RESUMO
Protein disulfide isomerase-A1 (PDIA1) is a master regulator of oxidative protein folding and proteostasis in the endoplasmic reticulum (ER). However, PDIA1 can reach the extracellular space, impacting thrombosis and other pathophysiological phenomena. Whether PDIA1 is externalized via passive release or active secretion is not known. To investigate how PDIA1 negotiates its export, we generated a tagged variant that undergoes N-glycosylation in the ER (Glyco-PDIA1). Addition of N-glycans does not alter its enzymatic functions. Upon either deletion of its KDEL ER-localization motif or silencing of KDEL receptors, Glyco-PDIA1 acquires complex glycans in the Golgi and is secreted. In control cells, however, Glyco-PDIA1 is released with endoglycosidase-H sensitive glycans, implying that it does not follow the classical ER-Golgi route nor does it encounter glycanases in the cytosol. Extracellular Glyco-PDIA1 is more abundant than actin, lactate dehydrogenase, or other proteins released by damaged or dead cells, suggesting active transport through a Golgi-independent route. The strategy we describe herein can be extended to dissect how select ER-residents reach the extracellular space.
RESUMO
The purpose of the present study was to test the hypothesis that doxorubicin (DX) and cyclophosphamide (CY) adjuvant chemotherapy (CHT) acutely impairs neurovascular and hemodynamic responses in women with breast cancer. Sixteen women (age: 47.0 ± 2.0 yr; body mass index: 24.2 ± 1.5 kg/m) with stage II-III breast cancer and indication for adjuvant CHT underwent two experimental sessions, saline (SL) and CHT. In the CHT session, DX (60 mg/m2) and CY (600 mg/m2) were administered over 45 min. In the SL session, a matching SL volume was infused in 45 min. Muscle sympathetic nerve activity (MSNA) from peroneal nerve (microneurography), calf blood flow (CBF; plethysmography) and calf vascular conductance (CVC), heart rate (HR; electrocardiography), and beat-to-beat blood pressure (BP; finger plethysmography) were measured at rest before, during, and after each session. Venous blood samples (5 ml) were collected before and after both sessions for assessment of circulating endothelial microparticles (EMPs; flow cytometry), a surrogate marker for endothelial damage. MSNA and BP responses were increased (P < 0.001), whereas CBF and CVC responses were decreased (P < 0.001), during and after CHT session when compared with SL session. Interestingly, the vascular alterations were also observed at the molecular level through an increased EMP response to CHT (P = 0.03, CHT vs. SL session). No difference in HR response was observed (P > 0.05). Adjuvant CHT with DX and CY in patients treated for breast cancer increases sympathetic nerve activity and circulating EMP levels and, in addition, reduces muscle vascular conductance and elevates systemic BP. These responses may be early signs of CHT-induced cardiovascular alterations and may represent potential targets for preventive interventions. NEW & NOTEWORTHY It is known that chemotherapy regimens increase the risk of cardiovascular events in patients treated for cancer. Here, we identified that a single cycle of adjuvant chemotherapy with doxorubicin and cyclophosphamide in women treated for breast cancer dramatically increases sympathetic nerve activity and circulating endothelial microparticle levels, reduces the muscle vascular conductance, and elevates systemic blood pressure.
Assuntos
Antineoplásicos/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/efeitos adversos , Doxorrubicina/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Adulto , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Micropartículas Derivadas de Células/efeitos dos fármacos , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/inervação , Nervo Fibular/fisiologiaRESUMO
The liver plays a capital role in the control of whole body energy homeostasis through the metabolization of dietary carbohydrates and lipids. However, under excess macronutrient uptake, those pathways overcharge nucleus-to-endoplasmic reticulum (ER) traffic pathways, leading to luminal overload of unfolded proteins which activates a series of adaptive signaling pathways known as unfolded protein response (UPR). The UPR is a central network mechanism for cellular stress adaptation, however far from a global nonspecific all-or-nothing response. Such a complex signaling network is able to display considerable specificity of responses, with activation of specific signaling branches trimmed for distinct types of stimuli. This makes the UPR a fundamental mechanism underlying metabolic processes and diseases, especially those related to lipid and carbohydrate metabolism. Thus, for a better understanding of the role of UPR on the physiopathology of lipid metabolism disorders, the concepts discussed along this chapter will demonstrate how several metabolic derangements activate UPR components and, in turn, how UPR triggers several metabolic adaptations through its component signaling proteins. This dual role of UPR on lipid metabolism will certainly foment the pursuit of an answer for the question: is UPR cause or consequence of lipid and lipoprotein metabolism disturbances?
