Effects of transplantation of peripheral blood mesenchymal stem cells with hypoxia preconditioning on postangioplasty restenosis in rabbits

AIM: To investigate the function and the mechanism of transplanting bone marrow derived peripheral blood mesenchymal stem cells (PBMSCs) on restenosis after carotid balloon angioplasty in the model of carotid atherosclerosis rabbits, and to determine if the functions of PBMSCs are enhanced after hypoxia preconditioning. METHODS: Bone marrow cells were mobilized by granulocyte colony-stimulating factor (G-CSF), and PBMSCs were collected through density gradient centrifugation and adherent culture, labeled with enhancement type green fluorescent protein (EGFP) genes. All animals with carotid atherosclerosis stenosis were randomly divided into three groups: hypoxia preconditioning group (n=24, received intravenous transplantation of PBMSCs with hypoxia preconditioning), non-hypoxia preconditioning group (n=24, received normal culture of PBMSCs) and control group (n=24, only received equal-volume of culture medium). Vascular endothelial growth factor (VEGF) was determined by enzyme linked immunosorbent assay (ELISA) at 7 d, 14 d and 28 d post-angioplasty, respectively. The vessel morphology, the homing of MSCs and the reendothelialization were analyzed with Weigert staining and immunohistochemistry. RESULTS: Compared to control group, the level of VEGF significantly increased in both hypoxia preconditioning group and non-hypoxia preconditioning group at all time points (P<0.01). The level of VEGF in hypoxia preconditioning group was higher than that in non-hypoxia preconditioning group (P<0.05) at 7 d and 14 d, but no difference at 28 d post-angioplasty was observed. At 7 d, GFP-positive cells were found both in hypoxia preconditioning group and non-hypoxia preconditioning group. Neointima thickening and the rate of restenosis were lower in hypoxia preconditioning group than those in non-hypoxia preconditioning group at 28 d (P<0.05), but both hypoxia preconditioning group and non-hypoxia preconditioning group were markedly lower than that in control group (P<0.01). The reendothelialization in hypoxia preconditioning group was outweigh than that in non-hypoxia preconditioning group (P<0.05), but both two groups were lower than that in control group (P<0.01). CONCLUSION: Intravenous transplantation of PBMSCs contributes to the reendothelialization, and attenuates neointima thickening after carotid balloon-induced injury in the rabbit model. Further, hypoxia preconditioning may strengthen the above function of MSCs, which is corelated with the increase in cytokines induced by hypoxia preconditioning to MSCs.     

Effect of granulocyte-colony stimulating factor on the reendothelialization and intima hyperplasia of ballon-injured rat carotid artery

AIM:To study the effect of mobilization of stem cells by exogenous recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the repairing process of reendothelialization and neointima hyperplasy on ballon injured rat carotid arteries.METHODS:Male Wistar rats were randomly divided into rhG-CSF group and NS+injury group.The animals were injected daily with 30 μg/kg rhG-CSF or 0.9% NaCl for 7 days,then underwent balloon angioplasty of the common carotid arteries which were harvested and processed for scanning electron microscopy (SEM),Evans blue staining,morphometric analysis of endothelialization and neointimal formation at 1 h,3 d,5 d,7 d,14 d after injury.Immunohistochemistry for proliferation cell nuclear antigen (PCNA) and RT-PCR for eNOS mRNA were also conducted for evaluating the proliferation of cells of the vessel wall and the possible mechanism of the repairing.RESULTS:SEM and Evan’s blue staining showed increased reendothelialization of the denuded vessels in rhG-CSF-treated animals compared with that NS+injury animals ［(60.6±7.3)% vs (41.6±3.3)%，P<0.01］.Neointima thickness was reduced by 37.3% in rhG-CSF group compared with NS+injury group 2 weeks after injury.Immunohistochemical staining for PCNA positive cells was less in rhG-CSF group compared with that in NS+injury group (42.6% vs 72.8%,P<0.01).CONCLUSION:rhG-CSF has beneficial effects on the reendothelialization and neointima thickness of the ballon-injured arteries.Mobilization of EPCs by exogenous granulocyte colony stimulating factor may be a potential therapeutic strategy for prevention of restenosis after percutenous coronary artery intervention.     

