Application of MTHFD2 in preparation of medicine for preventing and treating restenosis in coronary stent

Abstract

The invention discloses an application of MTHFD2 in preparation of a medicine for preventing and treating restenosis in a coronary stent. The MTHFD2 reduces pathological proliferation and migration of vascular smooth muscle cells, inhibits angiogenesis of neointima, reduces the thickness of neointima and improves the pathological process of vascular remodeling by regulating the level of NADPH, and effectively relieves oxidative stress and inflammatory response of an injured area, thereby achieving the purpose of treating restenosis in a coronary stent. Meanwhile, folic acid is added into the stent to supplement a metabolic substrate, and the function of MTHFD2 can be further enhanced, so that comprehensive repair of vascular injury is realized. The invention aims to explore how MTHFD2 maintains the phenotype of vascular smooth muscle cells by regulating NADPH metabolism, provides a targeted intervention strategy based on combination of MTHFD2 and folic acid, and provides a new technical means for prevention and treatment of restenosis in coronary stents.

Description

Technical Field

[0001] The present invention relates to the application of MTHFD2 in preparing a drug for preventing and treating coronary stent restenosis. The present invention belongs to the field of medical technology. Background Art

[0002] Coronary artery disease (CAD) has become one of the leading causes of death worldwide. Percutaneous coronary intervention (PCI) is one of the main treatments for CAD. While PCI significantly reduces the mortality rate of acute myocardial infarction (AMI), it still has some limitations, the most significant of which is that stent implantation can cause vascular restenosis, leading to new symptoms.

[0003] Coronary in-stent restenosis (ISR) is generally considered to be a stent-related restenosis lesion when coronary angiography reveals in-stent stenosis ≥50%, or new proliferative lesions appear within 5 mm of the stent edge. Despite significant progress in PCI, coronary ISR remains the most common cause of stent failure after PCI. Recent data show that coronary ISR-PCI accounts for 5% to 10% of all current PCI procedures, seriously affecting patients' quality of life and survival rate [GIUSTINO G, COLOMBO A, CAMAJ A, et al. Coronary in-stent restenosis: JACC state-of-the-art review [J]. J Am Coll Cardiol, 2022, 80(4): 348-372.].

[0004] Coronary in-stent restenosis is a major challenge in the treatment of cardiovascular diseases. Its core mechanism is closely related to the phenotypic transition of vascular smooth muscle cells (VSMCs). Under the stimulation of oxidative stress and excessive cytokines, intimal hyperplasia drives VSMCs to transform from a contractile phenotype to a synthetic phenotype with high migration and proliferation capabilities. This process directly leads to the formation of new intimal tissue and hinders the restoration of normal vascular function. Furthermore, the inflammation and oxidative stress associated with this VSMC phenotypic transition further exacerbate the progression of the disease.

[0005] Current research and treatment of vascular remodeling primarily focuses on the development of antiproliferative drugs and stent coating technologies. However, these technologies suffer from significant limitations. First, existing technologies primarily target VSMC proliferation, lacking effective control over phenotypic conversion, a key pathological process. VSMC phenotypic conversion is a core driver of neointimal formation and pathological vascular remodeling. Enhanced migration and proliferation exacerbate VSMC aggregation within lesions, making it difficult to halt lesion progression by simply inhibiting cell proliferation. Second, existing drugs have limited mechanisms of action and fail to target the complex metabolic and signaling networks involved in VSMC phenotypic conversion. For example, inflammatory factors and oxidative stress play a crucial role in VSMC phenotypic conversion, yet existing treatments have limited control over these pathological factors. Furthermore, long-term use of antiproliferative drugs such as paclitaxel and rapamycin can lead to chronic endothelial toxicity and delayed endothelialization, increasing the risk of stent thrombosis and resulting in a recurrence rate of 5%-10% within 5 to 10 years after angioplasty. This limitation is particularly pronounced in diabetic and high-risk patients, severely impacting the long-term efficacy of treatment.

