Chemotactic and cytotoxic effects of minocycline on human retinal pigment epithelial cells.
OBJECTIVE
To reveal the effects of minocycline, an anti-inflammatory and neuroprotective agent, on the viability and physiological properties of retinal pigment epithelial (RPE) cells and to compare the effects with those of triamcinolone acetonide.


METHODS
The proliferation of human RPE cells in vitro was investigated with a bromodeoxyuridine immunoassay; chemotaxis was examined with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. The gene expression of growth factors and MMP-9 was determined with real-time RT-PCR, and the secretion of VEGF was examined with ELISA. The phosphorylation of p38 MAPK and ERK1/2 proteins was determined with Western blot analysis.


RESULTS
Minocycline at low concentrations (50 nM-20 microM) stimulated chemotaxis and decreased the proliferation of RPE cells. Minocycline at high concentrations (above 5 microM) decreased the viability of RPE cells through the induction of cell necrosis. The chemotactic effect of minocycline was mediated by the stimulation of autocrine PDGF signaling and the activation of p38 MAPK. Minocycline promoted the expression of PDGF-B, HGF, VEGF, and MMP-9 and increased the amounts of phosphorylated p38 and ERK1/2 proteins in RPE cells. Triamcinolone reduced PDGF-evoked chemotaxis and VEGF expression and secretion and had no significant effects on cell viability and proliferation. Triamcinolone did not reverse the effects of minocycline on cell proliferation, chemotaxis, or viability or the expression of VEGF.


CONCLUSIONS
Low-dose minocycline induces the activation of RPE cells, as indicated by the activation of p38 and ERK1/2 and by enhanced chemotaxis mediated by autocrine PDGF signaling. High-dose minocycline induces RPE cell degeneration.
Hollborn
Margrit
M
Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany. hollbm@medizin.uni-leipzig.de
Wiedemann
Peter
P
Bringmann
Andreas
A
Kohen
Leon
L
D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008911:Minocycline / Q000494:pharmacology* / Q000633:toxicity; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D048053:Mitogen-Activated Protein Kinase 6 / Q000378:metabolism; D009336:Necrosis; D010766:Phosphorylation; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014222:Triamcinolone Acetonide / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D005938:Glucocorticoids; D036341:Intercellular Signaling Peptides and Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D048052:Mitogen-Activated Protein Kinase 3; D048053:Mitogen-Activated Protein Kinase 6; D048051:p38 Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9; D014222:Triamcinolone Acetonide; D008911:Minocycline