AT2R gene transfer to rat carotid arteries inhibits neointimal hyperplasia after balloon angioplasty

AIM: To study the effects of angiotensin Ⅱ type 2 receptor (AT2R) gene transfer on neointimal hyperplasia in rat carotid arteries after balloon angioplasty. METHODS:AT2R gene was transfected into rat carotid arteries with pAdCMV/AT2R after the establishment of rat carotid balloon injury restenosis model. The arteries were harvested at 7 days, 14 days and 21 days after gene transfer. The expression of AT2R, AT1R, PCNA in arteries and morphology analysis were evaluated by RT-PCR, immunohistochemistry and HE staining. The expressions of AT2R and PCNA were measured by double immunofluorescence staining and confocal microscope. RESULTS:pAdCMV/AT2R delivered into injured rat carotid arteries significantly up-regulated the levels of AT2R mRNA and protein in neointima from day 7 to day 21, but the levels of AT1R was not significantly different (P>0.05). pAdCMV/AT2R transfection significantly decreased the expression of PCNA in neointima at day 14 ［(27.29±5.81)% vs ( 72.25±4.47)%, (68.43±9.12)%，P<0.01］. At day 21, compared with no transfection group and pAd-GFP transfection group, pAdCMV/AT2R transfection reduced I/M (intimal/medial area) ratio significantly (0.78±0.06 vs 1.44±0.22, 1.36±0.21， respectively, P<0.01). No significant difference between pAd-GFP group and no transfection group was observed. CONCLUSION: Gene transfer of AT2R from lumen may effectively inhibit VSMC proliferation and neointimal hyperplasia in the rat carotid arteries after balloon angioplasty. The cross-talk between AT1R and AT2R may operate via signaling pathway, but not via counteraction of receptor expression.     

Effect of mesenchymal stem cells transfected with human heme oxygenase-1(HO-1)gene on myocardial apoptosis and angiogenesis

AIM: To investigate the effects of mesenchymal stem cells(MSCs)transfected with human heme oxygenase-1(HO-1)gene on myocardial apoptosis and angiogenesis. METHODS: MSCs were acquired from the bone marrow of adult rats. The cells were isolated, purified, cultured, and transfected with Adv-HO-1 in vitro before transplantation. At 1 h after left coronary artery ligation, Adv-HO-1-MSCs or MSCs were directly injected into the border of cardiac infarction in rats. Western blotting analysis was used to measure HO-1, and Bax protein expression in the border of cardiac infarction. ELISA was used to measure the expressions of VEGF and bFGF in the border of cardiac infarction. At 4 weeks after transplantation, the heart functions in survival rats were examined by the Buxco system. The rats were killed, then the myocardial infarct size was measured with Masson’s trichrome, and the expression of CD34 in myocardial infarction area was detected by immunohistochemical method. RESULTS: HO-1-MSCs exhibited increased HO-1 expression. The expression of HO-1, VEGF and bFGF in the border of cardiac infarction in the rats treated with HO-1-MSCs were higher than those in the rats treated with MSCs and PBS(P<0.01). However, the expression of apoptotic protein Bax was significantly lower than that in the rats treated with MSCs and PBS(P<0.01). The number of capillary vessels in the border of cardiac infarction in the rats treated with HO-1-MSCs was significantly higher than that in the rats treated with MSCs and PBS. The cardiac function in the rats treated with HO-1-MSCs was better than that in the rats treated with MSCs and PBS(P<0.01). CONCLUSION: The favorable effect on heart function appears to be a combined outcome of HO-1 and paracrine factors released by MSCs.     

