Objective: 

Hypoxia-inducible factor-1a (Hif-1a) protein level is mediated by cellular O2 concentration. It accumulates during hypoxia. Hif-1 induces the transcription of Vascular endothelial growth factor (VEGF), an important angiogenic stimulus. Furthermore, Hif-1 is essential for cell viability during ischemic processes. The endothelial Tyrosine Phosphatase SHP-1 is activated by VEGF and regulates VEGF dependent superoxide (O2-) formation. O2- have been shown to stabilize Hif-1a. However, it is unknown whether SHP-1 affects Hif-1a regulation during hypoxia. Thus we investigated the influence of SHP-1 on Hif-1a protein level in human microvascular endothelial cells (HMEC) during hypoxia.

Methods: 

HMEC were exposed to hypoxia (1% O2; 5% CO2; 94% N2) for 4 hours. Protein levels were assessed by Western Blotting and immunofluorescence staining. Knockdown of SHP-1 was reached by Magnetofection with Antisense Oligonucleotides (AS-Odn). Superoxide formation was measured by cytochrome-c reduction assay.

Results: 

HMEC exposed to hypoxia showed significantly increased Hif-1a protein levels in comparison to normoxia controls as shown by Western Blotting (n=4; p<0,05; HMEC) and immunofluorescence (n=3; HMEC). Moreover Hif-1a and SHP-1 were mainly localized in the nucleus during hypoxia as assessed by immunofluorescence staining. Knockdown of SHP-1 by AS-Odn (70nM) resulted in a significant rise of Hif-1a protein levels during hypoxia (n=4; p<0,05; HMEC) compared to cells treated with control AS-Odn. This effect could not be observed by specific pharmacological inhibition (Sodium Stibogluconate 10 mg/ml) of the SHP-1 phosphatase activity (n=6; HMEC). Knockdown of SHP-1 (70nM AS-Odn) resulted in increased O2- formation during normoxia (n=18; p<0,05; HMEC), but not during hypoxia (n=18; HMEC). However, immunoprecipitation showed a direct interaction between SHP-1 and Hif-1a (n=3; HMEC).

Conclusion: 

These results indicate that SHP-1 regulates Hif-1a protein levels and thus could be essential for hypoxia induced processes. This effect is neither mediated by the phosphatase activity of SHP-1 nor by endothelial superoxides, but rather by the direct interaction between SHP-1 and Hif-1a.