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 doxycycline on matrix metalloproteinase activity and vascular remodeling in balloon-injured rat carotid arteries

AIM:To examine the inhibition of matrix metalloproteinase (MMP) activity by doxycycline (Doxy) and its effect on vascular smooth muscle cells (SMCs) proliferation,neointimal hyperplasia and vascular remodeling.METHODS: The model of rat common carotid artery injury was established by balloon-dilatation.Doxy was administered to the animals of treatment group at dose of 30 mg·kg^-1·d^-1.The activity of MMPs in the tissue of injured carotid arteries was measured by gelatin zymography.The thickness and area of neointimal,lumen area and the proliferation of SMCs were measured by histological and morphometric analysis.RESULTS:1.After Doxy treatment,the activity of MMP-9 in the carotid arteries was reduced by 26.3% and 34.5% compared to that in rats without Doxy treatment at 24 hours and 3 days after balloon injury,respectively (P<0.01).The activity of MMP-2 was also reduced by 40.0% at 7 days after injury (P<0.01).2.The thickness of neointimal were significantly decreased by 32.0% and 38.8% (P<0.01) and the lumen area was increased by 58.0% and 90.4 % at 14 and 28 days after injury in the Doxy-treated rats compared to those in control rats,respectively (P<0.01).Doxy treatment significantly reduced intimal SMCs proliferation from 62.76%±1.02 % in the controls to 43.23%±1.06% at 7 days after injury (P<0.01).CONCLUSION: Doxy treatment inhibits the activity of MMPs,the SMCs proliferation of intimal,neointimal hyperplasia and vascular remodeling,suggesting that Doxy treatment is useful in preventing restenosis after PTCA.     

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 parthenolide on neointimal hyperplasia after balloon injury

AIM: To detect the effect and underlying mechanism of parthenolide (PN) on neointimal hyperplasia. METHODS: After 1 week of high-fat feeding, 30 male New Zealand white rabbits (2.0~2.3 kg) were randomly divided into 6 groups: sham+NS, rabbits received 0.9% normal saline after sham operation; sham+DMSO, rabbits received DMSO after sham operation; balloon injury(BI)+NS, rabbits received NS after balloon injury; BI+DMSO, rabbits received DMSO after balloon injury; BI+PN low, rabbits received PN at 1 mg/kg after balloon injury; BI+PN high, rabbits received PN at 2 mg/kg after balloon injury. The drugs were intraperitoneal injected once a day after the operation until sacrifice. After fed with high-fat diet for 4 weeks, the intima-media thickness, the expression of caspase-1, IL-1β, the levels of IL-8, TC, TG, LDL and HDL in the serum were measured. RESULTS: Compared with sham+DMSO group, the thickness of intima, the amount of caspase-1, IL-1β and IL-8 in BI+DMSO group were significantly increased (P<0.05). The levels of caspase-1, IL-1β and IL-8 were significantly decreased in BI+PN high group compared with BI+DMSO group (P<0.05). CONCLUSION: Neointimal hyperplasia is suppressed by PN after balloon injury, the potential mechanism may be associated with its anti-inflammatory role.     

Effect of atorvastatin on neointimal hyperplasia of venous autografts in rats

AIM: To investigate the effect of atorvastatin on neointimal hyperplasia of autogenous vein graft in rats. METHODS: The model of autogenous vein graft was prepared by transplanting the external jugular vein into aorta in Wistar rats. The rats were divided into three groups: sham operation group, graft control group and graft experimental group. From three days after transplantation, the rats of autograft experimental group were treated by atorvastatin at a dosage of 5 mg·kg-1·d-1. Four weeks after treatment, venous autografts were removed at autopsy and cut into 4 μm sections. Histopathological examination was carried out to analysis the neointimal hyperplasia of grafted veins. Immunohistochemical staining was conducted to evaluate SMα-actin and PCNA expression of neointimal cells in venous autografts. RESULTS: In venous autograft control and experimental groups, SMα-actin-positive smooth muscle cells were proliferated and accumulated excessively in venous autografts, which resulted in significant neointimal formation and vascular lumen narrowing. Neointima quantitative assay revealed that the neointimal hyperplasia of venous autografts was suppressed obviously in graft experimental group, and its neointimal area and NIA/MA ratio of venous autografts were significantly lower than those in graft control group (P<0.01). Immunohistochemical assay indicated that the PCNA labeling index of neointimal cells was significantly lower in graft experimental group than that in graft control group (P<0.01). CONCLUSION: Atorvastatin significantly inhibits the proliferation of neointimal smooth muscle cells and the development of neointimal hyperplasia of venous autografts in rats. Atorvastatin is a powerful inhibitor of restenosis after vascular reconstructive operation with a potential for therapeutic use.     

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.     

Effect of KLF4 on viability,apoptosis of colorectal cancer cells and chemosensitivity to cisplatin

AIM:To investigate the effect of Krüppel-like factor 4 (KLF4) on the viability, apoptosis and cisplatin chemosensitivity of colorectal cancer cells. METHODS:KLF4 expression in colorectal cancer cell lines Caco2, SW480 and HCT116 was detected by Western blot. The SW480 cells were divided into pcDNA3.1 group (transfected with pcDNA3.1 empty plasmid), pcDNA3.1-KLF4 group (transfected with pcDNA3.1-KLF4 expression plasmid) and pcDNA3.1-KLF4+cisplatin group (treated with 1 mg/L cisplatin for 48 h after pcDNA3.1-KLF4 was transfected into SW480 cells). The protein levels of KLF4, p-IκBα, cyclin D1 and survivin were determined by Western blot. The cell viability was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry. The content of reactive oxygen species(ROS) was measured by DCFH-DA probe. RESULTS:The expression of KLF4 in the colorectal cancer cells were significantly lower than that in the human colon mucosal epithelial NCM460 cells (P<0.05). Compared with pcDNA3.1 group, the protein expression of KLF4 in pcDNA3.1-KLF4 group was significantly increased (P<0.05). Compared with pcDNA3.1 group, the cell viability and the protein expression of cyclin D1 and survivin were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1 group (P<0.05). Compared with pcDNA3.1-KLF4 group, the cell viability and the expression of cyclin D1 and survivin proteins were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1-KLF4+cisplatin group (P<0.05). CONCLUSION:Upregulation of KLF4 gene expression in colorectal cancer cells reduces the cell viability, induces apoptosis and increases the chemosensitivity of the cells to cisplatin. The mechanism may be related to the enhancement of intracellular ROS content and down-regulaton of the phosphorylation level of IκBα, the key molecule of NF-κB signaling pathway.     

Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

Role of vascular remodeling for urotensin Ⅱ in rat thoracic aorta after balloon injury

AIM: To explore the possible effect of UII in the process of remodeling after vascular injury. METHODS: The rat model of balloon injury in thoracic aorta was established. Male rats were randomized to 4 groups (n=5), including sham injury group, injury group, UII group (UII pumped into the rats after thoracic aorta balloon injury at 1.0 nmol·kg-1·h-1) and urantide group (urantide pumped into the rats after thoracic aorta balloon injury at 10 nmol·kg-1·h-1). At 21 days, the thoracic aortas were taken out to measure the changes of pathology, the expression of UII, the proliferation of VSMC and the expression of collagen. RESULTS: (1) At the 21 days after operations, the systolic blood pressure was higher in UII group than that in injury group ［(140.0±10.0) mmHg vs (132.0±3.4) mmHg, P>0.05］. The systolic blood pressure was also obviously higher than that in urantide group ［(140±10.0) mmHg vs (128.0±2.4) mmHg, P<0.05］. (2) Urotensin Ⅱ was expressed strongly in the injured area after thoracic aorta injury. (3) In contrast to injury group, the intimal thicken in urotensin Ⅱ group enhanced, the decrease in lumen area was marked (0.13±0.05 vs 0.07±0.02, P<0.05), the cell proliferation index was markedly increased (0.74±0.16 vs 0.40±0.11, P<0.01), and the expression of collagen was also markedly increased (counted as IOD, 318±127 vs 78±26, P<0.01). (4) In contrast to injury group, the decrease in lumen area was not abolished (0.09±0.03 vs 0.07±0.02, P>0.05) after chronic infusion of urotensin Ⅱ receptor antagonist urantide, the cell proliferation index was markedly increased (0.73±0.15 vs 0.40±0.11, P<0.01) and the expression of collagen was not statistically increased (counted as IOD, 200±79 vs 78±26, P>0.05). CONCLUSION: Urotensin Ⅱ expresses strongly in the myointimal cells after thoracic aorta injury in rat. The extra UII enhances the proliferation of VSMC and expression of collagen in the myointimal, increases the stenosis of injured vasculature, indicating that UII might take part in the process of repairing after vessel injury.     

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.     

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.     

