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.     

Inhibitory effect of canstatin RNA transfection on the growth of cultured rabbit vascular smooth muscle cells

AIM: To investigate the effect of canstatin on cultured rabbit vascular smooth muscle cells(VSMC). METHODS: By means of cationic liposome mediated method, canstatin RNA was transferred into cultured VSMC. The proliferation quantity of VSMC were determined by the cell counting method and thymidine(-TdR) incorporation. RESULTS: Canstatin RNA could be effectively transferred into cultured primary rabbit aortic smooth muscle cells by the cationic liposome-Dosper and could markedly inhibit VSMC proliferation. CONCLUSION: Transfection of canstatin RNA could inhibit the growth of VSMC in vitro.     

Mechanism of vascular endothelial growth factor in diabetic rat retinopathy

AIM: To study the molecular mechanism of vascular endothelial growth factor (VEGF) in pathogenesis of diabetes in rats. METHODS: The diabetic rat model was established by using streptozocin. The animals were divided into normal control (C group), diabetic for one month (M₁ group), for three months (M₃ group) and for five months (M₅ group). In situ hybridization and immunohistochemistry were conduced to observe the expression of VEGF on retinal digest preparation and paraffin section. RESULTS: 1. On paraffin section: the positive rate of VEGF expression in M₅ group was 67% by in situ hybridization and 89% by immunohistochemistry. There was only 34% positive expression of VEGF in M₃ group by immunohistochemistry. 2. On retinal digest preparation: the VEGF positive expression rate in M₅ group was 34% by in situ hybridization and 56% by immunohistochemistry. CONCLUSION: Both endothelial cells and Mural cells and Müller cells express VEGF. The source of VEGF may be from the paracrine pathway in early stage of diabetic retinopathy.     

Effect of exogenous HGF gene transfection on pulmonary perfusion and pressure in pulmonary artery hypertension in rabbits

AIM: To investigate the feasibility of exogenous hepatocyte growth factor (HGF) gene transfection to promote pulmonary collateral angiogenesis, improve pulmonary perfusion and reduce pulmonary artery pressure in the rabbit model of pulmonary artery hypertension (PAH). METHODS: The model rabbits of PAH were randomly divided into control group, empty vector group and HGF gene transfection group. The rabbits in HGF gene transfection group were transfected with Ad-HGF via intratracheal instillation. Pulmonary hemodynamic indicators were monitored in the 4th week after HGF gene transfection. Density of pulmonary vessels was examined with double-labeling immunofluorescence (endothelial cells were labeled with anti-FⅧ and vascular smooth muscle cells were marked with anti-α-SMA). Double-labeling immunofluorescence of FITC-lectin and anti-α-SMA was also performed to evaluate the pulmonary blood perfusion. RESULTS: Four weeks after transfection, the density of pulmonary arterioles of the rabbits in HGF gene transfection group was higher than that in control group and empty vector group (P<0.05), which was confirmed by double-labeling immunofluorescence. Pulmonary blood perfusion in HGF group was significantly increased compared with that in the other two groups, in which pulmonary arterial stenosis and occlusion were observed. The mean pulmonary artery pressure in HGF transfection group was much lower than that in control group and empty vector group (P<0.05). CONCLUSION: Four weeks after intratracheal adenoviral-mediated HGF gene transfection, pulmonary collateral vessels and pulmonary perfusion increase, and the pulmonary artery pressure is effectively reduced.     

Protective effect of human insulin-like growth factor 1 gene transfection on rat skeletal myoblasts with ischemic/reperfusion injury

