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: Savolitinib (AZD6094, HMPL-504, volitinib) is an oral, potent, and highly selective MET receptor tyrosine kinase inhibitor. This series of studies aimed to develop a pharmacokinetic-pharmacodynamic (PK/PD) model to link inhibition of MET phosphorylation (pMET) by savolitinib with anti-tumour activity. Experimental approach: Cell line-derived xenograft (CDX) experiments using human lung cancer (EBC-1) and gastric cancer (MKN-45) cells were conducted in athymic nude mice using a variety of doses and schedules of savolitinib. Tumour pMET changes and growth inhibition were calculated after 28 days. Population PK/PD techniques were used to construct a PK/PD model for savolitinib. Key results: Savolitinib showed dose- and schedule-dependent anti-tumour activity in the CDX models, with more frequent, lower dosing schedules (e.g. twice daily) being more effective than intermittent, higher dosing schedules (e.g. 4 days on/3 days off or 2 days on/5 days off). There was a clear exposure–response relationship, with maximal suppression of pMET of >90%. Data from additional CDX and patient-derived xenograft (PDX) models overlapped, allowing the calculation of a single EC50 of 0.38 ng/mL. Tumour growth modelling demonstrated that prolonged, high levels of pMET inhibition (>90%) were required for tumour stasis and regression in the models. Conclusion and implications: High and durable levels of MET inhibition by savolitinib are needed for optimal monotherapy anti-tumour activity in preclinical models. The modelling framework developed here can be used to translate tumour growth inhibition from the mouse to human, and thus guide choice of clinical dose and schedule.
Many current animal models of heart failure are hampered by intrinsic methodological complexities, while other models yield only a subtle cardiac phenotype even after prolonged in vivo treatments. A new “chemogenetic” animal model of heart failure recapitulates a critical characteristic shared by many disease states that lead to heart failure in humans: an increase in redox stress in the heart. This “chemogenetic” approach exploits a recombinant yeast enzyme that can be dynamically and specifically activated in vivo to generate the reactive oxygen species (ROS) hydrogen peroxide (H2O2) in cardiac myocytes. Redox stress can be rapidly, selectively, and reversibly manipulated by chemogenetic generation of ROS in cardiac myocytes, yielding a new model of dilated cardiomyopathy. Treatment of animals with the angiotensin receptor blocker valsartan promotes recovery of ventricular function and resolution of adverse cardiac remodeling. This Mini-Review discusses in vivo chemogenetic approaches to manipulate and analyze oxidative stress in the heart.
Aldosterone binds to the mineralocorticoid receptor (MR), a transcription factor of the nuclear receptor family, present in the kidney and in various other non-epithelial cells including the heart and the vasculature (Cannavo et al., 2018). Indeed, extra-renal pathophysiological effects of this hormone have been characterized, extending its actions to the cardiovascular (CV) system (Messaoudi et al., 2012). A growing body of clinical and pre-clinical evidence suggests that MR activation plays an important pathophysiological role in CV remodeling by promoting cardiac hypertrophy, fibrosis, arterial stiffness, as well as in inflammation and oxidative stress (Bauersachs et al., 2015). The following review article outlines the role of MR in CV disease with a focus on myocardial remodeling and heart failure (HF) including clinical trials as well as cellular and animal studies.
Hyperexcitability-related diseases include epilepsies, pain syndromes, neuromuscular disorders, and cardiac arrhythmias. Sodium channel inhibitors can be used to treat these conditions, however, their applicability is limited by their nonspecific effect on physiological function. They act by channel block (obstructing ion conduction, since the binding site is within the channel pore), and by modulation (delaying recovery from the non-conducting inactivated state). Channel block inhibits healthy and pathological tissue equally, while modulation can preferentially inhibit pathological activity. Therefore, an ideal sodium channel inhibitor drug would act by modulation alone. Unfortunately, thus far no such drug has been known to exist. Here we present evidence that riluzole acts by this “ideal” mechanism, “non-blocking modulation” (NBM). We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the “fenestrations” (cavities connecting the central cavity to the membrane phase). Using precisely timed UV pulses to photolabel specific conformations of the channel, we show that association to the local anesthetic binding site requires prior inactivation. We discuss why kinetics of binding is crucial for selective inhibition of pathological activity, and how the NBM mechanism can be recognized using a special voltage- and drug application-protocol. Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential.
