CN119345368A

CN119345368A - Application of methylenetetrahydrofolate dehydrogenase 2 and its encoding gene in vascular remodeling

Info

Publication number: CN119345368A
Application number: CN202411476298.3A
Authority: CN
Inventors: 蔡菁菁; 孙雪菁; 伍俊儒; 袁洪; 彭丽萍; 翁春艳
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2024-10-22
Filing date: 2024-10-22
Publication date: 2025-01-24

Abstract

本发明公开了亚甲基四氢叶酸脱氢酶2及其编码基因在血管重构中的应用。本发明为预防、治疗和/或缓解血管重构和/或血管重构相关疾病提供了新思路。The present invention discloses the application of methylenetetrahydrofolate dehydrogenase 2 and its encoding gene in vascular remodeling, and provides a new idea for preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related diseases.

Description

Methylene tetrahydrofolate dehydrogenase 2 and application of encoding gene thereof in vascular remodeling

Technical Field

The invention belongs to the technical field of medicines, and relates to methylene tetrahydrofolate dehydrogenase 2 and application of a coding gene thereof in vascular remodeling.

Background

Methylene tetrahydrofolate dehydrogenase 2 (methylenetetrahydrofolate dehydrogenase (NADP+dependent) 2, MTHFD 2) is an important enzyme involved in cellular metabolism, whose function mainly includes play a key role in methylation and folate metabolic pathways. MTHFD2 is involved in folate (Folic Acid, FA) metabolism by reducing pentamethyltetrahydrofolate (5-Methyltetrahydrofolate, 5-MTHF) to tetrahydrofolate (Tetrahydrofolate, THF), providing a carbon unit for nucleotide synthesis. This is critical for DNA synthesis and cell proliferation. Furthermore MTHFD2 plays an important role in the energy metabolism and mitochondrial function of the cell.

Recent studies have shown that tertiary lymphoid organs (Tertiary Lymphoid Organ, TLO) (also known as tertiary lymphoid tissues, tertiary lymphoid structures) play a key role in the chronic inflammatory process of vascular lesions. TLO is a lymphoid structure formed in non-lymphoid tissues, and is involved in chronic inflammation and immune response, with important roles in immunomodulation and disease progression. Structurally, TLO has typical lymphoid tissue characteristics such as lymphoid follicles, T cell regions, B cell regions, etc. However, the mechanisms by which TLOs form in different vascular lesions vary, and the relationship between the modulation of T cell cytotoxicity and the balanced subpopulations in TLOs may limit the development of vascular lesions.

One-carbon metabolism affects the differentiation, function and survival of T cells by regulating key processes such as DNA synthesis, methylation modification and energy metabolism, and is critical to the normal operation of the immune system. Nucleotide synthesis required for rapid proliferation of T cells is indispensable. Although de novo purine metabolic signaling plays a key role in directing T cell differentiation and function, some studies identified MTHFD2 as a metabolic checkpoint and a potential target for the treatment of inflammatory diseases, no report was made of the relation of MTHFD2 to chronic progression of tertiary lymphoid organ formation and vascular remodeling.

Vascular remodeling is a common pathological basis for a variety of diseases such as restenosis and hypertension of blood vessels after atherosclerosis and angioplasty. Research shows that the reconstruction of blood vessels is a series of structural and functional abnormalities caused by the change of the internal and external environments of blood vessels, including proliferation, migration, apoptosis, change of mechanism components and the like, and on the basis of the series of dynamic changes, the stimulation of blood vessels to the internal and external environments of the blood vessels generates structural changes, such as thickening of the vessel walls, increase of the lumen ratio of the vessel walls and reduction of the number of tiny arteries, thereby causing abnormal blood vessel functions.

Disclosure of Invention

In vascular lesions, TLO is formed in the remodelled blood vessels where chronic inflammatory reactions occur. TLO appears in multiple reconstructed blood vessels such as atherosclerosis, aortic aneurysm, intimal hyperplasia, allograft and the like, recognizes and discovers the development regulation targets thereof, and has great significance for treating vascular lesions.

The inventor experiment research shows that the enzyme related to one-carbon metabolism is significantly up-regulated in the process of differentiating CD4 ⁺ T cells into key constitutive cell follicular helper T cells (Tfh cells) formed by TLO in a reconstructed blood vessel. Wherein, the expression of the mitochondrial one-carbon metabolism related enzyme gene is higher than that of cytoplasm and the expression enhancement of MTHFD coding gene is most obvious, compared with the CD4 ⁺ T cell, the mitochondria Mthfd2 in Tfh cells is increased by 8 times, which indicates that MTHFD2 plays an important role in the differentiation of the CD4 ⁺ T cells into the Tfh cells and the formation of TLO. In vitro studies have found that inhibition of MTHFD's mRNA expression by siRNA significantly reduced CD4 ⁺ T cell differentiation to Tfh cells. In vivo studies have found that MTHFD inhibitors can reduce TLO formation in the reconstituted vessel during allograft and can reduce the reconstituted vessel intima to media ratio and increase the reconstituted vessel lumen area. Experiments have shown that inhibition MTHFD2 can slow vascular remodeling by blocking Tfh cell differentiation and TLO formation.

In a first aspect, the present invention provides the use of an inhibitor of methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) for the preparation of a product for the prevention, treatment and/or alleviation of vascular remodeling and/or vascular remodeling related diseases.

In some embodiments, in the above-described applications, the MTHFD inhibitor has one or more of the following functions:

(1) Inhibit MTHFD protein expression and/or activity;

(2) Inhibit MTHFD expression of the gene encoding the protein.

In some embodiments, in any of the above uses, the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) is human MTHFD2, having an NCBI accession number np_006627.2 for its amino acid sequence, or an amino acid sequence having at least 80% sequence identity thereto and having methylene tetrahydrofolate dehydrogenase 2 activity, or an amino acid sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, compared thereto and having methylene tetrahydrofolate dehydrogenase 2 activity. In some embodiments MTHFD2 is mouse MTHFD2, the NCBI accession number of which is np_032664.1 for an amino acid sequence, or an amino acid sequence having at least 80% sequence identity thereto and having methylene tetrahydrofolate dehydrogenase 2 activity, or an amino acid sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared thereto and having methylene tetrahydrofolate dehydrogenase 2 activity.

In some embodiments, the MTHFD protein-encoding gene includes a DNA sequence encoding MTHFD protein, a pre-mRNA sequence transcribed from DNA encoding MTHFD protein (genomic DNA including introns and exons), and an mRNA sequence encoding MTHFD protein for any of the above uses. In some embodiments, the MTHFD mRNA is human MTHFD2 mRNA, the NCBI accession number of which is nm_006636.4 for the nucleic acid sequence, or a nucleic acid molecule having at least 80% sequence identity thereto and encoding a MTHFD2 protein of any one of the above. In some embodiments, the MTHFD mRNA is a mouse MTHFD mRNA, the NCBI accession number of which is nm_008638.2 for the nucleic acid sequence, or a nucleic acid molecule having at least 80% sequence identity thereto and encoding any of the MTHFD2 proteins described above.

In some embodiments, the MTHFD inhibitor is selected from one or more of an antibody to MTHFD2 or an antigen-binding fragment thereof, a nucleic acid (e.g., MTHFD siRNA as shown by SEQ ID NO: 2), a small molecule of a compound (e.g., MTHFD 2-specific targeting inhibitor DS 18561882), wherein the nucleic acid is a small interfering RNA, dsRNA, microRNA, antisense nucleic acid or gene editing vector (e.g., CRISPR-Cas9 gene editing vector or TALEN gene editing vector) targeting MTHFD2 or its transcript and capable of inhibiting MTHFD2 expression or gene transcription, and the small molecule of a compound may be those based on MTHFD structure or a small molecule inhibitor similar to the structure of the existing MTHFD inhibitor.

In some embodiments, in any of the above-described applications, the vascular remodeling is vascular remodeling associated with a vascular-related disease;

Preferably, the disease is selected from the group consisting of vascular wall injury (e.g., physical injury due to an interventional stent, vascular injury due to atherosclerosis, vascular injury due to hyperlipidemia, vascular injury due to hypertension, vascular injury due to diabetes, vascular injury due to autoimmune disease), post-injury vascular stenosis, post-injury blood flow dysfunction, thrombosis, PCI and Bypass post-operative vascular stenosis, coronary heart disease, myocardial ischemia, myocardial infarction, post-myocardial infarction, heart failure, post-myocardial infarction arrhythmia, aneurysm, atherosclerosis, cerebral infarction, graft vascular lesions after allograft, tumor angiogenesis, graft rejection, diabetic microvasculopathy, cerebrovascular lesions, and any combination thereof.

In a second aspect, the invention provides the following uses of methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) or a gene encoding it as biomarker or target, and/or reagents for detecting said methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) or a gene encoding it:

i. the application in screening vascular remodeling and/or vascular remodeling related diseases or preparing products for screening vascular remodeling and/or vascular remodeling related diseases;

Use in or for the manufacture of a product for assessing a vascular remodeling and/or a vascular remodeling related disorder;

use in the manufacture of a product for diagnosing vascular remodeling and/or a disease associated with vascular remodeling;

Use in screening and/or in the manufacture of a product for the prevention, treatment and/or alleviation of vascular remodeling and/or vascular remodeling related diseases;

v. use in monitoring the course of a vascular remodeling and/or a disease associated with vascular remodeling or in the manufacture of a product for monitoring the course of a vascular remodeling and/or a disease associated with vascular remodeling;

use in or for the preparation of a product for the analysis of a prognosis of a vascular remodeling and/or a disease associated with vascular remodeling;

use of a compound for inhibiting the differentiation of CD4 ⁺ T cells into Tfh cells and/or the formation of vascular tertiary lymphoid tissue or for the preparation of a product for inhibiting the differentiation of CD4 ⁺ T cells into Tfh cells and/or the formation of vascular tertiary lymphoid tissue.

In some embodiments, the agent is an agent for detecting the expression level and/or activity level of the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) and/or an agent for detecting the expression level of mRNA of a MTHFD encoding gene, e.g., an agent for detecting the expression level of mRNA of a MTHFD encoding gene (e.g., a primer composition for specifically amplifying the gene, a probe for specifically recognizing the gene), an agent for detecting the expression level of MTHFD protein (e.g., an antibody, an aptamer, a dye).

In some embodiments, the agent comprises an agent that detects the amount of expression of the biomarker by PCR, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, co-immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochromatography, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin techniques.

In some embodiments, in any of the above-described applications, the expression level of the MTHFD protein-encoding gene, the expression level or the activity of the MTHFD protein in a patient with vascular remodeling and/or vascular remodeling-related disease is increased over that of a normal control.

In some embodiments, in any of the above-described applications, the vascular remodeling is vascular remodeling associated with a vascular-related disease or condition;

Preferably, the disease or condition is selected from the group consisting of vascular wall injury (e.g., physical injury due to an interventional stent, vascular injury due to atherosclerosis, vascular injury due to hyperlipidemia, vascular injury due to hypertension, vascular injury due to diabetes, vascular injury due to autoimmune disease), post-injury vascular stenosis, post-injury blood flow dysfunction, thrombosis, PCI and Bypass post-operative vascular stenosis, coronary heart disease, myocardial ischemia, myocardial infarction, post-myocardial infarction, heart failure, post-myocardial arrhythmia, aneurysms, atherosclerosis, cerebral infarction, graft vascular lesions after allograft, tumor angiogenesis, graft rejection, diabetic microvasculopathy, cerebrovascular lesions, and any combination thereof.

In a third aspect, the present invention provides a method for screening a drug for preventing, treating and/or alleviating a vascular remodeling and/or vascular remodeling-related disease, comprising selecting a substance that inhibits methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) from the compounds to be screened as a drug for preventing, treating and/or alleviating a vascular remodeling and/or vascular remodeling-related disease.

In some embodiments, in the above methods, the substance that inhibits methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) has one or more of the following functions:

(1) Inhibit MTHFD protein expression and/or activity;

(2) Inhibit MTHFD expression of the gene encoding the protein.

In some embodiments, the method of any one of the above, the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) is human MTHFD, the NCBI accession number of which is np_006627.2 for the amino acid sequence, or for the amino acid sequence having at least 80% sequence identity thereto and methylene tetrahydrofolate dehydrogenase 2 activity, or for the amino acid sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, compared thereto and having methylene tetrahydrofolate dehydrogenase 2 activity. In some embodiments MTHFD2 is mouse MTHFD2, the NCBI accession number of which is np_032664.1 for an amino acid sequence, or an amino acid sequence having at least 80% sequence identity thereto and having methylene tetrahydrofolate dehydrogenase 2 activity, or an amino acid sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared thereto and having methylene tetrahydrofolate dehydrogenase 2 activity.

In some embodiments, the MTHFD protein-encoding gene comprises a DNA sequence encoding MTHFD protein, a pre-mRNA sequence transcribed from DNA encoding MTHFD protein (genomic DNA comprising introns and exons), and an mRNA sequence encoding MTHFD protein. In some embodiments, the MTHFD mRNA is human MTHFD2 mRNA, the NCBI accession number of which is nm_006636.4 for the nucleic acid sequence, or a nucleic acid molecule having at least 80% sequence identity thereto and encoding a MTHFD2 protein of any one of the above. In some embodiments, the MTHFD mRNA is a mouse MTHFD mRNA, the NCBI accession number of which is nm_008638.2 for the nucleic acid sequence, or a nucleic acid molecule having at least 80% sequence identity thereto and encoding any of the MTHFD2 proteins described above.

In some embodiments, the method of any one of the above, the vascular remodeling is vascular remodeling associated with a vascular-related disease or condition;

In a fourth aspect, the invention provides a product comprising or coated with an effective amount of a methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) inhibitor.

In some embodiments, in the above-described products, the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) inhibitor has one or more of the following functions:

(1) Inhibit MTHFD protein expression and/or activity;

(2) Inhibit MTHFD expression of the gene encoding the protein.

In some embodiments, in any of the above-described products, the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) is human MTHFD, having the NCBI accession number np_006627.2 for its amino acid sequence, or an amino acid sequence having at least 80% sequence identity thereto and having methylene tetrahydrofolate dehydrogenase 2 activity, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, compared thereto and having methylene tetrahydrofolate dehydrogenase 2 activity. In some embodiments MTHFD2 is mouse MTHFD2, the NCBI accession number of which is np_032664.1 for an amino acid sequence, or an amino acid sequence having at least 80% sequence identity thereto and having methylene tetrahydrofolate dehydrogenase 2 activity, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared thereto and having methylene tetrahydrofolate dehydrogenase 2 activity.

In some embodiments, the MTHFD protein-encoding gene comprises a DNA sequence encoding MTHFD protein, a pre-mRNA sequence transcribed from DNA encoding MTHFD protein (genomic DNA comprising introns and exons), and an mRNA sequence encoding MTHFD protein in any of the above described products. In some embodiments, the MTHFD mRNA is human MTHFD2 mRNA, the NCBI accession number of which is nm_006636.4 for the nucleic acid sequence, or a nucleic acid molecule having at least 80% sequence identity thereto and encoding MTHFD2 of any one of the above. In some embodiments, the MTHFD mRNA is a mouse MTHFD mRNA, the NCBI accession number of which is nm_008638.2 for the nucleic acid sequence, or a nucleic acid molecule having at least 80% sequence identity thereto and encoding any one of the MTHFD2 described above.

