Changes in retinal gene expression in proliferative vitreoretinopathy: glial cell expression of HB-EGF.
OBJECTIVE
To compare the gene expression pattern of control postmortem retinas with retinas from patients with proliferative vitreoretinopathy (PVR), to determine the expression of the heparin binding epidermal growth factor-like growth factor (HB-EGF) by glial cells in fibroproliferative membranes, and to examine whether cells of the human Müller cell line, MIO-M1, respond to HB-EGF with proliferation, migration, and secretion of the vascular endothelial growth factor (VEGF).


METHODS
To identify genes that were differently expressed in PVR and control retinas, the RNA from the neural retinas of seven postmortem donors and of two patients with PVR were analyzed for differential gene expression, by hybridization of labeled cRNA probes to an Affymetrix human genome microarray set. The results were validated by real time PCR experiments investigating RNA from 6 postmortem retinas and 4 PVR retinas. Epiretinal PVR membranes were immunohistochemically stained for colocalization of HB-EGF and the glial cell marker, glial fibrillary acidic protein (GFAP). The HB-EGF evoked proliferation of cultured Müller cells was investigated by a bromodeoxyuridine immunoassay, chemotaxis was assessed with a migration assay, and the release of VEGF was evaluated by ELISA.


RESULTS
Out of the 12,600 genes and expressed sequence tags investigated, the levels of 80 showed an increased expression, and 21 were expressed at decreased levels, in the retinas of PVR patients compared to the control retinas. The upregulated signals include genes for nuclear and cell cycle related proteins, extracellular secretory proteins, cytosolic signaling proteins, and proteins of the membrane and the extracellular matrix. The genes of the hepatocyte growth factor and of HB-EGF were found to be expressed in PVR retinas but not in control retinas. In epiretinal membranes of patients with PVR, HB-EGF immunoreactivity partially colocalized with GFAP. In cultured Müller cells, HB-EGF stimulated both proliferation and chemotaxis, and the secretion of VEGF, via activation of the extracellular signal regulated kinases 1 and 2 and of the phosphatidylinositol-3 kinase.


CONCLUSIONS
The development of PVR is accompanied by complex changes of the gene expression in the neural retina, with an upregulation of genes that support cell proliferation, cell signaling, cell motility, and extracellular matrix remodeling. HB-EGF is one of the factors that are significantly upregulated in PVR retinas. HB-EGF expression in fibroproliferative tissue and its stimulatory effect on glial cell proliferation, chemotaxis, and VEGF secretion suggest that HB-EGF may be a factor mediating glial cell responses during PVR.
Department of Ophthalmology, University Eye Hospital, University of Leipzig, Leipzig, Germany. hollbm@medizin.uni-leipzig.de
Margrit
M
Hollborn
Solveig
S
Tenckhoff
Karsten
K
Jahn
Ianors
I
Iandiev
Bernd
B
Biedermann
Ute E K
UE
Schnurrbusch
G Astrid
GA
Limb
Andreas
A
Reichenbach
Sebastian
S
Wolf
Peter
P
Wiedemann
Leon
L
Kohen
Andreas
A
Bringmann
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002633:Chemotaxis; D004247:DNA / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000494:pharmacology; D019773:Epiretinal Membrane / Q000378:metabolism / Q000473:pathology; D020224:Expressed Sequence Tags; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D017228:Hepatocyte Growth Factor / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D018630:Vitreoretinopathy, Proliferative / Q000378:metabolism* / Q000473:pathology
D005904:Glial Fibrillary Acidic Protein; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D004247:DNA