Abstract 280: Protein Tyrosine Phosphatase Oxidation and Redox Proteomics in Hypertension

Abstract

Aberrant signalling and vascular dysfunction in hypertension (HTN) have been associated with reactive oxygen species (ROS)-induced oxidation of redox-sensitive molecules. Protein tyrosine phosphatases (PTP) are susceptible to oxidation of thiols (-SH) at their active site, which leads to PTP inactivation. We hypothesize that oxidative stress is associated with reversible PTP oxidation and consequent reduced PTP activation in HTN. VSMCs from normotensive and hypertensive rats (WKY and SHRSP) were stimulated with AngII (10-7 M) and ET-1 (10-7 M). Protein oxidation was assessed by the oxyblot assay. PTP oxidation, TC-PTP and PTP1B expression levels were assessed by immunoblotting. Differential gel electrophoresis (DiGE) of CyDye-labelled cell lysates was employed for screening of oxidized proteome. Global-protein and PTP-specific oxidation was increased in VSMCs from SHRSP versus WKY (fold change (FC)=1.29 and FC=1.53, p<0.05, respectively). AngII stimulation did not affect global oxidation in either WKY or SHRSP, but increased PTP-oxidation in WKY (FC=1.47 at 5min, p<0.05) to levels similar to SHRSP. Stimulation with ET-1 increased total oxidation only in WKY (FC=1.52 at 15min, p<0.05) but not SHRSP, and had no effects on PTPs oxidation. Moreover, stimulation with AngII and ET-1 reduced protein expression of TC-PTP (FCAngII=0.8 at 2h for WKY; FCAngII=0.9 at 1h for SHRSP; FCET1=0.8 at 2h for WKY and 1h for SHRSP) and PTPB1 (FCAngII=0.87 at 1h for WKY and SHRSP; FCET1=0.9 at 2h for WKY and SHRSP), (p<0.05). DiGE proteomic data, filtered for FC>2, detected 1777 spots of which 1388 (78.1%) were equally oxidized across SHRSP and WKY, and 264 (14.9%) exhibited increased oxidation levels in SHRSP. Our findings demonstrate that oxidation of PTPs is increased in VSMCs from hypertensive rats and that vasoactive agents differentially regulate PTP oxidation and their expression levels in HTN. Furthermore, the oxidized state of 21.9% of the global proteome is altered in disease. Ongoing studies, in both rats and humans, are focusing on the characterization of a PTP-oxidation signature in HTN. Identification of specific PTPs which are deregulated in HTN could elucidate aberrant redox signalling in vascular dysfunction and highlight novel therapeutic targets.