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Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. This study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg.kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3ß and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.
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High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.
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Movimiento Celular , Endotelio Vascular , Células Endoteliales de la Vena Umbilical Humana , Peróxido de Hidrógeno , Ratones Endogámicos C57BL , NADPH Oxidasa 4 , Testosterona , Animales , Humanos , Masculino , Ratones , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Especies Reactivas de Oxígeno/metabolismo , Testosterona/farmacología , Testosterona/metabolismoRESUMEN
Prenatal exposure to maternal diabetes has been recognized as a significant cardiovascular risk factor, increasing the susceptibility to the emergence of conditions such as high blood pressure, atherosclerosis, and heart disease in later stages of life. However, it is unclear if offspring exposed to diabetes in utero have worse vascular outcomes on a high-salt (HS) diet. To test the hypothesis that in utero exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, we treated adult male wild-type offspring (DM_Exp, 6 mo old) of diabetic Ins2+/C96Y mice (Akita mice) with HS (8% sodium chloride, 10 days) and analyzed endothelial function via wire myograph and cyclooxygenase (COX)-derived prostanoids pathway by ELISA, quantitative PCR, and immunochemistry. On a regular diet, DM_Exp mice did not manifest any vascular dysfunction, remodeling, or inflammation. However, HS increased aortic contractility to phenylephrine and induced endothelial dysfunction (analyzed by acetylcholine-induced endothelium-dependent relaxation), vascular hydrogen peroxide production, COX2 expression, and prostaglandin E2 (PGE2) overproduction. Interestingly, ex vivo antioxidant treatment (tempol) or COX1/2 (indomethacin) or COX2 (NS398) inhibitors improved or reverted the endothelial dysfunction in DM_Exp mice fed a HS diet. Finally, DM_Exp mice fed with HS exhibited greater circulating cytokines and chemokines accompanied by vascular inflammation. In summary, our findings indicate that prenatal exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, primarily through the induction of oxidative stress and the generation of COX2-derived PGE2. This supports the concept that in utero exposure to maternal diabetes is a cardiovascular risk factor in adulthood.NEW & NOTEWORTHY Using a unique mouse model of prenatal exposure to maternal type 1 diabetes, our study demonstrates the novel observation that prenatal exposure to maternal diabetes results in a predisposition to high-salt (HS) dietary-induced vascular dysfunction and inflammation in adulthood. Mechanistically, we demonstrated that in utero exposure to maternal diabetes and HS intake induces vascular oxidative stress, cyclooxygenase-derived prostaglandin E2, and inflammation.
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Diabetes Gestacional , Endotelio Vascular , Efectos Tardíos de la Exposición Prenatal , Prostaglandinas , Animales , Femenino , Ratones , Embarazo , Ciclooxigenasa 2/metabolismo , Diabetes Gestacional/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Inflamación/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Prostaglandinas/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Cloruro de Sodio Dietético/metabolismoRESUMEN
BACKGROUND: Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown. METHODS: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction. RESULTS: Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5+/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5-/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NFκB (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NFκB, or CCR5. CONCLUSIONS: Our data demonstrate that CCL5/CCR5, through activation of NFκB and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess.
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Aldosterona , Quimiocina CCL5 , Hipertensión , Receptores CCR5 , Animales , Ratones , Aldosterona/farmacología , Células Endoteliales/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Inflamación , Receptores CCR5/genética , Receptores CCR5/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismoRESUMEN
Objective: Cardiovascular disease (CVD) is a global health crisis and a leading cause of mortality. The intricate interplay between vascular contractility and mitochondrial function is central to CVD pathogenesis. The progranulin gene (GRN) encodes glycoprotein progranulin (PGRN), a ubiquitous molecule with known anti-inflammatory property. However, the role of PGRN in CVD remains enigmatic. In this study, we sought to dissect the significance of PGRN in the regulation vascular contractility and investigate the interface between PGRN and mitochondrial quality. Method: Our investigation utilized aortae from male and female C57BL6/J wild-type (PGRN+/+) and B6(Cg)-Grntm1.1Aidi/J (PGRN-/-) mice, encompassing wire myograph assays to assess vascular contractility and primary aortic vascular smooth muscle cells (VSMCs) for mechanistic insights. Results: Our results showed suppression of contractile activity in PGRN-/- VSMCs and aorta, followed by reduced α-smooth muscle actin expression. Mechanistically, PGRN deficiency impaired mitochondrial oxygen consumption rate (OCR), complex I activity, mitochondrial turnover, and mitochondrial redox signaling, while restoration of PGRN levels in aortae from PGRN-/- mice via lentivirus delivery ameliorated contractility and boosted OCR. In addition, VSMC overexpressing PGRN displayed higher mitochondrial respiration and complex I activity accompanied by cellular hypercontractility. Furthermore, increased PGRN triggered lysosome biogenesis by regulating transcription factor EB and accelerated mitophagy flux in VSMC, while treatment with spermidine, an autophagy inducer, improved mitochondrial phenotype and enhanced vascular contractility. Finally, angiotensin II failed to induce vascular contractility in PGRN-/- suggesting a key role of PGRN to maintain the vascular tone. Conclusion: Our findings suggest that PGRN preserves the vascular contractility via regulating mitophagy flux, mitochondrial complex I activity, and redox signaling. Therefore, loss of PGRN function appears as a pivotal risk factor in CVD development.
