Lentiviral vector-mediated co-overexpression of VEGF and Bcl-2 improves mesenchymal stem cell survival and enhances paracrine effects in vitro

Abstract

Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for ischemic heart disease; however, the low survival rate of transplanted cells limits their therapeutic efficacy. The aim of this study was to investigate whether the dual genetic modification of vascular endothelial growth factor (VEGF) and B-cell lymphoma-2 (Bcl-2) confers a higher expression level of the target genes, better survival and a stronger paracrine effect in MSCs in an adverse environment than the modification of the individual genes. For this purpse, a lentiviral vector was constructed by using a self-cleaving T2A peptide sequence to link and achieve the co-overexpression of VEGF and Bcl-2. Rat MSCs were transfected to obtain cell lines that exhibited a stable overexpression. An in vitro model of oxygen glucose deprivation (OGD) was applied to mimic the ischemic microenvironment, and cell apoptosis, autophagy and the paracrine effects were then determined. Compared with the MSCs in which individual genes were modified and the control MSCs, the MSCs which were subjected to dual genetic modification had a higher expression level of the target genes, a more rapid proliferation, reduced apoptosis, decreased autophagy and an enhanced paracrine effect. Furthermore, the suppression of autophagy was found to contribute to the inhibition of apoptosis in this in vitro OGD model. On the whole, these data indicate that the co-overexpression of VEGF and Bcl-2 protects MSCs in an ischemic environment by inhibiting apoptosis, suppressing autophagy and enhancing the paracrine effects.

Bcl-2 Overexpression and Hypoxia Synergistically Enhance Angiogenic Properties of Dental Pulp Stem Cells

“…We demonstrated that Bcl-2 overexpression could enhance the survival of DPSCs by reducing the apoptosis both under in vitro serum-free conditions and following in vivo implantation. Our findings are in accordance with previous studies that demonstrated Bcl-2 overexpression promotes the survival by decreasing apoptosis in the setting of hypoxia induced stress in tumors [ 18 , 30 , 31 ] or mesenchymal stem cells [ 21 ].…”

“…DPSCs are considered a promising cell source with a therapeutic potential in dental pulp regeneration and other tissue engineering approaches. Inadequate post-implantation cell survival and timely vascularization are two major barriers that need to overcome in utilizing these cells in regenerative strategies [ 3 , 21 , 28 ]. In the current study, for the first time, we demonstrated that Bcl-2 overexpression significantly enhances the post-implantation cell viability and VEGF mediated angiogenic and vasculogenic potential of DPSCs.…”

“…It is localized at the outer membrane of mitochondria, where it plays a significant role in maintaining cell viability and inhibiting the actions of pro-apoptotic proteins. Subsequent studies demonstrated its effects on other aspects including cell differentiation, growth and angiogenesis [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Recombinant overexpression of the Bcl-2 protein has been demonstrated as a strategy to treat diseases associated with increased apoptosis such as cardiomyocyte death [ 15 ] and inherited cardiomyopathy [ 17 , 22 ].…”

Stem cell-based therapies for ischemic stroke: a systematic review and meta-analysis of clinical trials

“…Strategies to enhance the endurance and efficacy of grafted stem cells in ischemic conditions include treating with growth factors, pharmacological agents, ischemia/hypoxia, or electrical stimulation and have increased paracrine potentials [56][57][58]. Stem cells modified with exogenous growth factor genes such as vascular endothelial growth factor; brainderived neurotrophic factor by viral or non-viral delivery system can significantly increase the paracrine effects and decrease the mortality of mice [59,60]. Implantation of modified bone marrow-derived mesenchymal stem cells (SB623) transiently transfected with the human Notch-1 intracellular domain in a patient with stable chronic stroke is safe and is accompanied by improvements in clinical outcomes [61].…”

Improved therapeutics of modified mesenchymal stem cells: an update

“…Upon post transplantation analysis, these MSCs were associated with greatly improved heart function characterized by less cardiomyocyte apoptosis, improved cardiomyocytes proliferation and enhanced angiogenesis [54]. Other MSCs modifications known to enhance their survival include but not limited to overexpression of Gremlin1 [61] and protein kinase Cɛ [62], co-overexpression of Bcl-2 and VEGF [63], upregulation of TrkB [64], inhibition of mircoRNA-34a [65], Cripto stimulation [66], and tumor necrosis factor receptor (TNFR) gene transfection [67]. For instance, Bao and colleagues investigated the enhancement of MSCs survival by transfecting the cells with TNFR gene, causing overexpression of TNFR and the binding of TNF-alpha.…”