Objective To evaluate the effect of integrin-linked kinase (ILK) in the process of retinal neovascularization induced by vascular endothelial growth factor (VEGF). Methods The ILK activities of retinal choriodal endothelial cell line RF/6A were inhibited by LY294002 or siRNA knockdown. VEGF-induced changes of cell adhesion, proliferation, migration and endothelial cell tube-formation were measured then. The in-vivo effects of ILK were also assessed by intraperitoneal injection of LY294002 into an animal model of RNV. Results The cell adhesion measurements of control group, VEGF group, VEGF＋LY294002 group and VEGF＋siRNA group were 0.0726plusmn;0.01961, 0.1137plusmn;0.02631, 0.0837plusmn;0.01503 and 0.0853plusmn;0.02454 , respectively. The difference was statistically significant between VEGF group and control group（t =4.211，Plt;0.01）, and between (VEGF＋LY294002) group or (VEGF＋siRNA) group and control group (t =3.074, 2.91，Plt;0.01). The cell proliferation results of control group, VEGF group and VEGF＋LY294002 group were 0.4162plusmn;0.1392, 0.6412plusmn;0.2420, 0.4476plusmn;0.1834 , respectively. The difference was statistically significant between VEGF group and control group（t=2.608，Plt;0.05）, and between (VEGF＋LY294002) group and VEGF group（t=2.244，Plt;0.05）.The cell migration results of control group, VEGF group and VEGF＋LY294002 group were 83.66plusmn;30.283, 248plusmn;74.748, 138.5plusmn;38.167, respectively. The difference was statistically significant between VEGF group and control group（t=5.436，Plt;0.01）, and between (VEGF＋LY294002) group and VEGF group（t=3.682，Plt;0.01）. There was no obvious tube-formation after ILK activity was inhibited or knocked down. The non-perfusion areas were increased from （62798plusmn;16995.62）mu;m2 to （84722.65plusmn;10435.01）mu;m2 after intraperitoneal injection of LY294002 into animal model of RNV, the difference was statistically significant（t=3.476，Plt;0.01）. Conclusions ILK may play an important role in the process of VEGF-induced retinal neovascularization by regulating the cellular adhesion, proliferation, migration and tube-formation, as all those cellular functions were supressed obviously after the ILK activity was inhibited by LY294002 or the ILK expression was knocked down by siRNA.
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.
ObjectiveTo observe the expression of vascular endothelial growth inhibitor (VEGI, TL1A), vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in diabetes rats' serum, vitreous and retina, and discuss the role of VEGI in the pathogenesis of diabetic retinopathy (DR). MethodsA total of p70 adult male Wistar rats were randomly divided into 4 groups, the control group (10 rats), the diabetes mellitus (DM) 1 month group (20 rats), the DM 3 month group (20 rats) and the DM 6 month group (20 rats). Cytokines of serum and vitreous were determined by enzyme-linked immunosorbent assay (ELISA), and the concentrations of the cytokines in the retina were determined by immunohistochemistry on paraffin retinal sections. Hematoxylin-eosin (HE) staining of retina was used to estimate the pathological change of DR. The results were analyzed by one-way analysis of variances, independent samples t-test and LSD test. ResultsThe serum TL1A levels of the control group, the DM 1 month group, the DM 3 month group and the DM 6 month group rats were (92.09±2.05), (118.36±8.30), (85.90±7.51) and (78.90±4.88) ng/L respectively, the level of TL1A in serum of the DM 1 month group, the DM 3 month group and the DM 6 month group were significantly lower than that of the control group (F=77.405, P < 0.05). The concentration of serum TNF-α and IL-1β increased after DM model was established (F=3.508, 15.416; P < 0.05); the VEGF level in serum showed no difference between the groups (F=1.242, P > 0.05). The vitreous TL1A levels of the control group, the DM 1 month group, the DM 3 month group and the DM 6 month group were (91.50±8.18), (67.03±6.74), (47.44±4.92) and (46.01±4.62) ng/L respectively, every DM groups showed significant difference with the control group (F=114.777, P < 0.05); VEGF level in vitreous increased from 1 month after DM model was established (F=8.816, P < 0.05); TNF-α and IL-1β level in vitreous also showed an upward tendency (F=4.392, 3.635; P < 0.05). Paraffin section immunohistochemistry showed that the absorbance (also called optical density) of TL1A of the DM 1 month group and the DM 3 month group were significantly lower than that of the control group (t=6.851, 6.066; P < 0.05), but the DM 6 month group showed no difference with the control group (t=1.401, P > 0.05); the level of VEGF and TNF-α in DM groups were higher than that of the control group (tVEGF=-4.709, -16.406, -9.228; tTNF-α=-4.703, -6.583, -17.762; P < 0.05); the level of IL-1β were significantly higher in the DM 1 month group and the DM 6 month group (t=-4.108, -3.495; P > 0.05); but the DM 3 month showed no difference with the control group (t=-0.997, P > 0.05). HE staining of retina showed that the retina of the control group and the DM 1 month group had normal retinal structures, the DM 3 month group had retinal edema and disorganization, the DM 6 month group had severe retinal edema, deep stain of ganglion cells, and more neovascularization in inner plexiform layer. ConclusionVEGI is involved in the pathogenesis of DR, and it might interacts with VEGF, TNF-α and IL-1β to affect the development of DR.
Objective To investigate if lactic acid can promote the expression of vascular endothelial growth factor (VEGF) in the rat retinal explants.Methods The retinas of two-week neonatal SD rats were placed onto the culture plate inserts and incubated with Dulbeccoprime;s modified Eagleprime;s medium （DMEM） plus 2% fetal bovine serum (FBS) containing 10,20,30 mmol/L of lactic acid, respectively. Each group had 24 retinas. At 24 hours after incubation, the retinas were sectioned for light microscopy and the expression of VEGF was measured by real time PCR and Western blot. Results The cultured retinas maintained intact construction, and no cytolysis and apoptosis were observed under light microscope. RT-PCR showed the levels of VEGF mRNA were 0.74plusmn;0.06 for 10 mmol/L lactic acid group, 0.99plusmn;0.12 for 20 mmol/L group, and 1.45plusmn;0.17 for 30 mmol/L group respectively. VEGF expression was 0.34plusmn;0.15 for 10 mmol/L, 0.54plusmn;0.16 for 20 mmol/L, and 0.93plusmn;0.23 for 30 mmol/L group respectively by Western blot. Both PCR and Western blot showed 30 mmol/L of lactic acid significantly increased the levels of VEGF mRNA and VEGF expression. Conclusion The induction of retinal VEGF by lactic acid is concentration-dependent.
The intervention therapy targeting vascular endothelial growth factor (VEGF) has become a specific and effective method for the treatment of diabetic retinopathy (DR). However, some patients did not respond or responded poorly to anti-VEGF therapy, and its effects of eliminating edema and improving vision appear to be unstable in the same patient. Hypoxia-inducible factor-1α (HIF-1α), an important upstream transcriptional regulator of VEGF, is an oxygen concentration-sensitive protein expressed in tissues under hypoxia. It can simultaneously target many downstream target genes except VEGF, such as placental growth factor and angiopoietin-like protein 4, to cause blood-retinal barrier damage and neovascularization, and thus participate in various pathological changes of DR to promote the occurrence and development of DR. Therefore, direct intervention of HIF-1α or targeting one or more downstream target genes regulated by HIF-1α to treat DR may have better efficacy. In the future, the development of effective and safe HIF inhibitors or anti-VEGF with HIF-1α other target gene inhibitors may have broader clinical application prospects.
Proliferative vitreoretinopathy (PVR) is a common complication and major cause of blindness of ocular trauma. Many cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), participate in the process of the pathogenesis of traumatic PVR. VEGF competitively inhibits binding of PDGF to its receptor (PDGFRα), enables indirect activation of PDGFRα by non-PDGF ligands, resulting in reduced p53 expression, cell proliferation and migration, which is a key point in the pathogenesis of traumatic PVR.
Objective To observe the effects of dual targets intervention on the expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in diabetic rat retina. Methods Forty-eight Sprague -Dawley rats were randomly divided into control group (CON1 group) and diabetes mellitus group (DM group). The rats of DM group were induced with streptozotocin injection creating a diabetic model. Retinas were obtained at eight, 10, 12 weeks after DM induction from both groups. CTGF and VEGF mRNA levels were examined by realtime reverse transcriptionpolymerase chain reaction (RT-PCR). Based on the results of above experiments, 60 rats with same conditions were selected. Fifty rats were induced with streptozotocin injection creating a diabetic model, and 10 rats comprised the control group (CON2 group). Then the 50 diabetic rats were randomly divided into ranibizumab and CTGF shRNA dual targets intervention group, ranibizumab singletarget intervention group, CTGF shRNA singletarget intervention group and nonintervention group. Retinas were obtained at one week after intervention from all the groups. CTGF and VEGF mRNA levels were examined by RT-PCR. Results The levels of CTGF mRNA were significantly higher in DM group than that in CON1 group at the 8th weeks after DM induction, and this upregulation was maintained through the 12th week (t=-2.49, -2.67, -2.42;P＜0.05). There was no difference on VEGF mRNA levels between DM group and CON1 group at the 8th weeks after DM induction(t=-0.443,P=0.669). VEGF mRNA levels of DM group started to be significantly elevated over those in the CON1 group at the 10th week, and remained to be higher at the 12th week (t=-2.35, -2.57;P＜0.05). The VEGF mRNA of ranibizumab single-target intervention group was significantly lower than that in non-intervention group (t=-3.44,P=0.014), which was similar to CON2 group (t=-1.37,P＞0.05); however, the CTGF mRNA level was significantly increased as compared to the nonintervention group (t=2.48,P＜0.05). In the CTGF shRNA single-target intervention group, the levels of CTGF and VEGF mRNA were decreased as compared to the non-intervention group (t=0.23, -2.92;P＜0.05). In the ranibizumab and CTGF shRNA dual targets intervention group, the levels of CTGF and VEGF mRNA were decreased as compared to the non-intervention group (t=-6.09, -5.11;P＜0.001), which was similar to CON2 group (t=-1.16, 1.139; P＞0.05). Conclusions Both CTGF and VEGF gene expression are up-regulated in early diabetic rat retina, and the level of CTGF increased earlier than VEGF. Ranibizumab combined with CTGF shRNA could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina.