Autophagy mediates 2-methoxyestradiol-inhibited scleroderma collagen synthesis and endothelial-to-mesenchymal transition induced by hypoxia

Abstract

Objectives: To investigate whether autophagy mediates 2-methoxyestradiol (2-ME)-inhibited hypoxia-induced fibrosis and endothelial-to-mesenchymal transition (endoMT) in SSc.

Methods: Autophagy in the skin of SSc patients was assessed by transmission electron microscopy. SSc skin fibroblasts and human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic (1% O2) conditions with 2-ME or autophagy inhibitor. Collagen I and connective tissue growth factor (CTGF) in fibroblasts and vascular endothelial (VE)-cadherin, CD31, vimentin and α-smooth muscle actin (α-SMA) in HUVECs were examined by western blotting. Autophagic markers were evaluated by confocal microscopy and immunofluorescence.

Results: SSc skins presented increased autolysosomes, LC3-II, collagen I and CTGF. Hypoxia-challenged fibroblasts and HUVECs formed more autophagosomes and autolysosomes, with increased LC3 and decreased P62. Meanwhile, hypoxia increased collagen I and CTGF in fibroblasts and increased vimentin and α-SMA but decreased VE-cadherin and CD31 in HUVECs. Bafilomycin A1 increased LC3-II and P62 in fibroblasts and HUVECs and decreased collagen I and CTGF in fibroblasts and vimentin and α-SMA in HUVECs, while upregulating VE-cadherin and CD31. 3-methyladenine decreased autophagy and fibrosis in fibroblasts and endothelial-to-mesenchymal transition in HUVECs. 2-ME-treated HUVECs showed more autophagosomes and fewer autolysosomes while 2-ME-treated fibroblasts showed fewer of both. Moreover, 2-ME decreased LC3-II and increased P62 in fibroblasts and increased both in HUVECs. Inhibition of autophagy by 2-ME showed the same effect with bafilomycin A1 on fibroblast collagen synthesis as well as endothelial and mesenchymal markers in HUVECs.

Conclusion: Autophagy mediated hypoxia-induced fibroblast collagen synthesis and endoMT in SSc, which could be reversed by 2-ME.

Keywords: 2-methoxyestradiol; autophagy; collagen; endothelial-to-mesenchymal transition; hypoxia; systemic sclerosis.