Purinergic P2X7 Receptor Research Articles

Aerobic Exercise Inhibited P2X7 Purinergic Receptors to Improve Cardiac Remodeling in Mice With Type 2 Diabetes.

Background: Diabetic cardiomyopathy (DCM), the main complication of diabetes mellitus, presents as cardiac dysfunction by ventricular remodeling. In addition, the inhibition of P2X7 purinergic receptors (P2X7R) alleviates cardiac fibrosis and apoptosis in Type 1 diabetes. However, whether exercise training improves cardiac remodeling by regulating P2X7R remains unknown. Methods: Db/db mice spontaneously induced with type 2 diabetes and high-fat diet (HFD) and mice with streptozotocin (STZ)-induced type 2 diabetes mice were treated by 12-week treadmill training. Cardiac functions were observed by two-dimensional echocardiography. Hematoxylin-eosin staining, Sirius red staining and transmission electron microscopy were respectively used to detect cardiac morphology, fibrosis and mitochondria. In addition, real-time polymerase chain reaction and Western Blot were used to detect mRNA and protein levels. Results: Studying the hearts of db/db mice and STZ-induced mice, we found that collagen deposition and the number of disordered cells significantly increased compared with the control group. However, exercise markedly reversed these changes, and the same tendency was observed in the expression of MMP9, COL-I, and TGF-β, which indicated cardiac fibrotic and hypertrophic markers, including ANP and MyHC expression. In addition, the increased Caspase-3 level and the ratio of Bax/Bcl2 were reduced by exercise training, and similar results were observed in the TUNEL test. Notably, the expression of P2X7R was greatly upregulated in the hearts of db/db mice and HFD + STZ-induced DM mice and downregulated by aerobic exercise. Moreover, we indicated that P2X7R knock out significantly reduced the collagen deposition and disordered cells in the DM group. Furthermore, the apoptosis levels and TUNEL analysis were greatly inhibited by exercise or in the P2X7R−/− group in DM. We found significant differences between the P2X7R−/− + DM + EX group and DM + EX group in myocardial tissue apoptosis and fibrosis, in which the former is significantly milder. Moreover, compared with the P2X7R−/− + DM group, the P2X7R−/− + DM + EX group represented a lower level of cardiac fibrosis. The expression levels of TGF-β at the protein level and TGF-β and ANP at the genetic level were evidently decreased in the P2X7R−/− + DM + EX group. Conclusion: Aerobic exercise reversed cardiac remodeling in diabetic mice at least partly through inhibiting P2X7R expression in cardiomyocytes.

P2X Purinergic Receptors Are Multisensory Detectors for Micro-Environmental Stimuli That Control Migration of Tumoral Endothelium.

Simple SummaryExtracellular ATP is highly concentrated in tumor stroma. In this study, we investigated the effects of the synthetic ATP analog Benzoylbenzoyl-ATP, 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), an agonist for P2X receptors, on tumor-derived endothelial cells (TEC) obtained from three different human tumors (breast, kidney and prostate carcinomas, respectively, BTEC, RTEC and PTEC). Treatment with high BzATP concentrations (100 µM) significantly reduced migration of all TEC types, resulting ineffective on human normal microvascular endothelium (HMEC); intriguingly, both the functional effect and associated calcium signals are sensitive to some key biological parameters of tumor stroma that include pH, Ca2+ and Zn2+. The lack of calcium signals selectively observed in PTEC, in which BzATP still retains its functional effect, suggests variability of intracellular signaling among TEC. These findings provide novel insights into the role of extracellular ATP as a multisensory regulator of migratory potential in tumoral endothelium.The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.

Role of Etanercept and Infliximab on Nociceptive Changes Induced by the Experimental Model of Fibromyalgia.

Background: Fibromyalgia is a clinical condition that affects 1% to 5% of the population. No proper therapy has been currently found. It has been described that inflammation plays a central role in the nerve sensitizations that characterize the pathology. Methods: This paper aimed to evaluate the efficacy of etanercept and infliximab in the management of pain sensitization. Fibromyalgia was induced by three injections once a day of reserpine at the dose of 1 mg/kg. Etanercept (3 mg/kg) and infliximab (10 mg/kg) were administered the day after the last reserpine injection and then 5 days after that. Behavioral analyses were conducted once a week, and molecular investigations were performed at the end of the experiment. Results: Our data confirmed the major effect of infliximab administration as compared to etanercept: infliximab administration strongly reduced pain sensitization in thermal hyperalgesia and mechanical allodynia. From the molecular point of view, infliximab reduced the activation of microglia and astrocytes and the expression of the purinergic P2X7 receptor ubiquitously expressed on glia and neurons. Downstream of the P2X7 receptor, infliximab also reduced p38-MAPK overexpression induced by the reserpine administration. Conclusion: Etanercept and infliximab treatment caused a significant reduction in pain. In particular, rats that received infliximab showed less pain sensitization. Moreover, infliximab reduced the activation of microglia and astrocytes, reducing the expression of the purinergic receptor P2X7 and p38-MAPK pathway.

Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs’ production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy.

Soluble P2X7 Receptor Is Elevated in the Plasma of COVID-19 Patients and Correlates With Disease Severity

Inflammation is a tightly coordinated response against bacterial and viral infections, triggered by the production of pro-inflammatory cytokines. SARS-CoV-2 infection induces COVID-19 disease, characterized by an inflammatory response mediated through the activation of the NLRP3 inflammasome, which results in the production of IL-1β and IL-18 along with pyroptotic cell death. The NLRP3 inflammasome could be also activated by sterile danger signals such as extracellular ATP triggering the purinergic P2X7 receptor. Severe inflammation in the lungs of SARS-CoV-2-infected individuals is associated with pneumonia, hypoxia and acute respiratory distress syndrome, these being the causes of death associated with COVID-19. Both the P2X7 receptor and NLRP3 have been considered as potential pharmacological targets for treating inflammation in COVID-19. However, there is no experimental evidence of the involvement of the P2X7 receptor during COVID-19 disease. In the present study, we determined the concentration of different cytokines and the P2X7 receptor in the plasma of COVID-19 patients and found that along with the increase in IL-6, IL-18 and the IL-1 receptor antagonist in the plasma of COVID-19 patients, there was also an increase in the purinergic P2X7 receptor. The increase in COVID-19 severity and C-reactive protein concentration positively correlated with increased concentration of the P2X7 receptor in the plasma, but not with the IL-18 cytokine. The P2X7 receptor was found in the supernatant of human peripheral blood mononuclear cells after inflammasome activation. Therefore, our data suggest that determining the levels of the P2X7 receptor in the plasma could be a novel biomarker of COVID-19 severity.

Seizures in PPT1 Knock-In Mice Are Associated with Inflammatory Activation of Microglia.

Infantile neuronal ceroid lipofuscinosis (INCL), the most severe form of neuronal ceroid lipofuscinoses, is caused by mutations in the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). Typical symptoms of this disease include progressive psychomotor developmental retardation, visual failure, seizures, and premature death. Here, we investigated seizure activity and relevant pathological changes in PPT1 knock-in mice (PPT1 KI). The behavior studies in this study demonstrated that PPT1 KI mice had no significant seizure activity until 7 months of age, and local field potentials also displayed epileptiform activity at the same age. The expression levels of Iba-1 and CD68 demonstrated, by Western blot analysis, the inflammatory cytokine TNF-α content measured with enzyme-linked immunosorbent assay, and the number of microglia demonstrated by immunohistochemistry (IHC) were significantly increased at age of 7 months, all of which indicate microglia activation at an age of seizure onset. The increased expression of GFAP were seen at an earlier age of 4 months, and such an increase reached its peak at age of 6 months, indicating that astrocyte activation precedes microglia. The purinergic P2X7 receptor (P2X7R) is an ATP-sensitive ionic channel that is highly expressed in microglia and is fundamental to microglial activation, proliferation, cytokines release and epilepsy. We show that the ATP concentration in hippocampal tissue in PPT1 KI mice was increased using an enhanced ATP assay kit and demonstrated that the antagonist of P2X7R, A-438079, significantly reduced seizures in PPT1 KI mice. In contrast to glial cell activation and proliferation, a significant reduction in synaptic proteins GABAAR was seen in PPT1 KI mice. These results indicate that seizure in PPT1 KI mice may be associated with microglial activation involved in ATP-sensitive P2X7R signaling and impaired inhibitory neurotransmission.

Continuous administration of mirabegron has advantages in inhibition of central sensitization compared with short-term treatment cessation in a mouse model of overactive bladder.

