RESUMO
Diabetes mellitus accelerates vascular calcification (VC) and increases the risk of end-stage renal disease (ESRD). Nevertheless, the impact of VC in renal disease progression in type 2 diabetes mellitus (T2DM) is poorly understood. We addressed the effect of VC and mechanisms involved in renal dysfunction in a murine model of insulin resistance and obesity (ob/ob), comparing with their healthy littermates (C57BL/6). We analyzed VC and renal function in both mouse strains after challenging them with Vitamin D3 (VitD3). Although VitD3 similarly increased serum calcium and induced bone disease in both strains, 24-hour urine volume and creatinine pronouncedly decreased only in ob/ob mice. Moreover, ob/ob increased urinary albumin/creatinine ratio (ACR), indicating kidney dysfunction. In parallel, ob/ob developed extensive intrarenal VC after VitD3. Coincidently with increased intrarenal vascular mineralization, our results demonstrated that Bone Morphogenetic Protein-2 (BMP-2) was highly expressed in these arteries exclusively in ob/ob. These data depict a greater susceptibility of ob/ob mice to develop renal disease after VitD3 in comparison to paired C57BL/6. In conclusion, this study unfolds novel mechanisms of progressive renal dysfunction in diabetes mellitus (DM) after VitD3 in vivo associated with increased intrarenal VC and highlights possible harmful effects of long-term supplementation of VitD3 in this population.
Assuntos
Colecalciferol/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Suplementos Nutricionais , Resistência à Insulina , Nefropatias/patologia , Calcificação Vascular/complicações , Animais , Hormônios e Agentes Reguladores de Cálcio/farmacologia , Nefropatias/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/fisiopatologiaRESUMO
Approximately 20% of the world's population will be around or above 65 years of age by the next decade. Out of these, 40% are suspected to have cardiovascular diseases as a cause of mortality. Arteriosclerosis, characterized by increased vascular calcification, impairing Windkessel effect and tissue perfusion, and determining end-organ damage, is a hallmark of vascular pathology in the elderly population. Risk factors accumulated during aging affect the normal physiological and vascular aging process, which contributes to the progression of arteriosclerosis. Traditional risk factors, age-associated diseases, and respective regulating mechanisms influencing vascular calcification and vascular stiffness have been extensively studied for many years. Despite the well-known fact that aging alone can induce vascular damage, specific mechanisms that implicate physiological aging in vascular calcification, contributing to vascular stiffness, are poorly understood. This review focuses on mechanisms activated during normal aging, for example, cellular senescence, autophagy, extracellular vesicles secretion, and oxidative stress, along with the convergence of premature aging models' pathophysiology, such as Hutchinson-Gilford Progeria (prelamin accumulation) and Klotho deficiency, to understand vascular calcification in aging. Understanding the mechanisms of vascular damage in aging that intersect with age-associated diseases and risk factors is crucial to foster innovative therapeutic targets to mitigate cardiovascular disease. Visual Overview- An online visual overview is available for this article.
Assuntos
Envelhecimento/patologia , Calcificação Vascular/etiologia , Animais , Autofagia , Senescência Celular , Vesículas Extracelulares/fisiologia , Glucuronidase/deficiência , Glucuronidase/fisiologia , Humanos , Proteínas Klotho , Osteoporose/etiologia , Estresse Oxidativo , Progéria/complicações , Espécies Reativas de Oxigênio/metabolismoRESUMO
Objective- We hypothesized that ob/ob mice develop expansive vascular remodeling associated with calcification. Approach and Results- We quantified and investigated mechanisms of vascular remodeling and vascular calcification in ob/ob mice after vitamin D3(VD) stimulation or PBS (control), compared with C57BL/6 mice. Both ob/ob (OBVD [VD-treated ob/ob mice]) and C57BL/6 (C57VD [VD-treated C57BL/6 mice]) received 8×103 IU/day of intraperitoneal VD for 14 days. Control ob/ob (OBCT [PBS-treated ob/ob mice]) and C57BL/6 (C57CT [PBS-treated C57BL/6 mice]) received intraperitoneal PBS for 14 days. Hypervitaminosis D increased the external and internal elastic length in aortae from OBVD, resulting in increased total vascular area and lumen vascular area, respectively, which characterizes expansive vascular remodeling. OBVD decreased the aortic wall thickness, resulting in hypotrophic vascular remodeling. We demonstrated increased collagen deposition, elastolysis, and calcification in aortae from OBVD. Our results showed a positive correlation between expansive vascular remodeling and vascular calcification in OBVD. We demonstrated increased serum calcium levels, augmented Bmp (bone morphogenetic protein)-2 and osteochondrogenic proteins expression in OBVD aortae. Furthermore, aortae from OBVD increased oxidative stress, coincidently with augmented in situ MMP (matrix metalloproteinase) activity and exhibited no VDR (VD receptor) inhibition after VD. Conclusions- Our data provide evidence that obese and insulin-resistant mice (ob/ob) developed expansive hypotrophic vascular remodeling correlated directly with increased vascular calcification after chronic VD stimulation. Positive hypotrophic vascular remodeling and vascular calcification in this mouse model is possibly mediated by the convergence of absence VDR downregulation after VD stimulation, increased reactive oxygen species generation, and MMP activation.
