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

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.

Molecular control of nitric oxide synthesis through eNOS and caveolin-1 interaction regulates osteogenic differentiation of adipose-derived stem cells by modulation of Wnt/β-catenin signaling

“…It has been reported that both iNOS and eNOS play a role in osteogenesis of embryonic stem cells [57]. We [4] and others [58] have shown that MSCs do not express eNOS. Therefore, in order to investigate the role of eNOS in osteogenic differentiation of eASCs, in this study eASCs were genetically modified by lentiviral vector-based eNOS.…”

“…To construct the CMV promoter-driven eNOS expressing lentiviral vector, a codon optimized eNOS gene was synthesized [4] and subcloned into the pWPT-GFP lentiviral plasmid (Addgene, MA, USA) using Bam H1 and Sal 1 restriction endonucleases. Doxycycline (DOX) inducible eNOS construct was prepared by amplifying the eNOS gene using the forward (ATCA GAATTC ATGGGCAACCTGAA) and reverse primer (ATCA GAATTC TCATCAGGGGCTGT) by introducing Eco R1 restriction sites (underlined sequences in both forward and reverse primers) at both the 5’ and 3’ ends of the final PCR product, followed by subcloning the PCR product into FUW-TetO vector (Addgene).…”

“…Mesenchymal stem cells (MSCs) have been isolated from various tissues such as adipose [1], heart [2], bone marrow [3, 4], and blood [5–9], and have the potential to differentiate into different lineages, including osteoblasts, chondrocytes, and adipocytes [10, 11]. The osteoblast differentiation program of MSCs is switched on by cell recruitment, and timely expression of genes including Runx2, alkaline phosphatase (ALP), type I collagen (ColA1), and osteocalcin (OC) followed by extracellular matrix mineralization [12].…”

“…It can react within the cell where it is produced or penetrate cell membranes to affect adjacent cells [21]. NO exerts a variety of physiological effects such as regulating blood pressure via smooth muscle relaxation [22], mediating immune responses [23], controlling cell proliferation [24], modulating apoptosis [20], promoting growth factor-induced angiogenesis [4, 21], accelerating wound healing [4, 25], and functioning as a neurotransmitter [26]. These responses can be mediated through activating the primary NO effector soluble guanylyl cyclase to produce cGMP [27] by NO-based chemical modifications of proteins through S-nitrosylation [28] or through epigenetic modification [29].…”

Into the eyes of bone marrow-derived mesenchymal stem cells therapy for myocardial infarction and other diseases

“…Firstly, no standard method has been putted forward about isolation, purification and specific marker molecules for the identification of BM-MSCs. Secondly, the efficiency of BM-MSCs differentiation is not ideal, which is currently one of the research focus on how to induce BM-MSCs to differentiate to the single specific tissue and cells (46)(47)(48)(49). Thirdly, the signal transduction mechanism and the molecular basis of BM-MSCs' differentiation such as which transcription factors or gene were activated to make it for some specific differentiation is still not clear though it is generally considered to be related to reprogramming of BM-MSCs.…”