Loss of Endothelial Hypoxia Inducible Factor‐Prolyl Hydroxylase 2 Induces Cardiac Hypertrophy and Fibrosis

During development, the heart grows by cardiomyocyte proliferation and hypertrophy, whereas after birth, the cardiomyocytes lose proliferative potential, and heart growth is mainly via cardiomyocyte hypertrophy. Cardiac hypertrophy can happen in physiological and pathophysiological conditions. Pathological hypertrophy induced by hypertension, myocardial infarction, and/or cardiomyopathy results in ventricular remodeling, which is associated with systolic and diastolic dysfunction and interstitial fibrosis, and finally leads to deleterious outcomes such as heart failure.^{1, 2} Understanding the mechanistic molecular signaling in the event of physiological and pathological cardiac hypertrophy will lead to identifying novel therapeutic approaches for patients with heart failure. There are multiple cell types including cardiomyocytes, endothelial cells (ECs), fibroblasts, and smooth muscle cells in the heart. ECs lining the inner layer of the blood vessels account for the greatest number of cells in the heart. One of the major functions of cardiac vascular ECs is to supply oxygen and nutrients to support cardiomyocytes.^{3, 4} Previous studies have demonstrated that angiogenesis stimulated by VEGF (vascular endothelial growth factor)‐B or PIGF (placental growth factor) induces a marked increase of cardiac mass in rodents, whereas inhibition of angiogenesis results in decreased capillary density, contractile dysfunction, and impaired cardiac growth.^{5, 6} Cardiac vasculature rarefaction is associated with pathological cardiac hypertrophy and heart failure.⁷ Recent studies have also demonstrated the important role of EC‐derived paracrine factors such as endothelin‐1, apelin, neuregulin, and agrin in regulating cardiac hypertrophy, regeneration, and repair.^{8, 9} However, how ECs crosstalk with cardiomyocytes in the pathogenesis of cardiac hypertrophy and dysfunction is not fully understood.

HIFs (hypoxia‐inducible factors) are key transcriptional factors mediating cellular response to oxygen levels. The α subunit of HIF is delicately controlled by HIF–PHD1‐3 (prolyl‐4 hydroxylases 1‐3).^{10, 11, 12} Under normoxia conditions, PHDs (prolyl‐4 hydroxylases) hydroxylate HIF‐α (mainly HIF‐1α and HIF‐2α), then E3 ligase Von Hippel‐Lindau promotes degradation of hydroxylated HIF‐α through a proteasome‐degradation pathway. Under hypoxia conditions, PHD activities are inhibited, leading to stabilization of HIF‐α proteins that activate downstream target gene expression. Both HIF‐1a and HIF‐2α share similar DNA binding site or hypoxia response element.^{11, 13} Thus, some genes are coregulated by HIF‐1α and HIF‐2α, such as CXCL12 (C‐X‐C motif chemokine ligand 12). However, HIF‐1α and HIF‐2α also control some sets of unique downstream targets; for example, LDHA (lactate dehydrogenase A) is a HIF‐1α target, whereas OCT4 (octamer‐binding transcription factor 4) is an HIF‐2α target.¹⁴ HIF‐1α is ubiquitously expressed, whereas HIF‐2α is more restricted in certain cell types such as ECs and alveolar type 2 epithelial cells,¹⁵ suggesting that HIF‐α has a distinct function in different cell types under different (patho)physiological conditions. Our previous studies have shown that HIF‐2α but not HIF‐1α activation causes severe pulmonary hypertension in Egln1 (encoding PHD2) conditional knockout mice, whereas HIF‐1α induction and activation is important for endothelial regeneration and vascular repair following inflammatory injury.^{16, 17}

Egln1‐null mutant mice exhibit polycythemia and congestive heart failure.¹⁸ However, cardiomyocyte‐specific disruption of Egln1 causes only mild abnormality with the presence of occasional myocytes with increased hypereosinophilia and blurring of the cross‐striations,¹⁹ indicating that loss of PHD2 cells other than cardiomyocytes causes congestive heart failure. Fan et al show that EC deletion of both PHD2 and 3 induces spontaneous cardiomegaly because of enhanced cardiomyocyte proliferation and also prevents heart failure induced by myocardial infarction.²⁰ However, it is unknown whether these phenotypes are mediated by endothelial loss of either PHD2 or PHD3 alone. Studies have shown loss of both PHD2 and 3 but not PHD2 alone in cardiomyocytes induces ischemic cardiomyopathy.²⁰ Thus, it is important to determine whether loss of PHD2 alone in ECs affects cardiomyocyte and heart function in adult mice. To our surprise, we observed spontaneous left ventricular (LV) hypertrophy and cardiac fibrosis in tamoxifen‐inducible EC‐specific Egln1 knockout adult mice. Using the mice with endothelial deletion of Egln1 alone, Egln1 and Hif1a, or Egln1 and Hif2a, we found that loss of endothelial PHD2‐induced cardiac hypertrophy and fibrosis was mediated by HIF‐2α activation, and pharmacological inhibition of HIF‐2α inhibited LV hypertrophy and cardiac fibrosis. Analysis of single‐cell RNA sequencing data sets revealed that EGLN1 was mainly expressed in vascular ECs of the human heart under normal conditions. In patients with hypertrophic cardiomyopathy, EGLN1 expression in LV heart tissue (messenger RNA [mRNA]) and cardiac vascular ECs (protein) was markedly deceased, which validates the clinical relevance of our findings that endothelial PHD2 deficiency induces LV hypertrophy and cardiac fibrosis. Thus, targeting PHD2/HIF‐2α signaling could be a therapeutic approach for pathological cardiac hypertrophy leading to cardiomyopathy and heart failure.