Effects of Growth on Cell Adhesion and Modificationof Rac1 by Electrophiles in Vascular Endothelial Cells

  The Blood Pressure-Lowering Action of the Reductant Hydrogen Sulfide Is Paradoxically Mediated by the Oxidative Activation of Protein Kinase *,Į Daniel Stubbert1, Oleksandra Prysyazhna1, Olena Rudyk1, Joseph Robert Burgoyne1, and Philip Eaton1 1 .LQJ¶V&ROOHJH/RQGRQ&DUGLRYDVFXODU'LYLVLRQ the British Heart Foundation Centre of Excellence, United Kingdom Hydrogen sulfide (H2S) is an endogenous vasodilator; however the molecular basis of its blood pressure (BP)-lowering action is incompletely understood. Although H2S is a thiol reducing agent, we speculated that it may be able to react with endogenous oxidants such as molecular oxygen or peroxide to rapidly transition into disulfide or polysulfide species. Such events would be consistent with the pKa of H2S being 6.8. This means at physiological pH H2S significantly exists in the deprotonated thiolate (HS-) state which enhances its sensitivity to oxidants and formation of sulfides or polysulfides which have oxidant properties, especially by exchange reactions with reduced thiols. Indeed, in vitro spectrophotometric studies with NaSH confirmed H2S formed polysulfides when exposed to O2 or H2O2, but did not in their absence. Previously we reported that hydrogen peroxidedependent vasodilation and BP -lowering was mediated by it inducing interprotein disulfide protein kinase G (PKG) ID (i.e. oxidation), which directly activates the kinase independently of the nitric oxide-cGMP pathway. We found NaSH or the H2S donor GYY4137 caused dose-dependent oxidation of PKG ID in isolated smooth muscle cells, isolated mesenterics and purified recombinant protein in vitro. the physiological significance of H2Sinduced PKG ID oxidation was investigated by comparing the responses of C42S PKG 1D knock-in (KI) mice that lack the sensor thiol required for disulfide activation to wild-type (WT) littermate controls. Mesenteric vessels isolated from KI had impaired relaxation after constriction with U46619 in response to H2S, with their maximal relaxation ~25% less in KI mice compared to WT. the physiological significance of PKG ID oxidation by H2S was further substantiated in vivo as NaSH administered by Alzet micro-osmotic pumps reduced mean arterial pressure of WT mice by ~5-6 mmHg, but did not alter the BP of KI. to summarise; in the presence of oxidant molecules the reducing molecule H2S forms polysulfides with oxidant properties that promote PKG ID disulfide activation to lower BP.

 doi: 10.1016/j.freeradbiomed.2013.10.595

   Effects of Growth on Cell Adhesion and Modification of Rac1 by Electrophiles in Vascular Endothelial Cells Stephanie B. Wall1, Joo-Yeun Oh1, Karina C. Ricart1, Fen Zhou1, Praveen Vayalil Kumar1, Lauren Mitchell2, Sharon Campbell2, Matthew B. Renfrow1, and Aimee Landar1 1 University of Alabama at Birmingham, United States, 2University of North Carolina, Chapel Hill, United States. Endothelial function and dysfunction is relevant in disease processes such as angiogenesis, atherosclerosis, and formation of collateral flow in ischemic conditions. Endothelial cells (EC) must adjust dynamically to different conditions of blood flow and to chemical signals to maintain vasculature barrier integrity as well as formation of new blood vessels. in order to accomplish this, EC must be able to migrate and interact with other endothelial cells. the protein, Rac1, plays a vital role to support

6

these endothelial functions. Importantly, Rac1 is involved in a wide range of cellular functions, and is necessary for endothelial cell spreading and formation of cell-to-cell junctions. Our previous preliminary results showed that Rac1 is a target of the electrophile, 15-GHR[\ ¨12,14 Prostaglandin J2 (15d-PGJ2). However, it is not known how electrophile modification of Rac1 affects cell adhesion and also how growth state of endothelial cells or contact inhibition impacts Rac1 availability to be adducted by electrophiles. Therefore, we hypothesized that electrophile modification of Rac1 will be dependent on cellular growth, and that this modification will in turn inhibit endothelial cell adhesion. Cells were grown to either 50% or 100% confluency and treated for 4 hours by a dose range (0, 1, 2.5, 5, 7.5, 10 μM) of biotinylated 15d-PGJ2 under 0.5% or 10% fetal bovine serum. for each condition, cell viability and cell adhesion were monitored. the modification of Rac1 was monitored at each condition by biotin affinity precipitation and western analysis for Rac1 in the eluate. These results imply that Rac1 is an important target of endothelial cell function in different disease states and explores the conditions under which Rac1 may be modulated by electrophiles.

 doi: 10.1016/j.freeradbiomed.2013.10.596

   SIRT3 Deacetylates FOXO3 to Promote Mitochondrial Homeostasis in Endothelial Cells under Hypoxia Ling D Wang1,2 1 Inst. of Biomed. Sciences, Academia Sinica, Taipei, Taiwan, 2 Inst. of Medical Sciences, Tzu-Chi Univ., Hualien, Taiwan Endothelial cells (ECs), lining the vascular lumen, are vulnerable to stress such as hypoxia. Adaptation or remodeling of ECs under stress condition is essential for maintaining vascular integrity. Sirtuin-3 (SIRT3), a NAD+-dependent protein deacetylase, regulates mitochondrial function and metabolism. Hypoxia elicits a time-dependent increase in SIRT3 mRNA and protein expression in ECs. the forkhead box class O transcription factor FOXO3 is deacetylated by SIRT3 under the hypoxia. K271 and K290 of FOXO3 are identified to be targeted by SIRT3. the SIRT3mediated deacetylation of FOXO3 further reduces FOXO3 phosphorylation, ubiquitination and degradation, thereby stabilizing FOXO3 protein. as a result, a set of FOXO3-dependent mitochondrial antioxidant enzymes, including MnSOD, Prx3, Prx5 and Trx2, are upregulated to facilitate ROS removal in ECs, coupled with the improved mitochondrial bioenergetic function. Our results indicate that SIRT3 stabilizes FOXO3 via deacetylation, which then enhances the mitochondrial antioxidant defense system to increase the endothelial adaption during hypoxia. Thus, SIRT3-FOXO3 signaling axis plays an important role for mitochondrial homeostasis in ECs under oxidative stress condition.

 doi: 10.1016/j.freeradbiomed.2013.10.597

      

6)5%0