In some embodiments, the MTHFD inhibitor is selected from one or more of an antibody to MTHFD2 or an antigen-binding fragment thereof, a nucleic acid (e.g., MTHFD siRNA as shown by SEQ ID NO: 2), a small molecule of a compound (e.g., MTHFD 2-specific targeting inhibitor DS 18561882), wherein the nucleic acid is a small interfering RNA, dsRNA, microRNA, antisense nucleic acid or gene editing vector (e.g., CRISPR-Cas9 gene editing vector or TALEN gene editing vector) targeting MTHFD2 or a transcript thereof and capable of inhibiting MTHFD2 expression or gene transcription, and the small molecule of a compound may be those based on MTHFD structure or a structure similar to that of the existing MTHFD inhibitor.

In some embodiments, the product is a vascular stent or a catheter with a balloon, and the MTHFD inhibitor may be contained in a coating of the vascular stent or the balloon.

In some embodiments, the product is a pharmaceutical composition comprising the MTHFD inhibitor as an active ingredient, in addition to pharmaceutically acceptable excipients.

In some embodiments, the product is for preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related diseases;

the vascular remodeling is vascular remodeling associated with a vascular-related disease or condition;

Compared with the prior art, the invention has the following beneficial effects:

The invention discovers that the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) has potential targets for treating vascular remodeling and various cardiovascular diseases through researches. The inventors have found that MTHFD2 is significantly activated during the differentiation of follicular helper T cells (Tfh cells) in the reconstituted vessel, which is associated with the formation of organized lymphoid aggregate tertiary lymphoid Tissue (TLO) in the perivascular region and its inflammatory activation. In vivo studies have found that MTHFD inhibitors significantly reduce TLO formation in the reconstituted vessel and effectively alleviate graft vascular lesions. The invention provides a new thought for preventing, treating and/or relieving the vascular remodeling and/or vascular remodeling related diseases (especially organ transplantation rejection) by taking MTHFD as a biomarker for judging the progress of the vascular remodeling and vascular remodeling related diseases (especially organ transplantation rejection) or taking MTHFD as a target point for preventing, treating and/or relieving the vascular remodeling and vascular remodeling related diseases (especially organ transplantation rejection).

Drawings

In FIG. 1, panel A shows a thermal chart of enrichment analysis of metabolic pathways (rows) of various T cell subsets (columns) in a mouse allograft vessel obtained by Compass analysis, panel B shows a single cell transcriptome sequencing (scRNA-seq) analysis chart of CD4 positive T cells and follicular helper T cells in a mouse allograft vessel, and panel C shows a change chart of expression multiples of one-carbon metabolic cytoplasm and mitochondrial key enzyme genes in the follicular helper T cells compared with the CD4 positive T cells.

In FIG. 2, panel A is a graph of quantitative statistical analysis of mRNA expression levels of qPCR assay MTHFD2 in CD4 ⁺ T cells transfected with NC siRNA and MTHFD siRNA, and Panel B is a graph of quantitative statistical analysis of percentage of ICOS and CXCR5 double positive Tfh cells to total viable cells in flow cytometry assay in CD4 ⁺ T cells transfected with NC siRNA and MTHFD siRNA.

FIG. 3 is a graph showing quantitative statistical analysis of tertiary lymphoid structures in allograft blood vessels at week 4 of both control and inhibitor groups of mice.

In fig. 4, a-panels are representative pictures of H & E staining of 4 th week transplanted vessel sections of the vascular grafts of mice in the control group (left) and the inhibitor group (right), B-panels are the intima to media (I/M) ratio of the control group and the inhibitor group, and C-panels are the reconstituted vessel lumen areas of the control group and the inhibitor group.

Detailed Description

The present disclosure is further illustrated below in conjunction with specific embodiments. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. The present disclosure may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the disclosure, and therefore the disclosure is not to be limited to the specific embodiments disclosed below.

Unless otherwise indicated, all technical means used in the examples are routine in the art or according to the experimental methods suggested by the manufacturers of the kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.

Unless defined otherwise, 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 disclosure belongs. The terminology used in the description presented herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In this document, the terms "comprising," "including," or "containing" are to be construed as including the specified components, but not excluding any other components.

In this context, the term "about" means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, the value depending in part on how it is measured or determined (i.e., the limits of the measurement system). Or "about" may mean a range of up to ±20%, such as a range of ±10%, ±5% or ±1%. Unless otherwise indicated, when a particular value is found in this disclosure, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

The term "MTHFD gene encoding" or "MTHFD protein encoding gene" refers to any polynucleotide encoding MTHFD 2. For example, it includes a DNA sequence encoding MTHFD, a pre-mRNA sequence transcribed from DNA encoding MTHFD2 (genomic DNA including introns and exons), and an mRNA sequence encoding MTHFD 2. "MTHFD" mRNA "refers to mRNA encoding MTHFD protein.

The sequence of human MTHFD mRNA includes, but is not limited to, MTHFD mRNA shown by national center for biotechnology information (National Center for Biotechnology Information, NCBI) accession No. nm_ 006636.4. The sequence of mouse MTHFD mRNA includes, but is not limited to, MTHFD mRNA shown in NCBI accession No. nm_ 008638.2. Additional examples of MTHFD mRNA sequences are readily available using publicly available databases (e.g., NCBI Gene, ensembl).

As used herein, the term "MTHFD inhibitor" refers to a decrease in the amount of expression and/or activity of MTHFD protein, or a decrease in the amount of expression of the MTHFD encoding gene, in cells treated with the inhibitor under the same conditions, as compared to cells treated without the inhibitor, which may result in, for example, decreased differentiation of CD4 ⁺ T cells into Tfh cells, decreased formation of vascular tertiary lymphoid tissue, decreased vascular remodeling (e.g., decreased intima-to-media ratio, increased vascular lumen area), decreased graft rejection, and the like.

Herein, the phrase "inhibiting the expression and/or activity of MTHFD protein" encompasses inhibiting the expression and/or activity of any MTHFD protein (such as mouse MTHFD protein, rat MTHFD protein, monkey MTHFD2 protein, or human MTHFD protein) or mutant thereof, and the phrase "inhibiting the expression of a gene encoding MTHFD protein" encompasses inhibiting the expression of any gene encoding MTHFD protein, or mutant thereof.

Inhibition includes any level of inhibition. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

MTHFD2 encoding gene expression, such as MTHFD mRNA levels or MTHFD protein levels, can be assessed based on any variable level associated with MTHFD encoding gene expression. Inhibition may be assessed by a decrease in absolute or relative levels of one or more of these variables as compared to control levels. The control level may be any type of control level used in the art, such as a pre-dosing baseline level or a level determined from a similar untreated or control (e.g., buffer-only control or inert control) -treated subject, cell or sample.

Herein, the term "vascular remodeling associated with vascular-related diseases" refers to vascular-related diseases caused by vascular remodeling, or vascular remodeling caused by vascular-related diseases.

As used herein, the term "vascular remodeling" refers to structural and functional changes in a blood vessel to accommodate changes in the internal and external environment, including changes in the cellular biology of proliferation, hypertrophy, apoptosis, cell migration, production and degradation of extracellular matrix, etc. of the vessel wall cells. Vascular remodeling is an important pathological basis for the exacerbation of many vascular-related diseases (e.g., atherosclerosis and hypertension, etc.), and is the etiology of the development and progression of such diseases.

The terms "patient," "subject," and "individual" are used interchangeably herein to include humans and non-human animals, wherein non-human animals include mammals, such as monkeys, rats, mice, cows, pigs, goats, sheep, dogs, cats.

