Bruton’s tyrosine kinase (BTK) is a non-receptor kinase best known for its role in B lymphocyte development that is critical for proliferation, and survival of leukaemia cells in B cell malignancies. However, BTK is expressed in myeloid cells, particularly monocytes and macrophages where its inhibition has been reported to exhibit anti-inflammatory properties. Therefore, we explored the role of BTK on the migration of myeloid cells in vitro and in vivo. Using the zymosan induced peritonitis model of sterile inflammation we demonstrated that acute (1 h prior to zymosan) inhibition of BTK using a wide range of FDA (Ibrutinib and Acalabrutinib) and non-FDA approved inhibitors (ONO-4059, CNX-774, Olumatinib and LFM-A13) reduced neutrophil and monocyte recruitment. XID mice, which have a point mutation in the Btk gene had reduced neutrophil and monocyte recruitment to the peritoneum following zymosan challenge. To better understand the role of BTK in myeloid cell recruitment we investigated both chemotaxis and chemokine production in monocytes and macrophages. Pharmacological or genetic inhibition of BTK signalling substantially reduced human monocyte and murine macrophage chemotaxis to a range of chemoattractants (complement C5a and CCL2). We also demonstrated that inhibition of BTK in tissue resident macrophages significantly decreases chemokine secretion by reducing NF-kB activity and Akt signalling. Our work has identified a new role of BTK in regulating myeloid cell recruitment via two mechanisms, 1) reducing monocyte/macrophages’ ability to undergo chemotaxis, and 2) reducing chemokine secretion, via reduced NF-kB activity in tissue resident macrophages.

Background and Purpose Ca2+ influx via TRPV4 triggers Ca2+ release from the IP3-sensitive internal store to generate repetitive oscillations. While mitochondria are acknowledged regulators of IP3-mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+-induced Ca2+ release at IP3 receptors, to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. Experimental Approach TRPV4 evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. Key ResultsTRPV4 activation with GSK1016790A(GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+. The ATP synthase inhibitor oligomycin did not potentiate TRPV4 mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the phospholipase C (PLC) blocker U73122 and the inositol triphosphate receptor (IP3 R) blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. Conclusion and Implications These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 evoked Ca2+ release.

- Invited Commentary –

Since the start of the novel coronavirus SARS-Cov-2 pandemic, a disease that has become one of the world’s greatest global health challenges, the role of the immune system has been at the forefront of scientific studies. The pathophysiology of COVID-19 is complex, which is evident by those at higher risk for poor outcome. Multiple systems contribute to thrombosis and inflammation seen in COVID-19 patients, including neutrophil dysfunction, platelet activation, endothelial cell activation. Understanding how the immune system functions in different patient cohorts (particularly given recent emerging events with the Oxford/AstraZeneca vaccine) is vital to understanding the pathophysiology of this devastating disease and for subsequent development of novel therapeutic targets and expedite possible drug repurposing strategies that could benefit society on a global scale.

Pyroptosis, is a specialized form of inflammatory cell death which aids the defensive response against invading pathogens. Its tight regulation is lost during infection by the severe acute respiratory coronavirus 2 (SARS-CoV-2) and thus uncontrolled pyroptosis disrupts the immune system and the integrity of organs defining the critical conditions in patients with high viral load. Molecular pathways engaged downstream to the formation and stabilization of the inflammasome -required to execute the process- have been uncovered and drugs are available for their regulation. On the contrary, pharmacological inferring of the upstream events -which are critical to sense and interpret the initial damage by the pathogen- is far from being elucidated. This limits our capacity to identify early markers and targets to ameliorate SARS-CoV-2 linked pyroptosis. Here we aim to raise attention on mitochondria and pathways leading to its dysfunction with the goal to inform early steps of inflammasome and devise tools to interpret and counteract diseases by the SARS-CoV-2.

Cancer cachexia is one of the most common causes of death among cancer patients, no effective anti-cachectic treatment is currently available. In experimental cachectic animal models, aberrant activation of STAT3 in skeletal muscle has been found to contribute to muscle wasting. However, its clinical association, the factors regulating STAT3 activation, and the molecular mechanisms remain incompletely understood. Here, we show that an enhanced interaction between activated STAT3 and HSP90, which were observed in the skeletal muscle of cancer cachexia patients, is a crucial event for the development of cachectic muscle wasting. Administration of HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 tumor-bearing cachectic mice or the myotube atrophy of C2C12 cells induced by C26 conditional medium. A mechanistic study indicated that in cachectic skeletal muscle, prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner; Our results demonstrate that the HSP90/STAT3/FOXO1 axis plays a critical role in the cachectic muscle wasting, which might serve as potential therapeutic target for the treatment of cancer cachexia.

