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The neuropeptide CGRP (calcitonin gene-related peptide) is a potent vasodilator, with a cardioprotective role, although the precise mechanisms are unclear. Here we show the ability of endogenous and exogenous CGRP to restore blood pressure, when nitric oxide synthesis is blocked, in a model of cardiovascular disease associated with endothelial dysfunction and impaired nitric oxide production. Male wild-type and αCGRP knockout mice received L-nitro-arginine methyl ester (150 mg/kg in drinking water) to induce a sustained hypertension with evidence of cardiovascular remodeling. The hypertensive response was exacerbated in L-nitro-arginine methyl ester-treated αCGRP knockouts, indicating that endogenous αCGRP acts in a protective manner, when nitric oxide production is diminished. Exogenous CGRP rescued αCGRP knockout mice from both hypertension and cardiovascular remodeling. Further studies using a nonrecovery protocol with a CGRP receptor antagonist (BIBN4096 BS) revealed that CGRP acts via the canonical CGRP receptor (CLR [calcitonin-like receptor]/RAMP1 [receptor activity-modifying protein]); with no effect of an antagonist (AC187) of a second CGRP-responsive receptor (the amylin-1 receptor, CTR [calcitonin receptor]/RAMP1). Blood flow, in resistance vessels of the exteriorised mesentery, was investigated. Noradrenaline–induced vasoconstriction with recovery, in L-nitro-arginine methyl ester-treated wild-type mice. However, αCGRP knockout, or BIBN4096 BS-treated wild-type mice demonstrated a similar constrictor response to noradrenaline, but significantly impaired blood flow recovery. The combined findings highlight that αCGRP protects against cardiovascular dysfunction, signaling via the canonical CGRP receptor and acting when nitric oxide production is lost, such as in endothelial dysfunction associated with vascular disease. These in vivo results support the proposal that CGRP provides a novel treatment for cardiovascular disease.

The treatment of hypertension and heart failure remains a major challenge to healthcare providers. Despite therapeutic advances, heart failure affects more than 26 million people worldwide and is increasing in prevalence due to an ageing population. Similarly, despite an improvement in blood pressure management, largely due to pharmacological interventions, hypertension remains a silent killer. This is in part due to its ability to contribute to heart failure. Development of novel therapies will likely be at the forefront of future cardiovascular studies to address these unmet needs. Calcitonin gene-related peptide (CGRP) is a 37 amino acid potent vasodilator with positive-ionotropic and -chronotropic effects. It has been reported to have beneficial effects in hypertensive and heart failure patients. Interestingly, changes in plasma CGRP concentration in patients after myocardial infarction, heart failure, and in some forms of hypertension, also support a role for CGRP on hemodynamic functions. Rodent studies have played an important role thus far in delineating mechanisms involved in CGRP-induced cardioprotection. However, due to the short plasma half-life of CGRP, these well documented beneficial effects have often proven to be acute and transient. Recent development of longer lasting CGRP agonists may therefore offer a practical solution to investigating CGRP further in cardiovascular disease in vivo. Furthermore, pre-clinical murine studies have hinted at the prospect of cardioprotective mechanisms of CGRP which is independent of its hypotensive effect. Here, we discuss past and present evidence of vascular-dependent and -independent processes by which CGRP could protect the vasculature and myocardium against cardiovascular dysfunction.

Ageing is associated with increased vulnerability to environmental cold exposure. Previously, we identified the role of the cold-sensitive transient receptor potential (TRP) A1, M8 receptors as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor system may become dysfunctional in ageing. We show that behavioural and vascular responses to skin local environmental cooling are impaired with even moderate ageing, with reduced TRPM8 gene/protein expression especially. Pharmacological blockade of the residual TRPA1/TRPM8 component substantially diminished the response in aged, compared with young mice. This implies the reliance of the already reduced cold-induced vascular response in ageing mice on remaining TRP receptor activity. Moreover, sympathetic-induced vasoconstriction was reduced with downregulation of the α2c adrenoceptor expression in ageing. The cold-induced vascular response is important for sensing cold and retaining body heat and health. These findings reveal that cold sensors, essential for this neurovascular pathway, decline as ageing onsets.

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA’s anti-inflammatory and analgesic effects.

