Production and accumulation of thrombospondin-1 in human retinal pigment epithelial cells.
OBJECTIVE
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 neovascularization.
Miyajima-Uchida
H
H
Department of Ophthalmology, School of Medicine, Fukuoka University, Japan. hhayashi@fukuoka-u.ac.jp
Hayashi
H
H
Beppu
R
R
Kuroki
M
M
Fukami
M
M
Arakawa
F
F
Tomita
Y
Y
Kuroki
M
M
Oshima
K
K
D000911:Antibodies, Monoclonal; D002452:Cell Count; D002455:Cell Division; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019700:Thrombospondin 1 / Q000096:biosynthesis* / Q000235:genetics*
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D019700:Thrombospondin 1