Assuntos
Colecalciferol/farmacologia , Resistência à Insulina , Obesidade/complicações , Calcificação Vascular/induzido quimicamente , Remodelação Vascular/efeitos dos fármacos , Animais , Cálcio/sangue , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Receptores de Calcitriol/fisiologia , Remodelação Vascular/fisiologiaRESUMO
BACKGROUND: Coronary artery calcification (CAC) and atherosclerotic inflammation associate with increased risk of myocardial infarction (MI). Vascular calcification is regulated by osteogenic proteins (OPs). It is unknown whether an association exists between CAC and plasma OPs and if they are affected by atherothrombotic inflammation. We tested the association of osteogenic and inflammatory proteins with CAC and assessed these biomarkers after MI. METHODS: Circulating OPs (osteoprotegerin, RANKL, fetuin-A, Matrix Gla protein [MGP]) and inflammatory proteins (C-reactive protein, oxidized-LDL, tumoral necrosis factor-α, transforming growth factor [TGF]-ß1) were compared between stable patients with CAC (CAC ≥ 100 AU, n = 100) and controls (CAC = 0 AU, n = 30). The association between biomarkers and CAC was tested by multivariate analysis. In patients with MI (n = 40), biomarkers were compared between acute phase and 1-2 months post-MI, using controls as a baseline. RESULTS: MGP and fetuin-A levels were higher within individuals with CAC. Higher levels of MGP and RANKL were associated with CAC (OR 3.12 [95% CI 1.20-8.11], p = 0.02; and OR 1.75 [95% CI 1.04-2.94] respectively, p = 0.035). After MI, C-reactive protein, OPG and oxidized-LDL levels increased in the acute phase, whereas MGP and TGF-ß1 increased 1-2 months post-MI. CONCLUSIONS: Higher MGP and RANKL levels associate with CAC. These findings highlight the potential role of these proteins as modulators and markers of CAC. In addition, the post-MI increase in OPG and MGP, as well as of inflammatory proteins suggest that the regulation of these OPs is affected by atherothrombotic inflammation.
Assuntos
Proteínas de Ligação ao Cálcio/sangue , Doença da Artéria Coronariana/metabolismo , Proteínas da Matriz Extracelular/sangue , Infarto do Miocárdio/metabolismo , Osteoprotegerina/sangue , Ligante RANK/sangue , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/complicações , Feminino , Humanos , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/etiologia , Fator de Crescimento Transformador beta1/sangue , Fator de Necrose Tumoral alfa/sangue , alfa-2-Glicoproteína-HS/metabolismo , Proteína de Matriz GlaRESUMO
Vascular calcification is a tightly regulated process that increases during ageing and occurs mainly in patients with diabetes and chronic renal failure. Exosomes are small membrane vesicles that are synthesized in a particular population of endosomes, also called multivesicular bodies, by inside budding into the lumen of the compartment. After fusion of exosome with the plasma membrane, these internal vesicles are secreted. Exosomes have a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate. Investigators implicated microvesicles/exosomes as a specific signaling mechanism to induce vascular mineralization during vascular smooth muscle cells phenotypic transition. Vascular wall from healthy individual exhibit exosomes loaded with calcification inhibitors such as Fetuin A and MGP. Conversely, calcifying conditions induce secretion of exosomes, characterized by decreased calcifying inhibitors and increased phosphatidyl serine and Annexin A6 content, which serves as a nidus for vascular calcification.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Exossomos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Calcificação Vascular/metabolismo , Animais , Artérias/metabolismo , Artérias/patologia , Exossomos/patologia , Humanos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Calcificação Vascular/patologiaRESUMO
