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

BACKGROUND: Catalase (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. METHODS: H9c2 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. RESULTS: PEP-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. CONCLUSION: Our 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.

Plasma exchange plus glucocorticoids in the treatment of immune checkpoint inhibitor-induced myocarditis: A case series and review.

Immune checkpoint inhibitors (ICIs), including antiprogrammed cell-death (PD)-1/anti-PD-ligand (PDL-1) monoclonal antibodies, are effective at improving the prognosis of patients with cancer. Among immune-related adverse events, myocarditis associated with anti-PD-1/anti-PD-L1 antibodies is rare but lacks effective treatment and mortality is very high. In this study, the authors extracted data from the previous 8 years from electronic medical records housed in the hospital information system to identify patients hospitalized with myocarditis putatively caused by anti-PD-1/anti-PD-L1 tumor therapy. Clinical data from these patients are reported. Four patients who developed myocarditis after undergoing treatment with anti-PD-1/anti-PD-L1 antibodies for malignant tumors, all of whom responded favorably to therapy consisting of plasma exchange and glucocorticoids for myocarditis, and all patients improved and were discharged from hospital. Plasma exchange plus systemic glucocorticoids may be effective for treating anti-PD-1/anti-PD-L1 antibody-induced myocarditis in patients with cancer.

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.

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

BACKGROUND: Our 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. METHODS: SOD1, 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. RESULTS: In 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. CONCLUSION: This 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.

Combination of chemokine and angiogenic factor genes and mesenchymal stem cells could enhance angiogenesis and improve cardiac function after acute myocardial infarction in rats.

Gene and stem-cell therapies hold promise for the treatment of ischemic cardiovascular disease. Combined stem cell, chemokine, and angiogenic growth factor gene therapy could augment angiogenesis, and better improve heart function in the infarcted myocardium. In order to prove this action, we established the animal model of myocardial infarction (MI) was by occlusion of the left anterior descending artery in rats. Seven days after surgery, 5.0 x 10(6) Ad-EGFP-MSC, 5.0 x 10(6) Ad-SDF-1-MSC, 5.0 x 10(6) Ad-VEGF-MSC, or 5.0 x 10(6) Ad-SDF-VEGF-MSC (Ad-SDF-1-VEGF-MSC) suspension in 0.2 ml of serum-free medium was injected into four sites in the infarcted hearts. Results showed that MSCs transfected with Ad-VEGF and Ad-SDF-1 produced more SDF-1 and VEGF protein than MSCs alone, the increased protein levels of VEGF and SDF-1 activated Akt in MSCs transfected with Ad-VEGF and Ad-SDF-1, and improved the survival capability of the MSCs in vitro and in vivo. These transplanted cells showed that the characteristic phenotype of cardiomyocyte (e.g., cTnt) and endothelial cells (e.g., CD31). Four weeks after transplantation, reduced infarct size and fibrosis, greater vascular density, and a thicker left ventricle wall were observed in Ad-SDF-VEGF-MSC group. Measurement of hemodynamic parameters showed an improvement in left ventricular performance in Ad-SDF-VEGF-MSC group compared with other groups. These results demonstrated that combination of chemokine and angiogenic factor gene and stem cells could enhance angiogenesis and improves cardiac function after acute myocardial infarction in rats.

Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions.

UNLABELLED: Stromal cell-derived factor 1 alpha (SDF-1) is not only a major chemotactic factor, but also an inducer of angiogenesis. The effects of SDF-1 alpha on the left ventricular remodeling in a rat myocardial infarction (MI) model were analyzed. Myocardial infarction was induced by ligation of the left coronary artery in rats. 0.5 x 10(10) pfu/ml AdV-SDF-1 or 0.5 x 10(10) pfu/ml Adv-LacZ were immediately injected into the infarcted myocardium, 120 microl cell-free PBS were injected into the infarcted region or the myocardial wall in control, and sham group, respectively. We found that AdV-SDF-1 group had higher LVSP and +/-dP/dt(max), lower LVEDP compared to control or Adv-LacZ group. The number of c-Kit(+) stem cells, and gene expression of SDF-1, VEGF and bFGF were obviously increased, which was associated with reduced infarct size, thicker left ventricle wall, greater vascular density and cardiocytes density in infarcted hearts of AdV-SDF-1 group. Furthermore, the expression of collagen type I and type III mRNA, and collagen accumulation in the infarcted area was lower, which was associated with decreased TGF-beta1, TIMP-1 and TIMP-2 expression in AdV-SDF-1 group. CONCLUSION: SDF-1 alpha could improve cardiac structure and function after Myocardial infarction through angiogenic and anti-fibrotic actions.