[0006] In summary, current technologies lack the ability to intervene in vascular remodeling through metabolic regulation and phenotypic transformation, and relying solely on antiproliferative drug-coated stents cannot fully address this pathological problem. Therefore, a new treatment strategy is urgently needed.

[0007] Recombinant human methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) is a key enzyme in the folate metabolic pathway, playing an important role in cell proliferation, antioxidant activity, and metabolic regulation. MTHFD2 plays a key role in regulating mitochondrial and cellular redox homeostasis and metabolic adaptation by regulating intracellular NADPH levels. Furthermore, by adding folic acid to the scaffold to supplement metabolic substrates, the function of MTHFD2 can be further enhanced, thereby achieving comprehensive repair of vascular damage. Summary of the Invention

[0008] The purpose of the present invention is to explore how MTHFD2 regulates the phenotype of vascular smooth muscle cells and to propose a targeted intervention strategy based on the combination of MTHFD2 and folic acid to address the limitations of current treatment options.

[0009] In order to achieve the above object, the present invention adopts the following technical means:

[0010] On the one hand, the present invention proposes the use of MTHFD2 in preparing a drug for preventing and treating coronary in-stent restenosis.

[0011] Among them, preferably, the MTHFD2 reduces the pathological proliferation and migration of vascular smooth muscle cells by regulating the level of NADPH, inhibits the proliferation of angiogenic intimal hyperplasia, reduces the thickness of the neointima, improves the pathological process of vascular remodeling, and effectively alleviates the oxidative stress and inflammatory response in the damaged area, thereby achieving the purpose of preventing and treating restenosis in coronary stents.

[0012] On the other hand, the present invention also proposes the use of a combination of MTHFD2 and folic acid in the preparation of a drug for preventing and treating coronary stent restenosis.

[0013] Among them, preferably, MTHFD2 is used in combination with folic acid to further stabilize the microenvironment, reduce neointimal hyperplasia, and not affect the endothelialization process, thereby reducing the risk of intra-stent thrombosis.

[0014] In another aspect, the present invention further provides a pharmaceutical composition for treating coronary in-stent restenosis, wherein the pharmaceutical composition comprises MTHFD2 and folic acid.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention proposes an innovative therapeutic strategy based on the regulatory mechanism of MTHFD2, aiming to achieve comprehensive regulation of vascular remodeling through targeted intervention in the phenotypic transition process of VSMCs. MTHFD2 is a key enzyme in the folate cycle, playing a central role in maintaining intracellular NADPH levels and redox homeostasis. This invention systematically elucidates for the first time the key mechanism by which MTHFD2 regulates phenotypic transition of vascular smooth muscle cells and vascular remodeling through NADPH, and simultaneously enhances its effects by combining it with a folic acid supplementation strategy. The present invention provides a new technical approach for the prevention and treatment of in-stent restenosis in coronary arteries. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A vascular injury model was established in WT mice and the dynamic changes of MTHFD2 expression were observed;

[0018] (A) C57BL / 6J wild-type mice underwent unilateral femoral artery guidewire injury and unilateral carotid artery complete ligation, respectively. Sham surgery was performed on the contralateral side. Postoperatively, the corresponding vascular surgical sites were harvested to verify neointimal thickening. (B) Representative images of hematoxylin and eosin staining and quantification of neointimal area at 3, 7, 14, and 28 days after femoral artery guidewire injury and carotid artery ligation (n=6). (C) Histochemical staining images of MTHFD2, α-SMA, and Collagen III at the vascular surgical sites after femoral artery guidewire injury and carotid artery ligation (scale bar: 500 μm).