AIM: To observe the protective effect of human insulin-like growth factor 1 (hIGF-1) on rat skeletal myoblasts with ischemic/reperfusion injury. METHODS: Myoblasts were isolated from SD rats, cultured, purified, and transfected with plasmid pLghIGF-1SN or pLgGFPSN. The myoblasts were divided into insulin-like growth factor (IGF) group (myoblasts transfected with pLghIGF-1SN), green fluorescent protein (GFP) group (myoblasts transfected with pLgGFPSN), and control group (untransfected myoblasts). The expression of hIGF-1 in myoblasts was investigated by immunocytochemistry, RT-PCR and ELISA. The proliferation rate of myoblasts 14 days after transfection was detected. To observe the protective effect of IGF-1 gene on skeletal myoblasts with ischemic/reperfusion injury 7 days after transfection, the apoptotic myoblasts were detected by the method of in situ TdT-mediated dUTP nick end labeling (TUNEL). The expression of bax and bcl-2 mRNA was detected by RT-PCR, and the expression of caspase-3 was determined by Western blotting. RESULTS: The expression of hIGF-1 in myoblasts transfected with pLghIGF-1SN was detected by immunocytochemistry, RT-PCR and ELISA, but not in myoblasts transfected by pLgGFPSN and untransfected myoblasts. The proliferation rate of myoblasts in IGF group was higher than that in other groups (P<0.05). The results of RT-PCR showed that the expression of bax mRNA significantly decreased and bcl-2 mRNA significantly increased in IGF group compared with GFP group (P<0.05). The results of Western blotting showed that the expression of caspase-3 significantly decreased in IGF group compared with GFP group (P<0.05). CONCLUSION: The transfection of hIGF-1 gene mediated by a retroviral vector produces a protective effect in rat skeletal myoblasts with ischemic/reperfusion injury. The mechanisms may be associated with down-regulating the expression of Bax and caspase-3 and up-regulating Bcl-2 expression.     

Effects of testosterone on endothelial function and intimal proliferation after balloon injury in male rabbit abdominal aorta

AIM: To investigate the effects of testosterone on endothelial function and intimal proliferation after balloon injury in male rabbit abdominal aorta. METHODS: 24 male New Zealand white rabbits were divided randomly into three groups: control group (n=8, sham castration), hypotestosteronemia group (n=8,castration) and testosterone replacement group (n=8,castration +testosterone undecanoate intramuscular injection,14mg/kg). Abdominal aorta was injured with 3 mm PTCA balloon after testosterone undecanoate had been injected for three days. Two weeks later, blood samples were obtained for detection of plasma testosterone, lipids, metabolic product of nitric oxide (NO₂^-／NO₃^-), superoxide dismutase(SOD) and malondialdehyde (MDA),and all the abdominal aorta were excised to be analyzed by computer. RESULTS: The intimal area of hypotestosteronemia group were significantly larger than that of other two groups(P＜0.01). plasma NO₂^-／NO₃^- and SOD levels were significantly decreased, while the total cholesterol(TC),triglycerides(TG), low density lipoprotein(LDL) and plasma MDA were significantly increased. No difference was observed between control group and testosterone replacement group in all parameters. CONCLUSION: Testosterone, at physiological level,had the effects of inhibiting the intimal proliferation and of protecting the endothelial function after balloon injury in male rabbit abdominal aorta.     

Protective effects of ulinastatin on kidney injury in rabbits after cardiopulmonary resuscitation

AIM: To investigate the effect of ulinastatin (UTI) on inflammatory cell infiltration, expression of tumor necrosis factor-α (TNF-α) and concentration of oxidative stress product malondialdehyde (MDA) in the kidney medulla after cardiopulmonary resuscitation (CPR) in rabbits and to access its potential mechanisms in kidney injury after CPR.METHODS: Twenty-four male New Zealand adult rabbits were randomly divided into control group and UTI group after return of spontaneous circulation (ROSC) from 5 min ventricular fibrillation induced by alternating current. The UTI at the dose of 2.5×10⁴ U/kg was administered immediately after ROSC to the animals in UTI group, the same volume of saline were injected in control group. The urinary output was recorded and the serum concentration of BUN and creatinine were detected every 4 h after ROSC. The animals were sacrificed 24 h after ROSC and the kidney medullas were obtained to observe the degree of inflammatory cell infiltration, the expression of TNF-α and the concentration of MDA.RESULTS: Six rabbits in control group and 6 animals in UTI group survived to the end point of experiment. The urinary output was decreased gradually to the lowest 8 h-12 h after ROSC and then increased in both groups. The urinary output in UTI group was more than that in control group 8 h after ROSC (P<0.05). The serum concentrations of BUN and creatinine were significantly lower in UTI group than those in control group 4 h after ROSC (P<0.05). The myeloperoxidase-positive cells in control group were much higher than that in UTI group (P<0.05). The expression of TNF-α and concentration of MDA in the kidney medullas in UTI group were lower than those in control group (P<0.05, P<0.01).CONCLUSION: The standard dose of UTI (2.5×10⁴ U/kg) administered in rabbits suffered from ventricular fibrillation for 5 min may alleviate the degree of inflammatory cell infiltration, decrease the expression of TNF-α and reduce the concentration of MDA in kidney medulla. UTI has protective effects on the renal function after CPR.     

Role of nitric oxide in proliferation and secretion effect of vascular endothelial cells induced by vascular endothelial growth factor

AIM: To investigate the role of nitric oxide in proliferation and secretion of vascular endothelial cells induced by vascular endothelial growth factorr (VEGF). METHODS: The in vitro cultured vascular endothelial cells of rabbit aorta were divided into control group, VEGF-treated group and VEGF+L-NAME treated group, the absorbance (A) value of vascular endothelial cells, endothelin-1(ET-1) and von Willebrand factor (vWF) in the supernatant were examined by WST-1 assay, radioimmunoassay and ELISA. RESULTS: The A value in VEGF and VEGF+L-NAME treated group were higher than that in control group (P＜0.01). A value in VEGF group was higher than that in VEGF+L-NAME group the ET-1 and vWF were markedly decreased in VEGF group compared with the control and VEGF+L-NAME treated group (P＜0.05, P＜0.01). These results indicated that VEGF promoted the proliferation and inhibited the secretion of ET-1 and vWF in vascular endothelial cells, and L-NAME inhibited the effect of VEGF. CONCLUSION:Nitric oxide is an important mediator in the process of stimulating proliferation and regulating secretion of vascular endothelial cells by VEGF.     

Effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts in rabbits

AIM: To investigate the effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts. METHODS: The external jugule veins were autografted into common carotid arteries in the same side in 20 New Zealand rabbits, which were divided evenly into experimental and control group randomly. The transplanted veins of experimental group were immersed in the adenovirus-mediated p21 gene solution for 15 minutes just before anastomosis and coated with c-fos antisense oligoneucleotide glue gel just after anastomosis, while the control was only treated with empty vector. The transplanted vascular sample were taken at 2 weeks after operation. The intimal thickness (IT), degree of restenosis (DR), expression of proliferating cell nuclear antigen (PCNA), quantity of VSMC were determined by immunohischemistry. RESULTS: The IT, DR and expression of PCNA, VSMC were decreased, compared to control group. CONCLUSION: Transfection of c-fos antisense oligoneuleotide and p21 gene inhibits the intimal proliferation of venous antografs.     

Effect of L-arginine on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats

AIM:To investigate the effect of L-arginine (L-Arg) on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats. METHODS:Rats were divided into three groups :sham operation group, balloon injury group(this group included balloon 48 h,7 d and 14 d subgroup) and balloon+L-Arg group. Neointima area were calculated morphologiocally. The expression of cyclin dependent kinase-2(CDK2),cyclin E and proliferation cell nuclear antigen (PCNA) were measured by means of immunohistochemical technique and computer image analyzer. RESULTS:After vascular balloon injury, the level of plasma NO decreased, CDK2、cyclin E and PCNA expressed in the media at 48 h and in the neointima at 7 d and 14 d but with low and undetected expression in the media, the expression of CDK2, cyclin E and PCNA increased with the intima thickening. Compared with balloon 14 d group, the plasma NO level increased (P＜0.01), the neointima area reduced by 59.1%(P＜0.01) and the positive expression indexes of CDK2, cyclin E and PCNA decreased by 36.1%, 46.3% and 76.2% respectively in balloon+L-Arg group (P all＜0.01). CONCLUSION:L-Arg can effectively repress intima proliferation after vascular injury, which may be associated with its inhibiting the proliferation of vascular smooth muscle cell through downregulating the excessive expression of CDK2, cyclin E and PCNA.     

