Background: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. Methods: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. Results: Wnt/β-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/β-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (β-catenin-activated transgene driving expression of nuclear β-galactosidase) reporter mice RVs exhibited an increase in β-catenin expression compared with their respective controls. Genetic inhibition of β-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. Conclusions: Wnt- β-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

ATP citrate lyase (ACLY), a crucial enzyme in de novo lipid synthesis and histone acetylation, plays a key role in regulating vascular smooth muscle cell (VSMC) proliferation and survival. We found that human coronary and pulmonary artery tissues had up-regulated ACLY expression during vascular remodeling in coronary artery disease and pulmonary arterial hypertension. Pharmacological and genetic inhibition of ACLY in human primary cultured VSMCs isolated from the coronary arteries of patients with coronary artery diseases and from the distal pulmonary arteries of patients with pulmonary arterial hypertension resulted in reduced cellular proliferation and migration and increased susceptibility to apoptosis. These cellular changes were linked to diminished glycolysis, reduced lipid synthesis, impairment in general control nonrepressed protein 5 (GCN5)–dependent histone acetylation and suppression of the transcription factor FOXM1. In vivo studies using a pharmacological inhibitor and VSMC-specific Acly knockout mice showed that ACLY inhibition alleviated vascular remodeling. ACLY inhibition alleviated remodeling in carotid injury and ligation models in rodents and attenuated pulmonary arterial hypertension in Sugen/hypoxia rat and mouse models. Moreover, ACLY inhibition showed improvements in vascular remodeling in human ex vivo models, which included cultured human coronary artery and saphenous vein rings as well as precision-cut lung slices. Our results propose ACLY as a novel therapeutic target for treating complex vascular diseases, offering promising avenues for future clinical intervention.

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.

BACKGROUND: Pulmonary arterial hypertension (PAH) and breast cancer disproportionately affect women. BMPR2 (bone morphogenetic protein receptor type 2) mutations, the most common genetic cause of heritable PAH, also exert tumor-suppressive functions, but their role in linking these diseases remains unclear. METHODS: We combined bioinformatic, epidemiologic, and experimental approaches. Public cancer datasets were mined for BMPR2 alterations. In vivo, mammary tumor development and pulmonary hemodynamics were assessed in female Bmpr2 ^+/Δ71 rats with or without carcinogen (7,12-dimethylbenz[a]anthracene) exposure. Pulmonary arterial smooth muscle cells were exposed to tumor-conditioned media to test inflammatory proliferation. Finally, associations between breast cancer and PAH were examined in the French National Healthcare Database (9964 patients with PAH). RESULTS: BMPR2 expression was markedly reduced in human breast tumors, with recurrent somatic variants and deep deletions identified. Bmpr2 ^+/Δ71 rats exhibited spontaneous mammary tumors and, following 7,12-dimethylbenz[a]anthracene exposure, developed exacerbated pulmonary hypertension with increased vascular remodeling and inflammation. Tumor-bearing Bmpr2 ^+/Δ71 rats showed elevated lung IL-1β and NF-κB activation. In vitro, conditioned media from Bmpr2 ^+/Δ71 tumors induced proliferation of Bmpr2 ^+/Δ71 pulmonary arterial smooth muscle cells via IL-1β–dependent signaling, while neutralization of IL-1β attenuated this effect. Human pulmonary arterial smooth muscle cells carrying BMPR2 mutations similarly displayed heightened IL-1β–induced proliferation. Epidemiologically, breast cancer incidence was more than doubled in patients with PAH compared with the general population, and PAH incidence was increased nearly 9-fold among patients with breast cancer, indicating a bidirectional relationship. CONCLUSIONS: These findings identify a reciprocal association between breast cancer and PAH mediated by defective BMPR2 signaling and tumor-associated inflammation. Breast cancer may act as a “second hit,” unmasking BMPR2 -related susceptibility to PAH, underscoring BMPR2 as a shared molecular vulnerability with implications for surveillance of at-risk populations.