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.

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.

[The role of PEP-1-SOD1 fusion protein on ischemia-reperfusion injury in isolated perfused rat hearts.].

OBJECTIVE: The transduction efficiency of the purified PEP-1-SOD1 fusion protein and the effects of PEP-1-SOD1 fusion protein on ischemia reperfusion injury in the isolated perfused rat hearts were investigated. METHODS: The constructed pET15b-SOD1 and pET15b-PEP-1-SOD1 were transformed into BL21 (DE3) for expression and purification of SOD1 and PEP-1-SOD1, respectively. Isolated perfused rat hearts were subjected to 60 min of global ischemia and 30 min of reperfusion and treated with vehicle, 100 micromol/L SOD1 and 25, 50, 100 micromol/L PEP-1-SOD1, respectively. The transduction efficiency was evaluated with immunofluorescent microscopy and Western blot. The enzyme activity of the transduced PEP-1-SOD1 was measured with commercial SOD detection kit. The MDA content in myocardial tissue and the CK activity in coronary exudate at 15 min after reperfusion were also measured. Cardiomyocyte apoptosis was detected with TUNEL. The infarct size was determined in isolated hearts 60 min after reperfusion with TTC staining. RESULTS: Immunofluorescent microscopy and Western blot demonstrated PEP-1-SOD1 was transduced into myocardial tissue in a dose-dependent manner, whereas SOD1 could not be detected in SOD1 group. SOD activity in control, SOD1 group, 25, 50, 100 micromol/L PEP-1-SOD1 groups was (10.06 +/- 0.77) U/mg prot, (10.59 +/- 0.71) U/mg prot, (32.29 +/- 1.42) U/mg prot, (43.16 +/- 1.16) U/mg prot, (55.14 +/- 1.59) U/mg prot, respectively. MDA content in corresponding groups was (1.48 +/- 0.19) nmol/mg prot, (1.39 +/- 0.11) nmol/mg prot, (1.01 +/- 0.14) nmol/mg prot, (0.73 +/- 0.13) nmol/mg prot, (0.50 +/- 0.06) nmol/mg prot, respectively. CK activity in corresponding groups was (1.73 +/- 0.58) U/mg prot,(1.68 +/- 0.14) U/mg prot,(1.40 +/- 0.28) U/mg prot,(0.97 +/- 0.39) U/mg prot, (0.61 +/- 0.56) U/mg prot, respectively. Cardiomyocyte apoptotic index in corresponding groups was (17.25 +/- 0.75)%, (16.63 +/- 1.07)%, (11.50 +/- 0.57) U/mg prot, (6.50 +/- 0.63) U/mg prot, (4.13 +/- 0.52)%, repectively. The percentage of myocardial infarction area was (55.13 +/- 2.18)%, (52.13 +/- 2.59)%, (33.88 +/- 2.06)%, (25.50 +/- 2.16)%, (15.38 +/- 1.14)%, respectively. Compared with control group and SOD1 group, all P < 0.01 These results demonstrated the enzyme activity of the transduced PEP-1-SOD1 was significantly increased in a dose-dependent manner and the MDA content, CK activity, the cardiomyocyte apoptotic index and the infarct size was decreased siginificantly in PEP-1-SOD1 pretreatment groups compared with SOD1 group. CONCLUSION: The native, biologically active form of PEP-SOD1 fusion protein could be effectively transduced into the isolated rat hearts subjecting ischemia reperfusion injury in a dose-dependent manner. The transduced PEP-1-SOD1 has protective effects on ischemia reperfusion injury in the isolated rat hearts.

[Cell-penetrating peptide PEP-1-mediated transduction of enhanced green fluorescent protein into human umbilical vein endothelial cells].

OBJECTIVE: To investigate the penetrating ability of fusion protein PEP-1-EGFP with human umbilical vein endothelial cells. METHODS: Two prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 (DE3) to express EGFP and fusion protein PEP-1-EGFP, respectively. The expressed EGFP and PEP-1-EGFP were purified with Ni(2+) -resin affinity chromatography, and their capabilities of transduction into human umbilical vein endothelial cells were evaluated. The time- and dose-dependent transduction of the fusion protein PEP-1-EGFP and its stability in the human umbilical vein endothelial cells were observed. The toxicity of the fusion protein PEP-1-EGFP was detected by MTT method. RESULTS: EGFP failed to be transduced into human umbilical vein endothelial cells, whereas PEP-1-EGFP fusion protein was transduced into cells shortly in 5 minutes. Its transduction was time- and dose-dependent and the fluorescence in the cells were detected even 27 hours later. No cytotoxicity of the fusion protein PEP-1-EGFP to human umbilical vein endothelial cells was detected even when the dose reached up to 200 micromol/L. CONCLUSION: PEP-1-EGFP fusion protein can efficiently transduce the target protein into human umbilical vein endothelial cells, which provides a basis for future researches on the transduction of antioxidant enzymes mediated by the cell-penetrating peptide, PEP-1, in ischemia-reperfusion injury therapy.

