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OBJECTIVE: To establish an echocardiographic technique in koi carp (Cyprinus carpio), compare cardiopulmonary parameters under manual restraint versus anesthesia, and provide a gross anatomical and histologic cardiac description. METHODS: A randomized, crossover echocardiography study was performed in 40 clinically healthy adult, unknown sex, privately owned koi carp on May 10 and 11 through June 26 and 27, 2021. Echocardiography was examined for each koi under manual restraint and isoeugenol at 50 ppm, with 3 measurements per examination performed by a radiologist and cardiologist. Two koi were euthanized for gross anatomic and histologic cardiac evaluation. RESULTS: Mean ejection fraction (EF), stroke volume (SV), and cardiac output (CO) were significantly lower, mean heart rate (HR) was significantly higher, and opercular rate (OPR) was decreased significantly in anesthetized compared to manually restrained koi. Poor reproducibility for EF and SV was observed. CONCLUSIONS: Echocardiography was feasible in both manually restrained and anesthetized koi; however, this technique may best be applied to monitoring trends over time in individual fish due to low reproducibility. Significant differences in multiple cardiopulmonary parameters, including HR, EF, SV, CO, and OPR, were present between manually restrained and anesthetized koi. A gross anatomic and histologic cardiac description is provided for this species to pair with the echocardiographic images. CLINICAL RELEVANCE: This study provides the first description of echocardiography, cardiac gross anatomy, and histology in koi. The results support echocardiography as a safe and practical noninvasive diagnostic for cardiac assessment in koi under both manual restraint and anesthesia.
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OBJECTIVE: To determine antibiotic levels in plasma and interstitial fluid (ISF) after SC placement of compounded florfenicol (FF) calcium sulfate beads (CSBs) in New Zealand White rabbits (Oryctolagus cuniculus). ANIMALS: 6 juvenile female rabbits (n = 5 treatment and 1 control). METHODS: An ultrafiltration probe and CSBs were placed SC in 6 rabbits (n = 5 for FF CSBs and 1 for control CSBs). Plasma (3, 6, 12, 24, and 48 hours and 7, 14, and 21 days) and ISF (daily for 21 days) samples were collected, and FF was measured by HPLC for pharmacokinetic analysis. Hematology, biochemistry, and histopathology were assessed. RESULTS: Means ± SD for the area under the curve, maximum concentration, time of maximum concentration, terminal half-life, and mean residence time to the last data point for plasma and ISF were 16.63 ± 8.16 and 17,902 ± 7,564 h·µg/mL, 0.79 ± 0.38 and 245 ± 223 µg/mL, 2.90 ± 0.3 and 59 ± 40 hours, 30.81 ± 16.9 and 27.3 ± 9.39 hours, 23.4 ± 10 and 73.7 ± 13 hours, respectively. Plasma FF was < 2 µg/mL at all time points. The ISF FF remained > 8 µg/mL for 109.98 to 231.58 hours. One rabbit death occurred during treatment, but the cause of death was undetermined. Local tissue inflammation was present, but no clinically significant systemic adverse effects were found on hematology, biochemistry, or histopathology in the remaining rabbits. CLINICAL RELEVANCE: Florfenicol CSBs maintained antibiotic concentrations in ISF at levels likely to be effective against bacteria sensitive to > 8 µg/mL for 5 to 10 days while maintaining low (< 2 µg/mL) plasma levels. Florfenicol CSBs may be effective for local antibiotic treatment in rabbit abscesses.
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Antibacterianos , Sulfato de Calcio , Tianfenicol , Animales , Conejos , Tianfenicol/análogos & derivados , Tianfenicol/farmacocinética , Tianfenicol/administración & dosificación , Antibacterianos/farmacocinética , Antibacterianos/administración & dosificación , Femenino , Sulfato de Calcio/química , Líquido Extracelular/química , Semivida , Implantes de Medicamentos , Área Bajo la CurvaRESUMEN
Dental disease in sugar gliders (Petaurus breviceps) is understudied, with only limited clinical and pathological data available. An approximately 7-year-old female sugar glider presented to its primary care veterinarian for a decline in food intake, rapid weight loss and a mass involving the rostral mandible. At necropsy, the mandibular mass effaced most of the rostral mandible and adjacent musculature. Histologically, the mandible was disrupted by nodular infiltrates of variably degenerate neutrophils and macrophages encased in granulomatous inflammation and fibrous connective tissue. Within the neutrophilic cell population were segments of fragmented, necrotic bone and cloud-like colonies of gram-positive cocci. Aerobic culture yielded a heavy, pure growth of a gram-positive coccus morphologically consistent with those identified in the lesions, which was identified as a presumptively novel Kocuria sp by polymerase chain reaction and sequencing of the 16S rRNA gene. This is the first description of Kocuria infection in association with clinically significant pathology in an animal. Although isolated as a pure growth, Kocuria sp cannot be confirmed as the sole cause of lesion formation due to the case chronicity and potential for unculturable, polymicrobial infections. This report adds to our understanding of the clinical and pathological aspects of dental disease in sugar gliders.
