Pulmonary arterial hypertension (PAH) is rare disease that is categorized as idiopathic (IPAH) when no underlying cause can be identified. Lungs of most patients with IPAH contain increased numbers of T cells and dendritic cells (DCs), suggesting involvement of the immune system in its pathophysiology. However, our knowledge on circulating immune cells in IPAH is rather limited. We used flow cytometry to characterize peripheral blood DCs and T cells in treatment-naive IPAH patients, compared with connective-tissue disease-PAH (CTD-PAH) patients and healthy controls (HCs). At diagnosis, T-helper (Th) cells of IPAH patients were less capable of producing TNFα, IFNγ, IL-4 and IL-17 compared to HCs. IPAH patients showed a decreased frequency of Th2 cells and significantly enhanced expression of the CTLA4 checkpoint molecule in naive CD4⁺ T cells and both naive and memory CD8⁺ T cells. Frequencies and surface marker expression of circulating DCs and monocytes were essentially comparable between IPAH patients and HCs. Principal component analysis (PCA) separated IPAH patients—but not CTD-PAH patients—from HCs, based on T-cell cytokine profiles. At 1-year follow-up, the frequencies of IL-17⁺ production by memory CD4⁺ T cells were increased in IPAH patients and accompanied by increased proportions of Th17 and Tc17 cells, as well as decreased CTLA4 expression. Treatment-naive IPAH patients displayed a unique T-cell phenotype that was different from CTD-PAH patients and was characterized by reduced cytokine-producing capacity. These findings point to involvement of adaptive immune responses in IPAH, which may have an implication for the development of therapeutic interventions. Full article

Background: Pulmonary artery denervation (PADN) is an evolving interventional procedure capable to reduce pulmonary artery (PA) pressure. We aimed to compare PA nerve distribution in different specimens and assess the feasibility of an ovine model for a denervation procedure and evaluate the acute changes induced by laser energy. Methods: The experiment was divided into two phases: (1) the analysis of PA nerve distribution in sheep, pigs, and humans using histological and immunochemical methods; (2) fiberoptic PADN in sheep and postmortem laser lesion characteristics. Results: PA nerve density and distribution in sheep differ from humans, although pigs and sheep share similar characteristics, nerve fibers are observed in the media layer, adventitia, and perivascular tissue in sheep. Necrosis of the intima and focal hemorrhages within the media, adventitia, and perivascular adipose tissue were evidenced post laser PADN. Among the identified lesions, 40% reached adventitia and could be classified as effective for PADN. The use of 20 W ablation energy was safer and 30 W-ablation led to collateral organ damage. Conclusions: An ovine model is suitable for PADN procedures; however, nerve distribution in the PA bifurcation and main branches differ from human PA innervation. Laser ablation can be safely used for PADN procedures. Full article

Pulmonary arterial hypertension (PAH) is a progressive condition with an unmet need for early diagnosis, better monitoring, and risk stratification. The receptor for advanced glycation end products (RAGE) is activated in response to hypoxia and vascular injury, and is associated with inflammation, cell proliferation and migration in PAH. For the adult cohort, we recruited 120 patients with PAH, 83 with idiopathic PAH (IPAH) and 37 with connective tissue disease-associated PAH (CTD-PAH), and 48 controls, and determined potential plasma biomarkers by enzyme-linked immunoassay. The established heart failure marker NTproBNP and IL-6 plasma levels were several-fold higher in both adult IPAH and CTD-PAH patients versus controls. Plasma soluble RAGE (sRAGE) was elevated in IPAH patients (3044 ± 215.2 pg/mL) and was even higher in CTD-PAH patients (3332 ± 321.6 pg/mL) versus controls (1766 ± 121.9 pg/mL; p < 0.01). All three markers were increased in WHO functional class II+III PAH versus controls (p < 0.001). Receiver-operating characteristic analysis revealed that sRAGE has diagnostic accuracy comparable to prognostic NTproBNP, and even outperforms NTproBNP in the distinction of PAH FC I from controls. Lung tissue RAGE expression was increased in IPAH versus controls (mRNA) and was located predominantly in the PA intima, media, and inflammatory cells in the perivascular space (immunohistochemistry). In the pediatric cohort, plasma sRAGE concentrations were higher than in adults, but were similar in PH (n = 10) and non-PH controls (n = 10). Taken together, in the largest adult sRAGE PAH study to date, we identify plasma sRAGE as a sensitive and accurate PAH biomarker with better performance than NTproBNP in the distinction of mild PAH from controls. Full article