There is no clear pathophysiologic evidence determining how long overactive bladder (OAB) medication should be continued. We, therefore, investigated the effect of mirabegron using cessation (CES) or continuation (CON) treatment in an OAB animal model. Female C57BL/6 mice were divided into four groups (N = 8 each): Sham, OAB, CES, and CON groups. The OAB-like condition was induced by three times weekly intravesical instillations of KCl mixture with hyaluronidase. After the last intravesical instillation for inducing OAB, mirabegron (2 mg/kg/day) was administered in CES and CON groups for 10 and 20 days, respectively. Final experiments were carried out on 20 days from the last intravesical instillation in all groups. After cystometry, mRNA levels of bladder muscarinic, β-adrenergic, and P2X purinergic receptors were measured to investigate bladder efferent and afferent activity. In addition, mRNA levels of CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG) were measured to assess afferent sensitization. Immunofluorescent staining of CX3CR1, GFAP, and CCR2 in the L6 spinal cord was also conducted to investigate glial activation and central sensitization. OAB mice showed bladder overactivity evidenced by decreased intercontraction interval (3.56 ± 0.51vs. 5.76 ± 0.95 min in sham mice), increased non-voiding contractions (0.39 ± 0.11vs. 0.13 ± 0.07/min in sham mice), and inefficient voiding (72.1 ± 8.6% vs. 87.1 ± 9.5% in sham mice). Increased M2, M3, β2, β3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG indicate bladder efferent and afferent hyperexcitability. In addition, CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in OAB mice. However, theCON group exhibited reduced β2, β3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder, reduced CCL2 and CCR2 in DRG, which are markers of afferent hyperexcitability, and reduced immunoreactivities of CX3CR1, GFAP, and CCR2 in the L6 spinal cord, which are markers of the central sensitization. Moreover, theCON group showed better improvements in nonvoiding contractions (0.16 ± 0.09vs. 0.44 ± 0.17/min) and voiding efficiency (93.9 ± 7.4% vs. 76.5 ± 13.1%) and reductions in bladder β3 receptors and CCL2 of L6-S1 DRG, and immunoreactivities of CX3CR1 and GFAP in the L6 spinal cord compared to theCES group. Continuous mirabegron treatment seems to prevent central sensitization and, thus, might be desirable for long-term disease control of OAB.

Beyond monoamines: I. Novel targets and radiotracers for Positron emission tomography imaging in psychiatric disorders.

With the emergence of positron emission tomography (PET) in the late 1970s, psychiatry had access to a tool capable of non-invasive assessment of human brain function. Early applications in psychiatry focused on identifying characteristic brain blood flow and metabolic derangements using radiotracers such as [15 O]H2 O and [18 F]FDG. Despite the success of these techniques, it became apparent that more specific probes were needed to understand the neurochemical bases of psychiatric disorders. The first neurochemical PET imaging probes targeted sites of action of neuroleptic (dopamine D2 receptors) and psychoactive (serotonin receptors) drugs. Based on the centrality of monoamine dysfunction in psychiatric disorders and the measured success of monoamine-enhancing drugs in treating them, the next 30 years witnessed the development of an armamentarium of PET radiopharmaceuticals and imaging methodologies for studying monoamines. Continued development of monoamine-enhancing drugs over this time however was less successful, realizing only modest gains in efficacy and tolerability. As patent protection for many widely prescribed and profitable psychiatric drugs lapsed, drug development pipelines shifted away from monoamines in search of novel targets with the promises of improved efficacy, or abandoned altogether. Over this period, PET radiopharmaceutical development activities closely paralleled drug development priorities resulting in the development of new PET imaging agents for non-monoamine targets. Part one of this review will briefly survey novel PET imaging targets with relevance to the field of psychiatry, which include the metabotropic glutamate receptor type 5 (mGluR5), purinergic P2 X7 receptor, type 1 cannabinoid receptor (CB1 ), phosphodiesterase 10A (PDE10A), and describe radiotracers developed for these and other targets that have matured to human subject investigations. Current limitations of the targets and techniques will also be discussed.

Medullary astrocytes mediate irregular breathing patterns generation in chronic heart failure through purinergic P2X7 receptor signalling.

SummaryBackgroundBreathing disorders (BD) (apnoeas/hypopneas, periodic breathing) are highly prevalent in chronic heart failure (CHF) and are associated with altered central respiratory control. Ample evidence identifies the retrotrapezoid nucleus (RTN) as an important chemosensitivity region for ventilatory control and generation of BD in CHF, however little is known about the cellular mechanisms underlying the RTN/BD relationship. Within the RTN, astrocyte‐mediated purinergic signalling modulates respiration, but the potential contribution of RTN astrocytes to BD in CHF has not been explored.MethodsSelective neuron and/or astrocyte-targeted interventions using either optogenetic and chemogenetic manipulations in the RTN of CHF rats were used to unveil the contribution of the RTN on the development/maintenance of BD, the role played by astrocytes in BD and the molecular mechanism underpinning these alterations.FindingsWe showed that episodic photo-stimulation of RTN neurons triggered BD in healthy rats, and that RTN neurons ablation in CHF animals eliminates BD. Also, we found a reduction in astrocytes activity and ATP bioavailability within the RTN of CHF rats, and that chemogenetic restoration of normal RTN astrocyte activity and ATP levels improved breathing regularity in CHF. Importantly, P"X/ P2X7 receptor (P2X7r) expression was reduced in RTN astrocytes from CHF rats and viral vector-mediated delivery of human P2X7 P2X7r into astrocytes increases ATP bioavailability and abolished BD.InterpretationOur results support that RTN astrocytes play a pivotal role on BD generation and maintenance in the setting CHF by a mechanism encompassing P2X7r signalling.FundingThis study was funded by the National Research and Development Agency of Chile (ANID).

