Hypoxia/reoxygenation and TGF-beta increase alphaB-crystallin expression in human optic nerve head astrocytes.
Reactive astrocytes in glaucomatous optic nerve changes are characterized by an increased expression of alphaB-crystallin and transforming growth factor-beta (TGF-beta). In the pathogenesis of glaucomatous optic nerve damage, ischemia/reperfusion injury may play an important role. The goal of the present study was to determine the influence of hypoxia/reoxygenation and TGF-beta on the expression of alphaB-crystallin in cultured human astrocytes of the optic nerve head (ONH). Cultured human astrocytes were incubated under hypoxic conditions (1% O2 for 4-12 h) with subsequent reoxygenation (12-24 h). Additionally, cells were treated with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h. Expression of alphaB-crystallin was examined by Northern- and Western-blotting. Levels of TGF-beta1 and TGF-beta2 were analyzed by RT-PCR analysis and ELISA. The effect of TGF-beta blocking on the hypoxia/reoxygenation modulated expression of alphaB-crystallin was investigated by simultaneous incubation with neutralizing antibodies against TGF-beta during the reoxygenation phase. Hypoxia/reoxygenation increased the expression of alphaB-crystallin at the mRNA (2.8- to 3.1-fold) and protein level (1.8- to 2.1-fold). Treatment with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h markedly enhanced alphaB-crystallin mRNA expression approximately three- to fourfold. Using Western blot analysis, this increase ranged from 2 to 3 times. Both cytokines showed a twofold increase after 12 and 24 h of reoxygenation at the mRNA and a two- to threefold increase at the protein level. Simultaneous treatment with neutralizing antibodies against both TGF-beta isoforms prevented the hypoxia/reoxygenation-mediated elevation of alphaB-crystallin. The process of hypoxia/reoxygenation is capable of inducing the expression of alphaB-crystallin and TGF-ss in cultured ONH astrocytes. Therefore, optimization of conditions leading to hypoxia/reoxygenation in the ONH of glaucomatous patients may help to lower the incidence of characteristic changes in the optic nerve.
Yu
Alice L
AL
Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
Fuchshofer
Rudolf
R
Birke
Marco
M
Priglinger
Siegfried G
SG
Eibl
Kirsten H
KH
Kampik
Anselm
A
Bloemendal
Hans
H
Welge-Lussen
Ulrich
U
D000368:Aged; D000369:Aged, 80 and over; D000906:Antibodies / Q000276:immunology; D001253:Astrocytes / Q000276:immunology / Q000378:metabolism*; D015152:Blotting, Northern / Q000379:methods; D002478:Cells, Cultured; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D008875:Middle Aged; D009898:Optic Disk / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D010100:Oxygen / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000276:immunology*; D053773:Transforming Growth Factor beta1 / Q000032:analysis / Q000276:immunology; D053781:Transforming Growth Factor beta2 / Q000032:analysis; D038203:alpha-Crystallin B Chain / Q000032:analysis*
D000906:Antibodies; D012333:RNA, Messenger; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D038203:alpha-Crystallin B Chain; D010100:Oxygen