O50	Hypoxic angiogenic transcriptome in human keratinocytes and microvascular endothelial cells: macroarray and real-time PCR analysis.
1A.Jazwa, 1A.Loboda, 2G.Molema, 1A.Jozkowicz, 1J.Dulak
1Faculty of Biotechnology, Jagiellonian University, Krakow, PL; 2Department of Pathology and Laboratory Medicine, University of Groningen, Groningen, NL.

Background: Decreased oxygen tension is a crucial mediator of the formation of new capillaries from preexisting vascular structures. This process, termed angiogenesis, is a hallmark in the pathology of many diseases, including cancer and is also prerequisite for a proper wound healing. So far, however, limited data are available on the global analysis of angiogenic gene expression in skin cells, namely human keratinocytes (HaCaT) and human microvascular endothelial cells (HMEC-1). Results: Macroarray analysis has shown that hypoxia (1% O2) enhanced or decreased the expression of several genes involved in angiogenesis in HaCaT and HMEC-1 cells. In HaCaT keratinocytes hypoxia very strongly (> 5 times) up-regulated the expression of vascular endothelial growth factor-A (VEGF-A), the major angiogenic mediator. This effect was confirmed by RT-PCR for VEGF mRNA and ELISA for VEGF protein. Also the expression of integrin-α-5 and endoglin was enhanced more than 10 times and the expression of their ligands, fibronectin and TGFβ1, respectively, was increased. In HMEC-1 hypoxia strongly induced expression of VEGF-A and additionally augmented fibronectin, endoglin, TGFβR1, VEGF-D and prothrombin expression. In both cell lines the expression of maspin, anti-angiogenic mediator was decreased by hypoxia but real-time analysis showed different level of mRNA for this factor in HMEC-1 and HaCaT cells. Ct value (threshold cycle) for maspin in HaCaT keratinocytes was ~ 21 whereas in HMEC-1 it was ~ 34 indicating that maspin plays crucial role in HaCaT but not in HMEC-1 cells. Moreover, thrombospondin-1 (TSP-1) expression was down-regulated in both cell lines. Interestingly, hypoxia augmented by 15-fold the expression of placenta growth factor (PlGF) in HaCaT cells, as shown by real-time PCR. In contrast we did not observe such an effect in HMEC-1. Conclusions: Hypoxia significantly influences expression of several angiogenic mediators in human keratinocytes and endothelial cells. The pattern of genes affected by decreased oxygen tension is different in these two cell lines. Present data points to potential targets for treatment of cutaneous diseases which are related to enhancement (eg. psoriasis, cancer) or impairment (eg. chronic diabetic wounds) of angiogenesis. Supported by grant PBZ-KBN 107/P04/2004 and 2 P04B 016 26

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