated genes compared to CF. Among these, endothelial cell and fibroblast-specific genes were the focus. Mouse cardiac endothelial cell markers and mouse cardiac fibroblast markers from the CellMarker website were used to explore the transcriptome differences between the two groups. The results showed that, compared with the control CF, iECs exhibited increased expression of endothelial cell-specific genes Flt1, Fapp4, and Psrc1, while the expression of fibroblast-specific genes Col1a2 and Co3a1 was decreased. Figure 4 A).

[0035] (4) To investigate whether induced endothelial cells acquire endothelial cell fate and function, the Top 20 significantly enriched KEGG pathway entries were presented. Figure 4 B): Activation and upregulation of pathways such as focal adhesion, ECM-receptor pathway, tight junctions, and gap junctions are observed in the iEC, all of which are related to endothelial cell function. Focal adhesion is the connection structure between cells and the ECM. When ECs are exposed to blood flow, integrins sense changes in tension and transmit them to the cytoskeleton through focal adhesion. Tight junctions and gap junctions are both related to the adhesion and migration functions of endothelial cells.

[0036] The above data indicate that knocking down HLDH of four genes successfully induced the transdifferentiation of cardiac fibroblasts into endothelial cells under simulated ischemic conditions in vitro.

[0037] Example 2: HLDH-induced transdifferentiation of mouse in vivo cardiac fibroblasts into endothelial cells (1) Tamoxifen-induced fibroblast lineage tracer mice: H11-CAG-RSR-tdTomato and Col1a2-2A-DreERT2 genetically engineered mice were crossed, and F0 generation Col1a2DreERT:H11-RSRtdTomato mice were obtained after genotyping. Figure 5A). The obtained F0 generation mice were self-crossed for extended breeding. The genotypes of the offspring mice were determined by genotyping. Mice carrying the Col1a2DreERT gene and the homozygous H11-RSRtdTomato gene were used for subsequent experiments. Figure 5 B). After mice reached a weight of 20 g, they were induced to develop Dre enzyme by intraperitoneal injection of 75 mg / kg tamoxifen for 5 consecutive days. Subsequent experiments were conducted one week after induction. Figure 5 C). To verify the successful construction of the gene-modified mouse model, tissue samples were collected one week after induction, embedded in OCT, and prepared into frozen sections. To determine whether Col1a2DreERT:H11-RSRtdTomato mice could spontaneously express the red fluorescent protein TdTomato in fibroblasts in the presence of the Dre enzyme initiated by the tissue-specific promoter, successful TdTomato expression was observed directly under a fluorescence microscope after OCT embedding and frozen section preparation, without staining. Figure 5 D).

[0038] (2) HLDH-induced fibroblast transdifferentiation and participation in vascular composition: To perform in situ knockdown of the Huwe1, Ltbp3, Dysf, and Hivep3 genomes in cardiac fibroblasts, AAV-9 (AAV9-Tcf21-shHLDH) coated with 4in1 shRNA (sh-Huwe1, sh-Ltbp3, sh-Dysf, sh-Hivep3) and carrying the fibroblast-specific promoter Tcf21 was constructed. AAV-9 without the promoter Tcf21 was injected intramyocardially to achieve viral infection. During surgery, the left anterior descending coronary artery was ligated with a 7-0 suture needle under ventilator support. The experimental group (AAV-shHLDH group) received multiple injections of 20 μl AAV9 at the ischemic white border area of ​​the myocardium using a Halmilton 25 μl micro-injection needle, while the control group received AAV9-blank. The ligation was loosened after 30 min to restore myocardial perfusion. Cardiac function was monitored by echocardiography 1 day before surgery, 1 day, 7 days, 14 days after surgery, and 4 and 8 weeks after surgery. Heart samples were collected 8 weeks later. Figure 6 A).

[0039] Among them, shRNA is designed as si-RNA targeting the sequences shown in Table 2, and its sequence is shown in Table 3.

[0040] Table 3 shRNA sequences To better visualize colocalization, heart tissue was collected and frozen sections were prepared. tdTomato and endothelial cell markers CD31, vWF, and the nucleus were labeled using immunofluorescence to examine the colocalization of the fibroblast lineage marker tdTomato and the endothelial cell markers. Immunofluorescence results showed that tdTomato colocalized with endothelial cell markers CD31 and vWF in the AAV9-delivered shRNA knockdown group (also known as the AAV-shHLDH group). Figure 6 B), the AAV-shHLDH group showed a significant increase compared to the AAV9-blank group, with an increase of 4.53 ± 0.51% in the proportion of CD31++Tdtomato+ cells and an increase of 4.52 ± 0.85% in the proportion of vWF++Tdtomato+ cells. Figure 6 E); Figures 6 C and D show magnified CD31+tdtomato colocalization and vWF+Tdtomato colocalization in the AAV-shHLDH group, where endothelial cell markers expressed by fibroblast lineage cells can also be observed in the vessel wall. Figure 6 (D) indicates that HLDH knockout may, to some extent, promote the transdifferentiation of fibroblasts into endothelial cells in vivo and participate in angiogenesis.

[0041] (3) HLDH promotes angiogenesis: To further clarify the effect of shRNA delivered in situ via AAV9 on vascular implantation, the vascular area and cardiac function after myocardial ischemia were examined. It was observed that, compared with the AAV-blank group, the percentage of vascular area in the myocardial infarction border region after AAV9-shHLDH injection was significantly increased. After normalization by the proportion of VE-CAD positive immunohistochemical cells occupying nucleated cells, the increase in vascularity in the AAV9-shHLDH group was found to be statistically significant. Figure 7 A, B). Furthermore, myocardial infarction increases ventricular wall pressure and ventricular compliance, which may lead to cardiac systolic dysfunction. Therefore, I also characterized the cardiac function of the tracer mice. The cardiac function of both groups of mice was significantly lower than the baseline before I / R surgery, but the decline was significantly less in the AAV9-shHLDH group compared to the AAV-blank group, with a relatively preserved ejection fraction of (6.250 ± 2.797)% and a relatively preserved shortening fraction of (3.502 ± 1.354)% compared to the AAV-blank group. This is consistent with the results of fibroblast transdifferentiation into endothelial cells and increased vascular area in the heart of the AAV9-shHLDH group. Figure 7 D).

[0042] (4) Preservation of long-term cardiac function after myocardial ischemia in HLDH: C57BL / 6J mice underwent cardiac ultrasound examinations 1 day before myocardial ischemia-reperfusion surgery, 1 day after surgery, and at 1, 2, 4, 8, and 12 weeks after surgery. Heart samples were collected from mice at 8 and 12 weeks. Figure 8 A). Cardiac function was monitored by short-axis M-mode echocardiography of mice. Four weeks before viral injection, there was no statistically significant difference in cardiac function between the experimental and control groups; eight and twelve weeks after viral injection, the cardiac function of the knockdown group was significantly preserved compared to the control group. Figure 8 (B, C) suggests that in situ knockdown of HLDH combination in cardiac fibroblasts by AAV9 preserves late cardiac function after myocardial ischemia-reperfusion, which is a long-term protective measure.

[0043] By weighing the heart, body weight, and tibia length of mice, the heart-to-body weight ratio and heart-to-tibia length ratio were calculated. The knockdown group mice showed significantly lower heart-to-body weight ratio and heart-to-tibia length ratio, which was statistically significant. Figure 8 D). Detailed data on cardiac structural and functional parameters after ischemia in mice are shown in Table 4, with significant improvements in central output and left ventricular mass. These results indicate that AAV9-mediated knockdown of the HLDH combination in cardiac fibroblasts preserves late-stage cardiac function after myocardial ischemia-reperfusion and improves the adverse ventricular remodeling and myocardial hypertrophy phenotype.

[0044] Table 4. Effects of HLDH knockdown on cardiac structural and functional parameters in I / R model mice. Note: Left ventricular mass correction value: Heart mass after weight correction; This indicates a statistically significant difference compared to the PBS group (P<0.05).

[0045] (4) HLDH improves blood perfusion and increases angiogenesis after myocardial ischemia: To further investigate whether AAV9-shHLDH treatment is beneficial for the recovery of angiogenesis after myocardial ischemia, laser microcirculation technology was used to detect the macroscopic precordial blood perfusion in live mice after myocardial ischemia. Under anesthesia and respiratory support, the precordial region of the mouse heart was exposed, taking care not to damage the large blood vessels of the anterior chest wall during exposure to avoid excessive blood loss affecting precordial perfusion. After the blood perfusion changes stabilized, data were recorded, and the average perfusion percentage was calculated. The results showed that the average precordial blood perfusion in the AAV9-shHLDH group was significantly higher than that in the AAV-blank group ( Figure 9 (A, B) suggests that AAV9-shHLDH treatment is beneficial for restoring blood perfusion 12 weeks after myocardial ischemia.

