Abstract

Volume.118 Number.3

Identification of Molecular Targets for Intraocular Proliferative Diseases Using Genomic Approaches
Shigeo Yoshida
Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University

Intraocular proliferative diseases such as diabetic retinopathy (DR), age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) are a leading cause of decreased vision and blindness in Japan. In those diseases, retinal fibro (vascular) membrane formation above and beneath the retina plays a pivotal role in the primary pathology. The fibro (vascular) membrane formation reflects a wound healing response, but can be refractory when occuring excessively in the eye. In recent years, anti-vascular endothelial growth factor (VEGF) therapy has been introduced for the treatment of DR and AMD, and has revolutionized the treatment of intraocular neovascular diseases. However, the development of the additional molecular targeting therapy based on the exact pathogenesis of intraocular proliferation is still an unattained goal. Recent technological advancements in genomics have given investigators new opportunities to identify global gene expression in specific tissues in the eye. Therefore, we sought to develop a novel molecular targeting drug based on the gene expression profiling of human epiretinal membranes (ERMs).
I. Genome-wide gene expression profiling of human epiretinal membranes
In order to identify genes responsible for intraocular proliferation, we first determined the gene expression profiling of human retina, ERMs associated with proliferative diabetic retinopathy (PDR-ERMs), PVR (PVR-ERMs), or secondary ERMs. We next determined "highly expressed genes in PDR- and in PVR-ERMs" by comparing the gene expression profiles between PDR-, PVR-ERMs and the retina, and "genes that determine aggressiveness of ERMs" by comparing the gene expression profiles between PVR-ERMs and less-aggressive secondary ERMs. The former was subdivided by functional subsets of genes related to extracellular matrix, cell adhesion, differentiation, proliferation and other functions, and the latter related to cell adhesion and proliferation, etc. Subsequent analyses identified periostin, a matricellular protein, as an important molecule whose expression is enhanced specifically in proliferating ERMs compared to the retina, and facilitates their growth activity, because it was extracted at both comparison procedures.
II. Functional analysis of periostin
We found increased periostin expression in the vitreous of patients with both PDR and PVR. Immunohistochemical analysis showed colocalization of periostin and α-SMA in PDR- and PVR-ERMs. In vitro, periostin increased proliferation, adhesion, migration and collagen production in RPE cells. Periostin blockade suppressed migration and adhesion induced by transforming growth factor-β2 (TGF-β2) and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on experimental retinal and choroidal fibrovascular formation, and progression of experimental PVR without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for ERM formation as well as a promising therapeutic target for PVR.
III. Genome-wide expression profiling of ischemic retina
Because retinal ischemia is an important intermediate step in the pathogenesis of many intraocular proliferative diseases, we next sought to determine a profile of gene expression in ischemic retinas of a mouse model of oxygen-induced retinopathy (OIR). The differentially expressed genes in the hypoxic postnatal 12.5 retinas were broadly clustered into the development, inflammation, metabolism, signaling, antiapoptosis, cellular component, transport and glycolysis groups. The most upregulated gene among the differentially expressed genes in hypoxic retina was the macrophage inflammatory protein 1β (Mip-1β). Therefore, we next investigated the role played by MIP-1β in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a murine OIR. Our results showed that MIP-1β was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing antibody (Ab) against MIP-1β reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1β-regulated pathological neovascularization strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1β is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas.
IV. Development of innovative periostin-targeting ribonucleic acid drugs
Because the vitreous concentrations of periostin were not significantly correlated with those of VEGF in the PDR patients, it may be inferred that a periostin-targeting therapy could be added to anti-VEGF treatments. By employing a novel class of single-stranded RNAi agents that self-anneal into a unique helical structure, we determined the optimized sequence of periostin-targeting single-stranded RNAi. The optimized sequence had the significant inhibitory effect on experimental retinal and choroidal fibrovascular formation. Thus, the novel RNAi agent could be a potential therapeutic strategy for inhibiting the progression of intraocular proliferative diseases including DR and AMD.
Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 118: 241-282, 2014.

Key words
Intraocular proliferative disease, Genome-wide gene expression profiling, Expressed sequence tag analysis, Gene microarrays, Proliferative diabetic retinopathy, Proliferative vitreoretinopathy, Age-related macular degeneration, Epiretinal membranes, Periostin, Retinal ischemia, Macrophage inflammatory protein-1β, Open innovation
Reprint requests to
Shigeo Yoshida, M.D. Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University. 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan