RESUMO
Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFß signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.
Assuntos
Células Endoteliais/citologia , Mesoderma/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Proteína 7 Relacionada à Autofagia , Bleomicina/toxicidade , Linhagem Celular , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Fibrose Pulmonar/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
Autophagy regulates cellular homeostasis and integrates the cellular pro-survival machinery. We investigated the role of autophagy in the natural history of murine abdominal aortic aneurysms (AAA). ApoE(-/-) mice were implanted with saline- or angiotensin II (Ang-II)-filled miniosmotic pumps then treated with either the autophagy inhibitor chloroquine (CQ; 50 mg·(kg body mass)(-1)·day(-1), by intraperitoneal injection) or saline. Ang-II-elicited aneurysmal expansion of the suprarenal aorta coupled with thrombus formation were apparent 8 weeks later. CQ had no impact on the incidence (50% for Ang-II compared with 46.2% for Ang-II + CQ; P = NS) and categorical distribution of aneurysms. The markedly reduced survival rate observed with Ang-II (57.1% for Ang-II compared with 100% for saline; P < 0.05) was unaffected by CQ (61.5% for Ang-II + CQ; P = NS compared with Ang-II). CQ did not affect the mean maximum suprarenal aortic diameter (1.91 ± 0.19 mm for Ang-II compared with 1.97 ± 0.21 mm for Ang-II + CQ; P = NS). Elastin fragmentation, collagen accumulation, and smooth muscle attrition, which were higher in Ang-II-treated mice, were unaffected by CQ treatment. Long-term CQ administration does not affect the natural history and prognosis of experimental AAA, suggesting that global loss of autophagy is unlikely to be a causal factor in the development of aortic aneurysms. Manipulation of autophagy as a mechanism to reduce AAA may need re-evaluation.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/tratamento farmacológico , Cloroquina/farmacologia , Angiotensina II , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Autofagia/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Elastina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.
Assuntos
Apoptose/efeitos dos fármacos , Proteína BRCA2/genética , Doxorrubicina/toxicidade , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Miócitos Cardíacos/patologia , Animais , Antibióticos Antineoplásicos/toxicidade , Proteína BRCA2/deficiência , Insuficiência Cardíaca/epidemiologia , Camundongos , Camundongos Knockout , Miocárdio/patologia , Fenótipo , Fatores de Risco , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: MicroRNA are essential posttranscriptional modulators of gene expression implicated in various chronic diseases. Because microRNA-145 is highly expressed in vascular smooth muscle cells (VSMC) and regulates VSMC fate and plasticity, we hypothesized that it may be a novel regulator of atherosclerosis and plaque stability. METHODS AND RESULTS: Apolipoprotein E knockout mice (ApoE(-/-)) mice were treated with either a microRNA-145 lentivirus under the control of the smooth muscle cell (SMC)-specific promoter SM22α or a SM22α control lentivirus before commencing the Western diet for 12 weeks. The SMC-targeted microRNA-145 treatment markedly reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries. It also significantly increased fibrous cap area, reduced necrotic core area, and increased plaque collagen content. Cellular plaque composition analyses revealed significantly less macrophages in ApoE(-/-) mice treated with the SMC-specific microRNA-145. These mice also demonstrated marked increases in calponin levels and α-smooth muscle actin-positive SMC areas in their atherosclerotic lesions. Furthermore, lentiviral delivery of microRNA-145 resulted in reduced KLF4 and elevated myocardin expression in aortas from ApoE(-/-) mice, consistent with an effect of microRNA-145 to promote a contractile phenotype in VSMC. CONCLUSIONS: VSMC-specific overexpression of microRNA-145 is a novel in vivo therapeutic target to limit atherosclerotic plaque morphology and cellular composition, shifting the balance toward plaque stability vs plaque rupture.