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BACKGROUND: Renin-angiotensin (Ang II)-aldosterone system (RAAS) is crucial for the cardiovascular risk associated with excessive ethanol consumption. Disturbs in mitochondria have been implicated in multiple cardiovascular diseases. However, if mitochondria dysfunction contributes to ethanol-induced vascular dysfunction is still unknown. We investigated whether ethanol leads to vascular dysfunction via RAAS activation, mitochondria dysfunction, and mitochondrial reactive oxygen species (mtROS). METHODS: Male C57/BL6J or mt-keima mice (6-8-weeks old) were treated with ethanol (20% vol./vol.) for 12 weeks with or without Losartan (10 mg/kg/day). RESULTS: Ethanol induced aortic hypercontractility in an endothelium-dependent manner. PGC1α (a marker of biogenesis), Mfn2, (an essential protein for mitochondria fusion), as well as Pink-1 and Parkin (markers of mitophagy), were reduced in aortas from ethanol-treated mice. Disturb in mitophagy flux was further confirmed in arteries from mt-keima mice. Additionally, ethanol increased mtROS and reduced SOD2 expression. Strikingly, losartan prevented vascular hypercontractility, mitochondrial dysfunction, mtROS, and restored SOD2 expression. Both MnTMPyP (SOD2 mimetic) and CCCP (a mitochondrial uncoupler) reverted ethanol-induced vascular dysfunction. Moreover, L-NAME (NOS inhibitor) and EUK 134 (superoxide dismutase/catalase mimetic) did not affect vascular response in ethanol group, suggesting that ethanol reduces aortic nitric oxide (NO) and H2O2 bioavailability. These responses were prevented by losartan. CONCLUSION: AT1 receptor modulates ethanol-induced vascular hypercontractility by promoting mitochondrial dysfunction, mtROS, and reduction of NO and H2O2 bioavailability. Our findings shed a new light in our understanding of ethanol-induced vascular toxicity and open perspectives of new therapeutic approaches for patients with disorder associated with abusive ethanol drinking.
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Losartán , Lesiones del Sistema Vascular , Humanos , Ratones , Masculino , Animales , Losartán/farmacología , Receptor de Angiotensina Tipo 1/metabolismo , Etanol/toxicidad , Peróxido de Hidrógeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismoRESUMEN
Background: Aldosterone, a mineralocorticoid steroid hormone, has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of C-C Motif Chemokine Ligand 5 (CCL5) and its receptor, C-C Motif Chemokine Receptor 5 (CCR5), are well known in infectious diseases, but their roles in the genesis of aldosterone-induced vascular injury and hypertension are unknown. Methods: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg/day for 14 days) while receiving 1% saline to drink. Results: Here, we show that CCR5 plays a central role in aldosterone-induced vascular injury, hypertension, and renal damage. Long-term infusion of aldosterone in CCR5+/+ mice resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression. Aldosterone treatment also triggered vascular injury, characterized by endothelial dysfunction and inflammation, hypertension, and renal damage. Mice lacking CCR5 were protected from aldosterone-induced vascular damage, hypertension, and renal injury. Mechanistically, we demonstrated that CCL5 increased NADPH oxidase 1 (Nox1) expression, reactive oxygen species (ROS) formation, NFκB activation, and inflammation and reduced nitric oxide production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aortae incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking Nox1, NFκB, or with Maraviroc treatment. Conclusions: Our data demonstrate that CCL5/CCR5, through activation of NFkB and Nox1, is critically involved in aldosterone-induced vascular and renal damage and hypertension. Our data place CCL5 and CCR5 as potential targets for therapeutic interventions in conditions with aldosterone excess.
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Background The mechanisms determining vascular tone are still not completely understood, even though it is a significant factor in blood pressure management. Many circulating proteins have a significant impact on controlling vascular tone. Progranulin displays anti-inflammatory effects and has been extensively studied in neurodegenerative illnesses. We investigated whether progranulin sustains the vascular tone that helps regulate blood pressure. Methods and Results We used male and female C57BL6/J wild type (progranulin+/+) and B6(Cg)-Grntm1.1Aidi/J (progranulin-/-) to understand the impact of progranulin on vascular contractility and blood pressure. We found that progranulin-/- mice display elevated blood pressure followed by hypercontractility to noradrenaline in mesenteric arteries, which is restored by supplementing the mice with recombinant progranulin. In ex vivo experiments, recombinant progranulin attenuated the vascular contractility to noradrenaline in male and female progranulin+/+ arteries, which was blunted by blocking EphrinA2 or Sortilin1. To understand the mechanisms whereby progranulin evokes anticontractile effects, we inhibited endothelial factors. N(gamma)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) prevented the progranulin effects, whereas indomethacin (cyclooxygenase inhibitor) affected only the contractility in arteries incubated with vehicle, indicating that progranulin increases nitric oxide and decreases contractile prostanoids. Finally, recombinant progranulin induced endothelial nitric oxide synthase phosphorylation and nitric oxide production in isolated mesenteric endothelial cells. Conclusions Circulating progranulin regulates vascular tone and blood pressure via EphrinA2 and Sortilin1 receptors and endothelial nitric oxide synthase activation. Collectively, our data suggest that deficiency in progranulin is a cardiovascular risk factor and that progranulin might be a new therapeutic avenue to treat high blood pressure.
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Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , Masculino , Femenino , Ratones , Animales , Óxido Nítrico Sintasa de Tipo III/metabolismo , Presión Sanguínea , Progranulinas/farmacología , Óxido Nítrico/metabolismo , Células Endoteliales/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Arterias Mesentéricas/metabolismo , Endotelio Vascular/metabolismo , NorepinefrinaRESUMEN
BACKGROUND: Obesity is the number one cardiovascular risk factor for both men and women and is a complex condition. Although a sex dimorphism on vascular function has already been noted, the underlying processes remain unclear. The Rho-kinase pathway has a unique role in controlling vascular tone, and in obese male mice, hyperactivation of this system results in worsened vascular constriction. We investigated whether female mice exhibit decreased Rho-kinase activation as a protective mechanism in obesity. METHODS: We exposed male and female mice to a high-fat diet (HFD) for 14 weeks. At the end, energy expenditure, glucose tolerance, adipose tissue inflammation, and vascular function were investigated. RESULTS: Male mice were more sensitive to HFD-induced body weight gain, glucose tolerance, and inflammation than female mice. After establishing obesity, female mice demonstrated increase in energy expenditure, characterized by an increase in heat, whereas male mice did not. Interestingly, obese female mice, but not male, displayed attenuated vascular contractility to different agonists, such difference was blunted by inhibition of Rho-kinase, which was accompanied by a suppressed Rho-kinase activation, measured by Western blot. Finally, aortae from obese male mice displayed an exacerbated inflammation, whereas obese female demonstrated a mild vascular inflammation. CONCLUSION: In obesity, female mice demonstrate a vascular protective mechanism-suppression of vascular Rho-kinase-to minimize the cardiovascular risk associated with obesity, whereas male mice do not generate any adaptive response. Future investigations can help to understand how Rho-kinase becomes suppressed in female during obesity.
