RESUMO
Aims: The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Methods and results: Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Conclusion: Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.
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Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and ß3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-ß signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.
Assuntos
Plaquetas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Osteonectina/metabolismo , Trombina/metabolismo , Adulto , Animais , Plaquetas/citologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ativação Plaquetária , Agregação PlaquetáriaRESUMO
BACKGROUND: Cerebral vasospasm (CVS) is a frequent complication after subarachnoid hemorrhage (SAH), with no sufficient therapy and a complex pathophysiology. OBJECTIVE: To explore the vitamin D system as a potential treatment for CVS. METHODS: 25-vitamin D3 levels tested between 2007 and 2015 and data of SAH patients admitted during the months with a peak vs nadir of VitD3 values were analyzed, retrospectively. We prospectively correlated VitD3 and vasospasm/outcome data in SAH patients admitted in 2017. An experimental mice SAH model and cell culture model were used to investigate the effect of 1,25-dihydroxyvitamin D3 (1,25-VitD3). Additionally, the mediators acting in the VitD mechanism were researched and detected. RESULTS: Based on the retrospective analysis demonstrating an increased frequency of vasospasm in SAH patients during the low vitamin D period in winter, we started basic research experiments. Active 1,25-VitD3 hormone attenuated CVS, neurological deficit, and inflammation after intrathecal blood injection in mice. Deletion of the vitamin D receptor in the endothelium or in myeloid cells decreased the protective 1,25-VitD3 effect. Co-culture experiments of myeloid and endothelial cells with blood confirmed the anti-inflammatory 1,25-VitD3 effect but also revealed an induction of stroma-cell-derived factor 1α (SDF1α), vascular endothelial growth factor, and endothelial nitric oxide synthase by 1,25-VitD3. In mice, SDF1α mimicked the protective effect of 1,25-VitD3 against CVS. From bench to bedside, CVS severity was inversely correlated with vitamin D plasma level, prospectively. Patients with more severe CVS exhibited attenuated expression of SDF1α and 1,25-VitD3-responsive genes on circulating myeloid cells. CONCLUSION: 1,25-VitD3 attenuates CVS after SAH by inducing SDF1α. However, VitD administration should be tested as optional treatment to prevent CVS.
Assuntos
Calcitriol/administração & dosagem , Calcitriol/sangue , Estações do Ano , Vasoespasmo Intracraniano/sangue , Vasoespasmo Intracraniano/tratamento farmacológico , Adulto , Animais , Feminino , Seguimentos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Vasoespasmo Intracraniano/diagnóstico por imagem , Vitamina D/administração & dosagem , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico por imagem , Deficiência de Vitamina D/tratamento farmacológicoRESUMO
BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.
Assuntos
Cadeias beta de Integrinas/química , Compostos de Sulfidrila/química , Animais , Cromatografia Líquida de Alta Pressão , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Sulfeto de Hidrogênio/farmacologia , Cadeias beta de Integrinas/metabolismo , Mecanotransdução Celular , Camundongos , Resistência ao Cisalhamento , Espectrometria de Massas em Tandem , Vasodilatação/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1-/- mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.
Assuntos
Plaquetas/enzimologia , Calpaína/sangue , Micropartículas Derivadas de Células/enzimologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Angiopatias Diabéticas/enzimologia , Células Endoteliais/enzimologia , Receptor PAR-1/metabolismo , Vasculite/enzimologia , Proteína ADAM17/metabolismo , Adulto , Animais , Calpaína/genética , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/genética , Receptor de Proteína C Endotelial/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Receptor PAR-1/genética , Fator de Necrose Tumoral alfa/metabolismo , Vasculite/sangue , Vasculite/genéticaRESUMO
Diabetes is associated with platelet hyper-reactivity and enhanced risk of thrombosis development. Here we compared protein expression in platelets from healthy donors and diabetic patients to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analyses identified cyclin Y (CCNY) in platelets and its reduced expression in platelets from diabetic patients, a phenomenon that could be attributed to the increased activity of calpains. To determine the role of CCNY in platelets, mice globally lacking the protein were studied. CCNY-/- mice demonstrated lower numbers of circulating platelets but platelet responsiveness to thrombin and a thromboxane A2 analogue were comparable with that of wild-type mice, as was agonist-induced α and dense granule secretion. CCNY-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in "outside in" integrin signalling. This phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of ß3 integrin. Taken together we have shown that CCNY is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling associated with thrombin stimulation.
