Abstract 559: Machinery for Vitamin D Metabolism in Porcine Adipose--Derived Mesenchymal Stem Cells
Background: There is increasing evidence that vitamin D deficiency is an independent risk factor for cardiovascular disease. Cell-based therapy using adipose derived mesenchymal stem cells (MSCs) is an attractive option for endothelial layer regeneration post-angioplasty procedures. Vitamin D levels in circulating blood might regulate MSC based re-endothelialization of injured arteries. To our knowledge, the presence of vitamin D machinery in porcine MSCs is not yet reported. In this study, we investigated whether or not MSCs possess vitamin D machinery, including the expression levels of vitamin D receptor (VDR), vitamin D metabolizing enzymes (CYP24A1 and CYP27B1) after in vitro stimulation with active vitamin D, calcitriol.
Methods and Results: MSCs isolated from porcine adipose tissue were characterized by positive staining for MSC markers CD44, CD73, CD90, and negative staining for macrophage marker CD11b and hematopoietic stem cell markers, CD34 and CD45; and by tri-lineage differentiation to osteocytes, chondrocytes, and adipocytes. No cytotoxicity was observed when MSCs were stimulated with 0.1-10nM calcitriol. The MSCs were analyzed for mRNA and protein expression of CYP24A1, CYP27B1 and VDR by immunostaining, qPCR and ELISA (n=6). A significant increase (p<0.01) in mRNA transcripts of CYP24A1, CYP27B1 and VDR was observed after stimulation of MSCs with calcitriol (10nM). The in vitro time-dependent effect of calcitriol (10nM) on the vitamin D machinery on cultured MSCs was determined by qPCR after stimulation for 0, 3, 6, 12, and 24h. The VDR and CYP27B1 expression peaked at 3h, and CYP24A1 at 24h, respectively. The in vitro biosynthesis of 1,25(OH)2D3 by ADMSCs was analyzed by ELISA and Western blot. The expression of the active form of vitamin D was significantly decreased (p<0.01) upon inhibition of CYP enzymes.
Conclusion: Porcine MSCs possess vitamin D hydrolases and VDR to metabolize and respond to vitamin D. Hence, in vivo circulating 25-hydroxy vitamin D levels may have a significant role in regulating the differentiation of adipose MSCs into endothelial cells, and thus could be helpful in stem cell based re-endothelialization post-angioplasty.
Author Disclosures: Y. Llamas: None. D. Pankajakshan: None. D.K. Agrawal: None.
- © 2014 by American Heart Association, Inc.