Background and Purpose. Despite widespread abuse of cocaine, there are no approved treatments for cocaine use disorder. Chronic cocaine use is associated with upregulated dopamine D3 receptor (D3R) expression in the brain, and therefore, most D3R-based medication development has focused on D3R antagonists. However, D3R antagonists do not attenuate cocaine intake under “easy” self-administration conditions when response requirements are low. Here we evaluated a novel, highly selective and metabolically stable D3R partial agonist, VK4-40, for its efficacy in reducing cocaine intake and relapse to drug seeking. Experimental Approach. The impact of VK4-40 on cocaine intake and relapse were evaluated using intravenous self-administration procedures under a fixed-ratio 2 reinforcement schedule and cocaine-primed reinstatement conditions in rats. Optogenetic brain-stimulation reward procedures were used to evaluate the interaction of VK4-40 and cocaine in the mesolimbic dopamine system. Sucrose self-administration and a conditioned place preference paradigm was used to evaluate the abuse potential of VK4-40 alone and other unwanted effects. Key Results. VK4-40 dose-dependently reduced cocaine self-administration and cocaine-primed reinstatement of drug-seeking behavior. In addition, VK4-40 inhibited cocaine-enhanced brain-stimulation reward caused by optogenetic stimulation of dopamine neurons in the ventral tegmental area. VK4-40 alone decreased brain-stimulation reward, and produced neither conditioned place preference nor place aversion. This new D3R partial agonist also failed to alter oral sucrose self-administration. Conclusions and Implications. The novel D3R partial agonist, VK4-40, attenuates cocaine reward and relapse in rodents, without significant unwanted effects. These findings support further investigation of D3R partial agonists as putative treatments for cocaine use disorder.
Background and Purpose: Tacrolimus (Tac) induces pancreatic β cell dysfunction, causing new-onset diabetes mellitus (NODM) after transplantation. Reg3g is a member of the pancreatic regenerative gene family, as reported to improve type 1 diabetes by promoting β cell regeneration. Here, we aim to investigate the role and approach of Reg3g in reversing Tac-induced β cell dysfunction and NODM in mice. Experimental Approach: Circulating REG3A (the human homolog of mouse Reg3g) concentrations of patients treated with Tac after heart transplantation(HT) were detected. The glucose-stimulated insulin secretion (GSIS) and mitochondrial functions, including mitochondria membrane potential (MMP), mitochondria calcium uptake, ATP production, and oxygen consumption rate (OCR), were tested in β cells. Effects of Reg3g on Tac-induced NODM in mice were studied. Key Results: Circulating REG3A levels significantly decreased in NODM patients treated with Tac compared with those without diabetes. Tac down-regulated Reg3g via inhibiting STAT3-mediated transcription activation, while Reg3g protected against Tac-induced apoptosis of β cells. Besides, Reg3g restored GSIS suppressed by Tac in β cells via improving mitochondrial function, including increased MMP, mitochondria calcium uptake, ATP production, and OCR. Mechanically, Reg3g increased accumulation of pSTAT3(Ser727) in mitochondria by activating ERK1/2-STAT3 signaling pathway, leading to restoration of Tac-caused mitochondrial impairment. Moreover, Reg3g overexpression effectively ameliorated Tac-induced NODM in mice. Conclusion and Implications: Reg3g ameliorates Tac-induced pancreatic β cell dysfunction by restoring mitochondrial function via a pSTAT3(Ser727)-dependent way. Our observations identify a novel Reg3g-involved mechanism underlying the augmented incidence of Tac-induced NODM and reveal that Reg3g ameliorates Tac-induced β cell dysfunction.
Cysteinyl leukotrienes (CysLTs) are inflammatory lipid mediators that play a central role in the pathophysiology of several inflammatory diseases. Recently, there has been an increased interest in determining how these lipid mediators orchestrate tumor development and metastasis through promoting a pro-tumoral microenvironment. Upregulation of CysLTs receptors and CysLTs production is found in a number of cancers and has been associated with increased tumorigenesis. Understanding the molecular mechanisms underlying the role of CysLTs and their receptors in cancer progression will help investigate the potential of targeting CysLTs signaling for anti-cancer therapy. This review gives an overview of the biological effects of CysLTs and their receptors, along with current knowledge of their regulation and expression. It also provides a recent update on the molecular mechanisms that have been postulated to explain their role in tumorigenesis and on the potential of anti-CysLTs in the treatment of cancer.