Ageing is associated with increased vulnerability to environmental cold exposure. Previously, we identified the role of the cold-sensitive transient receptor potential (TRP) A1, M8 receptors as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor system may become dysfunctional in ageing. We show that behavioural and vascular responses to skin local environmental cooling are impaired with even moderate ageing, with reduced TRPM8 gene/protein expression especially. Pharmacological blockade of the residual TRPA1/TRPM8 component substantially diminished the response in aged, compared with young mice. This implies the reliance of the already reduced cold-induced vascular response in ageing mice on remaining TRP receptor activity. Moreover, sympathetic-induced vasoconstriction was reduced with downregulation of the α2c adrenoceptor receptor in ageing. The cold-induced vascular response is important for sensing cold and retaining body heat and health. These findings reveal that cold sensors, essential for this neurovascular pathway, decline as ageing onsets.

Platelet-associated complications including thrombosis, thrombocytopenia, and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis. Although several mechanisms that may contribute to the dysfunction of platelets during inflammatory diseases have been reported, knowledge on the primary molecules/mechanisms that underpin platelet-associated complications in such conditions is not fully established. Here, we report the significance of the mouse antimicrobial cathelicidin, mouse cathelicidin-related antimicrobial peptide (mCRAMP) (an orthologue of LL37 in humans), on the modulation of platelet reactivity during psoriasis using Imiquimod-induced psoriasis in mice as an inflammatory disease model for psoriasis vulgaris in humans. The activation of platelets during psoriasis is increased as evidenced by the elevated levels of fibrinogen binding and P-selectin exposure on the surface of platelets, and the level of soluble P-selectin in the plasma of psoriatic mice. The skin and plasma of psoriatic mice displayed increased levels of mCRAMP. Moreover, the plasma of psoriatic mice augmented the activation of platelets obtained from healthy mice. The effect of mCRAMP is partially mediated through formyl peptide receptor 2/3 (Fpr2/3, the orthologue to human FPR2/ALX) in platelets as a significant reduction in their activation was observed when FPR2/ALX-selective inhibitors such as WRW4 or Fpr2/3-deficient mouse platelets were used in these assays. Since the level of antimicrobial cathelicidin is increased in numerous inflammatory diseases such as psoriasis, atherosclerosis, and inflammatory bowel disease, the results of this study point towards a critical role for antimicrobial cathelicidin and FPR2/ALX in the development of platelet-related complications in such diseases.

The term ‘neurogenic inflammation’ is commonly used, especially with respect to the role of sensory nerves within inflammatory disease. However, despite over a century of research, we remain unclear about the role of these nerves in the vascular biology of inflammation, as compared with their interacting role in pain processing and of their potential for therapeutic manipulation. This chapter attempts to discuss the progress in understanding, from the initial discovery of sensory nerves until the present day. This covers pioneering findings that these nerves exist, are involved in vascular events and act as important sensors of environmental changes, including injury and infection. This is followed by discovery of the contents they release such as the established vasoactive neuropeptides substance P and CGRP as well as anti-inflammatory peptides such as the opioids and somatostatin. The more recent emergence of the importance of the transient receptor potential (TRP) channels has revealed some of the mechanisms by which these nerves sense environmental stimuli. This knowledge enables a platform from which to learn of the potential role of neurogenic inflammation in disease and in turn of novel therapeutic targets.

Cold exposure is directly related to skin conditions, such as frostbite. This is due to the cold exposure inducing a vasoconstriction to reduce cutaneous blood flow and protect against heat loss. However, a long-term constriction will cause ischaemia and potentially irreversible damage. We have developed techniques to elucidate the mechanisms of the vascular cold response. We focused on two ligand-gated transient receptor potential (TRP) channels, namely, the established “cold sensors” TRP ankyrin 1 (TRPA1) and TRP melastin (TRPM8). We used the anaesthetised mouse and measured cutaneous blood flow by laser speckle imaging. Two cold treatments were used. A generalised cold treatment was achieved through whole paw water immersion (10 °C for 5 min) and a localised cold treatment that will be potentially easier to translate to human studies was carried out on the mouse paw with a copper cold probe (0.85-cm diameter). The results show that TRPA1 and TRPM8 can each act as a vascular cold sensor to mediate the vasoconstrictor component of whole paw cooling as expected from our previous research. However, the local cooling-induced responses were only blocked when the TRPA1 and TRPM8 antagonists were given simultaneously. This suggests that this localised cold probe response requires both functional TRPA1 and TRPM8.

