ObjectiveTo investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. MethodsMüller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). ConclusionTargeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.
ObjectiveTo investigate the expressions of contactin-1 (CNTN-1), vascular endothelial growth factor-C (VEGF-C), and its receptor VEGFR-3 (Flt-4) in primary gastric cancer and to explore the relevance among them and their correlation with clinicopathologic features of gastric cancer. MethodsThe VEGF-C, VEGFR-3, and CNTN-1 protein expressions of tumor tissues and normal gastric mucosa tissues in 68 patients with primary gastric cancer were analyzed by immunohistochemistry. The Flt-4-positive vessel density (FVD) and lymphatic vessel density (LVD) were also analyzed by VEGFR-3positive and D2-40-positive staining, respectively. ResultsThe positivity rate of VEGF-C, VEGFR-3, and CNTN-1 protein expression in the primary tumor was 57.4% (39/68), 60.3% (41/68), and 55.9% (38/68), respectively, which was significantly higher than that in the normal gastric mucosa tissues 〔20.6% (14/68), 23.5% (16/68), and 16.2% (11/68)〕， P=0.000. The expressions of VEGF-C, VEGFR-3, and CNTN-1 protein were significantly correlated with TNM stage, lymphatic vessel invasion, and lymph node metastasis (Plt;0.05). The expression of CNTN-1 protein was significantly correlated with VEGF-C (r=0.372, P=0.002) and VEGFR-3 protein expression (r=0.308, P=0.011). In tumor tissues of sixtyeight patients the FVD was (10.41±9.38)/HP, which was significantly lower than LVD 〔(18.19±7.44)/HP〕, P=0.000. Elevated FVD and LVD was significantly found in patients with tumor characterized by later TNM stage, severer lymphatic vessel invasion, and severer lymph node metastasis (Plt;0.05). The FVD of tumor was significantly correlated with VEGF-C (P=0.029) and CNTN-1 protein expression (P=0.003). The LVD of tumor was not significantly correlated with CNTN-1 (P=0.727), VEGF-C (P=0.173), and VEGFR-3 protein expression (P=0.924). The patients with positive expression of VEGF-C, VEGFR-3, and CNTN-1 protein showed poorer prognosis (Plt;0.05). ConclusionsElevated expression of CNTN-1 protein is observed in primary gastric cancer and correlated with VEGF-C and VEGFR-3 protein expression, indicating that combined detection has great value in prediction of invasive potential and prognosis. VEGF-C-mediated CNTN-1 overexpression may promote lymphatic invasion via lymphangiogenesis pathway in patients with gastric cancer.
Objective To explore the clinical significance of estrogen receptor α( ERα) , estrogen receptor β( ERβ) in non-small cell lung cancer( NSCLC) .Methods EnVision method was used to detect the expressions of ERα, ERβ, vascular endothelial growth factor( VEGF) , and microvessel density( MVD) in 54 NSCLC patients, 10 patients with lung benign lesions, and 10 normal controls. The interrelation between ERα, ERβ, VEGF, and MVD was analyzed. Results No obvious expressions of ERα and ERβwere observed in the normal lung tissues and lung benign lesions. The positive expression rates of ERα, ERβ, and VEGF in NSCLC were 20. 4% ( 11/54) , 64. 8% ( 35/54) , and 64. 8% ( 35/54) , respectively. There were no significant differences between ERαin regard to clinical parameters of NSCLC. But the expression of ERβwas dependent on pathological classification and differentiation of NSCLC. The expression of ERβ was significantly higher in adenocarcinoma than in squamous cell carcinoma( P lt; 0. 05) . The expression rate of ERβin well differentiated group was significantly higher than that in low, moderately differentiated group( P lt;0. 05) . There were significant differences between VEGF in regard to lymph node metastasis and TNM stage. The expression of ERαinterrelated with VEGF and MVD with r value of 0. 4 and 0. 685 respectively ( P lt;0. 05) . There was little correlation between ERβ and VEGF, MVD( P gt; 0. 05) . Conclusion Theexpression of ERβ correlates with pathological classification and differentiation of NSCLC, suggesting its significance in evaluating the pathological classification and malignant degree of NSCLC. The expression of ERαcorrelates with VEGF and MVD, suggesting that ERαpossibly promote micro-angiogenesis of NSCLC by VEGF pathway.
