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Acta Physiologica 2012; Volume 204, Supplement 689
91st Annual Meeting of The German Physiological Society
3/22/2012-3/25/2012
Dresden, Germany
THE TYROSINE PHOSPHATASE SHP-1 REGULATES PROLIFERATION AND VEGF PRODUCTION OF HUMAN ENDOTHELIAL CELLS DURING HYPOXIA BY CONTROLLING HIF-1EXPRESSION
Abstract number: P207
Mannell1 *H., Alig1 S., Pircher2 J., Pohl1 U., Krotz2 F.
1Walter-Brendel-Centre for Experimental Medicine, LMU, Munich, Germany
2Medical Policlinic, LMU, Cardiology, Munich, Germany
Background:
The transcription factor Hif-1 plays a crucial role in angiogenesis and cell viability under hypoxic conditions. The endothelial tyrosine phosphatase SHP-1 has been shown to be involved in the regulation of proliferation and apoptosis in hypoxia. However, the underlying mechanism is not fully understood.
Methods:
Human microvascular endothelial cells were exposed to hypoxia (424h, pO2 36 mmHg ±1.5). VEGF protein was measured by FACS analysis, proliferation by increase in protein amounts. SHP-1 and HIF1a levels were assessed by Western Blotting and immunofluorescence staining. SHP-1 knockdown was achieved by antisense-Oligonucleotides (AS-ODN, 70 nM). Superoxide formation was measured by cytochrome-C assay, mRNA level by qRT-PCR.
Results:
During hypoxia,treatment with SHP-1 AS-ODN resulted in increased proliferation (p<0.05; n=18) and VEGF production (p<0.05; n=15) compared to control ODN treatment, which was blocked by inhibition of Hif-1 transcriptional activity (10ng/ml Echinomycin, p<0.05; n=11 and p<0.001; n=18 respectively). Interestingly, SHP-1 knockdown resulted in enhanced hypoxia induced Hif-1a expression (p<0.05; n=4) compared to control ODN treatment, whereas inhibition of SHP-1 catalytic activity (10mg/ml Sodium Stibogluconate) had no effect. SHP-1 AS-ODN treatment during hypoxia did not influence Hif-1a mRNA level (n.s.; n=10) but decreased Hif-1a protein translation after proteasome inhibition (p<0.05; n=4; 10mM MG132) as well as enhanced ROS formation (p<0.05; n=18). Finally, a direct interaction between SHP-1 and Hif-1a was observed, as HIF1a was co-immunoprecipitated with SHP-1 (n=3).
Conclusion:
SHP-1 negatively regulates endothelial proliferation and VEGF production during hypoxia by controlling Hif-1a degradation. Underlying mechanisms may be the regulation of ROS production or direct interaction between both proteins.
To cite this abstract, please use the following information:
Acta Physiologica 2012; Volume 204, Supplement 689 :P207