Background/Introduction Cardiovascular inflammation is associated with endothelial damage, resulting in leukocyte trafficking and oedema formation, which results in a positive-feedback loop of inflammatory events. Purpose The phosphoinositide 3-kinase (PI3K) pathway has been shown to be upregulated in vascular disease conditions whereby its inhibitors potentially have beneficial effects. Hence this study was designed to examine the actions of PI-103, a PI3K non-selective inhibitor, on inflammatory responses. Methods Permeability assays were undertaken to quantify the effect of TNF-a in the presence and absence of PI-103 using FITC-dextran (40 kDa, 0.1 mg/ml) to measure levels of permeability in trans-wells plates using human microvascular endothelial cells (HMVEC). In vivo analysis was carried out per the UK Home Office Animals (Scientific Procedures) Act 1986. CD1 mice were anaesthetised, to test PI-103 in a zymosan-induced model of dorsal skin inflammation on neutrophil accumulation (measured by myeloperoxidase) and oedema formation (measured by Evans Blue accumulation). All data were analysed using 1-way or 2-way ANOVA with post-hoc test. Results PI-103 significantly reduced TNF-α induced microvascular endothelial cell permeability (Graph), thus impeding in vitro cytokine-induced inflammation. Whilst there was no effect on neutrophil accumulation in vivo, there was significant reduction in weight of treated areas and oedema formation, which was inhibited by PI-103 (Table 1). Effect of PI-103 in vivo Control 30 mg/kg PI-103 Tyrode solution Zymosan (μg/ml) Tyrode solution Zymosan (μg/ml) 10 30 100 10 30 100 MPO/Protein (U/mg) 0.66±0.26 1.42±0.07 1.40±0.14 1.68±0.12* 0.60±0.11 1.23±0.32 1.36±0.25 1.59±0.21* Oedema Volume (mm3) 0.00±0.00 3.26±1.76 11.70±2.21 21.50±4.67 2.20±1.53 1.05±1.05 3.27±0.74 5.52±1.58 Mice were pre-treated with PI-103 i.p. for 30 min, i.d injected with zymosan (50 μl/site) for 4 hr, followed by ex vivo MPO assay, weight and oedema volume measurements. Oedema was quantified by measuring: width (x), height (y) and depth (z) from each of the i.d. sites; volume of oedema = ((π/6)xyz). N=6 independent experiments in duplicates; *p Effect of PI103 in vitro on permeability Conclusions Our findings show that the PI3K non-selective inhibitor, PI-103 (as well as a PI3K α-selective inhibitor), reduces endothelial activation and inhibits inflammatory oedema formation. We conclude that there is a potential for PI3K inhibitors to act as anti-oedema agents in cardiovascular-related inflammatory conditions. Acknowledgement/Funding British Heart Foundation

Increasing evidence suggests that nerve fibers responding to noxious stimuli (nociceptors) modulate immunity in a variety of tissues, including the skin. Yet, the role of nociceptors in regulating sterile cutaneous inflammation remains unexplored. To address this question, we have developed a detailed description of the sterile inflammation caused by overexposure to UVB irradiation (i.e., sunburn) in the mouse plantar skin. Using this model, we observed that chemical depletion of nociceptor terminals did not alter the early phase of the inflammatory response to UVB, but it caused a significant increase in the number of dendritic cells and αβ + T cells as well as enhanced extravasation during the later stages of inflammation. Finally, we showed that such regulation was driven by the nociceptive neuropeptide calcitonin gene–related peptide. In conclusion, we propose that nociceptors not only play a crucial role in inflammation through avoidance reflexes and behaviors, but can also regulate sterile cutaneous immunity in vivo.

