title: Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.
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 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.
  
  
  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.
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.|O'Brien Institute Department, St. Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia.
authors.forename: Nadeeka
authors.initials: N
authors.lastname: Bandara
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.
authors.forename: Saliya
authors.initials: S
authors.lastname: Gurusinghe
authors.affiliation: Central laboratory and key Laboratory of Oral and Maxillofacial-Head and Neck Medical Biology, Liaocheng People's Hospital, Liaocheng, 252000, PR China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Chen
authors.affiliation: Central laboratory and key Laboratory of Oral and Maxillofacial-Head and Neck Medical Biology, Liaocheng People's Hospital, Liaocheng, 252000, PR China.
authors.forename: Shuangfeng
authors.initials: S
authors.lastname: Chen
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.|Central laboratory and key Laboratory of Oral and Maxillofacial-Head and Neck Medical Biology, Liaocheng People's Hospital, Liaocheng, 252000, PR China.
authors.forename: Le-Xin
authors.initials: LX
authors.lastname: Wang
authors.affiliation: O'Brien Institute Department, St. Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia.|Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, VIC, 3002, Australia.
authors.forename: Shiang Y
authors.initials: SY
authors.lastname: Lim
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia. pstrappe@csu.edu.au.
authors.forename: Padraig
authors.initials: P
authors.lastname: Strappe
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002465:Cell Movement; D002478:Cells, Cultured; D004270:DNA, Circular / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis* / Q000235:genetics; D017207:Rats, Sprague-Dawley; D014162:Transfection
chemical_list: D004270:DNA, Circular; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III
keywords: Angiogenesis; DNA vector; Endothelial nitric oxide synthase; Mesenchymal stem cells; Minicircle; Nitric oxide; Transfection