Background and Purpose. The proalgesic transient receptor potential (TRP) ankyrin 1 (TRPA1) channel, expressed by a subpopulation of primary sensory neurons, has been implicated in various pain models in mice. However, evidence in rats indicates that TRPA1 conveys nociceptive signals elicited by channel agonists but not those associated with tissue inflammation or nerve injury. Here, in rats, we explored the TRPA1 role in mechanical allodynia associated with neurogenic inflammation and moderate (partial sciatic nerve ligation, pSNL) or severe (chronic constriction injury, CCI) sciatic nerve injury. Experimental Approach. Acute nociception and mechanical hypersensitivity associated with neurogenic inflammation and sciatic nerve injury (pSNL and CCI) were investigated in rats with TRPA1 pharmacological antagonism or genetic silencing. TRPA1 presence and function was analyzed in cultured rat Schwann cells. Key Results. Hind paw mechanical allodynia (HPMA), but not acute nociception, evoked by local injection of the TRP vanilloid 1 (TRPV1) agonist, capsaicin, or the TRPA1 agonist, allyl isothiocyanate, was mediated by calcitonin gene related peptide (CGRP) released from peripheral nerve terminals. CGRP-evoked HPMA was sustained by a reactive oxygen species (ROS)-dependent TRPA1 activation, probably in Schwann cells. HPMA evoked by pSNL, but not that evoked by CCI, was mediated by ROS and TRPA1 without the involvement of CGRP. Conclusions and Implications. As found in mice, TRPA1 mediates mechanical allodynia associated with neurogenic inflammation and moderate nerve injury in rats. The channel implication in mechanical hypersensitivity following inflammation and partial nerve damage is a common rodent feature and might be explored in humans.
Background and purpose: The pathophysiology of coronary artery spasm ( CAS), with its associated ischaemic crises, is currently poorly understood, and treatment is frequently ineffective. In view of increasing evidence that platelet- platelet based defects may occur in CAS patients,. we investigated platelet reactivity in CAS patients and whether symptomatic crises reflect activation of platelet-endothelial interactions. Experimental approach: CAS patients were evaluated during acute and/or chronic symptomatic phases, and compared with healthy control subjects. Inhibition of platelet aggregation with ADP by the nitric oxide (NO) donor sodium nitroprusside (SNP), . and plasma levels of syndecan-1 (glycocalyx shedding marker), tryptase (mast cell activation marker), and platelet microparticles were measured. Key Results: Inhibition of aggregation by SNP was impaired in chronic CAS, and tended to deteriorate further during symptomatic crises, while plasma levels of syndecan-1, tryptase and platelet microparticles increased. Infusion of high dose N-acetylcysteine (NAC) plus glyceryl trinitrate rapidly restored platelet responsiveness to SNP and decreased plasma syndecan-1 levels. The effect of NAC on platelet responsiveness to SNP was mimicked in vitro by the H2S donor NaHS. Conversely, inhibition of enzymatic release of H2S attenuated NAC effect. Conclusion and Implications: CAS is associated with substantial impairment of platelet NO signaling. During acute symptomatic exacerbations, platelet resistance to NO is aggravated, together with mast cell activation and damage to both vasculature and platelets. NAC reverses platelet resistance to NO via release of H2S, and reverses glycocalyx shedding during symptomatic crises: this suggests that H2S donors may correct the pathophysiological anomalies underlying CAS.
Background and Purpose: Hypertensive vascular remodeling (VR) is responsible for end-organ damage and is the result of increased extracellular matrix accumulation and excessive vascular smooth muscle cell (VSMC) proliferation. MicroRNA-26a (miR-26a), a non-coding small RNA, is involved in multiple cardiovascular diseases. We aimed to validate the effect and mechanisms of miR-26a in hypertensive VR. Experimental Approach: Spontaneously hypertensive rats (SHRs) were injected intravenously with recombinant adeno-associated virus-miR-26a. In vitro experiments, angiotensin II (AngII)-induced VSMCs were transfected with miR-26a mimic or inhibitor. Key Results: We found miR-26a downregulated in the thoracic aorta and plasma of SHRs. Overexpression of miR-26a inhibited extracellular matrix deposition by targeting connective tissue growth factor (CTGF) and mitigated VSMC proliferation by regulating the enhancer of zeste homolog 2 (EZH2)/p21 pathway both in vitro and in vivo. AngII-mediated Smad3 activation suppressed miR-26a expression, which in turn promoted Smad3 activation via targeted regulation of Smad4, leading to further downregulation of miR-26a. Conclusion and Implications: Our study reveals that AngII stimulates a Smads/miR-26a positive feedback loop, which further reduces miR-26a expression, leading to collagen production and VSMC proliferation and consequently, VR. MiR-26a has an antagonistic effect on hypertensive VR and can be a strategy for treating hypertensive VR.