[0019] Figure 2 The regulatory role of MTHFD2 in phenotypic switching in primary rat vascular smooth muscle cells;

[0020] (A) Western blot analysis of the protein levels of MTHFD2, α-SMA, SM22α, and Collagen III in VSMCs after PDGF-BB treatment (n=3); (B) Immunofluorescence analysis of the expression and quantification of MTHFD2 (green) and α-SMA (red) in VSMCs after PDGF-BB stimulation, with DAPI staining of cell nuclei (blue) (Scale bar: 500 μm) (n=6); (C) VSMCs were induced with PDGF-BB and treated with siMTHFD2, and the mRNA levels of MTHFD2, Acta2, Tagln, and COL3A1 in the cells were further quantitatively detected by qRT-PCR (n=6); (D) Western blot analysis of the protein levels of MTHFD2, α-SMA, SM22α, and Collagen III in VSMCs induced with PDGF-BB and treated with siMTHFD2 (n=3);

[0021] Figure 3 To construct conditional knockout mice and validate MTHFD2 function in vivo;

[0022] (A) Schematic diagram of the construction of conditional knockout mice; (B) Genetic identification to detect the knockout of MTHFD2 in VSMCs of conditional knockout mice and quantification of MTHFD2 protein expression in VSMCs (n=4); (C) Western blot analysis of MTHFD2, α-SMA, and Collagen III levels in VSMCs of MTHFD2Δ VSMC mice at 0, 3, 7, 14, and 28 days after ligation (n=3); (D) Immunofluorescence analysis of the expression levels and quantification of MTHFD2 (green) and α-SMA (red) in vascular endothelial cells of MTHFD2Δ VSMC mice after ligation, with DAPI staining of cell nuclei (blue) (Scale bar: 500 μm) (n=6);

[0023] Figure 4 To explore and functionally validate the therapeutic effects of targeting MTHFD2;

[0024] Among them, (A) qRT-PCR detection of MTHFD2 ΔVSMCmRNA levels of MTHFD2, Acta2, Tagln, and COL3A1 in primary mouse VSMCs after transfection with an MTHFD2-overexpressing plasmid (n=6); (B) LC / MS analysis of the expression levels of folate, tetrahydrofolate, MTHFD2, MTHFD1, SHMT2, and SHMT1 in WT mice fed a folic acid-treated diet (n=6); (C) Immunofluorescence analysis of the expression levels and quantification of MTHFD2 (green) and α-SMA (red) in blood vessels of WT mice fed a folic acid-treated diet and then ligated 28 days later, with DAPI staining the cell nuclei (blue) (scale bar: 500 μm) (n=6); (D) ΔVSMC Primary VSMCs from mice fed with or without folic acid were cultured in vitro and then stained with JC-1 to detect mitochondrial membrane potential; (E) WT and MTHFD2 ΔVSMC Primary VSMCs from mice fed a folic acid-supplemented diet were uniformly treated with PDGF-BB (20 ng / mL) to induce phenotypic transformation during in vitro culture. After 24 hours, the cells were subjected to a wound wound test. Representative images and quantification are shown (scale bar: 20 μm, n = 6). VSMC proliferation (green) was detected by EdU staining, and cell nuclei were stained with DAPI (n = 6). DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0026] Example 1

[0027] 1. Research Methods

[0028] 1.1 Animal samples

[0029] This study used C57BL / 6J wild-type (WT) mice, MTHFD2 flox / + Mice and SMMHC-Cre ERT2 Mice were provided by Saiye Biotechnology (Guangzhou, China). Male Sprague-Dawley (SD) rats (6–8 weeks old) were provided by the Laboratory Animal Center of the Second Hospital of Harbin Medical University.

[0030] 1.2 Construction of conditional knockout mice

[0031] MTHFD2 flox / flox MTHFD2 flox / +MTHFD2 mice were obtained by hybridization. flox / flox Female mice with SMMHC-Cre ERT2 Male mice mate to produce MTHFD2 flox / + SMMHC-Cre ERT2 These male mice were then exposed to MTHFD2 flox / flox Female mice mate and eventually acquire MTHFD2 flox / flox SMMHC-Cre ERT2 MTHFD2 ΔVSMC , i.e., MTHFD2 smooth muscle cell-directed knockout mice). ΔVSMC Mice were intraperitoneally injected with tamoxifen (100 mg / kg, for 5 consecutive days) and then received corresponding subsequent treatments after an interval of 2 days.