Purinergic P2X7 Receptor: A Therapeutic Target in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by upper and lower motor neuron loss. The pathomechanisms of ALS are still poorly understood with current hypotheses involving genetic mutations, excitotoxicity, and reactive oxygen species formation. In the absence of a disease-altering clinically approved therapeutic, there is an ever-increasing need to identify new targets to develop drugs that delay disease onset and/or progression. The purinergic P2X7 receptor (P2X7R) has been implicated widely across the ALS realm, providing a potential therapeutic strategy. This review summarizes the current understanding of ALS, the P2X7R and its role in ALS, the current landscape of P2X7R antagonists, and the in vivo potential of these antagonists in preclinical ALS models.

Uncovering Anti‐Arrhythmic Potential of Stellate Ganglion Purinergic Receptors

Cardiovascular disease affects over 30% of people worldwide and is one of the leading causes of death each year. Elevated sympathetic nerve activity is a common feature of cardiovascular disease, contributing to end‐organ damage, morbidity and mortality. Recent findings indicate that short‐circuiting sympathetic nerve overactivity by removal of the stellate ganglion can eradicate arrhythmias, emphasising the need for novel therapeutic targets to correct signalling non‐invasively.We have revealed upregulation of the P2X3 purinergic receptor in stellate ganglia of Spontaneously Hypertensive (SHR) compared to Wistar rats (16 week‐old), via RNASeq transcriptional profiling. We hypothesise that these purinergic receptors within cardiac stellate ganglia play a role in the excessive sympathetic drive to the heart in cardiovascular disease and can initiate cardiac arrhythmias. Thus, we have investigated the functional role of purinergic receptors in the stellate ganglion.Administration of ATP to acutely isolated post‐ganglionic sympathetic neurones from the stellate ganglia of Wistar rats (4‐6 week‐old) evokes a significant increase (Median; 0.21) in [Ca2+]i as measured by Fura‐2AM imaging (n=15, Wilcoxon matched‐pairs test; p<0.0001). This ATP‐induced calcium transient was inhibited by specific P2X3 receptor antagonism with either NF‐10 or AF‐130.Cardiac effects of stellate ganglion purinergic stimulation were investigated in the working heart‐brainstem preparation of Wistar rats (4‐6 week‐old). Increasing doses of ATP (50‐250 µg) delivered via microinjection (50‐250 µL) directly to the right stellate ganglion produced either a tachycardia (18 ± 6 bpm, n=6) or bradycardia (‐24 ± 12 bpm, n=5); these responses were typically found at distinct sites within the ganglion. Preliminary data from SHR suggest that higher doses of ATP (≥250µg) may be arrhythmogenic (n=3).P2X3 purinergic receptors are present in the sympathetic stellate ganglion and contribute to calcium ion flux and cardiac chronotropic regulation. Overexpression of P2X3 receptors is likely to contribute to excessive cardiac sympathetic activity and the development of hypertension and cardiac arrhythmias, making them a promising novel therapeutic target.

MP49-15 OVERACTIVE BLADDER MEDICATION; HOW LONG SHOULD IT BE CONTINUED? - MIRABEGRON INTERFERES WITH CENTRAL SENSITIZATION IN A MOUSE MODEL OF OVERACTIVE BLADDER

