Hongyan Dong

PEDF and PEDF-derived peptide 44mer protect cardiomyocytes against hypoxia-induced apoptosis and necroptosis via anti-oxidative effect

Pigment epithelium-derived factor (PEDF) has many biological activities. But its not known whether PEDF and its functional peptides could protect against hypoxia-induced cell death and the mechanisms are still unclear. We used cultured H9c2 cells and primary cardiomyocytes to show that apoptosis and necroptosis were significantly increased after hypoxia. Both PEDF and its fuctional peptides 44mer reduced apoptosis and necroptosis rates and inhibited the expression of cleaved caspase 3 and receptor-interacting protein 3 (RIP3). Furthermore, PEDF and 44mer could up-regulate super oxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels, promote clearing of reactive oxygen species (ROS) and malondialdehyde (MDA). While, 34mer, another functional peptides had no effect on cell apoptosis and necroptosis. Hereby this is the first evidence that PEDF and its functional peptide 44mer protect cultured H9c2 cells and primary cardiomyocytes against apoptosis and necroptosis under hypoxic condition via the anti-oxidative mechanism.

Hypoxic response elements control expression of human vascular endothelial growth factor165 genes transferred to ischemia myocardium in vivo and in vitro

Vascular endothelial growth factor (VEGF) gene transfer with recombinant adeno‐associated viral (rAAV) vector for ischemia heart disease therapy is being increasingly studied. However, uncontrolled long‐term expression of VEGF may cause some side effects. Therefore, an attempt to develop an effective gene control system for safeguarding against such side effects should be made. Pathphysiologically, an ideal control system for VEGF gene expression is letting it respond to hypoxia. We used nine copies of hypoxic response element (HRE) to regulate expression of hVEGF165 in the myocardium, and tried to elucidate the feasibility and safety of the application of the HIF‐1‐HRE system.

PEDF improves cardiac function in rats with acute myocardial infarction via inhibiting vascular permeability and cardiomyocyte apoptosis.

Pigment epithelium-derived factor (PEDF) is a pleiotropic gene with anti-inflammatory, antioxidant and anti-angiogenic properties. However, recent reports about the effects of PEDF on cardiomyocytes are controversial, and it is not known whether and how PEDF acts to inhibit hypoxic or ischemic endothelial injury in the heart. In the present study, adult Sprague-Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF-small interfering RNA (siRNA)-lentivirus (PEDF-RNAi-LV) or PEDF-LV was delivered into the myocardium along the infarct border to knockdown or overexpress PEDF, respectively. Vascular permeability, cardiomyocyte apoptosis, myocardial infarct size and animal cardiac function were analyzed. We also evaluated PEDF’s effect on the suppression of the endothelial permeability and cardiomyocyte apoptosis under hypoxia in vitro. The results indicated that PEDF significantly suppressed the vascular permeability and inhibited hypoxia-induced endothelial permeability through PPARγ-dependent tight junction (TJ) production. PEDF protected cardiomyocytes against ischemia or hypoxia-induced cell apoptosis both in vivo and in vitro via preventing the activation of caspase-3. We also found that PEDF significantly reduced myocardial infarct size and enhanced cardiac function in rats with AMI. These data suggest that PEDF could protect cardiac function from ischemic injury, at least by means of reducing vascular permeability, cardiomyocyte apoptosis and myocardial infarct size.

PEDF and 34-mer inhibit angiogenesis in the heart by inducing tip cells apoptosis via up-regulating PPAR-γ to increase surface FasL.

Pigment epithelial-derived factor (PEDF) is a potent anti-angiogenic factor whose effects are partially mediated through the induction of endothelial cell apoptosis. However, the underlying mechanism for PEDF and the functional PEDF peptides 34-mer and 44-mer to inhibit angiogenesis in the heart has not been fully established. In the present study, by constructing adult Sprague–Dawley rat models of acute myocardial infarction (AMI) and in vitro myocardial angiogenesis, we showed that PEDF and 34-mer markedly inhibits angiogenesis by selectively inducing tip cells apoptosis rather than quiescent cells. Peptide 44-mer on the other hand exhibits no such effects. Next, we identified Fas death pathway as essential downstream regulators of PEDF and 34-mer activities in inhibiting angiogenesis. By using peroxisome proliferator-activated receptor γ (PPAR-γ) siRNA and PPAR-γ inhibitor, GW9662, we found the effects of PEDF and 34-mer were extensively blocked. These data suggest that PEDF and 34-mer inhibit angiogenesis via inducing tip cells apoptosis at least by means of up-regulating PPAR-γ to increase surface FasL in the ischemic heart, which might be a novel mechanism to understanding cardiac angiogenesis after AMI.

PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation via PEDF-R.

