RESUMO
Streptozotocin (STZ) is widely used to induce experimental diabetes in murine models. However, the ability to induce diabetic nephropathy (DN) is more challenging. It has been recommended to inject STZ at multiple low doses within 15 min after dissolution due to its alleged instability. However, some studies suggest that STZ is stable for days due to equilibration of its two anomers (α and ß), 90 min after dissolution, and that this anomer-equilibrated STZ leads to higher survival rates and persistent hyperglycaemia with minimal weight loss. The aim of this study was to determine an optimal dose of anomer-equilibrated STZ to induce kidney tubular damage and compare it with the more commonly used freshly prepared STZ. We hypothesised that anomer-equilibrated STZ provides a better, reproducible experimental model of diabetes-induced kidney damage with improved animal welfare. Body measurements, fasting glycaemia, insulinemia and renal histology were assessed in male C57Bl/6J at two and six months of age treated with fresh (50 mg/kg) or anomer-equilibrated (dose ranging 35-50 mg/kg) STZ or vehicle control. We demonstrated a dose-dependent effect of anomer-equilibrated STZ on the induction of hypo-insulinaemia and hyperglycaemia, as well as body weight in two-month-old mice. Interestingly, in six-month-old mice STZ leads to body weight loss, independently of STZ preparation mode. Anomer-equilibrated STZ provoked moderate to severe kidney tubule structural damage, resulting in significant kidney hypertrophy, whereas freshly prepared STZ only caused mild alterations. In conclusion, our study proposes that anomer-equilibrated STZ provides a robust murine model of diabetes and early-stage diabetic nephropathy, which can be used to test therapeutic approaches to treat and/or prevent renal damage.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Hiperglicemia , Camundongos , Masculino , Animais , Nefropatias Diabéticas/patologia , Estreptozocina , Camundongos Endogâmicos C57BL , Diabetes Mellitus Experimental/patologia , Rim/patologia , Hiperglicemia/patologiaRESUMO
Recently, lipid nanoparticles (LNPs) have attracted attention due to their emergent use for COVID-19 mRNA vaccines. The success of LNPs can be attributed to ionizable lipids, which enable functional intracellular delivery. Previously, the authors established an automated high-throughput platform to screen ionizable lipids and identified that the LNPs generated using this automated technique show comparable or increased mRNA functional delivery in vitro as compared to LNPs prepared using traditional microfluidics techniques. In this study, the authors choose one benchmark lipid, DLin-MC3-DMA (MC3), and investigate whether the automated formulation technique can enhance mRNA functional delivery in vivo. Interestingly, a 4.5-fold improvement in mRNA functional delivery in vivo by automated LNPs as compared to LNPs formulated by conventional microfluidics techniques, is observed. Mechanistic studies reveal that particles with large size accommodate more mRNA per LNP, possess more hydrophobic surface, are more hemolytic, bind a larger protein corona, and tend to accumulate more in macropinocytosomes, which may quantitatively benefit mRNA cytosolic delivery. These data suggest that mRNA loading per particle is a critical factor that accounts for the enhanced mRNA functional delivery of automated LNPs. These mechanistic findings provide valuable insight underlying the enhanced mRNA functional delivery to accelerate future mRNA LNP product development.
Assuntos
COVID-19 , Nanopartículas , Humanos , Lipossomos , Nanopartículas/química , RNA Mensageiro/química , SARS-CoV-2RESUMO
AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.