It is supposed that TRPA1 receptor can be activated by hydrogen sulphide (H2S). Here, we have investigated the role of TRPA1 receptor in H2S-induced [Ca(2+)]i increase in trigeminal ganglia (TRG) neurons, and the involvement of capsaicin-sensitive sensory nerves in H2S-evoked cutaneous vasodilatation. [Ca(2+)]i was measured with ratiometric technique on TRG neurons of TRPA1(+/+) and TRPA1(-/-) mice after NaHS, Na2S, allylisothiocyanate (AITC) or KCl treatment. Microcirculatory changes in the ear were detected by laser Doppler imaging in response to topical NaHS, AITC, NaOH, NaSO3 or NaCl. Mice were either treated with resiniferatoxin (RTX), or CGRP antagonist BIBN4096, or NK1 receptor antagonist CP99994, or K(+) ATP channel blocker glibenclamide. Alpha-CGRP(-/-) and NK1 (-/-) mice were also investigated. NaHS and Na2S increased [Ca(2+)]i in TRG neurons derived from TRPA(+/+) but not from TRPA1(-/-) mice. NaHS increased cutaneous blood flow, while NaOH, NaSO3 and NaCl did not cause significant changes. NaHS-induced vasodilatation was reduced in RTX-treated animals, as well as by pre-treatment with BIBN4096 or CP99994 alone or in combination. NaHS-induced vasodilatation was significantly smaller in alpha-CGRP(-/-) or NK1 (-/-) mice compared to wild-types. H2S activates capsaicin-sensitive sensory nerves through TRPA1 receptors and the resultant vasodilatation is mediated by the release of vasoactive sensory neuropeptides CGRP and substance P.

Calcitonin gene-related peptide (CGRP) was discovered over about 35 years ago through molecular biological techniques. Its activity as a vasodilator and the proposal that it was involved in pain processing were then soon established. Today, we are in the interesting situation of having the approval for the clinical use of antagonists and antibodies that have proved to block CGRP activities and benefit migraine. Despite all, there is still much to learn concerning the relevance of the vasodilator and other activities as well as further potential applications of CGRP agonists and blockers in disease. This review aims to discuss the history and present knowledge and to act as an introductory chapter in this volume.

Platelet-associated complications including thrombosis, thrombocytopenia, and hemorrhage are commonly observed during various inflammatory diseases such as sepsis, inflammatory bowel disease, and psoriasis. Despite the reported evidence on numerous mechanisms/molecules that may contribute to the dysfunction of platelets, the primary mechanisms that underpin platelet-associated complications during inflammatory diseases are not fully established. Here, we report the discovery of formyl peptide receptor 2, FPR2/ALX, in platelets and its primary role in the development of platelet-associated complications via ligation with its ligand, LL37. LL37 acts as a powerful endogenous antimicrobial peptide, but it also regulates innate immune responses. We demonstrate the impact of LL37 in the modulation of platelet reactivity, hemostasis, and thrombosis. LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail bleeding time in mice. By utilizing a pharmacological inhibitor and Fpr2/3 (an ortholog of human FPR2/ALX)–deficient mice, the functional dependence of LL37 on FPR2/ALX was determined. Because the level of LL37 is increased in numerous inflammatory diseases, these results point toward a critical role for LL37 and FPR2/ALX in the development of platelet-related complications in such diseases. Hence, a better understanding of the clinical relevance of LL37 and FPR2/ALX in diverse pathophysiological settings will pave the way for the development of improved therapeutic strategies for a range of thromboinflammatory diseases.

Background The temporomandibular joints (TMJ) encompass the jawbones (mandibular condyle) and the skull region (temporal bone)1. Dysfunctions in these joints and surrounding muscles result in a condition known as TMJ disorder (TMD), which include mastication-related pain. 16%–59% of patients reported painful symptoms while 33%–86% of these patients showed clinical signs1. Currently, the etiology of TMD is unknown, although lavage samples from patients showed signs of synovitis, which may lead to the development of degenerative disorders, such as TMJ arthritis2. There are only a few murine models available, as the most commonly used animal model is TMJ arthritis in rats. Objectives We aimed to characterise a mouse model of TMJ arthritis by mimicking the formation of synovitis using zymosan. We sought to characterise the development of orofacial pain by performing various behavioural measurements and measure joint inflammation. Methods All in vivo procedures were carried out according to the UK Home Office Animals (Scientific Procedure) .Act 1986 Male CD1 mice (6–8 weeks) were anaesthetised transiently using 2% isoflurane. Zymosan (10, 30, or 100 µg; 10 µl), or saline was administered unilaterally into the TMJ as previously described3. Spontaneous pain behaviours were observed by counting the number of unilateral cheek wipes. All studies were terminated by cervical dislocation and the tissue was collected for ex vivo analyses. Mean ±SEM was calculated and data were analysed by unpaired Student’s T-test or 2-way repeated measures ANOVA with Bonferroni’s post hoc test. p Results A dose response study showed that 30 µg, but not 10 µg and 100 µg of zymosan, resulted in a maximal wiping response at 2 hours, which was maintained at 4 hours (table 1; ***p Table 1 A summary of zymosan-mediated spontaneous orofacial pain behaviours and inflammation. Conclusions We have established a mouse model of TMJ arthritis, which showed significant orofacial pain responses in vivo. Our data highlights an inflammatory profile typical of zymosan and shows a potential use for this model to investigate novel TMD treatments. References [1] Millam. J Musculoskel Neuron Interact2003;3(4):382–390. [2] Cheng and Israel. J Maxillofac Surg2005;63(6):761–765. [3] Kramer, et al. Physiol Behav2010;99(5):669–678. Acknowledgements XK and JV were funded by Arthritis Research UK. Disclosure of Interest None declared

Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis. Dual TRPC4/TRPC5 blockade by ML204 increased mortality and hypothermia in thioredoxin-treated LPS mice but preserved macrophage’s ability to phagocytose. TRPC5 deletion did not alter body temperature but promoted additional accumulation of peritoneal leukocytes and inflammatory mediator release in thioredoxin-administered LPS mice. Thioredoxin diminished macrophage-mediated phagocytosis in wild-type but not TRPC5 knockout animals. TRPC5 ablation did not affect LPS-induced responses. However, ML204 caused mortality associated with exacerbated hypothermia and decreased peritoneal leukocyte numbers and cytokines in LPS-injected mice. These results suggest that bacterial thioredoxin effects under LPS stimuli are mediated by TRPC4 and TRPC5, shedding light on the additional mechanisms of bacterial virulence and on the pathophysiological roles of these receptors.

Calcitonin gene-related peptide (CGRP) was discovered over about 35 years ago through molecular biological techniques. Its activity as a vasodilator and the proposal that it was involved in pain processing were then soon established. Today, we are in the interesting situation of having the approval for the clinical use of antagonists and antibodies that have proved to block CGRP activities and benefit migraine. Despite all, there is still much to learn concerning the relevance of the vasodilator and other activities as well as further potential applications of CGRP agonists and blockers in disease. This review aims to discuss the history and present knowledge and to act as an introductory chapter in this volume.

This study revealed the modulatory role of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) cation channels in the Aldara-induced (5% imiquimod) murine psoriasis model using selective antagonists and genetically altered animals. We have also developed a refined localized model to enable internal controls and reduce systemic effects. Skin pathology was quantified by measuring skin thickness, scaling, blood flow, and analyzing dermal cellular infiltrate, whereas nocifensive behaviors were also observed. Cytokine gene expression profiles were measured ex vivo. Psoriasiform dermatitis was significantly enhanced in TRPA1 knockout mice and with TRPA1 antagonist (A967079) treatment. By comparison, symptoms were decreased when TRPV1 function was inhibited. Imiquimod induced Ca2+ influx in TRPA1-, but not in TRPV1-expressing cell lines. Immunohistochemical studies revealed that CD4+ T helper cells express TRPA1 but not TRPV1 ion channels in mice skin. Compared with the TRPV1 knockout animals, additional elimination of the TRPA1 channels in the TRPV1/TRPA1 double knockout mice did not modify the outcome of the imiquimod-induced reaction, further supporting the dominant role of TRPV1 in the process. Our results suggest that the protective effects in psoriasiform dermatitis can be mediated by the activation of neuronal and nonneuronal TRPA1 receptors.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide. Discovered 30 years ago, it is produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP has two major forms (α and β). It belongs to a group of peptides that all act on an unusual receptor family. These receptors consist of calcitonin receptor-like receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for full functionality. CGRP is a highly potent vasodilator and, partly as a consequence, possesses protective mechanisms that are important for physiological and pathological conditions involving the cardiovascular system and wound healing. CGRP is primarily released from sensory nerves and thus is implicated in pain pathways. The proven ability of CGRP antagonists to alleviate migraine has been of most interest in terms of drug development, and knowledge to date concerning this potential therapeutic area is discussed. Other areas covered, where there is less information known on CGRP, include arthritis, skin conditions, diabetes, and obesity. It is concluded that CGRP is an important peptide in mammalian biology, but it is too early at present to know if new medicines for disease treatment will emerge from our knowledge concerning this molecule.

