Mesenchymal Stem Cells Ameliorated Glucolipotoxicity in HUVECs through TSG-6.

Jingping Liu,Yanrong Lu,Younan Chen,Zuyao Ni,Yujia Yuan,Ai Luo,Bo Chen,Lan Li,Dan Long,Xingxing An,Meimei Shi,Jingqiu Cheng

doi:10.3390/ijms17040483

Abstract

Glucolipotoxicity is one of the critical causal factors of diabetic complications. Whether mesenchymal stem cells (MSCs) have effects on glucolipotoxicity in human umbilical vein endothelial cells (HUVECs) and mechanisms involved are unclear. Thirty mM glucose plus 100 μM palmitic acid was used to induce glucolipotoxicity in HUVECs. MSCs and HUVECs were co-cultured at the ratio of 1:5 via Transwell system. The mRNA expressions of inflammatory factors were detected by RT-qPCR. The productions of reactive oxygen species (ROS), cell cycle and apoptosis were analyzed by flow cytometry. The tumor necrosis factor-α stimulated protein 6 (TSG-6) was knockdown in MSCs by RNA interference. High glucose and palmitic acid remarkably impaired cell viability and tube formation capacity, as well as increased the mRNA expression of inflammatory factors, ROS levels, and cell apoptosis in HUVECs. MSC co-cultivation ameliorated these detrimental effects in HUVECs, but no effect on ROS production. Moreover, TSG-6 was dramatically up-regulated by high glucose and fatty acid stimulation in both MSCs and HUVECs. TSG-6 knockdown partially abolished the protection mediated by MSCs. MSCs had protective effects on high glucose and palmitic acid induced glucolipotoxicity in HUVECs, and TSG-6 secreted by MSCs was likely to play an important role in this process.

Highlights

Diabetes is one of the major chronic diseases that is alarmingly increasing burdens for public health around the world
The results suggested that glucose combined with palmitic acid (100 and 200 μM) showed the synergistic effect to inhibit the cell viability in human umbilical vein endothelial cells (HUVECs) (Figure 1A)
Concomitant incubation of HUVECs and mesenchymal stem cells (MSCs) via a Transwell system showed that MSCs exerted protection of glucolipotoxicity in HUVECs by improving cell viability, cell proliferation, and tube formation, and repressing cell apoptosis and inflammation

Summary

Introduction

Diabetes is one of the major chronic diseases that is alarmingly increasing burdens for public health around the world. Obesity is a main cause of type 2 diabetes (T2DM), not all the obese patients will develop T2DM in their life time Based on this phenomena, the theory of “glucolipotoxicity” is proposed, that means glucotoxicity and lipotoxicity are interacted rather than separate adverse forces on the metabolic cells and tissues. Paracrine of a broad range of trophic factors or immune regulators has been considered as the primary mechanism of MSCs mediated protective effects observed in animal models of diabetic nephropathy, peripheral arterial diseases and ischemia, highlighting their capability to promote vascular regeneration [13]. We were aiming to explore the protective effects of MSCs on high glucose and high palmitic acid induced glucolipotoxicity in human umbilical vein endothelial cells (HUVECs), and reveal the relevant molecular mechanisms. Given that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) plays an important role in protection of inflammation, we used siRNA targeting TSG-6 in MSCs to investigate the role of TSG-6 in MSCs mediated amelioration of glucolipotoxicity in endothelial dysfunction

Results

MSCs Improved Capillary-Like Tube Formation Ability

The Effects of TSG-6 Knockdown on MSCs and MSCs Co-Cultured HUVECs

Discussion

Materials

Cell Cultures

Assessment of Cell Viability

Total RNA Extraction and Real-Time qPCR

Immunofluorescence Imaging

Endothelial Cell Capillary-Like Tube Formation Assay

Western Blot Analysis