[The protective effect of PEP-1-SOD1 preconditioning on hypoxia/reoxygenation injury in cultured human umbilical vein endothelial cells].

OBJECTIVE: To construct prokaryotic expression vector of pET15b-PEP-1-SOD1 and investigate whether PEP-1-SOD1 fusion protein could be transduced into human umbilical vein endothelial cells (HUVECs) and the effects on hypoxia/reoxygenation injury. METHODS: The recombinant plasmids pET15b-SOD1 and pET15b-PEP-1-SOD1 were constructed and transformed into E. coli BL21 (DE3) to express SOD1 and PEP-1-SOD1 with an N-terminal His-tag. The purified SOD1 and PEP-1-SOD1 were incubated with HUVECs and the viability (MTT assay) and the release of lactate dehydrogenase (LDH) in culture medium were determined in the hypoxia/reoxygenation injury model. The morphological changes were observed under an inverted phase contrast microscope. The content of malondialdehyde (MDA) in HUVECs was also determined with the method of thiobarbituric acid. RESULTS: PEP-1-SOD1 fusion protein could be transduced into cultured HUVECs in a time- and dose-dependent manner. The intracellular enzymatic activity of PEP-1-SOD1 after 30 min incubation with HUVECs was significantly higher than control group (60.88 U/ml +/- 6.73 U/ml vs. 41.06 U/ml +/- 4.19 U/ml, P < 0.01). The transduced PEP-1-SOD1 protein was enzymatically stable for 24 h within cells. After hypoxia/reoxygenation injury, control HUVECs shrunk, became round-shaped and intercellular space increased, while these morphological changes were not observed in PEP-1-SOD1 transduced HUVECs. PEP-1-SOD1 transduction also markedly increased the viability, decreased LDH leakage into culture media and reduced the content of MDA post hypoxia/reoxygenation. CONCLUSIONS: PEP-1-SOD1 fusion protein could be efficiently transduced into HUVECs in a natively active form, and the delivered enzymatically active PEP-1-SOD1 exhibits cellular protection against hypoxia/reoxygenation injury in HUVECs. The transduction of SOD1 mediated by cell-penetrating peptide, PEP-1, provides a basis for further research on the prevention of ischemia/reperfusion injury in vivo.

[The stable expression of human tissue-type plasminogen activator gene mediated by lipofectamine in human vein endothelial cell line cells].

We have established a human umbilical vein endothelial cell (HUVEC) line monoclonal cells with the stable expression of human tissue-type plasminogen activator (t-PA) gene to provide a basis for further study on the vascular tissue engineering. Recombinant plasmid pcDNA3. 1-Myc-His B (-)/t-PA was constructed by insertion of t-PAcDNA originated from PBS/t-PA into eukaryotic expression vector pcDNA3. 1-Myc-His B(-) and transfected into hUVEC line cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The transcription and expression of t-PA gene were investigated by RT-PCR and Western blotting respectively. The t-PA activity was measured by chromogenic substrate assay. The positive clone cells which transcripted the mRNA of t-PA gene was obtained by RT-PCR. Immunoreactive human t-PA of the medium was significantly increased in the group of transfected gene when compared with that in the controlled and transfected plasmid without t-PA gene group. The biological activity of the protein of the t-PA in the media was increased significantly in the positive clone cells with t-PA gene transfected. The HUVEC line monoclonal cells with the stable expression of t-PA gene was established successfully.

[The protective effect of PEP-1-CAT fusion protein on hydrogen peroxide-induced oxidative stress injury in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell (HUVECs) and the effects on hydrogen-peroxide (H2O2)-induced oxidative stress injury in these cells. METHODS: With the use of TA-cloning program and isocaudamer technique, the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) and the protein expression was induced by IPTG. The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni2+-NTA-resin column. The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro. The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro. The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity. The cells were treated with H2O2 (0.5 mmol/L) alone and in combination with PEP-1-CAT fusion protein for 4 h. Then, the cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) contents were measured. RESULTS: The PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose- and time-dependent manner and be stable for at least 48 h. After H2O2 administration, cell viability was significantly reduced compared with control group (37.23%+/-5.68% vs. 100%, P<0.05), while LDH leakage (849.3 U/L+/-95.1 U/L) and MDA (8.23 nmol/L+/-1.58 nmol/L) content were significantly higher than that in control group (540.6 U/L+/-65.7 U/L and 2.46 nmol/L+/-1.42 nmol/L, respectively, all P<0.05). Preincubation with PEP-1-CAT proteins at various concentrations (0.25-2 micromol/L) significantly attenuated H2O2-induced cell injury. CONCLUSION: The PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.