Assuntos
Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
Early-life environmental insults have been shown to promote long-term development of chronic non-communicable diseases, including metabolic disturbances and mental illnesses. As such, premature consumption of high-sugar foods has been associated to early onset of detrimental outcomes, whereas underlying mechanisms are still poorly understood. In the present study, we sought to investigate whether early and sustained exposure to high-sucrose diet promotes metabolic disturbances that ultimately might anticipate neurological injuries. At postnatal day 21, weaned male rats started to be fed a standard chow (10 % sucrose, CTR) or a high-sucrose diet (25 % sucrose, HSD) for 9 weeks prior to euthanasia at postnatal day 90. HSD did not alter weight gain and feed efficiency between groups, but increased visceral, non-visceral and brown adipose tissue accumulation. HSD rats demonstrated elevated blood glucose levels in both fasting and fed states, which were associated to impaired glucose tolerance. Peripheral insulin sensitivity did not change, whereas hepatic insulin resistance was supported by increased serum triglyceride levels, as well as higher TyG index values. Assessment of hippocampal gene expression showed endoplasmic reticulum (ER) stress pathways were activated in HSD rats, as compared to CTR. HSD rats had overexpression of unfolded protein response sensors, PERK and ATF6; ER chaperone, PDIA2 and apoptosis-related genes, CHOP and Caspase 3; but decreased expression of chaperone GRP78. Finally, HSD rats demonstrated impaired neuromuscular function and anxious behavior, but preserved cognitive parameters. In conclusion, our data indicate that early exposure to HSD promote metabolic disturbances, which disrupt hippocampus homeostasis and might precociously affect its neurobehavioral functions.
Assuntos
Comportamento Animal/efeitos dos fármacos , Sacarose Alimentar/administração & dosagem , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Síndrome Metabólica/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Ansiedade/metabolismo , Glucose/metabolismo , Hipocampo/metabolismo , Resistência à Insulina/fisiologia , Masculino , Ratos , Ratos Wistar , Triglicerídeos/metabolismoRESUMO
This study aimed at investigating the acute effects of aerobic exercise on endothelium-dependent vasomotor function of rat aorta, as well as mechanisms involved in endothelial nitric oxide (NO) bioactivity. Wistar rats were assigned to either a resting control (C, n = 21) or acutely exercised (E, n = 21) groups (60 min, 55-60% of maximum speed). After exercise, thoracic aorta was excised and cut into rings. Two rings were promptly applied to evaluate vasomotor function and the rest of aorta was used for additional measurements. Acute exercise significantly improved maximum ACh-induced relaxation (C, 91.6 ± 1.2 vs. E, 102.4 ± 1.7%, p < 0.001) and sensitivity to ACh (C, -7.3 ± 0.06 vs. E, -7.3 ± 0.02 log M, p < 0.01), and was accompanied by significantly increases on serine1177 eNOS phosphorylation, reflecting its enhanced activation. However, acute exercise also enhanced both superoxide and hydrogen peroxide production, as assayed by dihydroethidium oxidation, lucigenin chemiluminescence and Amplex Red assays. We also provided evidence for Nox2 NADPH oxidase (Nox) activation through gp91dstat-mediated inhibition of superoxide signals. Enhanced arterial relaxations associated with acute exercise were nearly-completely prevented by catalase, suggesting a role for paracrine hydrogen peroxide. Despite increased detectable oxidant generation, cellular oxidative stress was not evident, as suggested by unaltered GSH:GSSG ratio and lipid hydroperoxides. Collectively, these results demonstrate that one bout of moderate aerobic exercise improves endothelial function by increasing NO bioavailability, while superoxide and hydrogen peroxide are generated in a controlled fashion.
