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.

[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.

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.

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.

[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.

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.

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.

[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.

[Protective effect of preconditioning with PEP-1-CAT fusion protein against myocardial ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the transduction efficiency of purified PEP-1-CAT fusion protein into rat heart and the protective effect of the fusion protein against myocardial ischemia-reperfusion injury. METHODS: PEP-1-CAT or CAT (500 microg) was injected in SD rats via the caudal vein, using normal saline as the control, and the hearts were harvested at 0.5, 1, 2, 4, 8, and 24 h after the injection. The transduction efficiency was evaluated by immunofluorescence technique, and the CAT activity was measured. Forty rats were randomized into 5 groups, namely the sham-operated group, ischemia-reperfusion group, and 3 PEP-1-CAT -treated groups (100, 300, and 500 microg). The left main coronary artery was occluded for 1 h followed by a 2-h reperfusion, and at the end of reperfusion, serum LDH and CK and MDA content in the myocardium were measured. RESULTS: No green fluorescence was observed in saline group or CAT group. Bright green fluorescence was observed in PEP-1-CAT groups at different time points, most conspicuous at 8 h. No significant difference in CAT activity was found between CAT group and saline group (P>0.05); with the lapse of time, CAT activity in PEP-1-CAT group increased gradually, reaching the peak level at 8 h, which was 4.2 folds of that in the saline group. LDH ,CK and MDA were significantly lower in PEP-1-CAT- groups than in ischemia-reperfusion group (P<0.01). CONCLUSION: PEP-1 can mediate the transduction of CAT in rat heart in a time-dependent manner, and PEP-1-CAT preconditioning provides a protective effect against ischemia- reperfusion injury in rats.

[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.

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo.

AIM: To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS: We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS: The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was >or= 50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G(0)/G(1), while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION: p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G(0)/G(1). Therefore, p27mt can induce apoptosis in colorectal cells.

[Comparison of migration characteristics of MSCs in different assay systems].

The aim of this study was to explore the difference of MSC migration mediated by SDF-1/CXCR4 axis through Boyden chamber in vitro migration assay. The SDF-1 density-dependence of MSC migration was observed. Subsequently, the effects of different blocking agents on hSDF-MSC migration were observed after MSC were treated with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122, 126 micromol/L AMD3100 and 50 nmol/L verapamil respectively. The results showed the efficiency of MSC migration increased gradually with the increasing of hSDF-1 density. And after MSCs treatment with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122 and 126 micromol/L AMD3100 respectively, the ability of MSC migration decreased. The ability of MSCs migration obviously decreased when MSCs were treated with U73122, AMD3100. It is concluded that the SDF-1/CXCR4-mediated MSC migration may be related to mitogen-activated protein kinase (MAPK), phosphatidylinositol phospholipase C (PI-PLC) and protein kinase (PKC) signal pathways.

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.

[Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer promotes mesenchymal stem cell migration].

OBJECTIVE: To explore the role of stromal-derived factor-1 (SDF-1) in the migration of mesenchymal stem cells (MSCs) and the underlying signal transduction mechanism. METHODS: Rat bone marrow-derived MSCs were infected with 100 ml recombinant adenovirus containing human SDF-1alpha gene (Ad-hSDF-1alpha), and the cell migration changes were observed at 1, 2, and 3 days after the infection. Twelve hours after Ad-hSDF-1alpha transfection, the MSCs in separate cultures were treated with wortmannin (50 nmol/L), LY294002 (10 mmol/L), PD98059 (50 mmol/L), U73122 (10 mmol/L), AMD3100 (0.1 g/L), or verapamil (50 nmol/L), respectively, and the signal transduction pathways involved in MSC migration were analyzed. RESULTS: The MSCs grew in colonies after transfection with Ad-hSDF-1alpha, but this growth pattern was substantially attenuated after treatment with wortmannin, LY294002, PD98059, U73122, AMD3100 and verapamil, among which U73122 and AMD3100 treatments resulted in the most conspicuous inhibitory effects. CONCLUSION: The effect of SDF-1 in promoting MSC migration is related to mitogen-activated protein kinase, phosphatidylinositol phospholipase C, and protein kinase signal pathways.

[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.

[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.

[Cell-penetrating peptide PEP-1-mediated transduction of enhanced green fluorescent protein into human colorectal cancer SW480 cells].

BACKGROUND & OBJECTIVE: Cell-penetrating peptides, a class of small cationic peptides, could mediate macromolecules transduction into many cell lines. This study was to explore the penetrating ability of PEP-1 in the transduction of enhanced green fluorescent protein (EGFP) into human colorectal cancer cell line SW480. 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. Purified EGFP and PEP-1-EGFP were incubated with SW480 cells. The expression of EGFP in SW480 cells was observed under inverted fluorescent microscope. RESULTS: EGFP could not be transduced into SW480 cells, whereas PEP-1-EGFP fusion protein could be transduced into SW480 cells and distributed in cytoplasm and nuclei after 1-hour incubation. CONCLUSION: Mediated by PEP-1, EGFP could be transduced efficiently into SW480 cells.

[Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein].

OBJECTIVE: To construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-1-CAT. METHODS: Using pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2(+)-CAT plasmid, and the PCR product was added with "A" using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3' end was ligated with pGEM-T Easy vector and transformed into DH5alpha. The correct recombinant was identified by PCR and Sal I/Bgl II digestion and named as pGEM-T-CAT. Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho I-digested pET15b. The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and Sal I/Bgl II, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E. coli BL21(DE3) which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni(2+)-NTA-resin column. The fusion protein PEP-1-CAT was produced and confirmed by specific enzyme activity in vitro. RESULTS: Sequence analysis showed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase cDNA sequence in GenBank (accession No. AY028632), respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-1-CAT fusion protein with specific activity of 77.15 U/g. CONCLUSION: The prokaryotic expression plasmid pET15b-PEP-1-CAT has been constructed successfully, and the successful expression and purification of PEP-1-CAT provides a basis for prevention and therapy of various disorders related to oxidative stress.

[Expression and purification of PEP-1-EGFP fusion protein and its transduction into human umbilical vein endothelial cells].

OBJECTIVE: To construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) for evaluating the cell-penetrating capability of the cell-penetrating peptide PEP-1. METHODS: Two oligonucleotides encoding PEP-1 was synthesized and annealed to generate PEP-1-encoding DNA. The recombinant plasmid pET15b-pep-1-EGFP was constructed by inserting PEP-1-encoding DNA and enhanced green fluorescent protein (EGFP) cDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) was purified with Ni(2+)-resin affinity chromatography and transduced into human umbilical vein endothelial cells. RESULTS: Sequence analysis confirmed successful construction of the expression vector pET15b-pep-1-EGFP, and the fusion protein PEP-1-EGFP was expressed and purified efficiently with a yield of approximately 14.15 mg/100 ml bacteria medium. SDS-PAGE and Western blotting identified the purified protein as PEP-1-EGFP, and the cell-penetration assay verified that the fusion protein could be transduced into human umbilical vein endothelial cells. CONCLUSION: The successful expression and purification of PEP-1-EGFP and its efficient transduction into human umbilical vein endothelial cells provides a basis for PEP-1-mediated biomacromolecular transduction in protein therapy.