Identification and application of pulmonary arterial partial endothelial mesenchymal transformation subgroup of pulmonary arterial hypertension related to left heart disease

Abstract

The invention provides identification and application of a left heart disease-related pulmonary arterial hypertension pulmonary artery part endothelial mesenchymal transformation subgroup, and finds that according to the ratio of expression levels of endothelial cell markers (such as VWF and CD31) and mesenchymal cell markers (such as alpha-SMA, DCN and TAGLN) in an EndMT cell subgroup, the expression level of the endothelial cell markers (such as VWF and CD31) in the EndMT cell subgroup is determined, and the expression level of the mesenchymal cell markers (such as alpha-SMA, DCN and TAGLN) in the EndMT cell subgroup is determined. And the expression levels of TGF-beta1, TNFRSF1A, CXCL12, CCL14, CD74, TGFBR2 and CD44 in the EndMT cell subset can (i) be used for typing patients of PH-LHD according to different periods of mesenchymal transformation of endothelial cells; and / or (ii) is used for judging whether a patient suffering from the left heart disease related pulmonary hypertension (PH-LHD) is suitable for adopting a CD74 inhibitor, and / or a CD44 inhibitor is used as a target spot for treating the left heart disease related pulmonary hypertension (PH-LHD).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the identification and application of a pulmonary artery endothelial mesenchymal transition subpopulation in pulmonary artery of pulmonary hypertension related to left heart disease. BACKGROUND

[0002] Pulmonary hypertension (PH) is a severe progressive disease characterized by pulmonary vascular remodeling, right ventricular hypertrophy and failure. It can exist independently as a disease, or as a complication of various diseases in the later stage, with high morbidity and mortality, and is known as "the neglected killer" and "cancer of the heart-lung vascular system". Left heart disease (LHD) is the most common cause of PH, and about 2 / 3 of severe LHD will develop into PH. This PH caused by LHD is called pulmonary hypertension related to left heart disease (PH-LHD), which belongs to type II PH and is the most common type of PH in clinic. The pathogenesis of PH-LHD is unclear, and the pathophysiology is complex Figure 1 ), that is, LHD leads to increased left ventricular filling pressure, pulmonary venous reflux obstruction, and elevated pulmonary venous pressure, ultimately leading to pulmonary vascular remodeling and increased pulmonary vascular resistance. The treatment of PH-LHD includes LHD treatment and PH treatment. At the early stage of the disease, the increase in pulmonary vascular resistance is caused by pulmonary arteriolar spasm and contraction, and the pathological changes are functional and reversible. At this time, timely correction of LHD can achieve satisfactory results. In the later stage of the disease, pathological changes occur in the structure and function of the vascular wall, such as endothelial dysfunction, intimal hyperplasia, vascular smooth muscle cell proliferation, excessive deposition of collagen, middle layer thickening, and lumen stenosis and occlusion, i.e. "pulmonary vascular remodeling". At this time, the increase in pulmonary vascular resistance is irreversible, and even if LHD is cured, PH is difficult to alleviate, and patients eventually die of right heart failure. Therefore, PH-LHD has a higher morbidity and mortality than other types of PH. In summary, PH-LHD has a higher morbidity and mortality than other types of PH due to the lack of specific and effective treatment methods. Therefore, it is of great significance to the clinical treatment of PH-LHD to further explore the mechanism of pulmonary vascular remodeling and block the development of PH at the early stage of the disease.

[0003] EndMT refers to the process of endothelial cells gradually transforming into mesenchymal cells under the stimulation of external factors such as blood flow pressure, shear force, and immune inflammatory factor infiltration, characterized by the gradual disappearance of endothelial cell characteristics and the gradual acquisition of mesenchymal characteristics, and the English abbreviation is EndMT (Endothelial mesenchymal transformation). EndMT is an important pathophysiological mechanism of pulmonary vascular remodeling in pulmonary arterial hypertension. Previous studies have shown that EndMT is an all-or-nothing process, but recent research has found that EndMT is a gradual process, from endothelial cells to partial mesenchymal transformation, and finally to complete mesenchymal transformation. The specific regulatory mechanism of EndMT is unclear.

[0004] Pulmonary hypertension (PH) is a serious progressive disease, mainly manifested as abnormal contraction and increased resistance of pulmonary vessels, eventually leading to right heart failure and death. Global burden of disease research lists it as one of the major global health threats in 2021. Left heart disease (LHD) is the most common clinical cause of PH, and about 60% of LHD cases progress to PH. Left heart diseases such as valvular heart disease and left ventricular outflow obstruction gradually affect the systolic and diastolic function of the left heart. Passive backward filling from the left ventricle into the pulmonary circulation leads to pulmonary vascular remodeling, which is the main pathophysiological marker of the progression of pulmonary arterial hypertension caused by left heart disease (PH-LHD).

[0005] Currently, there is no good treatment for PH-LHD, and only drugs can be used to dilate blood vessels to relieve symptoms.

[0006] Therefore, there is an urgent need in the art to develop new methods that can effectively treat PH-LHD. SUMMARY

[0007] The purpose of the present application is to provide a new method that can effectively treat PH-LHD.

[0008] Another purpose of the present application is to provide markers for the early and late stages of pulmonary arterial endothelial mesenchymal transformation in PH-LHD disease, and to use different inhibitors for treatment after staging EndMT using different markers.

[0009] In the first aspect of the present application, a method for (i) typing patients with PH-LHD according to different stages of endothelial cell mesenchymal transformation; and / or (ii) using as a biomarker for determining whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease-related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the biomarker comprises: an EndMT cell subpopulation.

[0010] In another preferred embodiment, the EndMT cell subpopulation comprises an Endmtl cell subpopulation, an Endmt2 cell subpopulation, an Endmt3 cell subpopulation, an Endmt4 cell subpopulation, an Endmt5 cell subpopulation.

[0011] In another preferred embodiment, the EndMT cell subpopulation has one or more characteristics selected from the group consisting of:

[0012] (a) decreased expression of endothelial cell markers and increased expression of mesenchymal cell markers or increased ratio of mesenchymal cell markers / endothelial markers;

[0013] (b) up-regulated expression of TGF-β1, TNFRSF1A, CXCL12, CCL14 and CD74 at early stage of mesenchymal transition of endothelial cells, and up-regulated expression of CD44 at late stage of mesenchymal transition of endothelial cells.

[0014] In another preferred embodiment, the increased ratio refers to that the ratio of mesenchymal cell markers / endothelial markers in the patient with PH-LHD is increased by 2 folds, more preferably 3 folds, more preferably 4 folds, relative to the ratio of mesenchymal cell markers / endothelial markers in normal population.

[0015] In another preferred embodiment, the EndMT cell subpopulation comprises cells with increased ratio of mesenchymal cell markers / endothelial markers, and up-regulated expression of TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74 and TGFBR2.

[0016] In another preferred embodiment, the EndMT cell subpopulation comprises cells with increased ratio of expression levels of mesenchymal cell markers / endothelial markers, and up-regulated expression of CD44.

[0017] In another preferred embodiment, the EndMT cell subpopulation comprises a CD74+ EndMT subpopulation.

[0018] In another preferred embodiment, the EndMT subpopulation highly expresses CD74.

[0019] In another preferred embodiment, the EndMT subpopulation highly expresses CD74, indicating that the EndMT cell subpopulation is at early stage of mesenchymal transition of endothelial cells, and the patient with PH-LHD is an early-stage PH-LHD patient.

[0020] In another preferred embodiment, the endothelial cell markers comprise VWF, CD31.

[0021] In another preferred embodiment, the mesenchymal cell markers comprise a-SMA, DCN, TAGLN.

[0022] In another preferred embodiment, the ratio of mesenchymal cell marker / endothelial marker in the subpopulation of EndMT cells is increased and the expression of TGF-β1, TNFRSF1A, CXCL12, CCL14 and CD74 is upregulated, indicating that the subpopulation of EndMT cells is in an early stage of mesenchymal transformation of endothelial cells, and the patient with PH-LHD is an early stage PH-LHD patient.

[0023] In another preferred embodiment, when the patient with PH-LHD is judged to be an early stage PH-LHD patient, it is indicated that a CD74 inhibitor is suitable for treatment.

[0024] In another preferred embodiment, the CD74 inhibitor is administered to the early stage PH-LHD patient, effective in treating pulmonary vascular remodeling.

[0025] In another preferred embodiment, the CD74 inhibitor is administered to the early stage PH-LHD patient, effective in treating pulmonary vascular remodeling by reducing the degree of pulmonary arterial EndMT transformation.

[0026] In another preferred embodiment, the ratio of mesenchymal cell marker / endothelial marker in the subpopulation of EndMT cells is increased and the expression of CD44 is upregulated in a late stage of mesenchymal transformation of endothelial cells, indicating that the patient with PH-LHD is a middle-late stage PH-LHD patient.

[0027] In another preferred embodiment, when the patient with PH-LHD is judged to be a middle-late stage PH-LHD patient, it is indicated that a CD44 inhibitor is suitable for treatment.

[0028] In another preferred embodiment, the inhibitor comprises an shRNA.

[0029] In another preferred embodiment, the inhibitor comprises an antisense nucleic acid that inhibits the expression of the CD74 gene, and / or the CD44 gene.

[0030] In another preferred embodiment, the inhibitor is delivered by a viral vector, preferably by an adeno-associated viral vector or a lentiviral vector.

[0031] In another preferred embodiment, the adeno-associated viral vector is selected from the following serotypes: AAV9.

[0032] In another preferred embodiment, the biomarker is derived from a tissue, preferably pulmonary arterial tissue, more preferably the endothelial layer of the pulmonary arterial tissue.

[0033] The second aspect of the present application provides a method for (i) typing patients with PH-LHD according to different stages of endothelial cell mesenchymal transformation; and / or (ii) using as a reagent combination for determining whether a patient with left heart disease related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the reagent combination comprising a reagent for detecting the biomarker of the first aspect of the present application.

[0034] In another preferred embodiment, the reagent comprises a substance for detecting the biomarker of the first aspect of the present application by immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other known immunological routine detection / analysis techniques.

[0035] More preferably, the reagent comprises an antibody for detecting the biomarker of the first aspect of the present application.

[0036] In another preferred embodiment, the detection of the biomarker refers to the determination of the function of the EndMT cell subpopulation.

[0037] Preferably, the determination of the function of the EndMT cell subpopulation refers to the detection and quantification of the expression level of immune function molecule proteins in the EndMT cell subpopulation, and more preferably, the gene expression in the EndMT cell subpopulation sequencing data is scored for cell function according to the bioinformatics Addmodule algorithm, and the specific function of the cell subpopulation is determined according to the algorithm.