Herein, when referring to an animal, human, subject, cell, tissue, organ or biological fluid by "administering" and "treating" it is meant that the exogenous drug, therapeutic, diagnostic agent or composition is contacted with the animal, human, subject, cell, tissue, organ or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cell includes contacting the agent with the cell and contacting the agent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of cells, e.g., by agents, diagnostic agents, binding compositions, or by other cells.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount effective to prevent or slow down a disease or condition to be treated when MTHFD inhibitor is administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. A therapeutically effective amount further refers to an amount of the MTHFD inhibitor sufficient to cause a reduction in symptoms, such as treating, curing, preventing, or slowing the associated medical condition, or increasing the rate of treatment, cure, prevention, or slowing the condition. The effective amount for a particular subject can vary depending upon a variety of factors, such as the disease to be treated, the overall health of the patient, the route of administration, and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects. When administered to an individual an active ingredient administered alone, a therapeutically effective amount refers to the individual ingredient. When a combination is administered, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously. A therapeutically effective amount will generally alleviate symptoms by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40% and most preferably by at least 50%.

As used herein, the term "treating" includes amelioration or cessation of a disorder or symptoms thereof. Treatment includes inhibition, e.g., reducing the overall frequency of onset of the disorder or symptoms thereof.

As used herein, the term "alleviating" includes an improvement or alleviation of at least one indicator, sign or symptom of a disorder or a symptom thereof, such as a delay or slowing of the progression or severity of an improvement comprising one or more indicators of a disorder or a symptom thereof. The progress or severity of an indicator may be determined by subjective or objective measurements known to those skilled in the art.

Herein, the term "preventing" includes avoiding the onset of a disorder or symptoms thereof.

Herein, the term "pharmaceutically acceptable excipients" or "pharmaceutically acceptable carriers" refers to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of the use of auxiliary substances is to provide a pharmaceutical composition which is safe to use, stable in nature and/or has specific functionalities, and to provide a method so that the active ingredient can be dissolved at a desired rate after administration of the drug to a subject, or so that the active ingredient is effectively absorbed in the subject to whom it is administered. Pharmaceutically acceptable excipients may be inert fillers or may be functional ingredients that provide some function to the pharmaceutical composition (e.g., to stabilize the overall pH of the composition or to prevent degradation of the active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, binders, disintegrants, lubricants, anti-adherent agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweetening agents, and the like.

The pharmaceutical compositions of the present disclosure may be prepared using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes. In some embodiments, the pharmaceutical compositions of the present disclosure may be administered by any suitable route known in the art including, but not limited to, oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and/or cerebrospinal. The route of administration can be varied or adjusted in any suitable manner to meet the nature of the drug, the convenience of the patient and medical personnel, and other relevant factors.

Herein, "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, "antibody" is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primatized, chimeric antibodies, single chain antibodies, and the like.

As used herein, an "antigen binding fragment" refers to a portion of an antibody, such as F (ab ') 2, F (ab) 2, fab', fab, fv, scFv, and the like. Regardless of its structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antigen binding fragment" includes aptamers, stereoisomers, and diabodies. The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

Herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, each antibody comprising the population being identical. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include a plurality of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen.

Herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chains are derived from one source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

In this context, the term "encoding" refers to a polynucleotide that is referred to as "encoding" a protein if it can be transcribed and/or translated to produce mRNA of the protein and/or fragment thereof in its native state or when manipulated by methods well known to those skilled in the art.

In this context, the term "identity" may be assessed by the naked eye or by computer software such as the software program described in Ausubel et al eds. (2007) at Current Protocols in Molecular Biology. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The identity between two or more sequences may be expressed in percent (%), which may be used to evaluate the identity between related sequences. A polynucleotide sequence or amino acid sequence has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when the sequences are aligned, the percentage of bases or amino acids in the two sequences that are compared are identical.

In this context, "conservatively substituted variants" or "conservative amino acid substitutions" refer to amino acid substitutions known to those of skill in the art, which are made without generally altering the biological activity of the resulting molecule. In general, it is well recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Conservative substitutions may be made by amino acids containing chemically similar side chains, such as 1) aliphatic side chains of glycine, alanine, valine, leucine and isoleucine, 2) aliphatic hydroxyl side chains of serine and threonine, 3) amide-containing side chains of asparagine and glutamine, 4) aromatic side chains of phenylalanine, tyrosine and tryptophan, 5) basic side chains of lysine, arginine and histidine, 6) acidic side chains of aspartic acid and glutamic acid.

Methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) inhibitors for the treatment of vascular remodeling

By modeling mouse allogeneic vascular grafts and performing transcriptome sequencing and analysis, key one-carbon metabolic enzymes were found to be significantly upregulated during CD4 ⁺ T differentiation into Tfh cells, CD4 ⁺ T differentiation into Tfh cells likely was primarily dependent on mitochondrial one-carbon flux, mitochondrial one-carbon enzyme gene expression was higher than cytosol, and of which Mthfd2 was most significant.

Further, in vitro studies have found that inhibition of MTHFD's mRNA expression by siRNA significantly reduces CD4 ⁺ T cell differentiation to Tfh cells.

Further, in vivo studies have found that by injecting MTHFD inhibitor DS18561882 and detecting tertiary lymphoid tissue formation in a mouse allogeneic vascular graft model, the use of MTHFD inhibitor was found to significantly inhibit vascular tertiary lymphoid tissue formation. By staining the vascular samples, it was found that MTHFD inhibition slowed vascular remodeling by allograft, and reduced the intima to media ratio and increased the luminal area of the remodeling vessels, alleviating graft vascular lesions.

Based on the above findings, the present invention provides the use of an inhibitor of methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) for the preparation of a product for the prevention, treatment and/or alleviation of vascular remodeling and/or vascular remodeling related diseases.

In some embodiments, the MTHFD inhibitor is selected from one or more of an antibody to MTHFD2 or an antigen-binding fragment thereof, a nucleic acid, a small molecule of a compound.

In some embodiments, the nucleic acid is an siRNA, shRNA, antisense RNA, or gene editing vector, and in some embodiments, the gene editing vector is a CRISPR-Cas9 gene editing vector or a TALEN gene editing vector. It is well known in the art that siRNA, shRNA, antisense RNA or gene editing vectors can be used to interfere with or reduce expression of a target gene, and that these siRNA, shRNA, antisense RNA or gene editing vectors can be readily designed and selected by one of skill in the art using known methods and techniques depending on the target gene to be acted upon. In some embodiments, the nucleic acid is an siRNA, such as MTHFD siRNA herein as shown in SEQ ID NO. 2.

In some embodiments, the small molecule of the compound may be DS18561882.

Methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) and coding gene thereof as target for treating vascular remodeling

Based on the finding that the expression level of Mthfd gene, the expression level or activity of MTHFD2 protein is increased in patients with vascular remodeling and/or vascular remodeling related diseases compared to normal control, the present invention also provides the following uses of methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) or a gene encoding the same as a biomarker or target, and the following uses of reagents for detecting the methylene tetrahydrofolate dehydrogenase 2 (MTHFD 2) or a gene encoding the same:

In some embodiments, the reagent is a reagent for detecting the amount of expression and/or activity level of MTHFD2 and/or a reagent for detecting the amount of MTHFD mRNA expression.

All reagents for detecting the expression level and/or activity level of MTHFD and/or reagents for detecting the expression level of MTHFD mRNA can be used for the above-mentioned i-vi applications.

In some embodiments, the agent is an agent that detects the amount of expression of the biomarker in PCR, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, co-immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochromatography, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin techniques.

In some embodiments, the reagents are Primer compositions and probes for detecting MTHFD mRNA expression, e.g., forward primers as shown in SEQ ID NO. 8, reverse primers as shown in SEQ ID NO. 9, or primers or probes designed based on Primer-BLAST.

In some embodiments, the reagents are primary and secondary antibodies that detect MTHFD proteins, for example MTHFD2 (D8U 2I) rabit mAb (CELL SIGNALING Technology).

Vascular stents or catheters with balloons

The MTHFD inhibitors of the present invention can be coated on balloons in vascular stents and balloon-bearing catheters, which, by implanting such vascular stents and balloon-bearing catheters into a blood vessel, can maintain the lumen passageway of the blood vessel, improve blood flow and improve vascular remodeling.

Pharmaceutical composition

The invention also provides a pharmaceutical composition. Such compositions comprise an effective amount of MTHFD inhibitor and pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical compositions of the present invention may be administered by any suitable route known in the art including, but not limited to, oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and/or cerebrospinal.

In some embodiments, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous injection into the human body. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffers. The pharmaceutical composition may also contain a solubilizing agent and a local anesthetic such as lidocaine, thereby alleviating pain at the injection site. In general, the active ingredients are supplied individually or in admixture in unit dosage form, such as in the form of a dry lyophilized powder or dry concentrate, in a sealed container (e.g., ampoule or pouch) that is indicative of the amount of active agent. In the case of administration of the composition by infusion, the composition may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline may be used so that the active ingredients may be mixed prior to administration.

Combination therapy

The MTHFD inhibitors of the present invention may be used in combination (e.g., contained in the same pharmaceutical composition) with at least one additional therapeutic agent, such as an angiotensin converting enzyme inhibitor (e.g., enalapril, etc.), a statin (e.g., atorvastatin, etc.), a cholesterol absorption inhibitor, an antioxidant, etc., provided that such combination provides beneficial effects in preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related disorders.

EXAMPLE 1 significant activation of MTHFD2 in Tfh cell differentiation in reconstituted vessels

Experimental materials:

phosphate Buffered Saline (PBS), available from HyClone, cat# SH30258.01;

DMEM medium (containing sodium pyruvate) purchased from BI, cat# 06-1055-57-1ACS;

fetal Bovine Serum (FBS), purchased from Gibco under the accession number 10099141C;

papain, available from Sigma-Aldrich under the trade designation P4762-25MG.

The experimental method comprises the following steps:

1. A mouse allogeneic vascular transplantation model is established by using isoflurane to carry out inhalational continuous anesthesia (flow rate: 2L/min; concentration: 1%) on BALB/c donor mice (female, 8-12 weeks old, hunan Shekola laboratory animals Co., ltd.), fixing the donor mice on an operation plate, using forceps to apply cotton balls stained with alcohol to the abdomen of the mice, cutting the abdomen of the donor mice along the central line, pulling viscera aside to expose the inferior vena cava, and injecting heparin solution into the inferior vena cava. Separating aortic arch bifurcation, performing electric coagulation, cutting with micro-scissors, separating part of space at active bifurcation, passing through forceps with electric coagulator, performing electric coagulation on intercostal artery, cutting off appropriate blood vessel length, lavaging aorta until no blood exists in the cavity, and placing the lavaged aorta in a culture dish containing heparin solution. After C57BL/6J recipient mice (Male, 8-12 weeks old, hunan Szechwan laboratory animal Co., ltd.) were anesthetized, the neck skin tissue was cut off, the carotid artery was exposed, the surrounding carotid artery was carefully separated, and the carotid artery was ligated at both ends with 8-0 filaments, with the two ends being about 1mm apart, and cut off from the middle with scissors. The nylon sleeve is sleeved on the blood vessel, and one end of the sleeve is clamped and fixed by hemostasis. The scissors are tightly attached to the tying end to cut off the blood vessel, so that the vascular incision is cut flat, heparin washes residual blood in the blood vessel, the two ends of the blood vessel are clamped by using fine micro forceps, the blood vessel is turned over and sleeved on the nylon sleeve, and the blood vessel is tied and fixed by using 8-0 silk threads. Taking an appropriate length of aorta, sleeving a blood vessel on an arterial cannula, ligating with an 8-0 silk thread, and finally removing a hemostatic clamp to restore blood supply.

2. Vascular digestion and single cell suspension preparation, namely taking an aortic graft from a C57BL/6J receptor mouse at the 4 th week after vascular grafting operation, anaesthetizing the transplanted mouse by using 1% pentobarbital sodium according to 50mg/kg, fixing, cutting off the transplanted mouse from the abdomen by using scissors, exposing the heart by opening the chest, cutting off a small opening in the right atrium, pouring PBS (phosphate buffered saline) from the left ventricle to the liver for whitening, pulling off the viscera, exposing the aorta, separating the peripheral adipose tissues cleanly along the aorta by using forceps, separating the aorta, placing the separated aorta into a 50mL centrifuge tube containing 10mL of DMEM high-sugar medium with 10% FBS, observing under a split microscope, fixing the blood vessel by using curved forceps, and separating the blood vessel and the adventitia by using microscopic forceps. The medium and adventitia were placed separately in another clean DMEM high sugar culture dish containing 10% fbs, medium and adventitia vessels were fixed with microscopic forceps, the vessels were cut longitudinally with microscopic scissors, then crushed transversely, the supernatant in the dish was transferred with a pasteur pipette and placed in a 50mL centrifuge tube, and inserted on ice, at which time only undigested tissue pieces remained in the dish.

About 400. Mu.L (1 mg/mL) of papain was added to a dish containing chopped tissue pieces, the tissue was covered with the digest, the dish containing the tissue pieces was placed in a 37℃shaker and shaken at 135rpm for 10min, after 10min, the digested tissue was placed under a microscope to observe digestion, a grape-like cell mass was found, the pellet was gently blown several times with a pipette, and the supernatant in the dish was transferred to a 50mL centrifuge tube with a Pasteur pipette. The culture dish was then continuously added with 300. Mu.L (1 mg/mL) of papain, the tissue was minced as much as possible with a microtcut, and the same conditions were digested once to find that the number of mirrored grape bead-like cells and free cells increased, the supernatant was continuously transferred to the 50mL centrifuge tube with a Pasteur pipette, then 300. Mu.L (2 mg/mL) of papain was added, placed in a 37℃shaker, and shaken at 135rpm for 5min, and the supernatant in the dish was transferred to the 50mL centrifuge tube with a Pasteur pipette. Finally, the tissue was found to be loose and mostly free cells by pipetting several times and adding 300. Mu.L (1 mg/ml) of papain and shaking it in a 37℃shaker at 135rpm for 5 min. The supernatant was transferred to the 50mL centrifuge tube using a pasteur pipette, at which point the tube was filled with cell suspension to terminate digestion. The cell suspension with stopped digestion was filtered through a 40 μm screen to remove cell mass and incompletely digested tissue mass, and the screen was filtered as small as possible over a small area to reduce cell loss, and after filtration, the screen was rinsed 2 times with 200 μl PBS to obtain vascular single cell suspension.

3. Single cell transcriptome sequencing, namely collecting single cell suspension prepared from vascular tissue at 4 weeks after transplantation, and detecting the cell activity rate by trypan blue staining counting, so as to ensure that the cell activity rate is in the range of 60-90%. Following a standardized 10x Genomics single cell sequencing protocol, libraries were created using 10x Genomics Single Cell 3'v2 chemical kit (10X Genomics,1000265,4rxns) from Shanghai European biomedical sciences, expected to capture 8000-10000 cells for reverse transcription, expansion and purification. By dividing cells into gel bead emulsions, single cells with specific 10x barcodes and unique molecular identifiers can be generated. The cDNA sequence with the same 10x barcode was then considered to be the sequence from 1 cell. The library was generated and then sequenced on a single cell sequencing platform Illumina NovaSeq, 6000 using a paired end 150bp (PE 150) sequencing strategy to a depth of 30M data per cell. Single cell RNA sequencing raw data the raw data was decomposed using CELL RANGER (version 2.2.0) and a FASTQ file was generated, the read was matched to the mouse reference genome mm10 data to calculate UMI numbers and generate matrix data. Subsequent quality control and cluster analysis was performed using R package Seurat (version 2.3.0). Cells expressing more than 5000 genes were filtered to exclude non-cells or cell aggregates, and cells with a mitochondrial gene percentage greater than 5% were filtered. After quality control, the main component analysis is carried out to reduce the dimension after the logarithmic standardization data, and the subsequent analysis is carried out.

4. Cell cluster analysis and cell subpopulation marker gene identification normalization, clustering, differential gene expression analysis and visualization were performed using R software package Seurat.0.2. Cell clustering was performed using the cell population identification function in FindClusters function and visualized using RunUMAP function. A specific marker gene for a cell subpopulation was identified using Seurat' FINDMAKERS function. The specific method is to determine the differentially expressed genes of a specific cell subpopulation by comparing the cells of that cell subpopulation with all other cells using the Wilcoxon rank sum test (DEGs). Bonferroni-corrected p values was less than 0.05 as cut-off value for identifying DEGS of statistical significance. Genes whose average expression level in a specific cell subset is more than 2 times higher than that in other cell subsets are selected as marker genes.

5. Transcriptomic data for metabolic analysis scRNA-seq data can be combined with metabolic networks using Compass algorithm to infer the metabolic status of individual cells. Setting 3 parameters including an expression matrix (- -data), a process number (- -num-processes), a species (- -specialties), and using default values for the rest parameters, dividing cells into clusters by Compass through (- -microcluster-size), characterizing the clusters by the average value of the clusters, and generating a reaction.csv file in the current catalog after running the command. Wherein the value is a reaction penalty (reaction penalties), a high score indicates a lower likelihood of the reaction. The reaction code is then converted to the metabolic pathway name, the reaction penalty is converted (to a large number indicating high reactivity, plus 1, taken-log), and then the difference analysis is performed using Wilcoxon or other method.

6. The scMetabolism software scores each cell by using a VISION algorithm based on a conventional single cell matrix file, and finally obtains the activity score of the cell in each metabolic pathway. The metabolic scoring calculation method comprises the steps of performing metabolic scoring calculation by using a Vision algorithm, wherein the first column of an average scoring statistical table is the name of a metabolic item matched to a metabolic gene set, each column is the average scoring value of different groups, and in an average_keGG_ heatmap heat map drawn according to the score, the metabolic scores of which channels in different groups are higher and the overall score condition of an item of interest can be primarily known. After the metabolic score of each cell is obtained, a bimod test mode is used for carrying out differential analysis to obtain paths with obvious differences, so that the metabolic activity of different paths among different groups is evaluated, in the result, top 10_KEGG_Significa_results are path information with obvious differences, the obtained obvious metabolic paths are screened, and a heat map and a violin map are further drawn.

7. ScFEA takes a metabolic reconstruction diagram as a factor graph, captures complex information association between transcriptomes and metabolome based on network optimization solution, and uses a multi-layer neural network to reduce enzyme gene expression and reaction rate so as to realize calculation of cell metabolic flux. The basic calculation framework is characterized in that a predefined metabolic map is used as input by combining a network topology factor graph, significant nonzero gene expression and a secondary network input by user definition, after the factor graph of different metabolic modules is reconstructed by using flux balance analysis, a loss function L is defined by calculating a flux balance loss term, nonnegative flux, consistency between predicted flux and gene expression and flux scale constraint, and is used for deducing the cell metabolic flux from scRNA-seq data.

The experimental results of this example are shown in FIG. 1.

In FIG. 1, panel A is a heat map of enrichment analysis of various T cell subsets (columns) metabolic pathways (rows) within mouse allograft vessels obtained by Compass analysis. The results show that during the differentiation from the CD34 ⁺ progenitor state to the potent T cell state (e.g., CD4 ⁺ T cells and Tfh cells), various metabolic pathways, including carbohydrate active metabolism, phosphoglyceride metabolism, are highly activated. Notably, expression of the Tfh cell one-carbon metabolism related genes is particularly high compared to gene expression of other effector CD4 ⁺ T subsets (e.g., CD4 ⁺ T cells, th17 cells, treg cells). Significant activation of one-carbon metabolism also distinguishes Tfh cells from other CD4 ⁺ T cell subsets in terms of metabolic pathways.

In fig. 1, panel B is a single cell transcriptome sequencing (scRNA-seq) analysis of CD4 positive T cells and follicular helper T cells in mouse allograft vessels. The results show that the key one-carbon metabolizing enzyme is significantly upregulated during CD4 ⁺ T differentiation into Tfh cells. In contrast to the intracytoplasmic one-carbon metabolic pathway, CD4 ⁺ T differentiation into Tfh cells may depend primarily on mitochondrial one-carbon flux.

In FIG. 1, panel C is a graph showing fold change in expression of the genes of the key enzymes of the cytoplasm and mitochondria of the one-carbon metabolism in follicular helper T cells versus CD 4-positive T cells. The results show that the mitochondrial (green) one-carbon enzyme gene is expressed higher than cytosol (yellow), and that it is most pronounced at Mthfd. Mitochondrial Mthfd2 was increased 8-fold in Tfh cells compared to CD4 ⁺ T cells.

Example 2MTHFD interfering RNA inhibits differentiation of CD4 ⁺ T cells into Tfh cells

Experimental materials:

CD4 ⁺ T cell immunomagnetic bead sorting kit, available from Miltenyi Biotec, cat# 130-104-454;

Recombinant mouse IL-6 protein, purchased from Abcam, cat# ab198572;

recombinant mouse IL-21 protein, purchased from Abcam, cat# ab280343;

anti-IFNγ monoclonal antibodies, available from Invitrogen under the accession number 16-7311-81;

anti-TGF-beta monoclonal antibodies, available from Invitrogen under the designation 16-9243-85;

anti-CD 3 monoclonal antibodies, available from Invitrogen under the accession number 16-0032-82;

anti-CD 28 monoclonal antibodies, available from Invitrogen under the designation 16-0281-82;

ovalbumin, purchased from Sigma, cat# O1641;

Brilliant Violet 421 ^TM anti-mouse CD185 (CXCR 5) antibody, available from BioLegend under the accession number 107712;

APC anti-mouse CD278 (ICOS) antibodies, available from BioLegend, cat# 107711;

zombie Aqua ^TM can immobilize nuclear dye, available from BioLegend, cat# 423101.

The experimental method comprises the following steps:

1. Isolation of mouse CD4 ⁺ T cells to perform in vitro Co-culture experiments, ovalbumin (OVA) -specific CD4 ⁺ T cells were isolated from C57BL/6J background OT-II mice (Jackson Laboratory, 004194) by means of a AutoMACS (Miltenyi Biotec) fully automated magnetic cell sorter using anti-CD 4 microbeads and co-cultured with irradiated C57BL/6J mouse spleen Antigen Presenting Cells (APC) in the presence of the OVA323-339 peptide (amino acid sequence ISQAVHAAHAEINEAGR (SEQ ID NO: 1)). The spleen was homogenized by a plunger head of a 5mL syringe, filtered through a 40 μm filter and centrifuged at 1500rpm for 5 minutes at 4 ℃. After lysing the erythrocytes using ACK lysis buffer, the cells isolated from the spleen were washed twice with PBS and resuspended in RPMI1640 medium. CD4 ⁺ T cells were isolated using CD4 ⁺ T cell immunomagnetic bead sorting kit according to the recommended instructions. Purified CD4 ⁺ T cells (initial cell number 0.3 x10 ⁶) were treated in a co-culture system.