Background and Purpose: Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABAA receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal a5 subunit-containing GABAA receptors. The purpose of this study is to investigate tricyclic compounds in a5 subunit-containing receptor subtypes.Experimental Approach: Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABAA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the a5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings. Key Results: We show that the antipsychotic drugs clozapine and chlorpromazine exert functional inhibition on multiple GABAA receptor subtypes, including a5-containing ones. Based on a chlorpromazine binding site observed in a GABA-gated bacterial homologue, we identified a novel site in a5 GABAA receptor subunits and demonstrate differential usage of this and the orthosteric sites by these ligands.Conclusion and Implications: Despite high molecular and functional similarities among the tested ligands, they reduce GABA currents by differential usage of allosteric and orthosteric sites. The C C C C C C site we describe here is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology. Further studies in defined subtypes are needed to substantiate mechanistic links between the therapeutic effects of clozapine and its action on certain GABAA receptor subtypes.

Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmaceutical treatments targeting calcitonin gene-related peptide (CGRP) signalling molecules have provided safe and effective treatments; monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit the beneficious impact of these new drugs, it is important to understand the molecular mechanisms involved to better understand migraine pathophysiology and improve the therapy. Here we describe current views on the role of the CGRP family of peptides CGRP, calcitonin (CT), adrenomedullin (AM), amylin (AMY) and their receptors in the trigeminovascular system (TGV). All these molecules are present within the TGV system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets.

Sepsis causes multi-organ dysfunction and is a major cause of death in intensive care units, but there are no treatments that reverse the pathophysiological effects of sepsis. Vitamin C has antioxidant, anti-inflammatory, anticoagulant and immune modulatory actions, so is a potential treatment for sepsis. Recent clinical trials of high-doses of intravenous vitamin C (6-16 g/day) had variable effects. Since much higher doses are without side-effects in cancer and burns patients, we studied the effects of a mega-dose of intravenous sodium ascorbate (150 g/40 kg) in a clinically relevant ovine model of sepsis. This treatment dramatically improved the clinical state and over 3-7-h improved cardiovascular, pulmonary, hepatic and renal function and reduced body temperature. In a critically ill COVID-19 patient, intravenous sodium ascorbate (60 g) restored arterial pressure, improved renal function and increased arterial blood oxygen levels. Clinical trials are testing the effectiveness of mega-dose vitamin C in septic patients.

Alcohol use disorder (AUD) is one of the most common but still poorly treated psychiatric conditions. Developing new treatments requires a better understanding of the aetiology of symptoms and evaluation of novel therapeutic targets in preclinical studies. Recent developments in our understanding of the reinforcement-based cognitive biases (RBCBs) that contribute to the development of AUD and its treatment offer new opportunities for both clinical and preclinical research. In this review, we first briefly describe psychological and cognitive theories that implicate various aspects of reinforcement sensitivity in the development, maintenance, and recurrence of alcohol addiction. Furthermore, in separate sections, we describe studies investigating RBCBs and their neural, neurochemical, and pharmacological correlates, and we discuss possible interactions between RBCBs and trajectories of AUD. Finally, we describe how recent translational studies using state-of-the-art animal models can facilitate our understanding of the role of reinforcement sensitivity and RBCBs in various aspects of AUD.

Background and Purpose Liver failure is often associated with psychiatric alterations, partly resulting from the increased dopamine levels in brain. We aimed to investigate relationship between increased dopamine levels and mental abnormalities using bile duct ligation (BDL) rats and document mechanism that liver failure increased dopamine levels in SH-SY5Y cells. Experimental Approach Psychiatric alterations were operated following 14-day BDL. Dopamine and its metabolite levels in cortex, expressions of enzymes and transporters related to dopamine metabolism and transport in cortex and hippocampus were measured. SH-SY5Y cells were used to investigate whether NH4Cl, bile acids and bilirubin affected expression of tyrosine hydroxylase (TH) or not. TH expression in SH-SY5Y cells co-incubated with bilirubin and signal pathway inhibitors was measured. Key Results Open-field test results showed a remarkable increase in exploratory behavior following BDL. BDL increased dopamine levels and expression of TH protein in cortex. MAO-A and Mb-COMT slightly but significantly decreased. Data from SH-SY5Y cells showed that increased bilirubin levels was a factor in inducing TH expression. Both inhibitor of NF-κB pathway BAY117082 and silencing p65 remarkably reversed bilirubin-induced upregulation of TH protein. NF-κB activator TNF-α increased expression of TH protein. Roles of bilirubin in TH expression and increases in dopamine levels were documented using hyperbilirubinemia rats. Significant increases in dopamine levels, expressions of TH, p65 and p-p65 protein were observed in hyperbilirubinemia rats. Conclusion and Implications BDL significantly increased dopamine levels in rat cortex partly due to bilirubin-mediated TH induction. Increased bilirubin induced TH expression via activating NF-κB signaling pathway.