Assuntos
Aterosclerose/prevenção & controle , Terapia Genética , Vetores Genéticos/uso terapêutico , MicroRNAs/fisiologia , Actinas/genética , Animais , Aorta/citologia , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/genética , Aterosclerose/patologia , Artérias Carótidas/metabolismo , Células Cultivadas , Dieta Aterogênica , Genes Reporter , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Lentivirus/genética , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/fisiologia , Transdução GenéticaRESUMO
BACKGROUND: Endothelial dysfunction is an early event in the development and progression of a wide range of cardiovascular diseases. Various human studies have identified that measures of endothelial dysfunction may offer prognostic information with respect to vascular events. Microparticles (MPs) are a heterogeneous population of small membrane fragments shed from various cell types. The endothelium is one of the primary targets of circulating MPs, and MPs isolated from blood have been considered biomarkers of vascular injury and inflammation. CONTENT: This review summarizes current knowledge of the potential functional role of circulating MPs in promoting endothelial dysfunction. Cells exposed to different stimuli such as shear stress, physiological agonists, proapoptotic stimulation, or damage release MPs, which contribute to endothelial dysfunction and the development of cardiovascular diseases. Numerous studies indicate that MPs may trigger endothelial dysfunction by disrupting production of nitric oxide release from vascular endothelial cells and subsequently modifying vascular tone. Circulating MPs affect both proinflammatory and proatherosclerotic processes in endothelial cells. In addition, MPs can promote coagulation and inflammation or alter angiogenesis and apoptosis in endothelial cells. SUMMARY: MPs play an important role in promoting endothelial dysfunction and may prove to be true biomarkers of disease state and progression.
Assuntos
Doenças Cardiovasculares/etiologia , Micropartículas Derivadas de Células/fisiologia , Endotélio Vascular/metabolismo , Apoptose , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Proliferação de Células , Sobrevivência Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Estresse OxidativoRESUMO
BACKGROUND: Adropin is a recently identified protein that has been implicated in the maintenance of energy homeostasis and insulin resistance. Because vascular function and insulin sensitivity are closely related, we hypothesized that adropin may also exert direct effects on the endothelium. METHODS AND RESULTS: In vitro cell culture models were partnered with an in vivo murine injury model to determine the potential vascular effects of adropin. Adropin was expressed in human umbilical vein and coronary artery endothelial cells (ECs). Adropin-treated endothelial cells exhibited greater proliferation, migration and capillary-like tube formation and less permeability and tumor necrosis factor-α-induced apoptosis. In keeping with a vascular protective effect, adropin stimulated Akt Ser(473) and endothelial nitric oxide (NO) synthase Ser(1177) phosphorylation. The former was abrogated in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002, whereas the latter was attenuated by LY294002 and by mitogen-activated protein kinase kinase 1 inhibition with PD98059. Together, these findings suggest that adropin regulates NO bioavailability and events via the phosphatidylinositol 3-kinase-Akt and extracellular signal regulated kinase 1/2 signaling pathways. Adropin markedly upregulated vascular endothelial growth factor receptor-2 (VEGFR2) transcript and protein levels, and in VEGFR2-silenced endothelial cells, adropin failed to induce phosphorylation of endothelial NO synthase, Akt, and extracellular signal regulated kinase 1/2, supporting VEGFR2 as an upstream target of adropin-mediated endothelial NO synthase activation. Last, adropin improved murine limb perfusion and elevated capillary density following induction of hindlimb ischemia. CONCLUSIONS: We report a potential endothelial protective role of adropin that is likely mediated via upregulation of endothelial NO synthase expression through the VEGFR2-phosphatidylinositol 3-kinase-Akt and VEGFR2-extracellular signal regulated kinase 1/2 pathways. Adropin represents a novel target to limit diseases characterized by endothelial dysfunction in addition to its favorable metabolic profile.
Assuntos
Movimento Celular , Proliferação de Células , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Proteínas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
P53 protein levels are elevated by trastuzumab and the biologically similar rat ERBB2/HER2/NEU antibody; and that this coincides with enhanced apoptosis, increased cleaved caspase-3 levels and diminished cardiac function. We also demonstrate that MDM2 may be a regulatory target of anti-ERBB2 thereby implicating the MDM2/p53 axis as a potential molecular component for the undesirable cardiac outcomes noted with trastuzumab. Finally, we show that these MDM2/p53-mediated events are independent of both the ERK1/2 and Akt systems. In conclusion, our findings suggest that the adverse cardiac events observed with trastuzumab may stem from its negative regulation of MDM2 events which impairs p53 degradation resultantly promoting apoptosis leading to cardiac dysfunction. These observations may have important therapeutic implications since they suggest that anticancer agents that inhibit MDM2 and its downstream actions may curb tumor progression at the expense of increasing cardiac stress.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Coração/efeitos dos fármacos , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Trastuzumab , Proteína Supressora de Tumor p53/biossínteseRESUMO
Altered macrophage kinetics is a pivotal mechanism of visceral obesity-induced inflammation and cardiometabolic risk. Because monocytes can differentiate into either proatherogenic M1 macrophages or anti-inflammatory M2 macrophages, approaches that limit M1 while promoting M2 differentiation represent a unique therapeutic strategy. We hypothesized that adiponectin may prime human monocytes toward the M2 phenotype. Adiponectin promoted the alternative activation of human monocytes into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Adiponectin-treated cells displayed increased M2 markers, including the mannose receptor (MR) and alternative macrophage activation-associated CC chemokine-1. Incubation of M1 macrophages with adiponectin-treated M2-derived culture supernatant resulted in a pronounced inhibition of tumor necrosis factor-alpha and monocyte chemotactic protein-1 secretion. Activation of human monocytes into M2 macrophages by adiponectin was mediated, in addition to AMP-activated protein kinase and peroxisome proliferator-activated receptor (PPAR)-gamma, via PPAR-alpha. Furthermore, macrophages isolated from adiponectin knockout mice demonstrated diminished levels of M2 markers such as MR, which were restored with adiponectin treatment. We report a novel immunoregulatory mechanism through which adiponectin primes human monocyte differentiation into anti-inflammatory M2 macrophages. Conditions associated with low adiponectin levels, such as visceral obesity and insulin resistance, may promote atherosclerosis, in part through aberrant macrophage kinetics.