PEP-1-CAT-transduced mesenchymal stem cells acquire an enhanced viability and promote ischemia-induced angiogenesis.

OBJECTIVE: Poor survival of mesenchymal stem cells (MSC) compromised the efficacy of stem cell therapy for ischemic diseases. The aim of this study is to investigate the role of PEP-1-CAT transduction in MSC survival and its effect on ischemia-induced angiogenesis. METHODS: MSC apoptosis was evaluated by DAPI staining and quantified by Annexin V and PI double staining and Flow Cytometry. Malondialdehyde (MDA) content, lactate dehydrogenase (LDH) release, and Superoxide Dismutase (SOD) activities were simultaneously measured. MSC mitochondrial membrane potential was analyzed with JC-1 staining. MSC survival in rat muscles with gender-mismatched transplantation of the MSC after lower limb ischemia was assessed by detecting SRY expression. MSC apoptosis in ischemic area was determined by TUNEL assay. The effect of PEP-1-CAT-transduced MSC on angiogenesis in vivo was determined in the lower limb ischemia model. RESULTS: PEP-1-CAT transduction decreased MSC apoptosis rate while down-regulating MDA content and blocking LDH release as compared to the treatment with H(2)O(2) or CAT. However, SOD activity was up-regulated in PEP-1-CAT-transduced cells. Consistent with its effect on MSC apoptosis, PEP-1-CAT restored H(2)O(2)-attenuated mitochondrial membrane potential. Mechanistically, PEP-1-CAT blocked H(2)O(2)-induced down-regulation of PI3K/Akt activity, an essential signaling pathway regulating MSC apoptosis. In vivo, the viability of MSC implanted into ischemic area in lower limb ischemia rat model was increased by four-fold when transduced with PEP-1-CAT. Importantly, PEP-1-CAT-transduced MSC significantly enhanced ischemia-induced angiogenesis by up-regulating VEGF expression. CONCLUSIONS: PEP-1-CAT-transduction was able to increase MSC viability by regulating PI3K/Akt activity, which stimulated ischemia-induced angiogenesis.

[Effect of conditioned medium of mesenchymal stem cells on proliferation, migration and adhesion of human umbilical vein endothelial cells].

The aim of this study was to explore the effect of mesenchymal stem cell (MSC) conditioned medium (MSC-CM) on proliferation, migration and adhesion of human umbilical vein endothelial cell (CRL1730) and its mechanism. Isolation and purification of MSC were performed with the classic adhering method, the surface markers (CD29, CD90, CD45 and CD34) in MSC were detected by flow cytometry. MSC were treated and cultured for 3 d, the MSC-CM or MSC overexpressing stem cell-derived factor-1 (SDF-1) conditioned medium (Ad-SDF-1-MSC-CM) were collected. Subsequently, CRL1730 cells were treated respectively with 2% FBS-DMEM, 15% FBS-DMEM (control group), MSC-CM or Ad-SDF-1-MSC-CM for 24 h, the proliferation of CRL1730 cells was detected by MTT method. CRL1730 cell migration in vitro was performed by using wound healing system. The adhesion ability of CRL1730 cells was analyzed by microscope. The results indicated that the CRL1730 cells treated with Ad-SDF-1-MSC-CM showed greater proliferative capacity than CRL1730 cells treated with MSC-CM. While adding with AMD3100 5 µmol/L, the blocker of CXCR4, the CRL1730 proliferation mediated by Ad-SDF-1-MSC-CM was significantly reduced. Meanwhile, compared with MSC-CM, Ad-SDF-1-MSC-CM had greater effects for promoting CRL1730 migration and enhancing adhesion ability of CRL1730 cells, these effects were significantly inhibited by AMD3100. It is concluded that MSC-CM promotes the migration and adhesion ability of CRL1730 cells through SDF-1 expressed by MSC.

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.

[Effect of vascular endothelial growth factor on bone marrow-derived mesenchymal stem cell proliferation and the signaling mechanism].