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Obesidad , Quinasas Asociadas a rho , Femenino , Ratones , Animales , Quinasas Asociadas a rho/metabolismo , Ratones Obesos , Obesidad/metabolismo , Inflamación/complicaciones , Glucosa , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BLRESUMEN
ABSTRACT: Kawasaki disease (KD) is a systemic vasculitis of childhood characterized by vascular damage in the acute stage, which can persist into the late stage. The vascular mechanisms in the cardiovascular risk of KD are not fully studied. We investigated the vascular function and blood pressure in a murine model of KD. We used the Candida albicans water-soluble (CAWS) fraction model. Mice were injected with 4 mg CAWS for 5 consecutive days and separated into three groups. Control, CAWS 7 days (C7), and CAWS 28 days (C28). Hearts and arteries were harvested for vascular characterization. Rat aortic smooth muscle cells were used to studies in vitro. C7 presented elevated inflammatory markers in the coronary area and abdominal aortas, whereas C28 showed severe vasculitis. No difference was found in blood pressure parameters. Vascular dysfunction characterized by higher contractility to norepinephrine in C7 and C28 in aortic rings was abolished by blocking nitric oxide (NO), reactive oxygen species, and cyclooxygenase (COX)-derived products. The CAWS complex increased COX2 expression in rat aortic smooth muscle cells, which was prevented by Toll-like receptor 4 antagonist. Our data indicate that the murine model of KD is associated with vascular dysfunction likely dependent on COX-derived products, oxidant properties, and NO bioavailability. Furthermore, vascular smooth muscle cell may present an important role in the genesis of vascular dysfunction and vasculitis via the Toll-like receptor 4 pathway. Finally, the CAWS model seems not to be appropriate to study KD-associated shock. More studies are necessary to understand whether vascular dysfunction and COXs are triggers for vasculitis.
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Síndrome Mucocutáneo Linfonodular , Vasculitis , Ratones , Animales , Ratas , Síndrome Mucocutáneo Linfonodular/metabolismo , Receptor Toll-Like 4 , Presión Sanguínea , Modelos Animales de Enfermedad , Aorta , Candida albicansRESUMEN
BACKGROUND: Chemokine (C- Cmotif) ligand 5 (CCL5) and its receptor C-C motif chemokine receptor 5 (CCR5), have been broadly studied in conjunction with infectious pathogens, however, their involvement in cardiovascular disease is not completely understood. NADPH oxidases (Noxs) are the major source of reactive oxygen species (ROS) in the vasculature. Whether the activation of Noxs is CCL5/CCR5 sensitive and whether such interaction initiates vascular injury is unknown. We investigated whether CCL5/CCR5 leads to vascular damage by activating Noxs. MATERIAL AND METHODS: We used rat aortic smooth muscle cells (RASMC) to investigate the molecular mechanisms by which CCL5 leads to vascular damage and carotid ligation (CL) to analyze the effects of blocking CCR5 on vascular injury. RESULTS: CCL5 induced Nox1 expression in concentration and time-dependent manners, with no changes in Nox2 or Nox4. Maraviroc pre-treatment (CCR5 antagonist, 40uM) blunted CCL5-induced Nox1 expression. Furthermore, CCL5 incubation led to ROS production and activation of Erk1/2 and NFkB, followed by increased vascular cell migration, proliferation, and inflammatory markers. Notably, Nox1 inhibition (GKT771, 10uM) blocked CCL5-dependent effects. In vivo, CL induced pathological vascular remodeling and inflammatory genes and increased Nox1 and CCR5 expression. Maraviroc treatment (25 mg/Kg/day) reduced pathological vascular growth and Nox1 expression. CONCLUSIONS: Our findings suggest that CCL5 activates Nox1 in the vasculature, leading to vascular injury likely via NFkB and Erk1/2. Herein, we place CCR5 antagonists and/or Nox1 inhibitors might be preeminent antiproliferative compounds to reduce the cardiovascular risk associated with medical procedures (e.g. angioplasty) and vascular diseases associated with vascular hyperproliferation.