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we exposed kcnk3^fl/fl animals to hypoxia, metabolic diet and low dose lipopolysaccharide (LPS) and performed molecular characterization of their tissue. We also used tissue samples from KCNK3 patients (skin fibroblast derived inducible pluripotent stem cells, blood, lungs, peripheral blood mononuclear cells) and performed microarray, immunohistochemistry (IHC) and mass cytometry time of flight (CyTOF) experiments. Although a hypoxic insult did not alter vascular tone in kcnk3^fl/fl mice, RNASeq study of these lungs implied that inflammatory and metabolic factors were altered, and the follow-up diet study demonstrated a dysregulation of bone marrow cells in kcnk3^fl/fl mice. Finally, a low dose LPS study clearly showed that inflammation could be a possible second hit driving PAH in kcnk3^fl/fl mice. Multiplex, IHC and CyTOF immunophenotyping studies on human samples confirmed the mouse data and strongly indicated that cell mediated, and innate immune responses may drive PAH susceptibility in these patients. In conclusion, loss of function KCNK3 mutation alters various physiological processes from vascular tone to metabolic diet through inflammation. Our data suggests that altered circulating immune cells may drive PAH susceptibility in patients with KCNK3 mutation. Full article

Chronic pulmonary hypertension (PH) is a fatal disease characterized by the persistent activation of pulmonary vascular cells that exhibit aberrant expression of genes including miRNAs. We and others reported that decreased levels of mature microRNA-124 (miR-124) plays an important role in modulating the activated phenotype of pulmonary vascular cells and HDAC inhibitors (HDACi) can restore the levels of mature miR-124 and reverse the persistently activated phenotype of PH vascular cells. In this study, we sought to determine the mechanisms contributing to reduced levels of miRNAs, as well as how HDACi restores the levels of reduced miRNA in PH vascular cells. We found that pulmonary artery fibroblasts isolated from IPAH patients (PH-Fibs) exhibit reduced levels of mature miR-124 and several other miRNAs including let-7i, miR-224, and miR-210, and that these reduced levels can be restored by HDACi. Using miR-124 expression in human PH-Fibs as a model, we determined that reduced miR-124 gene transcription, not decreased expression of miRNA processing genes, is responsible for reduced levels of mature miR-124 in human PH-Fibs. Using both DNase I Sensitivity and chromatin immunoprecipitation assays, we found that the miR-124-1 gene exhibits a more condensed chromatin structure in human PH-Fibs, compared to corresponding controls. HDACi relaxed miR-124-1 chromatin structure, evidenced by increased levels of the open chromatin mark H3K27Ac, but decreased levels of closed chromatin mark H3K27Me³. Most importantly, the delivery of histone acetyltransferase (HAT) via CRISPR-dCas9-HAT and guiding RNAs to the promoter of the miR-124-1 gene increased miR-124-1 gene transcription. Thus, our data indicate epigenetic events play important role in controlling miR-124 and likely other miRNA levels and epigenetic regulators such as HDACs appear to be promising therapeutic targets for chronic PH. Full article

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. Full article

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. Full article

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old Tnfaip3^DNGR1-KO mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 (Tnfaip3) gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of Tnfaip3^DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3^DNGR1-KO mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in Tnfaip3^DNGR1-KO mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (Bmpr2), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the Bmpr2 gene. Full article

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation^®12 platform. The 5-bromo-2′-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH. Full article

Although the response of the right ventricle (RV) to the increased afterload is an important determinant of the patient outcome, very little is known about the underlying mechanisms. Mast cells have been implicated in the pathogenesis of left ventricular maladaptive remodeling and failure. However, the role of mast cells in RV remodeling remains unexplored. We subjected mast cell-deficient WBB6F1-KitW/W-v (Kit^W/Kit^W-v) mice and their mast cell-sufficient littermate controls (MC^+/+) to pulmonary artery banding (PAB). PAB led to RV dilatation, extensive myocardial fibrosis, and RV dysfunction in MC^+/+ mice. In PAB Kit^W/Kit^W-v mice, RV remodeling was characterized by minimal RV chamber dilatation and preserved RV function. We further administered to C57Bl/6J mice either placebo or cromolyn treatment starting from day 1 or 7 days after PAB surgery to test whether mast cells stabilizing drugs can prevent or reverse maladaptive RV remodeling. Both preventive and therapeutic cromolyn applications significantly attenuated RV dilatation and improved RV function. Our study establishes a previously undescribed role of mast cells in pressure overload-induced adverse RV remodeling. Mast cells may thus represent an interesting target for the development of a new therapeutic approach directed specifically at the heart. Full article

