BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PAs) and progressive increase in pulmonary vascular resistance leading to right ventricular failure. Although several drugs are approved for the treatment of PAH, mortality rates remain high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. METHODS: The expression of the RGD (arginylglycylaspartic acid)–binding integrin α5β1 was assessed in PAs, PA smooth muscle cells, and PA endothelial cells from patients with PAH and controls using NanoString, immunoblotting, and Mesoscale Discovery assays. RNA sequencing was conducted to identify gene networks regulated by α5β1 inhibition in PAH PA smooth muscle cells. The therapeutic efficacy of α5β1 inhibition was evaluated using a novel small molecule inhibitor and selective neutralizing antibodies in Sugen/hypoxia and monocrotaline rat models, with validation by an external contract research organization. Comparisons were made against standard-of-care therapies (ie, macitentan, tadalafil) and sotatercept and efficacy was assessed using echocardiographic, hemodynamic, and histological assessments. Ex vivo studies using human precision-cut lung slices were performed to further assess the effects of α5β1 inhibition on pulmonary vascular remodeling. RESULTS: We found that the arginine-glycine-aspartate RGD-binding integrin α5β1 is upregulated in PA endothelial cells and PA smooth muscle cells from patients with PAH and remodeled PAs from animal models. Blockade of the integrin α5β1 or depletion of the α5 subunit downregulated FOXM1 (forkhead box protein M1)–regulated gene networks, resulting in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. We demonstrated that α5β1 integrin blockade safely attenuates pulmonary vascular remodeling and improves hemodynamics and right ventricular function and matched or exceeded the efficacy of standard of care and sotatercept in multiple preclinical models. Ex vivo studies further validated its potential in reversing advanced remodeling in human precision-cut lung slices. CONCLUSIONS: These findings establish α5β1 integrin as a pivotal driver of PAH pathology and we propose its inhibition as a novel, safe, and effective therapeutic strategy for PAH.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by a sustained elevation of pulmonary artery (PA) pressure, right ventricular failure, and premature death. Enhanced proliferation and resistance to apoptosis (as seen in cancer cells) of PA smooth muscle cells (PASMCs) is a major pathological hallmark contributing to pulmonary vascular remodeling in PAH, for which current therapies have only limited effects. Emerging evidence points toward a critical role for Enhancer of Zeste Homolog 2 (EZH2) in cancer cell proliferation and survival. However, its role in PAH remains largely unknown. The aim of this study was to determine whether EZH2 represents a new factor critically involved in the abnormal phenotype of PAH-PASMCs. We found that EZH2 is overexpressed in human lung tissues and isolated PASMCs from PAH patients compared to controls as well as in two animal models mimicking the disease. Through loss- and gain-of-function approaches, we showed that EZH2 promotes PAH-PASMC proliferation and survival. By combining quantitative transcriptomic and proteomic approaches in PAH-PASMCs subjected or not to EZH2 knockdown, we found that inhibition of EZH2 downregulates many factors involved in cell-cycle progression, including E2F targets, and contributes to maintain energy production. Notably, we found that EZH2 promotes expression of several nuclear-encoded components of the mitochondrial translation machinery and tricarboxylic acid cycle genes. Overall, this study provides evidence that, by overexpressing EZH2, PAH-PASMCs remove the physiological breaks that normally restrain their proliferation and susceptibility to apoptosis and suggests that EZH2 or downstream factors may serve as therapeutic targets to combat pulmonary vascular remodeling.

Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.

Pulmonary arterial hypertension (PAH) is defined by increased pulmonary artery (PA) pressure and vascular remodeling, leading to progressive hypertrophy (compensation) and failure (decompensation) of the right ventricle (RV). The histone acetyltransferases P300/CBP have been identified as central players in various cellular processes (proliferation/apoptosis and hypertrophy/fibrosis). Given their role in controlling gene transcription programs, we hypothesized that P300/CBP contributes to maladaptive remodeling in PAH. We show by western blot (WB) and immunofluorescence (IF) increased P300/CBP expression in isolated PA smooth muscle cells (PASMCs) and distal PAs from PAH patients compared to controls (p<0.01) as well as in monocrotaline (MCT) and sugen-hypoxia rat models (p<0.05). Similarly, P300 expression is up-regulated in remodeled RV from PAH patients, MCT- and pulmonary artery banding-subjected rats (WB). In vitro, P300/CBP inhibition (CCS-1477 or siRNA) decreases PAH-PASMC proliferation and resistance to apoptosis [WB (PCNA, PLK1, Survivin) IF (Ki67, AnexinV), p<0.01], prevents phenylephrine-induced hypertrophy (H9C2 cells, adult rat cardiomyocytes, p<0.05), and reduces activation (pSMAD2/3, aSMA, Fn, Col1A, MMP2) and proliferation (PCNA, Survivin) of activated RV fibroblasts. In vivo, administration of CCS-1477 reduces pulmonary vascular remodeling (Elastica Van Gieson, p<0.05), improves pulmonary hemodynamics (p<0.01) and attenuates RV fibrosis (Masson’s Trichrome, p<0.05) in MCT rats with established PAH. Our finding provide evidence that targeting P300/CBP may represent a promising avenue to tackle both lung and RV maladaptive remodeling in PAH.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary artery (PA) pressure and vascular remodeling, resulting in compensated hypertrophy and decompensated failure of the right ventricle (RV). The histone acetyltransferases P300/CBP are critical regulators in various cellular processes such as proliferation, apoptosis, hypertrophy, and fibrosis. Considering their pivotal role in modulating gene transcription, we hypothesized that P300/CBP contributes to maladaptive remodeling observed in PAH. Using western blot (WB) and immunofluorescence (IF), we show increased P300/CBP expression in isolated PA smooth muscle cells (PASMCs) and PAs from PAH patients compared to controls (p<0.01) as well as in monocrotaline (MCT) and sugen-hypoxia rat (p<0.05). Similarly, P300 expression is elevated in remodeled RV from PAH patients, MCT- and pulmonary artery banding- rats (PAB). In vitro, P300/CBP inhibition (CCS-1477 or siRNA) decreases PAH-PASMC proliferation and resistance to apoptosis [WB (PCNA, PLK1, Survivin) IF (Ki67, AnexinV)], prevents phenylephrine-induced hypertrophy (H9C2 cells, adult rat cardiomyocytes), and reduces activation (pSMAD2/3, aSMA, Fn, Col1A, MMP2, WB) and proliferation (PCNA, WB) of RV fibroblasts. In vivo administration of CCS-1477 in MCT rats reduces pulmonary vascular remodeling (Elastica Van Gieson), improves pulmonary hemodynamics (mPAP, RVSP) and attenuates RV fibrosis (Masson’s Trichrome (MT)). In the PAB rat model, CCS-1477 improves RV function (TAPSE, CO, SV) and reduces fibrosis (MT). Our finding suggests that targeting P300/CBP may represent a promising avenue to tackle both lung and RV maladaptive remodeling in PAH.