Assuntos
Anticorpos/imunologia , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/imunologia , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Incretinas/metabolismo , Insulina/metabolismo , Camundongos , Biblioteca de PeptídeosRESUMO
Transient receptor potential vanilloid 1 (TRPV1) is involved in sensory nerve nociceptive signaling. Recently, it has been discovered that TRPV1 receptors also regulate basal body temperature in multiple species from mice to humans. In the present study, we investigated whether TRPV1 modulates basal sympathetic nervous system (SNS) activity. C57BL6/J wild-type (WT) mice and TRPV1 knockout (KO) mice were implanted with radiotelemetry probes for measurement of core body temperature. AMG9810 (50 mg/kg) or vehicle (2% DMSO/5% Tween 80/10 ml/kg saline) was injected intraperitoneally. Adrenoceptor antagonists or vehicle (5 ml/kg saline) was injected subcutaneously. In WT mice, the TRPV1 antagonist, AMG9810, caused significant hyperthermia, associated with increased noradrenaline concentrations in brown adipose tissue. The hyperthermia was significantly attenuated by the ß-adrenoceptor antagonist propranolol, the mixed α-/ß-adrenoceptor antagonist labetalol, and the α1-adrenoceptor antagonist prazosin. TRPV1 KO mice have a normal basal body temperature, indicative of developmental compensation. d-Amphetamine (potent sympathomimetic) caused hyperthermia in WT mice, which was reduced in TRPV1 KO mice, suggesting a decreased sympathetic drive in KOs. This study provides new evidence that TRPV1 controls thermoregulation upstream of the SNS, providing a potential therapeutic target for sympathetic hyperactivity thermoregulatory disorders.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Temperatura Corporal/fisiologia , Sistema Nervoso Simpático/fisiologia , Canais de Cátion TRPV/fisiologia , Acrilamidas/administração & dosagem , Acrilamidas/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1/administração & dosagem , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/farmacologia , Animais , Temperatura Corporal/genética , Regulação da Temperatura Corporal/genética , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Febre/genética , Febre/fisiopatologia , Humanos , Injeções Intraperitoneais , Injeções Subcutâneas , Labetalol/administração & dosagem , Labetalol/farmacologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prazosina/administração & dosagem , Prazosina/farmacologia , Propranolol/administração & dosagem , Propranolol/farmacologia , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/fisiologia , Sistema Nervoso Simpático/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Telemetria/métodosRESUMO
The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB4-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.
Assuntos
Leucócitos/metabolismo , Leucotrieno B4/farmacologia , Superóxidos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Anquirinas/farmacologia , Feminino , Leucócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Prurido/tratamento farmacológico , Prurido/metabolismo , Receptores do Leucotrieno B4/antagonistas & inibidores , Canais de Cátion TRPV/metabolismoRESUMO
Little information is available on the etiology and prevalence of viruses other than influenza viruses causing influenza-like illnesses (ILIs) in China. This study was conducted for simultaneous detection and identification of 14 respiratory viruses in Huizhou using real-time PCR. In total, viruses were detected in 48.66 % of ILI patient samples, in which influenza virus (19.98 %) was the most commonly detected, followed by rhinovirus (7.46 %), human coronaviruses (3.63 %), human metapneumovirus (3.06 %), parainfluenza virus (3.06 %), respiratory syncytial virus (2.39 %), adenovirus (2.29 %), and human bocavirus (1.43 %). Co-infections occurred in 5.35 % of all tested specimens and 11.00 % (56/509) of infected patients. Children under 5 years and adults older than 60 years were more likely to have one or more detectable viruses associated with their ILI (OR=1.75, 95 % CI: 1.37; 2.23). Influenza virus was detected during each month of each year, and increased viral activity was observed in 2013. Infections with adenovirus and human metapneumovirus had characteristic seasonal patterns. No significant differences were found in positive the rate between the gender groups, while significantly differences in positive rate were found among the different age groups (P-value<0.001). This study confirmed that multiple respiratory viruses may circulate concurrently in the population and play an important role in the etiology of ILI. The most frequent symptoms associated with respiratory viruses were sore throat, rhinorrhea and headache. This information needs to be considered by clinicians when treating patients presenting with ILI, and it could serve as a reference for government officers when designing and implementing effective intervention plans.