The physiopathology of pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) and endothelial cell (PAEC) dysfunction, contributing to pulmonary arterial obstruction and PAH progression. KCNK3 loss of function mutations are responsible for the first channelopathy identified in PAH. Loss of KCNK3 function/expression is a hallmark of PAH. However, the molecular mechanisms involved in KCNK3 dysfunction are mostly unknown. To identify the pathological molecular mechanisms downstream of KCNK3 in human PASMCs (hPASMCs) and human PAECs (hPAECs), we used a Liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach to identify the molecular pathways regulated by KCNK3. KCNK3 loss of expression was induced in control hPASMCs or hPAECs by specific siRNA targeting KCNK3. We found that the loss of KCNK3 expression in hPAECs and hPASMCs leads to 326 and 222 proteins differentially expressed, respectively. Among them, 53 proteins were common to hPAECs and hPASMCs. The specific proteome remodeling in hPAECs in absence of KCNK3 was mostly related to the activation of glycolysis, the superpathway of methionine degradation, and the mTOR signaling pathways, and to a reduction in EIF2 signaling pathways. In hPASMCs, we found an activation of the PI3K/AKT signaling pathways and a reduction in EIF2 signaling and the Purine Nucleotides De Novo Biosynthesis II and IL-8 signaling pathways. Common to hPAECs and hPASMCs, we found that the loss of KCNK3 expression leads to the activation of the NRF2-mediated oxidative stress response and a reduction in the interferon pathway. In the hPAECs and hPASMCs, we found an increased expression of HO-1 (heme oxygenase-1) and a decreased IFIT3 (interferon-induced proteins with tetratricopeptide repeats 3) (confirmed by Western blotting), allowing us to identify these axes to understand the consequences of KCNK3 dysfunction. Our experiments, based on the loss of KCNK3 expression by a specific siRNA strategy in control hPAECs and hPASMCs, allow us to identify differences in the activation of several signaling pathways, indicating the key role played by KCNK3 dysfunction in the development of PAH. Altogether, these results allow us to better understand the consequences of KCNK3 dysfunction and suggest that KCNK3 loss of expression acts in favor of the proliferation and migration of hPASMCs and promotes the metabolic shift and apoptosis resistance of hPAECs. Full article

Pulmonary arterial hypertension-congenital heart disease (PAH-CHD) is characterized by systemic to pulmonary arterial shunts and sensitively responds to volume overload and stretch of the vascular wall leading to pulmonary vascular remodeling. We hypothesized that the responses of pulmonary artery smooth muscle cells (PASMCs) to mechanical stress-associated volume overload may promote vascular remodeling in PAH-CHD. Here, we show that significantly increased collagen was in the PA adventitial layer by trichrome staining in PAH-CHD patients and an aortocaval fistula (ACF) rat model in which chronic vascular volume overload induced-PAH. We assessed the gene expression profiles of SMC markers, extracellular matrix, and collagen in isolated SMCs from pulmonary and thoracic vessels with cyclic stretch-triggered responses by real-time PCR analysis. The data corresponded to collagen deposition, which modulated pulmonary vascular remodeling in clinical and experimental PAH-ACF cases as well as in cyclic stretch-triggered SMCs in an in vitro model. We observe that collagen I A2 (COLIA2) is expressed in the control rat, but collagen I A1 (COLIA1) and Notchs remarkably increase in the lungs of ACF rats. Interestingly, closing the left-to-right shunt that leads to a reduced blood volume in the PA system of ACF rats (ACFRs) decreased the expression of COLIA1 and increased that of collagen I A2(COLIA2). This study contributes to the stretch-induced responses of SMCs and provides important future directions for therapies aimed at preventing abnormal matrix protein synthesis in volume overload-induced pulmonary hypertension (PH). Full article

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by obstructed pulmonary vasculatures. Current therapies for PAH are limited and only alleviate symptoms. Reduced levels of BMPR2 are associated with PAH pathophysiology. Moreover, reactive oxygen species, inflammation and autophagy have been shown to be hallmarks in PAH. We previously demonstrated that MnTBAP, a synthetic metalloporphyrin with antioxidant and anti-inflammatory activity, inhibits the turn-over of BMPR2 in human umbilical vein endothelial cells. Therefore, we hypothesized that MnTBAP might be used to treat PAH. Human pulmonary artery endothelial cells (PAECs), as well as pulmonary microvascular endothelial (MVECs) and smooth muscle cells (MVSMCs) from PAH patients, were treated with MnTBAP. In vivo, either saline or MnTBAP was given to PAH rats induced by Sugen 5416 and hypoxia (SuHx). On PAECs, MnTBAP was found to increase BMPR2 protein levels by blocking autophagy. Moreover, MnTBAP increased BMPR2 levels in pulmonary MVECs and MVSMCs isolated from PAH patients. In SuHx rats, MnTBAP reduced right ventricular (RV) afterload by reversing pulmonary vascular remodeling, including both intima and media layers. Furthermore, MnTBAP improved RV function and reversed RV dilation in SuHx rats. Taken together, these data highlight the importance of MnTBAP as a potential therapeutic treatment for PAH. Full article

Pulmonary arterial hypertension (PAH) results in hypertrophic remodeling of the right ventricle (RV) to overcome increased pulmonary pressure. This increases the O₂ consumption of the myocardium, and without a concomitant increase in energy generation, a mismatch with demand may occur. Eventually, RV function can no longer be sustained, and RV failure occurs. Beta-adrenergic blockers (BB) are thought to improve survival in left heart failure, in part by reducing energy expenditure and hypertrophy, however they are not currently a therapy for PAH. The monocrotaline (MCT) rat model of PAH was used to investigate the consequence of RV failure on myocardial oxygenation and mitochondrial function. A second group of MCT rats was treated daily with the beta-1 blocker metoprolol (MCT + BB). Histology confirmed reduced capillary density and increased capillary supply area without indications of capillary rarefaction in MCT rats. A computer model of O₂ flux was applied to the experimentally recorded capillary locations and predicted a reduction in mean tissue P_O2 in MCT rats. The fraction of hypoxic tissue (defined as P_O2 < 0.5 mmHg) was reduced following beta-1 blocker (BB) treatment. The functionality of the creatine kinase (CK) energy shuttle was measured in permeabilized RV myocytes by sequential ADP titrations in the presence and absence of creatine. Creatine significantly decreased the K_mADP in cells from saline-injected control (CON) rats, but not MCT rats. The difference in K_mADP with or without creatine was not different in MCT + BB cells compared to CON or MCT cells. Improved myocardial energetics could contribute to improved survival of PAH with chronic BB treatment. Full article

Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial proliferation and remodeling, resulting in a specific increase in right ventricle systolic pressure (RVSP) and, ultimately right ventricular failure. Recent studies have demonstrated that caffeic acid phenethyl ester (CAPE) exerts a protective role in NF-κB-mediated inflammatory diseases. However, the effect of CAPE on PAH remains to be elucidated. In this study, monocrotaline (MCT) was used to establish PAH in rats. Two weeks after the induction of PAH by MCT, CAPE was administrated by intraperitoneal injection once a day for two weeks. Pulmonary hemodynamic measurements and pulmonary artery morphological assessments were examined. Our results showed that administration of CAPE significantly suppressed MCT-induced vascular remodeling by decreasing the HIF-1α expression and PDGF-BB production, and improved in vivo RV systolic performance in rats. Furthermore, CAPE inhibits hypoxia- and PDGF-BB-induced HIF-1α expression by decreasing the activation of the AKT/ERK pathway, which results in the inhibition of human pulmonary artery smooth muscle cells (hPASMCs) proliferation and prevention of cells resistant to apoptosis. Overall, our data suggest that HIF-1α is regarded as an alternative target for CAPE in addition to NF-κB, and may represent a promising therapeutic agent for the treatment of PAH diseases. Full article

Background: Pulmonary hypertension (PH) is a common complication of idiopathic pulmonary fibrosis (IPF) that significantly contributes to morbidity and mortality. Macrophage migration inhibitory factor (MIF) is a critical factor in vascular remodeling of the pulmonary circulation. Objectives: We tested the effects of two small molecules targeting MIF on bleomycin (BLM)-induced collagen deposition, PH, and vascular remodeling in mouse lungs. Methods: We examined the distribution pattern of MIF, CD74, and CXCR4 in the lungs of patients with IPF-PH and the lungs of BLM-injected mice. Then, treatments were realized with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) and N-(3-hydroxy-4-fluorobenzyl)-5 trifluoromethylbenzoxazol-2-thione 31 (20 mg/kg/day per os for 3 weeks) started 24 h after an intratracheal BLM administration. Results: More intense immunoreactivity was noted for MIF, CD74, and CXCR4 in lungs from IPF-PH patients and BLM-injected mice. Furthermore, we found that treatments of BLM-injected mice with ISO-1 or compound 31 attenuated lung collagen deposition and right ventricular systolic pressure increase. Additionally, reduced pulmonary inflammatory infiltration and pulmonary arterial muscularization were observed in the lungs of BLM-injected mice treated with ISO-1 or compound 31. Conclusions: Treatments with ISO-1 or compound 31 attenuates BLM-induced inflammation and fibrosis in lung, and prevents PH development in mice, suggesting that MIF is an important factor for IPF-PH development. Full article

In pulmonary arterial hypertension (PAH), lung-angioproliferation leads to increased pulmonary vascular resistance, while simultaneous myocardial microvessel loss contributes to right ventricular (RV) failure. Endothelial colony forming cells (ECFC) are highly proliferative, angiogenic cells that may contribute to either pulmonary vascular obstruction or to RV microvascular adaptation. We hypothesize ECFC phenotypes (outgrowth, proliferation, tube formation) are related to markers of disease severity in a prospective cohort-study of 33 PAH and 30 healthy subjects. ECFC were transplanted in pulmonary trunk banded rats with RV failure. The presence of ECFC outgrowth in PAH patients was associated with low RV ejection fraction, low central venous saturation and a shorter time to clinical worsening (5.4 months (0.6–29.2) vs. 36.5 months (7.4–63.4), p = 0.032). Functionally, PAH ECFC had higher proliferative rates compared to control in vitro, although inter-patient variability was high. ECFC proliferation was inversely related to RV end diastolic volume (R² = 0.39, p = 0.018), but not pulmonary vascular resistance. Tube formation-ability was similar among donors. Normal and highly proliferative PAH ECFC were transplanted in pulmonary trunk banded rats. While no effect on hemodynamic measurements was observed, RV vascular density was restored. In conclusion, we found that ECFC outgrowth associates with high clinical severity in PAH, suggesting recruitment. Transplantation of highly proliferative ECFC restored myocardial vascular density in pulmonary trunk banded rats, while RV functional improvements were not observed. Full article

