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Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFβ and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFβ/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFβ/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFβ/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.

Pulmonary arterial hypertension (PAH) is a rapidly degenerating and devastating disease of increased pulmonary vessel resistance leading to right heart failure. Palliative modalities remain limited despite recent endeavors to investigate the mechanisms underlying increased pulmonary vascular resistance (PVR), i.e. aberrant vascular remodeling and occlusion. However, little is known of the molecular mechanisms responsible for endothelial proliferation, a root cause of PAH-associated vascular remodeling. Lung tissue specimens from PAH and non-PAH patients and hypoxia-exposed human pulmonary artery endothelial cells (ECs) (HPAEC) were assessed for mRNA and protein expression. Reactive oxygen species (ROS) were measured using cytochrome c and Amplex Red assays. Findings demonstrate for the first time an up-regulation of NADPH oxidase 1 (Nox1) at the transcript and protein level in resistance vessels from PAH compared with non-PAH patients. This coincided with an increase in ROS production and expression of bone morphogenetic protein (BMP) antagonist Gremlin1 (Grem1). In HPAEC, hypoxia induced Nox1 subunit expression, assembly, and oxidase activity leading to elevation in sonic hedgehog (SHH) and Grem1 expression. Nox1 gene silencing abrogated this cascade. Moreover, loss of either Nox1, SHH or Grem1 attenuated hypoxia-induced EC proliferation. Together, these data support a Nox1-SHH-Grem1 signaling axis in pulmonary vascular endothelium that is likely to contribute to pathophysiological endothelial proliferation and the progression of PAH. These findings also support targeting of Nox1 as a viable therapeutic option to combat PAH.

BackgroundVascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis.Methods and resultsIn SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis.ConclusionsOur findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH.

Pulmonary hypertension (PH) is common in advanced heart failure and often improves quickly after left ventricular assist device (VAD) implantation or orthotopic heart transplantation (OHT), but long-term effects and outcomes are not well-described. This study evaluated PH persistence after VAD as destination therapy (VAD-DT), bridge to transplant (VAD-OHT), or OHTalone. The study constituted a retrospective review of patients who underwent VAD-DT (n = 164), VAD-OHT (n = 111), or OHT-alone (n = 138) at a single tertiary-care center. Right heart catheterization (RHC) data was collected pre-, post-intervention (VAD and/or OHT), and 1-year from final intervention (latest-RHC) to evaluate the longitudinal hemodynamic course of right ventricular function and pulmonary vasculature. PH (Group II and Group I) definitions were adapted from expert guidelines. All groups showed significant improvements in mean pulmonary artery pressure (mPAP), pulmonary artery wedge pressure (PAWP), cardiac output, and pulmonary vascular resistance (PVR) at each RHC with greatest improvement at post-intervention RHC (post-VAD or post-OHT). PH was reduced from 98% to 26% in VAD-OHT, 92% -49% in VAD-DT, and 76%-28% in OHT-alone from preintervention to latest-RHC. At latest-RHC mPAP remained elevated in all groups despite normalization of PAWP and PVR. VAD-supported patients exhibited suppressed pulmonary artery pulsatility index (PaPi < 3.7) with improvement only posttransplant at latest-RHC. Posttransplant patients with PH at latest- RHC (n = 60) exhibited lower survival (HR: 2.1 [95% CI: 1.3-3.4], p < 0.001). Despite an overall significant improvement in pulmonary pressures and PH proportion, a notable subset of patients exhibited PH post-intervention. Postintervention PH was associated with lower posttransplant survival. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a rare yet devastating and incurable disease with few treatment options. The underlying mechanisms of PAH appear to involve substantial cellular proliferation and vascular remodeling, causing right ventricular overload and eventual heart failure. Recent evidence suggests a significant seminal role of the pulmonary endothelium in the initiation and promotion of PAH. Our previous work identified elevated reactive oxygen species (ROS)-producing enzyme NADPH oxidase 1 (NOX1) in human pulmonary artery endothelial cells (HPAECs) of PAH patients promoting endothelial cell proliferation in vitro. In this study, we interrogated chemokine CXCL12′s (aka SDF-1) role in EC proliferation under the control of NOX1 and specificity protein 1 (Sp1). We report here that NOX1 can drive hypoxia-induced endothelial CXCL12 expression via the transcription factor Sp1 leading to HPAEC proliferation and migration. Indeed, NOX1 drove hypoxia-induced Sp1 activation, along with an increased capacity of Sp1 to bind cognate promoter regions in the CXCL12 promoter. Sp1 activation induced elevated expression of CXCL12 in hypoxic HPAECs, supporting downstream induction of expression at the CXCL12 promoter via NOX1 activity. Pathological levels of CXCL12 mimicking those reported in human PAH patient serum restored EC proliferation impeded by specific NOX1 inhibitor. The translational relevance of our findings is highlighted by elevated NOX1 activity, Sp1 activation, and CXCL12 expression in explanted lung samples from PAH patients compared to non-PAH controls. Analysis of phosphofructokinase, glucose-6-phosphate dehydrogenase, and glutaminase activity revealed that CXCL12 induces glutamine and glucose metabolism, which are foundational to EC cell proliferation. Indeed, in explanted human PAH lungs, demonstrably higher glutaminase activity was detected compared to healthy controls. Finally, infusion of recombinant CXCL12 into healthy mice amplified pulmonary arterial pressure, right ventricle remodeling, and elevated glucose and glutamine metabolism. Together these data suggest a central role for a novel NOX1-Sp1-CXCL12 pathway in mediating PAH phenotype in the lung endothelium. [ABSTRACT FROM AUTHOR]

Sepsis is associated with an increase in reactive oxygen species (ROS), however, the precise role of ROS in the septic process remains unknown. We hypothesized that treatment with EUK-134 (manganese-3-methoxy N,N'-bis(salicyclidene)ethylene-diamine chloride), a compound with superoxide dismutase and catalase activity, attenuates the vascular manifestations of sepsis in vivo. Pigs were instrumented to measure cardiac output and blood flow in renal, superior mesenteric and femoral arteries, and portal vein. Animals were treated with saline (control), lipopolysaccharide (LPS; 10 µg·kg-1·h-1), EUK-134, or EUK-134 plus LPS. Results show that an LPS-induced increase in pulmonary artery pressure (PAP) as well as a trend towards lower blood pressure (BP) were both attenuated by EUK-134. Renal blood flow decreased with LPS whereas superior mesenteric, portal and femoral flows did not change. Importantly, EUK-134 decreased the LPS-induced fall in renal blood flow and this was associated with a corresponding decrease in LPS-induced protein nitrotyrosinylation in the kidney. PO2, pH, base excess and systemic vascular resistance fell with LPS and were unaltered by EUK-134. EUK-134 also had no effect on LPS-associated increase in CO. Interestingly, EUK-134 alone resulted in higher CO, BP, PAP, mean circulatory filling pressure, and portal flow than controls. Taken together, these data support a protective role for EUK-134 in the renal circulation in sepsis. [ABSTRACT FROM AUTHOR]

(1 SR ,4 RS )-3,3-Dimethyl-1,2,3,4-tetrahydro-1,4-(epiminomethano)naphthalenes were synthesized in 2-3 steps from commercially available materials and assessed for specificity and effectiveness across a range of Nox isoforms. The N -pentyl and N -methylenethiophene substituted analogs 11g and 11h emerged as selective Nox2 inhibitors with cellular IC 50 values of 20 and 32 μM, respectively.