Clinical Translational Research Program in Pulmonary Arterial Hypertension (PAH): Disease Mechanisms, Biomarkers, and Novel Therapeutic Targets

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Abstract

Pulmonary hypertension (PH) due to direct pulmonary vascular injury is collectively referred to as pulmonary arterial hypertension (PAH), & is distinct from other causes of PH such as left sided heart failure, parenchymal lung disease with hypoxemia & chronic thromboembolic disease. Idiopathic PAH (IPAH) is an unexplained form of PAH where the triggering insult to the endothelium is unclear. Several diseases may manifest PAH that is identical in histopathology to IPAH. Diseases resulting in PAH (referred to as disease-associated PAH) include autoimmune disorders (i.e. limited systemic sclerosis, mixed connective tissue disease, & systemic lupus erythematosus), HIV infection, congenital heart disease, & liver disease with portal hypertension. Contemporary paradigms of PAH pathobiology include endothelial damage and disruption, genetic & epigenetic contributions, metabolic derangements with a hyper-proliferative, anti-apoptotic cellular phenotype, & both systemically & locally dysregulated inflammation. In the absence of PAH-specific surrogate markers for assessing disease severity & prognosis, risk prediction continues to rely on subjective functional assessments & invasive hemodynamic measurements. There is continued interest in the discovery of novel, biologically relevant, non-invasive biomarkers that may simplify PAH risk stratification & serve as markers of ongoing disease progression &, ideally, response to therapy. In PAH, inflammation appears to drive the dysfunctional endothelial phenotype, propagating cycles of injury & repair. However, detailed phenotypic studies are lacking on the temporal evolution of this process & its contribution to RV & pulmonary vascular remodeling. At the NIH Clinical Center patients with WHO group 1 PAH are being enrolled in a natural history study (13-CC-0012) assessing patients at baseline, biannually in the 1st year and then annually to: 1) Characterize the contribution of inflammation to disease progression & long-term outcomes in PAH & 2) Identify non-invasive markers of vascular inflammation that add prognostic value to traditional measures of disease severity & suggest novel therapeutic targets for future research. In addition, to standard clinical testing, patients undergo serial assessments using innovative imaging techniques, flow cytometric analyses of circulating endothelial cells & bone marrow progenitor cells, genome-wide blood transcriptomic profiles and novel biomarkers such as plasma cfDNA. The collective data will be used to investigate the ability of blood markers of vascular inflammation and/or high-resolution cardiac CT & MRI to stage disease severity & predict clinically relevant outcomes. The study has enrolled 89 individuals (66 patients / 23 healthy volunteers). Total study population will be 150 adult PAH subjects & 50 age & gender matched controls (i.e. healthy volunteer matched to 3 PAH patients). Inflammation is recognized as a feature of the abnormal pulmonary arteries in PAH patients, & it has been hypothesized, but remains unknown as to whether drugs that block inflammation could be beneficial in patients with PAH. Mineralocorticoid receptor (MR) antagonists, like spironolactone, have been widely used in patients with left sided heart failure or LV dysfunction post-MI. Evidence suggests spironolactone improves endothelial function & reduces inflammation. Patients with WHO group 1 PAH are being enrolled in the Spironolactone Interventional Trial (SPIRIT-PAH, 12-CC-0211); a phase 1-2 randomized, double blinded, placebo-controlled 6-month study of spironolactone treatment in PAH. The trial examines the safety & tolerability of spironolactone & its efficacy as assessed by effects on exercise capacity & clinical worsening. We seek to determine if spironolactone provides benefits in PAH through anti-inflammatory effects & improvements in endothelial function. The study has enrolled 40 individuals & per statistical assessment plan targets at least 50. Our original PAH protocol (05-CC-0041: n = 31) assessed whether CECs and/or PBMC may serve as PAH biomarkers. The project used flow cytometry to develop a methodology for isolating relevant numbers of viable CECs from healthy & PAH subjects. CECs & PBMCs were obtained from peripheral blood (PB) specimens. A subset of subjects had a right heart catheterization to assess pulmonary pressures and obtain pulmonary blood specimens. Available data suggested no trend towards CEC enrichment in pulmonary vein blood compared to PB for healthy (4.4 vs 4.8 CEC/ml) & PAH (2.4 vs 3.0 CEC/ml) subjects. There was a trend towards CEC enrichment in pulmonary artery blood compared to PB for healthy (13.8 vs 4.8 CEC/ml) & PAH (3.3 vs 3.0 CEC/ml) subjects. We published a manuscript (Am J Physiol Lung Cell Mol Physiol, 318(1): L98-L111, 2019) containing our PBMC data as part of a larger meta-analysis. The meta-analysis defined a robust & generalizable transcriptomic signature in the blood of PAH patients that can help inform the identification of biomarkers & therapeutic targets. This protocol (1 of 3) is closed to enrollment & open for data analysis. Remaining bio-specimens include RNA, plasma, serum, circulating endothelial cells & T-Cells. We are further evaluating systemic inflammation in PAH by using CT to assess Coronary Artery Plaque Burden in Patients with PAH. Preliminary data was presented at the American Thoracic Society (ATS) International Conference (AJRCCM, 199:A6795, 2019). Compared to controls (n = 7) matched for traditional risk factors of coronary artery disease, PAH subjects (n = 7) tended to have a higher burden of coronary artery plaque as determined by CT angiography. These findings & their relevance to symptoms & functional capacity, need to be further investigated in a larger PAH cohort which we are accumulating. We also investigated whether or not PAH Patients display normal kinetics of clot Formation. Preliminary data was presented at the AHA Scientific Sessions (Circulation, 140:A10714, 2019) & the complete analysis subsequently published (Pulm Circ, 11(3):1-9, 2021). PAH patients on stable medical therapy did not demonstrate abnormal clotting kinetics or fibrinolysis by thrombelastography (TEG). Additionally, these patients did not demonstrate abnormal levels of hematologic markers associated with thrombosis & fibrosis. In addition, we investigated whether or not HSCs, CPCs & CECs are altered in PAH patients compared to healthy controls. Preliminary data was presented at the ATS International Conf (AJRCCM, 207:A3790, 2023). Analysis revealed an increased number of CECs in PAH patients thereby supporting the pathobiological paradigm of endothelial injury. In collaboration with the NHLBI Laboratory of Applied Precision Omics (APO) we are investigating Plasma Cell-free DNA (cfDNA) as a novel marker of disease severity in PAH. Preliminary data was submitted to the ACC 68th Annual Scientific Session (J Am Coll Cardiol, 73(9, S1): S1897, 2019). Based on the encouraging preliminary results, we entered into an MTA with PAH programs at Allegheny General Hospital & Tufts Medical Center, receiving 48 plasma samples from Allegheny (exploratory cohort) & 161 samples from tufts (validation cohort) along with clinical data. We not only measured cfDNA levels in patients & healthy volunteers (n=48), but also subjected a subset of the samples to bisulfite sequencing and a deconvolution algorithm to map tissue-specific sources of cfDNA. The complete analysis published during this reporting period (Circulation, 146(14):1033-1045, 2022) revealed that in patients with PAH, circulating cfDNA is elevated, correlates with disease severity & predicts worse survival. cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis, thus providing biologic plausibility for cfDNA as a PAH bio