Assuntos
Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Condicionamento Físico Animal/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação/fisiologia , Acetilcolina/metabolismo , Animais , Aorta/química , Aorta/metabolismo , Masculino , Óxido Nítrico/análise , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/análiseRESUMO
Non-alcoholic fatty liver disease (NAFLD) is intimately associated with insulin resistance and hypertriglyceridemia, whereas many of the mechanisms underlying this association are still poorly understood. In the present study, we investigated the relationship between microsomal triglyceride transfer protein (MTP) and markers of endoplasmic reticulum (ER) stress in the liver of rats subjected to neonatal monosodium L-glutamate (MSG)-induced obesity. At age 120 days old, the MSG-obese animals exhibited hyperglycemia, hypertriglyceridemia, insulin resistance, and liver steatosis, while the control (CTR) group did not. Analysis using fast protein liquid chromatography of the serum lipoproteins revealed that the triacylglycerol content of the very low-density lipoprotein (VLDL) particles was twice as high in the MSG animals compared with the CTR animals. The expression of ER stress markers, GRP76 and GRP94, was increased in the MSG rats, promoting a higher expression of X-box binding protein 1 (XBP-1), protein disulfide isomerase (PDI), and MTP. As the XBP-1/PDI/MTP axis has been suggested to represent a significant lipogenic mechanism in the liver response to ER stress, our data indicate that hypertriglyceridemia and liver steatosis occurring in the MSG rats are associated with increased MTP expression.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Fígado Gorduroso/metabolismo , Hipertrigliceridemia/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/síntese química , Fígado Gorduroso/induzido quimicamente , Ácido Glucurônico , Hipertrigliceridemia/induzido quimicamente , Masculino , Obesidade/induzido quimicamente , Estresse Oxidativo , Ratos , Ratos Wistar , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/síntese química , Proteína 1 de Ligação a X-BoxRESUMO
Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with ß2-AR signaling in mediating this protection. Ventricular superoxide (O2â») and hydrogen peroxide (H2O2) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O2â» and H2O2 production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H2O2 and O2â» production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in ß2-AR expression associated with coupling change to Gi; ß2-ARs-S-nitrosation (ß2-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with ß2-ARs overexpression and ß2-AR-SNO via an anti-apoptotic pathway.
Assuntos
Acetilcisteína/análogos & derivados , Regulação da Expressão Gênica , Nitrogênio/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores de LDL/genética , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose , Dislipidemias , Retículo Endoplasmático/metabolismo , Peróxido de Hidrogênio/química , Hipertrofia Ventricular Esquerda , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , SuperóxidosRESUMO
BACKGROUND: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux. METHODS: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI). RESULTS: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux.
Assuntos
Retículo Endoplasmático , Chaperonas Moleculares , Chaperonas Moleculares/metabolismo , Retículo Endoplasmático/metabolismo , Citosol/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , OxirreduçãoRESUMO
Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.
Assuntos
Hipertensão , Músculo Liso Vascular , NADPH Oxidase 1 , Isomerases de Dissulfetos de Proteínas , Ratos Endogâmicos SHR , Regulação para Cima , Animais , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , NADPH Oxidase 1/metabolismo , NADPH Oxidase 1/genética , Hipertensão/fisiopatologia , Hipertensão/genética , Hipertensão/metabolismo , Ratos , Músculo Liso Vascular/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Ratos Wistar , Transcrição GênicaRESUMO
BACKGROUND AND AIMS: Redox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection. METHODS: Initially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days. RESULTS: Transgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mortality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%-50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echocardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation. CONCLUSIONS: Our data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially uncovers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling.
RESUMO
OBJECTIVES: We have examined the impact of changes in modifiable risk factors on CVD mortality in 26 Brazilian states from 2005 to 2017. METHODS: Data were acquired from the Global Burden of Diseases study (GBD) and official sources of the Brazilian government, totalling 312 state-year observations. Population attributable fractions (PAFs) were calculated to determine the number of deaths attributed to changes in each risk factor. Fixed-effects multivariable linear regression models were performed, adjusting for income, income inequality, poverty and access to healthcare. RESULTS: Between 2005 and 2017, CVD deaths reduced by 21.42%, accompanied by a decrease in smoking (-33%) and increases in hyperglycaemia (+9.5%), obesity (+31%) and dyslipidaemia (+5.2%). Reduction in smoking prevented or postponed almost 20,000 CVD deaths in this period, while increased hyperglycaemia exposure resulted in more than 6,000 CVD deaths. The association between hyperglycaemia and CVD mortality was 5 to 10 times higher than those found for other risk factors, especially in women (11; 95%CI 7 to 14, deaths per 1-point increase in hyperglycaemia exposure). Importantly, the association between hyperglycaemia and CVD mortality was independent of socioeconomic status and access to healthcare, while associations for other risk factors after the same adjustments. CONCLUSION: Reduction in smoking was the risk factor that led to the highest number of CVD deaths prevented or postponed, while hyperglycaemia showed the most deleterious association with CVD mortality. Health policies should aim to directly reduce the prevalence of hyperglycaemia to mitigate the population burden of CVD in Brazil in the future.
