Hypoxia Supports Epicardial Cell Differentiation in Vascular Smooth Muscle Cells through the Activation of the TGFβ Pathway.

Jiayi Tao,Michiko Watanabe,Diana Ramírez-Bergeron,Joey Barnett

doi:10.3390/jcdd5020019

Abstract

Epicardium-derived cells (EPDCs) are an important pool of multipotent cardiovascular progenitor cells. Through epithelial-to-mesenchymal-transition (EMT), EPDCs invade the subepicardium and myocardium and further differentiate into several cell types required for coronary vessel formation. We previously showed that epicardial hypoxia inducible factor (HIF) signaling mediates the invasion of vascular precursor cells critical for patterning the coronary vasculature. Here, we examine the regulatory role of hypoxia (1% oxygen) on EPDC differentiation into vascular smooth muscle cells (VSMCs). Results: Hypoxia stimulates EMT and enhances expression of several VSMC markers in mouse epicardial cell cultures. This stimulation is specifically blocked by inhibiting transforming growth factor-beta (TGFβ) receptor I. Further analyses indicated that hypoxia increases the expression level of TGFβ-1 ligand and phosphorylation of TGFβ receptor II, suggesting an indispensable role of the TGFβ pathway in hypoxia-stimulated VSMC differentiation. We further demonstrate that the non-canonical RhoA/Rho kinase (ROCK) pathway acts as the main downstream effector of TGFβ to modulate hypoxia’s effect on VSMC differentiation. Conclusion: Our results reveal a novel role of epicardial HIF in mediating coronary vasculogenesis by promoting their differentiation into VSMCs through noncanonical TGFβ signaling. These data elucidate that patterning of the coronary vasculature is influenced by epicardial hypoxic signals.

Highlights

Mesenchyme production from the epicardium is a critical developmental event during the generation of coronary vessels [1]
TGFβ1–3 are a set of pleiotropic cytokines involved in the proliferation, differentiation, migration and epithelial-to-mesenchymal transition (EMT) of various cell types and the signaling is transduced to the nucleus through the formation of heterodimeric receptor complexes between Type I [TβRI, termed activin receptor-like kinase-5 (ALK-5)] and Type II (TβRII) II serine/threonine kinase receptors [29]
Since we previously reported that the forced expression of activated hypoxia inducible factor (HIF)-1α ex vivo or in ovo in the epicardium of developing avian embryos promoted epicardial EMT, we used a well-established immortalized mouse epicardium-derived cells (EPDCs) culture system to closely examine the contribution of hypoxia during these developmental events

Summary

Introduction

Mesenchyme production from the epicardium is a critical developmental event during the generation of coronary vessels [1]. We have shown that forced expression of caHIF-1α in the epicardium of avian embryos resulted in reduced invasion of these cells into the myocardium due to the increased expression of the antagonistic receptor to Vascular Endothelial Growth Factor (VEGF), VEGFR1, which inhibits VEGF signaling [39]. These findings support multiple roles for HIF-1α in coronary development, homeostasis and diseases requiring further investigation. Revealing the mechanisms controlling coronary development could direct the design of new diagnostic therapies and treatments related to cardiovascular anomalies and diseases

Cell Culture

Growth Factors and Inhibitors

Transfection and Virus Infection

Immunohistochemistry

Western Blotting

Results