[0038] Preferably, the method for detecting the biomarker comprises one or more of the following: immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other known immunological routine detection / analysis techniques, preferably Western blotting and immunofluorescence.

[0039] In another preferred embodiment, the immune function molecule comprises VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, and CD44.

[0040] In another preferred embodiment, when the ratio (E1 / E2) between the expression level E1 of the immune function molecule α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the endothelial layer of the tissue of the detection subject is higher than the reference value, it indicates that the detection subject has EndMT, and the reference value is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the same endothelial layer of the tissue of the normal population.

[0041] In another preferred embodiment, when the ratio (E1 / E2) between the expression level E1 of the immune function molecule α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the endothelial layer of the tissue of the detection subject is higher than the reference value C1, and the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, it indicates that the patient is an early PH-LHD patient, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C2 is the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue of the normal population.

[0042] In another preferred embodiment, when the ratio (E1 / E2) between the expression level E1 of the immune function molecule α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the endothelial layer of the tissue of the detection subject is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, it indicates that the patient is a middle-late PH-LHD patient, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population.

[0043] In another preferred embodiment, the detection subject is a human, preferably a patient with PH-LHD.

[0044] The third aspect of the application provides a kit (i) for typing a patient with PH-LHD according to different stages of mesenchymal transition of endothelial cells; and / or (ii) for determining whether a patient with pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for treating pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the kit comprising: specific tools or reagents for measuring the function of the subpopulation of EndMT cells in the biological sample.

[0045] In another preferred embodiment, the reagents include antibodies for detecting the immune function molecule protein in the subpopulation of EndMT cells.

[0046] In another preferred embodiment, the kit further comprises a negative control sample, which is calibrated to represent the function value of the subpopulation of EndMT cells in healthy subjects; preferably, the negative control sample is from one or more healthy subjects.

[0047] In another preferred embodiment, the kit further comprises instructions for use, indicating the following:

[0048] (i) when the ratio (E1 / E2) between the expression level E1 of the immune function molecule α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the endothelial layer of the tissue of the detection object is higher than the reference value C1, and the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, it indicates that the patient is an early PH-LHD patient, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C2 is the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue of the normal population;

[0049] (ii) when the ratio (E1 / E2) between the expression level E1 of the immune function molecule α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the endothelial layer of the tissue of the detection object is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, it indicates that the patient is a late PH-LHD patient, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population.

[0050] In another preferred embodiment, the subject is a human, preferably a patient suffering from PH-LHD.

[0051] The fourth aspect of the present application provides a use of the biomarker of the first aspect of the present application for the manufacture of a reagent or a kit for (i) typing a patient suffering from PH-LHD according to different stages of mesenchymal transition of endothelial cells; and / or (ii) determining whether a patient suffering from pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for treating pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor.

[0052] In another preferred embodiment, the typing or determining comprises the following steps:

[0053] (1) providing a sample derived from the subject to be tested, detecting the biomarker of the first aspect of the present application in the sample, preferably, detecting the expression levels of the immune function molecules VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, and CD44 in the EndMT cell subpopulation;

[0054] (2) if the ratio (E1 / E2) between the expression level E1 of the immune function molecule a-SMA, DCN or TAGLN protein in the EndMT cell subpopulation and the expression level E2 of the VWF or CD31 protein is higher than the reference value C1, and the expression level of TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, it indicates that the patient is an early PH-LHD patient, the reference value C1 is the ratio (E0 / E0’) between the expression level E0 of a-SMA, DCN or TAGLN protein and the expression level E0’ of VWF or CD31 protein in the endothelial layer of the same tissue in the normal population, and the reference value C2 is the expression level of TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue in the normal population, then it indicates that the patient is suitable for treatment with a CD74 inhibitor; or

[0055] If the ratio (E1 / E2) between the expression level E1 of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker measured in step (1) is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, the patient is a patient with advanced PH-LHD, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population, indicating that the CD44 inhibitor is suitable for treatment.

[0056] In another preferred embodiment, the sample is a tissue, preferably pulmonary arterial tissue, more preferably the endothelial layer of the pulmonary arterial tissue.

[0057] The fifth aspect of the present application provides a method for typing patients with PH-LHD according to different stages of the mesenchymal transformation of endothelial cells; and / or a method for determining whether a patient with left heart disease related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, comprising:

[0058] (1) providing a sample derived from the subject to be tested, detecting the biomarker of the first aspect of the present application in the sample, and the detection comprises detecting the function of the EndMT cell subpopulation, preferably, detecting the expression level of the immune function molecule protein VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the EndMT cell subpopulation;

[0059] (2) if the ratio (El / E2) between the expression level El of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker determined in step (1) is higher than the reference value Cl, and the expression level of the TGF-βΙ, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, the patient is an early stage PH-LHD patient, the reference value Cl is the ratio (E0 / E0) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0 of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C2 is the expression level of the TGF-βΙ, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue of the normal population, then a CD74 inhibitor is indicated for treatment; or

[0060] if the ratio (El / E2) between the expression level El of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker determined in step (1) is higher than the reference value Cl, and the expression level of the CD44 protein is higher than the reference value C3, the patient is a middle-late stage PH-LHD patient, the reference value Cl is the ratio (E0 / E0) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0 of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population, then a CD44 inhibitor is indicated for treatment.

[0061] In another preferred embodiment, the sample is a tissue, preferably a pulmonary arterial tissue, more preferably an endothelial layer of a pulmonary arterial tissue.

[0062] In another preferred embodiment, the subject to be tested is a human or a non-human mammal, preferably a patient suffering from PH-LHD.

[0063] In another preferred embodiment, the sample is a tissue sample of a patient suffering from PH-LHD.

[0064] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0065] The sixth aspect of the present application provides a method for typing patients with PH-LHD according to different stages of endothelial cell mesenchymal transition; and / or for use as a model for determining whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease-related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, characterized in that the method comprises the step of identifying a differential biomarker in a biological sample between patients with left heart disease-related pulmonary arterial hypertension (PH-LHD) and healthy controls, wherein the differential biomarker comprises the biomarker of the first aspect of the present application.

[0066] The seventh aspect of the present application provides a system for typing patients with PH-LHD according to different stages of endothelial cell mesenchymal transition; and / or for use as a model for determining whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease-related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the system comprising:

[0067] (a) a feature receiving module, the feature receiving module being configured to receive feature data from a sample of a subject to be tested; the feature data comprising: the level of the biomarker as described in the first aspect of the present application in the sample of the subject to be tested, the functional value of the EndMT cell subpopulation, preferably the expression level of the immune function molecule proteins VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the EndMT cell subpopulation;

[0068] (b) a judging module, which compares the received characteristic data with reference values, thereby obtaining a diagnosis or evaluation result, wherein when the ratio (E1 / E2) between the expression level E1 of the α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the characteristic data is higher than the reference value C1, and the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, it is suggested that the subject is an early PH-LHD patient, and it is indicated that a CD74 inhibitor is suitable for treatment, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C2 is the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue of the normal population; or when the ratio (E1 / E2) between the expression level E1 of the α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the characteristic data is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, it is suggested that the subject is a late PH-LHD patient, and it is indicated that a CD44 inhibitor is suitable for treatment, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population; and

[0069] (c) a result output module, which is used for receiving and outputting the evaluation result.

[0070] In another preferred embodiment, the subject is a human.

[0071] In another preferred embodiment, the subject includes men and women.

[0072] In another preferred embodiment, the subject includes teenagers or adults.

[0073] In another preferred embodiment, the detection method of the level of the biomarker includes immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other known immunological routine detection / analysis techniques.

[0074] In another preferred embodiment, the characteristic receiving module includes a sample collection instrument and a characteristic signal input end.

[0075] In another preferred embodiment, the computing processing module comprises a processor, and a storage, wherein the storage stores the level data of the biomarker according to the first aspect of the present application from normal population, including the functional value of the EndMT cell subpopulation in the same tissue endothelial layer of normal population, preferably the expression level of the immune functional molecule proteins VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the EndMT cell subpopulation.

[0076] In another preferred embodiment, the output module comprises any terminal, preferably a display, a printer, a tablet (PAD), a smart phone.

[0077] In another preferred embodiment, each module is connected by wired or wireless mode.

[0078] The eighth aspect of the present application provides a method, wherein the biomarker according to the first aspect of the present application is used for (i) typing patients with PH-LHD according to different stages of mesenchymal transformation of endothelial cells; and / or (ii) as an indicator for judging whether a patient with left heart disease related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor,

[0079] The method comprises determining the level of the biomarker in the sample from the subject, including determining the function of the EndMT cell subpopulation, preferably determining the expression level of the immune functional molecule proteins VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the EndMT cell subpopulation.

[0080] In another preferred embodiment, said method uses the comparison of the level of said biomarker, including the functional value of the EndMT cell subpopulation, preferably the ratio (E1 / E2) between the expression level E1 of the immunomodulatory protein a-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation, with the reference value C1, and the expression level of the immunomodulatory protein TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein with the reference value C2, as a marker for (ii) stratifying patients with PH-LHD according to the different stages of the mesenchymal transition of endothelial cells; and / or (iii) as a marker for determining whether a patient with pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for the treatment of pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the reference value C1 being the ratio (E0 / E0') between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue in a normal population, the reference value C2 being the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue in a normal population.

[0081] In another preferred embodiment, said method uses the comparison of the level of said biomarker, including the functional value of the EndMT cell subpopulation, preferably the ratio (E1 / E2) between the expression level E1 of the immunomodulatory protein a-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation, with the reference value C1, and the expression level of the immunomodulatory protein CD44 protein with the reference value C3, as a marker for (ii) stratifying patients with PH-LHD according to the different stages of the mesenchymal transition of endothelial cells; and / or (iii) as a marker for determining whether a patient with pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for the treatment of pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the reference value C1 being the ratio (E0 / E0') between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue in a normal population, the reference value C3 being the expression level of the CD44 protein in the endothelial layer of the same tissue in a normal population.

[0082] In another preferred embodiment, the level of the biomarker, including the functional value of the EndMT cell subpopulation, preferably the ratio (E1 / E2) between the expression level E1 of the immune functional molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation is higher than the reference value C1, and the expression level of the immune functional molecule protein TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 is higher than the reference value C2, indicating that the subject to be tested is an early PH-LHD patient, indicating that a CD74 inhibitor is suitable for treatment, the reference value C1 is the ratio (E0 / E0’) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0’ of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C2 is the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue of the normal population.