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Marsupiales , Osteomielitis , Enfermedades Estomatognáticas , Femenino , Animales , ARN Ribosómico 16S , Osteomielitis/veterinaria , Enfermedades Estomatognáticas/veterinaria , AzúcaresRESUMEN
OBJECTIVE To examine the effect of 24 hours of refrigeration on urine samples collected from dogs with signs of urinary tract infection (UTI). DESIGN Prospective cross-sectional study. ANIMALS 104 dogs with signs consistent with UTI that had a urine sample collected via cystocentesis as part of their diagnostic workup. PROCEDURES A 1-mL aliquot of each urine sample was refrigerated at 5°C for 24 hours in a plain glass tube, then processed for quantitative bacterial culture (QBC). A 0.5-mL aliquot was added to 3 mL of tryptic soy broth (TSB) and refrigerated at 5°C for 24 hours, then processed for QBC. The remaining portion was immediately processed for QBC, with results reported as numbers of bacterial colony-forming units (CFUs). Sensitivity of the QBC for detection of bacteria (and therefore UTI) was determined for sample refrigeration in the 2 conditions, compared with immediate processing (reference standard). RESULTS Bacterial growth was identified in 35.6% (n = 37), 33.7% (35), and 31.7% (33) of the immediately processed, refrigerated, and refrigerated-in-TSB urine samples, respectively. Sample refrigeration without TSB resulted in no significant difference in CFU counts relative to immediate processing; however, the sensitivity of this method was 95% (35/37). Sample refrigeration with TSB resulted in significantly lower CFU counts, and sensitivity was only 89% (33/37). CONCLUSIONS AND CLINICAL RELEVANCE Canine urine samples collected for bacterial culture should be immediately submitted for testing. Although CFU counts for refrigerated and immediately processed samples were statistically similar in this study, sample refrigeration in enrichment broth resulted in imperfect sensitivity for UTI detection and is not recommended.
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Enfermedades de los Perros/orina , Urinálisis/veterinaria , Infecciones Urinarias/veterinaria , Animales , Recuento de Colonia Microbiana/veterinaria , Estudios Transversales , Pruebas Diagnósticas de Rutina/veterinaria , Enfermedades de los Perros/microbiología , Perros , Femenino , Masculino , Estudios Prospectivos , Refrigeración/veterinaria , Sensibilidad y Especificidad , Manejo de Especímenes/veterinaria , Infecciones Urinarias/orinaRESUMEN
Epidermal growth factor receptor tyrosine kinase (EGFRtk) and endoplasmic reticulum (ER) stress are important factors in cardiovascular complications. Understanding whether enhanced EGFRtk activity and ER stress induction are involved in cardiac damage, and microvascular dysfunction in type 1 diabetes mellitus is an important question that has remained unanswered. Cardiac fibrosis and microvascular function were determined in C57BL/6J mice injected with streptozotocin only or in combination with EGFRtk inhibitor (AG1478), ER stress inhibitor (Tudca), or insulin for 2 weeks. In diabetic mice, we observed an increase in EGFRtk phosphorylation and ER stress marker expression (CHOP, ATF4, ATF6, and phosphorylated-eIF2α) in heart and mesenteric resistance arteries, which were reduced with AG1478, Tudca, and insulin. Cardiac fibrosis, enhanced collagen type I, and plasminogen activator inhibitor 1 were decreased with AG1478, Tudca, and insulin treatments. The impaired endothelium-dependent relaxation and -independent relaxation responses were also restored after treatments. The inhibition of NO synthesis reduced endothelium-dependent relaxation in control and treated streptozotocin mice, whereas the inhibition of NADPH oxidase improved endothelium-dependent relaxation only in streptozotocin mice. Moreover, in mesenteric resistance arteries, the mRNA levels of Nox2 and Nox4 and the NADPH oxidase activity were augmented in streptozotocin mice and reduced with treatments. This study unveiled novel roles for enhanced EGFRtk phosphorylation and its downstream ER stress in cardiac fibrosis and microvascular endothelial dysfunction in type 1 diabetes mellitus.