Pigment epithelial-derived factor (PEDF) is a multifunctional secreted glycoprotein, which could protect against hypoxia-induced cell death related to its anti-oxidative effect in cultured cardiomyocytes. However, the pathway mediating this cytoprotective process has not been fully established. Here we confirmed that PEDF bound to pigment epithelial-derived factor receptor (PEDF-R) expressed on the membrane of H9c2 cells. Under hypoxic condition, PEDF increased the ratio of MDM2:p53, so as to inhibited p53 mitochondrial translocation via PEDF-R. As a result, mitochondrial outer membrane permeabilization (MOMP) and mitochondrial permeability transition pore (MPTP) opening were inhibited, meanwhile cleaved caspase-3, PARP and the release of HMGB1 were reduced. Accordingly, apoptosis and necrosis were attenuated simultaneously. We conclude that PEDF-R mediates PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation.

Efficient cardiomyogenic differentiation of bone marrow mesenchymal stromal cells by combination of Wnt11 and bone morphogenetic protein 2

Wnt11 and bone morphogenetic protein 2 (BMP-2) are key signaling factors for stem cell differentiation into functional cardiomyocytes (CMs). In this study, we elucidate the biological effect of BMP-2 and Wnt11 on bone marrow mesenchymal stromal cells (BM-MSCs) that differentiate into myocardial-like cells in a simulated myocardial microenvironment in vitro. A cell co-culture system was established with recombinant Wnt11 treatment of NIH/3T3 cells and CMs. BMP-2 was added in a diverse schedule to induce cardiomyogenic differentiation of BM-MSCs co-cultured under various conditions. The levels of cardiac-specific markers Nkx2.5, α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC) and cardiac troponin I (cTnI) were determined by reverse transcriptase polymerase chain reaction and immunocytochemistry to evaluate cardiomyogenic differentiation. Wnt11 or BMP-2 used on their own to differentiate BM-MSCs resulted in no expression of α-MHC and cTnI. Wnt11 alone in a myocardial microenvironment enhanced cardiomyogenic differentiation. BMP-2 demonstrated a dose-dependent effect on BM-MSC differentiation into myocardial-like cells. Addition of BMP to BM-MSCs at various time points resulted in varying effects on cardiomyogenic differentiation. The combination of Wnt11 and BMP-2 treatment in a temporal manner significantly enhanced cardiomyogenic differentiation of BM-MSCs, with high expressions of α-MHC, β-MHC, Nkx2.5 and cTnI upon co-culture with CMs. Our study demonstrates that the combination of Wnt11 and BMP-2 effectively promotes cardiomyogenic differentiation of BM-MSCs in vitro. The synergistic effect of Wnt11 and BMP-2 on the cardiomyogenic differentiation of BM-MSCs is further enhanced in a myocardial microenvironment.

Angiogenesis induced by hVEGF165 gene controlled by hypoxic response elements in rabbit ischemia myocardium.

Hypoxic response element (HRE) offers satisfactory control over expression of hVEGF165 in cell levels. However, the characteristics of regenerated blood vessels induced by long-term expression of transferred hVEGF165 under control of HRE in vivo remain unknown. This study aims to investigate the effect of HRE on control of long-term expression of rAAV-delivered hVEGF165 gene to ischemic myocardium and evaluate characteristics of angiogenesis induced by hVEGF165 in vivo. Rabbit ischemic heart models were established surgically, rAAV-9HRE-hVEGF165 was transfected to ischemia hearts subjected to 12 week ischemia. Molecular biological and immunohistochemistry were employed to determine expressions of HIF-1α and hVEGF165. Microvessel densities of CD31+ and α-SMA+ regenerated vessels were also evaluated. Expressions of both hVEGF165 mRNA and protein were upregulated following over-expression of endogenous HIF-1α early after ischemia, peaked at 4–6 weeks post-MI, declined, and approached pre-ischemia level at the end of 12 weeks of ischemia (P < 0.01). The significantly upregulated CD31 in hVEGF165-treated hearts presented from 8 to 12 weeks of ischemia compared with the control (P < 0.01). However, α-SMA expression was rapidly downregulated after ischemia and remained lower than the control level by the end of 12 weeks post-MI (P < 0.01). Overexpression of hVEGF165 controlled by HIF-1α-HRE system shows a stably regional angiogenic efficacy in vivo. But, VEGF, as an early angiogenic cytokine, is inadequate for mediating histologically mature vessels in ischemia myocardium.

