PPARγ/ETV2 Regulate Endothelial‐to‐Mesenchymal Transition in the Hypoxia/Sugen Rat Model of Pulmonary Hypertension

Abstract

RationalePulmonary hypertension (PH) is a chronic cardiopulmonary disorder that causes significant morbidity and mortality. Endothelial‐to‐mesenchymal transition(EndoMT) plays an important role in initiation and progression of PH, suggesting that EndoMT may be a novel and effective therapeutic target in PH. However, the molecular mechanisms regulating EndoMT and how they promote PH remains largely unknown. Our lab and others have established the importance of peroxisome proliferator‐activated receptor gamma (PPARγ) in PH. Our studies indicate that PPARγ regulates the transcription factor, ETS variant 2 (ETV2), which is in dispensable for cardiovascular system development and serves as a master regulator for endothelial cell generation and function. Thus, we hypothesize that the PPARγ/ETV2 axis regulates EndoMT in PH.MethodsMale Sprague Dawley rats received a single injection of Sugen 5416 (SU, 20 mg/kg) followed by exposure to 3 weeks of hypoxia (10% O2) or normoxia(NOR, 21% O2) followed an additional 2 weeks of NOR. Right ventricular systolic pressure (RVSP)and right ventricular hypertrophy (RVH) were measured to confirm PH. To further examine the functional significance of ETV2 in PH, we measured RVSP and RVH in ETV2+/− mice. Levels of EndoMT markers in lungs of HYP/SU rats or ETV2+/− mice were compared to NOR/SU rats or wild‐typemice using qRT‐PCR and Western blot analysis. An ETV2 promoter activity assay was used to determine if PPARγ transcriptionally regulates ETV2 expression. Further, to determine if PPARg or ETV2 signaling regulates the levels of EndoMT markers, PPARγ or ETV2 loss or gain of function strategies were employed. To model in vivo EndoMT in PH, induced‐EndoMT (i‐EndoMT) cells were induced by treating human pulmonaryartery endothelial cells (HPAECs) with IL1β, TNFα, and TGFβ, and then examined functional alterations using collagen cell contraction and wound healing assays.ResultsAs expected, HYP/SU treatment and ETV2+/− mice resulted in PH. HYP/SU treatment and ETV2+/− mice also increased lung levels of mesenchymal markers, including desmin, TGFβ1, SLUG, and αSMA. Luciferase‐promoter assays showed that PPARγ transcriptionally activates the ETV2 promoter. HPAECs lost cobblestone morphology to a more elongeted, spindle‐shaped myofibroblastic cell morphology, indication the activation of EndoMT. I‐EndoMT cells significantly enhanced basal cellular contraction and migratory response compared with HPAECs. Levels of PPARγ and ETV2 were decreased in i‐EndoMT cells whereas markers of EndoMT were increased. Importantly, knockdown of PPARγ or ETV2 in HPAEC increased levels of EndoMT markers whereas overexpression of PPARγ/ETV2 axis led to reduced expression of EndoMT markers.ConclusionsCollectively, these studies establish that the PPARγ/ETV2 axis contributes to regulation of EndoMT and maythere by participate in the pathogenesis of PH. These findings suggest novel molecular pathways involved in EndoMT and PH pathogenesis that merit further exploration.Support or Funding InformationNIH HL102167 (RLS and CMH), NIH HL119291 (CP), and NIH HL133053 (BYK).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.