cross-sectional area, wall thickness and wall/lumen ratio. Data are represented as mean6SEM; indicates the number of animals in each group P,0.05 vs control; P,0.05 vs ANG II. doi:10.1371/journal.pone.0111117.t001 5 Angiotensin II and Kinin Vascular Interaction ANG II and B1R agonist have synergistic effects on NADPH oxidase-induced superoxide anion Salvianic acid A generation in aortic VSMC In order to determine if there is a synergistic effect between AT1 and B1 receptors signaling, primary cultures of aortic VSMC were treated with ANG II PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19720342 and DABK in a low concentration. At this concentration, neither ANG II or DABK alone induced NADPH oxidase activation, but when applied together a significant increase of NADPH oxidase-derived superoxide anion generation was observed. This effect was inhibited by LOS and DAL, as well by Tiron, a superoxide anion scavenger. In the absence of DABK, a B1R agonist, ANG II at a high concentration increased superoxide anion generation when compared with vehicle. This effect was inhibited by LOS, but not by DAL, a B1R antagonist. ANG II and B1R agonist have synergistic effects on ERK1/ 2 phosphorylation, protein synthesis and PCNA expression in VSMC ANG II and DABK at low concentration increased ERK1/2 phosphorylation only when added together and this effect was abolished by LOS, DAL and Tiron. On the other hand, at a high concentration ANG II and DABK induced ERK1/2 phosphorylation when applied alone and no additional effect was observed when the peptides were applied simultaneously. PCNA expression and leucine incorporation were assessed as molecular markers of cell growth. As demonstrated in 6 Angiotensin II and Kinin Vascular Interaction expression when compared with vehicle. This effect was not changed by DAL, a B1R antagonist, but was inhibited by LOS, AT1 receptor antagonist. ANG II and DABK at low concentration increased leucine incorporation only when added together PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717794 and LOS plus DAL abolished this effect. ANG II at high concentration increased leucine incorporation when compared with the control cells. Discussion Here we have demonstrated for the first time by in vivo and in vitro studies that B1R is upregulated in hypertensive ANG IIinfused rats and contributes to vascular hypertrophy through a redox-sensitive ERK1/2 pathway. We previously demonstrated that ANG II has a modulatory effect on B1R protein expression in the cardiovascular system. In addition, the increase in vascular B1R expression induced by ANG II relies on AT1 receptor activation, superoxide anion 
production by NADPH oxidase, PI3-kinase and NF-kB activation. ERK1/2 plays a role in NF-kB activation by Angiotensin II and Kinin Vascular Interaction inflammatory stimulus that up regulates B1R. However, ERK1/2 does not seem to be involved in ANG II-induced B1R up-regulation, since B1R antagonism reduced ERK1/2 activation but did not interfere with aortic B1R expression. ANG II plays an important role in the etiology of cardiovascular diseases and also in the pathophysiology of cardiac and vascular hypertrophy. A growing body of evidence suggests that ANG II effects on vascular structure are mediated by ROS production, through NADPH oxidase, and MAPK activation, which can mutually stimulate each other. In this study, we show that B1R activation, by endogenous DABK, contributes to vascular hypertrophy in ANG II-induced hypertension, by a mechanism involving ROS generation and ERK1/2 activation. In fact, it has been reported in isolated VSMC that DABK inc