Abstract
Cell-based therapy that can rejuvenate the endothelium with stimulated adipose-derived mesenchymal stem cells (AMSCs) is a promising therapeutic strategy for the re-endothelialization of denuded arteries at the stenting site. Previously, we have shown that silencing of MMP-2 and MMP-14 inhibits vascular endothelial growth factor receptor type 2 (VEGFR2) cleavage, and induces differentiation of AMSCs toward the endothelial cell (EC) lineage. In this study, we examined the underlying signaling pathways that regulate differentiation of AMSCs to ECs in vitro through VEGFR2.
AMSCs were isolated from porcine abdominal adipose tissue. The isolated AMSCs were characterized by positive expression of CD29, CD44, and CD90 and negative expression of CD11b and CD45. The isolated MSCs were transfected with siRNA to silence MMP-2, MMP-14, and angiotensin receptor 2 (ATR2). Cells were suspended either in endothelial basal media (EBM) or endothelial growth media (EGM) with various treatments. Flow cytometry was performed to examine the expression of EC markers, and western blot analysis was performed to examine the expression and activity of various kinases. Scratch assay was performed to examine the cell migration. Data were analyzed by ANOVA using PRISM GraphPad.
After 10 days of stimulation for EC differentiation, the morphology of AMSCs changed to a morphology similar to that of ECs. Silencing MMP-2 and MMP-14 resulted in significant decrease in the number of migrated cells compared with the EGM-only group. ATR2 siRNA transfection did not affect the migration and differentiation of AMSCs to ECs. Stimulation of AMSCs for EC differentiation with or without MMP-2 or MMP-14 siRNA resulted in significant increase in p-ERK, and significant decrease in p-JNK. There was no significant change in p-p38 in all three groups compared with the EBM group. ERK inhibition resulted in significant decrease in the expression of EC markers in the EGM, EGM + MMP-2 siRNA, and EGM + MMP-14 siRNA groups. The VEGFR2 kinase inhibitor induced a dose-dependent inhibition of ERK.
The ERK signaling pathway is critical for VEGF-A/VEGFR2-induced differentiation of AMSCs into ECs. These findings provide new insights into the role of the ERK signaling pathway in AMSC differentiation to ECs for potential clinical use in cardiovascular diseases.