Effect of exosomes secreted by human umbilical mesenchymal stem cells on high glucose-induced human umbilical vascular endothelial cells

Abstract

Objective To explore the effect of exosomes secreted by human umbilical mesenchymal stem cells (hUMSCs)on high glucose-induced human umbilical vascular endothelial cells (hUVECs). Methods hUMSCs and hUVECs were primary cultured by enzyme digestion method and the surface markers of P3 generation was detected by using flow cytometry (FCM). The serum-free medium of hUMSCs was collected to extract exosomes and then observed their ultrastructure was observed under an electron microscope and the surface markers were detected by using FCM again.The P3 generation hUVECs were divided into 4 groups in vitro: high glucose (20 mmol/L) group, exosomes with high glucose (20 mmol/L) group, miR-22 inhibitors (50 nmol/L) with high glucose (20 mmol/L) group, and a control group.After 24 hours′ incubation, Annexin V/PI staining method and FCM assay was used to evaluate cell apoptosis, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) and Transwell assay were used to detect the ability of cell proliferation as well as migration and tube formation assay to determine endothelial cell functions. Results Surface markers like CD105, CD73, CD90 had a high expression rate on hUMSCs while others like CD309, CD31, CD34 had a relatively high expression rate on hUVECs.Besides, CD63, CD105, CD73, CD90 and CD44 were positive in the exosomes, which suggested that separately-cultured hUMSCs and hUVECs had specificity phenotypic characteristics of mesen-chymal stem cells(MSCs) and endothelial cells(ECSs). Ball-like membranous structures with uneven sizes were seen in the exosomes under the scanning electron microscope.Cell viability in the control group, high glucose group, miR-22 inhibitors with high glucose group, exosomes with high glucose group were(99.0±1.6)%, (75.0±2.2)%, (66.0±1.8)%, (95.0±2.6)%, respectively, by MTT method.Compared with the control group, high glucose group and miR- 22 inhibitors with high glucose group cell proliferation were significantly decreased, the difference was statistically significant(t=28.271, P 0.05). Cell apoptosis rate in the control group, high glucose group, miR-22 inhibitors with high glucose group, exosomes with high glucose group were 16.44%, 52.67%, 45.92%, 17.58%, respectively, by FCM.Compared with the control group, high glucose group and miR- 22 inhibitors with high glucose group cell apoptosis rate were significantly higher, the difference was statistically significant(t=-13.756, -16.489, all P 0.05). Cell migration in the control group, high glucose group, miR-22 inhibitors with high glucose group, exosomes with high glucose group were 834.5 number, 295.8 number, 252.2 number, 779.5 number, respectively, by Transwell assay.Compared with the control group, high glucose group and miR- 22 inhibitors with high glucose group cell migration significantly reduced, the difference was statistically significant(t=18.280, 27.266, all P 0.05). Tube formation number in the control group, high glucose group, miR-22 inhibitors with high glucose group, exosomes with high glucose group were 29.8 number, 13.3 number, 11.3 number, 29.6 number, respectively, by tube formation assay.Compared with the control group, high glucose group and miR- 22 inhibitors with high glucose group tube formation number significantly reduced, the difference was statistically significant(t=9.030, P 0.05). Conclusions Exosomes of hUMSCs do have a protective effect on the injured hUVECs induced by high glucose, which can inhibit the apoptosis of hUVECs, promote their proliferation as well as keep their migration and tube formation functions.This study has provided an experimental basis for a novel therapeutic target of diabetic microvascular lesions. Key words: Human umbilical mesenchymal stem cells; Human umbilical vascular endothelial cells; Exosomes; High glucose; Apoptosis