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  • Interaction between neutrophils and ECs represents the


    Interaction between neutrophils and ECs represents the first step of the cellular phase of the inflammatory response, and provide the best condition for the transfer of the PMNL-derived LTA4 to the ECs, leading to the transcellular formation of cysteinyl-LTs. It is intriguing therefore to think that, as described, LTB4 induces PMNL activation and adhesion to the endothelium, leading to a secondary biosynthesis of cysteinyl-LTs through transcellular biosynthesis, that may contribute to the alteration of the vascular permeability. Indeed, locally formed cysteinyl-LTs resulting from transcellular biosynthesis have been shown to affect coronary [3], [4], [5] and cerebral vessels [6], resulting in myocardial ischemia and significant alteration of vascular permeability. Furthermore, a number of papers have reported the involvement of either CysLT2 [52] or both CysLT1 and CysLT2 receptors [53], [54], [55] in the inflammatory process subsequent to a NVP-LCQ195 vascular insult, such as vascular ischemia or oxygen deprivation. Taking advantage of a novel model of PMNL-perfused isolated rat brain we provide evidence that this alteration of vascular permeability is the result of Cys-LT2R activation, further supporting the notion that this receptor may represent an important target for pharmacological interventions aimed at modulating endothelial dysfunction in CV pathologies. With the present work we also demonstrate that the molecular events leading to cellular contraction and increased endothelial permeability are shared with thrombin, a well-known activator and contractor of ECs [56]. Indeed, LTC4- and LTA4-induced activation of CysLT2R in HUVEC increase [Ca2+]i and induce MLC20 phosphorylation to a level comparable to that observed upon activation with thrombin. Phosphorylation of MLC20 stimulates stress fiber formation and triggers actomyosin contraction as a result of the activation of Rho and Rho kinase. In agreement with this line of events we confirmed that inhibition of Rho kinase prevents LTC4-induced cytoskeleton rearrangement and HUVEC contraction. In addition, we demonstrated that Ca2+-triggered PKC activation is also involved in LTC4-induced rearrangement of EC cytoskeleton, extending previous results on Rho activation [57]. PKC, likely through the phosphorylation of a Rho GDI (Guanine nucleotide Dissociation Inhibitor) and a resulting increase of Rho activity [34], and/or through phosphorylation of the inositol 3 phosphate (IP3) receptor [58], will, in turn, increase Ca2+ availability within the contractile apparatus. Finally, the evaluation of cellular impedance changes associated with LTC4 challenge resulted consistent with the activation of a Gq-associated seven transmembrane receptor, and, again, were abolished by the pretreatment with a selective CysLT2R antagonist. Studies with CysLT receptor antagonists have always provided a controversial picture on the role for cysteinyl-LTs in the development of ischemic damage and in cardiac dysfunction, with conclusions often contradicting each other. Indeed, recent evidence showed that montelukast, zafirlukast and pranlukast may also antagonize the effects of nucleotides acting at different P2Y receptors [59], [60], inhibiting proinflammatory reactions that are not mediated by the known CysLT receptors. Furthermore for most of the cysteinyl-LTs antagonists used in original studies dealing with potential CV activities, little was known about their ability to act at the CysLT2R. Thus, taking advantage of a constitutive and of a recombinant system solely expressing the CysLT2R, we characterized the activity of a large panel of antagonists developed, and mainly used, as CysLT1R antagonists (LTRA). The results obtained showed that zafirlukast (Accolate), pranlukast (Onon), iralukast and pobilukast (widely used LTRAs for in vitro and animal studies) also interact with the CysLT2R with potencies that might have contributed to previously observed activities of these antagonists. Their interaction with the former ‘LTC4 receptor’, i.e. CysLT2R, was suggested for pobilukast and pranlukast in guinea pig trachea [61], [62], and for pobilukast in human lung tissue where it was able to displace both LTC4 and LTD4 binding [63]. This observation is consistent with the presence in the lung tissue of a high amount of ECs and thus of the presence of both CysLT1 (involved in parenchyma contraction) and CysLT2 (involved in vascular homeostasis) receptors, as exactly envisaged at that time [63]. On the other side, montelukast (Singulair), as previously reported [23], [41], appeared as a highly selective CysLT1R antagonist, and did not show any cross-reactivity at the CysLT2R. However, it should be taken into consideration that CysLT1R can be immune modulated by inflammatory stimuli [64], while CysLT1R activation may also have CV consequences, as has recently emerged from both experimental [65], [66] and clinical/epidemiological studies [67].