[0083] In another preferred embodiment, the level of the biomarker, including the functional value of the EndMT cell subpopulation, preferably the ratio (E1 / E2) between the expression level E1 of the immune functional molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation is higher than the reference value C1, and the expression level of the immune functional molecule protein CD44 is higher than the reference value C3, indicating that the subject to be tested is a late PH-LHD patient, indicating that a CD44 inhibitor is suitable for treatment, the reference value C1 is the ratio (E0 / E0’) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0’ of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population.

[0084] The ninth aspect of the present application provides a method for determining a treatment regimen, comprising:

[0085] a) providing a test sample from a subject;

[0086] b) detecting the expression level of the immune functional molecule protein VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the EndMT cell subpopulation in the test sample; and

[0087] c) determining a treatment regimen based on the expression levels of VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in said sample.

[0088] In another preferred embodiment, said sample comprises a tissue sample.

[0089] In another preferred embodiment, said subject is a human or a non-human mammal.

[0090] In another preferred embodiment, when the ratio (E1 / E2) between the expression level E1 of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of VWF or CD31 in the EndMT cell subpopulation in said sample is higher than the reference value C1, and the expression level of CD44 protein is higher than the reference value C3, it indicates that the patient is a late PH-LHD patient, then it indicates that the patient is suitable for treating PH-LHD with a CD44 inhibitor, said treatment regimen comprising a CD44 inhibitor.

[0091] In another preferred embodiment, when the ratio (E1 / E2) between the expression level E1 of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of VWF or CD31 in the EndMT cell subpopulation in said sample is higher than the reference value C1, and the expression level of CD44 protein is higher than the reference value C3, it indicates that the patient is a late PH-LHD patient, then it indicates that the patient is suitable for treating PH-LHD with a CD44 inhibitor, said treatment regimen comprising a CD44 inhibitor. BRIEF DESCRIPTION OF DRAWINGS

[0092] Figure 1 : Pathophysiological mechanisms of PH-LHD.

[0093] Figure 2 : Endothelial cell UMAP clustering of PH-LHD patients (right panel), LHD without PH patients group (middle panel) and Control group (left panel).

[0094] Figure 3 : PH-LHD group has more EndMT subpopulation cells than LHD without PH patients, Control group (12.6% VS 6.3%).

[0095] Figure 4 : Heatmap showing gene expression of endothelial cell subpopulation TOP10.

[0096] Figure 5Immunofluorescence of pulmonary arteries in PH-LHD patients, LHD patients without PH, and the Control group demonstrated the presence of EndMT in the tissues. In the figure, CD31 is the marker gene of endothelial cells, and α-SMA is the marker gene of smooth muscle cells. The two genes are co-localized in the smooth muscle layer, indicating that the EndMT phenomenon has occurred in endothelial cells.

[0097] Figure 6 A: UMAP subset map of all pulmonary artery cells from spatial transcriptome sequencing, divided into C1-C10; B: Heatmap showing the top 5 genes of each C1-C10 subset, where C1-C4 simultaneously express VWF and α-SMA, indicating that C1-C:4 represents the endothelial-mesenchymal transition state; C: HE staining images of pulmonary arteries from PH-LHD patients, LHD patients without PH, and the Control group from spatial transcriptome sequencing; D: Spatial location map of C1-C10 subsets. E. By deconvolution, the EndMT subset from single-cell data was mapped to spatial transcriptome data for visualization. The results showed that EndMT-state cells gradually migrated towards the outer membrane in PH-LHD disease. F. The C1-C3 subset was significantly increased in PH-LHD, indicating that the increase in endothelial-mesenchymal transition cells may be related to the pathogenesis of PH-LHD. G. Gene spatial localization analysis showed that CD74, CD44, and CD31 shared common localizations in the pulmonary arteries of the three groups, with CD74 and CD31 showing better co-localization.

[0098] Figure 7 A: Will Figure 6 Pseudo-temporal analysis of the endothelial-mesenchymal transition subsets C1-C4 in spatial transcriptome data showed that C1 gradually differentiated into C4 (i.e., the differentiation process from early to late stages corresponds to C1-C2-C3-C4), and immune-related genes (TNF3SF1A, CXCL12, CCL14), CD74, and TGFβ1 were upregulated in the early stages of differentiation, while CD44 was upregulated in the late stages. B: [The remaining text appears to be a fragment and doesn't translate coherently.] Figure 6 A violin plot of genes from the C1-C4 subsets in the spatial transcriptome data revealed that endothelial marker genes (VWF, PECAM1) gradually decreased from C1 to C4, while mesenchymal marker genes (ACTA2, TAGLN) gradually increased, indicating that C1-C4 represent different stages of EndMT. Furthermore, TNF3SF1A, CXCL12, CCL14, TGFBR2, and CD74 gradually decreased from C1 to C4, while CD44 gradually increased, suggesting that TNF3SF1A, CXCL12, CCL14, TGFBR2, and CD74 were upregulated in the early stages of EndMT, while CD44 was upregulated in the late stages of EndMT.

[0099] Figure 8 :exist Figure 7After identifying the regulatory mechanisms of EndMT at different stages in spatial transcriptome data, further validation was performed using single-cell data. Figure 2 The EndMT cells in the single-cell data were further divided into 5 EndMT subsets, named Endmt1-5. A: UMAP clustering of the 5 EndMT subsets in PH-LHD patients (right image) and healthy controls (left image); B: Heatmap showing the top 5 genes of the Endmt1-5 subsets; C: Violin plot of the genes of the Endmt1-5 subsets shows that endothelial marker genes (VWF, PECAM1) gradually decrease from C1 to C4, while mesenchymal marker genes (ACTA2, TAGLN) gradually increase, indicating that C1-C4 represent different stages of EndMT. TNF3SF1A, CXCL12, CCL14, TGFBR2, and CD74 gradually decrease from C1 to C4, while CD44 gradually increases, indicating that TNF3SF1A, CXCL12, CCL14, TGFBR2, and CD74 are upregulated in the early stage of EndMT, while CD44 is upregulated in the late stage of EndMT. This demonstrates, based on multi-omics data from single-cell transcriptional ensemble spatial transcriptomics, that the regulatory mechanisms at different stages of EndMT are the same. D: The Addmodule endothelial-mesenchymal transition score for EndMT subgroups 1-5 shows a gradual increase in mesenchymal transition between subgroups 1-5.

[0100] Figure 9 Following the previous identification of regulatory mechanisms at different stages of EndMT in single-cell and spatial transcriptome data, in vitro validation was performed using pulmonary artery endothelial cells (PAECs). The results showed that after different periods of mechanical stretching (0h, 12h, 24h, 36h), the levels of mesenchymal marker genes (ACTA2, TAGLN) in PAECs gradually increased, indicating that C1-C4 represent different stages of EndMT. Meanwhile, TGFBR2 and CD74 gradually decreased from C1 to C4, while CD44 gradually increased, indicating that TGFBR2 and CD74 were upregulated in the early stages of EndMT, while CD44 was upregulated in the late stages. This further validates the reliability of the single-cell and spatial transcriptome data analysis results in the in vitro cell model.

[0101] Figure 10 : A schematic diagram of the Banding model.

[0102] Figure 11 Next, we will go through Figure 10Banding operation, using titanium clips to perform "aortic constriction above the coronary artery" (which is an internationally recognized method of modeling PH-LHD rats), to simulate the progression of PH-LHD disease in rats. Divided into Control group, PH-LHD 5 weeks group, PH-LHD 7 weeks group, PH-LHD 9 weeks group. At each time node, measure the right ventricular pressure (RVSP, which can replace pulmonary artery pressure), left atrial pressure (LAP), right ventricular hypertrophy (RV W / BW), left ventricular hypertrophy (LV+S W / BW). A, B, C, D, E, the pulmonary artery pressure of PH-LHD rats increases with the extension of the disease course after modeling (5 weeks-9 weeks), and the right ventricular pressure, left atrial pressure, right ventricular hypertrophy, left ventricular hypertrophy also increase. F, G, HE staining of lung tissue also shows that the degree of pulmonary vascular remodeling also increases with the extension of the disease course after modeling. H, I, J, K, L, M, immunofluorescence and Western blot experiments show that the degree of endothelial mesenchymal transformation also increases with the extension of the disease course after modeling. All statistical analyses in the figure use one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001.

[0103] Figure 12: At the same time of modeling PH-LHD rats, CD74 and CD44 genes were knocked down by intratracheal instillation of AAV virus to verify the therapeutic effect of CD74 and CD44 in PH-LHD. A, B, C, D, E, F, G, H, After CD74 gene knockdown treatment, the right ventricular pressure (RVSP), left atrial pressure (LAP), and right ventricular hypertrophy degree (RV W / BW) of PH-LHD rats at different modeling weeks (5 weeks, 7 weeks, and 9 weeks) were improved to different degrees, while the left ventricular hypertrophy degree (LV+SW / BW) had no obvious effect, indicating that the therapeutic target of CD74 may be in the pulmonary vessels rather than the left ventricle; after CD44 knockdown treatment, the right ventricular pressure (RVSP), left atrial pressure (LAP), right ventricular hypertrophy degree (RV W / BW), and left ventricular hypertrophy degree (LV+SW / BW) of PH-LHD rats at different modeling weeks (5 weeks, 7 weeks, and 9 weeks) had no obvious effect. K, Immunofluorescence showed that the co-localization of CD31 and a-SMA in pulmonary arterioles of PH-LHD rats decreased after CD74 treatment at 5 weeks, 7 weeks, and 9 weeks; L, Immunofluorescence showed that the co-localization of CD31 and a-SMA in pulmonary arterioles of PH-LHD rats decreased only at 9 weeks after CD44 treatment; M, N, O, Columnar charts show the statistical analysis of the co-localization area of CD31 and a-SMA in PH-LHD rats at 5 weeks, 7 weeks, and 9 weeks; P, P1, Q, Q, Western blot experiments again confirmed that CD74 gene knockdown was effective at 5 weeks, 7 weeks, and 9 weeks after PH-LHD modeling, while CD44 was only effective at 9 weeks; R, R1, R2, Cell scratch experiment showed that CD74 and CD44 overexpression could enhance the migration ability of pulmonary arterial endothelial cells; S, S1, S2, Cell scratch experiment showed that CD74 and CD44 knockdown could reduce the migration ability of pulmonary arterial endothelial cells. DETAILED DESCRIPTION

[0104] The inventors have made extensive and in-depth studies and for the first time found that, according to the ratio between the expression levels of endothelial cell markers (such as VWF, CD31) and mesenchymal cell markers (such as a-SMA, DCN, TAGLN) in the EndMT cell subpopulation, and the expression levels of TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, TGFBR2, and CD44 in the EndMT cell subpopulation, (i) can be used for typing patients with PH-LHD according to different periods of mesenchymal transformation of endothelial cells; and / or (ii) can be used for judging whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease-related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor and / or a CD44 inhibitor. On this basis, the inventors completed the present application.

