title: Opticin production is reduced by hypoxia and VEGF in human retinal pigment epithelium via MMP-2 activation.
abstract: Opticin, a small leucine rich repeat protein (SLRP) contributes to vitreoretinal adhesion. This study was conducted to investigate the effects of hypoxia and vascular endothelial growth factor (VEGF) on matrix metalloproteinase (MMP) mediated opticin production in retinal pigment epithelium (RPE) cells. Primary cultured human RPE cells were treated with hypoxia (low oxygen and cobalt chloride) or VEGF (0-100 ng/mL). The mRNA levels of opticin and the protein levels of intra and extracellular opticin in RPE cells were examined by RT-PCR and Western blot assay, respectively. Furthermore, the MMP activity was analyzed by zymography, and EDTA was used as an MMP inhibitor. Analysis of the effect of MMP-2 on opticin was performed by recombinant human (rh) MMP-2 stimulation in RPE cultures and by human vitreous sample digestion with activated rhMMP-2. Our results showed that opticin was expressed by primary cultured human RPE cells. Hypoxia and VEGF stimulation did not alter opticin mRNA and protein expression in RPE cells, but markedly decreased the protein levels of extracellular opticin following increased latent MMP-2 activity. The VEGF- and hypoxia induced opticin degradation in the culture medium was blocked by EDTA. Together, opticin levels in the culture medium were also reduced after rhMMP-2 treatment. In addition, opticin in human vitreous samples could be cleaved by rhMMP-2. These results reveal that VEGF and hypoxia could decrease opticin protein levels in the human RPE secretome, and that opticin may be an enzymatic substrate for MMP-2.
authors.affiliation: Eye Center, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jifang Road, Hangzhou 310009, China. zj2majin@yahoo.com.cn
authors.forename: Jin
authors.initials: J
authors.lastname: Ma
authors.forename: Tie Pei
authors.initials: TP
authors.lastname: Zhu
authors.forename: Morten C
authors.initials: MC
authors.lastname: Moe
authors.forename: Panpan
authors.initials: P
authors.lastname: Ye
authors.forename: Ke
authors.initials: K
authors.lastname: Yao
mesh_terms: D015687:Cell Hypoxia / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics; D059748:Proteolysis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D018709:Statistics, Nonparametric; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology; D014822:Vitreous Body / Q000187:drug effects / Q000378:metabolism
chemical_list: D016326:Extracellular Matrix Proteins; C404108:OPTC protein, human; D061988:Proteasome Inhibitors; D011509:Proteoglycans; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D046988:Proteasome Endopeptidase Complex