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 (4–24h, 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.