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Diabetes Mellitus Tipo 1/fisiopatología , Estrés del Retículo Endoplásmico/fisiología , Receptores ErbB/metabolismo , Corazón/fisiopatología , Vasodilatación/fisiología , Animales , Western Blotting , Colagogos y Coleréticos/farmacología , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Fibrosis/prevención & control , Expresión Génica/efectos de los fármacos , Hipoglucemiantes/farmacología , Insulina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocardio/patología , Fosforilación/efectos de los fármacos , Quinazolinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estreptozocina , Ácido Tauroquenodesoxicólico/farmacología , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Tirfostinos/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
OBJECTIVE: Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In the present study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension. METHODS AND RESULTS: Mice were infused with angiotensin II (400 ng/kg per minute) with or without ER stress inhibitors (taurine-conjugated ursodeoxycholic acid and 4-phenylbutyric acid) for 2 weeks. Mice infused with angiotensin II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen I content, transforming growth factor-ß1 (TGF-ß1) activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGF-ß1 activity in aorta but not in MRA, and reduced endothelial NO synthase phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGF-ß1 activity, enhanced endothelial NO synthase phosphorylation, and improved EDR. The inhibition of TGF-ß1 pathway improved EDR in aorta but not in MRA, whereas the reduction in reactive oxygen species levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGF-ß1-dependent mechanism in aorta and reactive oxygen species-dependent mechanism in MRA. CONCLUSIONS: ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore, ER stress could be a potential target for cardiovascular diseases.
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Estrés del Retículo Endoplásmico/fisiología , Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Miocardio/patología , Animales , Fibrosis , Hipertensión/patología , Masculino , Arterias Mesentéricas/fisiopatología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo III/fisiología , Estrés Oxidativo , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta1/genética , Resistencia Vascular , VasodilataciónRESUMEN
Type 2 diabetes mellitus (T2DM) is associated with microvascular dysfunction. We hypothesized that increased poly(ADP-ribose) polymerase 1 (PARP-1) activity contributes to microvascular dysfunction in T2DM. T2DM (db(-)/db(-)) and nondiabetic control (db(-)/db(+)) mice were treated with 2 different PARP-1 inhibitors (INO-1001, 5 mg/kg per day and ABT-888, 15 mg/kg per day) for 2 weeks. Isolated coronary arterioles were mounted in an arteriograph. Pressure-induced myogenic tone was significantly potentiated, whereas endothelium-dependent relaxation was significantly attenuated in diabetic mice compared with control mice. These results were associated with decreased endothelial NO synthase phosphorylation and cGMP level and increased PARP-1 activity in coronary arterioles from diabetic mice compared with control mice. Interestingly, PARP-1 inhibitors significantly reduced the potentiation of myogenic tone, improved endothelium-dependent relaxation, restored endothelial NO synthase phosphorylation and cGMP, and attenuated cleaved PARP-1. These results were supported by in vitro studies indicating that downregulation of PARP-1 in mesenteric resistance arteries using PARP-1 short hairpin RNA lentiviral particles significantly improved endothelium-dependent relaxation in mesenteric resistance arteries from diabetic mice compared with control mice. The inhibition of NO synthesis by N(G)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the endothelium-dependent relaxation in coronary arterioles and mesenteric resistance arteries from control and diabetic mice treated with PARP-1 inhibitors and PARP-1 short hairpin RNA lentiviral particles. In addition, we demonstrated that enhanced cleaved PARP-1, its binding to DNA, and DNA damage were reduced after PARP-1 inhibition in cultured endothelial cells stimulated with high glucose. We provide evidence that T2DM impairs microvascular function by an enhanced PARP-1 activity-dependent mechanism. Therefore, PARP-1 could be a potential target for overcoming diabetic microvascular complications.
