PARP1-PKM2 Axis Mediates Right Ventricular Failure Associated With Pulmonary Arterial Hypertension.

The authors show that increased poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) and pyruvate kinase muscle isozyme 2 (PKM2) expression is a common feature of a decompensated right ventricle in patients with pulmonary arterial hypertension and animal models. The authors find in vitro that overactivated PARP1 promotes cardiomyocyte dysfunction by favoring PKM2 expression and nuclear function, glycolytic gene expression, activation of nuclear factor κB-dependent proinflammatory factors. Pharmacologic and genetic inhibition of PARP1 or enforced tetramerization of PKM2 attenuates maladaptive remodeling improving right ventricular (RV) function in multiple rodent models. Taken together, these data implicate the PARP1/PKM2 axis as a critical driver of maladaptive RV remodeling and a new promising target to directly sustain RV function in patients with pulmonary arterial hypertension.

Potential for inhibition of checkpoint kinases 1/2 in pulmonary fibrosis and secondary pulmonary hypertension.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterised by exuberant tissue remodelling and associated with high unmet medical needs. Outcomes are even worse when IPF results in secondary pulmonary hypertension (PH). Importantly, exaggerated resistance to cell death, excessive proliferation and enhanced synthetic capacity are key endophenotypes of both fibroblasts and pulmonary artery smooth muscle cells, suggesting shared molecular pathways. Under persistent injury, sustained activation of the DNA damage response (DDR) is integral to the preservation of cells survival and their capacity to proliferate. Checkpoint kinases 1 and 2 (CHK1/2) are key components of the DDR. The objective of this study was to assess the role of CHK1/2 in the development and progression of IPF and IPF+PH. METHODS AND RESULTS: Increased expression of DNA damage markers and CHK1/2 were observed in lungs, remodelled pulmonary arteries and isolated fibroblasts from IPF patients and animal models. Blockade of CHK1/2 expression or activity-induced DNA damage overload and reverted the apoptosis-resistant and fibroproliferative phenotype of disease cells. Moreover, inhibition of CHK1/2 was sufficient to interfere with transforming growth factor beta 1-mediated fibroblast activation. Importantly, pharmacological inhibition of CHK1/2 using LY2606368 attenuated fibrosis and pulmonary vascular remodelling leading to improvement in respiratory mechanics and haemodynamic parameters in two animal models mimicking IPF and IPF+PH. CONCLUSION: This study identifies CHK1/2 as key regulators of lung fibrosis and provides a proof of principle for CHK1/2 inhibition as a potential novel therapeutic option for IPF and IPF+PH.

Implication of EZH2 in the Pro-Proliferative and Apoptosis-Resistant Phenotype of Pulmonary Artery Smooth Muscle Cells in PAH: A Transcriptomic and Proteomic Approach.

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.

17ß-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease.

RATIONALE: Pulmonary hypertension (PH) due to left heart disease (LHD), or group 2 PH, is the most prevalent form of PH worldwide. PH due to LHD is often associated with metabolic syndrome (MetS). In 12% to 13% of cases, patients with PH due to LHD display vascular remodeling of pulmonary arteries (PAs) associated with poor prognosis. Unfortunately, the underlying mechanisms remain unknown; PH-targeted therapies for this group are nonexistent, and the development of a new preclinical model is crucial. Among the numerous pathways dysregulated in MetS, inflammation plays also a critical role in both PH and vascular remodeling. OBJECTIVE: We hypothesized that MetS and inflammation may trigger the development of vascular remodeling in group 2 PH. METHODS AND RESULTS: Using supracoronary aortic banding, we induced diastolic dysfunction in rats. Then we induced MetS by a combination of high-fat diet and olanzapine treatment. We used metformin treatment and anti-IL-6 (interleukin-6) antibodies to inhibit the IL-6 pathway. Compared with sham conditions, only supracoronary aortic banding+MetS rats developed precapillary PH, as measured by both echocardiography and right/left heart catheterization. PH in supracoronary aortic banding+MetS was associated with macrophage accumulation and increased IL-6 production in lung. PH was also associated with STAT3 (signal transducer and activator of transcription 3) activation and increased proliferation of PA smooth muscle cells, which contributes to remodeling of distal PA. We reported macrophage accumulation, increased IL-6 levels, and STAT3 activation in the lung of group 2 PH patients. In vitro, IL-6 activates STAT3 and induces human PA smooth muscle cell proliferation. Metformin treatment decreased inflammation, IL-6 levels, STAT3 activation, and human PA smooth muscle cell proliferation. In vivo, in the supracoronary aortic banding+MetS animals, reducing IL-6, either by anti-IL-6 antibody or metformin treatment, reversed pulmonary vascular remodeling and improve PH due to LHD. CONCLUSIONS: We developed a new preclinical model of group 2 PH by combining MetS with LHD. We showed that MetS exacerbates group 2 PH. We provided evidence for the importance of the IL-6-STAT3 pathway in our experimental model of group 2 PH and human patients.