High diesel exhaust particle levels are associated with increased health effects; however, knowledge on the impact of its chemical contaminant 1,2-naphthoquinone (1,2-NQ) is limited. We investigated whether postnatal and adult exposures to 1,2-NQ influence allergic reaction and the roles of innate and adaptive immunity. Male neonate (6 days) and adult (56 days) C57Bl/6 mice were exposed to 1,2-NQ (100 nM; 15 min) for 3 days, and on day 59, they were sensitized and later challenged with ovalbumin (OVA). Airway hyper-responsiveness (AHR) and production of cytokines, immunoglobulin E (IgE) and leukotriene B4 (LTB4) were measured in the airways. Postnatal exposure to 1,2-NQ activated dendritic cells in splenocytes by increasing expressing cell surface molecules (e.g., CD11c). Co-exposure to OVA effectively polarized T helper (Th) type 2 (Th2) by secreting Th2-mediated cytokines. Re-stimulation with unspecific stimuli (PMA and ionomycin) generated a mixed Th1 (CD4(+)/IFN-γ(+)) and Th17 (CD4(+)/IL-17(+)) phenotype in comparison with the vehicle-matched group. Postnatal exposure to 1,2-NQ did not induce eosinophilia in the airways at adulthood, although it evoked neutrophilia and exacerbated OVA-induced eosinophilia, Th2 cytokines, IgE and LTB4 production without affecting AHR and mast cell degranulation. At adulthood, 1,2-NQ exposure evoked neutrophilia and increased Th1/Th2 cytokine levels, but failed to affect OVA-induced eosinophilia. In conclusion, postnatal exposure to 1,2-NQ increases the susceptibility to antigen-induced asthma. The mechanism appears to be dependent on increased expression of co-stimulatory molecules, which leads to cell presentation amplification, Th2 polarization and enhanced LTB4, humoral response and Th1/Th2 cytokines. These findings may be useful for future investigations on treatments focused on pulmonary illnesses observed in children living in heavy polluted areas.

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-β) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-β stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-β receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-β but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

Patients with rheumatoid arthritis (RA) suffer from pain and joint disability. The transient receptor potential ankyrin 1 (TRPA1) channel expressed on sensory neurones and non-neuronal cells mediates pain transduction and inflammation and it has been implicated in RA. However, there is little information on the contribution of TRPA1 for human disease. Here, we investigated the expression of TRPA1 on peripheral blood leukocytes and the circulating levels of its endogenous activators 4-hydroxynonenal (4-HNE) and hydrogen peroxide (H2O2) in RA patients treated or not with the anti-rheumatic leflunomide (LFN) or the anti-TNFα adalimumab (ADA). We also assessed whether TRPA1 expression correlates with joint pain and disability, in addition to the immune changes in RA. TRPA1 expression on peripheral blood leukocytes correlated with pain severity and disability. TRPA1 levels on these cells were associated with the numbers of polymorphonuclear and the activation of CD14+ cells. No correlations were found between the lymphocyte population and TRPA1 expression, pain or disability. Patients recently diagnosed with RA expressed increased levels of TRPA1 on their leukocytes whilst treatment with either LFN or ADA down-regulated this receptor probably by reducing the numbers of polymorphonuclears and the activation of CD14+ cells. We suggest that the activation levels of CD14+ cells, the numbers of PMNs in the peripheral blood and the expression of TRPA1 on peripheral blood leukocytes correlate with RA progression, affecting joint pain sensitivity and loss of function.

