PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines.
OBJECTIVE
The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines.


METHODS
Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively.


RESULTS
PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance.


CONCLUSIONS
These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.
Li
Rong
R
National Institutes of Health, National Eye Institute, Bethesda, Maryland 20892-2510, USA.
Maminishkis
Arvydas
A
Wang
Fei E
FE
Miller
Sheldon S
SS
D000199:Actins / Q000378:metabolism; D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D002829:Choroid / Q000166:cytology; D016207:Cytokines / Q000494:pharmacology*; D005333:Fetus; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008222:Lymphokines / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing / Q000187:drug effects
D000199:Actins; D016207:Cytokines; D008222:Lymphokines; C427493:PDGFD protein, human; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; C411423:platelet-derived growth factor C; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta