Production and accumulation of thrombospondin-1 in human retinal pigment epithelial cells

PURPOSE. To investigate the production and release of thrombospondin-1 (TSP -1), a natural inhibitor of angiogenesis, by human retinal pigment epitheli al (RPE) cells to clarify the possible role of TSP-1 in maintaining intraoc ular angiogenesis. METHODS. Human RPE cells were isolated from a human cadaveric eye and cultu red in medium with 5% newborn calf serum. TSP-1 messages in the purified RN A of RPE cells were analyzed by reverse transcription-polymerase chain reac tion (RT-PCR). Intracellular TSP-1 peptides were detected by cytofluorograp hic analysis. TSP-1 peptides in the culture medium on RPE cells were measur ed by sandwich enzyme-linked immunosorbent assay (ELISA). TSP-1 specific im munofluorescent staining was tested in RPE cells cultured on glass slides a nd in a human retinal tissue specimen. RESULTS. mRNA specific for TSP-1 was found in RT-PCR products from RPE cell s, and it showed a time-dependent increase from the beginning of the cultur e. Intracellular staining for TSP-1 was identified by flow cytometry. The s andwich ELISA identified a time-dependent increase of TSP-1 peptides in the culture medium of RPE cells. Immunostaining for TSP-1 was observed in the cytoplasm of RPE cells cultured on glass slides. Positive immunostaining of TSP-1 was observed in the cytoplasm of the RPE layer in the human retinal tissue specimen. CONCLUSIONS. RPE cells can produce and release TSP-1 in vitro, and TSP-1 ac cumulates in the cytoplasm of RPE cells in vivo as well as in vitro. The pr oduction of TSP-1 by RPE cells is influenced by the state of proliferation and/or cell density. TSP-1 appears to be an important control factor in ret inal and choroidal neovascularization.