Transfection of mesenchymal stem cells with human heme oxygenase-1 gene improves survival under conditions of serum-free and hypoxia

AIM: To investigate the effects of human heme oxygenase-1 (HO-1) gene transfection on mesenchymal stem cells (MSCs) survival under the conditions of serum-free and hypoxia. METHODS: MSCs were acquired from the bone marrow of adult rats. The cells were isolated, purified, cultured, and transfected with Adv-HO-1. The expression of GFP was detected by immunofluorescence. Cell apoptosis was detected by nuclear DAPI staining and FACS. The concentrations of VEGF, HGF and b-FGF in the culture supernatant were measured by ELISA. The caspase-3 protein level and activity of cardiomyocytes cultured with the supernatants from different MSCs under the condition of serum-free and hypoxia were assayed by Western blotting and fluorimetry, respectively. RESULTS: HO-1-MSCs exhibited strong expression of GFP. The fragmented or condensed chromatin diminished in HO-1-MSCs compared with the MSCs lacking exogenous transfection of HO-1 gene. A lower proportion of apoptosis was observed in HO-1-MSCs compared with MSCs under the conditions of serum-free and hypoxia (P<0.01). The expressions of VEGF, HGF and b-FGF in the supernatants of HO-1-MSCs were higher than those in MSCs (P<0.01). A significant reduction of caspase-3 level and activity in the cardiomyocytes treated with the supernatants from HO-1-MSCs was observed, compared to that treated with supernatants from MSCs (P<0.01). CONCLUSION: HO-1 improves the MSCs survival under the conditions of serum-free and hypoxia. Several cytokines released by HO-1-MSCs may protect the cardiomyocytes against apoptosis.     

The feasibility of reendothelialization of the injured arterial wall by autologus endothelial cell transplantation and their effects on neointima proliferation

AIM: To investigate the feasibility of reendothelialization of the injured arterial wall by autologous endothelial cell transplantation and their influences on neointima proliferation. METHODS: New Zealand white rabbits (n=30) were subjected to bilateral iliofemoral artery balloon injury. Cultured, autologous venous endothelial cells were immediately transplanted into one vessel(transplantation group), whereas the contralateral artery received medium only(control group). Reendothelialization of the injured arterial wall was analysed 4 hours or 4 days after cell transplantation by fluorescent tracing、scanning electron microscope(SEM) and Evans blue staining. Pathology analysis was employed 28 days after cell transplantation to evaluate neointima proliferation. RESULTS: The transplanted endothelial cells had adhered into the aterial wall 4 hours after transplantation and began to attach and spread 4 days later. A number of fluorescent labeling endothelial cells were observed in the endothelial injured arterial wall. The vessels in control group were stained nearly completely by Evans blue, whereas about 60% area was not stained in transplantation group. Pathological examination demostrated that neointimal area and maximal intima thickness in transplantation group significant decreased than those in control. CONCLUSION: Autologus endothelial cells were effectively transplanted into the injured arterial wall by balloon catheter, and it can relieve neointima proliferation in the long time.     

Am80 inhibits neointima hyperplasia by promoting interaction of KLF4 with RARα

AIM: To investigate the inhibitory effect of Am80 on neointima hyperplasia in carotid arteries after balloon injury and to observe the interaction between Krüppel-like factor 4 (KLF4) and retinoic acid receptor α (RARα).METHODS: Neointima hyperplasia in carotid arteries was observed by hemotoxylin and eosin staining. The expression of KLF4 and cyclin D1 was examined by immunostaining and Western blotting analysis. To detect the interaction between KLF4 and RARα in the vascular tissue, the injured arteries were harvested, and the protein extracts were prepared and subjected to co-immunoprecipitation assay.RESULTS: Compared with injured group, Am80 significantly reduced neointimal hyperplasia and the thickness ratio of intima to media. Am80 not only up-regulated KLF4 or RARα expression in caro-tid arteries, but also increased the interaction between KLF4 and RARα at tissue levels.CONCLUSION: Am80 inhibits neointima hyperplasia in carotid arteries after balloon injury by promoting the interaction between KLF4 and RARα.     

