Khadija M. Alawi

The paradoxical role of the transient receptor potential vanilloid 1 receptor in inflammation.

The transient potential receptor vanilloid 1 (TRPV1) receptor is a non-selective cation channel that is chemically activated by capsaicin, the pungent component of hot peppers. In addition, endogenous compounds, in particular the endogenous cannabinoid receptor activator, anandamide, have been demonstrated to activate TRPV1 in vivo. TRPV1 receptors are also activated by temperatures within the noxious range (>43 degrees C) and low pH (<pH 6.0). TRPV1 receptors are predominantly expressed in primary afferent fibres which are peptidergic sensory neurones, such as the thinly myelinated A-delta and unmyelinated C-fibres. TRPV1 receptors have also been demonstrated to be present in non-neuronal cells. Historically, TRPV1 has been considered as a pro-inflammatory receptor due to its key role in several conditions, including neuropathic pain, joint inflammation and inflammatory bowel disease, amongst others. However, the purpose of this review is to underline the emerging new evidence which demonstrate paradoxical, protective functions for this unique receptor in vivo. For example, in experimentally induced sepsis, TRPV1 null mice demonstrated elevated levels of pathological markers in comparison to wild-type mice. In addition to the pro-inflammatory and protective roles of TRPV1 in pathophysiological states, TRPV1 has also been shown to have important functions under normal physiological conditions, for example in urinary bladder function, thermoregulation and neurogenesis. The emerging functions of TRPV1 highlight the necessity for further research in light of increasing reports of potential TRPV1 antagonists undergoing pre-clinical experimentations.

TRPA1 is essential for the vascular response to environmental cold exposure

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α2C-adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates.

The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature

Transient receptor potential vanilloid 1 (TRPV1) is involved in sensory nerve nociceptive signaling. Recently, it has been discovered that TRPV1 receptors also regulate basal body temperature in multiple species from mice to humans. In the present study, we investigated whether TRPV1 modulates basal sympathetic nervous system (SNS) activity. C57BL6/J wild‐type (WT) mice and TRPV1 knockout (KO) mice were implanted with radiotelemetry probes for measurement of core body temperature. AMG 9810∗∗∗ (50 mg/kg) or vehicle (2% DMSO/5% Tween 80/10 ml/kg saline) was injected intraperitoneally. Adrenoceptor antagonists or vehicle (5 ml/kg saline) was injected subcutaneously. In WT mice, the TRPV1 antagonist, AMG9810, caused significant hyperthermia, associated with increased noradrenaline concentrations in brown adipose tissue. The hyperthermia was significantly attenuated by the β‐adrenoceptor antagonist propranolol, the mixed α‐/β‐adrenoceptor antagonist labetalol, and the α1‐adrenoceptor antagonist prazosin. TRPV1 KO mice have a normal basal body temperature, indicative of developmental compensation. D‐Amphetamine (potent sympathomimetic) caused hyperthermia in WT mice, which was reduced in TRPV1 KO mice, suggesting a decreased sympathetic drive in KOs. This study provides new evidence that TRPV1 controls thermoregulation upstream of the SNS, providing a potential therapeutic target for sympathetic hyperactivity thermoregulatory disorders.—Alawi, K. M., Aubdool, A. A., Liang, L., Wilde, E., Vepa, A., Psefteli, M.‐P., Brain, S. D., Keeble, J. E. The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature. FASEB J. 29, 4285‐4298 (2015). www.fasebj.org

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy and Heart Failure

Background: Research into the therapeutic potential of &agr;-calcitonin gene–related peptide (&agr;-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated &agr;-CGRP analogue (&agr;Analogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. Methods: The ability of the &agr;Analogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the &agr;Analogue on angiotensin II–induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the &agr;Analogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. Results: The angiotensin II–induced hypertension was attenuated by cotreatment with the &agr;Analogue (50 nmol·kg–1·d–1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The &agr;Analogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the &agr;Analogue reversed angiotensin II–induced hypertension and associated vascular and cardiac damage. The &agr;Analogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the &agr;Analogue was well tolerated with neither signs of desensitization nor behavioral changes. Conclusions: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized &agr;Analogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freunds adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.

Tail‐Cuff Technique and Its Influence on Central Blood Pressure in the Mouse

Background Reliable measurement of blood pressure in conscious mice is essential in cardiovascular research. Telemetry, the “gold‐standard” technique, is invasive and expensive and therefore tail‐cuff, a noninvasive alternative, is widely used. However, tail‐cuff requires handling and restraint during measurement, which may cause stress affecting blood pressure and undermining reliability of the results. Methods and Results C57Bl/6J mice were implanted with radio‐telemetry probes to investigate the effects of the steps of the tail‐cuff technique on central blood pressure, heart rate, and temperature. This included comparison of handling techniques, operators sex, habituation, and influence of hypertension induced by angiotensin II. Direct comparison of measurements obtained by telemetry and tail‐cuff were made in the same mouse. The results revealed significant increases in central blood pressure, heart rate, and core body temperature from baseline following handling interventions without significant difference among the different handling technique, habituation, or sex of the investigator. Restraint induced the largest and sustained increase in cardiovascular parameters and temperature. The tail‐cuff readings significantly underestimated those from simultaneous telemetry recordings; however, “nonsimultaneous” telemetry, obtained in undisturbed mice, were similar to tail‐cuff readings obtained in undisturbed mice on the same day. Conclusions This study reveals that the tail‐cuff technique underestimates the core blood pressure changes that occur simultaneously during the restraint and measurement phases. However, the measurements between the 2 techniques are similar when tail‐cuff readings are compared with telemetry readings in the nondisturbed mice. The differences between the simultaneous recordings by the 2 techniques should be recognized by researchers.

