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

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

Neonatal hyperoxic lung injury favorably alters adult right ventricular remodeling response to chronic hypoxia exposure

The development of pulmonary hypertension (PH) requires multiple pulmonary vascular insults, yet the role of early oxygen therapy as an initial pulmonary vascular insult remains poorly defined. Here, we employ a two-hit model of PH, utilizing postnatal hyperoxia followed by adult hypoxia exposure, to evaluate the role of early hyperoxic lung injury in the development of later PH. Sprague-Dawley pups were exposed to 90% oxygen during postnatal days 0-4 or 0-10 or to room air. All pups were then allowed to mature in room air. At 10 wk of age, a subset of rats from each group was exposed to 2 wk of hypoxia (Patm = 362 mmHg). Physiological, structural, and biochemical endpoints were assessed at 12 wk. Prolonged (10 days) postnatal hyperoxia was independently associated with elevated right ventricular (RV) systolic pressure, which worsened after hypoxia exposure later in life. These findings were only partially explained by decreases in lung microvascular density. Surprisingly, postnatal hyperoxia resulted in robust RV hypertrophy and more preserved RV function and exercise capacity following adult hypoxia compared with nonhyperoxic rats. Biochemically, RVs from animals exposed to postnatal hyperoxia and adult hypoxia demonstrated increased capillarization and a switch to a fetal gene pattern, suggesting an RV more adept to handle adult hypoxia following postnatal hyperoxia exposure. We concluded that, despite negative impacts on pulmonary artery pressures, postnatal hyperoxia exposure may render a more adaptive RV phenotype to tolerate late pulmonary vascular insults

Loss of Estrogen Receptor Alpha (ERα) Exacerbates Experimental Pulmonary Arterial Hypertension (PAH)

Background and Hypothesis: PAH is a sexually dimorphic cardiopulmonary disease characterized by excessive vasoconstriction and pulmonary artery remodeling, leading to right ventricular (RV) failure and death. While women are more likely to develop PAH, they exhibit more favorable hemodynamics and increased survival compared to men. These improved outcomes in women with PAH have been linked to protective effects of the sex steroid 17ß-estradiol (E2). While E2’s receptor ERα is protective in the systemic vasculature, its function in the cardiopulmonary system has not been explored. We hypothesized that loss of ERα exacerbates PAH. Experimental Design: Studies were performed in male and female wild type (WT) or ERα loss-of-function mutant (ERαmut) rats with monocrotaline (MCT)-induced PAH as well as disease-free controls. We quantified hemodynamics (RV catheterization), RV structure and function (echocardiography) and pulmonary artery remodeling (Verhoff-van Giesson staining). Lung tissues were analyzed for expression of pulmonary vascular homeostatic regulators BMPR2 and apelin and pro-survival regulator ERK (Western blot). P<0.05 (ANOVA) was considered significant. Results: ERαmut rats did not differ hemodynamically from WT controls. However, after MCT administration, ERαmut rats exhibited more severe disease than WT MCT rats (demonstrated by increased RV hypertrophy, RV systolic pressure, total pulmonary resistance index, as well as decreased cardiac index and stroke volume index (p<0.05). Interestingly, female ERαmut MCT rats exhibited more severe disease than their male counterparts. Apelin expression decreased in ERαmut MCT lungs compared to WT and ERαmut controls (p<0.05). Furthermore, female WT MCT lungs exhibited preserved apelin expression compared to male WT MCT (p<0.05). BMPR2 expression in ERαmut MCT lungs decreased compared to WT and ERαmut controls, as well as WT MCT (p<0.05). Conclusion: Loss of ERα aggravates MCT-PAH, indicating that ERα exerts protective effects in the cardiopulmonary system. Harnessing ERα signaling may represent a novel treatment strategy for women and men with PAH

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

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