You have accessJournal of UrologyCME1 May 2022MP49-15 OVERACTIVE BLADDER MEDICATION; HOW LONG SHOULD IT BE CONTINUED? - MIRABEGRON INTERFERES WITH CENTRAL SENSITIZATION IN A MOUSE MODEL OF OVERACTIVE BLADDER Joonbeom Kwon, Eun-Ju Lee, Hye-Ri Park, Hyun-Jung Cho, Ji-Ae Jang, Donghwi Park, and Naoki Yoshimura Joonbeom KwonJoonbeom Kwon More articles by this author , Eun-Ju LeeEun-Ju Lee More articles by this author , Hye-Ri ParkHye-Ri Park More articles by this author , Hyun-Jung ChoHyun-Jung Cho More articles by this author , Ji-Ae JangJi-Ae Jang More articles by this author , Donghwi ParkDonghwi Park More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The etiology of overactive bladder (OAB) is multifactorial, and bladder afferent hyperexcitability has been proposed to be one of the contributing factors. In addition, chronic afferent hyperexcitability may induce neural remodeling in the central nervous system (CNS). However, there has been no clear pathophysiologic evidence determining how long OAB treatments should be continued. We, therefore, investigated the CNS effect of mirabegron using an OAB animal model and compared the therapeutic effect between treatment cessation (CES) and continuation (CON) groups. METHODS: Female C57BL/6 mice were divided into 4 groups (N=8 in each): Sham, OAB, CES, and CON groups. OAB was induced by 3-times weekly intravesical instillations of KCl and hyaluronidase. Mirabegron (2mg/kg) was administered for 10 and 20 days in CES and CON groups, respectively. Cystometry was performed, and mRNA levels of bladder muscarinic, β-adrenergic, and P2X purinergic receptors were measured. CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG), as afferent markers, and CX3CR1, GFAP, and CCR2 in the L6 spinal cord, as markers of glial activation and central sensitization, were also evaluated by RT-PCR and immunohistology. RESULTS: OAB mice showed bladder overactivity and inefficient voiding (Fig 1-a) with increased M2, M3, P2X2, P2X3, P2X4, and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG, indicating afferent hyperexcitability (Fig 1-b). CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in the OAB group (Fig 1-c). However, CON mice exhibited improvements of all these parameters of afferent hyperexcitability and central sensitization. Also, CON mice showed better voiding efficiency and reduced CNS changes compared with CES mice. CONCLUSIONS: Central sensitization may be an important pathophysiological mechanism of OAB. Continuous treatment of OAB with mirabegron seems to prevent the process of central sensitization via improvement of CNS neural remodeling. Therefore, continuous OAB medication may be desirable for long-term disease control. Source of Funding: Astellas Pharma. Co.Ltd. (ISR005728) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e859 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joonbeom Kwon More articles by this author Eun-Ju Lee More articles by this author Hye-Ri Park More articles by this author Hyun-Jung Cho More articles by this author Ji-Ae Jang More articles by this author Donghwi Park More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF DownloadLoading ...

PD08-07 ANTIFIBROSIS TREATMENT IMPROVES BLADDER DYSFUNCTION AND PAIN PERCEPTION IN A MOUSE MODEL OF BLADDER PAIN WITH CENTRAL SENSITIZATION MIMICKING INTERSTITIAL CYSTITIS

You have accessJournal of UrologyCME1 May 2022PD08-07 ANTIFIBROSIS TREATMENT IMPROVES BLADDER DYSFUNCTION AND PAIN PERCEPTION IN A MOUSE MODEL OF BLADDER PAIN WITH CENTRAL SENSITIZATION MIMICKING INTERSTITIAL CYSTITIS Joonbeom Kwon, Hye-Ri Park, Eun-Ju Lee, Ji-Ae Jang, Hyun-Jung Cho, and Naoki Yoshimura Joonbeom KwonJoonbeom Kwon More articles by this author , Hye-Ri ParkHye-Ri Park More articles by this author , Eun-Ju LeeEun-Ju Lee More articles by this author , Ji-Ae JangJi-Ae Jang More articles by this author , Hyun-Jung ChoHyun-Jung Cho More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002527.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies reported the possibility that the interruption of fibrosis may improve interstitial cystitis (IC). In addition, our previous study with nintedanib, which has an antifibrotic effect and inhibits the receptors of VEGF, FGF, and PDGF, demonstrated the improvement of bladder overactivity and C-fiber hyperexcitability in spinal cord injured mice. Therefore, we investigated whether antifibrotic therapy using nintedanib can improve the IC-like pathologic conditions. METHODS: Female C57BL/6 mice were divided into 3 groups (each N=8): (A) Sham, (B) IC mice treated with vehicle, and (C) IC mice treated with nintedanib. For inducing an IC-like model, intravesical instillation of hyaluronidase mixed with lipopolysaccharide was conducted 3 times once a week. At the last instillation, vehicle or nintedanib (50mg/kg) was administered daily for 3 weeks. Then, pain assessment using Von-Frey filaments and cystometry was conducted. Trichrome staining, RT-PCR in the bladder, and immunohistochemistry of L6-S1 dorsal root ganglia (DRG) and L6 spinal cord were performed to investigate the improvement of bladder fibrosis, inflammation, nociception, and central sensitization. RESULTS: In pain assessment, 50% thresholds of target force were significantly reduced in group B vs. A, but recovered in group C vs. B. In cystometry, non-voiding contractions and voiding efficiency were worsened in group B, but restored in group C (Fig 1-a). Bladder fibrosis increased in group B were improved in group C in trichrome staining (Fig.1-b). mRNA levels in the bladder related to inflammation (IFN-r, CCR2), nociception (TACR2), P2X purinergic (P2X3, P2X4, P2X7), muscarinic (M2), and β-adrenergic receptors (β2, β3) were increased in group B, but decreased in group C (Fig 1-c). In immunohistochemistry, TRPV1 in L6-S1 DRG and CX3CR1, GFAP, and CCR2 in L6 spinal cord were upregulated in group B, but reduced in group C (Fig 1-d). CONCLUSIONS: Antifibrosis treatment using nintedanib improved bladder pain and bladder overactivity in the IC model. Therapeutic effects seem to be mediated by inhibitions of bladder afferent hyperexcitability as well as central sensitization. Source of Funding: National Research Foundation of Korea (2020R1C1C1012208) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e107 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joonbeom Kwon More articles by this author Hye-Ri Park More articles by this author Eun-Ju Lee More articles by this author Ji-Ae Jang More articles by this author Hyun-Jung Cho More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF DownloadLoading ...

Structural Changes Induced in Hsp90 by Nitration lead to a Pathological Gain‐of‐function

Heat Shock Protein (Hsp90) is a pro‐survival molecular chaperone essential for the proper folding of a wide range of cellular proteins. It is highly abundant in most cell types and exists as a dimer that interacts with the client proteins. It is well described that the activity of Hsp90 is regulated by post‐translational modifications. We showed that in pathological conditions in which the powerful oxidant peroxynitrite is produced, Hsp90 undergoes nitration at 5 out of 24 tyrosine residues (Y) in its sequence. Further, nitration at Y33 and/or Y56 induces a pathological gain‐of‐function in Hsp90, leading to decreased mitochondrial activity and activation of the purinergic P2X7 receptor, which in motor neurons induces cell death. This is the first nitrated protein with a pathological function described to date. Nitration of Hsp90 is of particular relevance in amyotrophic lateral sclerosis (ALS), a motor neuron degenerative condition with an elusive pathogenesis and scant treatment options. We discovered that Hsp90 is endogenously nitrated in motor neurons in the spinal cord of ALS patients, the cell type that is compromised in this disease process. Establishing the structural changes induced in Hsp90 by nitration that lead to the pathological toxic function is crucial for the development of novel therapeutic strategies for ALS treatment. Here, using a combination of biophysical approaches we show that nitration of Hsp90 at Y33 and Y56, the residues relevant to the toxic function, had a profound impact on Hsp90 structure. To dissect the contribution of nitration at Y33 and/or Y56 on Hsp90 structural changes, we performed analytical ultracentrifugation sedimentation velocity experiments using recombinant Hsp90, peroxynitrite‐treated Hsp90 (fully oxidized protein), and site‐specific nitrated Hsp90 produced by genetic code expansion, carrying nitrotyrosine at either position 33 or 56, or simultaneously at both positions as the sole modification in the protein. Following peroxynitrite treatment, Hsp90 dimer was destabilized, showing a significant increase in the number of monomers, and the formation of oligomeric species. These results were confirmed by negative stain cryo‐electron microscopy, and size exclusion chromatography followed by multiangle light scattering. Site‐specific nitration at Y33 or Y56 was enough to destabilize the dimer conformation, while simultaneous nitration at Y33 and Y56 most closely resembled the structural changes observed after peroxynitrite treatment. In contrast, replacement of Y33 and Y56 by nitration‐resistant phenylalanine decreased dimer destabilization and prevented oligomer formation after peroxynitrite treatment. Together, these results suggest that nitration at Y33 and Y56 is enough to significantly affect the global structure of Hsp90, and that these changes may be responsible for nitrated Hsp90's toxic gain‐of‐function. Thus, targeting these structural changes may lead to the development of drugs that selectively inhibit nitrated Hsp90's pathological activity in ALS, without affecting the function of the unmodified chaperone in normal tissues.

La Esclerosis lateral amiotrófica no es solo una enfermedad de la motoneurona: impacto del eje simpatoadrenal

