Emerging role of angiogenesis in adaptive and maladaptive right ventricular remodeling in pulmonary hypertension

Right ventricular (RV) function is the primary prognostic factor for both morbidity and mortality in pulmonary hypertension (PH). RV hypertrophy is initially an adaptive physiological response to increased overload; however, with persistent and/or progressive afterload increase, this response frequently transitions to more pathological maladaptive remodeling. The mechanisms and disease processes underlying this transition are mostly unknown. Angiogenesis has recently emerged as a major modifier of RV adaptation in the setting of pressure overload. A novel paradigm has emerged that suggests that angiogenesis and angiogenic signaling are required for RV adaptation to afterload increases and that impaired and/or insufficient angiogenesis is a major driver of RV decompensation. Here, we summarize our current understanding of the concepts of maladaptive and adaptive RV remodeling, discuss the current literature on angiogenesis in the adapted and failing RV, and identify potential therapeutic approaches targeting angiogenesis in RV failure

Hypoxia Upregulates Estrogen Receptor β in Pulmonary Artery Endothelial Cells in a HIF-1α-Dependent Manner

17β-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension (HPH) through estrogen receptor (ER)-dependent effects, including inhibition of hypoxia-induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia-inducible factor 1α (HIF-1α)-dependent increases in ERβ expression. Sprague-Dawley rats and ERα or ERβ knockout mice were exposed to hypobaric hypoxia for 2-3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ERβ, but not ERα, in lungs from HPH rats as well as in rat and human PAECs. ERβ mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF-1α/HIF-2α stabilization increased PAEC ERβ, whereas HIF-1α knockdown decreased ERβ abundance in hypoxic PAECs. In turn, ERβ knockdown in hypoxic PAECs increased HIF-2α expression, suggesting a hypoxia-sensitive feedback mechanism. ERβ knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ERβ activation increased PHD2 and decreased both HIF-1α and HIF-2α, suggesting that ERβ regulates the PHD2/HIF-1α/HIF-2α axis during hypoxia. Whereas hypoxic wild-type or ERα knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF-1α after hypoxia compared with untreated hypoxic mice, ERβ knockout mice exhibited increased HIF-2α and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF-1α, increases ERβ expression and the E2-ERβ axis targets PHD2, HIF-1α, and HIF-2α to attenuate HPH development

BMPR2 mutations and endothelial dysfunction in pulmonary arterial hypertension (2017 Grover Conference Series)

Despite the discovery more than 15 years ago that patients with hereditary pulmonary arterial hypertension (HPAH) inherit BMP type 2 receptor ( BMPR2) mutations, it is still unclear how these mutations cause disease. In part, this is attributable to the rarity of HPAH and difficulty obtaining tissue samples from patients with early disease. However, in addition, limitations to the approaches used to study the effects of BMPR2 mutations on the pulmonary vasculature have restricted our ability to determine how individual mutations give rise to progressive pulmonary vascular pathology in HPAH. The importance of understanding the mechanisms by which BMPR2 mutations cause disease in patients with HPAH is underscored by evidence that there is reduced BMPR2 expression in patients with other, more common, non-hereditary form of PAH, and that restoration of BMPR2 expression reverses established disease in experimental models of pulmonary hypertension. In this paper, we focus on the effects on endothelial function. We discuss some of the controversies and challenges that have faced investigators exploring the role of BMPR2 mutations in HPAH, focusing specifically on the effects different BMPR2 mutation have on endothelial function, and whether there are qualitative differences between different BMPR2 mutations. We discuss evidence that BMPR2 signaling regulates a number of responses that may account for endothelial abnormalities in HPAH and summarize limitations of the models that are used to study these effects. Finally, we discuss evidence that BMPR2-dependent effects on endothelial metabolism provides a unifying explanation for the many of the BMPR2 mutation-dependent effects that have been described in patients with HPAH

Estrogen receptor-dependent attenuation of hypoxia-induced changes in the lung genome of pulmonary hypertension rats