BACKGROUND AND AIMS: Obesity and diabetes potentiate vascular calcification by increasing vascular smooth muscle cells osteoblastic differentiation mediated by the transcription factor Msx2 and bone morphogenetic protein-2 signaling. However, Bmp-2/Msx2 crosstalk to induce VSMC osteogenic phenotype transition and calcification is poorly understood in diabetes. We aimed to investigate mechanisms underlying Bmp-2-driven VSMC osteogenic differentiation and calcification in leptin-deficient ob/ob mice. METHODS: We incubated VSMC from ob/ob mice and wild type C57BL/6 littermates with or without Bmp-2. We used loss-of-function experiments to investigate the role of Msx2 in Bmp-2-induced ob/ob VSMC osteochondrogenic differentiation and calcification by transfecting Msx2 siRNA into VSMC. RESULTS: Baseline ob/ob VSMC and aorta showed increased Msx2, Runx2, alkaline phosphatase mRNA and protein expression, which further increased in Bmp-2-incubated ob/ob VSMC, therefore augmenting ob/ob VSMC calcification in comparison to wild type VSMC. Accordingly, signaling pathways to induce VSMC osteogenic differentiation, such as Smad1/5 phosphorylation increased in ob/ob versus wild type aorta. In comparison to wild type VSMC, Msx2 siRNA transfected VSMC decreased Bmp-2-dependent osteochondrogenic differentiation response by abrogating Msx2, Runx2, Alpl expression in ob/ob but not in wild type VSMC. Nonetheless, Msx2 inhibition did not decrease calcification in Bmp-2 stimulated ob/ob VSMC in vitro. CONCLUSIONS: Our data support a crucial role of Msx2 for ob/ob VSMC osteochondrogenic differentiation, however, Msx2 signaling alone is not sufficient for ob/ob VSMC calcification after Bmp-2 stimulation in vitro. These findings can be translated into novel perspectives for the understanding of the mechanisms and to provide therapeutic targets underlying vascular calcification in type 2 diabetes.
Assuntos
Doenças da Aorta/metabolismo , Proteínas de Homeodomínio/metabolismo , Resistência à Insulina , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Obesidade/metabolismo , Osteoblastos/metabolismo , Osteogênese , Calcificação Vascular/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 2/genética , Células Cultivadas , Modelos Animais de Doenças , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Resistência à Insulina/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Obesidade/genética , Obesidade/patologia , Osteoblastos/patologia , Fenótipo , Interferência de RNA , Transdução de Sinais , Transfecção , Calcificação Vascular/genética , Calcificação Vascular/patologiaAssuntos
Calcinose , Doenças Cardiovasculares , Pesquisa Translacional Biomédica , Calcificação Vascular , Animais , Biomarcadores/metabolismo , Calcinose/diagnóstico , Calcinose/genética , Calcinose/metabolismo , Calcinose/terapia , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/terapia , Predisposição Genética para Doença , Humanos , Fenótipo , Prognóstico , Fatores de Risco , Transdução de Sinais , Calcificação Vascular/diagnóstico , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/terapiaRESUMO
Vascular calcification in coronary artery disease is gaining importance, both in scientific research and in clinical and imaging applications. The calcified plaque is considered the most relevant form of atherosclerosis within the coronary artery tree and is frequently a challenge for percutaneous intervention. Recent studies showed that plaque calcification is dynamic and is strictly related to the degree of vascular inflammation. Several inflammatory factors produced during the different phases of atherosclerosis induce the expression and activation of osteoblastic cells located within the arterial wall, which, in turn, promote the deposit of calcium. The vascular smooth muscle cells have an extraordinary capacity to undergo osteoblastic phenotypical differentiation. There is no doubt that the role of these factors, as well as the elements of genomics and proteomics, could be a vital strategic point in prevention and treatment. Within this context, we conducted an updating review on coronary calcification focused on pathophysiology, experimental models, and clinical implications of vascular calcification.