2. MTHFD2 siRNA transfection and activation induction of mouse CD4 ⁺ T cells purified CD4 ⁺ T cells were transfected with MTHFD2 specific siRNA (sequence: CACTCCTATGTTCTCAACAAA (SEQ ID NO: 2)) at a final concentration of 30nM using Lipofectamine 3000, shRNA sequence ：Top strand:GATCCGCACTCCTATGTTCTCAACAAACTCGAGTTTGTTGAGAACATAGGAGTGTTTTTTG(SEQ ID NO:3),Bottom strand:AATTCAAAAAACACTCCTATGTTCTCAACAAACTCGAGTTTGTTGAGAACATAGGAGTGCG(SEQ ID NO:4)), and transfected with NC siRNA (sequence: TTCTCCGAACGTGTCACGTAA (SEQ ID NO: 5)) at the same concentration, the group of shRNA sequence ：Top strand:GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC(SEQ ID NO:6),Bottom strand:AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG(SEQ ID NO:7)) served as a blank control group after 48 hours incubation at 37℃under 5% CO ₂, total RNA of the cells was extracted, mRNA levels of MTHFD2 (forward primer: 5'-TGGAGGAGGGACAGAACGAG-3' (SEQ ID NO: 8), reverse primer: 5'-ACGGACAGGTCTTCTGCTGA-3' (SEQ ID NO: 9)) were detected by qPCR to evaluate transfection efficiency and gene knockdown effects by adding recombinant mouse IL-6 protein, recombinant mouse IL-21 protein, anti-IFN gamma monoclonal antibody and anti-TGF-beta monoclonal antibody at 50ng/mL for 20 hours, and then preparing an anti-CD 3 monoclonal antibody (10. Mu.g/CD 28. Mu.g) and after 16. Mu.mL monoclonal antibody stimulation of the cells.

3. Flow cytometry to detect the proportion of Tfh cells single cell suspensions were first incubated with Zombie Aqua ^TM fixable nuclear dye for 30min, then with Brilliant Violet 421 ^TM anti-mouse CD185 (CXCR 5) antibody and APC anti-mouse CD278 (ICOS) antibody for 30min. Absolute cell counts were detected by flow cytometry (BD FACSCalibur, USA) and analyzed using FlowJo software version X (Tree Star inc., USA) to detect ICOS and CXCR5 double positive Tfh cell ratios.

The experimental results of this example are shown in FIG. 2.

In fig. 2, panel a is a graph of quantitative statistical analysis of mRNA expression levels of qPCR assay MTHFD2 in CD4 ⁺ T cells transfected with NC siRNA and MTHFD siRNA. Experimental results show that the mRNA level of MTHFD is significantly reduced in CD4 ⁺ T cells transfected with MTHFD siRNA, and that the B diagram is a quantitative statistical analysis diagram of the percentage of ICOS and CXCR5 double positive Tfh cells in total living cells in flow cytometry detection in CD4 ⁺ T cells transfected with NC siRNA and MTHFD siRNA. The results showed that the proportion of Tfh cells in CD4 ⁺ T cells transfected with MTHFD siRNA was significantly lower than in the blank.

Example 3MTHFD2 inhibitors reduce TLO formation in reconstituted vessels

Experimental materials:

MTHFD 2A specific targeting inhibitor DS18561882, available from Med Chem Express, cat# HY-130251;

phosphate Buffered Saline (PBS), available from HyClone, cat# SH30258.01;

4% paraformaldehyde fixative, available from Shanghai Biyun biotechnology Co., ltd., product number: P0099-500ml;

CUBIC-L solution, available from TCI, cat# T3740;

CUBIC-R+ solution, available from TCI, cat# T3741;

anti-CD 3 monoclonal antibodies, available from Abcam, cat# ab237721;

AF488 fluorescent goat anti-rabbit IgG antibody, purchased from Abcam, cat# ab150077;

APC fluorescent anti-mouse/human CD45R/B220 antibodies were purchased from BioLegend, cat# 103212.

The experimental method comprises the following steps:

1. mice MTHFD were injected with inhibitors the experiment was performed using DS18561882 to inhibit MTHFD. A mouse allogeneic vascular graft model was established according to step 1 of example 1, the post-operative mice were randomly assigned to one of two groups, each group consisting of 6 mice using a computer-generated random number generator, the first group being a control group (i.e., a solvent group) to which the mice were intraperitoneally injected with a carrier solution at a dose of 10. Mu.L/g of mouse body weight, the carrier solution being a control solvent solution containing 10vol% DMSO, 10vol% PEG400, and 5vol% Tween-80 in physiological saline, and the second group being an inhibitor group to which DS18561882 dissolved in a solvent (the carrier solution described above) was intraperitoneally injected at a dose of 100mg/kg of mouse body weight. The solutions for each group were injected once every 35 hours until the materials were obtained.

2. Tissue permeabilization and 3D imaging detection of tertiary lymph node formation, namely collecting blood vessels at the 4 th week after blood vessel transplantation, performing tissue permeabilization, carefully removing a sleeve and an external suture at an interface after material drawing, fixing with 4% paraformaldehyde for 2 hours, washing with PBS for 3 times each for 20 minutes, washing off the paraformaldehyde, transferring the sample into a 5mL centrifuge tube which is preloaded with a tissue transparentizing reagent CUBIC-L, permeabilizing at 37 ℃ overnight, washing with PBS for three times each for 1 hour, washing off the CUBIC-L, shaking and observing that no large amount of foam is generated in the rinse liquid, after washing is completed, sealing with 1% BSA at normal temperature for 2 hours, taking out the sample, slightly sucking sealing liquid with filter paper, and performing tertiary lymphocyte staining including T cells and B cells. The specific antibodies are T cells marked by using an anti-CD 3 monoclonal antibody as a primary antibody pair CD3, and B cells marked by using APC fluorescent anti-mouse/human CD45R/B220 as a primary antibody pair B220. Incubation overnight with primary antibody working solution at normal temperature, washing with PBS for 3 times for 2 hours each time, transferring into secondary antibody (AF 488 fluorescence goat anti-rabbit IgG antibody) working solution, incubating overnight, washing with PBS for 3 times for 2 hours each time, transferring into CUBIC-R+, and immediately making the blood vessel of the mouse enter into CUBIC-R+ to become completely transparent. And then fixing the gasket on the glass slide by using an inert adhesive, dropwise adding a proper amount of CUBIC-R+ into the inner hole CUBIC-R+ solution, supplementing the CUBIC-R+ until the liquid level is slightly higher than the height of the gasket, sealing the inner hole by using a cover glass, and carefully sealing the adhesive to ensure that the CUBIC-R+ does not exude. The CUBIC-R+ can precipitate a large amount of crystals after evaporating water in the air, and the exuded CUBIC-R+ solution is carefully wiped off and then blocked by using an adhesive. Whole bright field and fluorescence images of aortic grafts and adjacent tissues were acquired under 5x (0.1 Numerical Aperture (NA)) illumination using a ZEISS Lightsheet Z.1light Sheet fluorescence microscope (17002189, ZEISS, oberkochen, germany), 3D imaging using illumination objective 5x (0.1 NA) and imaging objective transparency objective 5x (0.16 NA), data analysis was performed using Imaris software (version 9.5.1), and tertiary lymphoid tissue formation was detected.

The experimental results of this example are shown in FIG. 3.

FIG. 3 is a graph showing quantitative statistical analysis of tertiary lymphoid tissue in allograft vessels at week 4 in both control and inhibitor groups of mice. The results show that MTHFD of inhibition can inhibit the formation of vascular tertiary lymphoid tissue, and that mice injected with the inhibitor group of DS18561882, which is the inhibitor for MTHFD2, do not observe the formation of vascular tertiary lymphoid tissue after allogeneic vascular grafting, and are significantly less than the control group.

Example 4 inhibition MTHFD2 remission of graft vascular lesions

Experimental materials:

phosphate Buffered Saline (PBS), available from HyClone, cat# SH30258.01;

hematoxylin dye, purchased from Sigma-Aldrich, cat# GHS216;

eosin dye, available from Sigma-Aldrich, cat# 48900;

xylene, available from Sigma-Aldrich under the trade designation 534056-500ML.

The experimental method comprises the following steps:

1. Blood vessel sample section a control group (i.e. solvent group) and inhibitor group were set as per step 1 of example 3, blood vessel tissue at week 4 after blood vessel grafting was collected and fixed with 4% paraformaldehyde, then the tissue was dehydrated overnight in 30% sucrose aqueous solution, the tissue was embedded in OCT and frozen at-80 ℃, and sections with a thickness of 4-6 μm were made using CryoStar Cryostat (Thermo Scientific). When frozen slicing is carried out, the temperature of the slicing machine is reduced to reach the preset temperature (-20 ℃) in advance, the sample holder and the sample are fixed at the position of the quick freezing frame until the sample is completely frozen, the pre-cooled sample is inserted into the sample head, the anti-rolling plate is folded to one side, the pre-cooled slice is inserted, the fastening lock rod on the base is opened, the slicing knife or the blade holder approaches the sample, then the lock rod is closed again, the coarse push button approaches the slicing knife, the hand wheel is slowly rotated, and the sample is subjected to slice repairing, slicing and pasting. Intimal hyperplasia area and lumen area were detected by H & E staining.

2. H & E staining, namely placing the prepared frozen section at room temperature for about 20min, gently flushing for about 3-5min by tap water, removing OCT glue, fixing for 15min by 4% paraformaldehyde, and then washing for 3 times by PBS for 5min each time. The cell nuclei were stained by incubation with hematoxylin working solution for 5min, then differentiated with 1% alcoholic hydrochloric acid for 2s, rinsed 3-4 times with tap water, and then reversed blue with Scott blue-forming solution for 4s, and rinsed 3-4 times with tap water. Soaking in 85% alcohol and 95% alcohol for 4min, dehydrating, incubating with eosin dye solution for 4min, staining cell slurry, dehydrating with anhydrous alcohol for 3 times and 4min each time, soaking the dehydrated tissue sample slice with xylene for 2 times and 4min each time, sealing the tissue sample with neutral resin, and photographing.

The experimental results of this example are shown in FIG. 4.

In fig. 4, panel a is a representative picture of H & E staining of the 4 th week graft vessel section of the vascular grafts in the control group (left) and the inhibitor group (right) mice. The results show that MTHFD inhibition slowed vascular remodeling due to allograft.

In fig. 4, panel B shows the intima-to-media (I/M) ratio (intima-to-media (I/M) ratio = intima-to-media thickness) for the control and inhibitor groups, and panel C shows the reconstructed vessel lumen area in pixels (Pixels) for the control and inhibitor groups. The results show MTHFD that inhibition of MTHFD reduces the intima to media ratio and increases the area of the lumen of the reconstituted vessel, alleviating graft vascular lesions.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples represent only a few embodiments of the present disclosure, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that variations and modifications can be made by those skilled in the art without departing from the spirit of the disclosure, which are within the scope of the disclosure. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. Use of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) inhibitors in the preparation of products for preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related diseases.

2. The use according to claim 1, characterized in that: the MTHFD2 inhibitor is selected from one or more of the following: antibodies or antigen-binding fragments thereof, nucleic acids, and small molecule compounds against MTHFD2.

3. The use according to claim 1 or 2, characterized in that: the vascular remodeling is vascular remodeling associated with vascular-related diseases;

Preferably, the disease is selected from vascular wall damage, vascular stenosis after injury, blood flow dysfunction after injury, thrombosis, vascular stenosis after PCI and Bypass surgery, coronary heart disease, myocardial ischemia, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, aneurysm, atherosclerosis, cerebral infarction, transplant vascular lesions after allogeneic transplantation, tumor angiogenesis, transplant rejection, diabetic microangiopathy, cerebrovascular disease and any combination thereof.

4. The following uses of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) or its encoding gene as a biomarker or target, and/or a reagent for detecting said methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) or its encoding gene:

i. Application in screening for vascular remodeling and/or diseases related to vascular remodeling or in preparing products for screening for vascular remodeling and/or diseases related to vascular remodeling;

ii. Application in evaluating vascular remodeling and/or vascular remodeling-related diseases or in preparing products for evaluating vascular remodeling and/or vascular remodeling-related diseases;

iii. Application in the preparation of products for diagnosing vascular remodeling and/or vascular remodeling-related diseases;

iv. Application in screening and/or preparing products for preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related diseases;

v. Application in monitoring the course of vascular remodeling and/or vascular remodeling-related diseases or in preparing products for monitoring the course of vascular remodeling and/or vascular remodeling-related diseases;

vi. Application in analyzing vascular remodeling and/or the prognosis of diseases related to vascular remodeling or in preparing products for analyzing vascular remodeling and/or the prognosis of diseases related to vascular remodeling;

vii. Use in inhibiting the differentiation of CD4 ⁺ T cells into Tfh cells and/or the formation of tertiary vascular lymphoid tissue or in preparing a product that inhibits the differentiation of CD4 ⁺ T cells into Tfh cells and/or the formation of tertiary vascular lymphoid tissue.

5. The use according to claim 4, characterized in that: the reagent is a reagent for detecting the expression amount and/or activity level of the MTHFD2 and/or a reagent for detecting the mRNA expression amount of the MTHFD2 encoding gene;

Preferably, the reagents include reagents for detecting the expression amount of the biomarker by PCR, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunocoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance, immuno-PCR or biotin-avidin technology.

6. The use according to claim 4 or 5, characterized in that the expression level of the MTHFD2 protein encoding gene, the expression level and/or activity of the MTHFD2 protein in patients with vascular remodeling and/or vascular remodeling-related diseases are increased compared with those in a normal control group.

7. The use according to any one of claims 4 to 6, characterized in that: the vascular remodeling is a vascular remodeling associated with a vascular-related disease or condition;

Preferably, the disease or condition is selected from vascular wall damage, vascular stenosis after injury, blood flow dysfunction after injury, thrombosis, vascular stenosis after PCI and Bypass surgery, coronary heart disease, myocardial ischemia, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, aneurysm, atherosclerosis, cerebral infarction, transplant vascular disease after allogeneic transplantation, tumor angiogenesis, transplant rejection, diabetic microangiopathy, cerebrovascular disease and any combination thereof.

8. A method for screening drugs for preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related diseases, comprising: selecting a substance that inhibits methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) from the compounds to be screened as a drug for preventing, treating and/or alleviating vascular remodeling and/or vascular remodeling-related diseases.

9. The method according to claim 8, characterized in that: the vascular remodeling is vascular remodeling associated with a vascular-related disease or condition;

10. A product comprising or coated with an effective amount of a methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) inhibitor;

Preferably, the MTHFD2 inhibitor is selected from one or more of the following: antibodies or antigen-binding fragments thereof, nucleic acids, small molecules against MTHFD2;

Preferably, the product is a vascular stent or a catheter with a balloon; or the product is a pharmaceutical composition comprising the MTHFD2 inhibitor as an active ingredient and further comprising a pharmaceutically acceptable excipient.