BACKGROUND AND PURPOSENO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available L-arginine, reducing NO and increasing superoxide (O2 .-) production via NOS uncoupling. Elevated O2 .-combines with NO to form peroxynitrite (ONOO -), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide-induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase (ROCK) pathway. EXPERIMENTAL APPROACHArginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO -generator (SIN-1) or H2O2. Pretreatment with inhibitors of Rho kinase (Y-27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation. KEY RESULTSExposure to SIN-1 (25 mM, 24 h) or H2O2 (25 mM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Gö6976 or siRNA to p115-Rho GEF. There was an early activation of p115-Rho GEF (SIN-1, 2 h; H2O2, 1 h) and Rho A (SIN-1, 4 h; H2O2, 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN-1 and H2O2 also reduced NOS activity, which was blocked by pretreatment with p115-RhoGEF siRNA. CONCLUSIONS AND IMPLICATIONSOur data indicate that the oxidative species ONOO -and H2O2 increase arginase activity/expression through PKC-mediated activation of RhoA/Rho kinase pathway.

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.

Gold nanorods have promising applications in drug delivery and cancer treatment and are generally administered via direct injection into circulation. Thus it is necessary to evaluation their potential adverse effects on blood vessels. Herein we use gold nanorods with various surface modifications to evaluate the toxicity and cellular uptake of gold nanorods into vascular endothelial and smooth muscle cells of isolated rat aortic rings. Surfactant-capped gold nanorods (GNRs) were synthesized and either: 1) coated with polyelectrolytes (PE) in order to prepare PE-GNRs; or 2) modified with thiolated polyethylene glycol (PEG) in order to prepare PEG-GNRs. Using toxicity assays, small vessel myography, fluorescence microscopy and electron microscopy, we show that therapeutic concentrations of PE-GNRs but not PEG-GNRs are toxic to the vascular endothelium, which leads to impaired relaxation function of aortic rings. However, no toxicity to smooth muscles was observed. Moreover, electron microscopy analysis confirmed the cellular uptake of PE-GNRs but not PEG-GNRs into the endothelium of exposed aortic rings. The difference in toxicity and cellular uptake for PE-GNRs versus PEG-GNRs is explained and linked to free surfactant molecules and protein adsorption, respectively. Our results indicate that toxicity and cellular uptake in vascular endothelium in blood vessels are potential adverse effects of systemically administered gold nanorod solutions, which can be prevented by appropriate surface functionalization.

Introduction Angiotensin II (AngII) activates p38 mitogen-activated protein kinase (MAPK) and elevates arginase activity in endothelial cells. Upregulation of arginase activity has been implicated in endothelial dysfunction by reducing NO bioavailability. However, signaling pathways activated by AngII in the penis are largely unknown. Aim We hypothesized that activation of p38 MAPK increases arginase activity and thus impairs penile vascular function in AngII-treated mice. Methods Male C57BL/6 mice were implanted with osmotic minipumps containing saline or AngII (42 μg/kg/h) for 14 days and co-treated with p38 MAPK inhibitor, SB 203580 (5 μg/kg/day), beginning 2 days before minipump implantation. Systolic blood pressure (SBP) was measured. Corpus cavernosum (CC) tissue was used for vascular functional studies and protein expression levels of p38 MAPK, arginase and constitutive NOS, and arginase activity. Main Outcome Measures Arginase expression and activity; expression of phospho-p38 MAPK, -eNOS and nNOS proteins; endothelium-dependent and nitrergic nerve-mediated relaxations were determined in CC from control and AngII-infused mice. Results AngII increased SBP (22%) and increased CC arginase activity and expression (~2-fold), and phosphorylated P38 MAPK levels (30%) over control. Treatment with SB 203580 prevented these effects. Endothelium-dependent NO-mediated relaxation to acetylcholine was significantly reduced by AngII and this effect was prevented by SB 203580 (P<0.01). AngII (2-week) did not alter nitrergic function. However, SB 203580 significantly increased nitrergic relaxation in both control and AngII tissue at lower frequencies. Maximum contractile responses for phenylephrine and electrical field stimulation were increased by AngII (56% and 171%, respectively), and attenuated by SB 203580 treated. AngII treatment also decreased eNOS phosphorylation at Ser-1177 compared to control. Treatment with SB 203580 prevented all these changes. Conclusion p38 MAPK inhibition corrects penile arginase activity and protects against erectile dysfunction caused by AngII.