大鼠视网膜色素上皮细胞分离的改良及其MMP表达

目的探索和优化大鼠视网膜色素上皮(RPE)细胞分离培养的方法,评价RPE细胞的存活状态及细胞基质金属蛋白酶(MMP)的表达,为相关眼底疾病的研究提供细胞来源。方法采用改良的三步酶消化法分离大鼠RPE细胞,并进行细胞体外培养。倒置显微镜观察细胞形态,细胞生长曲线评价不同培养代数的RPE细胞的增殖活力。免疫荧光检测CRALBP和角蛋白表达鉴定RPE细胞,并观察不同培养代数RPE细胞中多种基质金属蛋白酶的表达。结果分离培养的RPE细胞可呈梭形、六角形,并维持RPE细胞特征性蛋白CRALBP和角蛋白表达,但细胞内色素成分随着细胞分裂和传代次数的增多逐渐减少。基质金属蛋白酶MMP2、MMP3、MMP9和MMP10在第1代和第3代RPE细胞中均表达阳性,且表达强度未见明显改变。结论应用改良的三步酶消化法可以成功的分离培养大鼠RPE细胞,并在第1代和第3代RPE细胞维持基质金属蛋白酶MMP2、MMP3、MMP9和MMP10的阳性表达。体外培养的大鼠RPE细胞为研究视网膜相关疾病提供了细胞模型。     

Adrenomedullin in the eye

Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. We showed the presence of high concentrations of adrenomedullin-immunoreactivity in the vitreous fluid, and the levels were elevated in patients with proliferative vitreoretinopathy. Furthermore, adrenomedullin mRNA expression levels were elevated in the tissues of intraocular tumors and orbital tumors. Adrenomedullin is produced and secreted by cultured human retinal pigment epithelial (RPE) cells. Inflammatory cytokines and hypoxia are strong stimulators for the adrenomedullin expression in retinal pigment epithelial cells. Adrenomedullin stimulated the proliferation of retinal pigment epithelial cells both under normoxia and hypoxia. Dexamethasone (DEX) increased the adrenomedullin expression in two cultured cell lines of human retinal pigment epithelial cells; ARPE-19 cells and D407 cells, while it had no noticeable effects on the cytokine-induced adrenomedullin expression. These findings suggest that adrenomedullin is involved in the pathophysiology of inflammatory and neoplastic eye diseases as an autocrine or paracrine growth stimulator. The findings on glucocorticoid-induced AM expression raise the possibility that it may be related to the pathogenesis of some eye diseases, such as central serous chorioretinopathy and multifocal posterior pigment epitheliopathy, which are frequently seen in patients treated with high doses of glucocorticoids.     

Phenotypic change regulates monocyte chemoattractant protein-1 (MCP-1) gene expression in human retinal pigment epithelial cells

We investigated the expression profile of monocyte chemoattractant protein-1 (MCP-1) in human retinal pigment epithelial (RPE) cells under different culture conditions and evaluated the molecular mechanism responsible for MCP-1 gene expression in RPE cells. After cellular confluence, total RNA was extracted and used for RT-PCR. Medium conditioned by RPE was used for ELISA and Western blotting. The result showed that RPE cells cultured on plastic expressed MCP-1 constitutively in the absence of any stimuli. On the other hand, growing human RPE on laminin-coated flasks instead of plastic reduced the production of MCP-1. In the RPE cells cultured on plastic, IkappaB was degraded and A20 protein increased concomitantly. MCP-1 upregulation in RPE cells on plastic was attenuated by the addition of MG-132, a proteasome inhibitor. Also, the addition of pyrolidine dithiocarbonate (PDTC) and hypoxic conditions (0.5% O2) decreased MCP-1 production in these cells. These findings suggested that the expression profile of MCP-1 is regulated by phenotypic alterations of the RPE cells. And the increased MCP-1 expression in RPE cells cultured on plastic is caused via spontaneous activation of NFkappaB induced by susceptibility to oxidative stress.     

Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells

We previously demonstrated that differentiated retinal pigment epithelial (RPE) cells express high levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), and a critical balance between VEGF and PEDF is important to prevent the development of choroidal neovascularization. We report here that VEGF secreted by RPE cells upregulates PEDF expression via VEGFR-1 in an autocrine manner. PEDF mRNA and protein expression was downregulated by neutralizing antibody against VEGF in differentiated human RPE cells. VEGFR-1 neutralization decreased PEDF mRNA and protein expression whereas anti-VEGFR-2 antibody had no effect. Addition of placenta growth factor (PlGF) restored PEDF expression in the presence of anti-VEGF antibody. These results demonstrate a regulatory interaction between angiogenesis stimulators and inhibitors to maintain homeostasis in normal human retina.     

Induction of retinol esterification in retinal pigment epithelial cells by butyrate

Butyrate induced marked morphological changes in cultured human retinal pigment epithelial (RPE) cells. The cells assumed a flattened structure within six hours of exposure to butyrate. The butyrate-treated retinal pigment epithelial cells possessed an enhanced capacity to esterify retinol. Among all short chain organic acids tested, butyrate was by far the most effective, followed by pentanoate and hexanoate. The inductive effect of butyrate was specific for retinol esterification, since the incorporations of fatty acid into phosphatidyl choline and cholesteryl ester were not enhanced. Time-dependent, butyrate-enhanced retinol esterification may be related to the inhibition of cell proliferation. This represents the first report on the induction of retinol esterification in cultured retinal pigment epithelial cells.     

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).     

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.     

Platelet-derived growth factor gene expression in cultured human retinal pigment epithelial cells.

Gene expression of platelet-derived growth factor (PDGF) and its receptors in cultured human retinal pigment epithelial (RPE) cells was studied by using semiquantitative polymerase chain reaction. The RPE cells were found to express PDGF A- and B-chain genes as well as alpha- and beta-receptor genes with dominant expression of B-chain and beta-receptor isoforms. Phorbol myristate acetate (PMA) and thrombin increased the expression of PDGF B-chain gene to 19.8 +/- 1.75 and 15.9 +/- 1.84 fold (n = 3) of the control without affecting beta-receptor gene expression. PDGF produced by the RPE cells may play an important role in the pathogenesis of some ocular proliferative diseases.     

Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.     

The tyrosinase related protein-1 (Tyrp1) promoter in transgenic experiments: targeted expression to the retinal pigment epithelium.

Transgenic experiments targeting gene expression to the retinal pigment epithelium (RPE) require use of a pigment cell-specific promoter. We have chosen 1.4 kb and 4 kb from the promoter of the tyrosinase-related protein 1 gene (Tyrp1) for RPE-specific expression, since Tyrp1 mRNA and protein are detected already at midgestation in this epithelial layer. In eyes of transgenic embryos, expression of the Tyrp1-lacZ fusion construct led to strong and specific expression of beta-galactosidase in the RPE from day E10.5 onwards. The promoter thus proved useful to target expression of two different oncogenes to the RPE, a constitutively active tyrosine kinase receptor (Rfp/Ret) and SV40 T antigen (Tag). Tyrp1-Rfp/Ret transgenic mice developed microphthalmia, primarily induced by changes in the developing RPE. In addition, Tyrp1-Rfp/Ret expression induced proliferation of RPE cells leading to benign RPE tumors in the adult. Tyrp1-Tag transgenic mice developed malignant eye tumors of RPE origin, which invaded the optic nerve and led to metastasis into lymph nodes and spleen.     

Opticin production is reduced by hypoxia and VEGF in human retinal pigment epithelium via MMP-2 activation

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.     

Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway

Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway.

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N-(4-hydroxyphenyl)retinamide

ARPE-19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N-(4-hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE-19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide-induced neuronal differentiation was dose and time dependent, started within 1-2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde-binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two-dimensional gel followed by matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated that heat-shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.