Increased growth and proliferation of distal pulmonary artery vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary arterial hypertension (PAH). Transforming Growth Factor-β (TGF-β) superfamily plays a critical role in PAH, but relative impacts of self-secreted Activin A, Gremlin1, and TGF-β on PAH PAVSMC growth and proliferation are not studied. Here we report that hyper-proliferative human PAH PAVSMC have elevated secretion of TGF-β1 and, to a lesser extent, Activin A, but not Gremlin 1, and significantly reduced Ser^465/467-Smad2 and Ser^423/425-Smad3 phosphorylation compared to controls. Media, conditioned by PAH PAVSMC, markedly increased Ser^465/467-Smad2, Ser^423/425-Smad3, and Ser^463/465-Smad1/5 phosphorylation, up-regulated Akt, ERK1/2, and p38 MAPK, and induced significant proliferation of non-diseased PAVSMC. Inhibitory anti-Activin A antibody reduced PAH PAVSMC growth without affecting canonical (Smads) or non-canonical (Akt, ERK1/2, p38 MAPK) effectors. Inhibitory anti-TGF-β antibody significantly reduced P-Smad3, P-ERK1/2 and proliferation of PAH PAVSMC, while anti-Gremlin 1 had no anti-proliferative effect. PDGF-BB diminished inhibitory effects of anti-Activin A and anti-TGF-β antibodies. None of the antibodies affected growth and proliferation of non-diseased PAVSMC induced by PAH PAVSMC-secreted factors. Together, these data demonstrate that human PAH PAVSMC have secretory, proliferative phenotype that could be targeted by anti-Activin A and anti-TGF-β antibodies; potential cross-talk with PDGF-BB should be considered while developing therapeutic interventions. Full article

Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included TFAM (−0.45-fold), suggesting impaired mitochondrial biogenesis, CYP2E1 (−3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (DHRS7C) (−2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (GALNT13), a known pulmonary hypertension (PH) biomarker (−2.7-fold). The most up-regulated gene encodes Periostin (POSTN; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (LTBP2), thrombospondin4 (THBS4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, ANNEXIN A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation. Full article

Endothelial dysfunction is a known consequence of bone morphogenetic protein type II receptor (BMPR2) mutations seen in pulmonary arterial hypertension (PAH). However, standard 2D cell culture models fail to mimic the mechanical environment seen in the pulmonary vasculature. Hydrogels have emerged as promising platforms for 3D disease modeling due to their tunable physical and biochemical properties. In order to recreate the mechanical stimuli seen in the pulmonary vasculature, we have created a novel 3D hydrogel-based pulmonary vasculature model (“artificial arteriole”) that reproduces the pulsatile flow rates and pressures seen in the human lung. Using this platform, we studied both Bmpr2^R899X and WT endothelial cells to better understand how the addition of oscillatory flow and physiological pressure influenced gene expression, cell morphology, and cell permeability. The addition of oscillatory flow and pressure resulted in several gene expression changes in both WT and Bmpr2^R899X cells. However, for many pathways with relevance to PAH etiology, Bmpr2^R899X cells responded differently when compared to the WT cells. Bmpr2^R899X cells were also found not to elongate in the direction of flow, and instead remained stagnant in morphology despite mechanical stimuli. The increased permeability of the Bmpr2^R899X layer was successfully reproduced in our artificial arteriole, with the addition of flow and pressure not leading to significant changes in permeability. Our artificial arteriole is the first to model many mechanical properties seen in the lung. Its tunability enables several new opportunities to study the endothelium in pulmonary vascular disease with increased control over environmental parameters. Full article

Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP₂ receptor, the role of which is unknown in PAH. We hypothesised that EP₂ receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP₂ receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP₂ (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP₂ receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP₂ receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP₂ receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP₂ receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH. Full article

Pulmonary arterial hypertension (PAH) is characterized by occlusion of lung arterioles, leading to marked increases in pulmonary vascular resistance. Although heritable forms of PAH are known to be driven by genetic mutations that share some commonality of function, the extent to which these effectors converge to regulate shared processes in this disease is unknown. We have causally connected extracellular matrix (ECM) remodeling and mechanotransduction to the miR-130/301 family in a feedback loop that drives vascular activation and downstream PAH. However, the molecular interconnections between factors genetically associated with PAH and this mechano-driven feedback loop remain undefined. We performed systematic manipulation of matrix stiffness, the miR-130/301 family, and factors genetically associated with PAH in primary human pulmonary arterial cells and assessed downstream and reciprocal consequences on their expression. We found that a network of factors linked to heritable PAH converges upon the matrix stiffening-miR-130/301-PPARγ-LRP8 axis in order to remodel the ECM. Furthermore, we leveraged a computational network biology approach to predict a number of additional molecular circuits functionally linking this axis to the ECM. These results demonstrate that multiple genes associated with heritable PAH converge to control the miR-130/301 circuit, triggering a self-amplifying feedback process central to pulmonary vascular stiffening and disease. Full article