Expression of vascular endothelial growth factor in chronic inflammatory mucosa of lacrimal sac

AIM: To study the expression of vascular endothelial growth factor (VEGF) in inflammatory mucosa of lacrimal sac. METHODS: Immunohistochemical S-P method was used to examine the expression of VEGF in the mocusa from 12 patients with chronic dacryocystitis and 8 volunteers. RESULTS: The positive rates of VEGF expression in different parts of the mocusa were: basal lamina: 44.3%±7.6％; surface epithelium: 16.9%±4.6％; connective tissue: 15.2%±4.9％, all normal mocusa of 8 cases were negative. There was a significant difference between the two groups (P<0.01), a significant difference among each part of the chronic inflammatory mocusa of lacrimal sac. CONCLUSION: VEGF may play an important role in hyperplasia of inflammatory mucosa of lacrimal sac.     

Effects of inhibition of Cripto gene siRNA on vascular endothelial growth factor of colon cancer cell line LS-174T

AIM:To study the effects of Cripto gene on vascular endothelial growth factor (VEGF) of colon carcinoma cells.METHODS:Cripto siRNA was designed and constructed. Colon cancer LS-174T cells were divided into 4 groups: control group and different dose (3.125, 6.25 and 12.5 nmol/L) of siRNA groups. After transfected for 24, 48 and 72 h, colon cancer cells were harvested to carry on the next tests. Expression of Cripto mRNA was determined with real-time PCR, and immunofluorescence isothiocyanate (FITC) labeling assay and Northern blotting were performed to examine the expression of protein and mRNA of VEGF, respectively. The cells in control group and cells transfected with 12.5 nmol/L siRNA were inoculated into nude mice respectively. 30 days after inoculated, the mice of two groups were executed, and immunohistochemical (ICH) assay was used to evaluate the VEGF protein of mice tumor. RESULTS:siRNA down-regulated the Cripto mRNA in a dose and time dependent manner. Protein and mRNA of VEGF in transfected cells reduced in a dose and time dependent manner. Compared to control, the expression of VEGF protein from ICH assay was lowered significantly (P<0.05).CONCLUSION:Cripto gene might contribute to the regulation of angiogenesis of colon carcinoma. The down-regulation of Cripto gene by siRNA can suppress angiogenesis of human colon cancer.     

Effects of angiotensin-converting enzyme 2 gene transfection on the expression of macrophage migration inhibitory factor in human endothelial cells

AIM: To implore the effects of recombinant angiotensin- converting enzyme 2 (ACE2) gene transfection on the expression of macrophage migration inhibitory factor (MIF) induced by angiotensin (Ang) II in cultured human endothelial cells. METHODS: A recombinant plasmid encompassing human ACE2 gene (pACE2) was constructed and transfected into human endothelial cells. Endothelial cells were stimulated with Ang II or Ang IV in the presence and absence of pACE2 gene transfer. The mRNA and protein levels of MIF in endothelial cells were determined by real-time PCR and Western blotting, respectively. RESULTS: The mRNA and protein expressions of MIF were strikingly enhanced after exposures of endothelial cells to 100 nmol/L Ang Ⅱ and 100 nmol/L Ang Ⅳ (P<0.01, respectively). However, significant downregulations of MIF mRNA and protein expression were observed in endothelial cells pretreated with pACE2 gene transfer (P<0.05, respectively). CONCLUSION: ACE2 gene overexpression contributes to diminishments of inflammation mediator MIF expression in endothelial cells, suggesting that ACE2 gene has anti-inflammatory properties to some extent and may provide novel therapeutic strategies for the inflammation-related diseases such as atherosclerosis.     

