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

Invest Ophthalmol Vis Sci. 2000 Feb;41(2):561-7.

Abstract

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 neovascularization.

MeSH terms

  • Antibodies, Monoclonal
  • Cell Count
  • Cell Division
  • Cells, Cultured
  • Cytoplasm / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression
  • Humans
  • Pigment Epithelium of Eye / cytology
  • Pigment Epithelium of Eye / metabolism*
  • RNA, Messenger / metabolism
  • Retina / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Thrombospondin 1 / biosynthesis*
  • Thrombospondin 1 / genetics*

Substances

  • Antibodies, Monoclonal
  • RNA, Messenger
  • Thrombospondin 1