[0001] REFERENCE TO GOVERNMENT GRANTS

[0002] This invention was made with government support under HL 142638 awarded by National Institutes of Health. The Government has certain rights in the invention.

[0003] FIELD OF THE INVENTION

[0004] The general field of the present disclosure are novel approaches to the treatment of pulmonary hypertension and methods for measuring or assessing the effectiveness of such treatments.

[0005] BACKGROUND

[0006] Pulmonary hypertension (PH) is a progressive illness often presenting with nonspecific symptoms including dyspnea, dizziness, lower extremity edema, and decreased exercise tolerance. At the cellular level, it is characterized by endothelial cell dysfunction and increased contractility of the small pulmonary arteries, which lead to abnormal intimal and smooth muscle proliferation together with resistance to apoptosis. Pulmonary vascular remodeling is a prominent feature of PH independent of the etiology. This remodeling increases pulmonary vascular resistance (PVR), which eventually leads to failure of the right ventricle (RV) due to rising afterload. Many symptoms of PH, including lower extremity edema and dyspnea, arise from RV failure. See Hensley et al., “Emerging therapeutics in pulmonary hypertension,” (2018) Am J Physiol Lung Cell Mol Physiol 314: L769-L781.

[0007] Specifically, PH is defined by end-expiratory’ mean pulmonary artery pressure >20 mmHg and PVR >3 Wood units at rest. Id. PH is a nonspecific umbrella term, which covers elevated pulmonary artery pressure regardless of the etiology’. The initial clinical classification of PH has arisen from a World Health Organization-sponsored international meeting in 1973. PH has been subdivided into five groups based on the disease pathology’ and specific cause. Pulmonary arterial hypertension (PAH; Group 1 PH) specifically refers to disease processes, which result in vasoconstriction and stiffening of the small arteries in the lungs secondary to cell proliferation, fibrosis, as well as the development of in situ thrombi or plexiform lesions. This pathology’ both defines PAH and unifies the multiple etiologies, which may lead to the development of the disease. PAH can be idiopathic, can be heritable, and can be associated with connective tissue disease, HIV, drug use, etc. There are other pathologies in which PH presents as a secondary disease, including left heart disease (Group 2), chronic lung diseases and/or hypoxia (Group 3), chronic thromboembolic pulmonary hypertension (CTEPH, Group 4), and miscellaneous or multi- factorial etiologies (Group 5). See Id. at Figure 1.

[0008] PH attributable to left heart disease or left ventricular diastolic dysfunction, also referred to as PH associated with heart failure with preserved ejection fraction (PH-HFpEF), is clearly the most common. See Lai et al., "Insights into the pulmonary vascular complications of heart failure with preserved ejection fraction,” (2019) J Physiol 597.4: pp. 1143-11 6. Although the true prevalence of PH-HFpEF remains largely unknown, the range in the reported prevalence of PH is between 23% and 83%. Heart failure with preserved ejection fraction (HFpEF) is characterized by abnormal active relaxation and increased passive stiffness of the left ventricle (LV), resulting in elevated LV filling pressure. Passive backward transmission of the elevated LV filling pressure eventually leads to an increase in pulmonary' (both venous and arterial) remodeling and pressures, which, over time, contribute to elevated pulmonary vascular resistance and right heart failure. See Rosenkranz et al.. “Left ventricular heart failure and pulmonary hypertension.” (2016) Eur Heart J. 37: pp. 942-954; Lai et al. 2019.

[0009] Accordingly, Group 2 PH is a growing public health problem that is increasing in prevalence, affecting approximately 1.6 million patients in the United States alone. Many patients with Group 1 PH due to pulmonary hypertension have many comorbidities including obesity’, hypertension, diabetes mellitus, and hypercholesterolemia, all clinical features of the metabolic syndrome (MS) which may itself predispose patients to PH. See Robbins et al., “Association of the Metabolic Syndrome with Pulmonary’ Venous Hypertension,” (2009) CHEST 136: pp. 31-36. [0010] However, no approved specific medication or consensus therapeutic strategy' for PH- HFpEF is available at present. As obesity, the major comorbidity of metabolic syndrome, is predicted to reach 18% in 2025 with an estimated health services cost of US$990 billion per year globally (see Ward et al., “Projected U.S. State-Level Prevalence of Adult Obesity and Severe Obesity,” (2019) N Engl J Med 381 : pp. 2440-2450. understanding of the pathophysiology processes and identifying biomarkers that can critically inform clinical diagnosis and risk stratification of PH-HFpEF are of important.

[0011] Sodium glucose cotransporter 2 (SGLT2) inhibitors have recently been approved for the treatment of HFpEF. See Anker et al., “Empagliflozin in Heart Failure with a Preserved Ejection Fraction,” (2021) N Engl J Med. 385: pp. 1451-1461. Although SGLT2 inhibitors have been reported to improve PH in SU5416-exposed obese ZSF1 (Ob-Su) rats, patients with type 2 diabetes, or HF, the search for effective therapies for PH-HFpEF remains. See Satoh et al., “Metabolic Syndrome Mediates ROS-miR-193b-NFYA-Dependent Down Regulation of sGC and Contributes to Exercise-Induced Pulmonary Hypertension in HFpEF,” (2021) Circulation 144: pp. 615-637; Kayano et al., “Dapagliflozin Influences Ventricular Hemodynamics and Exercise- Induced Pulmonary Hypertension in Type 2 Diabetes Patients - A Randomized Controlled Trial,” (2020) Circ J. 84: pp. 1807-1817; Nassif et al.. “Empagliflozin Effects on Pulmonary Artery Pressure in Patients With Heart Failure: Results From the EMBRACE-HF Trial,” (2021) Circulation 143: pp. 1673-1686. However, a deeper insight into pathophysiological processes and molecular mechanisms may reveal new candidate target(s) for early identification and effective management of PH-HFpEF.

[0012] Apart from the gold standard right heart catheterization (RHC) and/or echocardiographic data, B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are at present the only guideline-recommended biomarkers for the diagnosis and risk stratification of HF and PH. See Galie et al., “2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT),” (2015) Eur Respir J. 46: pp. 903-975; Ponikowski et al., “2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HF A) of the ESC,” (2016) Eur Heart J. 37: pp. 2129-2200; Leuchte et al., “Risk stratification strategy and assessment of disease progression in patients with pulmonary arterial hypertension: Updated Recommendations from the Cologne Consensus Conference.” (2018) Int J Cardiol. 272S: pp. 20-29. High circulating levels of BNP and NT-proBNP appear to correlate well with increased frequency of HF hospitalizations, impaired RV function, and severe exercise intolerance in PH-HFpEF patients. See Anjan et al., “Prevalence, clinical phenotype, and outcomes associated with normal B-type natriuretic peptide levels in heart failure with preserved ejection fraction,” (2012) Am J Cardiol 110: pp. 870-876; Gorter et al., “Right ventricular-vascular coupling in heart failure with preserved ejection fraction and pre- vs. post-capillary pulmonary hypertension,” (2018) Eur Heart J Cardiovasc Imaging 19: pp. 425-432; van Wezenbeek et al., “Right Ventricular and Right Atrial Function Are Less Compromised in Pulmonary' Hypertension Secondary to Heart Failure With Preserved Ejection Fraction: A Comparison With Pulmonary Arterial Hypertension With Similar Pressure Overload,” (2022) Circ Heart Fail. 15: e008726. However, disproportionately low BNP and NT-proBNP levels have been reported among patients with obesity, which coexists in up to 50% of patients with PH-HFpEF, complicating the usefulness of these factors in identifying at-risk subjects and predicting patient outcomes. See Obokata et al., “Evidence Supporting the Existence of a Distinct Obese Phenotype of Heart Failure With Preserved Ejection Fraction,” (2017) Circulation 136: pp. 6-19. Elevated plasma levels of endothelin-1 (ET-1), vascular endothelial growth factor-D (VEGF-D), leptin, and adiponectin, as well as reduced serum levels of microRNA-206, have been associated with PH severity in humans and in animal models of PH-HFpEF. See Obokata et al., “The neurohormonal basis of pulmonary ⁷ hypertension in heart failure with preserved ejection fraction,” (2019) Eur Heart J. 40: pp. 3707- 3717; Saleby et al., “Plasma receptor tyrosine kinase RET in pulmonary arterial hypertension diagnosis and differentiation,” (2019) ERJ Open Res. 5: 00037-2019; Jin et al., “The Circulating MicroRNA-206 Level Predicts the Severity of Pulmonary Hypertension in Patients with Left Heart Diseases,” (2017) Cell Physiol Biochem. 41 : pp. 2150-2160: Lai et al., “SIRT3-AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction,” (2016) Circulation 133: pp. 717-731; Ranchoux et al., “Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease,” (2019) Circ Res. 125: pp. 449-466; Todd et al., “Current Understanding of Circulating Biomarkers in Pulmonary Hypertension Due to Left Heart Disease.” (2020) Front Med (Lausanne) 7: p. 570016. However, their usefulness in the characterization and/or determination of disease management of PH-HFpEF remain unclear.

[0013] Furthermore, what is needed are clear biomarkers that can be identified early to allow ⁷ pharmacologic and other interventions to treat and prevent the effects of PH and its associated morbidities. The present invention addresses these needs.

[0014] SUMMARY OF THE INVENTION

[0015] The inventors have recently discovered that beta 2-microglobulin (B2M) is present at higher levels in patients with PH-HFpEF compared to control subjects. B2M is a component of major histocompatibility complex class I (MHC I) and elevated levels of B2M have been implicated in cardiac fibrosis, atherosclerosis, kidney diseases, aging - all known risk factors for HFpEF. Serum B2M concentration is positively correlated with patients with dialysis-related amyloidosis, chronic kidney disease (CKD), type 2 diabetes, COPD, acute coronary syndrome, atherosclerosis, multiple myeloma, and HIV. Circulating B2M levels are also associated with coronary heart disease (CHD) and all-cause mortality. Other studies have shown that cerebrospinal fluid (CSF) B2M levels are elevated in hypoxic-ischemic encephalopathy and have been implicated in age-related impairment of neurogenesis, declined glomerular filtration rate (GFR), cardiac fibrosis, inflammation in animals.

[0016] Using plasma proteomics, the inventors have identified high protein abundance levels of B2M in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle sirtuin-3 (SIRT3) deficiency or high-fat diet (HFD)-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure (PCWP) and right atrial pressure (RAP) at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells (PAVECs), but it also increased proliferating cell nuclear antigen (PCNA) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells (PAVSMCs). Finally, B2M deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered pulmonary hypertension, and attenuated RV hypertrophy in mice with HFD-induced PH-HFpEF.

[0017] These findings reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary' vascular proliferative remodeling and PH-HFpEF. These data also suggest that circulating and skeletal muscle B2M can be promising targets for identification and management of PH-HFpEF.

[0018] Accordingly, the present disclosure provides novel approaches to the diagnosis/prognosis and treatment of PH-HFpEF. As PH-HFpEF is largely associated with metabolic syndrome, including diabetes, hypertension, kidney disfunction, and heart failure with preserved ejection fraction (HFpEF), this invention provides potential diagnostic/prognostic value and treatment for metabolic syndrome and HFpEF.

[0019] More specifically, current invention provides inhibitors targeting B2M, B2M receptors, and regulators of B2M receptors for the treatment of Group 1-5 PH.

[0020] In other embodiments, the invention provides therapeutic approaches using the administration of inhibitors of B2M, B2M receptors, and regulators of B2M receptors for the treatment of Group 1-5 PH, metabolic syndrome, and HFpEF.

[0021] In still other embodiments, the current invention provides methods of treatment of PH- HFpEF by reducing B2M from the blood stream in a patient with elevated levels of B2M or other biomarkers.

[0022] In other embodiments, the current invention provides methods of treatment of PH- HFpEF by administration of antagonists for B2M receptors to patients in need thereof. [0023] In some embodiments, the current invention provides methods of treatment of PH- HFpEF by various types of receptor inhibition therapy.

[0024] In yet other embodiments, the current invention provides methods of treatment of PH- HFpEF skeletal muscle-specific gene therapy.

[0025] In any embodiment of the invention, is provided methods of determining the severity in PH-HFpEF patients by measuring the amount of B2M and related biomarker of PH-HFpEF severity' and prognosis.

[0026] More specifically, in embodiments of the current invention are provided methods of inhibiting, treating, or preventing the effects of elevated B2M in patients comprising inhibiting, neutralizing or depleting B2M from the patient.

[0027] In some embodiments, the invention provides methods of depleting B2M in addition to one or more regulators of B2M gene expression or synthesis.

[0028] Thus, in any of the methods provided in the current invention, B2M can be inhibited, neutralized or depleted by the administration of and agent to the patient where the agent comprises an adeno-associated virus (AAV) or lentovirus-containing an a short-hairpin RNA (shRNA) against B2M.

[0029] In some embodiments, the shRNA is commercially available and can be attached to or part of any vector known in the art including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0030] In other embodiments, the agent comprises a monoclonal or polyclonal antibody directed against B2M or a B2M receptor. In yet other embodiments, the agent comprises a monoclonal or polyclonal antibody directed against B2M or a B2M receptor. In still other embodiments, the agent is an siRNA or antisense oligonucleotide that targets B2M or a B2M receptor.

[0031] In still other embodiments, the agent is an antagonist that binds to a B2M-mediated receptor and prevents the binding of B2M.

[0032] These and other embodiments and features of the disclosure will become more apparent through reference to the following description, the accompanying figures, and the claims. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and permutations. [0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1A-D depicts the changes in plasma protein abundance profiles in patients with PH-HFpEF. FIG. 1A: Schematic overview of mass spectrometry -based plasma proteomic analysis. FIG. IB: Volcano plot of the fold change and statistical significance for protein abundance levels. FIG. 1C: Changing protein abundances in PH-HFpEF [Age: 69.8 ± 7.9; male sex: 8; BMI: 39.1 ± 10.4; mean pulmonary artery pressure (mPAP): 39.4± 8. 1 mmHg; pulmonary capillary wedge pressure (PCWP): 20.2 ± 4.5 mmHg; WHO function class II: 1 (6%), III: 14 (88%), and IV: 1 (6%)]. FIG. ID: Protein abundance ratio of B2M. Data are mean ± SEM. P value was determined by Mann-Whitney U test.

[0035] FIG. 2A-F show the circulating levels of B2M are elevated in patients with PH-HFpEF. Circulating levels of B2M are elevated in patients with PH-HFpEF. FIG. 2A: Circulating levels of B2M were measured by ELISA in plasma of the validation cohort of control subjects, HFpEF patients without PH, and PH-HFpEF patients [confirmed diagnosis by RHC when the resting mPAP > 25 mmHg, PCWP > 15 mmHg, and transpulmonary pressure gradient (TPG) > 12 mmHg OR during exercise mPAP > 30 mmHg, PCWP > 20 mmHg, and total pulmonary resistance (TPR) > 3] . Correlation between circulating levels of B2M and resting mPAP (FIG. 2B), PCWP (FIG. 2C), TPG (FIG. 2D), pulmonary vascular resistance (FIG. 2E), or right atrial pressure (RAP, FIG. 2F) in PH-HFpEF patients whose plasma samples were collected within a month of RHC. Spearman r is shown. Data are mean ± SEM. P value was analyzed by Mann-Whitney Gtest.

[0036] FIG. 3A-F shows the skeletal muscle levels of B2M are increased in mice with skeletal muscle SIRT3 deficiency or HFD-induced PH-HFpEF. FIG. 3A and 3B: Skeletal muscle (FIG. 3A) and plasma (FIG. 3B) levels of B2M in wild-type (WT) and skeletal muscle-specific SIRT3 knockout (Sirt3 ^skn1 ’ ¹ ’) mice. FIG. 3C: Plasma B2M levels correlate positively with right ventricular systolic pressures (RVSP) in S/rt3 ^skm '' mice. FIG. 3D and 3E: Skeletal muscle (FIG. 3D) and plasma (FIG. 3E) levels of B2M in mice fed a regular diet (RD, 10% lipids/kcal) or high-fat diet (HFD, 60% lipids/kcal) for 16 weeks. FIG. 3F: Plasma B2M levels correlate positively with RVSP in HFD-exposed mice. Spearman r is shown. Data are mean ± SEM. P value was analyzed by Mann- Whitney U test.

[0037] FIG. 4A-C shows that muscle expression levels of B2M are increased in patients with PH-HFpEF. FIG. 4A: B2M expression levels were measured in muscle biopsies of PH-HFpEF patients and control subjects by Western blots. FIG. 4B and 4C: Correlation between skeletal muscle B2M expression with resting PCWP (FIG. 4B) or RAP (FIG. 4C). Spearman r is shown. Data are mean ± SEM. P value w as analyzed by Mann-Whitney t/test. [0038] FIG. 5A-D shows that treatment with B2M induces PAVECs migration/proliferation and promotes PAVSMCs proliferation. FIG. 5 A and 5B: Human PAVECs were administered with exogenous B2M (10 mg/ml) for 4 days. Representative images of cell migration and related quantitative data (FIG. 5A). Cell proliferation assessed by cell counts (FIG. 5B). FIG. 5C and 5D: Human PAVSMCs were exposed to B2M (10 mg/ml) for 5 days. Representative images of cell numbers and cell proliferation assessed by cell counts (FIG. 5C). Representative Western blots for PCNA protein expression levels (FIG. 5D). Data are mean ± SEM. P value was analyzed by Student’s I test.

[0039] FIG. 6A-I sho s that B2M whole-body KO mice protects against metabolic syndrome- associated PH-HFpEF. FIG. 6A: 8-week-old WT and whole-body B2M knockout mice (82m ¹ ') were fed a RD or HFD for 16 w eeks. At w eek 16, body w eights (FIG. 6B), glucose tolerant abilities (FIG. 6C), RVSP (FIG. 6D), and left ventricular end-diastolic pressure (LVEDP, FIG. 6G) were measured. Weights of RV (FIG. 6E) and LV+S (FIG. 6F) normalized to tibial length were used as index of ventricular hypertrophy. FIG. 6H: Representative images of lung sections stained with a- smooth muscle actin (a-SMA) and quantification of wall thickness from the mean of 5-6 vessels per lung section from 3 mice/group. Scale bar, 30 jam. FIG. 61: PCNA levels were analy zed by Western blot in PAVSMCs of 82m' ¹ ' and WT mice. Data are mean ± SEM. P value was analyzed by One-Way ANOVA followed by Tukey’s post hoc test. For glucose tolerance test, two-way ANOVA followed by Bonferroni’s post hoc test was performed.

[0040] FIG. 7A-B: Correlation between circulating B2M levels and mPAP (FIG. 7A) or PCWP (FIG. 7B) measured during cardiopulmonary exercise testing in PH-HFpEF patients. Spearman r is shown.

[0041] FIG. 8A-D shows protein levels of B2M assessed by Western blots in kidney (FIG. 8A), LV (FIG. 8B), adipose tissue (FIG. 8C), and RV (FIG. 8D) of HFD-exposed mice. Data are mean ± SEM. P value was analyzed by Mann-Whitney U test.

[0042] DETAILED DESCRIPTION

[0043] The inventors have recently discovered that beta 2-microglobulin (B2M) is present at higher levels in patients with PH-HFpEF compared to control subjects. B2M is a component of major histocompatibility complex class I (MHC I) and elevated levels of B2M have been implicated in cardiac fibrosis, atherosclerosis, kidney diseases, aging - all known risk factors for HFpEF. Serum B2M concentration is positively correlated with patients with dialysis-related amyloidosis, chronic kidney disease (CKD), type 2 diabetes, COPD, acute coronary syndrome, atherosclerosis, multiple myeloma, and HIV. Circulating B2M levels are also associated with coronary heart disease (CHD) and all-cause mortality. Other studies have shown that cerebrospinal fluid (CSF) B2M levels are elevated in hypoxic-ischemic encephalopathy and have been implicated in age-related impairment of neurogenesis, declined glomerular filtration rate (GFR), cardiac fibrosis, and inflammation in animals.

[0044] The inventors have found that plasma B2M has a significant positive correlation with disease severity in PH-HFpEF patients, demonstrating clinical relevance of B2M in PH-HFpEF pathogenesis and suggesting B2M as a relevant biomarker of PH-HFpEF severity and prognosis.

[0045] Additionally. B2M-deficient mice are protected from metabolic syndrome-associated PH-HFpEF, further suggesting B2M as a clinically meaningful molecular target for the treatment of the disease. Moreover, the disclosed data show that B2M is regulated by sirtuin-3 (SIRT3), a mitochondria deacetylase, deficiency in skeletal muscle. SIRT3 deficiency and its downstream reactive oxygen species (ROS) production have been associated with the development of pulmonary arterial hypertension (PAH, Group 1 PH), PH associated with hypoxia and lung disease (Group 3 PH), chronic thromboembolic PH (Group 4 PH), and PH associated with multifactorial causes (Group 5 PH). Together, by identifying/designing compounds/small molecules targeting B2M, B2M receptors (hemochromatosis protein, transferrin receptor complex 1. and MHC I), as well as regulators of B2M receptors (antigen processing 1). this invention provides potential treatment for Group 1-5 PH.

[0046] Accordingly, the present disclosure provides:

[0047] —novel approaches to the diagnosis/prognosis and treatment of PH-HFpEF including PH-HFpEF associated with metabolic syndrome, including diabetes, hypertension, kidney disfunction, and heart failure with preserved ejection fraction (HFpEF);

[0048] — inhibitors targeting B2M, B2M receptors, and regulators of B2M receptors for the treatment of Group 1-5 PH;

[0049] —therapeutic approaches using the administration of inhibitors of B2M, B2M receptors, and regulators of B2M receptors for the treatment of Group 1-5 PH, metabolic syndrome and HFpEF;

[0050] —methods of treatment of PH-HFpEF by reducing B2M from the blood stream in a patient with elevated levels of B2M or other biomarkers;

[0051] —methods of treatment of PH-HFpEF by administration of antagonists for B2M receptors to patients in need thereof;

[0052] —methods of treatment of PH-HFpEF by various types of receptor inhibition therapy; [0053] — methods of treatment of PH-HFpEF skeletal muscle-specific gene therapy;

[0054] —methods of determining the severity ⁷ in PH-HFpEF patients by measuring the amount of B2M and related biomarker of PH-HFpEF severity and prognosis;

[0055] —methods of inhibiting, treating, or preventing the effects of elevated B2M in patients comprising inhibiting, neutralizing or depleting B2M from the patient;

[0056] —methods of depleting B2M in addition to one or more regulators of B2M gene expression or synthesis;

[0057] —methods wherein B2M can be inhibited, neutralized or depleted by the administration of and agent to the patient where the agent comprises an adeno-associated virus (AAV) or lentovirus-containing an a short-hairpin RNA (shRNA) against B2M; the shRNA is commercially available and can be attached to or part of any vector known in the art including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes;

[0058] —methods wherein the agent comprises a monoclonal or polyclonal antibody directed against B2M or a B2M receptor;

[0059] -methods wherein the agent comprises a monoclonal or polyclonal antibody directed against B2M or a B2M receptor;

[0060] -methods wherein the agent is an siRNA or antisense oligonucleotide that targets B2M or a B2M receptor;

[0061] -methods wherein the agent is an antagonist that binds to a B2M-mediated receptor and prevents the binding of B2M.

[0062] Definitions

[0063] Various quantities, such as amounts, sizes, dimensions, proportions, and the like, are presented in a range format throughout this disclosure. It should be understood that the description of a quantity in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as all individual numerical values within that range unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3. from 1 to 4, from 1 to 5. from 2 to 4, from 2 to 6. from 3 to 6 etc., as well as individual values within that range, for example, 1.1 , 2, 2.3, 4.62, 5, and 5.9. This applies regardless of the breadth of the range. The upper and low er limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0064] The terminology used herein is to describe particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” "an". and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes,” “comprises,” “including” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A. B, or C” can mean (A); (B); (C): (A and B); (B and C); (A and C); or (A, B, and C).

[0065] Unless expressly stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0066] In any of the embodiments disclosed herein, the terms “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

[0067] In any of the embodiments disclosed herein, the term “patient” refers to a human.

[0068] B2M Inhibitors

[0069] The current invention contemplates that B2M can be neutralized or inhibited by several different non-limiting methods. For example, as described herein, B2M can be neutralized or inhibited by administration of a therapeutically effective amount of an agent where the agent comprises an adeno-associated virus (AAV) or lentivirus-containing an a short-hairpin RNA (shRNA) against B2M (sh-“B2M”). In some embodiments, the sh-“B2M” is commercially available and can be attached to or part of any vector known in the art including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0070] Alternatively, as described herein, B2M can be neutralized or inhibited by administration of a therapeutically effective amount of an agent where the agent comprises an antibody, bivalent antibody or a monoclonal antibody directed against B2M, a B2M receptor or other B2M modulators.

[0071] Further, as described herein, B2M can be neutralized or inhibited by administration of a therapeutically effective amount of an agent where the agent comprises an siRNA or antisense oligonucleotide that targets B2M, a B2M receptor or other B2M modulators.

[0072] Also, as contemplated herein, B2M, a B2M receptor or other B2M modulators can be neutralized or inhibited by administration of a therapeutically effective amount of an agent where the agent comprises an antagonist that binds to B2M, a B2M receptor or other B2M modulator and prevents the binding of B2M.

[0073] The target B2M, a B2M receptor or other B2M modulator inhibitor or a composition therein can be administered once per day, two or more times daily or once per week. The target inhibitor or inhibitors or composition containing the same can occur by any conventional means including orally intramuscularly, intraperitoneally or intravenously into the subject. If injected, they can be injected at a single site per dose or multiple sites per dose.

[0074] B2M Antibodies and Related Inhibitors

[0075] More specifically a B2M inhibitor is an antibody directed against B2M, a B2M receptor or other B2M modulator as disclosed herein. Examples of suitable antibodies directed against one or more targets are disclosed herein and known to those of skill in the art. The B2M antibody can also include an antibody fragment or a bivalent antibody or fragment thereof, inhibiting one or more of B2M, a B2M receptor or other B2M modulator. As described herein, the B2M inhibitor may be part of a pharmaceutical composition where the composition may include either an antibody or fragment thereof for one or more of B2M, a B2M receptor or other B2M modulator.

[0076] The anti-B2M antibodies described herein can be made or obtained by any means known in the art, including commercially. It is also contemplated that an antibody can be specifically reactive with a particular B2M protein or polypeptide may also be used as an antagonist. An anti-B2M antibody herein may be an antibody or fragment thereof that binds to a B2M or a bivalent antibody that binds to B2M, a B2M receptor or other B2M modulator.

[0077] As used herein, the term “antibody’' refers to an immunoglobulin (Ig) whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen-binding domain. The term further includes “antigen-binding fragments” and other interchangeable terms for similar binding fragments such as described below. [0078] Native antibodies and native immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (“VH” or “VH”) followed by a number of constant domains (“CH” or “CH”). Each light chain has a variable domain at one end (“VL” or “VL”) and a constant domain (“CL” or “CL”) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0079] The cytokine inhibitors as described herein can be a “synthetic polypeptide” derived from a “synthetic polynucleotide” derived from a “synthetic gene,” meaning that the corresponding polynucleotide sequence or portion thereof, or amino acid sequence or portion thereof, is derived, from a sequence that has been designed, or synthesized de novo, or modified, compared to an equivalent naturally occurring sequence. Synthetic polynucleotides (antibodies or antigen binding fragments) or synthetic genes can be prepared by methods known in the art. including but not limited to, the chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes are typically different from naturally occurring genes, either at the amino acid, or polynucleotide level, (or both) and are typically located within the context of synthetic expression control sequences. Synthetic gene polynucleotide sequences, may not necessarily encode proteins with different amino acids, compared to the natural gene; for example, they can also encompass synthetic polynucleotide sequences that incorporate different codons but which encode the same amino acid (i.e., the nucleotide changes represent silent mutations at the amino acid level).

[0080] With respect to anti-B2M antibodies, the term “antigen” refers to the any of the B2M, a B2M receptor or other B2M modulator proteins disclosed herein, respectively or any fragment of the protein molecules thereof.

[0081] The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” “antibody fragment” or a “functional fragment of an antibody” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to one or more of B2M, a B2M receptor or other B2M modulator cytokines.