[0105] Definitions

[0106] The terms used in connection with the present application have the meaning commonly understood by a person of ordinary skill in the relevant art. However, for a better understanding of the present application, the following explanations of some definitions and related terms are provided:

[0107] According to the present application, the term "individual" refers to an animal, in particular a mammal, such as a primate, most preferably a human.

[0108] According to the present application, the terms "a", "an", and "the" refer to both the singular and plural of them, and include the general class of which a specific instance can be referred to where particular embodiments are discussed.

[0109] As used herein, the term "about" when used in the context of a recited numerical value means that the value can vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0110] As used herein, the term "comprising" or "including" can be open, semi-closed or closed. In other words, the term also includes "consisting essentially of or "consisting of".

[0111] It is noted that the explanations of the terms provided herein are only provided to better enable those skilled in the art to understand the present application and are not intended to be limiting.

[0112] As used herein, the AAV9-shCD74 and AAV9-shCD44 sequences are as follows:

[0113]

[0114] As used herein, the endothelial cell markers are VWF, PECAM1 (also known as CD31).

[0115] As used herein, the mesenchymal markers are ACTA2 (also known as a-SMA), TAGLN.

[0116] As used herein, the immune and inflammatory signaling molecules are CXCL12, CCL14, TNFRSF1A.

[0117] It was discovered for the first time that CD74 is a marker of early EndMT.

[0118] The application first discovers the CD74+ EndMT subpopulation in PH-LHD patients, and this special subpopulation can be used as a future treatment direction. In the application, the EndMT subpopulation in PH-LHD patients highly expresses CD74, indicating that the EndMT cell subpopulation is in the early stage of the mesenchymal transformation of endothelial cells, and then the PH-LHD patient is an early PH-LHD patient, which is suitable for treatment with a CD74 inhibitor, and can effectively treat pulmonary vascular remodeling.

[0119] Pulmonary hypertension associated with left heart disease (PH-LHD)

[0120] Pulmonary hypertension (PH) is a serious progressive disease characterized by pulmonary vascular remodeling, right ventricular hypertrophy and failure, which can exist independently as a disease or develop into a complication of various diseases in the later stage, has a high morbidity and mortality, and is known as the "neglected killer" and "cardio-pulmonary vascular system cancer". Left heart disease (LHD) is the most common cause of PH, and about 2 / 3 of severe LHD will develop into PH. This PH caused by LHD is called pulmonary hypertension associated with left heart disease (PH-LHD), which belongs to type II PH and is the most common type of PH in clinical practice. The pathogenesis of PH-LHD is unclear, and the pathophysiology is complex Figure 1 ), that is, LHD leads to increased left ventricular filling pressure, blocked pulmonary venous return, and elevated pulmonary venous pressure, ultimately leading to pulmonary vascular remodeling and increased pulmonary vascular resistance. The treatment of PH-LHD includes LHD treatment and PH treatment. At the beginning of the disease, the increase in pulmonary vascular resistance is caused by the spasm and contraction of pulmonary arterioles, and the pathological changes are functional and reversible. At this time, timely correction of LHD can achieve satisfactory results. In the later stage of the disease, pathological changes occur in the structure and function of the blood vessel wall, such as endothelial dysfunction, intimal hyperplasia, vascular smooth muscle cell proliferation, excessive deposition of collagen, middle layer thickening, lumen stenosis and occlusion, i.e. "pulmonary vascular remodeling". At this time, the increase in pulmonary vascular resistance is irreversible, and even if LHD is cured, PH is difficult to alleviate, and patients eventually die of right heart failure. Therefore, PH-LHD has a higher morbidity and mortality than other types of PH. In summary, PH-LHD has a higher morbidity and mortality than other types of PH due to the lack of specific and effective treatment methods. Therefore, it is of great significance to the clinical treatment of PH-LHD to further explore the mechanism of pulmonary vascular remodeling and block the occurrence and development of PH in the early stage of the disease.

[0121] EndMT cell subpopulation

[0122] Endothelial-mesenchymal transition (EndMT) refers to the phenotypic transformation of endothelial cells, which lose their characteristic cobblestone appearance and acquire the elongated shape typical of mesenchymal cells. This transformation is accompanied by increased proliferation and migration abilities, but weakened barrier function. At the molecular level, this is manifested by decreased expression of endothelial cell markers (CD31, VWF, PLVAP, etc.) and increased expression of mesenchymal markers (ACTA2, TAGLN, DCN, etc.).

[0123] In this invention, we utilized scRNA-seq and spatial transcriptomics data from human pulmonary arteries to discover for the first time an EndMT subset primarily distributed in the PH-LHD patient group. Figures 1-4 ). Immunofluorescence staining of human pulmonary arteries in vivo ( Figure 5 This further validated the reliability of our findings. In spatial transcriptomics data, based on the gradient expression of endothelial and mesenchymal marker genes in different subpopulations (…), Figure 6 AG) and pseudo-time series analysis showed that EndMT cell subsets were at different differentiation stages. Therefore, we believe that partial EndMT transformation at different stages coexists within the pulmonary artery, characterized by a gradual decrease in endothelial properties and a gradual increase in mesenchymal characteristics. Furthermore, pseudo-time series analysis indicated that different stages of mesenchymal transition are regulated by different signaling pathways. Immune and inflammatory signaling molecules CD74 and TGFβ are upregulated in the initial stage of EndMT, while these signaling pathways are downregulated as mesenchymal transition progresses. Conversely, CD44 signaling molecules are gradually upregulated throughout the terminal MT process. Figure 7 Furthermore, the trends in the expression of these signaling molecules were further confirmed in our scRNA-seq and cell experiments. Figure 8 AD, Figure 9 AD).

[0124] To validate the results of single-cell combined spatial transcriptome data, we constructed PH-LHD rat models at different time gradients (5 weeks, 7 weeks, and 9 weeks). The results showed that with the extension of modeling time, the degree of pulmonary vascular remodeling in PH-LHD rats significantly increased. Figure 11 AG), and the conversion rate of EndMT also increases accordingly ( Figure 11 HM). Furthermore, CD74 knockdown treatment reduced pulmonary vascular remodeling and EndMT in PH-LHD rats at all time points; however, CD44 only reduced EndMT in PH-LHD rats at 9 weeks, but could not reverse pulmonary vascular remodeling in PH-LHD rats. Figure 12 ).

[0125] In summary, by scRNA-seq and spatial transcriptomic data, we first demonstrate the existence of different stages of EndMT in PH-LHD at single-cell resolution. Immune and inflammatory signals and CD74 might play an inducing role in the early stage of EndMT, and have a good therapeutic effect on PH-LHD pulmonary vascular remodeling. In contrast, as the endothelial cell characteristics gradually weaken, CD44 molecules are increasingly involved in late EndMT, promoting further differentiation to mesenchymal characteristics. Therefore, this finding provides an important therapeutic target for the treatment of PH-LHD.

[0126] Patient typing according to different stages of endothelial cell mesenchymal transformation in PH-LHD

[0127] Endothelial mesenchymal transformation refers to the process of vascular endothelial cells being stimulated by external factors such as blood flow pressure, shear force, immune and inflammatory factor infiltration, etc., leading to the gradual transformation of vascular endothelial cells to mesenchymal cells, characterized by the gradual disappearance of endothelial cell characteristics and the gradual acquisition of mesenchymal characteristics, and the English abbreviation is EndMT (Endothelial mesenchymal transformation). EndMT is an important pathophysiological mechanism of pulmonary vascular remodeling in pulmonary arterial hypertension. Previous studies have shown that EndMT is an all-or-nothing process. Recent studies have found that EndMT is a gradual process, i.e., from endothelial cell state, to partial mesenchymal transformation, to complete mesenchymal transformation. The specific regulatory mechanism of EndMT is unclear.

[0128] The present application first found that different stages of mesenchymal transformation are regulated by different signal pathways. Immune and inflammatory signal molecules TNFRSF1A, CXCL12, CCL14, and CD74 and TGFβ1 are up-regulated in the initial stage of EndMT, while these signal pathways are down-regulated as mesenchymal transformation progresses. In contrast, CD44 signal molecules are gradually up-regulated throughout the entire EndMT process. In addition, the trend of expression of these signal molecules is confirmed again in our scRNA-seq and cell experiments.

[0129] In summary, by scRNA-seq and spatial transcriptomic data, we first demonstrate the existence of different stages of EndMT in PH-LHD at single-cell resolution. Immune and inflammatory signals TNFRSF1A, CXCL12, CCL14, and CD74 and TGFβ1 might play an inducing role in the early stage of EndMT. In contrast, as the endothelial cell characteristics gradually weaken, CD44 molecules are increasingly involved in late EndMT, promoting further differentiation to mesenchymal characteristics.

[0130] Furthermore, to further explore the spatial distribution of EndMT within the pulmonary arteries, we performed spatial transcriptomic sequencing of the pulmonary arteries for the first time. The results showed that EndMT mainly occurred in the medial and adventitial layers of the pulmonary arteries. In PH-LHD patients, the transition of the pulmonary arterial endometrium was characterized by gradual migration from the intima to the adventitia. In addition, consistent with the scRNA-seq results, the MHC class ii-related molecule CD74 was upregulated in EndMT cells. The CD74 molecule has been shown to promote EC angiogenesis in PAH lung tissue, and part of the EndMT process can lead to abnormal and pathological angiogenesis. CD44 is considered a stem cell marker in cancer and has recently been shown to be highly expressed in EndMT cells in the neointimal lesions of PH patients. Notably, the natural ligand of CD74 is macrophage migration inhibitory factor (MIF), which, upon binding, induces the formation of a CD44 complex. This complex is internalized into the endosomal compartment, releasing fragments of the intracellular domain of CD74, thereby activating downstream signaling pathways, indicating a sequential activation relationship between CD74 and CD44. These studies further support our findings, however, although these signaling molecules show potential roles in PH-related endometrial mt, the mechanisms by which they specifically regulate part of the endometrial mt process remain to be fully elucidated.