Transient receptor potential canonical 5 channels plays an essential role in hepatic dyslipidemia associated with cholestasis

Transient receptor potential canonical 5 (TRPC5), a calcium-permeable, non-selective cation channel is expressed in the periphery, but there is limited knowledge of its regulatory roles in vivo. Endogenous modulators of TRPC5 include a range of phospholipids that have an established role in liver disease, including lysophosphatidylcholine (LPC). Cholestasis is characterized by impairment of excretion of bile acids, leading to elevation of hepatic bile acids. We investigated the contribution of TRPC5 in a murine model of cholestasis. Wild-type (WT) and TRPC5 knock-out (KO) mice were fed a diet supplemented with 0.5% cholic acid (CA) for 21 days. CA-diet supplementation resulted in enlargement of the liver in WT mice, which was ameliorated in TRPC5 KO mice. Hepatic bile acid and lipid content was elevated in WT mice, with a reduction observed in TRPC5 KO mice. Consistently, liver enzymes were significantly increased in cholestatic WT mice and significantly blunted in TRPC5 KO mice. Localized dyslipidaemia, secondary to cholestasis, was investigated utilizing a selected lipid analysis. This revealed significant perturbations in the lipid profile following CA-diet feeding, with increased cholesterol, triglycerides and phospholipids, in WT, but not TRPC5 KO mice. Our results suggest that activation of TRPC5 contributes to the development of cholestasis and associated dyslipidemia. Modulation of TRPC5 activity may present as a novel therapeutic target for liver disease.

Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice

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 macrophages 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.

AB0095 Investigating The Role of Transient Receptor Potential Canonical 5 (TRPC5) in Complete Freund's Adjuvant (CFA)-Induced Arthritis

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by inflammation of diarthrodial joints. Sensory neurons expressing transient receptor potential (TRP) channels, a wide family of non-selective cation channels, have been shown to play a role in the pathogenesis of RA1. Transient receptor potential 5 (TRPC5) is a member of the canonical family of TRPC channels and is abundantly expressed in the central nervous system and periphery2, including CD55+ fibroblast-like synoviocytes3, but the role of TRPC5 in arthritis is unknown. Objectives To evaluate the for the first time, the contribution of TRPC5 in an in vivo model of arthritis and investigate the role of TRPC5 in the modulation of hyperalgesia and joint inflammation. Methods All experiments were conducted under the guidelines of the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. Age-matched, male 129S1/SvImJ wildtype (WT) and TRPC5 knockout (KO) mice littermates bred from heterozygotic mice provided by Prof. D.E. Clapham, USA2. Induction of arthritis using CFA was performed as previously described1; inflammation was allowed to progress to 14 days. Nociceptive tests were carried out weekly. Synovial blood flow was assessed in anaesthetised WT and TRPC5 KO mice using a full-field laser perfusion imager (FLPI)4 on day 14. Ex vivo analysis included assessment of cellular infiltration in the knee joint, and determination of cytokine concentrations. In separate studies, WT and TRPC5 KO mice received the TRPC4/5 antagonist, ML2045(2mg/kg, i.p., n=6) or vehicle (2% DMSO in saline, n=4) daily following i.art. injection of CFA. Results are presented as mean ± s.e.m. and analysed by 2-way ANOVA and Bonferonni post hoc test. Results WT mice developed primary hyperalgesia with a significant reduction in withdrawal threshold compared to baseline responses (334.7±25.7 vs 452.2±8.2 gram force (gf); p<0.01]). Primary hyperalgesia was exacerbated in TRPC5 KO mice compared to WT mice on day 14 (267.4±34.2 vs 334.7±25.4gf; p<0.05]). This was associated with similar deficits in weight-bearing on the ipsilateral hind-limb which was significantly reduced compared to WT mice. Ipsilateral synovial blood flow was significantly increased in TRPC5 KO mice compared to the contralateral synovium (291.3±50.2 vs 198.2±21.4 flux units (AU); p<0.01), and WT ipsilateral synovium (p<0.001). Pharmacological antagonism of TRPC5 in WT mice corroborated the results observed in TRPC5 KO mice, where hyperalgesia and joint inflammation was significantly exacerbated compared to vehicle treated WT mice. Conclusions Our results reveal a protective ability of TRPC5 in inflammatory joint conditions in vivo. Genetic deletion or pharmacological antagonism of TRPC5 resulted in an exacerbated inflammatory response highlighted by heightened nociceptive responses, increased cellular infiltration and cytokine concentrations in the knee joint. References Fernandes, E.S., et al. 2011. Arthritis Rheum 63, 819–829. Riccio, A., et al. 2009. Cell 137, 761–772. Xu, S.Z., et al. 2008. Nature 451, 69–72 Fernandes, E.S., et al. 2016. Arthritis Res Ther 18(1), 7 Miller, M., et al. 2011. J Biol Chem 286, 33436–46. Acknowledgement Supported by Arthritis Research UK. Disclosure of Interest None declared