[0082] It is contemplated that the B2M antibodies may also include “diabodies” which refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary' domains of another chain and create two antigen-binding sites. See for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993).

[0083] It is contemplated that the cytokine antibodies may also include “chimeric” forms of non-human (e.g., murine) antibodies include chimeric antibodies which contain minimal sequence derived from a non-human Ig. For the most part, chimeric antibodies are murine antibodies in which at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin are inserted in place of the murine Fc. See for example, Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

[0084] It is contemplated that the cytokine antibodies may also include a “monoclonal antibody” which refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which can include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a hybridoma method, recombinant DNA methods, or isolated from phage antibody.

[0085] As used herein, “immunoreactive” refers to binding agents, antibodies or fragments thereof that are specific to a sequence of amino acid residues on a B2M, a B2M receptor or other B2M modulator protein (“binding site” or “epitope”), yet if are cross-reactive to other peptides/proteins, are not toxic at the levels at which they are formulated for administration to human use. The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions under physiological conditions and including interactions such as salt bridges and water bridges and any other conventional binding means. The term “preferentially binds” means that the binding agent binds to the binding site with greater affinity' than it binds unrelated amino acid sequences. [0086] As used herein, the term '‘affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as Kd. Affinity of a binding protein to a ligand such as affinity of an antibody for an epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinities can be determined by methods such as an enzyme linked immunosorbent assay (ELISA) or any other technique familiar to one of skill in the art. Avidities can be determined by methods such as a Scatchard analysis or any other technique familiar to one of skill in the art.

[0087] '‘Epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction with the variable region binding pocket of an antibody.

[0088] The term “specific” refers to a situation in which an antibody will not show any significant binding to molecules other than the antigen containing the epitope recognized by the antibody. The term is also applicable where, for example, an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the antibody will be able to bind to the various antigens carry ing the epitope. The terms “preferentially binds” or “specifically binds” mean that the antibodies bind to an epitope with greater affinity than it binds unrelated amino acid sequences, and, if cross-reactive to other polypeptides containing the epitope, are not toxic at the levels at which they are formulated for administration to human use.

[0089] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions under physiological conditions and includes interactions such as salt bridges and water bridges, as well as any other conventional means of binding.

[0090] As contemplated herein, a target inhibitor for B2M, a B2M receptor or other B2M modulator may be generated through gene expression technology . The term “RNA interference” or “RNAi” refers to the silencing or decreasing of gene expression by siRNAs. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene. The gene may ⁷ be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi may also be considered to inhibit the function of a target RNA; the function of the target RNA may be complete or partial. [0091] The term "siRNAs" refers to short interfering RNAs. In some embodiments, siRNAs comprise a duplex, or double-stranded region, of about 18-25 nucleotides long; often siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand. At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the “antisense strand;” the strand homologous to the target RNA molecule is the “sense strand,” and is also complementary to the siRNA antisense strand. siRNAs may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures. siRNAs appear to function as key intermediaries in triggering RNA interference in invertebrates and in vertebrates, and in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants.

[0092] It is also contemplated that any B2M, a B2M receptor or other B2M modulator gene can be silenced or “turned” off’ through the use of CRISPR technology as known to those of skill in the art.

[0093] General Methods

[0094] Human Subjects and Ethical Considerations

[0095] De-identified plasma samples and muscle biopsies of PH-HFpEF subjects participating in clinical trials (NCT03629340 and NCT01431313) were collected at baseline within a month of confirmed diagnosis by RHC under protocols approved by the University of Pittsburgh Institutional Review Board and/or University' of California San Francisco Institutional Review Board. De-identified plasma samples of PH-HFpEF subjects and HFpEF patients without PH were collected more than a month of RHC and/or echocardiogram under protocols approved by the University of Illinois at Chicago Institutional Review Board. In addition, de-identified plasma samples of control subjects were collected under protocols approved by the Indiana University Institutional Review Board.

[0096] Mass spectrometry experiments:

[0097] Sample preparation and nanoLC-MS analysis: Plasma samples (10 pL of each sample) were depleted of abundant plasma proteins using High Select Top 14 abundant protein depletion spin columns (Thermo Fisher Scientific. Waltham, MA). Individual samples were then processed using EasyPep mini-MS sample preparation kits (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s procedure, which transformed protein-level samples into reduced/alkylated try ptic peptides. Samples were broken up into five separate batches; each batch was labeled with a separate set of TMTl l-plex reagents. Pre-labeling pooled samples (-20% of each individual sample pooled) were used as bridge channels to enable quantitative comparison across the sample batches. Following the labeling, samples in each batch were mixed and the combined group samples were each fractionated into eight fractions by high pH reversed phase using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Waltham, MA). Hence, the total of 40 separate samples for LC-MS (five batches with eight fractions each) were prepared. Each sample (-1 pg loaded on column) was analyzed separately by nano-liquid chromatography mass spectrometry (nanoLC-MS) using Thermo Scientific Ultimate 3000 nanoLC system and Orbitrap Fusion Tribrid Mass Spectrometer equipped with an EasySpray source. Briefly, chromatography was performed on a 50cm C18 nanoLC column with a 2-hour effective chromatography gradient and mass spectrometry w as performed using an optimized SPS MS acquisition method to attain the highest number of accurate quantifiable protein identifications.

[0098] Processing and analysis of mass spectrometry data: Raw data files were processed using Proteome Discoverer v2.3 (Thermo Scientific) and the tandem MS data were then searched using SEQUEST algorithms against a human UniProt (Swiss-Prot only) database (released June- 2019) with common contaminant proteins. The search parameters included trypsin as the protease with maximum of two missed cleavages allowed; oxidation of methionine (+15.9949 Da), and deamidation of asparagine and glutamine (+0.9848 Da) were set as a dynamic modification while static modifications included carbamidomethyl (+57.0215 Da) at cysteine and TMT as a static modification of lysine residues and peptide N-termini (+229.1629 Da). Precursor mass tolerance was set at 10 ppm and fragment mass tolerance w as set at 0.6 Da. Peptide confidence w as estimated with the Percolator node. Peptides were filtered at q-value <0.01 based on a decoy database search. Reporter ions for TMT labeled peptides w ere quantified using the Reporter Ions Quantifier Node included a TMT 11 pl ex quantification method in Proteome Discoverer with a peak integration tolerance of 20 ppm and an integration method based on the most confident centroid peak at the MS3 level. Only unique peptides w ere used for quantification, with protein groups considered for peptide uniqueness. Peptides with an average reporter signal-to-noise ratio >10 were used for protein quantification. Correction for the isotopic impurity of reporter quantification values was applied. The normalization was performed in two steps. First, peptide reporter ion signal-to-noise (S/N) values were normalized to the total sum per channel. Second, TMT batch effects were reduced by row -wise normalization based on median intensities. No imputation for missing values was performed. Once the normalization has been completed, the proteins.txt output file was imported into Perseus v. 1.6.13.0 software for further statistical analysis and data visualization.

The Perseus output was used for generating volcano plot.

[0099] Animal Studies

[00100] B2m knockout (B2m' ^l ~) and wild-type (WT) mice were purchased from Jackson Laboratories (002087 and 000664, respectively; Bar Harbor, ME). Beginning at 8 weeks of age, B2in and WT mice were randomly assigned to high-fat diet (HFD; 60% lipids/kcal; Research Diets, New Brunsw ick. NJ) or regular diet (RD; 10% lipids/kcal) exposure for 16 weeks. As female mice are protective in experimental models of PH and HFpEF, ²⁰²¹ only male mice were used in this study. See Tong et al., “Female Sex Is Protective in a Preclinical Model of Heart Failure with Preserved Ejection Fraction, "’ (2019) Circulation 140: pp. 1769-1771; Shah et al., “High-sensitivity C-reactive protein and parameters of left ventricular dysfunction/’ (2006) J Card Fail. 12: pp. 61- 65. All animals were maintained in a normoxic environment. All experimental procedures were approved by Indiana University School of Medicine Institutional Animal Care and Use Committee.

[00101] Hemodynamic Measurements

[00102] Mice were weighed and anesthetized with isoflurane (5% for induction, 2% during surgery', and 1% while performing pressure measurements). Right ventricular systolic pressure (RVSP) and LV end-diastolic pressure (LVEDP) were measured using a Millar catheter. Weights of RV and LV+septum normalized to tibial length were used as indexes of ventricular mass.

[00103] ELISA

[00104] Blood samples were collected using EDTA for anticoagulation and centrifuged for 15 minutes at 2500rpm. Plasma aliquots were immediately stored at -80°C. Plasma levels of human B2M (Abeam, #99977) and mouse B2M (Abeam, #223590) were quantified by commercially available ELISA kits according to manufacturer’s instructions. Blinded data analysis was performed.

[00105] Western blot analysis

[00106] Total protein extracts from tissues were homogenized (Bio-Gen 200 Homogenizer and 7X95 mm Saw-tooth Generator Probe; VWR International) in freshly prepared T-PER tissue protein extraction buffer (Thermo Fisher Scientific, Waltham, MA) with protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA). Supernatants were separated by centrifugation at 14000g for 10 min at 4°C. Cells were washed with ice-cold PBS and lysed with universal nucleases-contained lysis buffer (Thermo Scientific. Waltham, MA) and protease/phosphatase inhibitor cocktail. Supernatants were separated by centrifugation at 13200rpm for 10 min at 4°C. After protein concentration measurement done using Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA), aliquots of total lysates (4-6 pg) were subjected to immunoblotting with antibodies recognizing B2M, SIRT3, PCNA, vinculin, and GAPDH (see Table 1).