Arthritis is a condition characterised by mainly pain, reduced joint movement and signs of inflammation, such as swelling. The disorder has many different types, of which osteoarthritis (a degenerative joint disease) and rheumatoid arthritis (a chronic autoimmune disease) are the two most common forms. There are >6 million sufferers in the UK and both conditions have a huge potential to impair capabilities and contribute to social and economic burdens. Whilst there are a wide range of arthritic therapies available, many patients under treatment complain of poor pain relief. Thus there is a need for novel therapeutic approaches, and the transient receptor potential (TRP) family of receptor channels has been investigated. One particular area of recent research has been the ligand-gated transient receptor potential vanilloid 1 (TRPV1) channel. Findings from numerous pre-clinical models and scientific studies have shown that TRPV1 desensitisation, or the use of TRPV1 antagonists alleviates pain and some inflammatory aspects. New findings have started to unveil the potential of other TRP channels in mediating arthritic pain and inflammation. With the understanding that the currently available treatments for arthritis are limited, researchers have looked into the exciting prospect that TRP receptor antagonists may be developed into effective, specific drugs, which would potentially protect against the complications of arthritis. These antagonists are still under development, although only data from studies from pre-clinical models are currently available. This review acts to summarize knowledge of the potential influence of TRP receptors in arthritis to date.

Obesity induced by Western diets is associated with type 2 diabetes mellitus and cardiovascular diseases, although underlying mechanisms are unclear. We investigated a murine model of diet-induced obesity to determine the effect of transient potential receptor vanilloid 1 (TRPV1) deletion on hypertension and metabolic syndrome. Wild-type and TRPV1 knockout mice were fed normal or high-fat diet from 3 to 15 weeks. High-fat diet-fed mice from both genotypes became obese, with similar increases in body and adipose tissue weights. High-fat diet-fed TRPV1 knockout mice showed significantly improved handling of glucose compared with high-fat diet-fed wild-type mice. Hypertension, vascular hypertrophy, and altered nociception were observed in high-fat diet-fed wild-type but not high-fat diet-fed TRPV1 knockout mice. Wild-type, but not high-fat diet-fed TRPV1 knockout, mice demonstrated remodeling in terms of aortic vascular hypertrophy and increased heart and kidney weight, although resistance vessel responses were similar in each. Moreover, the wild-type mice had significantly increased plasma levels of leptin, interleukin 10 and interleukin 1β, whereas samples from TRPV1 knockout mice did not show significant increases. Our results do not support the concept that TRPV1 plays a major role in influencing weight gain. However, we identified a role of TRPV1 in the deleterious effects observed with high-fat feeding in terms of inducing hypertension, impairing thermal nociception sensitivity, and reducing glucose tolerance. The observation of raised levels of adipokines in wild-type but not TRPV1 knockout mice is in keeping with TRPV1 involvement in stimulating the proinflammatory network that is central to obesity-induced hypertension and sensory neuronal dysfunction.

This review highlights the progress from the initial finding of neurogenic inflammation up to the most recent development in the field of sensory nerves research, focusing on their roles in the microvasculature and the skin. Recent discovery of Transient Receptor Potential (TRP) channels highlight their important roles in detecting a range of environmental stimuli, including chemical and temperature. This provides us novel mechanisms for driving neurogenic inflammation upstream of neuropeptide release in addition to promising potential therapeutic targets in various diseases, including pain, itching and skin inflammation.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B₄ (LTB₄) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B₄ receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB₄-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.

The transient receptor potential vanilloid 1 (TRPV1) is primarily localized to sensory nerve fibers and is associated with the stimulation of pain and inflammation. TRPV1 knockout (TRPV1KO) mice show enhanced LPS-induced sepsis compared with wild type (WT). This implies that TRPV1 may have a key modulatory role in increasing the beneficial and reducing the harmful components in sepsis. We investigated immune and inflammatory mechanisms in a cecal ligation and puncture (CLP) model of sepsis over 24 h. CLP TRPV1KO mice exhibited significant hypothermia, hypotension, and organ dysfunction compared with CLP WT mice. Analysis of the inflammatory responses at the site of initial infection (peritoneal cavity) revealed that CLP TRPV1KO mice exhibited: 1) decreased mononuclear cell integrity associated with apoptosis, 2) decreased macrophage tachykinin NK1-dependent phagocytosis, 3) substantially decreased levels of nitrite (indicative of NO) and reactive oxygen species, 4) increased cytokine levels, and 5) decreased bacteria clearance when compared with CLP WT mice. Therefore, TRPV1 deletion is associated with impaired macrophage-associated defense mechanisms. Thus, TRPV1 acts to protect against the damaging impact of sepsis and may influence the transition from local to a systemic inflammatory state.