Amyotrophic lateral sclerosis (ALS), an adult onset, fatal neurodegenerative disease, has as a cardinal pathogenic feature the selective death of motor neurons (MNs) at the cortex, brainstem, and spinal cord. In this review we focus on four aspects: (i) the hypothesis of disease propagation through the cerebrospinal fluid (CSF); (ii) the distortion of the exocytotic release of neurotransmitters at the sympathoadrenal axis; (iii) the ultrastructural and functional alterations of mitochondria from adrenal medullary chromaffin cells (CCs); and (iv) the purinergic P2X7 receptor (P2X7R) as a potential target for neuroprotection. Concerning disease propagation from one to another area of the central nervous system (CNS), the pattern of clinical progression suggests that the disease spreads centrifugally. This indicates that a kind of toxin agent may be released and propagated through the CSF. In our laboratory we found that CSF from ALS patients exerted toxic effects on cultured cortical MNs. In CCs, we found deep distortions of the exocytotic kinetics and the exocytotic fusion pore in the process of catecholamine release, in the SOD1G93A mouse model of ALS. Furthermore, we found that these alterations could be related to the accumulation of mutated SOD1 into mitochondria; this resulted in mitochondrial depolarization, excess production of reactive oxygen species and deficiency in oxidative phosphorylation. Finally, we discuss recent data on the potential therapeutic effect of compound JNJ-47965567, a blocker of P2X7Rs known to be central-stage in neuroinflammation. Upon its chronic administration to SOD1G93A, we found that the compound delayed disease onset but only in females mice. In conclusion, why MN selectively die in ALS disease, remains a mystery; On the other hand, it seems that other cell types are also affected, particularly at the sympathoadrenal axis. As disease pathogenesis remains obscure, the search of therapeutic targets to slow disease progression in ALS, remains puzzling.

Berberine alleviates NLRP3 inflammasome induced endothelial junction dysfunction through Ca2+ signalling in inflammatory vascular injury

BackgroundBerberine has received rising attention for its application in cardiovascular disease because of its relationship with inflammation. The endothelial NLRP3 inflammasome triggers inflammatory vascular injury which would lead to cardiovascular disease. Endothelial calcium signalling plays a crucial role in both the activation of NLRP3 inflammasome and endothelial cells dysfunction. However, the efficacy of BBR on the endothelial NLRP3 inflammasome in inflammatory vascular injury remains unknown. PurposeIn this study, we focused on the NLRP3 pathway to determine whether BBR regulates endothelial junction function in inflammatory vascular injury. MethodsThe integrity of the junction proteins VE-cadherin (VEC) and zonula occludens‐1 (ZO‐1) detected by immunofluorescence and immunoblotting was used to determine the therapeutic effect of BBR (50, 100, or 200 mg/kg/day) in LPS (100 μg/kg/day)-induced inflammatory vascular injury in mice and mouse microvascular endothelial cells (MECs) treated with LPS (1 μLPS ) and ATP (5 mM). Endothelial permeability was assessed by FITC-labelled dextran and trans-endothelial electrical resistance (TEER) in vitro. The assembly and activation of NLRP3 inflammasomes were detected by western blotting and immunofluorescence. Pharmacophore-based virtual molecular docking studies and calcium imaging analyses were used to determine the interaction of BBR with the ATP-gated Ca2+ channel P2X7R (purinergic P2X receptor 7) in the context of inflammatory vascular injury. ResultsBBR recovered the expression of ZO‐1 and VEC and inhibited endothelial NLRP3 inflammasome activation in coronary microvascular endothelium and in MECs. These results suggested a crucial role of the NLRP3 inflammasome in BBR-regulated endothelial integrity. Further analysis demonstrated that BBR treatment suppressed the binding of TXNIP (thioredoxin interacting protein) with NLRP3. Intriguingly, eliminating extracellular Ca2+ showed a similar effect as BBR. Virtual docking analysis indicated that R574 of P2X7R is a potential target for BBR binding. Ca2+ imaging showed that BBR inhibited the Ca2+ influx in response to ATP, supporting the potential interaction of BBR with P2X7R. ConclusionsThese findings suggest that BBR exhibits potential and specific therapeutic value by targeting calcium signals and the endothelial NLRP3 inflammasome in inflammatory vascular injury.

Protective Effect of Purinergic P2X7 Receptor Inhibition on Acrolein-Induced Urothelial Cell Damage.

Patients undergoing chemotherapy with cyclophosphamide experience cystitis due to excretion of a toxic metabolite, acrolein. Cystitis, an inflammation of the bladder, is associated with damage to the integrity of the urothelial barrier. The purinergic P2X7 receptor (P2X7R) is increasingly recognized for its role in inflammation and cell death. P2X7R is expressed abundantly on the bladder urothelium. The aim of this study was to investigate the role of P2X7R in acrolein-induced inflammatory damage in primary cultured porcine bladder urothelial cells. Confluent urothelial cells in culture were treated with acrolein to induce damage; also, with the P2X7R selective antagonist, A804598. Cell viability assay, immunocytochemistry, and trans-epithelial electrical resistance (TEER) studies were carried out to investigate the effect of treatments on urothelial cell function. Acrolein induced a significant reduction in urothelial cell viability, which was protected by the presence of A804598 (10 µM). The urothelial barrier function, indicated by TEER values, was also significantly reduced by acrolein, whereas pre-incubation with P2X7R antagonist significantly protected the urothelial cell barrier from acrolein-induced TEER reduction. The structure of urothelial cell tight junctions was similarly impacted by acrolein treatment, showing the fragmentation of zona occludens-1 (ZO-1) immunoreactivity. Pre-treatment of cells with A804598 countered against the actions of acrolein and maintained ZO-1 expression level and cell structure. The damaging effect of acrolein on urothelial cells integrity could be impaired by inhibition of P2X7R, therefore P2X7R blockade may be a possible therapy in patients with bladder cystitis caused by cyclophosphamide treatment.