PEDF and PEDF-derived peptide 44mer inhibit oxygen–glucose deprivation-induced oxidative stress through upregulating PPARγ via PEDF-R in H9c2 cells

Pigment epithelial-derived factor (PEDF) is a glycoprotein with broad biological activities including inhibiting oxygen-glucose deprivation(OGD)-induced cardiomyocytes apoptosis through its anti-oxidative properties. PEDF derived peptide-44mer shows similar cytoprotective effect to PEDF. However, the molecular mechanisms mediating cardiomyocytes apoptosis have not been fully established. Here we found that PEDF and 44mer decreased the content of ROS. This content was abolished by either PEDF-R small interfering RNA (siRNA) or PPARγ antagonist. The level of Lysophosphatidic acid (LPA) and phospholipase A2 (PLA2) was observed as drawn from the ELISA assays. PEDF and 44mer sequentially induced PPARγ expression was observed both in qPCR and Western blot assays. The level of LPA and PLA2 and PPARγ expression increased by PEDF and 44mer was significantly attenuated by PEDF-R siRNA. However, PEDF and 44mer inhibited the H9c2 cells and cultured neonatal rat myocardial cells apoptosis rate. On the other hand, TUNEL assay and cleavage of procaspase-3 showed that PEDF-R siRNA or PPARγ antagonist increased the apoptosis again. We conclude that under OGD condition, PEDF and 44mer reduce H9c2 cells apoptosis and inhibit OGD-induced oxidative stress via its receptor PEDF-R and the PPARγ signaling pathway.

Biphasic effect of EGb761 on simulated ischemia-induced rat BMSC survival in vitro and in vivo

AIMS The standardized extract from the leaves of Ginkgo biloba (EGb761) is applied as a phyto-pharmacon in therapy of diverse cardiovascular disorders. However, the effects of EGb761 on bone-marrow mesenchymal stem cells (BMSCs) transplanted into the ischemic myocardium currently remain uncertain. In this study, the dosage-effects of EGb761 on BMSC survival in vitro and in vivo were investigated. MAIN METHODS The ischemic microenvironment of rat BMSCs was simulated by hypoxia/serum deprivation (SD) and the rat myocardial infarction model was established. The rat BMSCs were cultured under hypoxia/SD or transplanted into the animal ischemic heart. The BMSC apoptosis was determined by FACS and TUNEL assay. Each apoptotic signal molecules activity was assayed by immunoblot. KEY FINDINGS EGb761 showed a biphasic effect on the hypoxia/SD-induced BMSC apoptosis. Low concentration of EGb761 (10-100μg/ml) aggravated hypoxia/SD-induced apoptosis via Akt inactivation and an enhancement of caspase-9 and caspase-3 expressions, whereas high concentration of EGb761 (500-2000μg/ml) significantly prevented hypoxia/SD-induced BMSC apoptosis via the activated Akt and the inactivated caspase-9 and caspase-3. The animal study also indicated that the apoptotic index (AI) in the high concentration of EGb761 group was significantly lower than the low concentration of EGb761 group. SIGNIFICANCE The biphasic effect of EGb761 is closely related to the PI3K-Akt and caspase-9 signaling pathways. The therapeutic concentration of EGb761 may be one of the vital factors determining the specific action of EGb761 on cell apoptosis. It is of significant clinical implication to investigate the mechanisms of the biphasic effect of EGb761.

Differentiation of Reprogrammed Mouse Cardiac Fibroblasts into Functional Cardiomyocytes

Fibroblasts can be reprogrammed by ectopic expression of reprogramming factors to yield induced pluripotent stem (iPS) cells that are capable of transdifferentiating into diverse types of somatic cell lines. In this study, we examined if functional cardiomyocytes (CMs) can be produced from mouse cardiac fibroblasts (CFs), using iPS cell factor-based reprogramming. CFs were isolated from Oct4-GFP-C57 mice and infected with a retrovirus expressing the Yamanaka reprogramming factors, Oct4, Sox2, Klf4, and c-Myc to reprogram the CFs into a CF-iPS cell line. Primary mouse embryonic fibroblast cells (MEFs) were used as a control. We found that the dedifferentiated CF-iPS cells showed similar biological characteristics (morphology, pluripotent factor expression, and methylation level) as embryonic stem cells (ESs) and MEF-iPS cells. We used the classical embryoid bodies (EBs)-based method and a transwell CM co-culture system to simulate the myocardial paracrine microenvironment for performing CF-iPS cell cardiogenic differentiation. Under this simulated myocardial microenvironment, CF-iPS cells formed spontaneously beating EBs. The transdifferentiated self-beating cells expressed cardiac-specific transcription and structural factors and also displayed typical myocardial morphology and electrophysiological characteristics. CFs can be dedifferentiated into iPS cells and further transdifferentiated into CMs. CFs hold great promise for CM regeneration as an autologous cell source for functional CM in situ without the need for exogenous cell transplantation in ischemic heart disease.