Regulatory Mechanisms Underlying the Differentiation of Mesenchymal Stem Cells to Endothelial Cells
Ikhapoh, Izuagie P. A.
Cardiovascular disease is the foremost cause of mortality in the United States. Myocardial infarction often results from atherosclerotic occlusion of coronary arteries. Coronary angioplasty is performed to restore cardiac blood flow and function. Angioplasty is the most widely used interventional technique for coronary occlusion events. However, long term benefits are curtailed by vascular endothelium injury. Endothelial denudation often leads to the processes of thrombosis, inflammation, and restenosis that subject patients to reoccurrence of myocardial infarction. The current clinical therapies prescribed to patients with coronary artery disease are a combination of drugs used to control blood pressure, lipid levels and glucose levels. Unfortunately, none of these treatment regimens are designed to repair or replace damaged endothelium. |Bone marrow (BM)-derived mesenchymal stem cells (MSCs) can differentiate into endothelial cells (ECs) in the presence of vascular endothelial growth factor (VEGF) — 165 in vitro. Translational models have demonstrated that differentiated BM-derived MSCs can be transplanted into the site of angioplasty-induced endothelial injury. However, re-endothelialization by VEGF-induced BM-derived MSCs remains a complex clinical challenge. Following intracoronary or transendocardial delivery of the BM-derived MSCs, only ~10% of the differentiating cells engraft, and the majority of those persist as immature ECs. Currently, the mechanisms underlying EC-engraftment and re-endothelialization are not well understood. In patients undergoing coronary intervention, the mechanical process involved in performing angioplasty itself is known to stimulate atherogenesis. The goal of this study is to decipher a potential role of angioplasty-induced atherogenic mediators on the differentiation of BM-derived MSCs into ECs. The central hypothesis of these studies is that pro-atherogenic mediators, which are triggered after an angioplasty, inhibit VEGF-165-induced differentiation of BM-derived MSCs into ECs. |In this study, immunofluorescence was used to examine the expression levels of atherogenic mediators in left circumflex artery (LCX) samples from microswine. Microswine designated for angioplasty were maintained on a high cholesterol diet. After six months, the animals were subjected to angioplasty. Control microswine maintained on the same high cholesterol diet received no angioplasty. Atherogenic mediators interleukin (IL)-6, tumor necrosis factor (TNF) α, and Angiotensin (Ang) II were discovered to be upregulated at the interventional site. Subsequent experiments were performed to decipher the molecular mechanism governing VEGF-induced differentiation. Fluorescence activated cell sorting (FACS) analysis revealed that naïve BM-derived MSCs cultured in differentiation media containing VEGF demonstrated increased expression of EC-specific markers—Von Willebrand factor(vWF), platelet endothelial cell adhesion molecule (PECAM)-1, and vascular endothelial(VE)-cadherin), VEGF R2, Sox18, and enhanced endothelial tube formation. siRNA-mediated-knockdown of Sox18 inhibited EC differentiation whereas Sox18 overexpression induced VEGF R2 expression. Lastly, the inhibitory effects of the identified atherogenic mediators on VEGF-induced differentiation were examined via polymerase chain reaction (PCR) and western blot analyses. The results demonstrate that in comparison to VEGF alone, VEGF in combination with Ang II supported a robust increase in EC-specific marker expression and endothelial tube formation. VEGF-mediated EC differentiation was markedly suppressed in the presence of IL-6 and/or TNFα. Addition of Ang II to VEGF and IL-6 or TNFα was sufficient to rescue induction of the EC phenotype. Thus, Ang II promotes but IL-6 and/or TNFα inhibit VEGF-induced differentiation of BM-derived MSCs into ECs. Here, I demonstrate that VEGF mediates BM-derived MSC differentiation into EC through the induction of Sox18, a transcription factor that promotes EC differentiation. |These results suggest that the local production of atherogenic mediators has a large effect on EC phenotype. These findings have important clinical translational implications for cell-based therapies designed to re-endothelialize coronary arteries following intervention. Treatments such as anti-inflammatory drugs may be useful in promoting re-endothelialization with BM-derived MSC transplant in coronary arteries.
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