Pulmonary hypertension is a co-morbidity, which strongly participates in morbi-mortality in patients with chronic obstructive pulmonary disease (COPD). Recent findings showed that bromodomain-containing proteins, in charge of reading histone acetylation, could be involved in pulmonary arterial hypertension. Our aim was to study the effect of I-BET151, an inhibitor of bromodomain and extra-terminal domain (BET), on the right ventricle hypertrophy and pulmonary hypertension, induced by a combination of chronic hypoxia and pulmonary inflammation, as the two main stimuli encountered in COPD. Adult Wistar male rats, exposed to chronic hypoxia plus pulmonary inflammation (CHPI), showed a significant right ventricle hypertrophy (+57%, p < 0.001), an increase in systolic pressure (+46%, p < 0.001) and in contraction speed (+36%, p < 0.001), when compared to control animals. I-BET151 treated animals (CHPI-iB) showed restored hemodynamic parameters to levels similar to control animals, despite chronic hypoxia plus exposure to pulmonary inflammation. They displayed lower right ventricle hypertrophy and hematocrit compared to the CHPI group (respectively −16%, p < 0.001; and −9%, p < 0.05). Our descriptive study shows a valuable effect of the inhibition of bromodomain and extra-terminal domain proteins on hemodynamic parameters, despite the presence of chronic hypoxia and pulmonary inflammation. This suggests that such inhibition could be of potential interest for COPD patients with pulmonary hypertension. Further studies are needed to unravel the underlying mechanisms involved and the net benefits of inhibiting adaptations to chronic hypoxia. Full article

Right ventricular (RV) hypertrophy is characterized by cardiac fibrosis due to endothelial–mesenchymal transition (EndMT) and increased collagen production in pulmonary arterial hypertension (PAH) patients, but the mechanisms for restoring RV function are unclear. Prostanoid agonists are effective vasodilators for PAH treatment that bind selective prostanoid receptors to modulate vascular dilation. The importance of prostanoid signaling in the RV is not clear. We investigated the effects of the EP4-specific agonist L-902,688 on cardiac fibrosis and TGF-β-induced EndMT. EP4-specific agonist treatment reduced right ventricle fibrosis in the monocrotaline (MCT)-induced PAH rat model. L-902,688 (1 µM) attenuated TGF-β-induced Twist and α-smooth muscle actin (α-SMA) expression, but these effects were reversed by AH23848 (an EP4 antagonist), highlighting the crucial role of EP4 in suppressing TGF-β-induced EndMT. These data indicate that the selective EP4 agonist L-902,688 attenuates RV fibrosis and suggest a potential approach to reducing RV fibrosis in patients with PAH. Full article

Pulmonary hypertension (PH) is a progressive complex fatal disease of multiple etiologies. Hyperproliferation and resistance to apoptosis of vascular cells of intimal, medial, and adventitial layers of pulmonary vessels trigger excessive pulmonary vascular remodeling and vasoconstriction in the course of pulmonary arterial hypertension (PAH), a subgroup of PH. Multiple gene mutation/s or dysregulated gene expression contribute to the pathogenesis of PAH by endorsing the proliferation and promoting the resistance to apoptosis of pulmonary vascular cells. Given the vital role of these cells in PAH progression, the development of safe and efficient-gene therapeutic approaches that lead to restoration or down-regulation of gene expression, generally involved in the etiology of the disease is the need of the hour. Currently, none of the FDA-approved drugs provides a cure against PH, hence innovative tools may offer a novel treatment paradigm for this progressive and lethal disorder by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. Here, we review the effectiveness and limitations of the presently available gene therapy approaches for PH. We provide a brief survey of commonly existing and currently applicable gene transfer methods for pulmonary vascular cells in vitro and describe some more recent developments for gene delivery existing in the field of PH in vivo. Full article

Recognition of and response to pathogens and tissue injury is driven by the innate immune system via activation of pattern recognition receptors. One of the many patterns recognized is RNA and, while several receptors bind RNA, Toll-like receptor 3 (TLR3) is well placed for initial recognition of RNA molecules due to its localization within the endosome. There is a growing body of work describing a role for TLR3 in maintenance of vascular homeostasis. For example, TLR3 deficiency has been shown to play repair and remodeling roles in the systemic vasculature and in lung parenchyma. A hallmark of pulmonary arterial hypertension (PAH) is pulmonary vascular remodeling, yet drivers and triggers of this remodeling remain incompletely understood. Based on its role in the systemic vasculature, our group discovered reduced endothelial TLR3 expression in PAH and revealed a protective role for a TLR3 agonist in rodent models of pulmonary hypertension. This review will provide an overview of RNA signaling in the vasculature and how it relates to PAH pathobiology, including whether targeting double-stranded RNA signaling is a potential treatment option for PAH. Full article

Pulmonary arterial hypertension (PAH) is a rare disease caused by pulmonary vascular remodeling. Current vasodilator treatments have substantially improved patients’ survival. This improved survival has led to the appearance of complications related to conditions previously underdiagnosed or even ignored, such as pulmonary artery aneurysm (PAA). The presence of a dilated pulmonary artery has been shown to be related to an increased risk of sudden cardiac death among PAH patients. This increased risk could be associated to the development of left main coronary artery compression or pulmonary artery dissection. Nevertheless, very little is currently known about the molecular mechanisms related to PAA. Thoracic aortic aneurysm (TAA) is a well-known condition with an increased risk of sudden death caused by acute aortic dissection. TAA may be secondary to chronic exposure to classic cardiovascular risk factors. In addition, a number of genetic variants have been shown to be related to a marked risk of TAA and dissection as part of multisystemic syndromes or isolated familial TAA. The molecular pathways implied in the development of TAA have been widely studied and described. Many of these molecular pathways are involved in the pathogenesis of PAH and could be involved in PAA. This review aims to describe all these common pathways to open new research lines that could help lead to a better understanding of the pathophysiology of PAH and PAA and their clinical implications. Full article

Pulmonary arterial hypertension (PAH) is a debilitating and progressive disease that predominantly develops in women. Over the past 15 years, cumulating evidence has pointed toward dysregulated metabolism of sex hormones in animal models and patients with PAH. 17β-estradiol (E2) is metabolized at positions C2, C4, and C16, which leads to the formation of metabolites with different biological/estrogenic activity. Since the first report that 2-methoxyestradiol, a major non-estrogenic metabolite of E2, attenuates the development and progression of experimental pulmonary hypertension (PH), it has become increasingly clear that E2, E2 precursors, and E2 metabolites exhibit both protective and detrimental effects in PH. Furthermore, both experimental and clinical data suggest that E2 has divergent effects in the pulmonary vasculature versus right ventricle (estrogen paradox in PAH). The estrogen paradox is of significant clinical relevance for understanding the development, progression, and prognosis of PAH. This review updates experimental and clinical findings and provides insights into: (1) the potential impacts that pathways of estradiol metabolism (EMet) may have in PAH; (2) the beneficial and adverse effects of estrogens and their precursors/metabolites in experimental PH and human PAH; (3) the co-morbidities and pathological conditions that may alter EMet and influence the development/progression of PAH; (4) the relevance of the intracrinology of sex hormones to vascular remodeling in PAH; and (5) the advantages/disadvantages of different approaches to modulate EMet in PAH. Finally, we propose the three-tier-estrogen effects in PAH concept, which may offer reconciliation of the opposing effects of E2 in PAH and may provide a better understanding of the complex mechanisms by which EMet affects the pulmonary circulation–right ventricular interaction in PAH. Full article

Pulmonary hypertension (PH) is a potentially fatal condition with a prevalence of around 1% in the world population and most commonly caused by left heart disease (PH-LHD). Usually, in PH-LHD, the increase of pulmonary pressure is only conditioned by the retrograde transmission of the left atrial pressure. However, in some cases, the long-term retrograde pressure overload may trigger complex and irreversible biomechanical and biological changes in the pulmonary vasculature. This latter clinical entity, designated as combined pre- and post-capillary PH, is associated with very poor outcomes. The underlying mechanisms of this progression are poorly understood, and most of the current knowledge comes from the field of Group 1-PAH. Treatment is also an unsolved issue in patients with PH-LHD. Targeting the molecular pathways that regulate pulmonary hemodynamics and vascular remodeling has provided excellent results in other forms of PH but has a neutral or detrimental result in patients with PH-LHD. Therefore, a deep and comprehensive biological characterization of PH-LHD is essential to improve the diagnostic and prognostic evaluation of patients and, eventually, identify new therapeutic targets. Ongoing research is aimed at identify candidate genes, variants, non-coding RNAs, and other biomarkers with potential diagnostic and therapeutic implications. In this review, we discuss the state-of-the-art cellular, molecular, genetic, and epigenetic mechanisms potentially involved in PH-LHD. Signaling and effective pathways are particularly emphasized, as well as the current knowledge on -omic biomarkers. Our final aim is to provide readers with the biological foundations on which to ground both clinical and pre-clinical research in the field of PH-LHD. Full article

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) is a debilitating disease, for which the underlying pathophysiological mechanisms have yet to be fully elucidated. Occurrence of a pulmonary embolism (PE) is a major risk factor for the development of CTEPH, with non-resolution of the thrombus being considered the main cause of CTEPH. Polymorphisms in the α-chain of fibrinogen have been linked to resistance to fibrinolysis in CTEPH patients, and could be responsible for development and disease progression. However, it is likely that additional genetic predisposition, as well as genetic and molecular alterations occurring as a consequence of tissue remodeling in the pulmonary arteries following a persistent PE, also play an important role in CTEPH. This review summarises the current knowledge regarding genetic differences between CTEPH patients and controls (with or without pulmonary hypertension). Mutations in BMPR2, differential gene and microRNA expression, and the transcription factor FoxO1 have been suggested to be involved in the processes underlying the development of CTEPH. While these studies provide the first indications regarding important dysregulated pathways in CTEPH (e.g., TGF-β and PI3K signaling), additional in-depth investigations are required to fully understand the complex processes leading to CTEPH. Full article

Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are fatal diseases; however, their pathogenesis still remains to be elucidated. We have recently screened novel pathogenic molecules and have performed drug discovery targeting those molecules. Pulmonary artery smooth muscle cells (PASMCs) in patients with PAH (PAH-PASMCs) have high proliferative properties like cancer cells, which leads to thickening and narrowing of distal pulmonary arteries. Thus, we conducted a comprehensive analysis of PAH-PASMCs and lung tissues to search for novel pathogenic proteins. We validated the pathogenic role of the selected proteins by using tissue-specific knockout mice. To confirm its clinical significance, we used patient-derived blood samples to evaluate the potential as a biomarker for diagnosis and prognosis. Finally, we conducted a high throughput screening and found inhibitors for the pathogenic proteins. Full article

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant inherited disease characterised by multisystemic vascular dysplasia. Heritable pulmonary arterial hypertension (HPAH) is a rare but severe complication of HHT. Both diseases can be the result of genetic mutations in ACVLR1 and ENG encoding for proteins involved in the transforming growth factor-beta (TGF-β) superfamily, a signalling pathway that is essential for angiogenesis. Changes within this pathway can lead to both the proliferative vasculopathy of HPAH and arteriovenous malformations seen in HHT. Clinical signs of the disease combination may not be specific but early diagnosis is important for appropriate treatment. This review describes the molecular mechanism and management of HPAH and HHT. Full article

Pulmonary arterial hypertension (PAH) is a multifactorial and severe disease without curative therapies. PAH pathobiology involves altered pulmonary arterial tone, endothelial dysfunction, distal pulmonary vessel remodeling, and inflammation, which could all depend on ion channel activities (K⁺, Ca²⁺, Na⁺ and Cl⁻). This review focuses on ion channels in the pulmonary vasculature and discusses their pathophysiological contribution to PAH as well as their therapeutic potential in PAH. Full article

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of the pulmonary arteries, increased pulmonary infiltrates, loss of vascular cross-sectional area, and elevated pulmonary vascular resistance. Despite recent advances in the management of PAH, there is a pressing need for the development of new tools to effectively treat and reduce the risk of further complications. Dysregulated immunity underlies the development of PAH, and macrophages orchestrate both the initiation and resolution of pulmonary inflammation, thus, manipulation of lung macrophage function represents an attractive target for emerging immunomodulatory therapies, including cell-based approaches. Indeed, mesenchymal stem cell (MSC)-based therapies have shown promise, effectively modulating the macrophage fulcrum to favor an anti-inflammatory, pro-resolving phenotype, which is associated with both histological and functional benefits in preclinical models of pulmonary hypertension (PH). The complex interplay between immune system homeostasis and MSCs remains incompletely understood. Here, we highlight the importance of macrophage function in models of PH and summarize the development of MSC-based therapies, focusing on the significance of MSC exosomes (MEx) and the immunomodulatory and homeostatic mechanisms by which such therapies may afford their beneficial effects. Full article

Since its association with familial pulmonary arterial hypertension (PAH) in 2000, Bone Morphogenetic Protein Receptor II (BMPR2) and its related signaling pathway have become recognized as a key regulator of pulmonary vascular homeostasis. Herein, we define BMPR2 deficiency as either an inactivation of the receptor, decreased receptor expression, or an impairment of the receptor’s downstream signaling pathway. Although traditionally the phenotypic consequences of BMPR2 deficiency in PAH have been thought to be limited to the pulmonary vasculature, there is evidence that abnormalities in BMPR2 signaling may have consequences in many other organ systems and cellular compartments. Revisiting how BMPR2 functions throughout health and disease in cells and organs beyond the lung vasculature may provide insight into the contribution of these organ systems to PAH pathogenesis as well as the potential systemic manifestation of PAH. Here we review our knowledge of the consequences of BMPR2 deficiency across multiple organ systems. Full article

Myeloid–derived suppressor cells (MDSCs) comprised a heterogeneous subset of bone marrow–derived myeloid cells, best studied in cancer research, that are increasingly implicated in the pathogenesis of pulmonary vascular remodeling and the development of pulmonary hypertension. Stem cell transplantation represents one extreme interventional strategy for ablating the myeloid compartment but poses a number of translational challenges. There remains an outstanding need for additional therapeutic targets to impact MDSC function, including the potential to alter interactions with innate and adaptive immune subsets, or alternatively, alter trafficking receptors, metabolic pathways, and transcription factor signaling with readily available and safe drugs. In this review, we summarize the current literature on the role of myeloid cells in the development of pulmonary hypertension, first in pulmonary circulation changes associated with myelodysplastic syndromes, and then by examining intrinsic myeloid cell changes that contribute to disease progression in pulmonary hypertension. We then outline several tractable targets and pathways relevant to pulmonary hypertension via MDSC regulation. Identifying these MDSC-regulated effectors is part of an ongoing effort to impact the field of pulmonary hypertension research through identification of myeloid compartment-specific therapeutic applications in the treatment of pulmonary vasculopathies. Full article