Jian-Ye Yang

VEGF/SDF-1 Promotes Cardiac Stem Cell Mobilization and Myocardial Repair in the Infarcted Heart

AIMS The objective of this study was to investigate whether vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSC) improves myocardial survival and the engraftment of implanted MSC in infarcted hearts and promotes recruitment of stem cells through paracrine release of myocardial stromal cell-derived factor-1α (SDF-1α). METHODS AND RESULTS VEGF-expressing MSC ((VEGF)MSC)-conditioned medium enhanced SDF-1α expression in heart slices and H9C2 cardiomyoblast cells via VEGF and the vascular endothelial growth factor receptor (VEGFR). The (VEGF)MSC-conditioned medium markedly promoted cardiac stem cell (CSC) migration at least in part via the SDF-1α/CXCR4 pathway and involved binding to VEGFR-1 and VEGFR-3. In vivo, (VEGF)MSC-stimulated SDF-1α expression in infarcted hearts resulted in massive mobilization and homing of bone marrow stem cells and CSC. Moreover, VEGF-induced SDF-1α guided the exogenously introduced CSC in the atrioventricular groove to migrate to the infarcted area, leading to a reduction in infarct size. Functional studies showed that (VEGF)MSC transplantation stimulated extensive angiomyogenesis in infarcted hearts as indicated by the expression of cardiac troponin T, CD31, and von Willebrand factor and improved the left ventricular performance, whereas blockade of SDF-1α or its receptor by RNAi or antagonist significantly diminished the beneficial effects of (VEGF)MSC. CONCLUSION Exogenously expressed VEGF promotes myocardial repair at least in part through SDF-1α/CXCR4-mediated recruitment of CSC.

Mesenchymal stem cells over-expressing SDF-1 promote angiogenesis and improve heart function in experimental myocardial infarction in rats.

BACKGROUND In addition to its multipotent capability, the mesenchymal stem cell (MSC) can secrete and supply a large amount of vascular endothelial growth factor (VEGF). The stromal-derived factor-1 alpha (SDF-1alpha) plays an important role in the homing of stem cells to the injured tissues of the heart. Therefore, the MSCs over-expressing SDF-1alpha could augment the angiogenesis pathway. METHODS In vitro, the differentiation of the MSCs into endothelial-like cells was induced by cultivation of cells in 10% foetal calf serum and 50 ngml(-1) SDF-1alpha or in specific inhibitors for endothelial nitrous oxide synthase (eNOS). In vivo, the rat model of myocardial infarction was established by occlusion of the left anterior descending coronary artery. Seven days following surgery, 5.0 x 10(9)pfu Ad-SDF-1alpha (adenoviral vector containing human SDF-1alpha gene under the control of the rous sarcoma virus (RSV) promoter), 5.0 x 10(6) Ad-LacZ-MSC or 5.0 x 10(6) Ad-SDF-MSC suspension in a 0.2-ml serum-free medium was injected into four sites in infarcted areas (0.05 ml per site). The rats receiving Ad-SDF-MSC also received the nitrous oxide (NO) synthesis inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) in drinking water (1 mgkg(-1)). The rats in the control group received the same volume of cell-free medium. Four weeks following transplantation, the heart function was assessed, and histological and molecular analyses were conducted. RESULTS The MSCs could differentiate into endothelial cells in the presence of SDF-1alpha, and the effect could be inhibited by l-NAME in vitro and in vivo. Western Blotting revealed an increased expression of VEGF, Akt and eNOS. Four weeks following transplantation, a reduced infarct size and fibrosis, greater vascular density and thicker left ventricular wall were observed in the Ad-SDF-MSC group. The measurement of haemodynamic parameters showed an improvement in the left ventricular performance in the Ad-SDF-MSC group as compared with other groups. CONCLUSION The MSCs over-expressing the SDF-1alpha can produce effective angiogenesis, resulting in the prevention of progressive heart dysfunction after a myocardial infarction.

Vascular endothelial growth factor promotes cardiac stem cell migration via the PI3K/Akt pathway

