Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media

Peter A. Campochiaro, Roberta Sugg, Gary Grotendorst, Leonard M Hjelmeland

Research output: Contribution to journalArticle

102 Citations (Scopus)

Abstract

Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbecco's modified Eagle's medium. Conditioned media were exhaustively dialyzed against 0·5 n acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18·5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.

Original languageEnglish (US)
Pages (from-to)217-227
Number of pages11
JournalExperimental Eye Research
Volume49
Issue number2
DOIs
StatePublished - 1989

Fingerprint

Retinal Pigments
Conditioned Culture Medium
Platelet-Derived Growth Factor
Epithelial Cells
Immunoglobulin G
Proteins
Fibroblasts
Chemotaxis
Goats
Eagles
Antibodies
Autoradiography
Acetic Acid
Methionine
Thymidine
Immune Sera
Polyacrylamide Gel Electrophoresis
Cell Culture Techniques
Western Blotting
Acids

Keywords

  • platelet-derived growth factor
  • proliferative vitreoretinopathy
  • retinal pigment epithelium
  • wound healing

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media. / Campochiaro, Peter A.; Sugg, Roberta; Grotendorst, Gary; Hjelmeland, Leonard M.

In: Experimental Eye Research, Vol. 49, No. 2, 1989, p. 217-227.

Research output: Contribution to journalArticle

Campochiaro, Peter A. ; Sugg, Roberta ; Grotendorst, Gary ; Hjelmeland, Leonard M. / Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media. In: Experimental Eye Research. 1989 ; Vol. 49, No. 2. pp. 217-227.
@article{9a432c3a226a4799bb1e71d564726695,
title = "Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media",
abstract = "Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbecco's modified Eagle's medium. Conditioned media were exhaustively dialyzed against 0·5 n acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18·5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.",
keywords = "platelet-derived growth factor, proliferative vitreoretinopathy, retinal pigment epithelium, wound healing",
author = "Campochiaro, {Peter A.} and Roberta Sugg and Gary Grotendorst and Hjelmeland, {Leonard M}",
year = "1989",
doi = "10.1016/0014-4835(89)90092-4",
language = "English (US)",
volume = "49",
pages = "217--227",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media

AU - Campochiaro, Peter A.

AU - Sugg, Roberta

AU - Grotendorst, Gary

AU - Hjelmeland, Leonard M

PY - 1989

Y1 - 1989

N2 - Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbecco's modified Eagle's medium. Conditioned media were exhaustively dialyzed against 0·5 n acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18·5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.

AB - Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbecco's modified Eagle's medium. Conditioned media were exhaustively dialyzed against 0·5 n acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18·5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.

KW - platelet-derived growth factor

KW - proliferative vitreoretinopathy

KW - retinal pigment epithelium

KW - wound healing

UR - http://www.scopus.com/inward/record.url?scp=0024416686&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024416686&partnerID=8YFLogxK

U2 - 10.1016/0014-4835(89)90092-4

DO - 10.1016/0014-4835(89)90092-4

M3 - Article

VL - 49

SP - 217

EP - 227

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

IS - 2

ER -