Hypoxia/reoxygenation induces CTGF and PAI-1 in cultured human retinal pigment epithelium cells.
Early age-related macular degeneration (AMD) is characterized by thickening of Bruch's membrane due to the accumulation of extracellular matrix (ECM). This finding could be related to hypoxia of the retinal pigment epithelium (RPE). In the present study, we investigated the effects of hypoxia and reoxygenation on the expression of connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), collagen type IV (Col IV) and fibronectin (Fn) in cultured human RPE cells. Cultured human RPE cells were kept for 12-36h under hypoxic conditions (1% O(2)). Reoxygenation was conducted for 24h. Hypoxia-mediated CTGF and PAI-1 expression were analyzed by using immunohistochemistry, Northern and Western blot analysis. Actinomycin D was added to examine whether hypoxia induces the transcription of CTGF and PAI-1 mRNA. Furthermore, cells were transfected with siRNA against hypoxia-inducible factor-1alpha (HIF-1alpha) and kept under hypoxic conditions. The effects of antioxidants on hypoxia/reoxygenation-mediated CTGF and PAI-1 expression were tested by real-time PCR analysis. Production of Col IV and Fn were investigated by real-time PCR and Western blot analysis. Both hypoxia and hypoxia/reoxygenation increased the expression of CTGF, PAI-1, Col IV and Fn. Actinomycin D prevented the new transcription of CTGF and PAI-1 mRNA by hypoxia. Using siRNA against HIF-1alpha, the hypoxia-mediated increase of CTGF and PAI-1 was inhibited. Antioxidants attenuated the reoxygenation-mediated increase of CTGF and PAI-1. The process of hypoxia/reoxygenation in the RPE may lead to an increase of ECM in the RPE and thus may contribute to the accumulation of ECM in Bruch's membrane.
Fuchshofer
Rudolf
R
Department of Anatomy, University of Regensburg, Regensburg, Germany.
Yu
Alice L
AL
Teng
Hiang-Hwa
HH
Strauss
Rupert
R
Kampik
Anselm
A
Welge-Lussen
Ulrich
U
D000293:Adolescent; D000328:Adult; D000368:Aged; D000975:Antioxidants / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000096:biosynthesis / Q000235:genetics; D055513:Connective Tissue Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics; D008268:Macular Degeneration / Q000378:metabolism / Q000473:pathology; D008875:Middle Aged; D010100:Oxygen / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D055815:Young Adult
D000975:Antioxidants; D024141:Collagen Type IV; D005353:Fibronectins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C051321:SERPINE1 protein, human; D055513:Connective Tissue Growth Factor; D010100:Oxygen