[00107] Table 1: Western Blot Antibodies

[00108] Cell cultures and isolation of PAVSMCs

[00109] Primary human PAVECs were purchased from LONZA (Basel, Switzerland; 64-year- old male donor) and cultured in Endothelial Cell Basal Medium-2 (EBM-2, Lonza) supplemented with supplements and growth factors (EGM-2 MV; complete medium, Lonza). Primary human PAV SMCs (LONZA; 51-year-old male donor) were cultured in Smooth Muscle Cell Growth Basal Medium (SmBM, Lonza) supplemented with Smooth Muscle Cell Growth Medium-2 supplements and growth factors (SmGM-2; complete medium, Lonza). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Passages 4-9 were used in the study.

[00110] Primary PAVSMCs were isolated from mice using modified protocols as described previously. See Satoh et al. 2021. Isolated PAVSMCs were cultured in complete SmBM consisting of SmGM-2 supplements and maintained at 37 C in 5% CCh. Isolated PAVSMCs were characterized by immunostaining with a-SMA antibody while the endothelial cell marker CD31 antibody was used for negative control. [00111] Cell proliferation assays

[00112] Cells (1X10 ⁵ ) were adhered overnight and administered with exogenous B2M for indicated time. The contents of the wells were trypsinized and counted using a hemocytometer.

[00113] Cell migration assays

[00114] After human PAVECs were administered with exogenous B2M (10 mg/ml) for 3 days, cells were re-seeded in a 2- well ibidi silicone insert (lX10 ⁴ /well, complete medium) overnight to form monolayer, then the insert was removed to form a cell-free gap (black dashed line shown in the figures; Oh). Migration of the cells toward the gap (red solid lines shown in the figures) was recorded after 16 hours. Images were taken on a Leica DMi8 inverted microscope (Wetzlar, Germany). The wound area measurement was performed using Image J. Wounding area (%) = [(wound area at Oh) - (wound area at 16h)] / (wound area at Oh) x 100.

[00115] Analy sis of metabolites

[00116] For glucose tolerance testing, mice were fasted for 6 h before being subjected to intraperitoneal injection with 1.8 mg/g dextrose in 0.9% NaCl. Blood samples were taken at different time points as indicated, and blood glucose levels were measured with a portable glucose meter (ACCU-CHECK Aviva; Roche, Basel, Switzerland).

[00117] Lung histology

[00118] Lungs were perfused and fixed with 50% OCT-PBS. Frozen sections (7 pm) were stained with a-SMA. Images of terminal arterioles were captured w ith a fluorescence microscope digital camera system (Leica Microsystems, Wetzlar, Germany). The external diameter and internal diameter were measured using Image J in 5-6 muscular arteries (ranging in size from 30- 80 pm in external diameter) per lung section from 3 mice per group. Wall thickness (%) = [(external diameter-internal diameter)/extemal diameter] x 100. Blinded data analysis was performed.

[00119] Statistical Analysis

[00120] Statistical analyses were performed using Prism 10.0.2 software (La Jolla, CA). Statistical comparison between two groups were performed using the Mann- Whitney G-test. For in vitro studies, unpaired Student’s /-test was performed after testing for normality with Shapiro- Wilk test. Comparison among > 3 groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. For differences in blood glucose levels during the glucose tolerance test, two-way ANOVA followed by Bonferroni’s post hoc test was performed. Values of < 0.05 were considered statistically significant.

[00121] Further reference is made to the following experimental examples.

[00122] EXAMPLES

[00123] The following examples are provided for the purpose of illustrating various embodiments of the invention and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are provided only as examples, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

[00124] EXAMPLE 1

[00125] Mass spectrometry-based plasma proteomics identifies high protein abundance levels of B2M in patients with PH-HFpEF

[00126] The inventors used mass spectrometr -based bottom-up proteomics as a sensitive and comprehensive hypothesis-generating discovery’ technique to profile proteins in control subjects and patients with PH-HFpEF. Using nano-capillary liquid chromatography electrospray ionization mass spectrometry (nLC-ESI-MS) and tandem mass tag (TMT) isobaric labels, plasma from human patients with PH-HFpEF (17=16, Table 2) and random control subjects (h=17) were processed for a mass spectrometry experiment in which plasma proteomes were characterized and quantitatively compared (FIG. 1A).

[00127] Table 2: Clinical information of plasma samples used for proteomic analysis.

mPAP: mean pulmonary artery’ pressure; PCWP: pulmonary capillary’ wedge pressure; BMP body mass index. [00128] FIG. 1A-D depicts the changes in plasma protein abundance profiles in patients with PH-HFpEF. FIG. 1A: Schematic overview of mass spectrometry-based plasma proteomic analysis. FIG. IB: Volcano plot of the fold change and statistical significance for protein abundance levels. FIG. 1C: Changing protein abundances in PH-HFpEF [Age: 69.8 ± 7.9; male sex: 8; BMI: 39.1 ± 10.4; mPAP: 39.4 ± 8.1 mmHg; PCWP: 20.2 ± 4.5 mmHg; WHO function class II: 1 (6%), III: 14 (88%), and IV: 1 (6%)]. FIG. ID, Protein abundance ratio of B2M. Data are mean ± SEM. P value was determined by Mann- Whitney U test.

[00129] Twenty-five proteins were found to have statistically significant higher or lower abundance levels in patients with PH-HFpEF compared to control subjects (FIG. IB and 1C). Consistent with a previous report, protein abundance levels of a common biomarker of inflammation and cardiovascular distress, C-reactive protein (CRP), were also elevated in our PH- HFpEF patient cohort. See Shah et al. 2006. In contrast, the inventors did not observe any significant change in protein abundance of galectin-3 and interleukin-6 (IL-6), inflammation markers which have been associated with PH-HFpEF pathogenesis, in the study cohort. See Ranchoux et al., “Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease,” (2019) Circ Res. 125: pp. 449-466; Mazurek et al., “Galectin-3 Levels Are Elevated and Predictive of Mortality in Pulmonary Hypertension,” (2017) Heart Lung Circ. 26: pp. 1208-1215. Among the identified proteins, we selected B2M for further validation because of its relatively high abundance and its reported association with atherosclerosis, pulmonary fibrosis, cancers, and aging. See Smith et al., “Beta2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis,” (2015) Nat Med. 21: pp. 932-937; Prizment et al., “Circulating Beta- 2 Microglobulin and Risk of Cancer: The Atherosclerosis Risk in Communities Study (ARIC).” (2016) Cancer Epidemiol Biomarkers Prev. 25: pp. 657-664; Amighi et al., “Beta 2 microglobulin and the risk for cardiovascular events in patients with asymptomatic carotid atherosclerosis,” (2011) Stroke 42: pp. 1826-1833; Wu et al., “Beta2 -microglobulin as a biomarker of pulmonary fibrosis development in COPD patients,” (2020) Aging (Albany NY) 13: pp. 1251-1263.

[00130] EXAMPLE 2

[00131] Plasma B2M levels are significantly elevated in patients with PH-HFpEF and are associated with disease severity.

[00132] The inventors next measured B2M levels by ELISA in plasma obtained from a validation cohort of PH-HFpEF patients [n=15, age: 64 ± 11.1, without co-occurring kidney dysfunction, confirmed diagnosis by RHC of a mPAP > 25 mmHg, PCWP > 15 mmHg, and TPG > 12 mmHg OR during exercise mPAP > 30 mmHg, PCWP > 20 mmHg, and TPR > 3, Table 3], HFpEF patients without PH (w=9, age: 55.4 ± 17.9, mPAP < 20 mmHg by RHC or tricuspid regurgitation velocity (TRV) < 3 m/s measured by echocardiogram), and random control subjects free of HF. PH, chronic kidney disease (CKD), cancers, and a few other rare diseases (n=44, age: 65.5 ± 7.2).

[00133] Table 3: Clinical information of plasma samples used for B2M measurement by ELISA

mPAP: mean pulmonary' artery pressure; PCWP: pulmonary capillary wedge pressure; TRV: tricuspid regurgitation velocity.

[00134] FIG. 2A-F show the circulating levels of B2M are elevated in patients with PH-HFpEF. Circulating levels of B2M are elevated in patients with PH-HFpEF. FIG. 2A: Circulating levels of B2M were measured by ELISA in plasma of the validation cohort of control subjects, HFpEF patients without PH, and PH-HFpEF patients [confirmed diagnosis by RHC when the resting mPAP > 25 mmHg, PCWP > 15 mmHg, and TPG > 12 mmHg OR during exercise mPAP > 30 mmHg, PCWP > 20 mmHg, and TPR > 3], Correlation between circulating levels of B2M and resting mPAP (FIG. 2B), PCWP (FIG. 2C), TPG (FIG. 2D), PVR (FIG. 2E), or RAP (FIG. 2F) in PH-HFpEF patients whose plasma samples were collected within a month of RHC. Spearman r is show n. Data are mean ± SEM. P value was analyzed by Mann-Whitney U test.

[00135] In agreement with the results obtained from proteomic analysis (FIG. ID), ELISA assays demonstrated that circulating concentrations of B2M were higher in the validation cohort of PH-HFpEF patients compared to HFpEF patients without PH and/or control subjects (FIG. 2A). Importantly, the inventors found a positive correlation between plasma B2M levels and resting mPAP (P = 0.0333) in PH-HFpEF patients whose plasma samples were collected within a month of RHC (FIG. 2B).

[00136] FIG. 7A and 7B: Correlation between circulating B2M levels and mPAP (FIG. 7A) or PCWP (FIG. 7B) measured during cardiopulmonary exercise testing in PH-HFpEF patients. Spearman r is shown.

[00137] A positive correlation between plasma B2M levels with resting PCWP (P = 0.0209), TPG (P = 0.0397), PVR P = 0.0457), or (P = 0.0005) was also observed (FIG. 2C, 2D. 2E, 2F). Notably, no difference between plasma B2M levels and exercise mPAP or PCWP was observed in patients with PH-HFpEF (FIG. 7A-B).

[00138] EXAMPLE 3

[00139] Skeletal muscle levels of B2M are increased in mice with skeletal muscle SIRT3 deficiency or HFD-induced PH-HFpEF

[00140] The inventors have previously reported that defects in skeletal muscle sirtuin-3 (SIRT3), the mitochondrial deacetylase which is critical for modulating metabolic syndrome, PH, and aging, are associated with increased pulmonary vascular remodeling and pulmonary ⁷ pressures in Ob-Su rat model of PH-HFpEF. See Hirschey et al.. “SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome,” (2011) Mol Cell. 44: pp. 177-190; Paulin et al., “Sirtuin 3 deficiency is associated with inhibited mitochondrial function and pulmonary arterial hypertension in rodents and humans,” (2014) Cell Metab. 20: pp. 827-839; McDonnell et al., “SIRT3 regulates progression and development of diseases of aging,” (2015) Trends Endocrinol Metab. 26: pp. 486-492; Lai et al., ^L ‘SIRT3-AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction,” (2016) Circulation 133: pp. 717-731.

[00141] To evaluate whether skeletal muscle SIRT3 deficiency affects induction and secretion of B2M, the inventors measured skeletal muscle expression and circulating B2M levels by Western blot and ELISA, respectively, in soleus and plasma obtained from skeletal muscle-specific Sirt3 knockout mice (A7 73 ^skll ’“ “).

[00142] FIG. 3A-F shows the skeletal muscle levels of B2M are increased in mice with skeletal muscle SIRT3 deficiency or HFD-induced PH-HFpEF. FIG. 3A and 3B: Skeletal muscle (FIG. 3 A) and plasma (FIG. 3B) levels of B2M in WT and SirtS^'' ¹ ' mice. FIG. 3C: Correlation between plasma B2M levels with RVSP in WT and SirtS ⁵ ^' mice. FIG. 3D and 3E: Skeletal muscle (FIG. 3D) and plasma (FIG. 3E) levels of B2M in mice fed a RD or HFDfor 16 weeks. FIG. 3F: Correlation between plasma B2M levels with RVSP in mice fed a RD or HFD. Spearman r is shown. Data are mean ± SEM. P value was analyzed by Mann-Whitney U test.

[00143] The data showed skeletal muscle expression levels of B2M were higher in SzrG ^skm ' ^/ " mice compared to WT mice (FIG. 3A). B2M levels were significantly higher in plasma obtained from Sirt3 ^{sksa l} ~ mice, which positively correlated with elevated RVSP (FIG. 3B and FIG. 3C). Subsequently, we measured skeletal muscle expression and circulating levels of B2M in mice fed with a HFD, which reduces SIRT3 in skeletal muscle and induces a metabolic syndrome-associated PH-HFpEF phenotype. See Meng et al., “Development of a Mouse Model of Metabolic Syndrome, Pulmonary Hypertension, and Heart Failure with Preserved Ejection Fraction.” (2017) Am J Respir Cell Mol Biol. 56: pp. 497-505; Wang et al., “Treatment With Treprostinil and Metformin Normalizes Hyperglycemia and Improves Cardiac Function in Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction,” (2020) Arterioscler Thromb Vase Biol. 40: pp. 1543-1558; Kelly et al., “Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor,” (2017) Am J Respir Cell Mol Biol. 56: pp. 488-496.

[00144] As shown in FIG. 3D and 3E, skeletal muscle expression and plasma concentration of B2M levels were increased in HFD-exposed mice. Of note, a positive correlation betw een plasma B2M levels and RVSP was observed in HFD-exposed mice (FIG. 3F). Similarly, expression of B2M was found to be increased in kidney, but remained unchanged in LV, RV, and adipose tissue after 16 weeks of HFD exposure (FIG. 8A-D). FIG. 8A-D shows protein levels of B2M assessed by Western blots in kidney (FIG. 8A), LV (FIG. 8B), adipose tissue (FIG. 8C), and RV (FIG. 8D) of HFD-exposed mice. Data are mean ± SEM. P value was analyzed by Mann- Whitney U test. [00145] Together, these data suggest the link between skeletal muscle SIRT3 deficiency and the induction/secretion of B2M.

[00146] EXAMPLE 4

[00147] Skeletal muscle expression levels of B2M are increased in PH-HFpEF patients and correlated significantly with resting PCWP and RAP.

[00148] To further assess the clinical relevance of findings in animals, the inventors measured B2M expression levels in muscle biopsies from PH-HFpEF patients and control subjects free of HF and PH (Table 4).

[00149] Table 4: Clinical information of muscle biopsies used for B2M measurements. mPAP: mean pulmonary artery' pressure; PCWP: pulmonary' capillary w edge pressure; BMI: body mass index.

[00150] FIG. 4A-C shows that muscle expression levels of B2M are increased in patients with PH-HFpEF. FIG. 4A: B2M expression levels were measured in muscle biopsies of PH-HFpEF patients and control subjects by Western blots. FIG. 4B and 4C: Correlation between skeletal muscle B2M expression with resting PCWP (FIG. 4B) or RAP (FIG. 4C). Spearman r is shown. Data are mean ± SEM. P value was analyzed by Mann-Whitney’ U test. [00151] As shown in FIG. 4A, muscle biopsies of PH-HFpEF patients had higher protein expression levels of B2M compared to that of control subjects. Notably, skeletal muscle B2M expression levels significantly correlated with resting PCWP (P = 0.0044; FIG. 4B) and resting RAP (P = 0.0338; FIG. 4C), demonstrating an association and clinical relevance of skeletal muscle B2M in PH-HFpEF.

[00152] EXAMPLE 5

[00153] Treatment with B2M increases PAVECs migration/proliferation and promotes PAVSMCs proliferation.

[00154] While B2M has been associated with pulmonary fibrosis, emphysema, cardiac fibroblast activation, atherosclerosis, and aging, it is currently unknown whether B2M can regulate pulmonary vascular remodeling. See Smith et al., “Beta2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis,’' (2015) Nat Med. 21: pp. 932-937; Amighi et al.. ‘“Beta 2 microglobulin and the risk for cardiovascular events in patients with asymptomatic carotid atherosclerosis,” (2011) Stroke 42: pp. 1826-1833; Wu et al., ‘'Beta2-microglobulin as a biomarker of pulmonary fibrosis development in COPD patients,” (2020) Aging (Albany NY) 13: pp. 1251-1263; Gao et al., “Beta(2)-Microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence,” (2017) Am J Physiol Lung Cell Mol Physiol. 312: pp. L669-L677; Molenaar et al., "Single-cell transcriptomics following ischemic injury identifies a role for B2M in cardiac repair,” (2021 ) Commun Biol. 4: p. 146. To assess whether B2M affects endothelial abnormalities and medial wall thickness, the inventors administered exogenous B2M to cultured human pulmonary arterial vascular endothelial cells (PAVECs) and pulmonary arterial vascular smooth muscle cells (PAVSMCs).

[00155] FIG. 5A-D shows that treatment with B2M induces PAVECs migration/proliferation and promotes PAVSMCs proliferation. FIG. 5 A and 5B: Human PAVECs were administered with exogenous B2M (10 mg/ml) for 4 days. Representative images of cell migration and related quantitative data (FIG. 5A). Cell proliferation assessed by cell counts (FIG. 5B). FIG. 5C and 5D: Human PAVSMCs were exposed to B2M (10 mg/ml) for 5 days. Representative images of cell numbers and cell proliferation assessed by cell counts (FIG. 5C). Representative Western blots for PCNA protein expression levels (FIG. 5D). Data are mean ± SEM. P value was analyzed by Student’s / test

[00156] The data showed that B2M exposure increased cell migration and proliferation in PAVECs (FIG. 5 A and 5B). Similarly, the data showed that administration of B2M increased cell proliferation in correlation with elevated expression levels of proliferating cell nuclear antigen (PCNA) in PAVSMCs (FIG. 5C and 5D). Collectively, the current findings suggest a pathogenic role of B2M in the regulation of pulmonary vascular remodeling.

[00157] EXAMPLE 6

[00158] B2M whole-body KO mice are protected from HFD-induced PH-HFpEF

[00159] To evaluate the impact of B2M on metabolic syndrome-associated PH-HFpEF, B2m~ ¹ ' and WT mice were exposed to HFD for 16 weeks (FIG. 6A).

[00160] FIG. 6A-I shows that B2M whole-body KO mice protects against metabolic syndrome- associated PH-HFpEF. FIG. 6A: 8-week-old WT and whole-body B2M knockout mice (B2m' ¹ ') were fed a RD or HFD for 1 weeks. At week 1 , body weights (FIG. 6B), glucose tolerant abilities (FIG. 6C), RVSP (FIG. 6D), LVEDP (FIG. 6G) were measured. Weights of RV (FIG. 6E) and LV+S (FIG. 6F) normalized to tibial length were used as index of ventricular hypertrophy. FIG. 6H: Representative images of lung sections stained with a-smooth muscle actin (a-SMA) and quantification of wall thickness from the mean of 5-6 vessels per lung section from 3 mice/group. Scale bar, 30 pm. FIG. 61: PCNA levels were analyzed by Western blot in PAVSMCs of B2m' ¹ ' and WT mice. Data are mean ± SEM. P value was analyzed by One-Way ANOVA followed by Tukey’s post hoc test. For glucose tolerance test, two-way ANOVA followed by Bonferroni’s post hoc test was performed.

[00161] Consistent with previous observations, HFD-exposed WT mice exhibited significantly higher body weights, glucose intolerance, RVSP, LVEDP, and bi-ventricular hypertrophy than RD-exposed WT mice (FIG. 6B-G). While no difference in body weights, LV hypertrophy, or LVEDP was observed, HFD-exposed B2m' ^l ~ mice exhibited significantly improved glucose intolerance, lowered RVSP, and reduced RV hypertrophy compared to WT mice fed with a HFD (FIG. 6B-G). In addition, HFD-exposed B2m' ¹ ' mice had a lower percentage of wall thickness in comparison with HFD-exposed WT mice (FIG. 6H). Correspondingly, improvement of pulmonary vascular remodeling was associated with lower PCNA expression in PAVSMCs of HFD-exposed B2m’ ¹ ' mice (FIG. 61). Taken together, these data highlight the potential benefit of reduced B2M on metabolic syndrome-associated PH-HFpEF. [00162] EXAMPLE 7

[00163] Diagnostic and prognostic potential of plasma B2M in patients with PH-HFpEF

[00164] Further studies will be done to assess the diagnostic and prognostic potential of plasma B2M in patients w ith PH-HFpEF. The inventors will measure plasma B2M levels using a human B2M ELISA kit in baseline plasma samples from a larger cohort of PH-HFpEF patients and random control subj ects. They will also examine the correlation between plasma B2M levels and markers of PH-HFpEF severity (e.g. hemodynamic parameters obtained by RHC at rest and during cardiopulmonary exercise testing, functional class, VO2 max, cardiac output). In addition, the inventors will compare plasma B2M levels in PH-HFpEF patients and HFpEF patients without PH to assess whether plasma B2M levels may be useful for stratifying patients into sub-populations.

[00165] In other studies the inventors will compare plasma B2M levels collected at baseline and after 12 weeks of metformin treatment, which has been shown to improve metabolic syndrome and increase skeletal muscle SIRT3 expression, to evaluate if B2M levels are associated with treatment outcomes/clinical benefits (e.g. changes in hemodynamics during submaximal exercise and at rest, differences in six-minute walk distance, and Doppler-echocardiology assessments of LV and RV function between placebo and metformin at week 12, as well as baseline compared to posttreatment).

[00166] Additional studies will evaluate if plasma B2M levels can be used to characterize an individual's specific response to treatment. The diagnostic and prognostic potential of plasma B2M in patients with metabolic syndrome and Group 1-5 PH will be assessed with the same approach. The long-term goal is to employ machine learning methods to facilitate of screening, diagnosis, prognosis, and refine of specific patient phenotype for PH-HFpEF, metabolic syndrome, HFpEF, and Group 1 -5 PH.

[00167] EXAMPLE 8

[00168] Diagnostic and prognostic potential of muscle expression levels of B2M in PH-HFpEF patients and HFpEF patients without PH

[00169] Using Western blots with specific B2M antibody, the inventors will assess the diagnostic and prognostic potential of muscle expression levels of B2M in PH-HFpEF patients, HFpEF patients without PH, and random control subjects as described above. Associations of muscle B2M expressions with severity markers and treatment outcomes, as well as the potential of muscle B2M expression levels in identifying patient sub-populations, will also be evaluated as described above. The diagnostic and prognostic potential of skeletal muscle B2M in patients with metabolic syndrome and Group 1-5 PH will be assessed with the same approach as well.

[00170] EXAMPLE 9

[00171] Novel B2M Inhibitors

[00172] At present there are no commercially available B2M inhibitors. Therefore, the inventors will explore potential therapeutical approaches as following: a. designing peptides/small molecule antagonists for B2M receptors. B2M has been reported to interact with hemochromatosis protein (HFE) and transferrin receptor complex 1 (TFRC1) to regulate iron homeostasis and epithelial-mesenchymal transition of cancer cells. See Josson et al., “Beta2-microglobulin induces epithelial to mesenchymal transition and confers cancer lethality and bone metastasis in human cancer cells,” (2011) Cancer Res. 71: pp. 2600-2610.

[00173] B2M has also been shown to impair neurogenesis and cognitive function via reduced cell surface expression of MHC I and transporter associated with antigen processing 1 (TAPI). See Smith et al. 2015. Additionally, B2M has been shown to activate monocyte via through transforming growth factor beta receptor 2 (TGFPR2) signaling. See Hilt et al., “Platelet-derived beta2M regulates monocyte inflammatory responses,” (2019) JCI Insight 4. Using coimmunoprecipitation-coupled mass spectrometry, the inventors also identified cadherins desmoglein-1 (DSG1), desmoglein-2 (DSG2) and desmocollin- 1 (DSC 1), that may mediate B2M signaling through non-canonical junction-independent effects. Thus, in further studies, the inventors will determine whether HFE, TFRC1, MHC I, TAPI, TGF|3R2, DSG1, DSG2, and DSC1 are involved in B2M-mediated cell proliferation in PASMCs and PAECs by suppressing them individually in the PASMCs and PAECs. They will also use crosslinking mass spectrometry, computational prediction, and structure-based approach to design peptides/small molecule antagonists to block the interactions of these proteins with B2M. They will then test these peptides/small molecule antagonists in in vitro and in vivo models of diseases.

[00174] EXAMPLE 10

[00175] Reducing B2M from the blood stream

[00176] Recently, Lixelle". a B2M apheresis column, has been approved for the treatment of dialysis-related amyloidosis by FDA. See Tsuchida et al., “Direct hemoperfusion by using Lixelle® column for the treatment of systemic inflammatory response syndrome,” (2002) Int J Mol Med. 10: pp. 485-488. However, dialysis is not for every one, particularly for the elder and those with severe medical conditions. Thus, the inventors will develop therapeutic B2M antibodies for the treatment of PH-HFpEF, metabolic syndrome, HFpEF, and Group 1-5 PH. The effect of B2M therapeutic antibodies will be tested in animal models of diseases.

[00177] EXAMPLE 11

[00178] Development of skeletal muscle-specific gene therapy

[00179] Further studies are contemplated in order to develop skeletal muscle gene therapy based upon inhibition of B2M.

[00180] Discussion

[00181] As described herein, the inventors show that 1) B2M is present at higher levels in patients with PH-HFpEF; 2) skeletal muscle SIRT3 deficiency is associated with induction and secretion of B2M in animal models and human subjects with PH-HFpEF; 3) circulating and skeletal muscle expression levels of B2M correlate with PH-HFpEF severity; 4) B2M increases PAVECs migration/proliferation and promotes PAVSMCs proliferation; and 5) loss of B2M improves metabolic syndrome-associated PH-HFpEF. These findings not only reveal a previously unknown pathogenic role for B2M in PH-HFpEF, but also suggest the potential of using circulating and skeletal muscle B2M levels as biomarkers for PH-HFpEF.

[00182] B2M comprises the light chain of MHC I that forms an active part of the adaptive immune system. As it is noncovalently associated with the light chain and has no direct attachment to the cell membrane, free B2M circulates in the blood as a result of shedding and/or intracellular release. See Wilson et al., '‘Beta2-microglobulin as a biomarker in peripheral arterial disease: proteomic profiling and clinical studies,” (2007) Circulation 116: pp. 1396-1403. The net concentration of B2M is determined by its generation/secretion into circulation and its elimination by the kidneys. As such, in people without kidney disease, elevated B2M has been considered as a marker of altered cell proliferation. See Prizment et al. 2016. Increased circulating levels of B2M have been implicated in various cancers and associated with cancer progression with poor prognosis. See Id. and Zhang et al., "B2M overexpression correlates with malignancy and immune signatures in human gliomas,” (2021) Sci Rep. 11 : p. 5045. B2M has also been associated with increased colorectal cancer risk. See Prizment et al. 2016. Additionally, there is evidence to correlate elevated serum B2M concentrations with the development and progression of pulmonary fibrosis in patients with chronic obstructive pulmonary disease (COPD) and emphysema. See Wu et al. 2020; Molenaar et al. 2021. B2M was also found to be associated with adverse cardiovascular outcomes in patients with asymptomatic carotid atherosclerosis. See Amighi et al. 2011. Furthermore, plasma proteome studies found B2M to be a risk marker for coronary heart disease in postmenopausal women and to correlate with disease severity in patients with peripheral arterial disease (PAD) or HF with reduced ejection fraction (HFrEF). See Wilson et al. 2007; Prentice et al., “Novel proteins associated with risk for coronary heart disease or stroke among postmenopausal women identified by in-depth plasma proteome profiling,” (2010) Genome Med. 2: p. 48; Brioschi et al., “Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure,” (2021) Int J Mol Sci. 22. The inventors’ data show that B2M is present at higher levels in patients with PH-HFpEF. When analyzing clinical data, the observed a significant correlation between increased circulating B2M and pulmonary vascular hemodynamics, such as PCWP, mPAP, and RAP, at rest, but not during exercise, in PH-HFpEF patients whose plasma samples were collected around the same time of RHC. Additionally, they observed that patients with diagnosis confirmed by resting hemodynamics alone have higher circulating B2M levels than those with diagnosis confirmed by exercise hemodynamics (FIG. 7A- D). These findings suggest that B2M is probably related to chronic pathophysiological processes and abnormalities in the left heart, pulmonary vasculature, and the right heart. Whether circulating B2M levels can be a useful non-invasive tool to facilitate screening, to define/ refine specific patient sub-phenotypes, and/or to identify patients in a high-throughput fashion requires further investigations.