Background and purpose: Regulator of G-protein signal 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signaling. Here we investigate whether RGS4 modulates nociceptin/orphanin FQ opioid (NOP) receptor signaling and whether this modulation has relevance for L-Dopa induced dyskinesia. Experimental approach: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with N/OFQ or AT-403 in the presence of the RGS4 inhibitor, CCG-203920, and D1-stimulated cAMP or pERK responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and levodopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. Key results: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons, and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403 mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels which was reversed by levodopa. Levodopa acutely upregulated RGS4 in the lesioned striatum. Conclusions and Implications: RGS4 physiologically inhibits NOP receptor signaling and an RGS4 inhibitor enhances NOP responses. Furthermore, an RGS4 inhibitor improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of upregulation of striatal RGS4 levels occurring during dyskinesia expression.

Background and Purpose: Hydroxychloroquine and chloroquine, alone or in combination with azithromycin, have been proposed as therapies for COVID-19. However, there is currently scant and inconsistent data regarding their proarrhythmic potential in these patients. Moreover, their risk profile in the setting of altered physiological states encountered in patients with COVID-19 (i.e. febrile state, electrolyte imbalances, and/or acidosis) is unknown. Experimental approach: Potency of hERG block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from three separate individuals. Key Results: Chloroquine and hydroxychloroquine blocked hERG with IC50 of 1.47±0.07 µM and 3.78±0.17 µM respectively, indicating proarrhythmic risk at concentrations effective against SARS-CoV-2 in vitro and proposed in COVID-19 clinical trials. Hypokalaemia and hypermagnesemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs (i.e. reduced proarrhythmic risk), whereas increased temperature decreased potency of chloroquine and hydroxychloroquine but increased potency for azithromycin. In silico simulations across genetically diverse populations predicted that 17% of individuals exhibit action potential durations >500 ms at the highest proposed therapeutic levels, equating to significant QT prolongation. Conclusion and Implications: Significant proarrhythmic risk is predicted for hydroxychloroquine and chloroquine at doses proposed to treat COVID-19. Clinicians should carefully consider the risk of such treatments, and implement long term QT interval monitoring in trials, particularly in patients with electrolyte imbalances.

Background and Purpose: Superoxide anions can reduce the bioavailability and actions of endothelium-derived NO. In human resistance-sized arteries, endothelium-dependent vasodilatation can be mediated by H2O2 instead of NO. We tested the hypotheses that in resistance arteries from patients with resistant cardiovascular disease (CVD), endothelium-dependent vasodilatation uses mechanisms that are either insensitive to oxidative stress or involve a reactive oxygen species. Experimental Approach: Small arteries were isolated from biopsies of the parietal pericardium of patients undergoing elective cardiothoracic surgery and were studied by immunohistochemical and organ chamber techniques. Key Results: NO-synthases 1, 2 and 3, superoxide dismutase 1 and catalase proteins were observed in the microvascular wall. Relaxing responses to bradykinin were endothelium dependent. During submaximal depolarization-induced contraction, these relaxations were inhibited by inhibitors of NO-synthases (NOS) and soluble guanylyl cyclase (sGC) but not by scavengers of NO or HNO, inhibitors of cyclooxygenases, neuronal NO-synthase, superoxide dismutase or catalase, or by exogenous catalase. During contraction stimulated by endothelin-1, these relaxations were not reduced by any of these interventions except DETCA which caused a small reduction. Conclusion and Implications: In resistance arteries from patients with resistant CVD, endothelium-dependent relaxations seem not to be mediated by NO, HNO or H2O2 although NOS and sGC can be involved. These vasodilator responses proceed during excessive oxidative stress.