[0032] 1.3 Gene identification

[0033] DNA extraction: take MTHFD2 ΔVSMC Approximately 2 mm of mouse tail tip tissue was digested overnight at 56°C in 200 μL of lysis buffer (containing proteinase K). After centrifugation, the supernatant was removed and an equal volume of isopropanol was added to precipitate DNA. The DNA was washed with 75% ethanol and then dissolved in TE buffer.

[0034] PCR amplification: The primer sequences for MTHFD2-flox gene identification are as follows:

[0035] F:5'-TTTTCAGCCACCGAGACCATCCT-3'

[0036] R:5'-GACAACGGCTTCATTTCTGCAG-3'

[0037] Reaction conditions: 95°C for 5 min; 35 cycles (95°C for 30 s, 60°C for 30 s, 72°C for 45 s); extension at 72°C for 10 min. Expected products: wild-type (436 bp), floxed allele (520 bp). Electrophoresis analysis: 5 μL of PCR product was run on a 1.5% agarose gel (100 V, 30 min) and photographed using a gel imaging system (Bio-Rad).

[0038] 1.4 Establishment of the femoral artery guidewire injury and carotid artery ligation mouse model

[0039] Male mice aged 8-12 weeks were anesthetized with 1.25% Avertin and then underwent surgery. The left femoral artery endothelial cells were removed using a guidewire, or the left common carotid artery was completely ligated using 6-0 silk suture. The contralateral side, with only the extravascular tissue left open and sutured, was then subjected to a sham operation to establish the femoral artery guidewire injury and carotid artery ligation mouse models. Vascular tissue from the surgical group and the contralateral sham-operated group was collected at different time points after surgery.

[0040] 1.5 High folic acid diet

[0041] Customized high-folate mouse diets were provided by Xietong Biotechnology (Jiangsu, China). Mice (6-8 weeks old) were fed a high-folate diet (20 mg / kg) for one month, which contains 10 times more folate than a standard diet (2 mg / kg). The basic diet formula remained unchanged until sacrifice after model establishment.

[0042] 1.6 Cell culture and transfection

[0043] Primary rat VSMCs (rat thoracic aortic smooth muscle cells) were isolated from the thoracic aorta of SD rats, and primary mouse VSMCs were isolated from WT mice or MTHFD2 mice. ΔVSMC VSMCs were isolated from mouse aorta and cultured in high-glucose DMEM (containing 10% FBS and 1% penicillin-streptomycin). siRNAMTHFD2 (siMTHFD2, purchased from Jima Biotechnology (Suzhou, China)) was used to knock down the expression of MTHFD2. VSMCs were cultured in 6-well culture plates until the confluence rate was approximately 30% to 40%. According to the instructions, we mixed siRNA and Lipofectamine TM 2000 reagent (Invitrogen, Carlsbad, CA, USA) was diluted with serum-free DMEM for 5 min at room temperature, and then Lipofectamine TM The mixture of 2000 and siRNA was incubated for 20 min and then added to the cell culture dish. After 6 h, serum was added for detection.

[0044] The siMTHFD2 sequence is:

[0045] F: 5'-GGAUCUUCUUCCAGGUGAUTT-3';

[0046] R: 5'-AUCACCUGGAAGAAGAUCCAG-3'

[0047] To construct an MTHFD2 overexpression model, we cloned the mouse MTHFD2 coding region into the eukaryotic expression vector pcDNA3.1(+) (Thermo Fisher, USA). The MTHFD2 coding sequence was obtained from the NCBI database. We designed the following primers based on the multiple cloning site and incorporated EcoRI and XhoI restriction endonuclease sites at the 5' end for subsequent cloning:

[0048] Forward primer (EcoRI): 5'-GAATTCATGGCGGCGGCGGCGG-3'

[0049] Reverse primer (XhoI): 5'-CTCGAGTCAGTCAGGGGTTGTCCTC-3'

[0050] After PCR amplification using a high-fidelity DNA polymerase, the MTHFD2 fragment and the pcDNA3.1(+) plasmid were digested with EcoRI and XhoI (New England Biolabs, USA), recovered, purified, and ligated using T4 DNA ligase. The ligation product was transformed into competent DH5α Escherichia coli, and positive clones were screened and sequenced to verify sequence accuracy. Correctly cloned plasmids were large-scale extracted using the EndoFree Plasmid Kit (Qiagen, Germany) and used for cell transfection experiments.