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Plaquetas/metabolismo , Ciclinas/genética , Integrinas/metabolismo , Adulto , Animais , Ciclinas/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Ativação Plaquetária/genética , Adesividade Plaquetária/genética , Agregação Plaquetária/genética , Transdução de Sinais/genética , Adulto JovemRESUMO
Calpain 2 (CAPN2) is a Ca2+-dependent cysteine-protease that is involved in different cellular processes. Despite its important role, little is known about how CAPN2 expression is regulated. This study addressed the potential regulation of CAPN2 by microRNAs (miRNAs) in human endothelial cells. Two miRNAs were found to regulate CAPN2 expression by two distinct mechanisms, one direct and the other indirect. MiR-223 directly targeted CAPN2 by binding to the CAPN2 3'-untranslated region. Mir-223 overexpression decreased CAPN2 protein levels in cultured cells and in mice miR-223 antagonism led to an increase in CAPN2 protein in lung tissue. MiR-145 overexpression also decreased CAPN2 expression but did not affect a CAPN2 luciferase construct, indicating that the effect was indirect. MiR-145 targets histone deacetylase (HDAC) 2, and HDAC inhibition transcriptionally regulated CAPN2 expression by hyperacetylation of the promoter of CAPN2 gene and a subsequent decrease in polymerase 2 binding. Indeed, down regulation of HDAC2 by miR-145 not only decreased CAPN2 protein expression and calpain activity, but also protected paxillin against calpain-dependent degradation. Thus, protein levels of CAPN2 are regulated by miR-223, acting directly on the 3'-untranslated region as well as by miR-145, which acts via an increase in HDAC2. ENZYMES: Calpain 2 (EC 3.4.22.53), histone deacetylase 2 (EC 3.5.1.98).
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Calpaína/genética , Células Endoteliais/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Regulação da Expressão Gênica/genética , Histona Desacetilase 2/genética , Humanos , Camundongos , Regiões Promotoras GenéticasRESUMO
Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.
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Substituição de Aminoácidos , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Piruvato Quinase/metabolismo , Animais , Células Cultivadas , Células Endoteliais , Homeostase , Humanos , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/genética , Oxirredução , Via de Pentose Fosfato , Ligação ProteicaRESUMO
BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.
Assuntos
Aterosclerose/enzimologia , Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/enzimologia , Cistationina gama-Liase/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Células Endoteliais/enzimologia , Sulfeto de Hidrogênio/metabolismo , Placa Aterosclerótica , Idoso , Idoso de 80 Anos ou mais , Animais , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/prevenção & controle , Catepsinas/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cistationina gama-Liase/deficiência , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Progressão da Doença , Proteína Semelhante a ELAV 1/genética , Células Endoteliais/patologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disease associated with insulin resistance and increased risk of cardiovascular diseases. However, a biomarker for potential cardiovascular disease in PCOS patients is not available. MATERIALS AND METHODS: Twenty-two patients with PCOS and 22 healthy controls were included in the present study and amino acid profiling was performed on fasting plasma samples. Circulating microparticles were characterized by FACS analysis and complemented with enzyme activity assays. RESULTS: The ratio of ornithine to arginine was significantly increased in plasma form PCOS patients and was associated with a significant increase in plasma arginase levels and activity. Platelet-derived microparticles were identified to be the main sources of the increased plasma arginase activity. CONCLUSIONS: Increased levels of arginase-bearing platelet-derived microparticles contribute to the alteration of the arginine metabolism in patients with polycystic ovary syndrome. Moreover, ornithine and arginine levels represent early biomarkers of potential cardiovascular disease in PCOS patients.
Assuntos
Arginase/sangue , Arginina/sangue , Plaquetas/enzimologia , Micropartículas Derivadas de Células/metabolismo , Síndrome do Ovário Policístico/sangue , Adulto , Plaquetas/ultraestrutura , Estudos de Casos e Controles , Feminino , Humanos , Ornitina/sangue , Síndrome do Ovário Policístico/metabolismo , Adulto JovemRESUMO
SIGNIFICANCE: Calpains (CAPNs) are a family of calcium-activated cysteine proteases. The ubiquitous isoforms CAPN1 and CAPN2 have been involved in the maintenance of vascular integrity, but uncontrolled CAPN activation plays a role in the pathogenesis of vascular diseases. Recent Advances: It is well accepted that chronic and acute overproduction of reactive oxygen species (ROS) is associated with the development of vascular diseases. There is increasing evidence that ROS can also affect the CAPN activity, suggesting CAPN as a potential link between oxidative stress and vascular disease. CRITICAL ISSUES: The physiopathological relevance of ROS in regulating the CAPN activity is not fully understood but seems to involve direct effects on CAPNs, redox modifications of CAPN substrates, as well as indirect effect on CAPNs via changes in Ca2+ levels. Finally, CAPNs can also stimulate ROS production; however, data showing in which context ROS are the causes or the consequences of CAPN activation are missing. FUTURE DIRECTIONS: Detailed characterization of the molecular mechanisms underlying the regulation of the different members of the CAPN system by specific ROS would help understanding the pathophysiological role of CAPN in the modulation of the vascular function. Moreover, given that CAPNs have been found in different cellular compartments such as mitochondria and nucleus as well as in the extracellular space, identification of new CAPN targets as well as their functional consequences would add new insights in the function of these enigmatic proteases.
