Abstracts Category : Other

Matrix Metalloproteinases Regulate The Differentiation Of Adipose-Derived Mesenchymal Stem Cells Into Endothelial Cells

by Sami G Almalki

Mesenchymal stem cells (MSCs) have a great potential as a source of cells for cell-based therapy because of their ability for self-renewal and differentiation into functional cells. Cell-based therapy that can rejuvenate the endothelium with stimulated adipose-derived MSCs (AMSCs) is a promising therapeutic strategy for re-endothelialization at the site of intravascular stenting to prevent restenosis. Matrix metalloproteinases (MMPs) have critical role in the differentiation of MSCs to different lineages. Identifying the molecular factors and mechanisms that regulate AMSCs differentiation to endothelial cells (ECs) is important in the promotion of a greater understanding of these highly useful cells. The goal of this study was to examine the regulatory roles of MMPs, and the underlying signaling pathways that promote the differentiation of AMSCs to ECs in vitro.|AMSCs were isolated from porcine abdominal adipose tissue, and characterized by immunostaining, flow cytometry and multi-lineage differentiation. The mRNA transcripts of different MMPs and TIMPs, enzyme activity and protein expression were analyzed by gelatin zymography, ELISA, and Western blot. Flow cytometry was performed to examine the expression of EC markers. Angiogenesis assay and acetylated-LDL uptake were performed to examine EC functionality of differentiated AMSCs.|Isolated AMSCs were characterized by positive staining for MSC markers, CD29, CD44 and CD90, and negative staining for CD11b and CD45. The plasticity of AMSCs was detected by multi-lineage differentiation. After 10 days of stimulation for EC differentiation, the morphology of AMSCs changed to a round-shaped morphology similar to that of ECs. The mRNA transcripts and protein expression of MMP-2 and MMP-14 were significantly increased during the differentiation of AMSCs into ECs. Interestingly, siRNA silencing of MMP-2 and MMP-14 showed significant increase in the expression of endothelial cell markers, formation of capillary tubes, acetylated-LDL uptake and significant decrease in the number of migrated cells. To investigate the underlying mechanism of this effect, immunostaining of VEGFR2 was performed with and without MMP-2 and MMP-14 siRNAs. Findings revealed that MMP-2 and MMP-14 knockdown decreased the cleavage of VEGFR2, and inhibition of VEGFR2 significantly decreased the expression of EC markers. However, siRNA knockdown of ATR2 neither affected the expression of EC markers nor the number of migrated cells. Silencing of MMP-2 and MMP-14 significantly increased p-ERK, and decreased p-JNK with no apparent change in p-p38. Moreover, inhibition of ERK resulted in significant decrease in the expression of EC markers. The VEGFR2 kinase inhibitor induced a dose-dependent inhibition of ERK during EC differentiation of AMSCs.|The findings of this study, for the first time, demonstrate that up-regulation of MMP-2 and MMP-14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibits the cleavage of VEGFR2 and induces the differentiation process.Advisors/Committee Members: Agrawal, Devendra K (advisor).