Wenzhen Yu

Association of C3 Gene Polymorphisms with Neovascular Age-Related Macular Degeneration in a Chinese Population

Purpose: The purpose of this study was to determine whether the genetic polymorphisms of complement factor 3 (C3) are associated with neovascular age-related macular degeneration (AMD) in the Chinese population. Methods: A total of 123 unrelated Chinese Han patients with neovascular AMD and 130 control subjects were recruited. Their six single-nucleotide polymorphisms (SNPs) in the C3 gene, one in the complement factor H (CFH) gene and two in the complement factor B (CFB) gene were characterized. Their genotypes, allele frequencies, and odds ratios were analyzed. Results: The G allele of the C3 IVS2 rs2250656, but not other tested C3 SNPs of rs2230205, rs10411506, rs2230199, rs339392, and rs163913, was significantly associated with a reduced risk for AMD in the Chinese population (OR 0.605, 95% CI 0.39–0.93, p = 0.023), even after adjusting for age, gender, smoking status, CFH rs1061170, CFB rs4151667, and CFB rs641153 allele status (OR 0.58, 95% CI 0.35–0.96, p = 0.033). However, the C3 haplotype of A-A-C-A-T-T was identified as a statistically significant risk factor for neovascular AMD (OR 1.41, 95% CI 1.02–1.94). Furthermore, the C allele of the CFH rs1061170, but not the CFB rs4151667 and rs641153, was significnatly associated with increased risk for AMD (OR 3.09, 95% CI 1.55–6.15, p < 0.001). Conclusion: The G allele of C3 IVS2 rs2250656 may be a significantly protective factor for neovascular AMD in the Chinese population. This, together with low MAF of C3 R102G, may be partially responsible for the low prevalence of AMD in the Chinese population.

Association of genetic polymorphisms with response to bevacizumab for neovascular age-related macular degeneration in the Chinese population

AIMS To determine whether there is an association between CFH, ARMS2, HTRA1, VEGF, SERPING1 or C3 genotypes and patient response to treatment with intravitreal bevacizumab for neovascular age-related macular degeneration (AMD). MATERIALS & METHODS This was a multicenter prospective study. One hundred and forty four patients with neovascular AMD treated with bevacizumab were recruited from 13 centers. Twelve SNPs were genotyped using Sequenom. Visual acuity score (VAS), central retinal thickness and maximum thickness of lesion were measured at each visit. RESULTS For the CFH rs800292 polymorphism, mean VAS changes were 4.4, 8.7 and 15.5 letters in the CC, CT and TT genotype carriers (p = 0.009). For ARMS2 rs10490924, mean VAS changes were 3.6, 12.1 and 9.6 letters for the TT, TG and GG genotypes (p = 0.001). For HTRA1 rs11200638, mean VAS changes were 3.6, 12.3 and 9.6 letters for the AA, AG and GG genotypes (p < 0.001). CONCLUSION CFH, ARMS2 and HTRA1 genotypes may influence patient response to treatment with intravitreal bevacizumab for neovascular AMD.

Induction of interleukin-8 gene expression and protein secretion by C-reactive protein in ARPE-19 cells

C-reactive protein (CRP) is an acute phase reactant and its level rises rapidly during inflammation. Recent studies have suggested the potential involvement of CRP in the pathogenesis of age-related macular degeneration (AMD). To delineate the functional roles of CRP in inflammatory response by the ocular posterior segments, the effects of CRP on ARPE-19, an immortalized human retinal pigment epithelia (hRPE) cell line, were investigated in the present study. Treatment of ARPE-19 cells with CRP resulted in enhanced NF-kB nuclear translocation and dose-dependent transient induction of IL-8 mRNA synthesis and protein secretion. Stimulated expression of VEGF, but not MCP-1 by CRP was also observed. The induced IL-8 expression was transient and peaked at 12h post stimulation. In the presence of inhibitors for NF-kB, p38, MEK and JNK, the CRP-induced IL-8 production was abolished by 99.5+/-2.3, 97.8+/-2.1, 55.3+/-2.5 and 37.3+/-1.3%, respectively. Neutralization of Fc gamma receptors by anti-CD32 and CD64 antibodies produced 39.9+/-1.6 and 59.5+/-2.6% reduction, respectively, of CRP-stimulated IL-8 secretion, whereas that by anti-CD16 antibody had no effect. This study suggests that the pro-inflammatory effects of CRP in ARPE-19 cells may contribute to the inflammatory retinal diseases by induction of pro-inflammatory cytokines such as IL-8. This induction is mediated by NF-kB and multiple MAPK pathways through Fc gamma receptors.

Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy

Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with beta1 and beta3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P<0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.

siRNA Targeting Mammalian Target of Rapamycin (mTOR) Attenuates Experimental Proliferative Vitreoretinopathy

Purpose: To investigate the effect of mammalian target of rapamycin (mTOR) specific siRNA on proliferative vitreoretinopathy (PVR). Methods: Cultured human retinal pigment epithelial (hRPE) cell line D407 was treated with three mTOR specific small interfering RNAs. Cell proliferation, attachment, spreading, and migration were performed. The impact of the mTOR specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of hRPE cells. Results: Decreasing mTOR expression by about 82% using small interfering RNA resulted in a significant decrease in cell spreading and migration. Whereas retinal detachment occurred in 100% of the control group animals, co-injection of the mTOR specific siRNA substantially reduced the severity and incidence (50%) of retinal detachments. Conclusions: Gene therapy with mTOR specific siRNA attenuates PVR in a rabbit model of the disease. This may be a new approach to preventing PVR in humans.

Different Hereditary Contribution of the CFH Gene Between Polypoidal Choroidal Vasculopathy and Age-Related Macular Degeneration in Chinese Han People

PURPOSE To investigate whether 11 variants in complement factor H gene contributed differently in patients with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) of Chinese descent. METHODS We performed a case-control study in a group of Chinese patients with nAMD (n = 344) or PCV (n = 368) and contrasted the results against an independent control group comprising 511 mild cataract patients without any evidence of age-related maculopathy. Association analysis of allele and genotype frequencies was performed for 11 haplotype-tagging single-nucleotide polymorphisms (SNPs) at the CFH locus (rs1061170, rs1329428, rs1410996, rs2284664, rs375396, rs529825, rs551397, rs7540032, rs800292, rs2274700, and rs1065489). Multinomial logistic regression analyses were performed to estimate and compare the effect of these 11 CFH polymorphisms on AMD and PCV, using the wild-type genotype as reference. Differences in the observed genotypic distributions between cases and controls were tested by using χ(2) tests, with age and sex adjusted for using logistic regression. RESULTS CFH rs1065489 was not significantly associated with the nAMD phenotype in Chinese collections either on univariate or multivariate analysis (P > 0.05 for all comparisons). The other 10 SNPs of CFH were significantly associated with the nAMD phenotype. As for PCV, all 11 SNP markers were significantly associated with risk of PCV before or after correction for age and sex differences. Eight of the 11 SNP markers showed significant evidence of heterogeneity between AMD and PCV (P < 0.05 for all comparisons). CONCLUSIONS Our data suggest that the genetic architecture at the CFH locus is complex with some markers showing significant skewing of the genotypes toward nAMD or PCV in Asians. This further supports the clinical observation that nAMD and PCV could have distinct pathogenesis mechanisms, which will require larger studies to accurately dissect.

Targeting of integrin-linked kinase with a small interfering RNA inhibits endothelial cell migration, proliferation and tube formation in vitro.

Purpose: To investigate the role of integrin-linked kinase (ILK) in endothelial cell migration, proliferation and tube formation in vitro. Materials and Methods: Cultured RF/6A cells were knocked down for ILK using small interfering RNA (siRNA). Cellular ILK expression was quantified by real-time quantitative PCR and Western blot analysis. Cell migration was measured by cell counting in modified Boyden chambers, while microfilament dynamics were assessed by immunofluorescence analysis. Cell cycling was determined using a FACS Calibur flow cytometer, and the expression of cyclin D1 was also measured by the Western blot assay. The secretion of vascular endothelial growth factor (VEGF) in culture medium samples was assessed by ELISA, and capillary/tube-like network formation assays were performed using matrigel. Results: Both ILK mRNA and protein levels were vir- tually undetectable after transfection with ILK siRNA. In addition, there was a significant accumulation of cells in the G₀–G1 phase and a marked decrease in cell numbers in the S phase in ILK-specific siRNA-transfected cells, and the expression of cyclin D1 was decreased after transfection. The knockdown of ILK significantly inhibited cell migration ability by disturbing F actin assembly, and VEGF secretion in conditioned medium was also reduced by 33%. Furthermore, treatment with ILK siRNA suppressed tube formation in RF/6A cells and significantly reduced the overall length of the tubes. Conclusions: Knockdown of ILK with siRNA effectively inhibits endothelial cell migration, proliferation and tube formation in vitro.