Effect of Fas ligand gene on reendothelialization in rat carotid artery with balloon withdrawal injury

AIM: To investigate the effect of Fas ligand on reendothelialization in the rat carotid artery with balloon withdrawl injury. METHODS: Balloon withdrawal injury was used to establish the denuded carotid artery rat model in twelve SD rats and then six SD rats were treated with β-gal virus supernatant (β-gal group), and the others were treated with FasL virus supernatant (FasL group). After fourteen days, Evans blue dying was conducted via injection through rat tail vein at 30 min before the animals were killed, then fixation perfusion in situ was performed with 10% formalin and specimens were obtained. Histologic observation and morphometric analysis were made. RESULTS: The reenothelialization area (RA) of arterial balloon injury and the ratio of the reenothelialization area to the total area (RA/TA) significantly increased in FasL group compared with those in β-gal group (P<0.05, respectively). CONCLUSION: FasL gene has an effect of accelerating reendothelialization on the injured vessels.     

Crocin promotes mobilization of EPCs and re-endothelialization of damaged blood vessels in mice with carotid artery injury

AIM: To study the effect of crocin on the mobilization of endothelial progenitor cells (EPCs) in the peripheral blood of the mice with carotid arterial injury and its mechanism.METHODS: The carotid artery injury model of the C57BL/6 mice was established by the method of wire injury. The animals were divided into sham operation group, saline-treated model group, and low dose, medium dose and high dose (10, 50 and 100 μmol·kg^-1·L^-1, respectively) of crocin treatment groups. The mobilization of the EPCs in peripheral blood of the mice with carotid artery injury was detected by flow cytometry at 3 d. The changes of vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and matrix metalloproteinase-9 (MMP-9) in the peripheral blood of the mice with carotid artery injury were detected by enzyme-linked immunosorbent assay at 7 d. The vascular re-endothelialization and intimal hyperplasia of the mice with carotid artery injury were detected by Evans blue and hematoxylin-eosin staining. At the same time, real-time PCR was used to detect the mRNA expression of vascular repair factor-related receptors, vascular endothelial growth factor receotor-2 (VEGFR-2), CXC chemokine receptor-4 (CXCR4), basic fibroblast growth factor receptor (bFGFR) and epidermal growth factor receptor (EGFR), in the injured segments of carotid arteries.RESULTS: Compared with sham group, the EPCs mobilization and the content of vascular repair factors VEGF, SDF-1, bFGF, EGF and MMP-9 in peripheral blood were increased in model group (P<0.05). The area of vascular endothelium was decreased, while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were increased significantly (P<0.05). The expression levels of VEGFR-2, CXCR4, bFGFR and EGFR were also increased in the injured segments of carotid arteries (P<0.05). Compared with model group, the EPCs mobilization and the content of vascular repair factors VEGF, SDF-1, bFGF, EGF and MMP-9 in peripheral blood were significantly increased in different concentrations of crocin-treated mice with carotid artery injury (P<0.05). The area of vascular endothelium was gradually increased, while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were gradually decreased (P<0.05). The expression levels of VEGFR-2, CXCR4, bFGFR and EGFR were also gradually increased in the injured segments of cartid arteries (P<0.05).CONCLUSION: Crocin promotes the mobilization of EPCs and the re-endothelialization of damaged blood vessels in the mice with carotid artery injury, thus repairing the injured vasculature.     

Intravenous transfusion of endothelial progenitor cells reduces neointima formation

AIM: To investigate the feasibility of transplanting endothelial progenitor cells (EPCs) obtained from spleen in vascular endothelium repairmen after vascular injury. METHODS: EPCs were isolated by using a Ficoll density gradient centrifugation, and were cultured in plate. The endothelial characteristics of EPCs were identified by immunochemical staining and fluorescent labeling. Dil-Ac-LDL labeled spleen-derived EPCs were transplanted into the rats by intravenous injection directly after induction of arterial injury and again 24 hours later. Rats received FITC-labeled lectin intravenously before euthanasia. The distribution of fluorescent labeled EPCs was traced. The morphology of arterial intima and media was studied by optical microscopy and image analysing system. RESULTS: The adherent cells were considered EPCs that showed spindle shape and form blood-siland-like structures during development. The adherent cells had many endothelial characteristics. Fluorescent labeling showed that the intravenously injected EPCs specifically restricted to the vascular injury site, and lectin binding confirmed the endothelial phenotype. The ratio of neointimal/media area in EPCs transplantation group was obviously reduced than that in injury group and M199 group (0.82±0.09 vs 1.52±0.21, 1.48±0.19, P<0.01). The PCNA positive expression cells were evidently decreased compared with injury group and M199 group (19.25±3.96 vs 31.42±5.23, 29.37±3.16, P<0.05). CONCLUSION: EPCs incorporate into the process of injured carotid reendothelialization. EPCs transplantation induces an increase in the circulating EPCs, accelerates the process of endothelial repairmen and reduces neointima formation.     

Inhibitory effects of Am80 on proliferation in vascular endothelial cells and neointima hyperplasia of carotid arteries

AIM: To explore the inhibitory effects of Am80 on the proliferation in the vascular endothelial cells (VECs) and neointima hyperplasia of the carotid arteries after balloon injury in the rats. METHODS: The proliferation of EA-hy926 cells were detected by cell counting and MTS assay after the cells were treated with various doses of Am80 for 24 h. The cell cycle was analyzed by flow cytometry after the cells were stained with PI. The mRNA expression of cyclinB1, P21 and matrix metalloproteinase (MMP)-2 in the EA-Hy926 cells was detected by real-time PCR. The changes of neointima hyperplasia in the carotid arteries were observed under microscope with hemotoxylin and eosin (HE) staining, and the expression of cyclinB1 was examined by the method of immunohistochemistry. RESULTS: The proliferation of EA-Hy926 cells was obviously inhibited in a dose-dependent manner when the cells were treated with various doses of Am80 for 24 h. The cell cycle was arrested at G₂/S stage in response to Am80 treatment. The mRNA expression of P21 was increased, however, the mRNA expression of cyclinB1 and MMP-2 was decreased when the cells were treated with Am80 at 4 μmol/L for various times. In addition, the vivo experiment demonstrated that Am80 not only significantly reduced neointimal hyperplasia and the thickness ratio of intima to tunicae media compared with injured group, but also inhibited cyclinB1 expression in the carotid arteries. CONCLUSION: Am80 inhibits the proliferation of VECs and neointima hyperplasia in the carotid arteries after balloon injury by promoting P21 expression and decreasing cyclinB1 expression.     

Effects of cotransplantation of umbilical cord blood and mesenchymal stem cells by intra-bone marrow injection on hematopoietic reconstitution in a rat model

AIM: To investigate the effects of cotransplantation of mesenchymal stem cells (MSCs) and umbilical cord blood (UCB) by intra-bone marrow (IBM) injection on the hematopoietic reconstitution and recovery of bone marrow MSCs in the recipients. METHODS: Wistar female rats were transplanted with fetal and neonatal peripheral blood (FNPB) and BrdU-labeled MSCs separated from BMNCs of F344 rats. The MSCs were infused by IBM injection in bilateral tibiae or intravenous injection (IV), while the FNPB was all via IBM route. The survival rate, reconstitution of hematopoietic and immunological function, engraftment level of HSCs and recovery of bone marrow (BM)-MSCs in recipients were monitored. The origins of BM-MSCs of recipients were examined by immunofluorescence assay. RESULTS: (1)The survival rate in the two cotransplantation groups was 100% at day 60, while that in FNPB group was only 66.7%. (2)The counts of peripheral blood cells and BM hematopoietic stem/progenitor cell colonies of the recipients were better in cotransplantation groups than those in FNPB group, especially in the FNPB (IBM)+MSC (IBM) group. (3)No significant difference between of engraftment level of HSCs in the two cotransplantation groups was observed. The percentage of RT1A1 cells subset in FNPB (IBM)+MSC (IBM) group was much higher than that in FNPB group (P<0.05). (4)At day 30, the growth characteristic of recipient BM-MSCs was still below normal, but that in FNPB (IBM)+MSC (IBM) group was the best of all the experiment groups (P<0.05). (5)The donor MSCs coexisted with host MSCs in only a few recipient rats. CONCLUSION: The cotransplantation of MSCs and FNPB can accelerate the recovery of recipient BM-MSCs and hematopoietic reconstitution, promote the engraftment level of HSCs. Cotransplantation by IBM route is safe and has better effects on hematopoietic reconstitution than by IV route.     

