Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells

Nadeeka Bandara, Saliya Gurusinghe, Haiying Chen, Shuangfeng Chen, Le-xin Wang, Shiang Y. Lim, Padraig Strappe

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Abstract

Background: Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS)
may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells
is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and
angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic
modification to express eNOS may promote NO production and subsequent cellular responses.
Methods: Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector
expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and
NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of
angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase
chain reaction.
Results: Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were
compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells
(55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC
(15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and PeNOS
gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRα and FGFR2, respectively.
Conclusions: A novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of
CD31, angiogenic growth factor, and receptor gene expression.
Original languageEnglish
Article number48
Pages (from-to)1-15
Number of pages15
JournalStem Cell Research and Therapy
Volume7
DOIs
Publication statusPublished - 2016

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