Robo1/robo4: different expression patterns in retinal development.

Two members of the roundabout (Robo) family, Robo1 and Robo4, serve as neuronal guidance receptors. During neurogenesis, Robo1 and Robo4 participate in axonal guidance by mediating a repulsive signal. It has been reported that Robo4 is mainly expressed in the vasculature and that Robo1 is expressed both in neural and non-neural tissues. However, the roles of these Robo proteins in the mammalian vasculature are still unclear. In this current study, the expression patterns of Robo1 and Robo4 in the retinal vasculature were determined using C57BL/6J mice at postnatal days (P) 1, P3, P5, P7, P9, P12, P14, P17, P21 and adult mice (1month). We found that Robo4 was expressed not only in the retinal vessels but also in the retinal ganglion cell and photoreceptor layers during retinal development. Robo4 expression peaked at P3 and P9, which suggest that Robo4 may function in stabilizing the retinal vasculature. Robo1 expression was observed in the retina neuronal cells and vessels. Both Robo1 mRNA and protein expression showed a typical expression pattern, which related to Robo1s roles in the different stages of retinal vascular development in the murine retina. Robo1 displayed high expression levels at P1 (correlated with superficial vascular plexus formation) and P7 (correlated with deep vascular plexus formation). The high levels of Robo1 during these two well-defined phases of retinal capillary plexus formation indicate that Robo1 is likely to play a part in retinal neovascularization.

Effects of Semaphorin 3A on Retinal Pigment Epithelial Cell Activity

PURPOSE Semaphorin 3A (Sema3A), a chemorepellant guidance protein, has been shown to be crucial for neural and vascular remodeling. This study is designed to examine the effects of Sema3A on RPE cell activity both in vitro and in vivo. METHODS Retinal pigment epithelial were incubated with Sema3A, or VEGF- and Sema3A-containing medium. Cell proliferation, migration, cell cycle, apoptosis, cocultured human umbilical vein endothelial cells tube formation, VEGF receptor 2 (VEGFR2) and neuropilin 1 (Nrp1) receptor expression, VEGF- and pigment epithelium-derived factor (PEDF) concentration, and c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38MAPK) signaling pathway studies were measured. A rabbit proliferative vitreoretinopathy (PVR) model was used for in vivo study. Subconfluent ARPE19 cells were injected intravitreously with or without Sema3A. Data were analyzed with Graphpad Prism 5.0 software. RESULTS In vitro, Sema3A not only induced RPE cell cycle arrest and inhibited RPE migration under normal culture conditions, but also inhibited exogenous and endogenous VEGF165-induced cell activities. These activities included proliferation, migration, cell cycle arrest, JNK and p38MAPK signaling pathway phosphorylation, and cocultured endothelial cell tube formation. It is shown that both VEGF165 and Sema3A induced the upregulation of VEGFR2 and Nrp1 receptors. Activity inhibition was mediated by impeding VEGF165 utilization and possibly mediated by competitive inhibition of VEGF165 binding to its receptor VEGFR2, but not by the suppression of VEGF165 secretion. In vivo, Sema3A inhibited PVR, which is induced by RPE proliferation. CONCLUSIONS These results suggested that Sema3A could be a useful therapeutic strategy for preventing RPE malfunction.

Is asthma related to choroidal neovascularization

Background Age-related degeneration(AMD) and asthma are both diseases that are related to the activation of the complement system. The association between AMD and asthma has been debated in previous studies. The authors investigated the relationship between AMD and asthma systemically. Principal Findings The epidemiological study showed that asthma was related to choroidal neovascularization(CNV) subtype(OR = 1.721, P = 0.023). However, the meta-analysis showed there was no association between AMD and asthma. In an animal model, we found more fluoresce in leakage of CNV lesions by FA analysis and more angiogenesis by histological analysis in rats with asthma. Western blot demonstrated an elevated level of C3α-chain, C3α’-chain and VEGF. After compstatin was intravitreally injected, CNV leakage decreased according to FA analysis, with the level of C3 and VEGF protein decreasing at the same time. Significance This study first investigated the relationship between AMD and asthma systematically, and it was found that asthma could be a risk factor for the development of AMD. The study may provide a better understanding of the disease, which may advance the potential for screening asthma patients in clinical practice.