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Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/enzimología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/enzimología , Angiopatías Diabéticas/tratamiento farmacológico , Angiopatías Diabéticas/enzimología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Bencimidazoles/farmacología , Glucemia/análisis , Determinación de la Presión Sanguínea , Western Blotting , Peso Corporal , Vasos Coronarios/patología , Angiopatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Inmunohistoquímica , Indoles/farmacología , Resistencia a la Insulina/fisiología , Ratones , Ratones Endogámicos , Poli(ADP-Ribosa) Polimerasa-1 , Reacción en Cadena de la Polimerasa/métodos , Distribución Aleatoria , Sensibilidad y EspecificidadRESUMEN
Type 2 diabetes is a key risk factor for ischemia-dependent pathology; therefore, a significant medical need exists to develop novel therapies that increase the formation of new vessels. We explored the therapeutic potential of epidermal growth factor receptor tyrosine kinase (EGFRtk) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibition in impaired ischemia-induced neovascularization in type 2 diabetes. Unilateral femoral artery ligation was performed in diabetic (db(-)/db(-)) and their control (db(-)/db(+)) mice for 4 weeks, followed by treatments with EGFRtk and ERK1/2 inhibitors (AG1478, 10 mg/kg/day and U0126, 400 µg/kg/day, respectively) for 3 weeks. Neovascularization, blood flow recovery, vascular and capillary density, and endothelial nitric oxide synthase activity were significantly impaired and were associated with enhanced EGFRtk and ERK1/2 activity in db(-)/db(-) mice. EGFRtk and ERK1/2 inhibitors did not have any effect in control mice, while in db(-)/db(-) mice there was a significant increase in neovascularization, blood flow recovery, vascular and capillary density, endothelial nitric oxide synthase activity, and were associated with a decrease in EGFRtk and ERK1/2 activity. Our data demonstrated that the inhibition of EGFRtk and ERK1/2 restored ischemia-induced neovascularization and blood flow recovery in type 2 diabetic mice. Thus, EGFRtk and ERK1/2 could be possible targets to protect from ischemia-induced vascular pathology in type 2 diabetes.
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Diabetes Mellitus Tipo 2/prevención & control , Angiopatías Diabéticas/prevención & control , Receptores ErbB/antagonistas & inhibidores , Miembro Posterior/irrigación sanguínea , Isquemia/prevención & control , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Velocidad del Flujo Sanguíneo/fisiología , Glucemia/metabolismo , Peso Corporal/fisiología , Capilares/fisiología , GMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Angiopatías Diabéticas/sangre , Angiopatías Diabéticas/fisiopatología , Insulina/metabolismo , Isquemia/sangre , Isquemia/fisiopatología , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Endoplasmic reticulum (ER) stress and inflammation are important mechanisms that underlie many of the serious consequences of type II diabetes. However, the role of ER stress and inflammation in impaired ischaemia-induced neovascularization in type II diabetes is unknown. We studied ischaemia-induced neovascularization in the hind-limb of 4-week-old db - /db- mice and their controls treated with or without the ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) and interleukin-1 receptor antagonist (anakinra, 0.5 µg/mouse per day) for 4 weeks. Blood pressure was similar in all groups of mice. Blood glucose, insulin levels, and body weight were reduced in db - /db- mice treated with TUDCA. Increased cholesterol and reduced adiponectin in db - /db- mice were restored by TUDCA and anakinra treatment. ER stress and inflammation in the ischaemic hind-limb in db - /db- mice were attenuated by TUDCA and anakinra treatment. Ischaemia-induced neovascularization and blood flow recovery were significantly reduced in db - /db- mice compared to control. Interestingly, neovascularization and blood flow recovery were restored in db - /db- mice treated with TUDCA or anakinra compared to non-treated db - /db- mice. TUDCA and anakinra enhanced eNOS-cGMP, VEGFR2, and reduced ERK1/2 MAP-kinase signalling, while endothelial progenitor cell number was similar in all groups of mice. Our findings demonstrate that the inhibition of ER stress and inflammation prevents impaired ischaemia-induced neovascularization in type II diabetic mice. Thus, ER stress and inflammation could be potential targets for a novel therapeutic approach to prevent impaired ischaemia-induced vascular pathology in type II diabetes.