Assuntos
Doenças Cardiovasculares , Hiperglicemia , Brasil/epidemiologia , Feminino , Carga Global da Doença , Humanos , Hiperglicemia/complicações , Hiperglicemia/epidemiologia , Fatores de Risco , Fatores SocioeconômicosRESUMO
Metabolic dysfunctions, such as hyperglycemia and insulin resistance, have been associated to cognitive impairment and dementia regardless of advanced age, although the underlying mechanisms are still elusive. Thus, this study investigates the deleterious effects of metabolic syndrome (MetS) induced by long-term exposure to a high-sucrose diet on motor and cognitive functions of male adult rats and its relationship with hippocampal endoplasmic reticulum (ER) stress. Weaned Wistar male rats were fed a high-sucrose diet until adulthood (HSD; 6 months old) and compared to both age-matched (CTR; 6 months old) and middle-aged chow-fed rats (OLD; 20 months old). MetS development, serum redox profile, behavioral, motor, and cognitive functions, and hippocampal gene/protein expressions for ER stress pro-adaptive and pro-apoptotic pathways, as well as senescence markers were assessed. Prolonged exposure to HSD induced MetS hallmarked by body weight gain associated to central obesity, hypertriglyceridemia, insulin resistance, and oxidative stress. Furthermore, HSD rats showed motor and cognitive decline similar to that in OLD animals. Noteworthy, HSD rats presented marked hippocampal ER stress characterized by failure of pro-adaptive signaling and increased expression of Chop, p21, and Parp-1 cleavage, markers of cell death and aging. This panorama resembles that found in OLD rats. In toto, our data showed that early and sustained exposure to a high-sucrose diet induced MetS, which subsequently led to hippocampus homeostasis disruption and premature impairment of motor and cognitive functions in adult rats.
RESUMO
In Marfan syndrome (MFS), dilation, dissection, and rupture of the aorta occur. Inflammation can be involved in the pathogenicity of aortic defects and can thus be a therapeutic target for MFS. Previously, we showed that the formulation of methotrexate (MTX) associated with lipid nanoparticles (LDE) has potent anti-inflammatory effects without toxicity. To investigate whether LDEMTX treatment can prevent the development of aortic lesions in the MFS murine model. MgΔloxPneo MFS (n = 40) and wild-type (WT, n = 60) mice were allocated to 6 groups weekly injected with IP solutions of: (1) only LDE; (2) commercial MTX; (3) LDEMTX (dose = 1mg/kg) between 3rd and 6th months of life. After 12 weeks of treatments, animals were examined by echocardiography and euthanatized for morphometric and molecular studies. MFS mice treated with LDEMTX showed narrower lumens in the aortic arch, as well as in the ascending and descending aorta. LDEMTX reduced fibrosis and the number of dissections in MFS but not the number of elastic fiber disruptions. In MFS mice, LDEMTX treatment lowered protein expression of pro-inflammatory factors macrophages (CD68), T-lymphocytes (CD3), tumor necrosis factor-α (TNF-α), apoptotic factor cleaved-caspase 3, and type 1 collagen and lowered the protein expression of the transforming growth factor-ß (TGF-ß), extracellular signal-regulated kinases ½ (ERK1/2), and SMAD3. Protein expression of CD68 and CD3 had a positive correlation with an area of aortic lumen (r 2 = 0.36; p < 0.001), suggesting the importance of inflammation in the causative mechanisms of aortic dilation. Enhanced adenosine availability by LDEMTX was suggested by higher aortic expression of an anti-adenosine A2a receptor (A2a) and lower adenosine deaminase expression. Commercial MTX had negligible effects. LDEMTX prevented the development of MFS-associated aortic defects and can thus be a candidate for testing in clinical studies.
RESUMO
ABSTRACT: Leukocyte Nox2 is recognized to have a fundamental microbicidal function in sepsis but the specific role of Nox2 in endothelial cells (EC) remains poorly elucidated. Here, we tested the hypothesis that endothelial Nox2 participates in the pathogenesis of systemic inflammation and hypotension induced by LPS. LPS was injected intravenously in mice with Tie2-targeted deficiency or transgenic overexpression of Nox2. Mice with Tie2-targeted Nox2 deficiency had increased circulating levels of TNF-α, enhanced numbers of neutrophils trapped in lungs, and aggravated hypotension after LPS injection, as compared to control LPS-injected animals. In contrast, Tie2-driven Nox2 overexpression attenuated inflammation and prevented the hypotension induced by LPS. Because Tie2-Cre targets both EC and myeloid cells we generated bone marrow chimeric mice with Nox2 deletion restricted to leukocytes or ECs. Mice deficient in Nox2 either in leukocytes or ECs had reduced LPS-induced neutrophil trapping in the lungs and lower plasma TNF-α levels as compared to control LPS-injected mice. However, the pronounced hypotensive response to LPS was present only in mice with EC-specific Nox2 deletion. Experiments in vitro with human vein or aortic endothelial cells (HUVEC and HAEC, respectively) treated with LPS revealed that EC Nox2 controls NF-κB activation and the transcription of toll-like receptor 4 (TLR4), which is the recognition receptor for LPS. In conclusion, these results suggest that endothelial Nox2 limits NF-κB activation and TLR4 expression, which in turn attenuates the severity of hypotension and systemic inflammation induced by LPS.
Assuntos
Células Endoteliais/fisiologia , Endotoxemia/etiologia , Hipotensão/etiologia , Inflamação/etiologia , NADPH Oxidase 2/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Interferon gama/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/fisiopatologia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Miócitos Cardíacos/patologia , Adulto JovemRESUMO
BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.
Assuntos
Peróxidos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Ácido Úrico/análogos & derivados , Domínio Catalítico , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Cromatografia Líquida/métodos , Cisteína/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Oxirredução , Peroxidases/metabolismo , Agregação Plaquetária , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem/métodos , Trombose/metabolismo , Ácido Úrico/metabolismoRESUMO
Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.
Assuntos
Síndrome de Marfan/patologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Actinas/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Feminino , Fibrilina-1/metabolismo , Adesões Focais/metabolismo , Adesões Focais/patologia , Masculino , Síndrome de Marfan/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Proteômica/métodosRESUMO
Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3ß and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.
Assuntos
Neoplasias do Colo/metabolismo , NADPH Oxidase 1/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células CACO-2 , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HCT116 , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (medianâ¯=â¯330â¯pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.
Assuntos
Células Endoteliais/metabolismo , Fenótipo , Isomerases de Dissulfetos de Proteínas/sangue , Proteoma , Proteômica , Adulto , Biomarcadores , Sobrevivência Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Oxirredução , Agregação Plaquetária , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
Syzygium cumini is used worldwide for the treatment of metabolic syndrome-associated outcomes. Previously, we described the antihypertriglyceridemic effect of the hydroethanolic extract of S. cumini leaf (HESc) in monosodium L-glutamate- (MSG-) induced obese rats. This study sought to investigate the molecular mechanisms underlying the antihypertriglyceridemic effect of HESc in MSG-obese rats. Newborn male Wistar rats were injected subcutaneously with MSG (4.0 g/kg/day, obese group) or saline 1.25% (1.0 mL/kg/day, lean group), from 2nd through 10th postnatal day. At 8 weeks old, obese rats started to be orally treated with HESc (0.5 or 1.0 g/kg/day, n = 7) or saline 0.9% (1 mL/kg/day, n = 7). Lean rats received saline solution (1 mL/kg/day, n = 7). Upon 8-week treatment, animals were euthanized for blood and tissue collection. Another set of adult nonobese Wistar rats was used for the assessment of HESc acute effects on Triton WR1339-induced hypertriglyceridemia. HESc reduced weight gain, as well as adipose tissue fat pads, without altering food intake of obese rats. HESc restored fasting serum glucose, triglycerides, total cholesterol, and free fatty acids, as well as insulin sensitivity, to levels similar to lean rats. Additionally, HESc halved the triglyceride content into very low-density lipoprotein particles, as well as healed liver steatosis, in obese rats. Hepatic protein expression of the endoplasmic reticulum chaperone GRP94 was decreased by HESc, which also downregulated the hepatic triglyceride secretion pathway by reducing the splicing of X-box binding protein 1 (XBP-1s), as well as protein disulfide isomerase (PDI) and microsomal triglyceride transfer protein (MTP) translational levels. This action was further corroborated by the acute inhibitory effect of HESc on triglyceride accumulation on Triton WR1339-treated rats. Our data support the downregulation of the XBP-1s/PDI/MTP axis in the liver of MSG-obese rats as a novel feasible mechanism for the antihypertriglyceridemic effect promoted by the polyphenolic phytocomplex present in S. cumini leaf.