Assuntos
Influenza Humana/virologia , Vírus/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , China/epidemiologia , Coinfecção/epidemiologia , Coinfecção/virologia , Feminino , Humanos , Lactente , Influenza Humana/epidemiologia , Masculino , Pessoa de Meia-Idade , Orthomyxoviridae/classificação , Orthomyxoviridae/genética , Orthomyxoviridae/isolamento & purificação , Filogenia , Vigilância de Evento Sentinela , Vírus/classificação , Vírus/genética , Adulto JovemRESUMO
The transient receptor potential vanilloid 1 (TRPV1) is primarily localized to sensory nerve fibers and is associated with the stimulation of pain and inflammation. TRPV1 knockout (TRPV1KO) mice show enhanced LPS-induced sepsis compared with wild type (WT). This implies that TRPV1 may have a key modulatory role in increasing the beneficial and reducing the harmful components in sepsis. We investigated immune and inflammatory mechanisms in a cecal ligation and puncture (CLP) model of sepsis over 24 h. CLP TRPV1KO mice exhibited significant hypothermia, hypotension, and organ dysfunction compared with CLP WT mice. Analysis of the inflammatory responses at the site of initial infection (peritoneal cavity) revealed that CLP TRPV1KO mice exhibited: 1) decreased mononuclear cell integrity associated with apoptosis, 2) decreased macrophage tachykinin NK(1)-dependent phagocytosis, 3) substantially decreased levels of nitrite (indicative of NO) and reactive oxygen species, 4) increased cytokine levels, and 5) decreased bacteria clearance when compared with CLP WT mice. Therefore, TRPV1 deletion is associated with impaired macrophage-associated defense mechanisms. Thus, TRPV1 acts to protect against the damaging impact of sepsis and may influence the transition from local to a systemic inflammatory state.
Assuntos
Síndrome de Resposta Inflamatória Sistêmica/imunologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Regulação para Cima/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/antagonistas & inibidores , Peritônio/imunologia , Peritônio/patologia , Peritônio/cirurgia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Síndrome de Resposta Inflamatória Sistêmica/genética , Canais de Cátion TRPV/biossíntese , Regulação para Cima/genéticaRESUMO
Introduction: Oxyntomodulin (Oxm) hormone peptide has a number of beneficial effects on nutrition and metabolism including increased energy expenditure and reduced body weight gain. Despite its many advantages as a potential therapeutic agent, Oxm is subjected to rapid renal clearance and protease degradation limiting its clinical application. Previously, we have shown that subcutaneous administration of a fibrillar Oxm formulation can significantly prolong its bioactivity in vivo from a few hours to a few days. Methods: We used a protease resistant analogue of Oxm, Aib2-Oxm, to form nanfibrils depot and improve serum stability of released peptide. The nanofibrils and monomeric peptide in solution were characterized by spectroscopic, microscopic techniques, potency assay, QCM-D and in vivo studies. Results: We show that in comparison to Oxm, Aib2-Oxm fibrils display a slower elongation rate requiring higher ionic strength solutions, and a higher propensity to dissociate. Upon subcutaneous administration of fibrillar Aib2-Oxm in rodents, a 5-fold increase in bioactivity relative to fibrillar Oxm and a significantly longer bioactivity than free Aib2-Oxm were characterized. Importantly, a decrease in food intake was observed up to 72-hour post-administration, which was not seen for free Aib2-Oxm. Conclusion: Our findings provides compelling evidence for the development of long-lasting peptide fibrillar formulations that yield extended plasma exposure and enhanced in vivo pharmacological response.
Assuntos
Peptídeo 1 Semelhante ao Glucagon , Glucagon , Ingestão de Alimentos/fisiologia , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Oxintomodulina/química , Oxintomodulina/farmacologia , Peptídeo Hidrolases , Peptídeos/farmacologia , Receptores de Glucagon/metabolismo , AnimaisRESUMO
mRNA lipid nanoparticles (LNPs) are at the forefront of nucleic acid intracellular delivery, as exemplified by the recent emergency approval of two mRNA LNP-based COVID-19 vaccines. The success of an LNP product largely depends on the systematic optimisation of the four lipidic components, namely the ionisable lipid, PEG lipid, structural and helper lipids. However, the in vitro screening of novel lipidic components and LNP compositions is limited by the low-throughput of LNP preparation. To address these issues, we herein present an automated high-throughput screening platform to select novel ionisable lipids and corresponding LNPs encapsulating mRNA in vitro. This high-throughput platform employs a lab-based automated liquid handling system, amenable to high-throughput (up to 384 formulations per plate and several plates per run) and allows precise mixing and reproducible mRNA LNP preparation which ensures a direct head-to-head comparison of hundreds and even thousands of novel LNPs. Most importantly, the robotic process has been successfully applied to the screening of novel LNPs encapsulating mRNA and has identified the same novel mRNA LNP leads as those from microfluidics-mixing technology, with a correlation coefficient of 0.8751. This high-throughput platform can facilitate to narrow down the number of novel ionisable lipids to be evaluated in vivo. Moreover, this platform has been integrated into a fully-automated workflow for LNP property control, physicochemical characterisation and biological evaluation. The high-throughput platform may accelerate proprietary lipid development, mRNA LNP lead optimisation and candidate selection to advance preclinical mRNA LNP development to meet urgent global needs.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19 , Nanopartículas , Vacinas Sintéticas/administração & dosagem , Vacinas de mRNA/administração & dosagem , COVID-19/prevenção & controle , Humanos , Lipossomos , RNA Interferente PequenoRESUMO
Peptide therapeutics are increasingly used in the treatment of disease, but their administration by injection reduces patient compliance and convenience, especially for chronic diseases. Thus, oral administration of a peptide therapeutic represents a significant advance in medicine, but is challenged by gastrointestinal instability and ineffective uptake into the circulation. Here, we have used glucagon-like peptide-1 (GLP-1) as a model peptide therapeutic for treating obesity-linked type 2 diabetes, a common chronic disease. We describe a comprehensive multidisciplinary approach leading to the development of MEDI7219, a GLP-1 receptor agonist (GLP-1RA) specifically engineered for oral delivery. Sites of protease/peptidase vulnerabilities in GLP-1 were removed by amino acid substitution and the peptide backbone was bis-lipidated to promote MEDI7219 reversible plasma protein binding without affecting potency. A combination of sodium chenodeoxycholate and propyl gallate was used to enhance bioavailability of MEDI7219 at the site of maximal gastrointestinal absorption, targeted by enteric-coated tablets. This synergistic approach resulted in MEDI7219 bioavailability of ~ 6% in dogs receiving oral tablets. In a dog model of obesity and insulin resistance, MEDI7219 oral tablets significantly decreased food intake, body weight and glucose excursions, validating the approach. This novel approach to the development of MEDI7219 provides a template for the development of other oral peptide therapeutics.
Assuntos
Doença Crônica , Sistemas de Liberação de Medicamentos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Peptídeos , Engenharia de Proteínas , Animais , Cricetinae , Humanos , Masculino , Camundongos , Administração Oral , Células CACO-2 , Química Farmacêutica/métodos , Ácido Quenodesoxicólico/administração & dosagem , Células CHO , Doença Crônica/tratamento farmacológico , Cricetulus , Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Células Secretoras de Insulina/citologia , Camundongos Endogâmicos C57BL , Peptídeos/química , Galato de Propila/administração & dosagem , Engenharia de Proteínas/métodos , Receptores de Glucagon/agonistas , Comprimidos com Revestimento EntéricoRESUMO
Transient potential vanilloid 1 (TRPV1) receptor is an ion channel receptor primarily localized on sensory nerves and activated by specific stimuli to initiate and amplify pain and inflammation, as typified by murine models of scald and arthritis. Little is known of the role of TRPV1 in sepsis, an infective disease associated with inflammation. Through use of a sublethal murine model of lipopolysaccharide-induced peritoneal sepsis, we provide novel evidence that genetic deletion of TRPV1 leads to an enhanced onset of various pathological components of systemic endotoxemia. Paired studies of TRPV1 knockout (KO) and wild-type mice demonstrate significantly enhanced hypotension (56+/-2% vs. 38+/-6% decrease in blood pressure, n=12), hypothermia (13+/-3% vs. 7+/-1% decrease in core temperature, n=6), and peritoneal exudate mediator levels (TNF-alpha, 0.78+/-0.2 vs. 0.38+/-0.1 ng/ml; nitrite, for NO, 35+/-10 vs. 15+/-3 microM; n=8) in TRPV1 KO mice, indicating loss of protective effect. Findings correlated with liver edema and raised plasma levels of aspartate aminotransferase in TRPV1 KO mice. These data suggest that TRPV1 may play an important regulatory role in sepsis independent of the major sensory neuropeptide substance P. The findings are relevant to developing strategies that increase the beneficial, and reduce the harmful, components of sepsis to prevent and treat this often fatal condition.
