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Objective This study was conducted to investigate the role of protease-activated receptor 2 (PAR2) in platelet-activating factor (PAF)-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Methods BALB/c mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin, a reversible inhibitor of serine proteases, prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL1) and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. Results C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. Conclusion This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Amitriptyline (AM) is a classical and typical tricyclic antidepressant drug. Despite its well-known effects on the nervous system, it has been described to work as a TLR4 antagonist and several clinical works suggested some unexpected cardiovascular effects. The role of amitriptyline on vascular tone is not clear, thus we hypothesized that amitriptyline has a double effect on vascular tone by both endothelial TLR4-dependent nitric oxide down-regulation and calcium channel blockade in smooth muscle cells.Changes in isometric tension were recorded on a wire myograph. NO production was evaluated by fluorescence microscopy and flow cytometry in the mouse aorta and EAhy926 cells using DAF fluorescence intensity. Calcium influx was evaluated in A7r5 cells by flow cytometry. Western blot was used to analyze eNOS and nNOS phosphorylation.AM reduced PE-induced contraction by calcium influx diminution in smooth muscle cells (F/FOur data show that amitriptyline impaired vascular function through two different mechanisms: blockade of TLR4 in endothelial cells and consequent decrease in NO production and calcium influx reduction in smooth muscle and endothelial cells. We also suggest, for the first time, nNOS activity reduction by AM in non-neuronal cells.

Leukocyte influx into the arterial wall plays a critical role in the development and progression of atherosclerosis. Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues plays a role in regulating leukocyte trafficking into the vascular wall during atherogenesis. Gene expression profiling revealed that members of the Netrin, Semaphorin and Ephrin family of guidance molecules are differentially regulated in athero-prone and athero-protected regions of the aortae of Ldlr-/- mice. Netrin-1 and Semaphorin 3A are highly expressed in the outer curvature of the aorta, and potently inhibit chemokine-directed migration of monocytes in vitro. Endothelial expression of these negative guidance cues is down-regulated by pro-atherogenic factors associated with monocyte entry into the vessel wall, including oscillatory shear stress and pro-inflammatory cytokines. Furthermore, using intravital microscopy we show that in vivo delivery of Netrin-1- or Semaphorin3A-blocking peptides increases leukocyte adhesion to the endothelium ~2-fold compared to the control peptides. Conversely, we show that a guidance cue that is highly expressed in atheroprone regions of the aorta, EphrinB2, is up-regulated in endothelium cells under pro-atherosclerotic flow conditions (eg. oscillatory shear stress, pro-inflammatory cytokines) and functions as a monocyte chemoattractant. These data suggest a new paradigm for leukocyte-endothelial interactions in which the coordinate regulation of chemo-attractive and -repulsive guidance cues regulates leukocyte trafficking in health and disease.