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Antiinflamatorios/farmacología , Vasos Sanguíneos/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Angiopatías Diabéticas/prevención & control , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Isquemia/tratamiento farmacológico , Músculo Esquelético/irrigación sanguínea , Ácido Tauroquenodesoxicólico/farmacología , Animales , Biomarcadores/sangre , Vasos Sanguíneos/inmunología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/sangre , Angiopatías Diabéticas/etiología , Angiopatías Diabéticas/inmunología , Angiopatías Diabéticas/patología , Modelos Animales de Enfermedad , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Miembro Posterior , Isquemia/sangre , Isquemia/complicaciones , Isquemia/inmunología , Isquemia/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Recuperación de la Función , Flujo Sanguíneo Regional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
OBJECTIVE: We previously demonstrated that a reduced number of CD(4+)CD(25+)-regulatory T cells (Tregs) was associated with microvascular dysfunction in hypertension. However, the underlying mechanism by which Tregs regulate vascular endothelial function remains unknown. METHODS AND RESULTS: Control and interleukin (IL)-10(-/-) knockout mice were infused with angiotensin II (400 ng/kg/min) for 2 weeks (hypertensive [HT] and HT-IL-10(-/-)). Endothelium-dependent relaxation (EDR) in response to acetylcholine was significantly reduced in mesenteric resistance artery (MRA) from HT and HT-IL-10(-/-) compared with control and IL-10(-/-) mice. Importantly, the incubation of MRA from HT mice with the conditioned media of cultured Tregs, isolated from control mice, reduced NADPH oxidase activity and improved EDR, whereas no effect was observed in MRA from control mice incubated with the same media. These effects were reversed when MRAs were preincubated with IL-10 antibody or IL-10 receptor antagonist, whereas incubation with transforming growth factor-ß receptor antagonist had no effect. The transfer of cultured Tregs, isolated from control mice, into HT-IL-10(-/-) mice reduced systolic blood pressure (SBP) and NADPH oxidase activity and improved EDR in MRA compared with untreated HT-IL-10(-/-) mice. In vivo treatment of HT mice with IL-10 (1000 ng/mouse) significantly reduced SBP and NADPH oxidase activity and improved EDR in MRA compared with untreated HT mice. The transfer of cultured Tregs, isolated from IL-10(-/-) mice, into HT mice did not reduce SBP or NADPH oxidase activity or improve EDR. The incubation of MRA from HT mice with apocynin improved EDR, whereas NADPH oxidase substrate attenuated EDR in MRA from control mice, which was reversed with exogenous IL-10. CONCLUSION: These data demonstrate that IL-10 released from Tregs attenuates NADPH oxidase activity, which is a critical process in the improvement of microvascular endothelial function in hypertension, suggesting that Tregs/IL-10 could be a therapeutic target for treatment of vasculopathy in hypertension.
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Antígenos CD4/metabolismo , Endotelio Vascular/fisiología , Hipertensión/metabolismo , Interleucina-10/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , Linfocitos T Reguladores/metabolismo , Acetilcolina/farmacología , Angiotensina II/efectos adversos , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Interleucina-10/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/fisiología , NADPH Oxidasas/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacologíaRESUMEN
Coronary artery disease in patients with hypertension is increasing worldwide and leads to severe cardiovascular complications. The cellular and molecular mechanisms that underlie this pathologic condition are not well understood. Experimental and clinical research indicates that immune cells and inflammation play a central role in the pathogenesis of cardiovascular diseases. Recently, it has been reported that CD4(+)CD25(+) regulatory T cells (Tregs) regulate heart fibrosis in hypertension. In this study, we determined the role of Tregs in coronary arteriolar endothelial dysfunction in angiotensin II-dependent hypertensive mice. Mice infused with angiotensin II had significantly increased blood pressure, as determined using telemetry, and apoptotic Treg numbers, as measured using flow cytometry. The mice displayed inflammation, assessed by macrophage activation/infiltration into coronary arterioles and the heart, and increased local tumor necrosis factor-α release, which participates in reduced coronary arteriolar endothelial-dependent relaxation in response to acetylcholine using an arteriograph. Hypertensive mice injected with Tregs isolated from control mice had significantly reduced macrophage activation and infiltration, reduced tumor necrosis factor-α release, and improved coronary arteriolar endothelium-dependent relaxation. Our novel data indicate that Tregs are important in the development of coronary arteriolar endothelial dysfunction in hypertension. These results suggest a new direction in the investigation of vascular disease in hypertension and could lead to a therapeutic strategy that involves immune system modulation using Tregs.