Microvessel density and expression of vascular endothelial growth factor in glomeruli of diabetic mice

AIM: To investigate the relationship between microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in glomeruli of diabetic mice. METHODS: Streptozotocin-induced diabetic mice as well as the control mice were involved in this study for 6 weeks. The body weight and blood glucose level of the mice in each group were weekly measured at certain time point. The morphological changes of the kidney were observed under light microscope, and the diameter, perimeter and area of the glomeruli were detected by an image analysis system. The expression of CD34 and VEGF in glomeruli was examined by immunohistochemistry method, and MVD and VEGF index were also calculated. RESULTS: In comparison with the control mice, the blood glucose level was significantly increased,and the body weight was decreased in diabetic mice(P<0.01). The diameter, perimeter and area of glomeruli in diabetic mice were significant greater than those in control mice (P<0.05). Increased expression of CD34 and VEGF in the glomeruli of diabetic mice was observed. Glomerular MVD of diabetic mice was significantly higher than that of the controls (P<0.01), and was positively correlated with the VEGF index (r=0.9979, P<0.05). CONCLUSION: VEGF may promote the angiogenesis in glomeruli of diabetic mice. The increase in VEGF expression may play a role in the pathogenesis of diabetic nephropathy.     

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Effect of microcapsulated catechin on expression of vascular endothelial growth factor in rats with adriamycin induced-nephrotic syndrome

AIM: To study the effect of microcapsulated catechin on vascular endothelial grower factor (VEGF) expression in rats with adriamycin induced-nephrotic syndrome.METHODS: 120 female SD rats were randomly distributed in control group,nephrotic group,dexamethason group,vitamin E group,catechin group and microcapsule group.Rat with nephrotic syndrome were induced by injection of adriblastine (5 mg/kg BW).VEGF concentrations in serum and urine were detected by ELISA assay.VEGF expression in kidney was measured by immunohistochemistry assay.RESULTS: At the end of 4th week and 6th week,VEGF concentration in other groups in kidney,serum and urine were higher than that in control (all P<0.01),and were lower than that in nephritic group (exclude Vit E group,all P<0.01).Serum and urine VEGF concentrations in microcapsule group were lower than those in catechin group (P<0.01,P<0.05,respectively).VEGF expression in microcapsule group in kidney was lower than that in catechin group,but it was not significant different.Urinary protein excretion at 24 h in microcapsule group was lower than that in catechin group (P<0.05),there was positive correlation among urine protein,serum VEGF level,urine VEGF concentration and renal VEGF expression at 24 h (all P<0.01).CONCLUSION: Catechin decreases urinary protein excretion in the rats with nephrotic syndrome through reducing the expression and secretion of VEGF.Microcapusulated catechin is benefit in decreasing the expression and secretion of VEGF.     

Effect of cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor gene transferred neonatal cardiomyocytes

AIM:To observe the secretion of vascular endothelial growth factor (VEGF) when adenovirus induced VEGF165 (AdVEGF165) gene transferred into neonatal cardiomyocytes in vitro,and to investigate the impact on heart function after transplanted the transferred cardiomyocytes into infarct myocardium in rats.METHODS:Neonatal cardiomyocytes were cultured in vitro and labeled with BrdU,then transferred by AdVEGF165.ELISA was applied to assay the expression and secretion of VEGF.Wistar rats,in which left descending branch of coronary artery was ligated,were randomly divided into four groups and transplanted into MI area with transferred cardiomyocytes (group Ⅰ),untransferred cardiomyocytes (group Ⅱ),AdVEGF165 (group Ⅲ) and culture medium (group Ⅳ),respectively.The echocardiograph was applied to evaluate the heart function before and after cell transplantation.Then the rats were executed and the hearts were harvested for histological (hematoxylin-eosin) and immunohistological (anti-BrdU dyeing) examinations.The vessels were also counted in injected area.RESULTS:ELISA indicated that the expression and secretion of VEGF in groupⅠwere higher than those in the rest (P<0.01).Echocardiograph revealed that the improvement of ejection fraction (EF) in groupⅠwas greater than that in other groups (P<0.01).Immunohistological study showed that the implanted cardiomyocytes survived in MI area.More blood vessels in groupⅠthan other groups were observed (P<0.01) by vessel-counting.CONCLUSIONS:AdVEGF165 gene transfer increased the VEGF-production,accelerated neovascularization in MI area and increased the blood flow.The method makes favor for the survival of implanted cells and greatly improves the heart function.