[0131] In the spatial transcriptomic data, based on the gradient expression of endothelial and mesenchymal marker genes in different subpopulations and pseudotemporal analysis showed that the EndMT cell subpopulations were at different stages of differentiation, therefore, we believe that different stages of partial EndMT transformation coexist within the pulmonary arteries, characterized by a gradual decrease in endothelial properties and an increase in mesenchymal traits. In addition, pseudotemporal and cell communication analysis studies showed that different stages of mesenchymal transition are regulated by different signaling pathways. The immune and inflammatory signaling molecules TNFRSF1A, CXCL12, CCL14, as well as CD74 and TGFβ1 were upregulated in the initial stage of EndMT, while these signaling pathways were downregulated as mesenchymal transformation progressed. In contrast, the CD44 signaling molecule was gradually upregulated throughout the endometrial mt process. In addition, the trend of expression of these signaling molecules was again confirmed in our scRNA-seq and cell experiments. In summary, through scRNA-seq and spatial transcriptomic data, we have for the first time demonstrated the existence of partial EndMT in PH-LHD at single-cell resolution. The immune and inflammatory signals TNFRSF1A, CXCL12, CCL14, as well as CD74 and TGFβ1 can play an inducing role in the early stages of EndMT. In contrast, as the endothelial cell characteristics gradually weaken, the CD44 molecule is increasingly involved in late EndMT, promoting further differentiation towards mesenchymal characteristics.

[0132] Therefore, according to the different differentiation stages of the EndMT cell subpopulation, the patient with PH-LHD can be typed, for example, if the EndMT cell subpopulation is at the initial stage of differentiation, i.e. the EndMT cell subpopulation is at the early stage of mesenchymal transformation of endothelial cells, the patient with PH-LHD is an early PH-LHD patient; if the EndMT cell subpopulation is at the terminal stage of differentiation, i.e. the EndMT cell subpopulation is at the late stage of mesenchymal transformation of endothelial cells, the patient with PH-LHD is a late PH-LHD patient.

[0133] In summary, based on single-cell sequencing and spatial transcriptomic sequencing technology, the present application first confirms the existence of endothelial mesenchymal transformation cell subpopulation in the pulmonary artery vessels of pulmonary hypertension, and the regulation mechanism of this EndMT transformation process is different, in the early stage of transformation, it is mainly regulated by CD74, and with the progress of transformation, CD44 occupies the main regulatory position. For the treatment of pulmonary hypertension, in the future, patients with short disease duration may use CD74 inhibitors for intervention, and patients with long disease duration may use CD44 for intervention.

[0134] Sample

[0135] The term "sample" or "specimen" as used herein refers to material specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. A sample can consist wholly or in part of biological material from the subject. A sample can also be material that has been in contact with the subject in a manner that allows testing of the sample to provide information about the subject. A sample can also be material that has been in contact with other material that is not the subject but that enables the first material to be subsequently tested to determine information about the subject, for example, a swab or scalpel rinse. A sample can be from a source of biological material other than the subject, as long as a person of ordinary skill in the art would still be able to determine information about the subject from the sample.

[0136] The sample is selected from, for example, a tissue, preferably a pulmonary artery tissue.

[0137] Expression

[0138] As used herein, the term "expression" refers to the presence of a cell surface marker (typically a protein) on the surface of a cell after the processes of RNA transcription, protein translation and intracellular transport. The level or intensity of expression of a cell surface marker can be measured by flow cytometry. Preferably, the "high" or "low" expression of a particular marker between two samples can be reflected by the "more" or "less" proportion of cells counted that express at or above a specified level or intensity. Preferably, the "high" or "low" expression of a particular marker between two samples can be reflected by the "high" (Hi) or "low" (Low) fluorescence signal intensity.

[0139] Reference value

[0140] The term "reference value" or "reference amount" or "reference level" as used herein refers to a value (or amount, or level) of a parameter or biomarker that is indicative of the status of a subject with respect to a particular disease (or illness, or condition). A suitable reference level of a parameter or biomarker can be quantified, determined or measured by detecting the parameter / biomarker in a number of suitable reference subjects. Such reference levels can be adjusted according to a particular population of subjects. A reference value or reference level can be an absolute value; a relative value; a value with an upper or lower limit; a range of values; an average value; a median value, mean value or value compared to a particular control or baseline value. A reference value can be based on the value of an individual sample, e.g. a value obtained from a sample from a subject under test but at an earlier time point. A reference level can be based on a large number of samples, such as a population of subjects from a cohort of real age-matched subjects, or based on a pool of samples including or not including the sample under test. Depending on the context, a reference level corresponds to the value of a parameter (or biomarker) quantified, or determined, or measured on a sample from the same tissue from a normal population.

[0141] Biomarkers of the application and uses thereof

[0142] In the present application, a biomarker and uses thereof are provided, which can be used for the preparation of a kit for (i) typing patients with PH-LHD according to different stages of the mesenchymal transition of endothelial cells; and / or (ii) for use as a judge of whether a patient with pulmonary arterial hypertension associated with left heart disease (PH-LHD) is suitable for treating pulmonary arterial hypertension associated with left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor.

[0143] In particular, the biomarker comprises a subpopulation of EndMT cells.

[0144] In one embodiment, the kit comprises, in the kit: binding molecules, specific antibodies, specific amplification primers, specific probes or chips, isotopes, enzyme-substrate complexes, or combinations thereof specific for each biomarker of the panel, in particular specific polyclonal or monoclonal antibodies, preferably monoclonal antibodies, or fragments or derivatives thereof can be mentioned.

[0145] In another preferred embodiment, each biomarker is detected or identified by one or more methods selected from the group consisting of immunoprecipitation, flow cytometry, Western blot, ELISA, ELISPOT, antibody microarray, immunohistology, dot blot, protein microarray, tissue microarray coupled to immunohistochemistry, or other well-known immunological routine detection / analysis techniques.

[0146] In a preferred embodiment, the present application provides a method of (i) typing a patient with pulmonary hypertension associated with left heart disease (PH-LHD) according to the different stages of endothelial-to-mesenchymal transition; and / or (ii) for use as a judge whether a patient with pulmonary hypertension associated with left heart disease (PH-LHD) is suitable for treating pulmonary hypertension associated with left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, said subject being a subject to be tested in a healthcare institution, preferably a subject to be tested in a hospital, more preferably a subject to be tested in an emergency room, a resuscitation room, an intensive care unit or a continuing care unit. The method comprises or even consists of the following steps:

[0147] (1) providing a sample derived from the subject to be tested, detecting the biomarkers of the first aspect of the application in the sample, including detecting the function of the subpopulation of EndMT cells, preferably determining the expression level of the immunological function molecules VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the subpopulation of EndMT cells;

[0148] (2) if the ratio (E1 / E2) between the expression level E1 of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker determined in step (1) is higher than the reference value C1, and the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, the reference value C1 being the ratio (E0 / E0’) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0’ of the VWF or CD31 protein in the endothelial layer of the same tissue in the normal population, and the reference value C2 being the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue in the normal population, it indicates that the patient is an early PH-LHD patient, and a CD74 inhibitor is suitable for treatment; or

[0149] if the ratio (E1 / E2) between the expression level E1 of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker determined in step (1) is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, the reference value C1 being the ratio (E0 / E0’) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0’ of the VWF or CD31 protein in the endothelial layer of the same tissue in the normal population, and the reference value C3 being the expression level of the CD44 protein in the endothelial layer of the same tissue in the normal population, it indicates that the patient is a middle-late PH-LHD patient, and a CD44 inhibitor is suitable for treatment.

[0150] The EndMT cell subpopulation of the application has one or more characteristics selected from the group consisting of:

[0151] (a) reduced expression of endothelial cell markers and increased expression of mesenchymal cell markers or increased ratio of mesenchymal cell markers / endothelial markers;

[0152] (b) up-regulation of TGF-β1, TNFRSF1A, CXCL12, CCL14 and CD74 at the early stage of mesenchymal transformation of endothelial cells, and up-regulation of CD44 at the late stage of mesenchymal transformation of endothelial cells.

[0153] The expression level of the immune function molecule is the expression level of the immune function molecule in the EndMT cell subpopulation, which is referred to as “EndMT cell subpopulation function value” in the application.

[0154] The immune function molecules are selected from one or more of VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44.

[0155] The detection of the various biomarkers according to the application using antibodies against the various cell surface antigen proteins / surface markers described above is a method well known to the person skilled in the art.

[0156] The method of the application is an in vitro or ex vivo method. For example, compared to etiologic detection and SOFA Score, the present application has the advantage of allowing to easily (i) classify patients with PH-LHD according to the different stages of the mesenchymal transition of endothelial cells; and / or (ii) be used as a marker to determine whether a patient with pulmonary arterial hypertension associated with left heart disease (PH-LHD) is suitable for the treatment of pulmonary arterial hypertension associated with left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor. The measurement of said marker is fully compatible with the performance by automated analysis machines or by test methods called rapid tests.

[0157] The sample on which the method of the application is performed is also called test sample.

[0158] The test sample is taken from a biological sample of a subject to be tested suffering from pulmonary arterial hypertension associated with left heart disease (PH-LHD).

[0159] In particular, the test sample is selected from a tissue, preferably a pulmonary arterial tissue.

[0160] In the context of the present application, the terms "detection" or "measurement" or "determination" are used interchangeably and have the same meaning. These terms mean the detection and quantification of the expression level values of the immune function molecules (detection and quantification of said function molecules at the protein level, referred to in the present application as "immune cell function values"). For this purpose, any detection and / or quantification method known to the person skilled in the art can be used for the implementation of the present application.

[0161] In particular, the determination of the expression level values of the immune function molecules (detection and quantification of said function molecules at the protein level) in the subpopulation of EndMT cells is performed using specific tools or reagents for the EndMT cell subpopulation and / or the expression level of the immune function molecules that allow to directly or indirectly determine their presence and / or quantify their expression level.

[0162] Among these tools or reagents that allow to detect and / or quantify the expression level values of the immune function molecules in the subpopulation of EndMT cells, specific polyclonal or monoclonal antibodies, preferably monoclonal antibodies, or fragments or derivatives thereof can be mentioned in particular.