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Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasa 1/metabolismo , Receptores CCR5/metabolismo , Lesiones del Sistema Vascular/metabolismo , Animales , Antagonistas de los Receptores CCR5/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Maraviroc/farmacología , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , NADPH Oxidasa 1/genética , Ratas , Receptores CCR5/agonistas , Receptores CCR5/genética , Proteínas Recombinantes/farmacología , Lesiones del Sistema Vascular/prevención & controlRESUMEN
Angiotensin-II (Ang-II), a major target for treatment of cardiovascular disease, promotes cardiovascular dysfunction by directly modulating structure and function of vascular cells. Inflammasome components are expressed in the vasculature and are activated by specific stimuli. However, whether Ang-II activates the inflammasome in vascular cells or inflammasome activation contributes to Ang-II-induced vascular damage is still not fully elucidated. We tested the hypothesis that Ang-II induces endothelial dysfunction, vascular remodeling, and high blood pressure via inflammasome activation. C57BL6/J wild type (WT) and Caspase-1 knockout (Casp1-/-) mice were infused with vehicle or Ang-II for two weeks (490 ng/Kg/day) to determine whether the inflammasome contributes to vascular damage induced by Ang-II. Rat Aortic Vascular Smooth Muscle cells (RASMC) were used to determine if the interaction between Ang-II and inflammasomes causes migration and proliferation of vascular smooth muscle cells. Ex vivo studies revealed that Ang-II infusion induced vascular oxidative stress, endothelial dysfunction and vascular remodeling in WT mice. Casp1-/- mice were protected against Ang-II-induced vascular injury. In vitro experiments, Ang-II activated the NLRP3 inflammasome in RASMC, i.e. Ang-II increased Caspase-1 (Casp1) activity and cleavage of pro-interleukin (IL)-1ß. MCC950 (NLRP3 receptor antagonist) prevented Ang-II-induced vascular migration and proliferation, but failed to reduce reactive oxygen species production. In conclusion, Ang-II leads to inflammasome activation in the vasculature contributing to endothelial dysfunction and vascular remodeling. Taken together, we place inflammasomes as a possible therapeutic target in conditions associated with increased Ang-II levels.
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Angiotensina II , Inflamasomas , Angiotensina II/farmacología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , RatasRESUMEN
Mycoplasma é um patógeno altamente contagioso, podendo causar mastite, pneumonia, artrite, entre outras enfermidades. Seu isolamento requer meios e condições específicas devido ao seu crescimento fastidioso. Devido à complexidade do seu diagnóstico, acredita-se que a real prevalência de casos de mastite por micoplasma seja subestimada. O objetivo do presente estudo foi identificar a prevalência de Mycoplasma bovis em diferentes rebanhos de bovinos leiteiros no estado de São Paulo. O estudo foi dividido em fase de triagem, na qual colheram-se amostras de 67 tanques de expansão e a coleta individual, na qual propriedades positivas para M. bovis foram visitadas e colhidas amostras de leite de todos os animais com mastite clínica e subclínica. O diagnóstico laboratorial foi feito por meio da PCR e cultivo microbiológico específico. A prevalência de M. bovis encontrada na fase de triagem foi de 1,4%. Na fase individual, todas as amostras de leite, procedentes de propriedade positiva para M. bovis no tanque de expansão, foram negativas, o que permite concluir pela baixa prevalência do agente nas condições do presente estudo.(AU)
Mycoplasma is a highly contagious pathogen, which can cause mastitis, pneumonia, arthritis, among other diseases. Its isolation requires specific means and conditions due to its fastidious growth. Due to the complexity of its diagnosis, it is believed that the real prevalence of mastitis cases by Mycoplasma is underestimated. The objective of the present study was to identify the prevalence of Mycoplasma bovis in different dairy herds in the state of São Paulo. The study was divided into a screening phase in which samples were collected from 67 expansion tanks and individual collection, in which positive properties for M. bovis were visited and collected milk samples from all animals with clinical and subclinical mastitis. The laboratory diagnosis was made through PCR and specific microbiological culture. The prevalence of M. bovis found in the screening phase was 1.4%. In the individual phase, all milk samples from M. bovis positive property in the expansion tank were negative, which allows to conclude the low prevalence of the agent under the conditions of the present study.(AU)
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Animales , Femenino , Bovinos , Bovinos/microbiología , Mycoplasma bovis/clasificación , Mycoplasma bovis/patogenicidad , Leche/microbiología , Mastitis BovinaRESUMEN
