Abstract
BackgroundWe hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart.Methods and ResultsMSCs from male rats were transduced with adenoviral vector encoding for PKG1α (PKG1αMSCs).Controls included native MSCs (NatMSCs) and MSCs transduced with an empty vector (NullMSCs). PKG1α activity was increased approximately 20, 5 and 16 fold respectively in PKG1αMSCs. PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD) which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in PKG1αMSCs compared to NatMSCs and NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, PKG1αMSCs group showed higher survival compared with NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and NullMSCs group, PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05) and attenuated infarct size (27.2±2.5%, p<0.01). Heart function indices including ejection fraction (52.1±2.2%, p<0.01) and fractional shortening (24.8%±1.3%, p<0.01) were improved significantly in PKG1αMSCs group.ConclusionOverexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the infarcted heart.
Highlights
Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been extensively used to treat cardiovascular ischemic disease due to MSCs’ regenerative potential in both experimental animal model and human [1,2,3,4,5,6,7,8]
Adenovirus transduction was evidenced by GFP expression in NullMSCs and PKG1aMSCs (Fig. 1A)
Compared to controls (NatMSCs and NullMSCs), PKG1aMSCs had a reduced cell damage after 8 h oxygen and glucose deprivation (OGD) shown by low level of lactate dehydrogenase (LDH) release (PKG1aMSCs 13.761.3% vs native MSCs (NatMSCs) 34.661.6% and NullMSCs 29.661.4%, p,0.01) (Fig. 2A), decreased caspase-3/7 activity (p,0.01)(Fig. 2B) and reduced number of Terminal dUTP nick-end labeling (TUNEL) positive cells
Summary
Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been extensively used to treat cardiovascular ischemic disease due to MSCs’ regenerative potential in both experimental animal model and human [1,2,3,4,5,6,7,8]. The Nterminus of PKG1 is encoded by two different exons resulting in the production of two isoforms, PKG1a and PKG1b. Both isoforms are present in heart and vessels [22,23,24]. In a previous study we showed that PDE5a inhibition with adenoviral short hairpin RNA could protect cardiomyocytes against anoxia, attenuate infarction size and improve cardiac remodeling and dysfunction [30]. We hypothesized that overexpression of cGMP-dependent protein kinase type 1a (PKG1a) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart
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