Therapeutic efficacy of Ang-1 gene-modified MSCs in cerebral infarction

AIM: To observe the therapeutic efficacy of Ang-1 gene-modified mesenchymal stem cells (MSCs) in cerebral infarction. METHODS: The constructed lentiviral vector carrying the Ang-1 gene was used to infect the rat mesenchymal stem cells (rMSCs) to establish the Ang-1 gene-modified rMSCs (Ang-rMSCs). Adult male F344 rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAO) with modified Zea Longa method. Phosphate buffered saline (PBS, 1 mL 0.1 mol/L, for control group), or Ang-rMSCs suspension (1 mL, for Ang-rMSCs group), or rMSCs suspension (1 mL, for pNL-rMSCs group), were infused into tail vein of rat respectively at 24 h after MCAO (n=8 in each group). Functional recovery measurements using the modified neurological severity scores (mNSS) were performed at 24 h post-MCAO and 1 week, 1 month and 3 months post-transplantation, respectively. The quantitative evaluation of blood-brain barrier permeability was performed at 1 week post-transplantation. The distribution, differentiation and malignant sign of grafted rMSCs were observed with immunofluorescence staining and histological method. RESULTS: Significant neurological function improvement was observed in groups treated with Ang-rMSCs or pNL-rMSCs at 1 week, 1 month post-transplantation compared with that in control group, as evidenced by mNSS (P<0.01). Better neurological function improvement was also found in Ang-rMSCs group than that in pNL-rMSCs group (P<0.01). The results of quantitative evaluation of blood-brain barrier permeability showed that the permeability in Ang-rMSCs group was the lowest compared to those in other two groups (P<0.01), and in the pNL-rMSCs group was the lower than that in control group (P<0.01). The grafted rMSCs survived in Ang-rMSCs and pNL-rMSCs groups, most were localized around the ischemic focus, and a few of them expressed NSE, NF and GFAP. The grafted rMSCs expressed BDNF abundantly in Ang-rMSCs group. These grafted rMSCs survived up to 3 months at least. No malignant sign was observed in these grafted cells. CONCLUSION: Ang-1 gene-modified MSCs transplantation has better therapeutic efficacy in cerebral infarction than that of MSC transplantation. The transplantation of cells with gene engineering is an effective therapeutic method for stroke patients.     

Effect of human β-NGF gene-modified bone marrow-derived mesenchymal stem cells on rotational behavior of rats with Parkinson disease

AIM：To investigate the effect of human β-nerve growth factor (β-NGF) gene-modified bone marrow-derived mesenchymal stem cells (MSCs) transplantation on the rotational behavior improvement in a rat model of Parkinson disease (PD). METHODS：The rat model of PD was established successfully and the animals were divided into 4 groups:β-NGF-MSCs group (transplanted with 5×105 β-NGF-engineered MSCs), MSCs group (transplanted with 5×105 MSCs), DMEM/F12 group (5 μL transplantation medium was injected in the right striatum of the rats) and PD model group (without transplantation). The rotational scores were assessed 2 weeks, 4 weeks and 6 weeks after transplantation. At different time points after transplantation, the rats were tested for apomorphine (APO)-induced rotational behavior and the brains of the PD model rats were examined by fluorescence microscopy and immunohistochemical staining. RESULTS：Transplantation of human β-NGF gene-modified MSCs effectively improved the behavioral performance in the rats. At the 2nd, 4th and 6th weeks after cell transplantation, the rotational frequencies after injection of APO decreased significantly in β-NGF-MSCs group compared with MSCs group and PD group (P<0.05). Both β-NGF gene-modified MSCs and MSCs survived in the brains of PD model rats, had good compatibility with the host cells, and showed no signs of destroying the host and the glial cicatrisation. The β-NGF gene-modified MSCs expressed β-NGF stablely in the brains of PD model rats, and showed obvious improvement of the rotational behavior in the PD model rats induced by APO compared with MSCs group. CONCLUSION:The behavior of the rats with PD is significantly improved by transplanting β-NGF-modified MSCs in right striatum, and β-NGF gene therapy has potential clinical value．     