Background and Purpose: Acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and pulmonary fibrosis remain major causes of morbidity, mortality and healthcare burden in the critically ill patient. There is an urgent medical need for identifying factors of susceptibility and prognosis and for designing new therapeutic tools for treating these disorders. Here, we evaluate the capacity of the immunomodulatory neuropeptide cortistatin to regulate pulmonary inflammation and fibrosis in vivo. Experimental Approach: ALI/ARDS and pulmonary fibrosis were induced experimentally in wild-type and cortistatin-deficient mice by pulmonary infusion of the bacterial endotoxin LPS or the chemotherapeutic drug bleomycin, and the histopathological signs, pulmonary leukocyte infiltration and cytokines and fibrotic markers were evaluated. Key Results: Partially-deficient mice in cortistatin showed exacerbated pulmonary damage, pulmonary inflammation, alveolar oedema and fibrosis, and subsequent increased respiratory failure and mortality when challenged to LPS or bleomycin, even at low doses. Treatment with cortistatin reversed these aggravated phenotypes and protected from progression to severe ARDS and fibrosis after high-exposition to both injury agents. Moreover, cortistatin-deficient pulmonary macrophages and fibroblasts showed exaggerated ex vivo inflammatory and fibrotic responses. The anti-fibrotic protective effect of cortistatin was also observed in experimental scleroderma, in which lack of cortistatin predisposes to develop more severe dermal lesions and associated pulmonary fibrosis. Conclusion and Implications: We identify to cortistatin as an endogenous break of pulmonary inflammation and fibrosis. Deficiency in cortistatin could be a marker of poor-prognosis in inflammatory/fibrotic pulmonary disorders. Cortistatin-based therapies emerge as attractive candidates to treat severe ALI/ARDS, including SARS-Cov-2-associated ARDS.

Emerging data shows pregnant women with COVID-19 are at significantly higher risk of severe outcomes compared to non-pregnant women of similar age. This review discusses the invaluable insight revealed from vaccine clinical trials in women who were vaccinated and inadvertently became pregnant during the trial period. It further explores a number of clinical avenues in their management and proposes a drug development strategy in-line with clinical trials for vaccines and drug treatments for the drug development community. Little is known of the long-term effects of COVID-19 on the mother and the baby. We provide a rationale for our hypothesis that COVID-19 predisposes pregnant women to cardiovascular diseases later in life, in a similar way, to preeclampsia and may increase the risk of preeclampsia in their subsequent pregnancy. This is an ever-evolving landscape and early knowledge for healthcare providers and drug innovators is offered to ensure benefits outweigh the risks.

BACKGROUND AND PURPOSE The cysteine674 (C674) thiol of Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is easily and irreversibly oxidized under atherosclerotic conditions. However, contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved. EXPERIMENTAL APPROACH Heterozygous SERCA2 C674S knock-in (SKI) mice in which half of the C674 was substituted by serine674 were used to mimic removal of the reactive C674 thiol which occurs under patholog-ical conditions. The whole aorta and aortic root were isolated for histological analysis. Bone marrow derived macrophages (BMDMs) and a cardiac endothelial cell line were used for intra-cellular Ca2+, macrophage adhesion and protein expression analysis. KEY RESULTS SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Cell cul-ture studies suggest the partial substitution of SERCA2 C674 increased intracellular calcium lev-els and ER stress in both BMDMs and ECs. The release of pro-inflammatory factors and macro-phage adhesion increased in SKI BMDMs. In normal ECs, the overexpression of C674S mutant induced endothelial inflammation and promoted macrophage recruitment. Additionally, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, prevented the increased atherosclerosis observed in SKI mice, and alleviated ER stress and inflammatory responses in BMDMs and ECs exposed to 4-PBA. CONCLUSIONS AND IMPLICATIONS The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by in-ducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox status in the context of atherosclerosis, and open up a novel therapeutic strategy to combat atherosclerosis.

Background and Purpose: Many pain-triggering nociceptor neurons express TRPV1 or TRPA1, cation-selective channels with large pores that enable permeation of QX-314, a cationic analogue of lidocaine. Co-application of QX-314 with TRPV1 or TRPA1 activators can silence nociceptors. We now describe BW-031, a novel more potent cationic sodium channel inhibitor, test whether its application alone can inhibit the pain associated with tissue inflammation, and whether this strategy can also inhibit cough. Experimental Approach: We characterized BW-031 inhibition of sodium channels and tested BW-031 in three models of inflammatory pain: rat paw inflammation produced by Complete Freund’s Adjuvant injection or surgical incision and a mouse paw UV burn model. We also tested the ability of BW-031 to inhibit cough induced by inhalation of dilute citric acid in guinea pigs. Key Results: BW-031 inhibited Nav1.7 and Nav1.1 channels with ~6-fold greater potency than QX-314 when introduced inside cells and entered capsaicin-activated TRPV1 expressing sensory neurons. BW-031 inhibited inflammatory pain in all three models, producing more effective and longer-lasting inhibition of pain than QX-314 in the mouse UV burn model. BW-031 was also effective in reducing cough counts by 78-90% when applied intratracheally under isoflurane anesthesia or by aerosol inhalation in awake guinea pigs with airway inflammation produced by ovalbumin sensitization. Conclusion and Implications: BW-031 a novel cationic sodium channel inhibitor can be applied locally as a single agent to inhibit inflammatory pain and also effectively inhibits cough in a guinea pig model of nociceptor-activated cough, suggesting a new clinical approach to treating cough.