Because FPR is expressed in VSMCs and its

Because FPR-1 is expressed in VSMCs and its role in non-hematopoietic cells is still unclear, we questioned whether FPR-1 would have physiological relevance for vascular behavior, in the same way as it does in neutrophils (Ca2+ homeostasis and SKF 81297 hydrobromide sale polymerization). First, we tested if FPR-1 is involved in arterial contraction. Indeed, here we observed that FPR-1 KO decreased acute and prolonged vascular contraction to receptor-dependent and -independent stimuli. Agonist-induced vasoconstriction in VSMCs has been attributed not only to a process in which receptor activation increase intracellular Ca2+ leading to myosin light-chain kinase activation and subsequent cycling of actin-myosin cross-bridges (acute contraction) in VSMCs, but recent evidence demonstrated that prolonged contraction is related to actin polymerization [4] Accordingly, Dr. Martinez-Lemus’s group (2013) has shown that exposure of isolated arterioles to vasoconstrictor agonists induces VSMCs actin polymerization via activation of small GTPases [4]. Also, several studies support the conclusion that VSMC contraction is accompanied by an increase in F-actin and a decrease in G-actin content [3]. Measurements of F- and G-actin ratio at rest and after stimulation by using adrenergic receptors (in vessels) or muscarinic receptors (in airways) agonists demonstrated that cellular actin undergoes polymerization [3]. Drs. Walsh and Cole Walsh and Cole [3] described that this response is consistent with the presence of two pools of cellular actin: one (‘contractile actin’) involved in the contractile machinery, associated with tropomyosin and stabilized in the filamentous form, and the other (‘cytoskeletal actin’) localized to the cell cortex, not associated with tropomyosin and undergoing reversible polymerization–depolymerization [3]. In the present study, we found that the direct activation of actin polymerization ameliorates contractility in arteries from FPR-1 KO. This result revealed that FPR-1 is essential for sustained contraction via actin polymerization. To further confirm that FPR-1 plays a role in actin polymerization in VSMCs, we observed that FPR-1 agonist, mitochondrial N-formyl peptides (F-MIT), increased F:G actin ratio, and specific FPR-1 inhibitors abolished this result. Supporting these findings, using confocal microscopy we demonstrated that F-actin bundles were disorganized in VSMCs from FPR-1 KO mice when compared to control. Another piece of supporting evidence that FPR-1 indeed plays a role in smooth muscle cell (SMC) contractility was seen in our previous study [13], where FPR activation leads to trachea, bronchus and bronchiole (no endothelial cell influence) contractile responses and activate cell division control protein 42 (CDC42). Actin cytoskeletal remodeling is an important component of airway smooth muscle contraction [30]. Up-regulation of a cytoskeletal recruitment in highly shortened airway smooth muscle has been shown to be an important mechanism of reduced airway distensibility [30]. Also, CDC42 is a member of Rho GTPase family, which regulates F-actin reorganization and induces actin polymerization, either by stimulating de novo actin nucleation or by stimulating the uncapping or severing of filaments [31]. Additionally, in a previous publication we observed that lipoxin A4 (an endogenous FPR agonist) induces acute and prolonged contraction in aorta from naïve animals. The contractions were abolished in the presence of Y27631 (RhoA/Rho kinase inhibitor). Therefore, these studies confirm our hypothesis that FPR plays a role in SMC contractility (acute and chronic), independent of developmental changes. In the present study, we observed that sustained vasoconstriction and actin polymerization via FPR-1 could also be via small GTPases, including RhoA/ROCK, given that VSMCs treated with FPR agonist induced MYPT1 phosphorylation. However, RhoA activator did not improve contraction in arteries from FPR-1 KO, this could be due to the presence of disruption of actin polymerization.