Proteomic and Transcriptomic Analyses of Retinal Pigment Epithelial Cells Exposed to REF-1/TFPI-2

Shibuya, Masabumi; Okamoto, H.; Nozawa, Takehiro; Utsumi, Jun; Reddy, Venkat N.; Echizen, Hirotoshi; Tanaka, Yasuhiko; Iwata, Takeshi

doi:10.1167/iovs.06-0434

Cited by 9 publications

(5 citation statements)

References 32 publications

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“…Additionally, WFDC1 , which encodes a protease inhibitor previously shown to be increased in macular RPE (29), demonstrated increased expression within the macula. Peripheral RPE cells were notable for increased expression of TFPI2 , which promotes RPE proliferation in vitro (30), and IGFBP5 , which has been associated with reduced neovascularization (31) (Fig. 2 B ).…”

Section: Resultsmentioning

confidence: 99%

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

Voigt

Mulfaul

Mullin

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

277

292

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The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

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Section: Resultsmentioning

confidence: 99%

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

Voigt

Mulfaul

Mullin

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

277

292

View full text Add to dashboard Cite

show abstract

“…Most of these studies have identified age-related alterations in key component proteins in metabolic pathways such as the MAPK (mitogen-activated protein kinase) cascade, lysosomal cathepsins, and stress responses markers [9, 30]. Several studies have also evaluated the mechanisms of RPE activation under oxidative stress, ageing, disease states, and normal growth [31] [26–28, 30, 32–34]. The current investigation employed a model of aged Bruch’s membrane to assess its impact on the RPE proteome.…”

Section: Discussionmentioning

confidence: 99%

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Glenn

Mahaffy

Dasari

et al. 2011

Graefes Arch Clin Exp Ophthalmol

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Purpose The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Methods Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Results Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. Conclusions This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.

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“…The western blotting for RB/pRB, p21 and p27, all of which are important cell cycle regulators, further supported the result of cell cycle analysis. Cell cycle regulation by TFPI‐2 was mainly found in normal cells, and its ability to affect cell cycle may be cell type‐specific. In malignant cells, TFPI‐2 was not found to affect cell cycle status.…”

Section: Discussionmentioning

confidence: 99%

Adenovirus‐mediated gene transfer of tissue factor pathway inhibitor‐2 inhibits gallbladder carcinoma growth in vitro and in vivo

et al. 2012

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Tissue factor pathway inhibitor‐2 (TFPI‐2) has been identified as a tumor suppressor gene in several types of cancers, but its role in gallbladder carcinoma (GBC) is yet to be determined. In the present study, TFPI‐2 expression in GBC tissues was examined, and its inhibitory activities against GBC growth were evaluated in vitro and in vivo after adenovirus‐mediated gene transfer of TFPI‐2 (Ad5‐TFPI‐2) was constructed to restore the expression of TFPI‐2 in GBC cell lines (GBC‐SD, SGC‐996, NOZ) and xenograft tumors. Immunohistochemical staining showed that TFPI‐2 was significantly downregulated in GBC tissue specimens. Ad5‐TFPI‐2 could significantly inhibit GBC growth both in vitro and in vivo. Apoptosis analysis and western blotting assay demonstrated that Ad5‐TFPI‐2 could induce the apoptosis of both GBC cell lines and tissues by promoting the activities of cytochrome c, Bax, caspase‐3 and ‐9 and suppressing Bcl‐2 activity. These data indicated that TFPI‐2 acts as a tumor suppressor in GBC, and may have a potential role in gene therapy for GBC. (Cancer Sci 2012; 103: 723–730)

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Proteomic and Transcriptomic Analyses of Retinal Pigment Epithelial Cells Exposed to REF-1/TFPI-2

Cited by 9 publications

References 32 publications

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Adenovirus‐mediated gene transfer of tissue factor pathway inhibitor‐2 inhibits gallbladder carcinoma growth in vitro and in vivo

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