[0163] In the method of the application, the level of expression of immune function molecules (immune cell function values) in the EndMT cell subpopulation can be detected and / or quantified, inter alia, using well-known analytical techniques such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarrays, immunoprecipitation, immunohistology, dot blotting, protein microarrays, or tissue microarrays coupled with immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopy or histochemical methods using single or multiple excitation wavelengths and applying any suitable optical method, such as electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy and radiofrequency methods, for example multipolar resonance spectroscopy, confocal and non-confocal, detecting fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance and birefringence or refractive index (for example surface plasmon resonance, ellipsometry, resonant mirror methods, grating coupler methods or interferometry), cell ELISA, radioisotopes, magnetic resonance imaging, polyacrylamide gel electrophoresis (SDS PAGE) analysis; HPLC-mass spectrometry; liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS). Preferably, the level of expression of immune function molecules in the EndMT cell subpopulation is identified, selected, sorted, quantified (and any combination thereof) by flow cytometry.

[0164] In the present application, the level of expression of immune function molecules (one or more of VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44) in the EndMT cell subpopulation is preferably "elevated" or "decreased" by, for example, measuring these EndMT cell subpopulation proportion values and / or the level of expression of immune function molecules by flow cytometry.

[0165] Optionally, for said EndMT cell subpopulation and / or for the immune function molecule (one or more of VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44), the level of the immune function molecule in said EndMT cell subpopulation can be measured as "increased" or "decreased" by measuring the amount of the immune function molecule in these EndMT cell subpopulations as "increased" or "decreased" by e.g. flow cytometry. The exact amount of the immune function molecule in said surface EndMT cell subpopulation is not important, what is important is to compare the level of the immune function molecule in the EndMT cell subpopulation in the biological sample to be tested with the level of the immune function molecule in the EndMT cell subpopulation in the control sample. In other words, it is not necessary to measure the actual quantitative "level" of the immune function molecule in the EndMT cell subpopulation, it is preferable to measure the "high" or "low" level of the immune function molecule in said EndMT cell subpopulation and then compare the level of the immune function molecule in the EndMT cell subpopulation with the level of the immune function molecule in the EndMT cell subpopulation in the control sample.

[0166] All indications and preferences above relating to measuring the level of the immune function molecule in said EndMT cell subpopulation apply equally to measuring the level in the test sample and in the reference sample.

[0167] The main advantages of the present application include:

[0168] (1) The present application is the first to find that, based on the ratio between the expression level of endothelial cell markers (such as VWF, CD31) and mesenchymal cell markers (such as a-SMA, DCN, TAGLN) in the EndMT cell subpopulation, and the expression level of TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, TGFBR2 and CD44 in the EndMT cell subpopulation, can be used to (i) classify patients with PH-LHD according to different stages of mesenchymal transformation of endothelial cells; and / or (ii) serve as a judge whether a patient with pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for treating pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor.

[0169] (2) The present application first proves the existence of different stages of EndMT in PH-LHD at single cell resolution. Immune and inflammatory signals, as well as CD74 and TGFβ, can play an inducing role in the early stage of EndMT. In contrast, CD44 molecules are increasingly involved in late EndMT as endothelial cell characteristics gradually weaken, promoting further differentiation to mesenchymal characteristics. Therefore, this finding provides an important therapeutic target for the treatment of PH-LHD.

[0170] (3) The present application first confirms the existence of a subpopulation of endothelial mesenchymal transition cells in the pulmonary artery vessels of pulmonary hypertension, and the regulatory mechanisms of this EndMT transformation process are different. In the early stage of transformation, CD74 plays a major regulatory role, and as the transformation progresses, CD44 occupies the dominant regulatory position. For the treatment of pulmonary hypertension, in the future, patients with short disease duration can be intervened using CD74 inhibitors, while patients with long disease duration can be intervened using CD44.

[0171] It should be noted that the explanations of the terms provided herein are only for better understanding of the present application by those skilled in the art, and are not intended to limit the present application.

[0172] The following specific examples illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present specification.

[0173] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application. The test methods in the following examples are not specified, and are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturers.

[0174] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified by the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0175] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are usually carried out according to the conventional conditions or the conditions suggested by the manufacturers. Unless otherwise specified, percentages and parts are by weight.

[0176] Unless otherwise specified, the reagents and materials used in the examples of the application are commercially available products.

[0177] Example 1

[0178] By collecting the pulmonary artery of heart transplant recipients and donors (obtained from the Affiliated Union Hospital of Fujian Medical University), single cell and spatial transcriptome sequencing were performed, vascular cells were divided into different subgroups at the single cell level, and each subgroup was functionally annotated using bioinformatics analysis. It was found that the proportion of endothelial cells increased from 20.2% in the normal group to 23.6% in the disease group. Therefore, we further analyzed the endothelial cell subgroups and found the endothelial mesenchymal transition subgroup (EndMT EC), which was first discovered at the single cell level that there was an EndMT subgroup in pulmonary hypertension.

[0179] Specific steps:

[0180] 1. Sample source: pulmonary artery tissue of heart transplant recipients and donors

[0181] 2. Instrument: Chromium 10x, single cell captured in droplet emulsion using GemCode single cell instrument (10x Genomics). scRNA-seq library was constructed using single cell 3’v2 Kit from 10x Genomics following 10x Genomics protocol. Briefly, pulmonary artery was chopped into small pieces and then digested in a lysis solution containing 2 mg / mL Collagenase II (Sigma) and 50 mg / mL Elastase, 37°C water bath, shaking every 15 minutes to ensure even digestion and viability assessment. After 1 hour and 15 minutes of digestion, the reaction was terminated and the single cell suspension was filtered through a 70 pm cell strainer and diluted with PBS. After centrifugation (500g, 3 min, 4°C), the cells were resuspended in red blood cell lysis buffer (Solarbio) and incubated on ice for 5 minutes and then further diluted with PBS. After another centrifugation (500g, 3 min, 4°C), the cell pellet was resuspended in DMEM containing 2% fetal bovine serum and ready for scRNA-seq cell capture. Single cells were captured in droplet emulsion using GemCode single cell instrument (10x Genomics) and single cell 3’v2 Kit following 10x Genomics protocol. In essence, gel beads with barcodes, cells and enzymes and oil were mixed to form gel bead-in-emulsion (GEMs) using the 10x Genomics Chromium platform and 8-channel microfluidic “double cross” system; water microsystem). After GEM formation, cells were lysed and gel beads were automatically dissolved, releasing multiple barcode sequences. Then mRNA was reverse transcribed into cDNA containing 10-fold barcode and unique molecular identifier (UMI) information. Subsequently, cDNA amplification and library quality assessment were performed. After capturing 10,000 target cells, the library was sequenced using Illumina NovaSeq 6000 sequencer (Beijing Kedbio Science and Technology Co., Ltd.).

[0182] Raw sequencing reads generated using the NovaSeq 6000 system were aligned to the human reference genome (https: / / cf.10xgenomics.com / supp / spatial-exp / refdata-gex-GRCh38-2020-A.tar.gz) and pre-processed using CellRanger. Ambient RNA contamination was corrected using the SoupX package (vl.6.1) [1]. Downstream analysis was performed using the Seurat package (v5.0.2) [2, 3]. Low-quality cells were removed according to the following criteria: (1) cells expressing < 500 or > 5000 genes, each gene must be expressed in at least 3 different cells were excluded; (2) cells with mitochondrial content > 10% were excluded; (3) predicted doublets were removed using the DoubletFinder package (v2.0.3) [4]. After quality control, the data were normalized and regression of technical variation was performed using the SCTransform method implemented in Seurat. Principal component analysis (PCA) was performed on high-quality single-cell data, with the inclusion of principal components based on cumulative contribution, accounting for more than 85% of the variance, and the difference between consecutive pcs was less than 0.1%. Batch effects were removed using the harmony package (v1.2.0) [5]. Cells were clustered in an unsupervised manner using the FindClusters function (resolution = 1.0). Marker genes for each cluster were calculated using the FindAllMarkers function and Wilcoxon rank-sum test, with the following conditions: (1) min.pct > 0.25; (2) lnFC > 0.5; (3) P < 0.05. Cell types were annotated by comparison with classical cell type markers. When performing cell subpopulation analysis, the subset function was used to isolate specific cell subpopulations, and the above steps were repeated to mitigate batch effects and identify clusters driven by biological differences. To quantify the tissue preference of each subpopulation, the ratio of observed cell counts to expected cell counts (Ro / e) was calculated for each cluster [6] in various tissues. The chi-square test was used to determine the expected cell count for each cell cluster and tissue combination.

[0183] 3. Cell subpopulation functional enrichment analysis

[0184] Genes were pre-selected for gene set enrichment analysis (GSEA) based on cell-corrected p-value < 0.05 and expression in > 10% of cells. These genes were pre-classified based on fold change and served as input for GSEA. GSEA analysis was performed using the “clusterProfiler” (v4.10.1) [7] package, which contains gene sets related to gene Ontology (GO).

[0185] EndMT score was assessed using the AddModuleScore function to evaluate the subpopulation pathway activity of different ECs subpopulations and different groups (patients and control groups). Endothelial-mesenchymal transition score was based on the "HALLMARK: epithelial-mesenchymal transition" gene set.

[0186] UMAP plot of single-cell data is shown in FIG. 1. Figure 2

[0187] The results show that the number of endothelial cells in the endothelial-mesenchymal transition state increases in PH-LHD patients, suggesting that EndMT is associated with the pathogenesis of PH-LHD.

[0188] Cell proportion diagram is shown in FIG. 2. Figure 3

[0189] Endothelial cell subpopulation classification and top 10 Marker gene heat map are shown in FIG. 3. Figure 4

[0190] The results show that each cell subpopulation has its own marker gene, indicating that the cell subpopulation result is reliable.

[0191] Example 2 Immunofluorescence and cell Western blot of tissues and cells verify the existence of EndMT subpopulations in pulmonary arterial hypertension

[0192] For formalin-fixed, paraffin-embedded human arterial tissues (obtained from the Affiliated Union Hospital of Fujian Medical University), they were dewaxed to water, and then heat-induced extracted in EDTA (pH 8.0) for 20 minutes. The tissues were incubated in 3% hydrogen peroxide at room temperature for 25 minutes to block endogenous peroxidase, and then blocked with 3% bovine serum albumin for 30 minutes. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated at room temperature for 1 hour. The immunofluorescence images were acquired using Pannoramic Scanner software (v3.0.3.139795RTM). Image analysis was performed using Slide Viewer software (v2.5) and ImageJ software (v2.14.0). The antibodies used include rabbit anti-human CD31 (1:20 00, Servicebio), mouse anti-human a-SMA (1:20 00, Servicebio), rabbit anti-human CD45 (1:20 00, Servicebio), enzyme-labeled goat anti-rabbit IgG (1:5000, Servicebio), and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:40 00, Servicebio).

[0193] ​​​Stretched and unstretched paec (pulmonary artery endothelial cells) were fixed in 4% paraformaldehyde (HRA2070, Herubio) for 10 minutes and washed the cells with PBS 3 times. Cells were incubated in PBS containing 0.1% Triton X-100 (HRD0141, Herubio) for 10 minutes and washed with PBS 3 times for 5 minutes each. Cells were incubated in PBST (PBS + 0.1% Tween 20) containing 1% BSA (HRD0111, Herubio) and 22.52 mg / mL glycine (HRA1011, Herubio) for 30 minutes. Stretched and unstretched paec (pulmonary artery endothelial cells) were incubated with diluted primary antibodies at 4°C overnight, respectively. Recovered primary antibodies, washed the cells with PBS 3 times for 5 minutes each. Cells were incubated with secondary antibodies at room temperature for 1 hour in the dark. The secondary antibody solution was poured out and the cells were washed with PBS 3 times for 5 minutes each in the dark. 0.5 pg / mL Hoechst was incubated for 1 minute and washed with PBS once. LAS X software (3.9.0) was used to obtain the immunofluorescence staining images. The antibodies used include CD31 rabbit antibody (1:200, ab clone), a-SMA mouse antibody (1:200, Proteintech), CD45 mouse antibody (1:200, Santa Cruz Biotechnology), goat anti-mouse IgG conjugated (1:100, ab clone) and goat anti-rabbit IgG conjugated (1:100, ab clone) mixed.

[0194] Western blot

[0195] Human pulmonary artery homogenate was quickly ground by tissue grinder (60HZ, 1 min, 2 times). Human pulmonary artery or paec was homogenized in frozen RIPA buffer (HRX0087, Heruibio) and added with protease and phosphatase inhibitor cocktail (P1046, Beyotime). The total protein concentration was determined by BCA method (ZJ102, Epizyme). SDS-PAGE was used to separate proteins, which were then transferred to a transfer membrane (IPVH00010, Millipore). The membrane was blocked in 5% skim milk (P0216, Beyotime) at room temperature for 1 hour. Then, the membrane was incubated with primary antibody diluted in 5% skim milk or primary antibody dilution buffer (P0256, Beyotime) at 4°C overnight. Subsequently, the secondary antibody was diluted 1:1 000 in 5% skim milk or secondary antibody dilution buffer (P0258, Beyotime) for 1 hour, and detected by ECL Kit (GK1008, Glpbio). The following primary antibodies were used: GAPDH (1:1000, A19056, Abeam), β-actin (1:1000, AC026, Abeam), a-SMA (1:1000, Abeam), TNF-a (1:5000, Abeam), TGF-βΙ (1:1000, Abeam), IL-6 (1:1000, Abeam), CD74 (1:1000, Genetex), CD44 (1:1000, Abeam), β-tubulin (1:1000, ab clone), goat anti-rabbit IgG (H+L) (1:10000, Abeam).

[0196] Results are shown in Figure 5

[0197] The results show that significant endothelial mesenchymal transition phenomenon occurs in both in vivo and in vitro experiments of PH-LHD patients, further proving that EndMT plays an important role in PH-LHD.

[0198] Example 3

[0199] After the first discovery of EndMT subpopulation in the data of pulmonary artery single cell sequencing, we further performed spatial transcriptome sequencing, and found that there is also a subpopulation of endothelial cells undergoing EndMT in the spatial transcriptome data, and they are mainly distributed in the middle layer and adventitia of the blood vessels.

[0200] As Figure 6 ​As shown, the C1, C2, C3, and C4 subsets (the Endmt subset separated from the idling data) in the spatial transcriptome data have the same characteristics as the EndMT subset in single-cell sequencing, both of which simultaneously express endothelial cell markers (VWF, CD31) and mesenchymal cell markers (α-SMA, DCN, TAGLN).

[0201] Meanwhile, the differentiation process from C1 to C4 occurs along with endothelial cell markers (such as... Figure 7 VWF and PECAM1 (also known as CD31) in mesenchymal cells gradually decrease, as do mesenchymal cell markers (such as... Figure 7 The gradual increase in ACTA2 (also known as α-SMA) and TAGLN in pulmonary artery endothelial cells (PH-LHD) is similar to that in partial EndMT. Furthermore, in partial EndMT, TGF-β1, TNFRSF1A, CXCL12, CCL14, and CD74 play a regulatory role in the early stages, while CD44 plays a regulatory role in the later stages. An increased mesenchymal cell marker / endothelial cell marker ratio indicates endothelial-mesenchymal transition (EMT). Based on this, increased expression of pulmonary artery endothelial immune signals (CXCL12, TNFRSF1A, CCL14) and CD74 and TGF-β1 indicates that the patient is in the early stage of EndMT, meaning the EndMT cell subset is in the early stage of endothelial-mesenchymal transition; therefore, PH-LHD patients are early-stage PH-LHD patients. Conversely, elevated CD44 in the pulmonary artery endothelial layer indicates that the patient is in the late stage of EndMT, meaning the EndMT cell subset is in the late stage of endothelial-mesenchymal transition; therefore, PH-LHD patients are late-stage PH-LHD patients.

[0202] To verify this finding, we returned to single-cell data for analysis and validation.

[0203] First, we further subdivided the EndMT subset of endothelial cells in the single-cell data into five subsets: Endmt1, 2, 3, 4, and 5, to verify the expression of endothelial cell markers and mesenchymal cell markers, such as... Figure 7 Endothelial cell markers (e.g., Endmt 1 to 5 subsets) also appeared. Figure 8 VWF and PECAM1 (also known as CD31) in mesenchymal cells gradually decrease, while mesenchymal markers (such as...) decrease. Figure 8ACTA2 (also known as a-SMA), TAGLN) and the regulatory signals (CXCL12, TNFRSF1A, CCL14) and CD74, TGFβ1 also play a role in the early stage of EndMT, while CD44 plays a role in the late stage of EndMT. The increase in the ratio of mesenchymal cell markers / endothelial cell markers indicates that endothelial mesenchymal transformation has occurred. Then, on this basis, the expression of endothelial layer signals (CXCL12, TNFRSF1A, CCL14) and CD74, TGFβ1 in the pulmonary artery indicates that the patient is in the early stage of EndMT, that is, the EndMT cell subpopulation is in the early stage of mesenchymal transformation of endothelial cells, and the patient with PH-LHD is an early PH-LHD patient; if the CD44 of the pulmonary artery endothelial layer is increased, it indicates that the patient is in the late stage of EndMT, that is, the EndMT cell subpopulation is in the late stage of mesenchymal transformation of endothelial cells, and the patient with PH-LHD is a late PH-LHD patient.

[0204] To further verify the reliability, we simulated the disease state of PH-LHD endothelial cells by mechanical stretching of pulmonary artery endothelial cells, as follows: the 3-4th generation of pulmonary endothelial cells (purchased from ATCC) were evenly seeded into 6-well plates (BF-3001C, Flexcell) at a density of 2.5x10 5 cells / well, and the cell culture medium (1001, Sciencell) contained 5% FBS (0025, Sciencell) and 1% endothelial cell growth factor (1052, Sciencell). When the cells grew to 70% confluence, the culture medium was replaced with serum-free medium (sigma), and the cells were cultured for 24 h to induce a resting period. The serum-free medium was replaced again before mechanical stretching stimulation. The paec was subjected to periodic stimulation using a Flexcell 5000 tension system. The 6-well plates were placed in a 37°C, 5% CO2 incubator. The main computer group was turned on, and an external vacuum pump was connected. The stress loading system parameters were preset. According to previous studies [8], the non-stretching group (control group): no stretching force stimulation, and the remaining culture conditions were the same as those of the stretching force group below. The 15% stretching group (patient group): mechanical stretching force stimulation, maximum stretching peak of 15%, stretching frequency of 1hz, and duration of 12h, 24h, and 36h. The cells were collected at 12h, 24h, and 36h.

[0205] The collected endothelial cells were used to verify the expression changes of regulatory signals mesenchymal cell markers and the expression trends of TGF-β1, CD74, and CD44 under mechanical stretching for 12h, 24h, and 36h using western blot, as shown in Figure 9 . Figure 9Mesenchymal cell marker (ACTA2 (also known as a-SMA)) gradually increased, and TGF-β1, CD74 played a regulatory role in the early stage, and CD44 played a regulatory role in the late stage).

[0206] Figure 9 The experiment was done in endothelial cells, so it only needs to prove that mesenchymal cell markers (a-SMA) slowly increase over time to prove that mesenchymal transformation is continuously progressing. In the 0h, 12h, 24h, 36h group, aSMA is gradually increasing, indicating that mesenchymal transformation is continuously progressing, and TGF-β1, CD74 gradually increase at 0, 12, 24, indicating that they play a role in the early stage, and decrease at 36h (late stage). CD44 gradually increases in this process, indicating that the later the CD44 in EndMT, the greater the role.

[0207] Then, we further verify the EndMT process in the PH-LHD basic at the animal level. Since EndMT is a gradual transformation process, we divide the PH-LHD rats into four groups: normal control group (Control), PH-LHD group 5 weeks (Banding operation), PH-LHD group 7 weeks, and PH-LHD group 9 weeks. The method of Banding operation to create a PH-LHD rat model is as follows.

[0208] 1. Construction of PH-LHD rat model

[0209] (1) Model making method:

[0210] After the rats (obtained from the Animal Medical Center of Fujian Medical University) were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (50ml / kg), they were fixed in a supine position on a heat preservation operating table and connected to a small animal respirator for mechanical ventilation. The second intercostal space on the left side of the sternum was separated layer by layer, the thymus was pushed aside, and the aorta and pulmonary artery were exposed. A self-made titanium clip with an inner diameter of 0.8mm was quickly fixed around the ascending aorta. After checking the tightness of the titanium clip and confirming that there was no obvious bleeding, the chest was closed at the end of inspiration, and the incision was sutured layer by layer with sterile surgical technique. After the rats woke up, they were put back into the animal center feeding cage. Penicillin was added to the drinking water for 3 days after the operation to prevent infection.

[0211] (2) Hemodynamic monitoring:

[0212] ① Heart Doppler ultrasound measurement: 42 days after Banding, a GE VIVID-7 DIMENSION ultrasonic diagnostic instrument (equipped with a 10S probe) was used to scan the long rectangular strong echo shadow at the distal end of the ascending aorta, confirm the position of the titanium clip, measure the pulmonary artery pressure value for 3 consecutive cardiac cycles, and take the average value.