OBJECTIVE: To observe the effect of vascular endothelial growth factor (VEGF) on bone marrow-derived mesenchymal stem cell (MSC) proliferation and explore the signaling mechanism involved. METHODS: MSC culture was performed following the classical whole bone marrow adhering method. The characteristics of MSC were identified by induction of multi-lineage differentiation and flow cytometry for surface marker analysis (CD34, CD45, CD29, and CD90). Following the addition of 50 nmol/L wortmannin, 50 µmol/L PD98059, 30 µmol/L SB203580, 10 µmol/L H89, 20 µmol/L Y27632, 1 µmol/L rapamycin, 10 µmol/L straurosporine, 6 nmol/L Go6976, or 50 µmol/L Pseudo Z inhibitors in the cell culture, the MSC were treated with 20 ng/ml VEGF and the changes of the cell proliferation rate was measured with MTT assay. RESULTS: Cultured MSC were capable of multi-linage differentiation and did not express VEGF-R, CD29 or CD90. Treatment with 20 ng/ml VEGF obviously promoted MSC proliferation, and this effect was inhibited partially by p38 mitogen-activated protein kinase (MAPK) inhibitor rapamycin, PD98059, SB203580, Go6976, and straurosporine. CONCLUSIONS: VEGF promotes MSC proliferation in close relation to the AKT-PKC pathway, in which PKC signal pathway may play the central role.

Mesenchymal stem cells modified with stromal cell-derived factor 1 alpha 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 21 days 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.

[VEGF promotes the proliferation of bone marrow derived mesenchymal stem cells through ERK1/2 signal pathway].

In order to explore the effect of VEGF on mesenchymal stem cell (MSC) proliferation and its possible signal transduction mechanism, MSC culture was performed with the classical bone marrow adhering method; characteristics of passage 3 rat MSC (P3MSC) was identified through multi-differentiation and surface marker assay (CD34, CD45, CD90, CD29); P3MSC were treated with 20 ng/ml VEGF, and the effect of VEGF on the MSC proliferation was measured during 12, 36 and 72 hours by MTT assay. Subsequently, P3MSC were treated with extracellular-signal regulated kinase (ERK1/2) inhibitor PD98059 (50 µmol/L) or p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580 (30 µmol/L) for 30 minutes, the culture medium was replaced with new medium including 20 ng/ml VEGF. After 72 hours, the effect of PD98059 or SB203580 on MSC proliferation mediated by VEGF was measured by MTT assay. The result showed that the cultured MSC expressed PDGFR-α, PDGFR-ß and NRP1, but did not express VEGF-R (Flk1 and Flt1). The MSC had the multi-differentiation ability and displayed the characteristics of CD90+ (96.7%), CD29+ (94.6%), CD34- (0.79%) and CD45- (0.84%). The MSC proliferation rate increased gradually with prolonging of the functioning time of 20 ng/ml VEGF, and MSC proliferation rate may reach to maximum value after treating with 20 ng/ml VEGF for 72 hours. The effect of VEGF on MSC proliferation was found to be abolished, even was under level of control group after treating with PD98059 or SB203580 for 30 minutes. Furthermore, the inhibitory effect of PD98059 on MSC proliferation was obviously higher than that of SB203580. It is concluded that the VEGF can promote MSC proliferation, and its possible mechanism may relate to ERK1/2 pathway.

[Effect of adenovirus-mediated stromal cell-derived factor-alpha gene transfer on ventricular remodeling in rats with myocardial infarction].

OBJECTIVE: To explore the effect of adenovirus-mediated human stromal cell-derived factor-1alpha (hSDF-1alpha) on ventricular remodeling in rats with myocardial infarction. METHODS: A recombinant adenoviral plasmid containing hSDF-1alpha cDNA was constructed using homologous recombination in bacteria and the recombinant adenovirus particles expressing hSDF-1alpha (AdV-SDF-1) were prepared. In rat models of myocardial infarction induced by left anterior descending artery occlusion, 1x10(10) PFU AdV-SDF-1 or PFU AdV-LacZ were injected at multiple sites into the infarcted myocardium 1 h after the operation, using 200 l cell-free PBS as the control. Four weeks after the injection, the cardiac function of the rats was analyzed, and the heart tissues were taken after the measurement of hemodynamics. On serial frozen sections, histological observation and morphometric measurement were carried out using a microscopic image analysis system, and the expression of hSDF-1alpha was detected by immunocytochemistry. RESULTS: Four weeks after AdV-SDF-1 injection, the myocardium in the infracted area showed significantly higher expression rates of hSDF-1alpha. The injection resulted in a obvious reduction in the infarct size and collagen content and a marked increase in the left ventricle wall, and the rats showed improved cardiac functions. CONCLUSION: SDF-1alpha can improve the cardiac structure and function in rats with myocardial infarction by inhibiting collagen synthesis and deposition in the infarcted area.