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Hiroko Miyajima–Uchida, Hideyuki Hayashi, Richiko Beppu, Motomu Kuroki, Mitsue Fukami, Fumiko Arakawa, Yoshihiro Tomita, Masahide Kuroki, Kenji Oshima; Production and Accumulation of Thrombospondin-1 in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(2):561-567.
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purpose. To investigate the production and release of thrombospondin-1 (TSP-1),
a natural inhibitor of angiogenesis, by human retinal pigment
epithelial (RPE) cells to clarify the possible role of TSP-1 in
maintaining intraocular angiogenesis.
methods. Human RPE cells were isolated from a human cadaveric eye and cultured
in medium with 5% newborn calf serum. TSP-1 messages in the purified
RNA of RPE cells were analyzed by reverse transcription–polymerase
chain reaction (RT-PCR). Intracellular TSP-1 peptides were detected by
cytofluorographic analysis. TSP-1 peptides in the culture medium on RPE
cells were measured by sandwich enzyme-linked immunosorbent assay
(ELISA). TSP-1 specific immunofluorescent staining was tested in RPE
cells cultured on glass slides and in a human retinal tissue specimen.
results. mRNA specific for TSP-1 was found in RT-PCR products from RPE cells,
and it showed a time-dependent increase from the beginning of the
culture. Intracellular staining for TSP-1 was identified by flow
cytometry. The sandwich 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 accumulates
in the cytoplasm of RPE cells in vivo as well as in vitro. The
production 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 retinal and choroidal
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