Application of SPP1 in treatment of pulmonary arterial hypertension

Abstract

The invention relates to the technical field of biological medicine, in particular to application of SPP1 in treatment of pulmonary arterial hypertension, and is technically characterized in that SPP1 serves as multifunctional extracellular matrix protein to prepare medicine for treating pulmonary arterial hypertension, and the medicine is an inhibitor; the expression profile of the SPP1 is highly overlapped with the malignant transformation of the PAH. The invention also provides application of the SPP1 inhibitor in preparation of drugs for treating pulmonary arterial hypertension. The SPP1 serving as a multifunctional extracellular matrix protein in the scheme not only participates in pathological processes such as cell adhesion, signal transduction and inflammatory response, but also is proved to play an important role in tumor evolution, bone growth, immunoregulation and the like; the invention discloses a pulmonary arterial hypertension malignant transformation mechanism, abnormal proliferation of the pulmonary arterial hypertension can be inhibited in a targeted manner, pulmonary vascular remodeling is blocked, and a key treatment strategy is provided for treatment of pulmonary arterial hypertension.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically, to the application of SPP1 in the treatment of pulmonary arterial hypertension. Background Art

[0002] Pulmonary arterial hypertension (PAH) is a fatal disease characterized by pulmonary vascular remodeling and right heart failure, and existing treatment methods are difficult to reverse the process of vascular remodeling. Although current research mainly focuses on vasoconstriction and inflammation regulation, its molecular mechanism has not been fully elucidated, and there is a lack of systematic analysis of the core molecular signaling pathways driving vascular remodeling. Research shows that the core pathological links of pulmonary artery structural remodeling include abnormal proliferation of vascular cells, functional imbalance, and persistent inflammation. Among them, pulmonary artery smooth muscle cells are the core functional units of the vascular wall, maintaining normal pulmonary artery blood pressure by dynamically regulating vascular tension and blood flow. Under pathological conditions, their abnormal proliferation and phenotypic malignant transformation directly lead to irreversible lumen stenosis, increased pulmonary vascular resistance, and increased right heart load. During the progression of pulmonary arterial hypertension, various pathological stimuli (such as hypoxia, inflammatory factors, and mechanical stress) drive the imbalance of the pulmonary vascular microenvironment homeostasis, forming a pro-remodeling malignant phenotype. Its core mechanisms include:

[0003] 1) Excessive proliferation of PASMCs causes persistent vasospasm and lumen stenosis;

[0004] 2) Activated PASMCs secrete excessive amounts of extracellular matrix (ECM) such as collagen and fibronectin, resulting in pathological thickening of the vascular wall;

[0005] 3) Abnormal deposition of ECM and imbalance of vascular tension form an inflammation-fibrosis cascade reaction, exacerbating endothelial dysfunction and vascular fibrosis;

[0006] 4) PASMCs can also secrete inflammatory factors such as Interleukin-6 (IL-6), inducing abnormal infiltration of immune cells (such as macrophages and T cells), remodeling the inflammatory microenvironment, and the chronic inflammatory microenvironment further leads to T cell functional exhaustion, forming a vicious cycle. In this pathological cascade reaction, the proliferation of PASMCs is the core driving factor. Revealing its malignant transformation mechanism and targeting and inhibiting its abnormal proliferation are the key treatment strategies for blocking pulmonary vascular remodeling.

[0007] Therefore, the present application provides the application of SPP1 in the treatment of pulmonary arterial hypertension to solve the above problems. Summary of the Invention

[0008] The purpose of the present invention is to solve the technical problems raised in the above background art and provide the application of SPP1 in the treatment of pulmonary arterial hypertension.

[0009] The above object of the present invention is achieved as follows:

[0010] The solution of the present invention provides the application of SPP1 as a multifunctional extracellular matrix protein in the preparation of drugs for treating pulmonary arterial hypertension.

[0011] Furthermore, the drug is an inhibitor.

[0012] Furthermore, the expression profile of SPP1 highly coincides with the malignant transformation of PAH.