P2x4 receptor promotes mammary cancer progression by sustaining autophagy and associated mesenchymal transition.

Metastatic progression is a major burden for breast cancer patients and is associated with the ability of cancer cells to overcome stressful conditions, such as nutrients deprivation and hypoxia, and to gain invasive properties. Autophagy and epithelial-to-mesenchymal transition are critical contributors to these processes. Here, we show that the P2X4 purinergic receptor is upregulated in breast cancer biopsies from patients and it is primarily localised in endolysosomes. We demonstrate that P2X4 enhanced invasion in vitro, as well as mammary tumour growth and metastasis in vivo. The pro-malignant role of P2X4 was mediated by the regulation of lysosome acidity, the promotion of autophagy and cell survival. Furthermore, the autophagic activity was associated with epithelial-to-mesenchymal transition (EMT), and this role of P2X4 was even more pronounced under metabolic challenges. Pharmacological and gene silencing of P2X4 inhibited both autophagy and EMT, whereas its rescue in knocked-down cells led to the restoration of the aggressive phenotype. Together, our results demonstrate a previously unappreciated role for P2X4 in regulating lysosomal functions and fate, promoting breast cancer progression and aggressiveness.

Derivation and Characterization of Murine and Amphibian Müller Glia Cell Lines.

PurposeMüller glia (MG) in the retina of Xenopus laevis (African clawed frog) reprogram to a transiently amplifying retinal progenitor state after an injury. These progenitors then give rise to new retinal neurons. In contrast, mammalian MG have a restricted neurogenic capacity and undergo reactive gliosis after injury. This study sought to establish MG cell lines from the regeneration-competent frog and the regeneration-deficient mouse.MethodsMG were isolated from postnatal day 5 GLAST-CreERT; Rbfl/fl mice and from adult (3–5 years post-metamorphic) X laevis. Serial adherent subculture resulted in spontaneously immortalized cells and the establishment of two MG cell lines: murine retinal glia 17 (RG17) and Xenopus glia 69 (XG69). They were characterized for MG gene and protein expression by qPCR, immunostaining, and Western blot. Purinergic signaling was assessed with calcium imaging. Pharmacological perturbations with 2’-3’-O-(4-benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) and KN-62 were performed on RG17 cells.ResultsRG17 and XG69 cells express several MG markers and retain purinergic signaling. Pharmacological perturbations of intracellular calcium responses with BzATP and KN-62 suggest that the ionotropic purinergic receptor P2X7 is present and functional in RG17 cells. Stimulation of XG69 cells with adenosine triphosphate–induced calcium responses in a dose-dependent manner.ConclusionsWe report the characterization of RG17 and XG69, two novel MG cell lines from species with significantly disparate retinal regenerative capabilities.Translational RelevanceRG17 and XG69 cell line models will aid comparative studies between species endowed with varied regenerative capacity and will facilitate the development of new cell-based strategies for treating retinal degenerative diseases.

Immune Response Studies Based on P2X7 Receptors: A Mini-Review.

Inflammation, as a complex immunopathological process, is the organism's natural defense response against harmful, foreign, and destructive immune or non-immune factors. It is the main pathological form of various diseases, such as tumors, neurodegenerative diseases, periodontitis, alcoholic steatohepatitis, asthma, and other diseases. The P2X7 receptor (P2X7R) is widely distributed in vivo and up-regulated in various inflammatory pathological states. Studies have shown that milder chronic inflammation is related to a deficiency or inhibition of P2X7R, which is an indispensable part of the pro-inflammatory mechanism in vivo. P2X7R, a unique subtype of seven purinergic P2X receptors, is an ATP-gated non-selective cationic channel. P2X7R will promote the influx of Ca2+ and the outflow of K+ after being stimulated. The influx of Ca2+ is essential for activating the body's innate immune response and inducing the production of inflammatory factors. This paper reviews the regulation of P2X7R in inflammation from the perspectives of innate immunity and adaptive immunity.