[00183] Despite reports implicating the increased circulating B2M concentrations in cancers, pulmonary fibrosis, emphysema, aging, and HFrEF, very little is known about the mechanism by which B2M contributes to these diseases. Beyond being a simple biomarker, exogenous B2M injections have been shown to promote age-related cognitive dysfunction and impaired neurogenesis via cell surface expression of MHC I. See Smith et al. 2015. B2M has also been shown to promote cancer cell growth and metastasis in cancers through increased inflammatory activities and activation of PI3K/Akt, ERK, or NLRP3 inflammasomes. See Zhang et al. 2021; Nomura et al., “Targeting beta2 -microglobulin mediated signaling as a novel therapeutic approach for human renal cell carcinoma,” (2007) J Urol. 178: pp. 292-300; Hofbauer et al., “Beta(2)- microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression,” (2021) Immunity 54: pp. 1772-1787. In addition, cardiomyocyte-secreted B2M contributes to fibroblasts activation after ischemia injury and platelet-derived B2M induces monocyte proinfl ammatory differentiation, highlighting a potential role of B2M as a signaling factor. See Molenaar et al. 2021; Hilt et al., “Platelet-derived beta2M regulates monocyte inflammatory responses,” (2019) JCI Insight 4; Hilt et al., “Beta2M Signals Monocytes Through Non-Canonical TGFB Receptor Signal Transduction,” (2021) Circ Res. 128: pp. 655-669. Our in vitro and in vivo studies indicate a pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling. As elevated circulating concentration of B2M is associated with increased protein expression of B2M in skeletal muscle, our data further suggest B2M may act as an endocrine signaling molecule to induce pulmonary vascular remodeling via increasing PAVECs migration/proliferation and PAVSMCs proliferation. While the data show that skeletal muscle expression levels of B2M correlate with PH-HFpEF severity, little is known about the pathological mechanism underlying increased skeletal muscle B2M in PH-HFpEF. Upregulation and prolonged expression of MHC I in muscle cells is a hallmark of inflammatory myopathies, which have been recognized as one of an extra-cardiopulmonary source of inflammation in HFpEF and PH. See Nagaraju et al.. “Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies,” (2000) Proc Natl Acad Sci USA 97: pp. 9209-9214; Shi et al., “Value of the HFA- PEFF diagnostic algorithms for heart failure with preserved ejection fraction to the inflammatory myopathy population,” (2023) Arthritis Res Ther. 25: p. 141; Sanges et al., “Pulmonary arterial hypertension in idiopathic inflammatory myopathies: Data from the French pulmonary hypertension registry and review- of the literature,” (2016) Medicine (Baltimore) 95: e4911; Hervier et al., "Pulmonary hypertension in antisynthetase syndrome: prevalence, aetiology and survival,” (2013) Eur Respir J. 42: pp. 1271-1282. Pro-inflammatory cytokines and viral infection increase surface expression of MHC I in muscle cells. Nagaraju et al. 2000. Interestingly, the current data data show that SIRT3 deficiency and HFD exposure, both of which are known to induce chronic metabolic inflammation, increase skeletal muscle B2M expression. It is possible that skeletal muscle B2M reflects and/or contributes to the chronic, systemic, metabolic inflammation in PH-HFpEF. Future studies are needed to determine whether skeletal muscle B2M contributes to PH-HFpEF pathogenesis and the interplay between immunological and metabolic processes, dependent or independent of MHC I, in this context.

[00184] Management of PH-HFpEF is challenging due to the lack of proven PH therapies in the setting of HFpEF. SGLT2 inhibitors were very recently approved for the treatment of HFpEF. See Anker et al., “Empagliflozin in Heart Failure with a Preserved Ejection Fraction,” (2021) N Engl J Med. 385: pp. 1451-1461. SGLT2 inhibitors have been reported to improve exercise-induced PH in Ob-Su rats and type 2 diabetes patients and reduce PA pressure in HF patients. See Satoh et al. 2021; Kayano et al. 2020; Nassif et al. 2021.

[00185] However, the search for effective therapies for PH-HFpEF remains. Using HFD model of metabolic syndrome-associated PH-HFpEF, the inventors demonstrate that loss of B2M exhibits beneficial effects on glucose intolerance, pulmonary vascular proliferative remodeling, pulmonary hypertension, and RV hypertrophy. This highlights B2M as a promising therapeutic candidate for the treatment of PH-HFpEF. More work is needed to determine whether B2M affects other cell types in PH-HFpEF pathogenesis and to evaluate if B2M inhibition can reduce or reverse PH- HFpEF in the more severely affected animal models. See Lai et al., “SIRT3-AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary ⁷ Hypertension Associated With Heart Failure With Preserved Ej ection Fraction,” (2016) Circulation 133: pp. 717-731; Ranchoux et al., “Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease,” (2019) Circ Res. 125: pp. 449-466. As skeletal muscle SIRT3 was found to be decreased in the rat model of pulmonary arterial hypertension (PAH, Group 1 PH) and SIRT3-deficient mice develop spontaneous PAH, the role of B2M in the pathogenesis of PAH merits future investigations. See Paulin et al., 2014; Goncharov et al., “Metformin Therapy for Pulmonary Hypertension Associated with Heart Failure with Preserved Ejection Fraction versus Pulmonary Arterial Hypertension,” (2018) Am J Respir Crit Care Med. 198: pp. 681-684. In addition, high B2M levels are associated with lung emphysema and pulmonary' fibrosis development in COPD patients. See Wu et al., 2020; Gao et al. 2017. B2M has also been shown to complex with hemochromatosis gene (HFE) protein to activate iron metabolism, such as hypoxia-inducible la (HIF-la) signaling, which induces epithelial cells to mesenchymal transition. See Gao et al. 2017; Josson et al. 2011. As hypoxia, inflammation, and loss of capillaries in severe emphysema are associated with PH development and pulmonary vascular remodeling in COPD (PH-COPD, Group 3 PH), the inventors’ findings may open anew avenue for approaches targeting B2M in the management of PH-COPD as well. See Vizza et al.. “Pulmonary Hypertension in Patients With COPD: Results From the Comparative, Prospective Registry' of Newly Initiated Therapies for Pulmonary ⁷ Hypertension (COMPERA),” (2021) Chest 160: pp. 678-689.

[00186] The current findings suggest the potential of using circulating and skeletal muscle B2M levels as biomarkers for PH-HFpEF to guide future research towards facilitation of screening, diagnosis, refinement of specific patient phenotype, and/or identification of patients in a more efficient manner. The findings also provide new ⁷ insights into the mechanistic basis of B2M in the regulation of pulmonary ⁷ vascular proliferative remodeling and PH-HFpEF. From a translational perspective, the data indicate that B2M is an an important therapeutic target for the treatment of PH-HFpEF in the future.

[00187] Thus, together, by identifying/designing compounds/small molecules targeting B2M, B2M receptors (hemochromatosis protein, transferrin receptor complex 1. MHC I, and etc), as well as regulators of B2M receptors (antigen processing 1), this invention provides potential treatment for Group 1-5 PH.

[00188] As will be appreciated from the descriptions herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, without limitation, the aspects and embodiments listed below:

[00189] Accordingly, the present disclosure provides:

[00190] —novel approaches to the diagnosis/prognosis and treatment of PH-HFpEF including PH-HFpEF associated with metabolic syndrome, including diabetes, hypertension, kidney disfunction, and heart failure with preserved ejection fraction (HFpEF);

[00191] — inhibitors targeting B2M, B2M receptors, and regulators of B2M receptors for the treatment of Group 1-5 PH;

[00192] —therapeutic approaches using the administration of inhibitors of B2M, B2M receptors, and regulators of B2M receptors for the treatment of Group 1-5 PH, metabolic syndrome, and HFpEF;

[00193] —methods of treatment of PH-HFpEF by reducing B2M from the blood stream in a patient with elevated levels of B2M or other biomarkers;

[00194] —methods of treatment of PH-HFpEF by administration of antagonists for B2M receptors to patients in need thereof;

[00195] —methods of treatment of PH-HFpEF by various types of receptor inhibition therapy;

[00196] — methods of treatment of PH-HFpEF skeletal muscle-specific gene therapy;

[00197] —methods of determining the severity in PH-HFpEF patients by measuring the amount of B2M and related biomarker of PH-HFpEF severity and prognosis;

[00198] —methods of inhibiting, treating, or preventing the effects of elevated B2M in patients comprising inhibiting, neutralizing or depleting B2M from the patient;

[00199] —methods of depleting B2M in addition to one or more regulators of B2M gene expression or synthesis;

[00200] —methods wherein B2M can be inhibited, neutralized or depleted by the administration of and agent to the patient where the agent comprises an adeno-associated virus (AAV) or lentivirus-containing an a short-hairpin RNA (shRNA) against B2M; the shRNA is commercially available and can be attached to or part of any vector known in the art including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes;

[00201] —methods wherein the agent comprises a monoclonal or polyclonal antibody directed against B2M or a B2M receptor;

[00202] -methods wherein the agent comprises a monoclonal or polyclonal antibody directed against B2M or a B2M receptor;

[00203] -methods wherein the agent is an siRNA or antisense oligonucleotide that targets B2M or a B2M receptor;

[00204] -methods wherein the agent is an antagonist that binds to a B2M-mediated receptor and prevents the binding of B2M.

[00205] While embodiments of the present disclosure have been described herein, it is to be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.