Effect of livin-modified BM-MSCs transplantation on cardiac function following acute myocardial infarction in a rat model

AIM: To study the effect of livin gene-modified bone marrow mesenchymal stem cells(BM-MSCs) transplantation on the cardiac function following acute myocardial infarction in a rat model and the expression of livin, caspase-3, caspase-7 and caspase-9 in the livin gene-modified BM-MSCs. METHODS: The MSCs were obtained by the whole bone marrow culture method, and the apoptosis of the MSCs after infection with adenovirus vector carrying enhanced green fluorescent protein(EGFP) gene and livin recombinant vector(rAd-livin) were detected by flow cytometry. The expression of livin, caspase-3, caspase-7 and caspase-9 was detected by Western blot. After permanent left anterior descending artery occlusion, the rats were randomized to receive intramyocardial injection of DMEM without cells(vehicle group), or containing MSCs(MSCs group), MSCs(EGFP)(rAd-control/MSCs group) or MSCs(livin)(rAd-livin/MSCs group). Left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), the maximum increased rate of left ventricular pressure(-dp/dt_max) and the maximum decline rate of left ventricular pressure(+dp/dt_max) were recorded for evaluating the cardiac functions. RESULTS: The apoptosis of rAd-livin/MSCs was significantly decreased as compared with MSCs and rAd-control/MSCs(P<0.05). Meanwhile, the expression of caspase-3, caspase-7 and caspase-9 was significantly downregulated as compared with the other 2 groups(P<0.05). The cardiac function in rAd-livin/MSCs group was significantly improved as compared with DMEM group, and those in the other 2 groups got the similar results, but the function in rAd-livin/MSCs group was better improved. Meanwhile, the number of surviving cells in rAd-livin/MSCs group was significantly improved as compared with the other 2 groups. CONCLUSION: The apoptosis of MSCs is decreased after rAd-livin transfection, and the expression of caspase-3, caspase-7 and caspase-9 is also significantly downregulated while the expression of livin is significantly upregulated. Transplantation of livin-modified BM-MSCs by lentiviral vector results in better prognosis for treating myocardial infarction by enhancing cell survival.     

Retrovirus-mediated cellular repressor of E1A-stimulated genes inhibits neointima formation in the rat carotid artery after balloon injury

AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes (CREG) mediated by retrovirus on neointima formation in injured rat carotid. METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM α-actin and Ki-67 were detected. RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM α-actin expression. CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.     

Effects of angiotensin converting enzyme inhibitor on the arterial elastase after balloon injury

AIM: To study the effects of angiotensin converting enzyme inhibitor (ACEI) on elastase after balloon injury. METHODS: The carotid arteries and aortas twelve-weeks-old Wistar male rats were injured by balloon catheter. The rats were divided into experimental and control groups in which ACEI (temocapril-HCl, 10 mg·kg^-1 ·d^-1) and the vehicle were administered 2 days before injury respectively and the animals were sacrificed on day 2, 3, 5 and 10, respectively. In situ hybridization, immunohistochemistry and elastase activity bioassay were used for studying elastase. RESULTS: The intimal area on day 10 in the experimental group was significantly suppressed compared to that in the control rats (P<0.01), positive cells for elastase mRNA, elastase and elastase activities in the media in the experimental groups, at 2, 3 and 5 days were significantly less than those in the control groups respectively (P<0.01, P<0.05). CONCLUSION: AECI (temocapril-HCl) obviously suppressed the elastase expressions and activities of the medial smooth muscle cells after arterial injury, it suggests that elastase expression is related to the involvement of ACE.     