[0051] VSMCs were placed in 6-well culture plates and cultured until the confluence rate was about 30% to 40%. According to the instructions, we injected pcDNA3.1-MTHFD2 plasmid and Lipofectamine TM 2000 reagents were diluted in serum-free DMEM and incubated at room temperature for 5 minutes. The two were then mixed and incubated for 20 minutes before adding to the cell culture dish. After 6 hours, the medium was replaced with serum-containing complete medium and cultured for 24-48 hours before subsequent testing.

[0052] 1.7 qRT-PCR

[0053] Total RNA was extracted using TRIzol reagent, 1 μg of RNA was reverse transcribed into cDNA, and SYBR Green qRT-PCR was performed on the StepOne Plus system with β-tubulin as the internal reference gene to calculate gene expression changes.

[0054] 1.8 Western Blot

[0055] Proteins were extracted using lysis buffer (containing protease and phosphatase inhibitors) and transferred to PVDF membranes after protein separation. After blocking, the membranes were incubated with primary antibodies including Anti-MTHFD2 (1:1000, Abcam, USA); Anti-α-SMA (1:1000, Abcam, USA); Anti-SM22 (1:1000, Abcam, USA); and Anti-Collagen III (1:1000, Abcam, USA), along with horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG (1:1000, Zsbio) or goat anti-rabbit IgG (1:1000, Zsbio), and developed using ECL.

[0056] 1.9 Histology and Immunostaining

[0057] After surgery, animals were sacrificed at various time points and vascular tissues from the injured and control sides were immediately removed, rinsed with PBS, and fixed in 4% paraformaldehyde for 24 hours. The tissues were then dehydrated, embedded, and paraffin sections (5 μm thick) were prepared for subsequent histological analysis.

[0058] Hematoxylin-eosin (HE) staining: After dewaxing and hydration of paraffin sections, HE staining was performed according to standard procedures, followed by hematoxylin staining, differentiation, bluing, and eosin staining. After dehydration and sealing, the vascular wall structure and neointimal formation were observed under a bright-field microscope.

[0059] Immunofluorescence staining: After antigen retrieval, paraffin sections were permeabilized with 0.3% Triton X-100 and blocked with 10% normal goat serum. Primary antibodies (e.g., anti-MTHFD2, α-SMA, Collagen III) were added and incubated overnight at 4°C. The next day, fluorescently labeled secondary antibodies (Alexa Fluor 488 / 594) were added and incubated for 1 hour at room temperature. Cell nuclei were counterstained with DAPI. After sections were mounted, images were acquired using a fluorescence or confocal microscope.

[0060] Immunohistochemical staining: After dewaxing, hydration, and high-temperature antigen retrieval, paraffin sections were blocked with 3% H₂O₂ to block endogenous peroxidase activity and then with 5% BSA. Primary antibodies (e.g., anti-MTHFD2, α-SMA, Collagen III) were added and incubated overnight at 4°C. The next day, HRP-conjugated secondary antibodies were used for incubation, and DAB was used for color development and counterstained with hematoxylin. After dehydration and mounting, sections were observed under a bright-field microscope.

[0061] All images were captured by microscope and quantitatively analyzed to compare the expression levels of MTHFD2 and phenotype-related proteins in each group.

[0062] 1.10 Cell function experiments

[0063] Scratch wound assay: VSMC migration was assessed using a scratch wound assay. VSMCs were grown in 6-well plates in DMEM / F12 supplemented with 10% fetal bovine serum. VSMCs were pretreated with serum-free DMEM for 48 hours. Floating cell debris was removed by manually placing a blank area on the cell plate with the tip of a 200 μL pipette. Images were captured at fixed positions at 0 and 12 hours.

[0064] EdU incorporation assay: The proliferation capacity of VSMCs was assessed using an EdU incorporation assay. EdU solution (Biyuntian EdU kit) was added to the culture medium for 4 hours according to the manufacturer's instructions to label VSMCs during DNA synthesis. Confocal microscopy was used for imaging and quantitative analysis.

