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.
The introduction of anti-vascular endothelial growth factor (VEGF) therapy represents a landmark in the management of wet age-related macular degeneration (AMD). However, as a new therapy, several problems such as durability of the therapeutic effects, medication side effects, and medication selection have emerged. We should make appoint of improving the therapeutic effect and safety by realizing the limitation of the therapy, monitoring the clinical potential adverse reactions of anti-VEGF agents, and recommending individualized treatment.
ObjectiveTo investigate the inhibitory effects of 5-lipoxygenase (5-LOX) on oxygen-induced retinal neovascularization in mice and to explore its possible mechanisms.
Methods7-day-old C57BL/6J mice were randomly divided into normal group, oxygen induced retinopathy (OIR) model group, large-dose group, small-dose group and control group with 12 mice in each group. The mice with their mothers were kept in (75±2)% of oxygen environment for 5 days and then returned to normoxia for 5 days to establish the OIR model except for normal group. From postnatal day 12 to 17, the large-dose group and small-dose group received intravitreous injection of 5-LOX at dose of 100 mg/kg and 50 mg/kg respectively, while the control group received the same volume of 1% dimethyl sulfoxide. The mice in the OIR group received no treatment. The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. The mRNA expression of 5-LOX, vascular endothelial growth factor (VEGF)-a, VEGF receptor 2 (VEGFR-2) on retinal tissue were detected by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of 5-LOX, VEGF-a, VEGFR-2 and phosphorylation extracellular signal-regulated kinase (P-ERK) 1/2 on retinal tissue were detected by Western blot.
ResultsThe number of vascular cell nuclei breaking through the ILM in the large-dose group and small-dose group decreased significantly compared with the OIR group and control group (F=73.390, P < 0.05). The mRNA expression and protein expression of 5-LOX, VEGFa, VEGFR-2 on retinal tissue were decreased significantly in the large-dose group and small-dose group as compared with the OIR group and control group (F=92.668, P < 0.05). The difference of VEGFR-2 protein expression between large-dose group and small-dose group was not significant (F=2.118, P > 0.05). The differences of 5-LOX, VEGF-a, P-ERK 1/2 protein expression between large-dose group and small-dose group were significant (F=86.490, 165.128, 139.424; P < 0.05).
ConclusionHypoxia may induce 5-LOX expression in the retina. Retinal neovascularization was significantly inhibited by selective inhibition of 5-LOX.
Objective The observe the effects of interferon-inducible protein-10 (IP-10) on proliferation, migration and capillary tube formation of human retinal vascular endothelial cells (HREC) and human umbilical vein endothelial cells (HUVEC). Methods The chemokine receptor (CXCR3) mRNA of HREC and HUVEC were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). In the presence of the different concentrations of IP-10, the difference in proliferation capacity of HREC and HUVEC were analyzed by cell counting kit-8 (CCK-8) methods. Wound scratch assay and threedimensional in vitro matrigel assay were used for measuring migration and capillary tube formation of HREC and HUVEC, respectively. Results RT-PCR revealed both HREC and HUVEC expressed CXCR3. The proliferation of HREC in the presence of IP-10 was inhibited in a dosagedependent manner (F=6.202,P＜0.05), while IP-10 showed no effect on the inhibitory rate of proliferation of HUVEC (F=1.183,P＞0.05). Wound scratch assay showed a significant reduction in the migrated distance of HREC and HUVEC under 10 ng/ml or 100 ng/ml IP-10 stimulation (F=25.373, 23.858; P＜0.05). There was no effect on the number of intact tubules formed by HREC in the presence of 10 ng/ml or 100 ng/ml IP-10. The number of intact tubules formed by HREC in the presence of 1000 ng/ml IP-10 was remarkably smaller. The difference of number of intact tubules formed by HREC among 10, 100, 1000 ng/ml IP-10 and nonintervention group was statistically significant (F=5.359,P＜0.05). Conclusion IP-10 can inhibit the proliferation, migration and capillary tube formation ability of HREC and the migration of HUVEC.
Myopic choroidal neovascularization (MCNV) is one of the main reasons of vision loss in working population in Asia, which has brought economical and social-psychological burdens with high incidence in China, The precise pathogenesis of MCNV is unclear. Metamorphosia is the main reported symptom in these patients. The lesions were usually with smaller area, less leakage and relatively slow progression. Currently, intravitreal anti-vascular endothelial growth factor agents are now the established standard of care for MCNV, which was a major breakthrough in the treatment of MCNV achieving visual acuity improvement. Since the natural history, clinical features and therapy response of this disease is significant different from that in choroidal neovascularization secondary to age-related macular degeneration, the treatment dosing, frequency, retreatment criteria and the follow-up interval should been considerately. Facing the myopia boom in China, there is a need for the development of a precise definition and a more detailed classification for pathogenic myopia, optimize the outcome assessment and follow-up strategy, which should benefit to the further basically and clinical studies.