In recent years increasing evidence suggests that commensal microbiota may play an important role in health and disease, including cerebrovascular disease. Gut microbes impact physiology, at least in part by metabolizing dietary factors and also host-derived substrates and then generating active compounds including toxins. The purpose of the current review is to highlight the complex interplay between microbiota, their metabolites and essential functions for human health ranging from regulation of the metabolism and the immune system to modulation of brain development and function. We also discuss the role of gut dysbiosis in cerebrovascular disease, specifically in acute and chronic stroke phases and the possible implication of intestinal microbiota in post-stroke cognitive impairment and dementia, and we identify potential therapeutic opportunities of targeting microbiota in this context.
Background and purpose: Chronic alcohol intake provoked unfavorable geometric and functional changes in the heart along with altered autophagy. Parkin, a cytosolic E3 ubiquitin ligase encoded by PARK2 gene, governs mitochondrial homeostasis and mitophagy although its role in alcoholic cardiomyopathy remains unclear. Experimental approach: This study was designed to examine the role of Parkin in alcohol-induced cardiomyopathy. Adult male wild-type (WT) and PARKIN2 knockout (Parkin-/-) mice were placed on alcohol (4%) or control diet for 8 weeks. Echocardiographic and cardiomyocyte mechanical properties, myocardial and mitochondrial morphology, autophagy and mitophagy were evaluated. GFP-LC3 puncta was employed to assess autophagosome formation. Key results: Our results revealed that alcohol intake led to unfavorable geometric and contractile changes (enlarged left ventricular chamber; decreased fractional shortening, ejection fraction, peak shortening and velocity of shortening/relengthening, prolonged relengthening duration), enlarged cardiomyocyte size and interstitial fibrosis, as well as mitochondrial swelling with cristae disarrangement and mitochondrial depolarization, the effects of which were exacerbated by Parkin deficiency. Alcohol consumption promoted autophagy and PINK1-Parkin-mediated mitophagy, the effects of which were cancelled off by Parkin knockout. Co-immunoprecipitation noted a tight interaction between Parkin and Ambra1 (autophagy and beclin1 regulator 1). In vitro study using neonatal rat cardiomyocytes revealed that Parkin transfection ameliorated ethanol-induced changes in autophagy. However, Ambra1 silencing negated Parkin-induced protection against ethanol-induced autophagy. Conclusions and implications: Taken together, these data suggest an integral role for Parkin in the face of alcoholic challenge possibly through its interaction with Ambra1 to promote autophagy and maintain mitochondrial homeostasis.
The Popeye Domain Containing Protein 1 (POPDC1), a tight junction-associated transmembrane protein with a unique binding site for cAMP, has been shown to act as a tumour suppressor in cancer cells. Through interaction with many downstream effectors and signalling pathways, POPDC1 promotes cell adhesion and inhibits uncontrolled cell proliferation, epithelial-to-mesenchymal transition, and metastasis. However, POPDC1 expression is downregulated in many types of cancer, thereby reducing its tumour-suppressive actions. This review discusses the role of POPDC1 in the progression of the malignant phenotype and highlights the broad range of benefits POPDC1 stabilisation may achieve therapeutically. Cancer stem cells (CSC) are a key hallmark of malignancies and commonly promote treatment resistance. This article provides a comprehensive overview of CSC signalling mechanisms, many of which have been shown to be regulated by POPDC1 in other cell types, thus suggesting an additional therapeutic benefit for POPDC1-stabilising anticancer drugs.
Background and Purpose Inactivation of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) by causing the accumulation of intracellular Ca2+ to activate calcineurin-mediated nuclear factor of activated T-lymphocytes (NFAT)/NF-κB pathways, resulted in the phenotypic modulation of smooth muscle cells (SMCs) to accelerate angiotensin II-induced aortic aneurysm. Our goal was to investigate the mechanism involved. Experimental Approach We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was substituted by serine, to represent partial irreversible oxidation of C674. The aortas of SKI mice and their littermate wild-type mice were collected for RNA sequencing, cell culture, protein expression, luciferase activity and aortic aneurysm analysis. KEY RESULTS Inactivation of C674 inhibited the promoter activity and protein expression of PPARγ, which could be reversed by inhibitors of calcineurin or NF-κB. Overexpression of PPARγ2 inhibited the phenotypic modulation of SKI SMCs. Pioglitazone, the activator of PPARγ, blocked the activation of NFAT/NF-κB, inhibited SMC phenotypic modulation, and ameliorated angiotensin II-induced aortic aneurysms in SKI mice. CONCLUSIONS AND IMPLICATIONS The inactivation of SERCA2 C674 promotes the development of aortic aneurysm by disrupting the balance between PPARγ and NFAT/NF-κB. Our study highlights the importance of C674 redox status in regulating PPARγ to maintain aortic homeostasis.
Background and Purpose: Myocardial infarction (MI) is the leading cause of mortality globally due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we demonstrated that overexpression of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (4F), induced cell division in ~20% of the post-mitotic CMs overexpressed 4F. The current study aims to identify a small molecule that augments 4F-induced CM cycle induction. Experimental Approach, Key Results: Screening of small molecules with a potential to augment 4F-induced cell-cycle induction in 60-day-old mature human induced pluripotent cardiomyocytes (hiPS-CMs) revealed N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC), which activates cell cycle progression in 4F-transduced hiPS-CMs. Autodock tool and Autodock vina computational methods showed that NDPPC has a potential interaction with the binding site at the human p38⍺ mitogen-activated protein kinase (p38⍺ MAP kinase), a critical negative regulator of the mammalian cell cycle. A p38⍺ MAP kinase activity assay showed that NDPPC inhibits its activity in a dose-dependent manner. Overexpression of p38⍺ MAP kinase in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect. Conclusion and Implications: NDPPC is a novel inhibitor for p38⍺ MAP kinase and is a promising drug to augment CM cell cycle response to the 4F. NDPPC could become an adjunct treatment with other cell cycle activators for heart failure treatment.
Background and purpose: Amphetamine use disorder is a serious health concern, but surprisingly little is known about the vulnerability to the moderate and compulsive use of this psychostimulant and its underlying mechanisms. Previous research showed that inherited serotonin transporter (SERT) down-regulation increases the motor response to cocaine, as well as moderate and compulsive intake of this psychostimulant. Here we sought to investigate whether these findings generalize to amphetamine and the underlying mechanisms in the nucleus accumbens. Experimental Approach: In serotonin transporter knockout (SERT−/−) and wild-type control (SERT+/+) rats we assessed the locomotor response to acute amphetamine (AMPH) and intravenous AMPH self-administration under short access (ShA: 1 hr daily sessions) and long access (LgA: 6 hr daily sessions) conditions. 24 hrs after AMPH self-administration we analysed the expression of glutamate system components in the nucleus accumbens shell and core. Key results: We found that SERT−/− animals displayed an increased AMPH-induced locomotor response and increased AMPH self-administration under LgA, but not ShA conditions. Further, we observed changes in the vesicular and glial glutamate transporters, NMDA and AMPA receptor subunits and their respective postsynaptic scaffolding proteins as function of serotonin transporter genotype, AMPH exposure (baseline, ShA and LgA) and nucleus accumbens sub region. Conclusion and implications: We demonstrate that SERT gene deletion increases the psychomotor and reinforcing effects of AMPH, and that the latter is potentially mediated, at least in part, by homeostatic changes in the glutamatergic synapse of the nucleus accumbens shell and/or core.
Background and Purpose Intra-islet heparan sulfate (HS) plays an important role in the maintenance of the pancreatic islet function. The aim of this study was to investigate the effect mechanism of HS loss on the functioning of islets in diabetic mice. Experimental Approach The hypoglycemic effect of a heparanase inhibitor, OGT2115, was tested in streptozotocin-induced diabetic mice. The islets of pancreas sections were also stained to reveal their morphology. An insulinoma MIN6 cell line and primary isolated murine islets were used to investigate the effect of OGT2115 in vitro. Key Results Intra-islet HS was clearly lost in streptozotocin-induced diabetic mice due to the increased heparanase expression in damaged islets. OGT2115 prevented intra-islet HS loss to improve the glucose profile and insulin secretion in streptozotocin-treated mice. The apoptosis of pancreatic beta cells, the infiltration of mononuclear macrophages, CD4 and CD8 positive T-cells in islets was reduced by OGT2115 in streptozotocin-treated mice, but OGT2115 did not alter the direct streptozotocin-induced damage in vitro. The expression of heparanase was increased in high glucose-treated isolated islets but not in response to direct streptozotocin stimulation. Further experiments showed that high glucose stimuli could decrease the expression of peroxisome proliferator-activated receptor gamma (PPARγ) in cultured islets, thereby relieving the PPARγ-induced inhibition of heparanase gene expression. Conclusion and Implications Hyperglycemia could cause intra-islet HS loss by elevating the expression of heparanase, thereby aggravating inflammatory cell infiltration and islet damage. Inhibition of heparanase might provide benefit for pancreatic beta cell protection in type 1 diabetes.