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Taiji Sakamoto, MD; Hitomi Sakamoto, MD; Todd L. Murphy; Christine Spee; Danilo Soriano, MD; Tatsuro Ishibashi, MD; David R. Hinton, MD; Stephen J. Ryan, MD

Arch Ophthalmol. 1995;113(4):512-520.

Abstract
Objective
To elucidate the mechanism of bovine choroidal endothelial (BCE) cell angiogenesis and, in particular, the role of retinal pigment epithelial (RPE) cells by use of an in vitro coculture assay system.

Methods
The BCE cells were isolated from choroidal tissues and cultured. They were embedded in type I collagen gel and incubated. The gel-embedded BCE cells were then covered with a monolayer of RPE cells, pericytes, choroidal fibroblasts, or additional BCE cells on culture day 0 or day 14. The BCE cells in culture formed a meshwork of tubelike structures. The length of the tubelike structures (micrometers per field) was quantified by image analysis, as an indicator of angiogenesis. The effect of RPE cells on normal and growth-arrested BCE cell tube formation was evaluated in this assay system. The mechanism of RPEinduced angiogenesis was studied by parallel experiments with the use of neutralizing antibodies against specific growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor β).

Results
Ultrastructural analysis revealed that the tubelike structures had features typical of choroidal endothelial cells. Cocultures initiated on day 0 revealed that BCE cell angiogenesis was promoted by overlying RPE cells and, to a lesser extent, by pericytes, choroidal fibroblasts, and additional BCE cells when compared with BCE cells without covering cells. In cocultures initiated after BCE tube formation (day 14), there was inhibition of BCE angiogenesis by overlying RPE cells when compared with cultures without overlay or with an overlay of BCE cells. The RPE cells stimulated tube formation of growth-arrested BCE cells less effectively than did normal BCE cells. Neutralizing antibody for basic fibroblast growth factor and vascular endothelial growth factor, but not transforming growth factor β, inhibited control and RPE-induced tube formation by BCE cells.

Conclusions
Overlying RPE cells stimulate the formation of tubelike structures by choroidal endothelial cells more effectively than do fibroblasts or pericytes but inhibit BCE tube formation in older cultures. The effect involves endothelial proliferation and differentiation. The stimulatory effect of overlying RPE cells can be inhibited by neutralizing antibodies to vascular endothelial growth factor and basic fibroblast growth factor, which suggests that these growth factors play an important role in this phenomenon.

Author Affiliations
From the Doheny Eye Institute and the Departments of Ophthalmology (Drs T. Sakamoto, H. Sakamoto, Soriano, and Ryan, Mr Murphy, and Ms Spee) and Pathology (Dr Hinton), University of Southern California School of Medicine—Los Angeles, and the Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka, Japan (Dr Ishibashi).; Dr T. Sakamoto is now affiliated with the Department of Ophthalmology, Kyushu University.

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