Effect of transplantation of hRAMP1 gene-modified bone marrow MSCs on postangioplasty neointima formation in rabbits

AIM: To explore the effect of transplantation of human receptor activity-modifying protein 1 ( hRAMP1 ) gene-modified bone marrow mesenchymal stem cells (MSCs) on neointima formation after carotid balloon angioplasty in carotid atherosclerosis rabbits. METHODS: MSCs were collected through density gradient centrifugation and adherent culture. MSCs were transfected with adenovirus vector carrying hRAMP1 gene to generate hRAMP1 gene-modified MSCs (hRAMP1-MSCs). All animals with carotid atherosclerosis and balloon angioplasty were randomly divided into hRAMP-MSCs group, MSCs group and control group. After the model was established, MSCs transfected with pAd2-EGFP-hRAMP1 or pAd2-EGFP and PBS were injected to the ear vein,respectively. The injured carotid arteries were harvested to detect the homing of MSCs,reendothelialization and neointima thickness 7 d, 14 d and 28 d after cell transplantation. The plasma samples were collected for detecting vascular endothelial growth factor (VEGF) by ELISA. The expression of endothelial nitric oxide synthase (eNOS) in injured carotid arteries was measured by Western blotting. RESULTS: The expression of CD31 and EGFP was observed in the neointima at different time points in hRAMP1-MSCs group and MSCs group. Compared to control group, the reendothelialization of carotid significantly increased in both hRAMP1-MSCs group and MSCs group at different time points (P<0.05), and that in hRAMP1-MSCs group showed better than that in MSCs group (P<0.05). The area of neointima and the rate of restenosis were lower in hRAMP1-MSCs group and MSCs group than those in control group, and those in hRAMP1-MSCs group were significantly lower than those in MSCs group. The plasma level of VEGF and the expression of eNOS in the injured carotid arteries were significantly higher in both hRAMP1-MSCs group and MSCs group than those in control group at different time points (P<0.05), and those in hRAMP1-MSCs group were better than those in MSCs group (P<0.05). In the injured carotid arteries, the expression level of proliferating cell nuclear antigen (PCNA) in hRAMP1-MSCs group was the lowest,with the middle level in MSCs group and the highest level in control group. CONCLUSION: The hRAMP1 gene-modified MSCs are better in promoting reendothelialization and attenuating neointima than natural MSCs. The recombinant hRAMP1 adenovirus vectors dont affect the differentiation potential of MSCs into endothelial cells.These findings indicate that the modified stem cells have the potency of more effective reendothelialization to decrease restenosis after angioplasty.     

Recombinant human interleukin-10 inhibits vascular smooth muscle cell proliferation by TNF-α and PDGF-BB in vitro

AIM: To determine the effects of recombinant human interleukin-10(rhIL-10) on proliferation of vascular smooth muscle cells(VSMC) by TNF-α and PDGF-BB and neointimal hyperplasia after rat carotid arterial injury.METHODS: Rat aortic VSMC was cultured and treated with rhIL-10 with or without tumor necrosis factor-α(TNF-α) and platelet-derived growth factor-BB (PDGF-BB), respectively.Proliferation of VSMC was quantified by colormetric assay.Cell cycle analysis was performed by flow cytomertry.SD rats were treated with recombinant human IL-10(rhIL-10) for 3 days after carotid arteries injury.Neointima to media area ratio at the site of arterial injury was measured at 28 days after balloon injury.RESULTS: Compared to control,both TNF-α and PDGF-BB stimulated VSMC proliferation. rhIL-10 alone had no effect on VSMC growth.With TNF-α or PDGF-BB stimulation,rhIL-10,at dose as low as 10 μg/L,inhibited VSMC growth( P <0.05) for both cases.Cell number in G 0/G 1 phase of PDGF-BB and rhIL-10 co-treatment group was higher than those of PDGF-BB treatment alone( P <0.01) by flow cytometry analysis.The same results were observed in TNF-α and rhIL-10 co-treatment group( P <0.01).Compared with the arterial injury group,neotima/media area ratio of recombinant human IL-10 group was reduced at 45%( P <0.01).CONCLUSIONS: The anti-inflammatory cytokine rhIL-10 inhibits TNF-α and PDGF-BB-induced VSMC proliferation,respectively.These results suggest the possibility that recombinant human IL-10 as a potential therapeutic approach prevents neointimal hyperplasia.     

Prevention and treatment of restenosis after PTA operation in rabbit artery by 3,3-diindolylmethane

AIM: To evaluate the safety and efficacy of 3,3-diindolylmethane (DIM) on neointimal proliferation of rabbit artery after balloon angioplasty. METHODS: Restenosis models of carotid artery after balloon injury was established in rabbits. 30 rabbits were randomly divided into 4 groups: sham-operated group, model group, low-dose DIM group and high-dose DIM group. DIM was given to the rabbits in low-dose treatment group (2 mg·kg-1·d-1) and high-dose treatment group (8 mg·kg-1·d-1) once a day from 3 d before operation to 4 weeks after operation. The two treatment groups were administered with intraperitoneal injection of emulsified DIM, while the other two groups with saline at the same volume. All rabbits were killed after 28 d and carotid arteries were removed. With HE staining, automatic image analysis and immunohistochemistry, artery morphology was observed and the thickness of arterial intima and media was measured. Platelet derived growth factor (PDGF) and transfer growth factor β1 (TGF-β1) were examined. The expression of 〖STBX〗bcl-2〖STBZ〗 gene was detected by in situ hybridization (ISH) with computer-assisted picture analysis system. RESULTS: No significant difference was found between low-dose DIM group and model group in intimal thickness, media thickness, luminal area and the expression of PDGF and TGF-β1 in low-dose DIM group (P>0.05). The intimal thickness was decreased in high-dose DIM group compared with model group and low-dose DIM group (P<0.01). The luminal area was significantly larger in high-dose DIM group than that in model group and low-dose DIM group (P<0.01). The expression of PDGF and TGF-β1 in low-dose DIM group was significantly reduced compared with model group and low-dose DIM group (P<0.01). CONCLUSION: The inhibition and treatment of DIM on arterial restenosis are safe and effective. The effect might be achieved by preventing neointimal proliferation and the expression of PDGF and TGF, enhancing apoptosis in the vascular smooth muscle cells.     

Effect of KLF6 gene on apoptosis of THP-1 cell-derived macrophages

AIM: To investigate the effects of Kruppel-like factor 6 (KLF6) over-expression on the viability, apoptosis, reactive oxygen species (ROS) level and AKT signaling pathway of THP-1 cell-derived macrophages. METHODS: Human monocyte cell line THP-1 was induced to differentiate into macrophages by phorbol myristate acetate (PMA), and the macrophages were randomly divided into pcDNA3.1 group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+pcDNA3.1 group and ox-LDL+pcDNA3.1-KLF6 group. pcDNA3.1 was transfected according to Lipofectamine^TM 2000 Kit. The cell viability, apoptotic rate and ROS level were detected by MTT assay, flow cytometry with Annexin V-FITC/PI double staining and H₂DCF-DA probing, respectively. The protein levels of Bcl-2, Bax and p-AKT were determined by Western blot. RESULTS: After pcDNA3.1-KLF6 was transfected into the macrophages, the expression of KLF6 was increased significantly (P<0.05). ox-LDL significantly inhibited the viability of the macrophages, induced apoptosis and ROS production, up-regulated the protein expression of Bax, and down-regulated the protein levels of Bcl-2 and p-AKT (P<0.05). Over-expression of KLF6 significantly reduced the effects of ox-LDL on cell viability, apoptosis, ROS level and the protein levels of Bcl-2, Bax and p-AKT (P<0.05). CONCLUSION: KLF6 significantly reduces the apoptosis of THP-1 cell-derived macrophages induced by ox-LDL, which may be related to the reduction of ROS level and activation of AKT signaling pathway.     

Cyclosporine A inhibits intima proliferation of rat abdominal aortas injured by balloon

AIM: To observe the effect of cyclosporine A on intima hyperplasia of rat abdominal aortas injured by balloon.METHODS: Thirty-six healthy adult male SD rats were randomly divided into 3 groups: sham group (n=12), balloon-injured group (n=12) and cyclosporine A treatment group (n=12).From the 3rd day before injury to the 30th day after injury, the rats in cyclosporine A treatment group were treated with cyclosporine A at a dose of 12.5 mg/kg everyday, while the rats in sham group and balloon-injured group were fed with the same volume of water.On the 30th day after injury, the specimens were obtained from the rats.HE staining and immunohistochemistry were used to measure the expression level of calcineurin (CaN) in arterial wall.Pathological changes of the arterial wall were observed under light microscope.The expression of CaN and nuclear factor 3 of activated T-cells(NFATc3) in the abdominal aortas was detected by the technique of real-time PCR.RESULTS: Intimal hyperplasia was observed in balloon-injured rats.The neointima was not uniform in thickness on the 30th day.The thickness of the intimal layers and the ratio of the intimal to the medial layers in cyclosporine A group were obviously lower than those in balloon group(P<0.05).Immunohistochemistry revealed that calcineurin expression increased after balloon injury, but the expression of calcineurin in cyclosporine A group was obviously decreased as compared with balloon group (P<0.05).The results of real-time PCR showed that the mRNA expression of calcineurin and NFATc3 in cyclosporine A group was significantly lower than that in balloon-injured group (P<0.05).CONCLUSION: Cyclosporine A attenuates restenosis by suppressing the CaN-NFATc signaling pathway.     

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.     

Effect of neural stem cells transfected with VEGF on expression of NSE in rat brain injury irradiated by radioaction

AIM: To explore the effect of neural stem cells (NSC) transfected with vascular endothelial growth factor (VEGF) on brain injury irradiated by radioaction. METHODS: Mature Sprague-Dawley (SD) rats were randomly divided into 4 groups: control group, radiation brain injury group, NSC therapeutic group, NSC transfected with VEGF (NSC/VEGF) therapeutic group. NSC transfected with VEGF was transplanted into the rat brain of therapeutic group after preparation of radiation encephalopathy model for 1 week. The brain tissue was taken at week 1, 2, 3, 4 and 6 after transplantation. Immunohistochemical method was used to assay the count of neuro-specific enolase (NSE) stained cells in brain tissue. Western blotting was used to detect the expression of NSE protein. RESULTS: Compared to control group, the count of NSE stained cells in brain of radiation brain injury group was decreased significantly (P<0.05). Compared to that in radiation brain injury group, the count of NSE stained cells in brains was enhanced in NSC therapeutic group and NSC/VEGF therapeutic group since 3 weeks after transplantation (P<0.05). Compared to that in NSC therapeutic group, the count of NSE stained cells in NSC/VEGF therapeutic group was more enhanced since 6 weeks after transplantation (P<0.05). Compared to that in radiation brain injury group, the expression of NSE protein was enhanced in NSC group and NSC/VEGF group since 3 weeks after transplantation (P<0.05). Compared to that in NSC group, the expression of NSE protein in NSC/VEGF group was more enhanced since 6 weeks after transplantation (P<0.05). CONCLUSION: Transplantation of NSC transfected with VEGF may increase the content of NSE in rat brain of radiation encephalopathy model.     

Effects of heterogously-expressed RON receptor tyrosine kinase on motile/invasive ability of human colorectal cancer cell line RKO

AIM: To investigate the roles of overexpression of RON receptor tyrosine kinase in motile/invasive ability of human colorectal cancer cell line RKO. METHODS: A eucaryotic expression vector pDR2 containing full-length wt-RON cDNA was transfected into the colorectal cancer cell line RKO and a stable expression clone was obtained. The motile/invasive ability was tested by wound healing test and the transwell migration assay. The expression of E-cadherin was measured by Western blotting. RESULTS: Motile ability of transfected RKO was greatly promoted by transwell chemotaxis assay (P<0.01). The wound healing time showed statistical difference as of (42.50±4.12) h, (69.50±2.52) h and (70.50±3.42) h, respectively in transfected group, untransfected group and vector control group. After knocking down RON by siRNA, the motile became less than that in control group (P<0.01). E-cadherin expression in transfected RKO was decreased significantly due to pDR2-wt-RON transfection. CONCLUSIONS: Overexpression of wt-RON led to the decrease in expression of E-cadherin and decreased cancer cell-cell adhension. At the same time, migration/invasion ability was promoted. Taken together, abnormal accumulation of RON might play potential roles in invasion/metastasis of colorectal cancer. RNAi can block motile/invasion ability mediated by RON.     

Inhibitory effects of local transfection of vascular endothelial growth factor 165 gene on intimal hyperplasia of artery in rabbits after operation injury

AIM: To observe the effects of local transfection of vascular endothelial growth factor 165 (VEGF165) gene on inhibiting intimal hyperplasia and restenosis of artery in rabbits after operation injury, and its possible mechanisms. METHODS: Microsurgery injury was used to establish the intimal injury model of right external iliac artery in rabbits. 105 male New Zealand rabbits were randomly divided into 3 groups (35 rabbits in each group). Group A was physiological saline control group, group B was pBudCE4.1-transfected group, group C was pBudCE4.1/VEGF165-transfected group. The physiological saline, pBudCE4.1 and pBudCE4.1/VEGF165 transfection solutions were injected into injured vessel walls of above-mentioned groups. The injured vascular specimen was harvested for pathologic examination, electric microscope observation, RT-PCR examining and immunohistochemical staining. RESULTS: Rabbit intimal thickness and area of vessel walls in group C at every time point after operation were significantly less than those in group A and group B (P<0.01). The stenosis ratio of vessels in group C at 28 days after operation decreased by 51.6% and 49.8%, respectively, as compared with groups A and B. The expression of VEGF165 mRNA and VEGF165 positive cells in Group C were increased significantly than those in group A and B at every time point after operation (P<0.01). CONCLUSION: Local transfection of VEGF165 gene restrains intimal hyperplasia and restenosis of vessels, which lays a foundation for future gene therapy of vascular intimal hyperplasia.