[0065] 1.11 Mitochondrial function and oxidative stress detection

[0066] JC-1 staining is used to measure mitochondrial membrane potential, which reflects intracellular ROS levels. Cells were seeded onto slides and incubated with an appropriate concentration of a fluorescent probe (JC-1: 5 μM). Incubation was performed in the dark for 30 minutes, followed by washing with PBS. The cells were then imaged and quantitatively analyzed using a confocal microscope.

[0067] 1.12 Liquid chromatography-mass spectrometry

[0068] To evaluate the effects of folic acid intervention on the expression of key metabolic enzymes, including MTHFD2, this study used liquid chromatography-mass spectrometry (LC-MS) to perform proteomic or metabolomic analysis on vascular tissue samples. WT mice and MTHFD2 conditional knockout mice were fed a standard diet or a folic acid diet for four weeks. Vascular tissue from the injured side was collected 28 days after surgery and rapidly cryopreserved in liquid nitrogen.

[0069] Tissue samples were lysed with pre-chilled lysis buffer, and after protein quantification using the BCA assay, trypsin digestion, desalting, drying, and resolubilization were performed before loading. Samples were separated using an ultra-high-performance liquid chromatography (UHPLC) system and connected to a high-resolution mass spectrometer (such as Orbitrap or QExactive) for MS / MS data acquisition. Mass spectrometry data were processed using MaxQuant or Proteome Discoverer software. This study focused on the differential expression of enzymes in the one-carbon metabolic pathway (such as MTHFD2, MTHFD1, and SHMT2) under different dietary conditions. Combined with changes in protein expression in vascular tissue, the effects of folic acid intervention on metabolic networks and phenotypic regulation were evaluated.

[0070] 1.13 Statistical Analysis

[0071] The data are expressed as mean ± SEM and analyzed using GraphPad Prism 9.0. The data between two groups were compared using t-test, and the data of multiple groups were compared using ANOVA. p < 0.05 was considered statistically significant.

[0072] 2. Experimental results

[0073] 2.1 Establishment of a vascular injury model in WT mice and observation of dynamic changes in MTHFD2 expression

[0074] This study first established two commonly used vascular injury models in C57BL / 6J wild-type mice, including a unilateral femoral artery guidewire injury model and a unilateral common carotid artery complete ligation model. Figure 1 As shown in A, both models injure the left vascular system, and the right side is a sham-operated control group. Samples were collected 28 days after surgery for subsequent analysis.

[0075] In order to dynamically observe the changes of related molecules and tissue structures during the process of vascular injury repair, multiple time points were set up immediately after surgery (0 days), 3 days, 7 days, 14 days and 28 days, and the morphological evolution of the neointimal layer was observed by HE staining ( Figure 1 B) Results showed that both models exhibited progressively more severe neointimal hyperplasia on the injured side, while the sham-operated side maintained a largely normal morphology. Quantitative analysis revealed that neointimal growth formed to varying degrees in both the femoral and carotid artery injury models at each time point, but the area of neointimal growth did not differ significantly between the two models.

[0076] At 28 days after surgery, the expression levels of MTHFD2, α-SMA, and Collagen III in the injured and control vessels were further detected by immunohistochemical staining ( Figure 1 C) Results showed that MTHFD2 expression was significantly upregulated in the neointimal region of injured vessels compared to the sham-operated side. Furthermore, expression of α-SMA, a marker for contractile vascular smooth muscle cells, was reduced in the injured area, while expression of Collagen III, a marker for synthetic smooth muscle cells, was enhanced, suggesting a significant phenotypic transformation of smooth muscle cells.

[0077] In summary, MTHFD2 showed a continuous increasing trend with the intimal repair process in the two vascular injury models, mainly localized in the neointimal area, and was consistent with the transformation of smooth muscle cells to a synthetic phenotype, suggesting that it may play an important regulatory role in the process of vascular remodeling.

[0078] 2.2 Regulatory Effects of MTHFD2 on Phenotypic Transition in Rat Primary Vascular Smooth Muscle Cells

[0079] To explore the functional role of MTHFD2 in the phenotypic transformation of VSMC, we further conducted in vitro experiments using rat primary aortic VSMC as a model. First, PDGF-BB was used to induce the cell phenotype transformation from contractile to synthetic. The results of Western blot and immunofluorescence staining were as follows: Figure 2 As shown in A and 2B. Under induction conditions, the expression level of MTHFD2 was significantly increased, the expression of contractile phenotype markers α-SMA and SM22α was downregulated, while the expression of synthetic marker Collagen III was significantly upregulated, suggesting that MTHFD2 is closely related to the phenotypic conversion of VSMCs induced by PDGF-BB.

[0080] Furthermore, siRNA was used to downregulate MTHFD2 expression to explore its functional role, e.g. Figure 2 As shown in C and 2D, knockdown of MTHFD2 under PDGF-BB induction significantly enhanced the phenotypic transformation trend, as shown by a further decrease in the expression levels of contractile markers Acta2 (α-SMA) and Tagln (SM22α), and a further increase in the expression of synthetic marker COL3A1 (Collagen III).

[0081] These results suggest that MTHFD2 expression is continuously elevated during phenotypic transformation of smooth muscle cells in vitro and is involved in regulating their phenotypic state. Inhibition of MTHFD2 significantly promotes the acquisition of a synthetic phenotype, suggesting that MTHFD2 may play a key role in phenotypic maintenance.

[0082] 2.3 Construction of conditional knockout mice and in vivo verification of MTHFD2 function

[0083] To clarify the function of MTHFD2 in maintaining VSMC phenotype in vivo, we further constructed VSMC-specific MTHFD2 conditional knockout mice (MTHFD2 △VSMC ).like Figure 3 As shown in A, through MTHFD2 fl / fl Mice and SMMHC CreERT2 Experimental animals were obtained by crossbreeding mice and 6 weeks after birth, Tamoxifen was injected to induce Cre recombination activation, thereby achieving specific knockout of MTHFD2 in VSMCs. Genotyping results showed that the knockout group mice successfully carried MTHFD2-flox and Cre genes ( Figure 3 B) Meanwhile, Western blot analysis showed that MTHFD2 protein was significantly downregulated in vascular tissue after tamoxifen treatment, indicating that MTHFD2 △VSMC The model was built successfully.

[0084] To further evaluate the effect of MTHFD2 deficiency on VSMC phenotype and vascular remodeling, we performed a double-blind analysis of WT and MTHFD2 △VSMC The mice were sampled on days 0, 3, 7, 14, and 28 after injury to detect changes in phenotypic marker expression in vascular intima tissue ( Figure 3 C) The results showed that in WT mice, MTHFD2 and α-SMA gradually increased over time, while CollagenIII showed a gradual upregulation trend. In contrast, MTHFD2 △VSMC The expression of α-SMA was significantly downregulated and the expression of Collagen III was significantly increased in mice, indicating that the vascular remodeling process was aggravated in the absence of MTHFD2. In addition, immunofluorescence staining further verified the above changes ( Figure 3 D): Compared with WT mice, MTHFD2 △VSMC The MTHFD2 fluorescence signal in mouse blood vessels was significantly weakened, the α-SMA signal was reduced, and the Collagen III signal was enhanced, further supporting the protective role of MTHFD2 in the process of vascular remodeling in vivo.

[0085] The above results indicate that MTHFD2 plays an important role in maintaining the contractile phenotype of VSMC and inhibiting its conversion to the synthetic type. Its loss can aggravate pathological vascular remodeling, providing an in vivo basis for it as a potential intervention target.

[0086] 3. Therapeutic exploration and functional verification of targeting MTHFD2

[0087] To explore the application value of intervention strategies targeting MTHFD2 in vascular remodeling, multiple in vitro and in vivo experiments were designed to evaluate its therapeutic potential in regulating the phenotypic transformation of smooth muscle cells. ΔVSMC Primary VSMCs from mice were transfected with MTHFD2 overexpression plasmid ( Figure 4 A) qRT-PCR results showed that exogenous MTHFD2 could re-enhance its expression level in the knockout background and reverse the phenotypic transformation trend of α-SMA downregulation and Collagen III elevation, suggesting that MTHFD2 has reversible regulatory ability in maintaining cell phenotype.