[0046] Immunohistochemical staining of paraffin sections with the vascular marker Ve-Cadherin was performed to visualize angiogenesis in the peri-infarction zone. The results showed that angiogenesis in the AAV9-shHLDH group was significantly more vigorous than in the control group, and this was statistically significant. Figure 9 (C, D). These results all validate that in situ injection of AAV9-shHLDH induces the transdifferentiation of cardiac fibroblasts into endothelial cells, increases angiogenesis, preserves cardiac function, and improves adverse ventricular remodeling.

[0047] (5) HLDH reduces myocardial fibrosis and adverse ventricular remodeling: Late ventricular remodeling occurs several weeks after myocardial ischemia, with myocardial cell hypertrophy, apoptosis, and diffuse fibrosis. Myocardial fibrosis leads to decreased ventricular wall stress, and as the disease progresses, left ventricular volume increases with thinning of the left ventricular wall and weakened motility, eventually leading to heart failure. HE staining showed that 12 weeks after viral injection, the knockdown group mice retained significantly thicker ventricular walls compared to the control group mice, with neat myocardial arrangement and significantly reduced inflammatory cell infiltration, indicating that the application of the HLDH strategy reduced the degree of adverse ventricular remodeling in the heart. Figure 10 A). Myocardial ischemia activates cardiac myofibroblasts to secrete extracellular matrix to fill the infarcted area. Therefore, I used the Masson trichrome staining assay to investigate the effect of the HLDH strategy on the area of ​​cardiac fibrosis in multiple transverse sections of the heart. Figure 10 B). The results showed that the cardiac fibrosis area in mice treated with AAV-shHLDH was significantly reduced by approximately 17% compared to the AAV-blank group (B). Figure 10 C). The above results indicate that the HLDH strategy can significantly reduce cardiac fibrosis and adverse ventricular remodeling after myocardial ischemia.

[0048] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A si-RNA composition for inducing the transdifferentiation of cardiac fibroblasts into endothelial cells, characterized in that, include: Targeting genes Huwe1 si-RNA; Targeting genes Ltbp3 si-RNA; Targeting genes Dysf si-RNA; Targeting genes Hivep3 si-RNA.

2. The si-RNA composition for inducing transdifferentiation of cardiac fibroblasts into endothelial cells according to claim 1, characterized in that, The gene Huwe1 The si-RNA has a sense strand with the sequence shown in SEQ ID NO: 1 and an antisense strand with the sequence shown in SEQ ID NO:

2.

3. The si-RNA composition for inducing the transdifferentiation of cardiac fibroblasts into endothelial cells according to claim 1, characterized in that, The gene Ltbp3 The si-RNA has a sense strand with the sequence shown in SEQ ID NO:3 and an antisense strand with the sequence shown in SEQ ID NO:

4.

4. The si-RNA composition for inducing transdifferentiation of cardiac fibroblasts into endothelial cells according to claim 1, characterized in that, The gene Dysf The si-RNA has a sense strand with the sequence shown in SEQ ID NO: 5 and an antisense strand with the sequence shown in SEQ ID NO:

6.

5. The si-RNA composition for inducing transdifferentiation of cardiac fibroblasts into endothelial cells according to claim 1, characterized in that, The gene Ltbp3 The si-RNA has a sense strand with the sequence shown in SEQ ID NO: 7 and an antisense strand with the sequence shown in SEQ ID NO:

8.

6. The si-RNA composition for inducing transdifferentiation of cardiac fibroblasts into endothelial cells as described in any one of claims 1-5, used in the preparation of a medicament for treating myocardial ischemia.

7. A vector comprising shRNA capable of expressing the si-RNA composition for inducing transdifferentiation of cardiac fibroblasts into endothelial cells as described in any one of claims 1-5.

8. The carrier according to claim 7, characterized in that, The vector is an adenovirus carrying a fibroblast-specific promoter.

9. The carrier according to claim 8, characterized in that, The fibroblast-specific promoter is Tcf21 .

10. The carrier as described in claim 7 is used in the preparation of a drug for treating myocardial ischemia.

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