17β-estradiol (E2) exerts complex and context-dependent effects in pulmonary hypertension. In hypoxia-induced pulmonary hypertension (HPH), E2 attenuates lung vascular remodeling through estrogen receptor (ER)-dependent effects; however, ER target genes in the hypoxic lung remain unknown. In order to identify the genome regulated by the E2-ER axis in the hypoxic lung, we performed a microarray analysis in lungs from HPH rats treated with E2 (75 mcg/kg/day) ± ER-antagonist ICI182,780 (3 mg/kg/day). Untreated HPH rats and normoxic rats served as controls. Using a false discovery rate of 10%, we identified a significantly differentially regulated genome in E2-treated versus untreated hypoxia rats. Genes most upregulated by E2 encoded matrix metalloproteinase 8, S100 calcium binding protein A8, and IgA Fc receptor; genes most downregulated by E2 encoded olfactory receptor 63, secreted frizzled-related protein 2, and thrombospondin 2. Several genes affected by E2 changed in the opposite direction after ICI182,780 co-treatment, indicating an ER-regulated genome in HPH lungs. The bone morphogenetic protein antagonist Grem1 (gremlin 1) was upregulated by hypoxia, but found to be among the most downregulated genes after E2 treatment. Gremlin 1 protein was reduced in E2-treated versus untreated hypoxic animals, and ER-blockade abolished the inhibitory effect of E2 on Grem1 mRNA and protein. In conclusion, E2 ER-dependently regulates several genes involved in proliferative and inflammatory processes during hypoxia. Gremlin 1 is a novel target of the E2-ER axis in HPH. Understanding the mechanisms of E2 gene regulation in HPH may allow for selectively harnessing beneficial transcriptional activities of E2 for therapeutic purposes

Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations

Isolated heart model demonstrates evidence of contractile and diastolic dysfunction in right ventricles from rats with sugen/hypoxia‐induced pulmonary hypertension

Although extensively used for the study of left ventricular function, limited experience exists with the isolated heart model in the evaluation of right ventricular (RV) function. In particular, no published experience exists with this tool in sugen/hypoxia‐induced pulmonary hypertension (SuHx‐PH), a frequently used model of severe and progressive PH. We sought to characterize markers of RV contractile and diastolic function in SuHx‐PH and to establish their relationship with markers of maladaptive RV remodeling. Hearts were excised from anesthetized Sprague Dawley rats with or without SuHx‐PH and perfused via the aorta using a Langendorff preparation. We explored the Frank–Starling relationship of RV function (RV developed pressure, dP/dt max, and dP/dt min; all normalized to RV mass) by increasing RV end‐diastolic pressure (RVEDP) from 0 to 40 mmHg. Functional studies were complemented by quantification of RV pro‐apoptotic signaling (bcl2/bax), procontractile signaling (apelin), and stress response signaling (p38MAPK activation). Pearson's correlation analysis was performed for functional and biochemical parameters. SuHx‐RVs exhibited severe RV dysfunction with marked hypertrophy and decreased echocardiographic cardiac output. For any given RVEDP, SuHx‐RVs demonstrated less developed pressure and lower dP/dt max, as well as less pronounced dP/dt min, suggestive of decreased contractile and diastolic function. SuHx‐RVs exhibited decreased bcl2/bax ratios, apelin expression, and p38MAPK activation. Bcl2/bax and apelin RNA abundance correlated positively with RV developed pressure and dP/dt max and negatively with dP/dt min. p38MAPK activation correlated positively with RV developed pressure. We conclude that SuHx‐RVs exhibit severe contractile and diastolic dysfunction. Increased pro‐apoptotic signaling and attenuated procontractile and stress response signaling may contribute to these functional alterations

Interaction of human tRNA-dihydrouridine synthase-2 with interferon-induced protein kinase PKR

PKR is an interferon (IFN)-induced protein kinase, which is involved in regulation of antiviral innate immunity, stress signaling, cell proliferation and programmed cell death. Although a low amount of PKR is expressed ubiquitously in all cell types in the absence of IFNs, PKR expression is induced at transcriptional level by IFN. PKR's enzymatic activity is activated by its binding to one of its activators. Double-stranded (ds) RNA, protein activator PACT and heparin are the three known activators of PKR. Activation of PKR in cells leads to a general block in protein synthesis due to phosphorylation of eIF2α on serine 51 by PKR. PKR activation is regulated very tightly in mammalian cells and a prolonged activation of PKR leads to apoptosis. Thus, positive and negative regulation of PKR activation is important for cell viability and function. The studies presented here describe human dihydrouridine synthase-2 (hDUS2) as a novel regulator of PKR. We originally identified hDUS2 as a protein interacting with PACT in a yeast two-hybrid screen. Further characterization revealed that hDUS2 also interacts with PKR through its dsRNA binding/dimerization domain and inhibits its kinase activity. Our results suggest that hDUS2 may act as a novel inhibitor of PKR in cells