[0088] Further mass spectrometry analysis of drug targets in the folate pathway that can be used to increase MTHFD2 expression revealed that folic acid dietary intervention in WT mice significantly increased the expression levels of multiple one-carbon metabolism-related enzymes, including MTHFD2 ( Figure 4 B) verified that nutritional regulation can be used as one of the strategies for activating MTHFD2. ΔVSMCMice were fed a normal diet or a folic acid diet for 1 month and then subjected to guidewire injury. Samples were collected and analyzed 28 days later. Immunofluorescence staining showed ( Figure 4 C), folic acid diet can significantly enhance MTHFD2 expression, maintain α-SMA expression and inhibit the upregulation of Collagen III, thereby alleviating vascular intimal thickening and improving VSMC phenotypic abnormalities.

[0089] At the level of therapeutic mechanism, JC-1 staining results ( Figure 4 D) shows that under PDGF-BB induction conditions, MTHFD2 ΔVSMC The mitochondrial membrane potential of primary mouse cells decreased, and folic acid supplementation could significantly alleviate this damage, indicating that MTHFD2 is involved in regulating and neutralizing reactive oxygen species to maintain mitochondrial homeostasis, and folic acid intervention has a metabolic repair effect. Figure 4 E) Further confirmation showed that the migration and proliferation abilities of VSMCs were significantly enhanced after MTHFD2 deficiency, while folic acid supplementation could significantly inhibit this phenomenon, suggesting that folic acid supplementation plays a negative regulatory role in the process of pathological phenotypic transformation by increasing MTHFD2 expression.

[0090] In summary, the expression of MTHFD2 in VSMC is regulated by one-carbon metabolism, and its function can be restored through nutritional intervention (such as folic acid supplementation), thereby alleviating phenotypic transformation and mitochondrial dysfunction, providing clear experimental evidence for it as a potential therapeutic target.

[0091] Application effect:

[0092] 1. Targeting MTHFD2 can effectively attenuate the pathological phenotypic transformation of VSMCs, inhibit their abnormal proliferation and migration behavior, thereby reducing the formation of neointima and improving the pathological process of vascular remodeling.

[0093] 2. The expression level of MTHFD2 can be significantly increased by folic acid supplementation. Folic acid intervention can reverse the VSMC dysfunction and phenotypic imbalance caused by MTHFD2 deficiency, alleviate abnormal changes in the damaged area, and improve the local microenvironment.

[0094] 3. While folic acid sustained-release stent treatment reduces neointimal hyperplasia, it does not affect the endothelialization process and reduces the risk of thrombosis in coronary stents.

Claims

1. Application of MTHFD2 (methylenetetrahydrofolate dehydrogenase (NADP+dependent)2, methenyltetrahydrofolate cyclohydrolase) in the preparation of drugs for the prevention and treatment of coronary in-stent restenosis (ISR).

2. The use according to claim 1, characterized in that The MTHFD2 reduces the pathological proliferation and migration of vascular smooth muscle cells (VSMC) by regulating NADPH levels, inhibits angiogenic intimal hyperplasia, reduces the thickness of the neointima, improves the pathological process of vascular remodeling, and effectively alleviates oxidative stress and inflammatory response in the damaged area, thereby achieving the purpose of treating coronary stent restenosis.

3. Application of a combination of MTHFD2 and folic acid in the preparation of drugs for preventing and treating coronary stent restenosis.

4. The use according to claim 3, characterized in that The combined use of MTHFD2 and folic acid further stabilizes the microenvironment, reduces neointimal hyperplasia without affecting the endothelialization process, and reduces the risk of thrombosis in coronary stents.

5. A pharmaceutical composition for treating coronary in-stent restenosis, characterized in that: The pharmaceutical composition consists of MTHFD2 and folic acid.

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