Assuntos
Aterosclerose/complicações , Calcificação Vascular/fisiopatologia , Animais , Aterosclerose/metabolismo , Modelos Animais de Doenças , Humanos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Fatores de Risco , Calcificação Vascular/etiologia , Calcificação Vascular/metabolismoRESUMO
A calcificação vascular na doença arterial coronária está ganhando importância, tanto em pesquisas científicas como em aplicações clínicas e de imagem. A placa calcificada é considerada a forma mais relevante de aterosclerose dentro da árvore arterial coronária e frequentemente apresenta um desafio para a intervenção percutânea. Estudos recentes têm demonstrado que a calcificação da placa é dinâmica e está estreitamente ligada ao grau de inflamação vascular. Vários fatores inflamatórios, produzidos durante as diferentes fases da aterosclerose, induzem a expressão e ativação de células osteoblásticas localizadas na parede arterial, que, por sua vez, promovem a deposição de cálcio. As células do músculo liso vascular possuem uma capacidade extraordinária de sofrer diferenciação fenotípica osteoblástica. Não há dúvida de que o papel desses fatores, bem como os elementos de genômica e proteômica, poderia ser um ponto estratégico fundamental na prevenção e no tratamento. Neste contexto, realizamos uma atualização sobre a calcificação coronária, com foco em fisiopatologia, modelos experimentais e implicações clínicas da calcificação vascular.
Vascular calcification in coronary artery disease is gaining importance, both in scientific research and in clinical and imaging applications. The calcified plaque is considered the most relevant form of atherosclerosis within the coronary artery tree and is frequently a challenge for percutaneous intervention. Recent studies showed that plaque calcification is dynamic and is strictly related to the degree of vascular inflammation. Several inflammatory factors produced during the different phases of atherosclerosis induce the expression and activation of osteoblastic cells located within the arterial wall, which, in turn, promote the deposit of calcium. The vascular smooth muscle cells have an extraordinary capacity to undergo osteoblastic phenotypical differentiation. There is no doubt that the role of these factors, as well as the elements of genomics and proteomics, could be a vital strategic point in prevention and treatment. Within this context, we conducted an updating review on coronary calcification focused on pathophysiology, experimental models, and clinical implications of vascular calcification.
Assuntos
Aterosclerose , Isquemia Miocárdica , Insuficiência Renal , Vitamina DRESUMO
Bone morphogenetic protein-2 (BMP-2) increases oxidant stress and endoplasmic reticulum (ER) stress to stimulate differentiation of osteoblasts; however, the role of these signaling pathways in the transition of smooth muscle cells to a calcifying osteoblast-like phenotype remains incompletely characterized. We, therefore, treated human coronary artery smooth muscle cells (HCSMC) with BMP-2 (100ng/mL) and found an increase in NADPH oxidase activity and oxidant stress that occurred via activation of the bone morphogenetic protein receptor 2 and Smad 1 signaling. BMP-2-mediated oxidant stress also increased endoplasmic reticulum (ER) stress demonstrated by increased expression of GRP78, phospho-IRE1α, and the transcription factor XBP1. Analysis of a 1kb segment of the Runx2 promoter revealed an XBP1 binding site; electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that XBP1 bound to the Runx2 promoter at this site in BMP-2-treated HCSMC. Inhibition of oxidant stress or ER stress decreased Runx2 expression, intracellular calcium deposition, and mineralization of BMP-2-treated HCSMC. Thus, in HCSMC, BMP-2 increases oxidant stress and ER stress to increase Runx2 expression and promote vascular smooth muscle cell calcification.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Calcinose/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Vasos Coronários/patologia , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Miócitos de Músculo Liso/patologia , Estresse Oxidativo , Proteína Morfogenética Óssea 2/farmacologia , Calcinose/genética , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