Objective
To determine the effect of posterior vitreous detachment on the prognosis of branch retinal vein occlusion (BRVO).
Methods
One hundred and sixteen patients (116 eyes) with BRVO who underwent vitreous examination were retrospectively studied.The relati onship of vitreous conditions to posterior segment neovascularization and macular edema was statistically investigated.
Results
In 40 ischemic cases,12 of 25 eyes (48.0%) with no posterior vitreous detachme nt (PVD) or partial PVD developed retinal or optic disc neovascularization ,or both,but only one of the 15 eyes (6.7%) with complete PVD developed neovasculariz ation during a mean follow-up period of 10.7plusmn;2.2 months (Plt;0.05) . Diffuse macular edema was found in 45 eyes (38.8%).The incidence o f macular edema was significantly higher in eyes with vitreomacular attachment (51.5%) than in those with vitreomacular separation (22.0%) (Plt;0.01).
Conclusion
It was suggest ed that compl ete PVD may play a role in protecting eyes with BRVO from posterior segment neov ascularization and macular edema.
(Chin J Ocul Fundus Dis, 2001,17:2-4)
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.
Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group,oxygen-induced retinopathy (OIR) group, empty vector group and treated group,14 mices in each group. Except for the control group,the mice in the other groups were exposed to (75plusmn;2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid.The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane（ILM） was observed and counted by optical microscope.Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group.The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups（F=39.006，Plt;0.001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinalneovascularization in OIR.
Objective To investigate the related factors of effects on distant visual acuity after photodynamic therapy (PDT) for choroidal neovascularization (CNV). Methods One hundred and thirty-five cases (135 eyes ) of CNV treated with PDT were observed. The gender, preoperative distant and near visual acuity, disease course, pathogeny, area of CNV, types of CNV ascertained by fundus fluorescein angiography (FFA), and changes of CNV in FFA were recorded. Multi-factor regression analysis of visual acuity within 1 month and 3 months after PDT was performed with SPSS statistics software. Results The distant visual acuity within 1 month postoperatively was related to the preoperative distant visual acuity, the area of CNV and the changes in the FFA（P=0.000,0.030,0.062）, and 3 months after PDT, it was related to the distant and near visual acuity preoperatively and the changes in the FFA（P=0.000,0.054,0.034）. The condition of distant visual acuity within 1 month postoperatively was related to the FFA type of CNV and the disease course（P=0.018,0.08）. Conclusion The smaller the area of CNV is, the better postoperative distant visual acuity would be. The proportion of improvement of visual acuity is relatively higher in patients with classic CNV. Early treatment for the patients with the indicatio may improve the visual acuity effectively. (Chin J Ocul Fundus Dis，2004，20：292-294)
Objective To observe the the inhibitory effect of recombined adenovirus mediated delivery of p21 (rAd-p21) on oxygen-induced retinal neovascularization in mice. Methods A total of 56 C57BL/6 mice at the age of seven days were divided into control group, phosphate buffer solution (PBS) group, rAd-p21 group and rAdno purpose gene control (rAd-NC) group, 14 mice in each group. The retinal neovascularization of PBS, rAd-p21and rAd-NC group were induced by oxygen, and received an intravitreal injection 1 mu;l PBS, rAd-p21 and rAd-NC at postnatal day 11, respectively.The rats of control group were not intervened. At postnatal day 17,RNV was determined by retinal flat mounts and retinal section; non-perfusion areas of retina were analyzed by Image-Pro plus 6.0 software; reverse transcription-polymerase chain reaction (RT-PCR) and Western blot was used to measure the mRNA and protein expression of p21 and CDK2. Results Compared with PBS and rAdNC groups, the retinal nonperfusion areas, neovascularization and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in rAd-p21 group were reduced significantly. Nonperfusion areas of retina in rAd-p21 group was less than that in PBS and rAd-NC groups, the difference among these three groups was significantly (F=101.634，P＜0.05). Compared with the other three groups, the level of p21 mRNA and protein in rAd-p21 group increased significantly (F=839.664, 509.817;P＜0.05); the level of CDK2 mRNA and protein in rAd-p21 group decreased significantly (F=301.858, 592.882;P＜0.05). Conclusion rAd-p21can inhibit oxygen-induced retinal neovascularization, up-regulated p21 expression and down-regulated CDK2 expression may be the mechanism.