Assuntos
Endotoxinas/toxicidade , Sepse/prevenção & controle , Canais de Cátion TRPV/fisiologia , Animais , Imuno-Histoquímica , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. METHODS: Mono-arthritis was induced by unilateral intra-articular injection of complete Freund's adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8-37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. RESULTS: Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. CONCLUSIONS: We provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions.
Assuntos
Artrite Experimental/metabolismo , Velocidade do Fluxo Sanguíneo/fisiologia , Temperatura Baixa/efeitos adversos , Adjuvante de Freund/toxicidade , Articulações/metabolismo , Canais de Potencial de Receptor Transitório/biossíntese , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Adjuvante de Freund/administração & dosagem , Membro Posterior/efeitos dos fármacos , Membro Posterior/metabolismo , Membro Posterior/patologia , Injeções Intra-Articulares , Articulações/efeitos dos fármacos , Articulações/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/deficiênciaRESUMO
Radiotelemetry was used to investigate the in vivo cardiovascular and activity phenotype of both TRPA1 (transient receptor potential ankyrin 1) wild-type (WT) and TRPA1 knockout (KO) mice. After baseline recording, experimental hypertension was induced using angiotensin II infusion (1.1 mg(-1) kg(-1) a day, for 14 days). TRPA1 WT and KO mice showed similar morphological and functional cardiovascular parameters, including similar basal blood pressure (BP), heart rate, size, and function. Similar hypertension was also displayed in response to angiotensin II (156 ± 7 and 165 ± 11 mmHg, systolic BP ± SEM, n = 5-6). TRPA1 KO mice showed increased hypertensive hypertrophy (heart weight:tibia length: 7.3 ± 1.6 mg mm(-1) vs. 8.8 ± 1.7 mg mm(-1)) and presented with blunted interleukin 6 (IL-6) production compared with hypertensive WT mice (151 ± 24 vs. 89 ± 16 pg mL(-1)). TRPA1 expression in dorsal root ganglion (DRG) neurones was upregulated during hypertension (163% of baseline expression). Investigations utilizing the TRPA1 agonist cinnamaldehyde (CA) on mesenteric arterioles isolated from näive mice suggested a lack of TRPA1-dependent vasoreactivity in this vascular bed; a site with notable ability to alter total peripheral resistance. However, mesenteric arterioles isolated from TRPA1 KO hypertensive mice displayed significantly reduced ability to relax in response to nitric oxide (NO) (P < 0.05). Unexpectedly, naïve TRPA1 KO mice also displayed physical hyperactivity traits at baseline, which was exacerbated during hypertension. In conclusion, our study provides a novel cardiovascular characterization of TRPA1 KO mice in a model of hypertension. Results suggest that TRPA1 has a limited role in global cardiovascular control, but we demonstrate an unexpected capacity for TRPA1 to regulate physical activity.
RESUMO
Obesity induced by Western diets is associated with type 2 diabetes mellitus and cardiovascular diseases, although underlying mechanisms are unclear. We investigated a murine model of diet-induced obesity to determine the effect of transient potential receptor vanilloid 1 (TRPV1) deletion on hypertension and metabolic syndrome. Wild-type and TRPV1 knockout mice were fed normal or high-fat diet from 3 to 15 weeks. High-fat diet-fed mice from both genotypes became obese, with similar increases in body and adipose tissue weights. High-fat diet-fed TRPV1 knockout mice showed significantly improved handling of glucose compared with high-fat diet-fed wild-type mice. Hypertension, vascular hypertrophy, and altered nociception were observed in high-fat diet-fed wild-type but not high-fat diet-fed TRPV1 knockout mice. Wild-type, but not high-fat diet-fed TRPV1 knockout, mice demonstrated remodeling in terms of aortic vascular hypertrophy and increased heart and kidney weight, although resistance vessel responses were similar in each. Moreover, the wild-type mice had significantly increased plasma levels of leptin, interleukin 10 and interleukin 1ß, whereas samples from TRPV1 knockout mice did not show significant increases. Our results do not support the concept that TRPV1 plays a major role in influencing weight gain. However, we identified a role of TRPV1 in the deleterious effects observed with high-fat feeding in terms of inducing hypertension, impairing thermal nociception sensitivity, and reducing glucose tolerance. The observation of raised levels of adipokines in wild-type but not TRPV1 knockout mice is in keeping with TRPV1 involvement in stimulating the proinflammatory network that is central to obesity-induced hypertension and sensory neuronal dysfunction.