[0013] The solution of the present invention also provides the application of an SPP1 inhibitor in the preparation of drugs for treating pulmonary arterial hypertension.

[0014] Furthermore, the inhibitor includes thalidomide.

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

[0016] 1. The solution of the present invention systematically analyzes the molecular switch and mechanism of action of the malignant transformation of PAH, and combines the previous single-cell multi-omics data of pulmonary arteries of PAH patients (scRNA-seq / bulk RNA-seq, GSE228644) by the inventors of the present application, and discovers and confirms that the expression profile of secreted phosphoprotein 1 (SPP1) is highly correlated with the malignant progression of the vascular microenvironment. Inhibiting its expression in vivo and in vitro can effectively relieve pulmonary vascular remodeling and reverse the PAH process;

[0017] 2. As a multifunctional extracellular matrix protein, SPP1 in the solution of the present invention not only participates in pathological processes such as cell adhesion, signal transduction and inflammatory response, but is also proven to play an important role in tumor progression, bone growth, immune regulation, etc. The present invention reveals the mechanism of malignant transformation of pulmonary arterial hypertension, can target and inhibit its abnormal proliferation, block pulmonary vascular remodeling, and provides a key treatment strategy for the treatment of pulmonary arterial hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows that the expression profile of SPP1 highly coincides with the malignant transformation of PAH in the embodiment of the present invention;

[0019] Figure 2 shows that the expression of SPP1 is significantly up-regulated in the malignant microenvironment of PAH in the embodiment of the present invention;

[0020] Figure 3 shows that the expression of SPP1 is up-regulated and promotes the malignant transformation of pulmonary artery smooth muscle cells in the embodiment of the present invention;

[0021] Figure 4In the embodiment of the present invention, SPP1 is specifically knocked out in vivo smooth muscle to hinder the progression of pulmonary hypertension. Detailed implementation manners

[0022] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0024] In the accompanying drawings of this embodiment, the same or similar reference numerals correspond to the same or similar components; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", etc. indicating the orientation or positional relationship, they are based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the accompanying drawings are only for illustrative purposes and cannot be understood as limiting the present invention. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific circumstances.

[0025] Refer to Figures 1-4 As shown, it is a preferred embodiment provided by the present invention.

[0026] Embodiment: The embodiment solution of the present invention provides the application of SPP1 in the treatment of pulmonary hypertension, specifically: the application of SPP1 as a multifunctional extracellular matrix protein in the preparation of drugs for the treatment of pulmonary hypertension; the drug is an inhibitor; wherein, the expression profile of SPP1 highly coincides with the malignant transformation of PAH. The solution of this embodiment also provides the application of SPP1 inhibitors in the preparation of drugs for the treatment of pulmonary hypertension. In this embodiment, the inhibitor includes but is not limited to thalidomide.

[0027] The following is the specific implementation process of the embodiment of the present invention:

[0028] (1) Analyze the data to screen for the key signaling pathways and molecular switches that regulate the malignant vascular microenvironment of PAH, and find that SPP1 plays an important regulatory role in the malignant transformation of pulmonary artery cells in PAH. In the early stage of this application, the single-cell sequencing data of pulmonary arteries from patients with primary PAH disease were analyzed. UMAP visualization was used, and a variety of subcellular marker genes were selected for cell clustering to reveal the cell components of the pulmonary artery vascular microenvironment. Pseudotime analysis was used to determine the dynamic changes of the pulmonary artery cell population. It was found that PASMC showed an explosive activation of the DNA damage repair pathway at the terminal differentiation stage. Through analysis, screening and experimental verification, it was found that the changes in the SPP1 gene highly coincided with the dynamic transformation process in the PAH pulmonary artery vascular microenvironment.

[0029] As Figure 1 shown, it is the highly coincidence of the SPP1 expression profile and the malignant transformation of PAH. Figure 1 Among them: A. Bioinformatics analysis of single-cell data of pulmonary arteries from PAH patients, and UMAP visualization of cell clustering. B & C. Pseudotime analysis of the subtype changes of the PAH pulmonary artery cell population. Pulmonary artery smooth muscle changes from benign (LRPPRC-Sirt2-BECN1-FKBPL-MAP3K20-CAVIN3-ACTA2+) to malignant proliferation / apoptosis-inhibiting type (CDK1+PCNA+FOXO1+NAT10+ITGB1+CELRP+HIF1α+ID4-). D - F. Pseudotime analysis of the dynamic changes of the PAH pulmonary artery cell population. The expression of SPP1 highly coincides with the malignant transformation process of PAH (F). G. Enrichment analysis of signaling pathways in the normal group and the PAH group. DNA damage, repair, and inflammatory responses were significantly enhanced in the PAH group, and were mainly localized in pulmonary artery smooth muscle cells. I. The expression of SPP1 was enhanced in the PAH group. J. Pseudotime analysis of the dynamic changes of SPP1 when cells transform from normal to malignant PAH.

[0030] (2) Detect the expression changes of SPP1 in the PAH malignant microenvironment in vivo and in vitro, and find that the expression of SPP1 is significantly increased in pulmonary artery smooth muscle cells and regulates various signaling pathways such as DNA damage and repair.

[0031] As Figure 2 shown, it is the obvious upregulation of SPP1 expression in the PAH malignant microenvironment. Figure 2Chinese: A. Venn diagram analysis reveals the intersection of differential genes in endothelial and smooth muscle cells, screening for genes regulating the PAH malignant microenvironment. B. Volcano plot of gene changes in PAH vascular endothelial cells, with SPP1 showing a significant upregulation in PAH endothelial cells. C. Volcano plot of gene changes in PAH vascular smooth muscle cells, with SPP1 showing a significant upregulation in PAH smooth muscle cells. D. SPP1 is significantly upregulated in the data of patients with idiopathic pulmonary arterial hypertension in GSE283520. E. SPP1 is significantly upregulated in the constructed hypoxia mouse and monocrotaline-induced pulmonary arterial hypertension models. F. ATAC-seq was performed on hypoxic pulmonary arterial hypertension mice, and the chromatin accessibility of the enhancer of SPP1 was significantly opened in hypoxic pulmonary arterial hypertension mice. G. Immunofluorescence shows that SPP1 is mainly localized in the pulmonary artery smooth muscle layer. H. qRT-PCR was used to detect the mRNA level of SPP1 in hypoxic and monocrotaline mouse models, and the transcriptional level of SPP1 was significantly increased in hypoxic and monocrotaline mouse models. I&J. Primary human pulmonary artery smooth muscle cells were treated with hypoxia, and the protein level (I) and transcriptional level (J) of SPP1 were significantly upregulated. K. KEGG analysis found that SPP1 can affect signaling pathways by binding to multiple proteins. L. GSEA analysis of the correlation between SPP1 and diseases found that SPP1 can affect cell proliferation, cellular response to hypoxia signaling, inflammatory response, and DNA repair.

[0032] (3) Overexpression of SPP1 promotes the transformation of pulmonary artery smooth muscle cells towards abnormal proliferation.

[0033] As Figure 3 shown, SPP1 expression is upregulated and promotes the malignant transformation of pulmonary artery smooth muscle cells. Figure 3 Chinese: A. Lentivirus overexpressing SPP1 was constructed and infected with pulmonary artery smooth muscle cells. Overexpression of SPP1 promoted the migration of pulmonary artery smooth muscle cells. B. CCK8 was used to detect the proliferation of pulmonary artery smooth muscle cells. Overexpression of SPP1 promoted the proliferation of pulmonary artery smooth muscle cells. C. Confocal microscopy was used to detect cell ki67. Overexpression of SPP1 promoted the expression of cell ki67. D. The supernatant of pulmonary artery smooth muscle cells was collected, ultra-concentrated, and WB was used to detect the expression of SPP1. The secretion of SPP1 increased under hypoxic conditions. E. RNA-seq was used to detect the RNA level of pulmonary artery smooth muscle cells. The expression of SPP1 increased the mRNA level of IL6. F. Inflammatory factor chip was used to detect the synthesis of inflammatory factors in pulmonary artery smooth muscle cells. The cells overexpressing SPP1 promoted the protein level of IL6. G. Immunoinfiltration analysis of the correlation between SPP1 and various immune cells indicated a positive correlation between SPP1 and CD8-positive T cells (cytotoxic T cells). H. Correlation analysis of the correlation between SPP1 and inflammatory factors in different types of PH showed a positive correlation between SPP1 and IL6, CCL5, and G-CSF in cells.

[0034] (4) Constructed SPP1 smooth muscle-specific transgenic knockout mice. After knocking out SPP1, the progression of pulmonary hypertension was inhibited.

[0035] In this application, a perfect vascular cell-specific gene knockout system has been constructed. Using the Cre-Loxp gene knockout system, SPP1 knockout gene Floxed mice are mated with Tg(Tagln-cre)1Her mice (vascular smooth muscle cell Cre tool mice). After induction with Tamoxifen, this Cre will be specifically activated in vascular smooth muscle cells. Knockout of SPP1 specifically in smooth muscle in vivo hinders the progression of pulmonary hypertension.

[0036] As Figure 4 shown, knockout of SPP1 specifically in smooth muscle in vivo hinders the progression of pulmonary hypertension. Figure 4 Among them: A. Roadmap for constructing sugen5416 and chronic hypoxia-induced pulmonary hypertension mice. B. Detecting the right ventricular systolic pressure of mice using a right heart catheter. Specific knockout of SPP1 reduced the increase in right ventricular systolic pressure in mice under hypoxic conditions. C. Detecting the effects of specific knockout of SPP1 on the cardiopulmonary function of mice using small animal ultrasound. Knockout of SPP1 restored cardiopulmonary function. RVFWT (Right Ventricular Free Wall Thickness) reflects right ventricular hypertrophy; the ratio of PAAT (Pulmonary Artery Acceleration Time) to PAET (Pulmonary Artery Ejection Time) reflects pulmonary artery pressure, and a decrease indicates an increase in pulmonary artery pressure; RV / LV+Septum refers to the ratio of the right ventricle (RV) to the left ventricle (LV) plus the septum (S), reflecting right ventricular hypertrophy; TASPE (Tricuspid Annular Plane Systolic Excursion) reflects right ventricular systolic function; RVOT AT (Right Ventricular Outflow Tract Acceleration Time), shortening of RVOT AT indicates an increase in right ventricular afterload and a decrease in pulmonary artery compliance. D. Pulmonary arteriography. The distal end of the pulmonary artery in pulmonary hypertension mice was significantly blocked, and blood flow in the distal pulmonary artery of mice could be restored after knocking out SPP1. E. Detecting vascular remodeling in mice by H&E staining. Knockout of SPP1 inhibited the thickening of the mouse vascular wall.

[0037] Through the above embodiments of the present invention, SPP1 in the present invention's solution, as a multifunctional extracellular matrix protein, not only participates in pathological processes such as cell adhesion, signal transduction, and inflammatory responses, but has also been proven to play an important role in aspects such as tumor progression, bone growth, and immune regulation; the present invention reveals the mechanism of malignant transformation of pulmonary hypertension, and the application of SPP1 provided by the present invention in the treatment of pulmonary hypertension can target and inhibit its abnormal proliferation and block pulmonary vascular remodeling, providing a key treatment strategy for the treatment of pulmonary hypertension.

[0038] The foregoing are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of SPP1 as a multifunctional extracellular matrix protein in the preparation of a medicament for treating pulmonary arterial hypertension.

2. The application according to claim 1, wherein The medicament is an inhibitor.

3. The application according to claim 1, characterized in that, The expression profile of SPP1 highly coincides with the malignant transformation of PAH.

4. Use of an SPP1 inhibitor in the preparation of a medicament for treating pulmonary arterial hypertension.

5. The application according to claim 1, wherein The inhibitor includes thalidomide.

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