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Vasos Sanguíneos/metabolismo , Calpaína/metabolismo , Oxirredução , Animais , Cálcio/metabolismo , Regulação da Expressão Gênica , Humanos , Espécies Reativas de Oxigênio/metabolismo , Doenças Vasculares/metabolismoRESUMO
AIMS: Nitroglycerine (NTG) given prior to an ischaemic insult exerts cardioprotective effects. However, whether administration of an acute low dose of NTG in a clinically relevant manner following an ischaemic episode limits infarct size, has not yet been explored. METHODS AND RESULTS: Adult mice were subjected to acute myocardial infarction in vivo and then treated with vehicle or low-dose NTG prior to reperfusion. This treatment regimen minimized myocardial infarct size without affecting haemodynamic parameters but the protective effect was absent in mice rendered tolerant to the drug. Mechanistically, NTG was shown to nitrosate and inhibit cyclophilin D (CypD), and NTG administration failed to limit infarct size in CypD knockout mice. Additional experiments revealed lack of the NTG protective effect following genetic (knockout mice) or pharmacological inhibition (L-NAME treatment) of the endothelial nitric oxide synthase (eNOS). The protective effect of NTG was attributed to preservation of the eNOS dimer. Moreover, NTG retained its cardioprotective effects in a model of endothelial dysfunction (ApoE knockout) by preserving CypD nitrosation. Human ischaemic heart biopsies revealed reduced eNOS activity and exhibited reduced CypD nitrosation. CONCLUSION: Low-dose NTG given prior to reperfusion reduces myocardial infarct size by preserving eNOS function, and the subsequent eNOS-dependent S-nitrosation of CypD, inhibiting cardiomyocyte necrosis. This novel pharmacological action of NTG warrants confirmation in clinical studies, although our data in human biopsies provide promising preliminary results.
Assuntos
Ciclofilinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Nitroglicerina/farmacologia , Adulto , Idoso , Animais , Ciclofilinas/genética , Ciclofilinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Necrose , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitrosação , Transdução de SinaisRESUMO
AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα-/-) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Fenilefrina/metabolismo , Fosforilação , Vasoconstrição/genética , Vasoconstrição/fisiologiaRESUMO
Platelets play an important role in vascular homeostasis through their interaction with circulating blood cells as well as the vascular wall. Platelet-mediated communication with other cells can take the form of direct cell-cell interactions via membrane receptors or indirectly through the release of different soluble factors stored in their granules as well as through the release of microparticles. The latter carry different proteins and RNAs which are transferred to the target cells. The aim of this review is to discuss the role of platelet-derived factors, adhesion molecules as well as RNAs as mediators of the cross-talk between platelets and the vessel wall.
Assuntos
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Leucócitos/metabolismo , RNA não Traduzido/genética , Animais , Plaquetas/citologia , Comunicação Celular , Células Endoteliais/citologia , Endotélio Vascular/citologia , Humanos , Leucócitos/citologia , Adesividade PlaquetáriaRESUMO
Diabetes is associated with a number of co-morbidities including an increased risk of developing cardiovascular diseases. The activation of Ca2+-activated proteases of the calpain family has been implicated in platelet activation associated with diabetes and this study aimed to determine the role of calpain activation in the development of endothelial dysfunction. Diabetes induction in mice attenuated acetylcholine-induced relaxation of mesenteric artery rings, an effect prevented in mice receiving a calpain inhibitor. A nitric oxide-independent but diclofenac-sensitive component of the relaxation-response was altered and correlated with a loss of prostacyclin (PGI2) generation and reduced vascular levels of PGI2 synthase. Calpain inhibition was also able to restore PGI2 synthase levels and PGI2 generation in arteries from diabetic animals. The effects of diabetes were reproduced in vitro by a combination of high glucose and palmitate, which elicited calpain activation, PGI2 synthase cleavage and inactivation as well as endothelial dysfunction in mesenteric arteries from wild-type mice. PGI2 cleavage was not observed in arteries from calpain 1-/- mice or mice overexpressing the endogenous calpain inhibitor calpastatin. Finally, proteomic analyses revealed that calpain 1 cleaved the C-terminal domain of PGI2 synthase close to the catalytic site of the enzyme. These data demonstrate that diabetes leads to the activation of calpain 1 in mesenteric arteries and can initiate endothelial dysfunction by cleaving and inactivating the PGI2 synthase. Given that calpain inhibition prevented diabetes-induced endothelial dysfunction in mesenteric arteries, calpains represent an interesting therapeutic target for the prevention of cardiovascular complication of diabetes.