Objective To observe the influence of rAAV-mediated antisense vascular endothelial growth factor (rAAV-aVEGF165) on the expression of retinal VEGF in diabetic rats. Methods 40 Sprague-Dawley rats induced diabetic rat model by intraperitoneal injection with streptozotocin (STZ). 32 rats were involved in study besides death and blood sugar recovery in experimental process, 16 spragud-Dawleg (SD) rats were received intravitreal injection with rAAV-aVEGF165 (1010 pfu) as experimental group, another group of Sprague-Dawleg (SD) rats were injected with phosphate buffered saline (PBS) as control group. One and five month after model establishment, the expression of retinal VEGF was evaluate by immunhistochemistry and Western blot; the retinal vasular was examined by transmission electron microscopy. Results On 1 month,the expression of retinal VEGF was lowest in each group. On 5 month, the expression of retinal VEGF was decreased in experimental group which compared to control, the difference are statistically significant （t=23.87,Plt;0.01）. The transmission electron microscopy results showed that retina has no obvious chages in experimental group, however,contral group showed fragmental thickening and splitting of basement membrane, swelling and deformation of endothelia cells,fingerlike prcess into the capillary cavity,and uneven distibution of heterochromatin in pericytes. Conclusion rAAV-aVEGF165 can reduce the expression of retinal VEGF thereby preventing occurrence and development of diabetic retinopathy. rAAV is an effective vectors of eye antisense gene. （Chin J Ocul Fundus Dis，2008，24：255-258）
ObjectiveTo analyze the regulative rule of mRNA of vascular endothelial growth factor (VEGF) in mice with oxygen-induced retinopathy, and to elucidate the possible mechanism of occurrence of neovascularization in retinopathy of prematurity (ROP).MethodsSixty 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group. In oxygen-induced retinopathy group, 36 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days; in control group, 24 mice were raised in room air. Vascular perfusion of fluorescein and retinal stretched preparation were used to observe the morphologic changes of retinal vessels. Reversal transcriptionpolymerase chain reaction (RT-PCR) was used to observe changes of VEGF mRNA in each group. ResultsIn oxygen-induced retinopathy group, the morphologic characteristics of retinal vessels were the unperfused area at the center of superficial and deepseated vessels, and the neovascularization appeared at mid-peripheral retina after 2 days in relative hypoxia condition. The results of RT-PCR showed space-time corresponding relation between expression of VEGF and neovascularization, which meant that the transcription of VEGF mRNA decreased in hyperxia conditionand increased in relative hypoxia condition. ConclusionHypoxia is the main reason of occurrence of retinal neovascularization; increased expression of VEGF caused by relative hypoxia after hyperxia might be effective in reducing the occurrence of neovascularization in ROP.(Chin J Ocul Fundus Dis, 2005,21:292-295)
Objective To observe the effect of panretinal photocoagulation (PRP) on the expression of cyclooxygenase-2 (COX-2), vascular endothelial cell growth factor (VEGF) in epiretinal membrane of proliferative diabetic retinopathy (PDR). Methods Atotal of 35 patients (35 eyes) with PDR and underwent plana vitrectomy were enrolled in this study. The patients were divided into non-PRP group (19 patients, 19 eyes) and PRP group (16 patients, 16 eyes) depends on if they had received PRP before surgery. The epiretinal membranes stripped during operation were collected for pathological examination. The histopathological features was observed by haematoxylin and eosin stain. The expression of CD34, COX-2 and VEGF, and microvessel density (MVD) were measured by immunohistochemistry method. Results Many new dispersed capillary blood vessels were found in the thick epiretinal membranes of non-PRP group, while scattered small blood vessels were found in the relatively thin epiretinal membranes of PRP group. MVD value was (7.42±1.39) in the non-PRP group and (4.56±1.22) in the PRP group, which was lower than the non-PRP group (t=6.41, P<0.01). The expression of CD34, COX-2 and VEGF in the tissues of epiretinal membrane in PRP group were obviously lower than the non-PRP group (t=6.147, 5.944, 7.445; P<0.01). Conclusion PRP can effectively inhibit the expression of COX-2 and VEGF in epiretinal membrane of PRP patients.
Objective To evaluate the efficacy and safety of anti-vascular endothelial growth factor (VEGF) agents for advanced renal cell carcinoma. Methods We searched MEDLINE, EMbase, The Cochrane Library, CBMdisc and China Academic Periodical database from the establishment of each database to April 2009. We included randomized controlled trials (RCTs) that evaluated anti-VEGF agents (sunitinib, sorafenib and bevacizumab). The quality of the included trials was evaluated by two reviewers independently. Meta-analyses were conducted by the Cochrane Collaboration’s RevMan 4.2 software. Results Four RCTs involving 2 320 patients were identified. According to the different interventions for advanced renal cell carcinoma, we divided the patients into two groups: anti-VEGF agents monotherapy and anti-VEGF agents plus interferon combination treatment. Our meta-analyses showed: monotherapy was superior to interferon on inhibition of tumor progression [OR=0.38, 95%CI (0.29, 0.51), Plt;0.01] and control of tumor [OR=2.53, 95%CI (1.87, 3.43), Plt;0.01], but was not significantly different from interferon on the overall effective rate [OR=1.97, 95%CI (0.20, 19.57), P=0.56] and serious side effects [OR=1.98, 95%CI (0.90, 4.34), P=0.09]. There were significant differences between anti-VEGF agents plus interferon and interferon alone on inhibition of tumor progression [OR=0.67, 95%CI (0.53, 0.84), P=0.000 5], overall effective rate [OR=2.65, 95%CI (1.94, 3.61), Plt;0.01], control of tumor [OR=2.14, 95%CI (1.65, 2.78), Plt;0.01] and serious side effects [OR=2.63, 95%CI (2.09, 3.31), Plt;0.01]. Conclusion Compared with interferon, anti-VEGF agents could inhibit tumor progression more effectively. Moreover, the combination therapy with interferon could offer a more favorable overall effective rate for advanced renal cell carcinoma, but then followed by more serious side effects. We need to weigh the merits and demerits of drugs before making a clinical decision for advanced renal cell carcinoma.
Objective To explore a new method of treating early avascular necrosis of femoral head (AVNFH). Methods Sixty-nine New Zealand adult rabbitswith a mean weight of 2.8 kg after AVNFH presenting were randomly divided into three groups. In group A, deproteinized bone(DPB) combined with the recombinant plasmid pcDNA3.1/vascular endothelial growth factor 165(VEGF165) was implanted in the drilled channel of the necrotic femoral head. In group B, only DPB was implanted. In group C, channel was drilled without DPB or plasmid implanted. Femoral head specimens were obtained 3 days, 1, 2, 4, 8 and 16 weeks after operation. The expression of VEGF165 was examined by RT-PCR, Western blot and immunohistochemical techniques. X-ray testedbone formation generally. Angiogenesis and repair of the femoral head were observed by histological and histomorphometric analysis. Results In group A, the expressions of VEGF165 mRNA and protein were detected 3 days postoperatively, reached apex 1 week and lasted more than 3 weeks after implantation. The ratios of IOD of collagen type Ⅰ were 0.29±0.11, 0.55±0.13 and 0.67±0.10 IOD/μm2 respectively at 2, 4 and 8 weeks postoperatively and the ratios of IOD of new capillary vessels were 0.33±0.10and 0.57±0.16 IOD/μm2 respectively at 2, 4 weeks postoperatively in group A, showing statistically significant difference (Plt;0.01) when compared with groups B and D. X-ray test indicated much bone callus formed early. Conclusion Transfection of the VEGF165 gene can enhance local angiogenesis at early stage andDPBVEGF165 compound can improve bone formation. Deproteinized bone combined with VEGF165 gene provides a potential method for therapy of osteonecrosis.
Abstract: Objective To study the pathophysiological mechanism of the morphological change of immature pulmonary vessels in the piglet model of congenital heart defect with decreased pulmonary blood flow established with balloon atrial septostomy and pulmonary artery banding. Methods Twenty piglets at an age of one to two months were divided into three groups with random number table. For the control group (group C,n=6), small incisions were carried out on the right chest to produce a transient reduction in the pulmonary blood; for the lowmedium pulmonary artery stenosis group (group T1, n=7), the balloon dilator was delivered through the surface of the right atrium and septostomy and pulmonary artery banding were performed, and the systolic transpulmonary artery banding pressure (Trans-PABP) was controlled to be 20.30 mm Hg; For the severe pulmonary artery stenosis group (group T2, n=7), the same surgical procedures with group T1 were performed while TransPABP was controlled to be more [CM(159mm]than 3050 mm Hg.At 2 months after surgery respectively,a lung tissue of 1.0 cm×0.8 cm×0.8 cm from the lateral segment of the right middle lobe was taken out to be observed under optic microscope. The morphological change of the distal arterioles was detected. Furthermore, the content of vascular endothelial growth factor (VEGF) and matrix metalloproteinase2( MMP2) were also examined by the method of enzymelinked immunosorbent assay (ELISA). Results The model was successfully established in all the survival piglets of the group T1 and group T2. Two months after operation, the inner diameter of the pulmonary arterioles in group T1 was significantly higher than that in group C (82.89±10.72 μm vs.74.12±9.28 μm;t=－5.892, Plt;0.05), so as group T2 (85.47±5.25 μm vs.74.12±9.28 μm;t=-6.325, Plt;0.05); the number of arterioles per square centimeter (NAPSC) of group T1 was significantly lower than that of the group C (229.70±88.00 entries/cm 2 vs. 431.50±40.60 entries/cm2; t=39.526, Plt;0.05), so as group T2 (210.00±40.30 entries/cm2 vs. 431.50±40.60 entries/cm2; t=67.858, Plt;0.05). Two months after operation, the lung expression of MMP -2 and VEGF in group T1 was significantly lower than that in group C (58.30±19.60 ng/ml vs. 81.20±16.70 ng/ml, t=14.261, Plt;0.05; 17.80±3.00 pg/ml vs. 21.40±3.80 pg/ml, t=8.482, P＜0.05), so does group T2 (42.10±15.20 ng/ml vs. 81.20±16.70 ng/ml, t=27.318, P＜0.05; 12.30±3.20 pg/ml vs. 21.40±3.80 pg/ml, t=15.139, P＜0.05). Conclusion Structural remodeling of pulmonary extracellular matrix is an important feature of the piglet model of congenital heart defect with decreased pulmonary blood flow. The arterioles show significant hypoplasia or degradation. Change in the structural proteins and cytokines during the reduction of blood in the lung is the key to structural remodeling.
ObjectiveTo observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV).MethodsForty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV.ResultsThe results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P＜0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000).ConclusionsThe expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.