The actions of glucocorticoids are mediated, in part, by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which amplifies their effects at the pre-receptor level by converting cortisone to cortisol. Glucocorticoids, such as dexamethasone, inhibit vascular smooth muscle cell proliferation; however, the role of 11ß-HSD1 in this response remains unknown. Accordingly, we treated human coronary artery smooth muscle cells (HCSMC) with dexamethasone (10(-9)-10(-6) mol/l) and found that after 72 h dexamethasone increased 11ß-HSD1 expression (14.16 ± 1.6-fold, P < 0.001) and activity (6.21 ± 1.2-fold, P < 0.001) in a dose- and time-dependent manner, which was dependent upon glucocorticoid receptor (GR) activation and C/EBPß and C/EBPδ signaling. As glucocorticoids are known to negatively regulate GR expression, we examined the effect of decreasing 11ß-HSD1 expression on GR expression. In HCSMC transfected with 11ß-HSD1 siRNA, GR expression was increased; this effect was associated with protein kinase A activation and CREB phosphorylation. To examine the role of 11ß-HSD1 in HCSMC proliferation, we decreased 11ß-HSD1 expression and stimulated cells with platelet-derived growth factor (PDGF) (10 ng/ml). Decreased 11ß-HSD1 expression was associated with increased cell proliferation in the absence of PDGF compared to scrambled control-transfected cells (236.10 ± 13.11%, n = 4, P < 0.001) and this effect was augmented by PDGF. Furthermore, the inhibitory effect of dexamethasone on cellular proliferation was abrogated in 11ß-HSD1 siRNA-transfected HCSMC. Downregulation of 11ß-HSD1 was associated with decreased p27(kip1) expression and increased phosphorylated retinoblastoma protein, consistent with a proliferative response. These findings suggest that 11ß-HSD1 plays a role in the effects of glucocorticoids on vascular smooth muscle cell phenotype.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Proliferação de Células/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Animais , Western Blotting , Células Cultivadas , Vasos Coronários/enzimologia , Vasos Coronários/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Receptores de Glucocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis. METHODS AND RESULTS: We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet +10(4) IU/d Vit.D2 for 12 weeks. Superoxide and H2O2 microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H2O2 signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol (P < or = 0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA. CONCLUSIONS: Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation.
Assuntos
Antioxidantes/farmacologia , Estenose da Valva Aórtica/patologia , Calcinose/patologia , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/enzimologia , Colesterol na Dieta/administração & dosagem , Modelos Animais de Doenças , Progressão da Doença , Ecocardiografia , Fluorescência , Humanos , Peróxido de Hidrogênio/efeitos adversos , Imuno-Histoquímica , NADPH Oxidases/metabolismo , Oxirredução , Estresse Oxidativo , Probabilidade , Coelhos , Distribuição Aleatória , Espécies Reativas de Oxigênio/efeitos adversos , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Técnicas de Cultura de TecidosRESUMO
Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies.
Assuntos
Vasos Sanguíneos/metabolismo , Etídio/análogos & derivados , NADPH Oxidases/metabolismo , Superóxidos/metabolismo , Animais , Vasos Sanguíneos/enzimologia , Cateterismo , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Etídio/metabolismo , Fluorescência , Artéria Ilíaca/lesões , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/metabolismo , Oxirredução , Peroxidase/metabolismo , Coelhos , Ferimentos e Lesões/enzimologia , Ferimentos e Lesões/etiologiaRESUMO
Accumulating evidence indicates that vascular dysfunction in atherosclerosis, hypertension, and diabetes is either caused by or accompanied by oxidative stress in the vessel wall. In particular, the role of redox processes as mediators of vascular repair and contributors to post-angioplasty restenosis is increasingly evident. Yet the pathophysiology of such complex phenomena is still unclear. After vascular injury, activation of enzymes such as NADPH oxidase leads to a marked increase in superoxide generation, proportional to the degree of injury, which rapidly subsides. Such early superoxide production is significantly greater after stent deployment, as compared to balloon injury. Recent data suggest the persistence of low levels of oxidant stress during the vascular repair reaction in neointimal and medial layers. Despite the compensatory increase in expression of iNOS and nNOS, nitric oxide bioavailability is reduced because of increased reaction rates with superoxide, yielding as by-products reactive nitrogen/oxygen species that induce protein nitration. Concurrently, the activity of vascular superoxide dismutases exhibits a sustained decrease following injury. This decreased activity appears to be a key contributor to vasoconstrictive remodeling and a major determinant of the occurrence of nitrative/oxidative stress. Replenishment of superoxide dismutase (SOD), as well as treatment with vitamins C and E or the lipid-lowering drug probucol and its analogs, led to decrease in constrictive remodeling and improved vessel caliber. Better understanding of the redox pathophysiology of vascular repair should help clarify the pathogenesis of many other vascular conditions and may provide novel therapeutic strategies to prevent vascular lumen loss.