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The Tyrosine Phosphatase, SHP-1, Is a Negative Regulator of Endothelial Superoxide Formation

Florian Krötz, MD^_*,,_*, Barbara Engelbrecht, MS^_*, Martin A. Buerkle, MD, Florian Bassermann, MD, Hanna Bridell, BS^_*, Torsten Gloe, PhD^_*, Justus Duyster, MD, Ulrich Pohl, MD^_* and Hae-Young Sohn, MD

^_* Institute of Physiology, Ludwig-Maximilians University, Munich, Germany
Institute of Cardiology, Medical Policlinic, Ludwig-Maximilians University, Munich, Germany
Clinic of Anesthesiology, Ludwig-Maximilians University, Munich, Germany
Department of Internal Medicine III, TU Munich, Germany

View larger version (25K): [in a new window]   Figure 1 SH2-domain containing tyrosine phosphatase-1 (SHP-1) is constitutively active in endothelial cells and degraded by antisense-magnetofection. Magnetofection of antisense oligodesoxynucleotide (AS-ODN) against SHP-1 into human umbilical vein endothelial cells (HUVEC) resulted in deprivation of its protein associated with a loss of SHP-1 activity. Note that there is remarkable activity of SHP-1 in HUVEC grown under control conditions (random ODN, similar activity observed in untransfected cells, see Fig. 4). **Significantly different vs. control at p < 0.01.

View larger version (16K): [in a new window]   Figure 2 Pharmacological inhibition of SH2-domain containing tyrosine phosphatase-1 by sodium stibogluconate (SS) increases basal endothelial superoxide (O2·–) production. Pretreatment with SS (10 µg/ml) significantly enhanced O2·– release from human umbilical vein endothelial cells monolayers. The effect of SS reached significant levels after 30 min of preincubation, indicating a nontranscriptional mechanism. **Significantly different vs. control at p < 0.01.

View larger version (21K): [in a new window]   Figure 3 Degradation of SH2-domain containing tyrosine phosphatase-1 (SHP-1) protein increases endothelial O2·– production. Human umbilical vein endothelial cells O2·– release was enhanced, when SHP-1 was inhibited by a pharmacological agent (sodium stibogluconate [SS] 10 µg/ml) and when its protein was degraded by antisense oligodesoxynucleotide (AS-ODN) or short interfering ribonucleic acid (siRNA). N-Acetylcysteine (NAC) reduced O2·– production. *, **Significantly different vs. control at p < 0.05 and p < 0.01, respectively.

View larger version (33K): [in a new window]   Figure 4 Influence of vascular endothelial growth factor (VEGF) on SH2-domain containing tyrosine phosphatase-1 (SHP-1) activity and superoxide production. (A) Stimulation with VEGF (10 ng/ml) enhanced basal phosphatase activity of SHP-1 in human umbilical vein endothelial cells (HUVEC). (B) VEGF (10 ng/ml) also increased HUVEC O2·– release, which was further enhanced when SHP-1 was knocked down using antisense oligodesoxynucleotide (AS-ODN). *, **Significantly different vs. control at p < 0.05 and p < 0.01, respectively; #significantly different vs. VEGF at p < 0.05.

View larger version (21K): [in a new window]   Figure 5 SH2-domain containing tyrosine phosphatase-1 (SHP-1) down-regulates reduced nicotinamide adenine dinucleotide (phosphate) (NAD[P]H)-oxidase activity. (A) Specific inhibitory peptides against NAD(P)H-oxidase (gp91ds-tat, 100 µmol/l) abrogated the increase in O2·– release in human umbilical vein endothelial cell (HUVEC) monolayers that was caused by sodium stibogluconate (SS). (B) NADH-dependent O2·– production in HUVEC protein lysates was increased 3.3-fold after treatment of cells with antisense oligodesoxynucleotide (AS-ODN) against SHP-1 but not with random ODN. *, **Significantly different vs. control at p < 0.05 and p < 0.01, respectively; ##significantly different vs. SS at p < 0.01.

View larger version (29K): [in a new window]   Figure 6 Phosphatidyl-inositol-3-kinase (PI3K) is a downstream target of endothelial SH2-domain containing tyrosine phosphatase-1 (SHP-1). (A) Inhibition of PI3K activity by wortmannin (10 nmol/l) abolished the increased O2·– production after treatment with SHP-1 antisense oligodesoxynucleotide (AS-ODN) or sodium stibogluconate (SS). (B) Co-staining of immunoprecipitates of the p85 regulatory subunit of PI3K showed increased tyrosine phosphorylation of p85 after knock out of SHP-1. Means of three independent experiments are shown. *, **Significantly different vs. random ODN at p < 0.05 or p < 0.01, respectively; #, ##significantly different vs. SHP-1 AS/SS at p < 0.05 or p < 0.01, respectively.

View larger version (37K): [in a new window]   Figure 7 Inhibition of SH2-domain containing tyrosine phosphatase-1 (SHP-1) enhances Rac1 activation mediated by phosphatidyl-inositol-3-kinase (PI3K). (Top) Pull-down assays of Rac1 human umbilical vein endothelial cell revealed increased amounts of active Rac1 (bound to GST-PAK) after sodium stibogluconate (SS) or antisense oligodesoxynucleotide (AS-ODN) against SHP-1; SS-induced Rac1-activation was inhibited by wortmannin (not shown). (Bottom) The ratio between blot densities of GTP-bound Rac1 to total Rac1 revealed significant induction of Rac1 activity by SHP-1 AS-ODN and by SS (means of three experiments).