Abstract
BackgroundNon-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.MethodsRat 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.ResultsMinicircle 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 P-eNOS 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.ConclusionsA 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.
Highlights
Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase may enhance production of nitric oxide and promote angiogenesis
Tri-lineage differentiation of the rat bone marrow-derived mesenchymal stem cell (rBMSC) was performed in the appropriate media to osteoblasts as demonstrated by Alzarin Red S staining of mineralised extracellular matrix (Fig. 1b), to chondrocytes by Alcian Blue staining of proteoglycans in three-dimensional pellet cultures (Fig. 1c) and to adipocytes as shown by Oil Red O staining of lipid vesicles (Fig. 1d)
MC-endothelial nitric oxide synthase (eNOS) gene transfer to rBMSCs induces endothelial CD31 gene expression We found a significant increase in CD31 mRNA expression by 0.42-fold in portion from the final construct (P-eNOS) transfected cells compared to P-green fluorescent protein (GFP), Minicircle vectors expressing GFP (MC-GFP) and un-transfected control, suggesting that eNOS gene transfer may promote endothelial differentiation of rBMSCs (Fig. 8)
Summary
Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Changes in eukaryotic gene expression may be altered due to the antibiotic resistance marker and immune responses to bacterial CpG sequences [1] These prokaryotic DNA sequences present in pDNA vectors may lower their biocompatibility and safety. During the intracellular trafficking of pDNA, the bacterial sequences of pDNA vectors are rapidly associated with histone proteins, packing the sequences into a dense heterochromatin structure If these are spread into the adjacent transgene in the vector, the sequences can become inaccessible by transcription factors, leading to reduced transgene expression through silencing of the eukaryotic promoter [5]. The removal of CpG islands by cloning out, or elimination of non-essential sequences, can reduce these undesirable responses but is timeconsuming and tedious
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