Assuntos
Calpaína/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Diabetes Mellitus Experimental/metabolismo , Oxirredutases Intramoleculares/metabolismo , Artérias Mesentéricas/metabolismo , Animais , Cardiomiopatias Diabéticas/metabolismo , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Espectrometria de Massas em TandemRESUMO
RATIONALE: Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. OBJECTIVE: We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. METHODS AND RESULTS: The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. CONCLUSIONS: NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aorta/metabolismo , Pressão Sanguínea/fisiologia , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/fisiologia , Frequência Cardíaca/fisiologia , Receptor Muscarínico M3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Animais , Aorta/química , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/análise , Endotélio Vascular/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Receptor Muscarínico M3/análiseRESUMO
RATIONALE: MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus. OBJECTIVE: This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function. METHODS AND RESULTS: Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins. CONCLUSIONS: Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus.
Assuntos
Plaquetas/enzimologia , Calpaína/sangue , RNA Helicases DEAD-box/sangue , Diabetes Mellitus Tipo 2/sangue , MicroRNAs/sangue , Agregação Plaquetária/fisiologia , Ribonuclease III/sangue , Adulto , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/fisiologia , Cálcio/farmacologia , Calpaína/deficiência , Proteínas do Citoesqueleto/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Fator XIII/metabolismo , Feminino , Humanos , Ionomicina/farmacologia , Masculino , Proteínas de Membrana/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Repetições de Microssatélites , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Agregação Plaquetária/efeitos dos fármacos , ProteomaRESUMO
Polycystic ovary syndrome (PCOS) is associated with decreased fertility, insulin resistance and an increased risk of developing cardiovascular disease. Treating PCOS patients with metformin improves fertility and decreases cardiovascular complications. Given that platelet activation contributes to both infertility and cardiovascular disease development, we assessed platelet reactivity in PCOS patients and the consequences of metformin treatment. Compared to washed platelets from healthy donors, platelets from PCOS patients demonstrated enhanced reactivity and impaired activation of the AMP-activated kinase (AMPK). PCOS platelets also demonstrated enhanced expression of mitochondrial proteins such as the cytochrome c reductase, ATP synthase and the voltage-dependent anion channel-1. However, mitochondrial function was impaired as demonstrated by a decreased respiration rate. In parallel, the phosphorylation of dynamin-related protein-1 (Drp-1) on Ser616 was increased while that on Ser637 decreased. The latter changes were accompanied by decreased mitochondrial size. In insulin-resistant PCOS patients (HOMA-IR> 2) metformin treatment (1.7 g per day for 4 weeks to 6 months) improved insulin sensitivity, restored mitochondrial integrity and function and normalised platelet aggregation. Treatment was without effect in PCOS patients with HOMA-IR< 2. Moreover, treatment of megakaryocytes with metformin enhanced mitochondrial content and in the same cells metformin enhanced the phosphorylation of the Drp-1 on Ser637 via an AMPKα1-dependent mechanism. In conclusion, the improvement of mitochondrial integrity and platelet reactivity may contribute to the beneficial effects of metformin on cardiovascular disease.
Assuntos
Plaquetas/efeitos dos fármacos , Metformina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Síndrome do Ovário Policístico/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/sangue , Proteínas Quinases Ativadas por AMP/genética , Adulto , Plaquetas/enzimologia , Plaquetas/ultraestrutura , Estudos de Casos e Controles , Linhagem Celular , Relação Dose-Resposta a Droga , Dinaminas , Ativação Enzimática , Feminino , GTP Fosfo-Hidrolases/sangue , Humanos , Resistência à Insulina , Proteínas Associadas aos Microtúbulos/sangue , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/sangue , Tamanho Mitocondrial/efeitos dos fármacos , Fosforilação , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/diagnóstico , Síndrome do Ovário Policístico/enzimologia , Síndrome do Ovário Policístico/genética , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Resultado do TratamentoRESUMO
BACKGROUND: Calpain has been associated with the pathophysiology of Alzheimer's disease (AD) and with apoptotic neuronal cell death leading to microparticles (MPs) formation. METHODS: A total of 64 patients with AD and 52 age- and gender-matched cognitively healthy elderly controls were included in the study. We measured calpain activity and levels of MPs, amyloid beta (Aß1-42), h-tau, and p-tau181. RESULTS: AD patients showed significantly increased calpain activity and higher levels of MPs in cerebrospinal fluid (CSF) and significantly decreased calpain activity and lower levels of MPs in serum and plasma compared with healthy controls. Combined assessment of calpain activity and Aß1-42 levels in CSF improved diagnostic accuracy as compared with singular or combined traditional CSF biomarkers of AD. CONCLUSIONS: This is the first study showing increased calpain activity and microparticle levels in CSF of AD patients. Calpain activity could represent a novel diagnostic and prognostic biomarker and promising treatment target for AD.