Inhibition of PHD2 Induces Endothelial‐to‐Mesenchymal Transition in Pulmonary Vascular Endothelial Cells
Authors: Babicheva Aleksandra , McDermott Kimberly M. , Tang Haiyang , Song Shanshan , Sun Xutong , Cordery Arlette G. , Desai Ankit A. , Black Stephen M. , Garcia Joe G.N. , Makino Ayako , Yuan Jason X.‐Y. 
Published: November 2023

RATIONALEPulmonary arterial hypertension (PAH) is a fatal disease that predominantly affect young women. Enhanced proliferation and/or reduced apoptosis of pulmonary vascular endothelial cells (PVEC) contribute to the concentric pulmonary vascular wall thickening and intraluminal obliteration in small pulmonary arteries in patients with PAH. However, the precise pathogenic mechanisms by which PVEC contribute to the formation of intimal lesions is not fully understood. Endothelial‐to‐mesenchymal transition (EndMT) may convert slowly grown PVEC to highly proliferative myofibroblasts and contribute to pulmonary vascular remodeling in PAH. We recently reported that EndMT of lung microvascular endothelial cells (LMVEC) from patients with PAH was associated with prolyl hydroxylase domain 2 (PHD2) downregulation and that mice with endothelial specific deletion of phd2 or egln1 developed severe pulmonary hypertension (PH) under normoxic conditions. In this study, we further investigate the mechanism by which PHD2 inhibition mediates the development and progression of EndMT in PH.METHODSWe utilized the monocrotaline (MCT)‐induced rat model of PH to define whether EndMT is present in lung tissue from MCT‐PH rats. Also we used endothelial specific deletion of phd2 in mice to detect whether EndMT is enhanced in phd2 knock‐out (KO) mice. Expression of the endothelial markers (Pecam11, Cdh55, Ctnnd1), the smooth muscle (Acta2, Tagln) and fibroblast (S100A4, Vim) markers, and the EndMT‐related transcription factors (Snai1/2) were quantified using real‐time RT‐PCR and ICC. PHD2 gene expression was measured by real‐time RT‐PCR and Western blot. To determine whether pharmacological inhibition of PHD2 stimulates the progression of EndMT in vitro, we treated LMVEC isolated from healthy subjects with PHD2 inhibitor (FG‐4592) at a dose of 100 μM for 7 days.RESULTSMCT injection results in severe PH in rats after 2–4 weeks. The increased right ventricular systolic pressure and right ventricular hypertrophy were closely associated with pulmonary vascular remodeling and enhanced EndMT in the endothelium of remodeled small pulmonary arteries in MCT‐PH rats. Additionally MCT treatment decreased PHD2 mRNA expression in rat lung extracts. The heterozygous (phd2EC+/−) and homozygous (phd2EC−/−) KO mice both exhibited PH at age of 10 weeks under normoxic conditions. Endothelial‐specific deletion of phd2 results in EndMT associated with obliterative pulmonary vascular lesions. Inhibition of PHD2 activity with FG‐4592 induced expression of Snai1, an EndMT‐related transcription factor, in normal LMVEC.CONCLUSIONSOur results suggest that genetic or pharmacological inhibition of PHD2 promotes EndMT and may contribute to elevated pulmonary arterial pressure by promoting concentric and obliterative pulmonary vascular remodeling in patients with PAH and rats with MCT‐induced PH.Support or Funding InformationThis work was supported in parts by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL 115014, HL066012 and HL125208).