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Vasos Coronarios/fisiopatología , Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Linfocitos T Reguladores/inmunología , Animales , Arteriolas/inmunología , Arteriolas/fisiopatología , Vasos Coronarios/inmunología , Endotelio Vascular/inmunología , Hipertensión/inmunología , Recuento de Linfocitos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND PURPOSE: we previously showed that epidermal growth factor receptor tyrosine kinase (EGFRtk) is essential in the development of myogenic tone. GRB2-SOS, protein kinase B (Akt), Janus kinase (JAK), and Signal Transducer and Activator of Transcription 3 (STAT3) are activated by stretch. Thus, we hypothesized that GRB2-SOS, Akt, JAK and STAT3 are downstream signaling of the EGFR and play role in myogenic tone. EXPERIMENTAL APPROACH: myogenic tone was determined in freshly isolated coronary arterioles from C57/BL6 mice with and without inhibitors. Pressurized coronary arterioles under 25 and 75mm Hg were subjected to Western blot analysis to determine signaling phosphorylation. Smooth muscle cells (SMC) stimulated with EGF were used to determine the interaction between signaling. KEY RESULTS: coronary arteriole myogenic tone was significantly reduced under EGFRtk, GRB2-SOS, JAK, and STAT3 inhibition (53.6 ± 2 vs. 83.4 ± 1.3; 82.8 ± 1; 83.6 ± 1; 86.1 ± 1% of passive diameter at 75mm Hg, p<0.05, respectively). However, Akt inhibition had no effect on coronary arteriole myogenic tone. Western blot analysis showed increased EGFRtk, STAT3, JAK, and Akt phosphorylation at 75mm Hg, which was significantly inhibited under EGFRtk inhibition. Interestingly, immunoprecipitation/Western blot analysis showed two intracellular complexes (ERK1/2-JAK-STAT3) involved in myogenic tone and (Akt-JAK-STAT3) not involved in myogenic tone. CONCLUSION AND IMPLICATIONS: these findings demonstrate that ERK1/2-JAK-STAT3 complex and GRB2-SOS, down stream signaling of the EGFRtk, are critical in the development of myogenic tone, thereby highlighting these signaling events as potential therapeutic targets in cardiovascular disease states associated with altered myogenic tone.
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Arteriolas/fisiología , Vasos Coronarios/fisiología , Receptores ErbB/metabolismo , Músculo Liso Vascular/fisiología , Transducción de Señal/fisiología , Animales , Arteriolas/efectos de los fármacos , Células Cultivadas , Vasos Coronarios/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Adaptadora GRB2/antagonistas & inhibidores , Quinasas Janus/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Nitroprusiato/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Cloruro de Potasio/farmacología , Presión , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinazolinas/farmacología , Factores de Transcripción STAT/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Son Of Sevenless/antagonistas & inhibidores , Tirfostinos/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Diabetes is increasing in the world and causes severe cardiovascular complications. Diabetes-induced limb ischemia leads to foot amputation and therapeutic remedies are urgently needed. Here we report that local injection of mesenchymal stem cells (MSCs) prestimulated with epidermal growth factor (EGF) restored blood flow and vasculogenesis in the ischemic hind-limb of type II diabetic (db(-)/db(-)) mice. Bone marrow cells from db(-)/db(-) mice are altered as evidenced by increased oxidative stress and reduced Akt and adhesion molecules when compared with control (db(-)/db(+)). Femoral artery ligation-induced ischemia was performed in the hind-limb of db(-)/db(-) and db(-)/db(+) mice for 28 days. Enhanced green fluorescent protein (EGFP)-MSCs stimulated+/-exogenous EGF for 24 h were injected locally into the ischemic muscle. Blood flow measured with MoorLDI-Laser and microangiography assessed with X-ray showed 100% recovery in db(-)/db(+) compared to 50% recovery in db(-)/db(-) mice. Interestingly, db(-)/db(-) mice had 60 and 96% blood flow recovery and 61 and 98% of vasculogenesis when treated with MSCs alone or MSCs modified with EGF, respectively. Western blot analysis of hind-limb muscles revealed an increase in Akt and vascular endothelial growth factor receptor phosphorylation and hypoxia-inducible factor) expression in db(-)/db(-) mice injected with MSCs or MSCs+EGF compared to db(-)/db(-) mice. Fluorescent microscopic images show that EGFP-MSCs differentiate into new microvessels. Adhesion and migration of MSCs on cultured endothelial cells were ICAM1-, VCAM1- and Akt-dependent mechanism and elevated when MSCs were prestimulated with EGF compared with nonstimulated MSCs. Our novel study data provide evidence that in type II diabetes, stimulated MSCs with EGF enhance the recovery of blood flow and angiogenesis.