Exogenous KLF4 inhibits neointimal hyperplasia after balloon injury in rat

AIM: To observe the neointimal hyperplasia and to investigate the effect and mechanism of exogenous krüppel-like factor 4 (KLF4) on neointimal formation induced by balloon injury. METHODS: Adenovirus vector encoding the KLF4 (pAd-KLF4) was constructed and transfected into the balloon-injured carotid artery in rat. Neointima thickening was assessed using HE staining. Expression of exogenous KLF4 and the marker genes were detected by immunohistochemistry and RT-PCR. RESULTS: pAd-KLF4 stably expressed KLF4 protein in the transfected vascular wall. Overexpression of KLF4 significantly inhibited neointimal hyperplasia induced by balloon injury. The ratio of intima-to-media area (I/M) in pAd-KLF4 group (2.48±0.38) was significantly less than that in pAd group (0.52±0.15) (P<0.05). Proliferating cell nuclear antigen (PCNA) and c-Jun were significantly reduced in pAd-KLF4 group, compared with the pAd group (P<0.05). CONCLUSION: Overexpression of KLF4 inhibits the phenotype remodeling of vascular smooth muscle cells and neointimal hyperplasia induced by balloon injury.     

Effect of mesenchymal stem cells on secreting function of T lymphocytes from patients with idiopathic thrombocytopenic purpura in vitro

AIM: To analyze the effect of mesenchymal stem cells (MSCs) on secreting cytokines by T lymphocytes from patients with idiopathic thrombocytopenic purpura (ITP) in vitro.METHODS: Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells. Allogeneic T lymphocytes of ITP were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column. Then the stromal feeder layers of different numbers (2×103, 1×104, 5×104 per well) of MSCs treated with mitomycin were co-cultured with above-mentioned T lymphocytes. The supernatant were respectively collected on day 2, 4 and 6 after co-culture, then the levels of IL-2, IFN-γ, IL-4, IL-10 secreted by T lymphocytes were measured by enzyme linked immunosorbent assay (ELISA) dynamically.RESULTS: The levels of IL-2 and IFN-γ secreted by T cells from ITP were higher than those from normal control (P<0.05, respectively). Inversely, IL-4 and IL-10 were lower than those in normal control (P<0.05, respectively). After co-cultured with T lymphocytes, MSCs significantly inhibited the cytokine levels of IL-2 and IFN-γ secreted by T lymphocytes from ITP or health adults (P<0.05, respectively) in a dose dependent manner (P<0.05, respectively), and the effect was more obvious when co-cultured for 4 days or 6 days than that for 2 days (P<0.05, respectively). However, MSCs significantly promoted the releases of IL-4 and IL-10 by T lymphocytes from ITP patients (P<0.05, respectively) in a dose dependent manner (P<0.05, respectively), and the effect on IL-10 was in a time dependent way (P<0.05), while the effect on IL-4 had no obvious difference among 2 d, 4 d and 6 d(P>0.05). As for health control group, when cell numbers exceeded above 1×104, MSCs obviously promoted IL-4 and IL-10 levels secreted by T lymphocytes (P<0.05) in a dose dependent manner (P<0.05), and both of the effects were more noticeable when co-cultured for 4 d or 6 d than that for 2 d(P<0.05, respectively).CONCLUSION: MSCs regulate the balance between Th1 and Th2 reaction and partly correct ITP Th1 polarization in vitro.     

Effect of diammonii glycyrrhizinatis on collagen synthesis induced by angioplasty in rabbits

AIM:To observe the effect of diammonii glycyrrhizinatis (DG) on collagen synthesis induced by angioplasty in rabbits. METHODS:The right common carotid artery of male rabbits were injured with 3.5F balloon catheter. Four weeks after operation, arterial tissure collagen content, serum procollagen type I(PCI), procollagen type Ⅲ(PCⅢ) concentration, neointimal thickness and the rate of stenosis were measured. RESULTS:Arterial tissue collagen content, serum PCI, PCⅢ concentration, neointimal thickness and the rate of stenosis of low and high dose DG group is lower than those of injured group. CONCLUSION:DG could inhibit collagen synthesis and neointimal proliferation of rabbits carotid artery induced by angioplasty. It suggests that DG might have clinical potential prespective in prevention and therapy of restenosis.