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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

Manuscript received August 21, 2004; revised manuscript received January 4, 2005, accepted February 1, 2005.

^_* Reprint requests and correspondence: Dr. Florian Krötz, Cardiology, Medical Policlinic, Ludwig-Maximilians-University, Ziemssenstr. 1, 80336 Munich, Germany. (Email: fkroetz{at}lmu.de).

OBJECTIVES: We investigated the role of SH2-domain containing phosphatase-1 (SHP-1) in endothelial reduced nicotinamide adenine dinucleotide (phosphate) (NAD[P]H)-oxidase-dependent oxidant production.

BACKGROUND: Superoxide (O₂^·–) generation by endothelial NAD(P)H-oxidase promotes endothelial dysfunction and atherosclerosis. Signaling pathways that regulate NAD(P)H-oxidase activity are, however, poorly understood.

METHODS: SH2-domain containing phosphatase-1 was inhibited using site-directed magnetofection of antisense oligodesoxynucleotides (AS-ODN) or short interfering ribonucleic acid (siRNA) in vitro in human umbilical vein endothelial cells (HUVEC) and in isolated hamster arteries; O₂^·– was measured by cytochrome c reduction in vitro. Activities of NAD(P)H-oxidase activity, phosphatidyl-inositol-3-kinase (PI3K), and SHP-1 were assessed by specific assays; Rac1 activation was assessed by a pull-down assay.

RESULTS: Basal endothelial O₂^·– release was enhanced after inhibition of endothelial SHP-1 (p < 0.01), which could be prevented by specific inhibition of NAD(P)H-oxidase (p < 0.01); SHP-1 activity was high under basal conditions, further increased by vascular endothelial growth factor (10 ng/ml, p < 0.05), and abolished by SHP-1 AS-ODN treatment (p < 0.01), which also increased NAD(P)H-oxidase activity 3.3-fold (p < 0.01). Vascular endothelial growth factor also induced O₂^·– release (p < 0.01), which was even more enhanced when SHP-1 was knocked down (p < 0.05). The effect of SHP-1 was mediated by inhibition of PI3K/Rac1-dependent NAD(P)H-oxidase activation (p < 0.01); SHP-1 AS-ODN augmented tyrosine phosphorylation of the p85 regulatory subunit of PI3K (p < 0.05) and Rac1 activation. The latter was prevented by wortmannin, a blocker of PI3K.

CONCLUSIONS: In HUVEC, SHP-1 counteracts basal and stimulated NAD(P)H-oxidase activity by negative regulation of PI3K-dependent Rac1 activation; SHP-1 thus seems to be an important part of endothelial antioxidative defense controlling the activity of the O₂^·–-producing NAD(P)H-oxidase.

Abbreviations and Acronyms   AS-ODN = antisense oligodesoxynucleotide   HUVEC = human umbilical vein endothelial cells   NAD(P)H = reduced nicotinamide adenine dinucleotide (phosphate)   O2·– = superoxide radical   PI3K = phosphatidyl-inositol-3-kinase   PTP = protein tyrosine phosphatase   AT = angiotensin   RIPA = radioimmunoprecipitation (buffer)   SHP-1 = SH2-domain containing tyrosine phosphatase-1   siRNA = short interfering ribonucleic acid   SS = sodium stibogluconate

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