[0213] ② PE catheter measurement method: After the rats were anesthetized, the trachea and internal jugular vein were exposed and bluntly separated. The PE catheter passed through the small incision of the blood vessel through the right internal jugular vein, superior vena cava, right atrium to the right ventricle, connected with BL-420S monitor, and the pulmonary arterial pressure was measured.

[0214] After modeling, we measured the right ventricular pressure (RVP) of each group of rats, left atrial pressure (LAP), calculated the percentage of right ventricular weight to body weight (RVW / BW), the percentage of left ventricular and septal weight to body weight (LV+S W / BW), and multiple data such as pulmonary artery media thickness calculated by pulmonary tissue HE staining to confirm the success of PH-LHD rat modeling Figure 11 A-G). In addition, it was confirmed by immunofluorescence that the degree of EndMT transformation in the pulmonary artery of rats increased with the extension of PH-LHD modeling time (5 weeks to 9 weeks) Figure 11 H-M).

[0215] Finally, in order to explore the role of CD74 and CD44 in EndMT of PH-LHD rats, we knocked down CD74 or CD44 in PH-LHD rats by intratracheal instillation of AAV9-shRNA. The results showed that after intratracheal instillation of CD74 inhibitor, the right ventricular pressure and pulmonary vascular remodeling of PH-LHD rats at 5 weeks, 7 weeks and 9 weeks decreased, while the left atrial pressure and left ventricular remodeling did not improve, indicating that CD74 mainly plays a therapeutic role in the pulmonary artery, not in the left ventricle. However, after intratracheal instillation of CD44 inhibitor, the above indexes did not improve Figure 12 A-J), CD44 can only alleviate the EndMT of PH-LHD rats at 9 weeks, but cannot reverse the pulmonary vascular remodeling of PH-LHD rats Figure 12 ).

[0216] In addition, the degree of EndMT transformation was detected by immunofluorescence and western blot detection (CD31 and a-SMA) expression, and the results showed that after intratracheal instillation of CD74 inhibitor, the degree of EndMT transformation of PH-LHD rats at 5 weeks, 7 weeks and 9 weeks decreased, while CD44 did not significantly improve Figure 12 K-S, R1, R2, S1, S2, P1, Q1).

[0217] Therefore, we believe that CD74 may play a therapeutic role in the early stage of PH-LHD patients by reducing the degree of pulmonary artery EndMT transformation, and is expected to become a target for future PH-LHD treatment.

[0218] References

[0219] [1] Young, MD, Behjati, S. SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data. Gigascience. 2020; 9(12).

[0220] [2] Hao, Y, Stuart, T, Kowalski, MH, et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis. NAT BIOTECHNOL. 2023; 42(2): 293-304.

[0221] [3] Macosko, EZ, Basu, A, Satija, R, et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. CELL. 2015; 161(5): 1202-1214.

[0222] [4] McGinnis, CS, Murrow, LM, Gartner, ZJ. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. CELLSYST. 2019; 8(4): 329-337.e4.

[0223] [5] Korsunsky, I, Millard, N, Fan, J, et al. Fast, sensitive and accurate integration of single-cell data with Harmony. NAT METHODS. 2019; 16(12): 1289-1296.

[0224] [6] Zhang, L, Yu, X, Zheng, L, et al. Lineage tracking reveals dynamic relationships of T cells in colorectal cancer. NATURE. 2018; 564(7735): 268-272.

[0225] [7] Wu, T, Hu, E, Xu, S, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (N Y). 2021; 2(3): 100141.

[0226] [8] Zhou Y, Tabib T, Huang M, et al. Molecular Changes Implicate Angiogenesis and Arterial Remodeling in Systemic Sclerosis-Associated and Idiopathic Pulmonary Hypertension. Arterioscler Thromb Vasc Biol. 2024 Aug; 44(8): e210-e225.

[0227] All documents referred to in this disclosure are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that these equivalents ype within the scope of the appended claims.

Claims

1. A use of (i) for the classification of a patient with PH-LHD according to different stages of endothelial cell mesenchymal transition; and / or (ii) as a biomarker for determining whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for the use of a CD74 inhibitor, and / or a CD44 inhibitor as a treatment for left heart disease-related pulmonary arterial hypertension (PH-LHD), characterized in that, The biomarker comprises: an EndMT cell subpopulation.

2. The biomarker of claim 1, wherein, The EndMT cell subpopulation has one or more characteristics selected from the group consisting of: (a) decreased expression of endothelial cell markers and increased expression of mesenchymal cell markers or increased ratio of mesenchymal cell markers / endothelial markers; (b) TGF-β1, TNFRSF1A, CXCL12, CCL14 and CD74 are up-regulated in the early stage of mesenchymal transformation of endothelial cells, and CD44 is up-regulated in the late stage of mesenchymal transformation of endothelial cells.

3. The biomarker of claim 1, wherein, The biomarker is derived from a tissue, preferably pulmonary arterial tissue, more preferably the endothelial layer of pulmonary arterial tissue.

4. A combination of agents for (i) typing a patient with PH-LHD according to the different stages of the mesenchymal transition of endothelial cells; and / or (ii) use as a judge of whether a patient with pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for treating pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, characterized in that, The reagent combination comprises reagents for detecting the biomarker of claim 1.

5. A kit (i) for the phenotyping of a patient with PH-LHD according to different stages of endothelial cell mesenchymal transition; and / or (ii) for use as a diagnostic to determine whether a patient with pulmonary arterial hypertension related to left heart disease (PH-LHD) is suitable for the treatment of pulmonary arterial hypertension related to left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, characterized in that, The kit comprises: specific tools or reagents for measuring the function of the EndMT cell subpopulation in the biological sample.

6. Use of the biomarker of claim 1, characterized in that, A reagent or kit for preparing a reagent or kit for (i) typing patients with PH-LHD according to different stages of mesenchymal transformation of endothelial cells; and / or (ii) determining whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease-related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor and / or a CD44 inhibitor.

7. A method for the stratification of patients with PH-LHD according to the different stages of the mesenchymal transition of endothelial cells; and / or for the use as a method for determining whether a patient with pulmonary arterial hypertension associated with left heart disease (PH-LHD) is suitable for the treatment of pulmonary arterial hypertension associated with left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, characterized in that, Comprising: (1) providing a sample derived from a subject to be tested, detecting the biomarker of claim 1 in the sample, wherein the detection comprises detecting the function of the EndMT cell subpopulation, preferably, determining the expression levels of the immune function molecules VWF, CD31, α-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74 and CD44 in the EndMT cell subpopulation; (2) if the ratio (E1 / E2) between the expression level E1 of the immune function molecule α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker determined in step (1) is higher than the reference value C1, and the expression level of TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, it indicates that the patient is an early PH-LHD patient, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue in the normal population, and the reference value C2 is the expression level of TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue in the normal population, then it indicates that the patient is suitable for treatment with a CD74 inhibitor; or If the ratio (E1 / E2) between the expression level E1 of the immune function molecule protein a-SMA, DCN or TAGLN and the expression level E2 of the VWF or CD31 protein in the EndMT cell subpopulation of the biomarker determined in step (1) is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, the patient is a patient with advanced PH-LHD, the reference value C1 is the ratio (E0 / E0’) between the expression level E0 of the a-SMA, DCN or TAGLN protein and the expression level E0’ of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population, indicating that the CD44 inhibitor is suitable for treatment.

8. A method of establishing (i) a model for the classification of patients with PH-LHD according to different stages of mesenchymal transition of endothelial cells; and / or (ii) a model for the use as a judge of whether a patient with left heart disease-related pulmonary arterial hypertension (PH-LHD) is suitable for the treatment of left heart disease-related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, characterized in that, The method comprises the step of identifying the difference biomarkers in the biological sample between the patients with left heart disease-related pulmonary arterial hypertension (PH-LHD) and the healthy controls, wherein the difference biomarkers comprise the biomarkers according to claim 1.

9. A method for classifying a patient with PH-LHD according to different stages of endothelial cell mesenchymal transition; and / or a system for determining whether a patient with pulmonary arterial hypertension associated with left heart disease (PH-LHD) is suitable for treating pulmonary arterial hypertension associated with left heart disease (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, characterized in that, The system comprises: (a) a feature receiving module, which is used for receiving feature data from the sample of the subject to be tested; the feature data comprises the level of the biomarker according to claim 1 in the sample of the subject to be tested, the function value of the EndMT cell subpopulation, and preferably the expression level of the immune function molecule protein VWF, CD31, a-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74 and CD44 in the EndMT cell subpopulation; (b) a discrimination processing module, which compares the received characteristic data with reference values, thereby obtaining a diagnosis or evaluation result, wherein when the ratio (E1 / E2) between the expression level E1 of the α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the characteristic data is higher than the reference value C1, and the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein is higher than the reference value C2, it is suggested that the subject is an early-stage PH-LHD patient, indicating that a CD74 inhibitor is suitable for treatment, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C2 is the expression level of the TGF-β1, TNFRSF1A, CXCL12, CCL14 or CD74 protein in the endothelial layer of the same tissue of the normal population; or when the ratio (E1 / E2) between the expression level E1 of the α-SMA, DCN or TAGLN protein and the expression level E2 of the VWF or CD31 protein in the characteristic data is higher than the reference value C1, and the expression level of the CD44 protein is higher than the reference value C3, it is suggested that the subject is a late-stage PH-LHD patient, indicating that a CD44 inhibitor is suitable for treatment, the reference value C1 is the ratio (E0 / E0') between the expression level E0 of the α-SMA, DCN or TAGLN protein and the expression level E0' of the VWF or CD31 protein in the endothelial layer of the same tissue of the normal population, and the reference value C3 is the expression level of the CD44 protein in the endothelial layer of the same tissue of the normal population; and (c) a result output module, which is used for receiving and outputting the evaluation result.

10. A method for using the biomarker of claim 1 in a subject to be tested for typing patients with PH-LHD according to different stages of the mesenchymal transformation of endothelial cells; and / or (ii) as an indicator for judging whether a patient with left heart disease related pulmonary arterial hypertension (PH-LHD) is suitable for treating left heart disease related pulmonary arterial hypertension (PH-LHD) with a CD74 inhibitor, and / or a CD44 inhibitor, the method comprising determining the level of the biomarker in a sample from the subject to be tested, including determining the function of the EndMT cell subpopulation, preferably, determining the expression level of the immune function molecule proteins VWF, CD31, α-SMA, DCN, TAGLN, TGF-β1, TNFRSF1A, CXCL12, CCL14, CD74, CD44 in the EndMT cell subpopulation.

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