Assuntos
Doenças Cardiovasculares/genética , Hipertensão/genética , Síndrome Metabólica/genética , Obesidade/genética , Canais de Cátion TRPV/genética , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Pressão Sanguínea/fisiologia , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/metabolismo , Dieta Hiperlipídica , Hipertensão/complicações , Hipertensão/metabolismo , Resistência à Insulina , Interleucina-10/sangue , Interleucina-1beta/sangue , Leptina/sangue , Síndrome Metabólica/complicações , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
The vasodilator and vascular regulatory peptide adrenomedullin (AM), a member of the calcitonin gene-related peptide family of peptides, is predicted to play a pivotal protective role in cardiovascular dysfunction. The principle AM (AM1) receptor is composed of a G protein-linked calcitonin receptor-like receptor and a receptor activity-modifying protein (receptor activity-modifying protein 2). There is little knowledge of the receptors via which AM acts in diseases. Using smooth muscle-targeted receptor activity-modifying protein 2 transgenic mice with increased vascular density of functional AM1 receptors, we demonstrate that receptor activity-modifying protein 2 transgenic mice are not protected against angiotensin II-induced hypertension or cardiac hypertrophy. However, vascular hypertrophy, together with vascular cell adhesion molecule 1 and monocyte chemotactic protein 1 expression, is significantly reduced in the aortic walls of transgenic mice, as determined by histological techniques. This indicates that the AM1 vascular smooth muscle receptor can mediate local protection in vivo. This is supported by proliferation studies in cultured smooth muscle cells. By comparison, levels of hypotension and inflammation in a shock model were similar to those in wild-type mice. Thus, a role of the AM1 receptor in the vasoactive component could not be detected, and evidence is provided to show that the hypotensive response to AM is subject to desensitization in vivo. The finding that the vascular smooth muscle AM1 receptor acts at a local level to protect against hypertension-induced vascular hypertrophy and inflammation provides evidence that targeting this receptor may be a beneficial therapeutic approach.
Assuntos
Angiotensina II/sangue , Hipertensão/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/patologia , Adrenomedulina/sangue , Animais , Pressão Sanguínea/fisiologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Hipertensão/metabolismo , Hipertrofia , Hipotensão/metabolismo , Hipotensão/fisiopatologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Modificadoras da Atividade de Receptores , Receptores de Adrenomedulina , Receptores de Peptídeos/metabolismo , Vasculite/metabolismo , Vasculite/fisiopatologiaRESUMO
Inflammatory diseases associated with pain are often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Recently, there has been considerable interest in the role of reactive oxygen species (ROS) in inflammatory disease, but little is known of the role of hydrogen peroxide (H(2)O(2)) in hyperalgesia. In the present study, intraplantar injection of H(2)O(2)-induced a significant dose- and time-dependent mechanical and thermal hyperalgesia in the mouse hind paw, with increased c-fos activity observed in the dorsal horn of the spinal cord. H(2)O(2) also induced significant nociceptive behavior such as increased paw licking and decreased body liftings. H(2)O(2) levels were significantly raised in the carrageenan-induced hind paw inflammation model, showing that this ROS is produced endogenously in a model of inflammation. Moreover, superoxide dismutase and catalase significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, providing evidence of a functionally significant endogenous role. Thermal, but not mechanical, hyperalgesia in response to H(2)O(2) (i.pl.) was longer lasting in TRPV1 wild type mice compared to TRPV1 knockouts. It is unlikely that downstream lipid peroxidation was increased by H(2)O(2). In conclusion, we demonstrate a notable effect of H(2)O(2) in mediating inflammatory hyperalgesia, thus highlighting H(2)O(2) removal as a novel therapeutic target for anti-hyperalgesic drugs in the clinic.