December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Hypoxia inhibits uPAI-1 Gene and Protein Levels in Cultured Human Corneal Epithelial Cells
Author Affiliations & Notes
  • Z Wang
    Anatomy and Cell Biology Wayne State University School of Medicine Detroit MI
  • M Kurpakus-Wheater
    Anatomy and Cell Biology Wayne State University School of Medicine Detroit MI
  • Footnotes
    Commercial Relationships   Z. Wang, None; M. Kurpakus-Wheater, None. Grant Identification: MidWest EyeBanks and Transplantation Center
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1667. doi:
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      Z Wang, M Kurpakus-Wheater; Hypoxia inhibits uPAI-1 Gene and Protein Levels in Cultured Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1667.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Hypoxia is known to affect the urokinase plasminogen activator (uPA) system in epithelium. In addition to uPA, components of the system include receptor for uPA (uPAR) and one of two inhibitors of uPA, urokinase plasminogen activator inhibitor-1 (uPAI-1). The purpose of this study is to test the hypothesis that in human corneal epithelial cells hypoxia inhibits both gene and protein expression of uPAI-1. Methods: Tertiary cultures of human corneal epithelial cells (HCEC) were purchased from Cascade Biologics (Portland, OR). HCEC were maintained in serum-free medium at either 20% oxygen (normoxic controls) or 2% oxygen (hypoxic experimental condition). Cells were cultured for 1, 3, 5 or 7 days. Relative uPAI-1 gene expression was analyzed from total RNA preparations using a gene profiling assay from SuperArray (Gaithersburg, MD) coupled with densitometry. Cell-surface uPAI-1 and uPA/uPAR/uPAI-1 complexes were analyzed using surface biotinylation, immunoprecipitation with antibodies to either uPA, uPAR, or uPAI-1, and densitometry. Results: mRNA levels of uPA and uPAR are not affected by hypoxia in cultured human corneal epithelial cells. In contrast, hypoxic HCEC demonstrate a 1.6-fold decrease in mRNA levels for uPAI-1 compared to normoxic controls. This supports our previous observation that less uPAI-1 protein is secreted into the conditioned medium of hypoxic cells compared to normoxic controls. Biotinylation and immunoprecipitation confirmed that uPAI-1 is complexed to uPA and uPAR on the cell surface in both hypoxic and normoxic HCEC. However, the amount of uPAI-1 associated with this complex is decreased in hypoxic HCEC compared to normoxic controls. Conclusion: We have shown that cell-associated uPA activity is increased in hypoxic HCEC. One explanation for this is that hypoxic cells produce and secrete less uPAI-1. A decrease in uPAI-1 available for cell-surface uPA/uPAR/uPAI-1 complex formation results in sustained and increased uPA activity in hypoxic human corneal epithelial cells compared to normoxic controls. We conclude that hypoxia-induced alterations in the uPA/uPAR/uPAI-1 system reside at the level of uPAI-1 gene expression in cultured human corneal epithelial cells.

Keywords: 428 hypoxia • 372 cornea: epithelium • 342 cell membrane/membrane specializations 

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