br Methods br Results It is well known

Methods
Results It is well known that during the onset of vasoconstriction there is an increase in intracellular Ca2+ which activates myosin light-chain kinase followed by cycling of actin-myosin cross-bridges in VSMCs. Recently, it has been recognized that CCG 203971 polymerization is also involved in the prolonged phase of vasoconstriction [4]. To determine if FPR-1 is required for acute and/or prolonged contraction, isometric force was measured in conductance (aorta) and resistance arteries [mesenteric resistance arteries (MRA) and intrarenal arteries - diameter < 300 μm] from FPR-1 KO and WT (C56BL/6) mice. The absence of FPR-1 decreased both the acute- (actin-myosin cross-bridges) as well as the prolonged-phase (actin polymerization) of KCl- induced contraction in all arterial beds studied (Fig. 1A–C). Similarly, phenylephrine-induced contraction was decreased in aorta (Figs. 1D and 2 C) and resistance arteries (Fig. 2A and B) from FPR-1 KO animals. These data revel a novel and previously unrecognized role underlying FPR-1 function in vascular contractility.
Discussion Our observations revealed that the innate immune system pattern recognition receptor FPR-1 is also required for vascular homeostasis, including myogenic behavior during physiological conditions. Here, we have determined that the function of FPR-1 is not only limited to its detection of pathogens and DAMPs in the bloodstream, but it is also significant for the dynamic plasticity of the vasculature. We observed that upon activation (by agonist or stretch), FPR-1 plays a role in myogenic tone via an integrated network with actin cytoskeleton (Fig. 9). Overall, these observations provide 1) a new mechanistic insight into how the immune and cardiovascular system form a network affecting vascular plasticity and 2) identify a mechanism by which interaction between cells of the immune system and arteries can mediate inter-tissue responses to danger. Although N-formyl peptides are the main ligands for FPR-1, there are only two sources of these molecules in the human body, bacteria and mitochondria [11]. Therefore, FPR-1 is fundamental for bacterial and mitochondrial clearance [11]. The FPR family, a subfamily of G protein-coupled receptors, is represented by at least three isoforms, FPR 1, 2, and 3 [[11], [12], [13]]. Formyl peptide receptors are expressed at high levels in neutrophils and monocytes, and when activated by binding of FMLP (bacterial peptide) or F-MIT (mitochondrial peptide) [12,13] they induce NADPH oxidase activation, ROS generation, and cell chemotaxis [28]. Besides being expressed in neutrophils, the FPR family is expressed in a range of somatic cells and tissues, including the endothelium, epithelium, spleen, lung, liver, skeletal, and smooth muscle [11,28]. Human FPR family receptors are intrinsically associated with the Gi protein subunit [28], and upon activation, FPR-1 triggers stimulation of phosphatidylinositol-3-kinase (PI3K), phospholipase C (PLC), protein kinase C (PKC), Ca2+ flux and mitogen-activated protein kinases (MAPKs) [28]. Also, FPR-1 activation is important for wound healing mechanism [28], and is associated with neutrophil migration, via actin polymerization, to sites of sterile and non-sterile inflammation [12,29,13]. As described above, Ca2+ signaling is critical for vascular contraction and, upon activation, FPR-1 triggers Ca2+ increase in neutrophils, which subsequently leads to migration, ROS generation and cytokines release [11,28]. Given the importance of Ca2+ influx to vascular contraction, we performed a concentration-response curve to Cav 1.2 activator, Bay K 8644. We did not observe differences in contraction induced by this compound in resistance arteries and aorta from WT and FPR-1 KO, suggesting that the Cav1.2-dependent Ca2+ influx is not impaired in arteries from FPR-1 KO. Also, we used a fluorescent indicator, fura-2-acetoxymethyl ester (fura-2 AM) to measure intracellular Ca2+ in VSMC in the presence or absence of FPR-1. Single-cell intracellular Ca2+ measurements on fura-2-loaded VSMC showed that baseline intracellular Ca2+ concentrations (absence of stimulus) is higher in VSMC from FPR-1-KO when compared to WT. Addition of 120 mM of KCl led to a rapid and transient increase in the intracellular Ca2+ in both groups. This increase was significantly higher in VSMC from FPR-1 KO when compared to WT. These data discard the fact that reduction of intracellular Ca2+ would be the main factor that leads to decreased contraction in FPR-1 KO arteries. Actually, it was the opposite then the expected, intracellular concentration of Ca2+ was higher in cells from FPR-1 KO. This could be a compensatory mechanism due to decrease of L-type Ca2+ (Cav1.2) expression. We do not know why Cav1.2 expression is decreased in FPR-1 KO, but it is possible to speculate that there is an interaction between FPR-1 and Cav1.2, or maybe a possible transactivation. We suggested that a possible link between FPR-1 and Cav 1.2. is a 700 kDa scaffolding protein called Ahnak. Recent evidence has demonstrated that this protein interacts directly with Cav 1.2. and this interaction is important for proper cardiac function [27]. Furthermore, it has been reported that Ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel [32], which means that Ahnak could directly influence actin polymerization and Cav 1.2. function. To further support our hypothesis that Ahnak could form a complex with FPR-1 and Cav 1.2, a recent study showed that Ahnak interacts with the annexin 2/S100A10 complex, and it is well known that annexin I receptors belong to FPR family [33]. Here, we demonstrated that loss of FPR-1 leads to significant reduction of Ahnak protein expression. Therefore, based on this result we can infer that Cav 1.2 is connected to FPR-1 via Ahnak, which could interact with cytoskeleton leading to actin polymerization and, subsequently this complex modulates contractility. However, this result is too preliminary and needs further investigation.