Identification of Novel Therapeutic Targets for Pulmonary Arterial Hypertension.

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.

Pulmonary arterial hypertension: New pathophysiological insights and emerging therapeutic targets.

Pulmonary arterial hypertension (PAH) encompasses a group of clinical entities characterized by sustained vasoconstriction and progressive vascular remodeling that act in concert to elevate pulmonary vascular resistance. The current treatments for PAH are mainly dedicated to target the process of vasoconstriction and do not offer a cure. There is now accumulating evidence that expansion of pulmonary artery cells due to increased proliferation and apoptotic evasion is a key pathological component of vascular remodeling that occurs in PAH. Thus, vascular lesions seen in advanced PAH patients present some cancer-like characteristics offering important avenues for exploration and expanding treatment options. In this review article, we will discuss recent advances into mechanisms underlying disease progression, with a focus on pulmonary artery smooth muscle cells.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary Hypertension: A Possible Novel Therapeutic Target.

RATIONALE: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. OBJECTIVE: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. METHODS AND RESULTS: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator-activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. CONCLUSIONS: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.

Protective Roles of Endothelial AMP-Activated Protein Kinase Against Hypoxia-Induced Pulmonary Hypertension in Mice.

RATIONALE: Endothelial AMP-activated protein kinase (AMPK) plays an important role for vascular homeostasis, and its role is impaired by vascular inflammation. However, the role of endothelial AMPK in the pathogenesis of pulmonary arterial hypertension (PAH) remains to be elucidated. OBJECTIVE: To determine the role of endothelial AMPK in the development of PAH. METHODS AND RESULTS: Immunostaining showed that endothelial AMPK is downregulated in the pulmonary arteries of patients with PAH and hypoxia mouse model of pulmonary hypertension (PH). To elucidate the role of endothelial AMPK in PH, we used endothelial-specific AMPK-knockout mice (eAMPK(-/-)), which were exposed to hypoxia. Under normoxic condition, eAMPK(-/-) mice showed the normal morphology of pulmonary arteries compared with littermate controls (eAMPK(flox/flox)). In contrast, development of hypoxia-induced PH was accelerated in eAMPK(-/-) mice compared with controls. Furthermore, the exacerbation of PH in eAMPK(-/-) mice was accompanied by reduced endothelial function, upregulation of growth factors, and increased proliferation of pulmonary artery smooth muscle cells. Importantly, conditioned medium from endothelial cells promoted pulmonary artery smooth muscle cell proliferation, which was further enhanced by the treatment with AMPK inhibitor. Serum levels of inflammatory cytokines, including tumor necrosis factor-α and interferon-γ were significantly increased in patients with PAH compared with healthy controls. Consistently, endothelial AMPK and cell proliferation were significantly reduced by the treatment with serum from patients with PAH compared with controls. Importantly, long-term treatment with metformin, an AMPK activator, significantly attenuated hypoxia-induced PH in mice. CONCLUSIONS: These results indicate that endothelial AMPK is a novel therapeutic target for the treatment of PAH.

Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure Overload in Mice.

OBJECTIVE: Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinases and promotes inflammation. However, the role of Bsg in the pathogenesis of cardiac hypertrophy and failure remains to be elucidated. We examined the role of Bsg in cardiac hypertrophy and failure in mice and humans. APPROACH AND RESULTS: We performed transverse aortic constriction in Bsg(+/-) and in wild-type mice. Bsg(+/-) mice showed significantly less heart and lung weight and cardiac interstitial fibrosis compared with littermate controls after transverse aortic constriction. Both matrix metalloproteinase activities and oxidative stress in loaded left ventricle were significantly less in Bsg(+/-) mice compared with controls. Echocardiography showed that Bsg(+/-) mice showed less hypertrophy, less left ventricular dilatation, and preserved left ventricular fractional shortening compared with littermate controls after transverse aortic constriction. Consistently, Bsg(+/-) mice showed a significantly improved long-term survival after transverse aortic constriction compared with Bsg(+/+) mice, regardless of the source of bone marrow (Bsg(+/+) or Bsg(+/-)). Conversely, cardiac-specific Bsg-overexpressing mice showed significantly poor survival compared with littermate controls. Next, we isolated cardiac fibroblasts and examined their responses to angiotensin II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion, and extracellular signal-regulated kinase/Akt/JNK activities in Bsg(+/+) cardiac fibroblasts, all of which were significantly less in Bsg(+/-) cardiac fibroblasts. Consistently, extracellular and intracellular Bsg significantly promoted cardiac fibroblast proliferation. Finally, serum levels of Bsg were significantly elevated in patients with heart failure and predicted poor prognosis. CONCLUSIONS: These results indicate the crucial roles of intracellular and extracellular Bsg in the pathogenesis of cardiac hypertrophy, fibrosis, and failure in mice and humans.