VEGF is a major inducer of angiogenesis. However, the homing role of VEGF for cardiac stem cells (CSCs) is unclear. In in vitro experiments, CSCs were isolated from the rat hearts, and a cellular migration assay was performed using a 24-well transwell system. VEGF induced CSC migration in a concentration-dependent manner, and SU5416 blocked this. Western blot analysis showed that the phosphorylated Akt was markedly increased in the VEGF-treated CSCs and that inhibition of pAkt activity significantly attenuated the VEGF-induced the migration of CSCs. In in vivo experiments, rat heart myocardial infarction (MI) was induced by left coronary artery ligation. One week after MI, the adenoviral vector expressing hVEGF165 and LacZ genes were injected separately into the infarcted myocardium at four sites before endomyocardial transplantation of 2x10(5) PKH26 labeled CSCs (50 muL) at atrioventricular groove. One week after CSC transplantation, RT-PCR, immunohistochemical staining, Western blot, and ELISA analysis were performed to detect the hVEGF mRNA and protein. The expression of hVEGF mRNA and protein was significantly increased in the infarcted and hVEGF165 transfected rat hearts, accompanied by an enhanced PI3K/Ak activity, a greater accumulation of CSCs in the infarcted region, and an improvement in cardiac function. The CSC accumulation was inhibited by either the VEGF receptor blocker SU5416 or the PI3K/Ak inhibitor wortmannin. VEGF signaling may mediate the migration of CSCs via activation of PI3K/Akt.

Mesenchymal stem cells modified with stromal cell-derived factor 1α improve cardiac remodeling via paracrine activation of hepatocyte growth factor in a rat model of myocardial infarction

Mesenchymal stem cells (MSCs) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether SDF-1 transfection improve MSC viability and paracrine action in infarcted hearts. We found SDF-1-modified MSCs effectively expressed SDF-1 for at least 21days after exposure to hypoxia. The apoptosis of Ad-SDF-1-MSCs was 42% of that seen in Ad-EGFP-MSCs and 53% of untreated MSCs. In the infarcted hearts, the number of DAPI-labeling cells in the Ad-SDF-1-MSC group was 5-fold that in the Ad-EGFP-MSC group. Importantly, expression of antifibrotic factor, HGF, was detected in cultured MSCs, and HGF expression levels were higher in Ad-SDF-MSC-treated hearts, compared with Ad-EGFP-MSC or control hearts. Compared with the control group, Ad-SDF-MSC transplantation significantly decreased the expression of collagens I and III and matrix metalloproteinase 2 and 9, but heart function was improved in d-SDF-MSC-treated animals. In conclusion, SDF-1-modified MSCs enhanced the tolerance of engrafted MSCs to hypoxic injury in vitro and improved their viability in infarcted hearts, thus helping preserve the contractile function and attenuate left ventricle (LV) remodeling, and this may be at least partly mediated by enhanced paracrine signaling from MSCs via antifibrotic factors such as HGF.

Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox- sensitive transcription factor regulating expression of a number of cytoprotective genes. Recently, Nrf2 has emerged as an important contributor to chemoresistance in cancer therapy. In the present study, we found that non-toxic dose of apigenin (APG) significantly sensitizes doxorubicin-resistant BEL-7402 (BEL-7402/ADM) cells to doxorubicin (ADM) and increases intracellular concentration of ADM. Mechanistically, APG dramatically reduced Nrf2 expression at both the messenger RNA and protein levels through downregulation of PI3K/Akt pathway, leading to a reduction of Nrf2-downstream genes. In BEL-7402 xenografts, APG and ADM cotreatment inhibited tumor growth, reduced cell proliferation and induced apoptosis more substantially when compared with ADM treatment alone. These results clearly demonstrate that APG can be used as an effective adjuvant sensitizer to prevent chemoresistance by downregulating Nrf2 signaling pathway.

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells.

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.

In vivo protein transduction: delivery of PEP-1-SOD1 fusion protein into myocardium efficiently protects against ischemic insult.

Myocardial ischemia-reperfusion injury is a medical problem occurring as damage to the myocardium following blood flow restoration after a critical period of coronary occlusion. Oxygen free radicals (OFR) are implicated in reperfusion injury after myocardial ischemia. The antioxidant enzyme, Cu, Zn-superoxide dismutase (Cu, Zn-SOD, also called SOD1) is one of the major means by which cells counteract the deleterious effects of OFR after ischemia. Recently, we reported that a PEP-1-SOD1 fusion protein was efficiently delivered into cultured cells and isolated rat hearts with ischemia-reperfusion injury. In the present study, we investigated the protective effects of the PEP-1-SOD1 fusion protein after ischemic insult. Immunofluorescecnce analysis revealed that the expressed and purified PEP-1-SOD1 fusion protein injected into rat tail veins was efficiently transduced into the myocardium with its native protein structure intact. When injected into Sprague-Dawley rat tail veins, the PEP-1- SOD1 fusion protein significantly attenuated myocardial ischemia-reperfusion damage; characterized by improving cardiac function of the left ventricle, decreasing infarct size, reducing the level of malondialdehyde (MDA), decreasing the release of creatine kinase (CK) and lactate dehydrogenase (LDH), and relieving cardiomyocyte apoptosis. These results suggest that the biologically active intact forms of PEP-1-SOD1 fusion protein will provide an efficient strategy for therapeutic delivery in various diseases related to SOD1 or to OFR.

Acetylcholine induces mesenchymal stem cell migration via Ca2+/PKC/ERK1/2 signal pathway

Acetylcholine (ACh) plays an important role in neural and non‐neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh‐induced MSC migration. InsP3Rs inhibitor 2‐APB that inhibits MAPK/ERK phosphorylation completely blocks ACh‐mediated MSC migration. Interestingly, intracellular Ca2+ ATPase‐specific inhibitor thapsigargin also completely blocks ACh‐induced MSC migration through the depletion of intracellular Ca2+ storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh‐induced MSC migration, but PKC‐ζ siRNA completely inhibits ACh‐induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca2+, PKC, and ERK1/2 signal pathways. J. Cell. Biochem. 113: 2704–2713, 2012.

The combined transduction of copper, zinc-superoxide dismutase and catalase mediated by cell-penetrating peptide, PEP-1, to protect myocardium from ischemia-reperfusion injury

BackgroundOur previous studies indicate that either PEP-1-superoxide dismutase 1 (SOD1) or PEP-1-catalase (CAT) fusion proteins protects myocardium from ischemia-reperfusion-induced injury in rats. The aim of this study is to explore whether combined use of PEP-1-SOD1 and PEP-1-CAT enhances their protective effects.MethodsSOD1, PEP-1-SOD1, CAT or PEP-1-CAT fusion proteins were prepared and purified by genetic engineering. In vitro and in vivo effects of these proteins on cell apoptosis and the protection of myocardium after ischemia-reperfusion injury were measured. Embryo cardiac myocyte H9c2 cells were used for the in vitro studies. In vitro cellular injury was determined by the expression of lactate dehydrogenase (LDH). Cell apoptosis was quantitatively assessed with Annexin V and PI double staining by Flow cytometry. In vivo, rat left anterior descending coronary artery (LAD) was ligated for one hour followed by two hours of reperfusion. Hemodynamics was then measured. Myocardial infarct size was evaluated by TTC staining. Serum levels of myocardial markers, creatine kinase-MB (CK-MB) and cTnT were quantified by ELISA. Bcl-2 and Bax expression in left ventricle myocardium were analyzed by western blot.ResultsIn vitro, PEP-1-SOD1 or PEP-1-CAT inhibited LDH release and apoptosis rate of H9c2 cells. Combined transduction of PEP-1-SOD1 and PEP-1-CAT, however, further reduced the LDH level and apoptosis rate. In vivo, combined usage of PEP-1-SOD1 and PEP-1-CAT produced a greater effect than individual proteins on the reduction of CK-MB, cTnT, apoptosis rate, lipoxidation end product malondialdehyde, and the infarct size of myocardium. Functionally, the combination of these two proteins further increased left ventricle systolic pressure, but decreased left ventricle end-diastolic pressure.ConclusionThis study provided a basis for the treatment or prevention of myocardial ischemia-reperfusion injury with the combined usage of PEP-1-SOD1 and PEP-1-CAT fusion proteins.

PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways

BackgroundCatalase (CAT) breaks down H2O2 into H2O and O2 to protects cells from oxidative damage. However, its translational potential is limited because exogenous CAT cannot enter living cells automatically. This study is aimed to investigate if PEP-1-CAT fusion protein can effectively protect cardiomyocytes from oxidative stress due to hypoxia/reoxygenation (H/R)-induced injury.MethodsH9c2 cardomyocytes were pretreated with catalase (CAT) or PEP-1-CAT fusion protein followed by culturing in a hypoxia and re-oxygenation condition. Cell apoptosis were measured by Annexin V and PI double staining and Flow cytometry. Intracellular superoxide anion level was determined, and mitochondrial membrane potential was measured. Expression of apoptosis-related proteins including Bcl-2, Bax, Caspase-3, PARP, p38 and phospho-p38 was analyzed by western blotting.ResultsPEP-1-CAT protected H9c2 from H/R-induced morphological alteration and reduced the release of lactate dehydrogenase (LDH) and malondialdehyde content. Superoxide anion production was also decreased. In addition, PEP-1-CAT inhibited H9c2 apoptosis and blocked the expression of apoptosis stimulator Bax while increased the expression of Bcl-2, leading to an increased mitochondrial membrane potential. Mechanistically, PEP-1-CAT inhibited p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways, resulting in blockade of Bcl2/Bax/mitochondrial apoptotic pathway.ConclusionOur study has revealed a novel mechanism by which PEP-1-CAT protects cardiomyocyte from H/R-induced injury. PEP-1-CAT blocks Bcl2/Bax/mitochondrial apoptotic pathway by inhibiting p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways.