Abstract
Cardiovascular disease is the leading cause of mortality and morbidity in developed countries. The main contributing factor to vascular disease is the formation of atherosclerotic plaques. The vascular endothelium is an important barrier against atherosclerosis. Endothelial dysfunction or endothelium loss promotes atherosclerosis progression and stenosis. Furthermore, endothelium loss permits restenosis to occur following interventional procedure. The human body’s stem cells serve as an endogenous repair mechanism for damaged endothelium. However, this repair system is not entirely effective, and vascular disease is still a major health problem. Over the past decade, stem cell therapy for treatment of cardiovascular disease has opened new perspectives on cardiac tissue repair with bone marrow-derived mesenchymal stem cells (BM-MSCs) being the most frequently used source. |Adipose-derived mesenchymal stem cells (ADMSC) are promising candidates for endothelium regeneration because of their superior proliferative capacity compared to bone-marrow-derived MSCs. However, most of the in vitro studies using mesenchymal stem cells (MSCs) report low rates of endothelial cell differentiation and low survival rates. The goal of this study was to examine the effects of vitamin D treatment of MSCs on their differentiation and therapeutic potential. |The central hypothesis is that 1,25-Dihydroxyvitamin D [1,25-(OH)2D3] (calcitriol) and VEGF enhance the differentiation of ADMSCs into endothelial cells (ECs) by dampening the Wnt pathway, and these differentiated cells are able to re-endothelialize injured coronary arteries.|In this study, the effect of calcitriol stimulation on the expression of vitamin D receptor (VDR) and vitamin D metabolizing enzymes, which include CYP24A1 and CYP27B1, was examined in MSCs isolated from porcine subcutaneous adipose tissue. Further, the in vitro biosynthesis was also measured following stimulation with calcidiol, (25(OH)D3). Next, ADMSCs were stimulated and differentiated into ECs with endothelial growth media and vascular endothelial growth factor (EGM and VEGF) and EGM media containing calcitriol (EGM and VEGF w/calcitriol) for 10 days. The expression of EC markers, platelet endothelial cell adhesion molecule (PECAM-1) and vascular endothelial cadherin (VE-cadherin), along with proteins in the Wnt pathway was examined after 10 days of differentiation using PCR. Also, EC function was examined using angiogenesis and acetylated low density lipoprotein (LDL) uptake assays after 10 days of differentiation. β-catenin and Kremen1 are two main mediators in the Wnt pathway. The effects of silencing β-catenin and Kremen1 on ADMSC differentiation, EC marker expression and EC function were assessed. Finally, the ability of differentiated ADMSCs to adhere on the luminal surface of a denuded coronary artery was examined ex vivo using a bioreactor system.|ADMSC express VDR and vitamin D metabolizing enzymes and possess the capability of converting inactive vitamin D to the active form of vitamin D. Treatment of ADMSCs with calcitriol significantly increased mRNA and protein levels of VDR and CYP24A1 while CYP27B1 was significantly decreased compared to control. Calcidiol treatment significantly increased the amount of calcitriol in the ADMSC and the use of CYP inhibitor, Ketoconazol, significantly reduced the amount of calcitriol that was produced. Calcitriol enhanced EGM and VEGF-induced differentiation of ADMSCs into ECs, as revealed by an increase in mRNA and protein expression of EC markers. Assessment of endothelial functionality showed significant increases in capillary tube sprouting, and LDL uptake by differentiated cells in response to EGM and VEGF with calcitriol. Findings from Wnt Pathway array revealed a decrease in β-catenin and an increase in Kremen1 protein in the cells treated with EGM and VEGF with calcitriol. Silencing of β-catenin resulted in a significant increase in the expression of EC markers, formation of capillary tubes, and LDL uptake. Silencing of Kremen1 had the opposite effect. The ex vivo study demonstrated that ADMSCs differentiated with VEGF and calcitriol or VEGF alone had the ability to adhere to an endothelium-denuded vascular graft and restore endothelium-dependent relaxation following a 24-hour incubation period in a bioreactor system. These data suggest that calcitriol works synergistically with VEGF through the Wnt pathway to significantly increase the differentiation of ADMSC into ECs. Thus, priming MSCs with VEGF and calcitriol or downregulating β-catenin can significantly contribute to the differentiation of ADMSCs. This translational study will give us an opportunity to explore inexpensive, new, and safe therapeutic applications of vitamin D-primed MSCs in the treatment of cardiovascular disease, which could provide enhanced cell survival efficiency, endothelial function and inhibition of restenosis, compared to MSC therapy alone.