21: Mesenchymal Stem Cells Alleviated Palmitic Acid Induced Endothelial to Mesenchymal Transition in HUVEC by Secreting STC1

Abstract

Background: Endothelial dysfunction (ED) is an early indicator of vascular complications in type 2 diabetes. Hyperlipidemia is believed to be a key causal factor promoting ED. Our previous study showed that mesenchymal stem cells (MSCs) were able to ameliorate palmitic acid (PA) induced ED, but the mechanisms involved are unclear. Endothelial-to-mesenchymal transition (EndMT) is demonstrated mechanistically related to ED in a variety of vascular diseases. Here we explored the effects of MSCs on PA induced EndMT and disclosed the mechanism in human umbilical vein endothelial cells (HUVECs). Methods: The primary HUVECs were treated by PA (50 μM) with or without human umbilical cord derived MSCs (UC-MSCs) for 24 h in Transwell system at a ratio of 5:1. EndMT related proteins expression were evaluated by western blot and immunofluorescence. The secretion of stanniocalcin 1 (STC1) was quantified using an ELISA kit. Recombinant STC1 (rSTC1) and siRNA targeting STC1 were used to evaluate the role of STC1 in MSCs mediated protective effects. Result: After PA treatment, the cell morphology of HUVECs changed from an endothelial-typical cobblestone-like appearance to a mesenchymal-typical spindle-shaped pattern, suggesting the occurrence of EndMT. However, coculture with MSCs alleviated the morphological change of HUVECs (Fig.1A). Western blot analysis revealed that PA significantly decreased endothelial marker CD31 and CD144 expression and increased fibroblast markers vimentin and α-SMA expression, which could be partly reversed by coculture with MSCs (Fig.B). Consistently, immunofluorescent staining for CD31 and α-SMA revealed a significant loss of CD31 and enhancement of α-SMA in PA group in comparison of control. However, MSCs effectively antagonized PA induced toxicity by increasing CD31 and decreasing α-SMA expression in HUVECs (Fig.1C). Next, we explored the underlying mechanisms focusing on the secretome of MSCs. We found that resting MSCs secreted more STC1 than HUVECs did, while, PA (50 μM) strongly stimulated MSCs to synthesize and release much more STC1 (Fig.2A). Furthermore, the cell viability of HUVECs declined to 49% of control after PA stimulation. However, the addition of rSTC1 showed similar protective effect of MSCs, and restored the cell viability to 89% (Fig.2B). rSTC1 also reversed the expression switch of EndMT- related proteins (Fig.2C). Additionally, silencing of STC1 gene by siRNA in MSCs abrogated above protective effects suggesting the critical role of STC1 in MSCs (Fig.2D-E). Conclusion: MSCs ameliorated PA-induced EndMT in HUVECs, in which the STC1 secreted by MSCs has played a critical role. National Natural Science Foundation of China (grant number 31571474 and 81370824).