In addition to Staphylococcus aureus nowadays other coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS), earlier considered of minor importance, are now accepted as relevant pathogens for humans and animals. The involvement of these microorganisms in bovine mastitis etiology and the possibility their transmission through milk to humans justify the requirement of developing reliable methods for identification of the most frequent species among them. The purpose of this study was to compare the phenotypic techniques with the genotypic method carried out by sequencing of the rpoB gene in identification of several species of the genus Staphylococcus isolated from bovine mastitis. A total of 300 staphylococci isolates of bovine mastitis cases from several Brazilian dairy herds were studied by phenotypic and genotypic techniques, respectively: 150 CoPS and 150 CoNS strains. A total of 18 CoNS different species and 4 CoPS species were identified. Among the CoNS the following species were recognized: 48 (32%) Staphylococcus warneri, 22(15%) S. epidermidis, 20(13%) S. hyicus, 10(7%) S. xylosus, 7(5%) S. haemolyticus, 6(4%) S. simulans, 6(4%) S. schleiferi subsp schleiferi, 6(4%) S. hominis, 5(3%) S. pasteuri, 4(2.7%) S. cohnii, 3(2%) S. saprophyticus subsp. saprophyticus 3(2%) S. chromogenes 3(2%) S. sciuri, 2(1%) S. saccharolyticus, 2(1%) S. lugdunensi, 1(0,7%) S. auricularis, 1(70%) S. saprophyticus subsp. bovis, 1(0.7%) S. capitis. And among the 150 CoPS were identified respectively: 105 (70%) S. aureus, 21(14%), S. hyicus, 19(13%) S. intermedius e 5(3%) S. schleiferi subsp coagulans. Considering the 150 CoNS isolates, the identifications performed by phenotypic and genotypic tests presented 96.7% of concordance, kappa coefficient of agreement = 0.933, SE (standard error) of kappa=0.021 (95% confidence interval: 0.893 to 0.974), Pearson's correlation coefficient (r) = 0.9977, (confidence interval 95%: 0.9938 a 0.9992) and in relation to 150 CPS isolates it was detected an agreement of 98.7%, kappa = 0.960, SE of kappa = 0.016, (95% confidence interval: 0.929 to 0.992) Pearson's correlation coefficient (r) = 0.9994 (95% confidence interval: 0.9681 to 1.0000). The verified agreement strength between the identification methods can be considered as excellent. These results assure that according to laboratory resources any of them will be suitable to perform the staphylococci identification.(AU)
Além de Staphylococcus aureus atualmente outros estafilococos coagulase positiva (SCP) e estafilococos coagulase-negativos (SCN), anteriormente considerados de menor relevância, são reconhecidos como importantes patógenos para humanos e animais. O envolvimento desses micro-organismos na etiologia da mastite bovina e a possibilidade da sua transmissão através do leite aos humanos justifica a utilização de métodos confiáveis para a identificação das espécies mais frequentes. O objetivo deste estudo foi comparar as técnicas fenotípicas com o método genotípico realizada por sequenciamento do gene rpoB na identificação de espécies do gênero Staphylococcus spp. isolados de mastite bovina. Um total de 300 estafilococos isolados de casos de mastite bovina em diferentes rebanhos leiteiros brasileiros foram estudados por técnicas fenotípicas e genotípicas, respectivamente: 150 linhagens de SCP e 150 linhagens de SCN. Foram identificados um total de 18 espécies de SCN e 4 espécies SCP. Entre os SCN as seguintes espécies identificadas: 48 (32%) Staphylococcus warneri, 22 (15%) S. epidermidis, 20 (13%) S. hyicus, 10 (7%) S. xylosus, 7 (5%) S. haemolyticus, 6 (4%) S. simulans, 6 (4%) S. schleiferi subsp schleiferi, 6 (4%) S. hominis, 5 (3%) S. pasteuri, 4 (2,7%) S. cohnii, 3 (2%) S. saprophyticus subsp. saprophyticus, 3 (2%) S. chromogenes, 3 (2%) S. sciuri, 2 (1%) S. saccharolyticus, 2 (1%) S. lugdunensi, 1 (0,7%) S. auricularis, 1 (70 %) S. saprophyticus subsp. bovis, 1 (0,7%) S. capitis. E entre as 150 SCP foram identificados, 105 (70%) S. aureus, 21 (14%), S. hyicus, 19 (13%) S. intermedius e 5 (3%) S. schleiferi subsp coagulans. Considerando-se os 150 SCN isolados, as identificações realizadas por testes fenotípicos e genotípicos apresentaram 96,7% de concordância, coeficiente de concordância kappa = 0,933, SE (erro padrão) de kappa = 0,021 (95% intervalo de confiança: 0,893-0,974), coeficiente de correlação de Pearson (r) = 0,9977, (intervalo de confiança de 95%: 0,9938 a 0,9992) e em relação a 150 SCP isolados foi observado uma concordância de 98,7%, kappa = 0,960, sE de kappa = 0,016, (95% de intervalo de confiança: 0,929 a 0,992) coeficiente de correlação de Pearson (r) = 0,9994 (95% intervalo de confiança: 0,9681-1,0000). A correlação entre os métodos de identificação pode ser considerada como excelente. Esses resultados demonstraram que de acordo com os recursos disponíveis no laboratório, poderia ser utilizada qualquer uma das metodologias.(AU)
Asunto(s)
Animales , Femenino , Bovinos , Secuencia de Bases , Genotipo , Mastitis Bovina/etiología , Fenotipo , Staphylococcus/genéticaRESUMEN
This paper presents data on fungal peritonitis (FP) in patients undergoing peritoneal dialysis (PD) at the University Hospital of Botucatu Medical School, São Paulo, Brazil. In a total of 422 patients, 30 developed FP, from which the medical records and the fungal isolates of 23 patient cases were studied. All patients presented abdominal pain, cloudy peritoneal effluent, needed hospitalization, had the catheter removed and were treated with fluconazole or fluconazole plus 5-flucitosine; six of them died due to FP. Concerning the agents, it was observed that Candida parapsilosis was the leading species (9/23), followed by Candida albicans (5/23), Candida orthopsilosis (4/23), Candida tropicalis (3/23), Candida guilliermondii (1/23), and Kodamaea ohmeri (1/23). All the isolates were susceptible to amphotericin B, voriconazole and caspofungin whereas C. albicans isolates were susceptible to all antifungals tested. Resistance to fluconazole was observed in three isolates of C. orthopsilosis, and dose-dependent susceptibility to this antifungal was observed in two isolates of C. parapsilosis and in the K. ohmeri isolate. Biofilm production estimates were high or moderate in most isolates, especially in C. albicans species, and low in C. parapsilosis species, with a marked variation among the isolates. This Brazilian study reinforces that FP in PD is caused by a diverse group of yeasts, most prevalently C. parapsilosis sensu stricto species. In addition, they present significant variation in susceptibility to antifungals and biofilm production, thus contributing to the complexity and severity of the clinical features.
Asunto(s)
Antifúngicos/farmacología , Biopelículas/crecimiento & desarrollo , Micosis/microbiología , Diálisis Peritoneal/efectos adversos , Peritonitis/microbiología , Saccharomycetales/efectos de los fármacos , Saccharomycetales/fisiología , Adulto , Anciano , Brasil , Farmacorresistencia Fúngica , Femenino , Hospitales Universitarios , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Estudios Retrospectivos , Saccharomycetales/clasificación , Saccharomycetales/aislamiento & purificación , Análisis de SupervivenciaRESUMEN
Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.
Asunto(s)
Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Biopelículas/crecimiento & desarrollo , Pared Celular/patología , Rojo Congo/farmacología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Presión Osmótica , Estrés Oxidativo , Fosforilación , Transducción de Señal , Sorbitol/farmacología , Estrés Fisiológico , VirulenciaRESUMEN
Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilm production of different species among the different sources of isolation: blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilm production was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilm production by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilm production was more frequent among bloodstream isolates than other clinical sources,in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilm production. The numbers of tandem-repeat copies per allele were not associated with biofilm production, suggesting the evolvement of other genetic determinants.
Asunto(s)
Humanos , Biopelículas/crecimiento & desarrollo , Candida/genética , Candida/fisiología , Candidiasis/microbiología , Proteínas Fúngicas/genética , Polimorfismo Genético , Brasil , Candida/clasificación , Candida/aislamiento & purificación , Hospitales Públicos , Centros de Atención TerciariaRESUMEN
We evaluated the frequency of enterococci from food and found 95.2% of positivity, being E. faecium and E. faecalis the most frequent species. High-level streptomycin resistance was observed, as well as gelatinase and hemolysis activity, showing the potential role of environmental strains as reservoir of virulence and resistance traits.
Asunto(s)
Enterococcus/clasificación , Enterococcus/aislamiento & purificación , Contaminación de Alimentos , Microbiología de Alimentos , Brasil , Farmacorresistencia Bacteriana , Enterococcus/fisiología , Gelatinasas/análisis , Hemólisis , Prevalencia , Factores de Virulencia/análisisRESUMEN
Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilm production of different species among the different sources of isolation: blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilm production was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilm production by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilm production was more frequent among bloodstream isolates than other clinical sources, in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilm production. The numbers of tandem-repeat copies per allele were not associated with biofilm production, suggesting the evolvement of other genetic determinants.