Assuntos
Adiponectina/metabolismo , Diferenciação Celular/fisiologia , Ativação de Macrófagos , Macrófagos/metabolismo , Monócitos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adiponectina/genética , Adiponectina/farmacologia , Análise de Variância , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Monócitos/efeitos dos fármacos , PPAR gama/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Conventional haemodialysis (CHD) is associated with a reduction in vascular smooth muscle cells (VSMCs) proliferation and an increase in apoptosis. METHODS: VSMC proliferation, migration, apoptosis and Runx2 expression were assessed under normal conditions (n = 4) and before and after conversion from CHD to NHD (n = 15). RESULTS: Compared to normal, CHD is associated with a reduction in VSMC proliferation [0.49 +/- 0.07 (CHD) versus 1.34 +/- 0.02 RFU, P < 0.01], an augmented caspase-3 activity [0.30 +/- 0.02 (CHD) versus 0.22 +/- 0.02 RFU, P = 0.014] and a 1.4 +/- 0.3 fold increase in Runx2 expression. After conversion to NHD, VSMC proliferation was higher than during CHD [from 0.49 +/- 0.07 (CHD) to 0.68 +/- 0.09 RFU, P = 0.006] and approached that of controls (1.34 +/- 0.02, P > 0.05). Caspase-3 activity was restored to similar values as controls [0.26 +/- 0.02 (NHD) versus 0.22 +/- 0.04 (normal), P > 0.05]. Runx2 expression decreased to similar levels as normal controls. NHD enhanced dialysis dose delivery, lowered blood pressure, plasma parathyroid hormone levels and normalized plasma phosphate (from 1.7 +/- 0.1 to 1.2 +/- 0.1 mmol/L, P < 0.01). The reduction in plasma phosphate correlated with the change in VSMC proliferation (r = -0.71, P = 0.007). CONCLUSIONS: We demonstrate that NHD is associated with restoration of abnormal VSMC biology in ESRD. Given the increasing importance of VSMCs in the pathogenesis of atherosclerosis and medial calcification, these data may have important implications for vascular risk in ESRD patients.
Assuntos
Falência Renal Crônica/terapia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Diálise Renal/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: Monocyte-derived microparticles (mono-MPs) are emerging as critical transducers of inflammatory signals, and have been suggested to link cardiovascular risk factors to vascular injury. Since adiponectin has been proposed to exert multiple anti-inflammatory and vasculoprotective effects, we hypothesized that it might serve to limit the production and/or action of mono-MPs. METHODS: Flow cytometry and western blot studies were conducted on THP-1 cells, THP-1-derived MPs, human umbilical vein endothelial cells (HUVECs), peripheral blood CD14+ monocytes and mice to evaluate the effects of adiponectin on mono-MPs. RESULTS: Adiponectin attenuated lipopolysaccharide (LPS)-evoked MP release from THP-1 monocytes (30% difference) and peripheral blood monocytes (both P < 0.05) as well as dampened LPS-induced mono-MP generation in vivo. Furthermore, peritoneal monocytes from Adipoq(-/-) mice generated significantly greater MPs than those from Adipoq(+/+) littermates in the absence (2.3 fold difference, P < 0.05) and presence (1.6 fold difference, P < 0.05) of LPS. LPS-induced MP expression of NLRP3 inflammasome and its key components, namely cleaved ASC, caspase-1 and IL-1ß (pro- and cleaved), were markedly attenuated by adiponectin. HUVECs incubated with MPs from LPS-treated THP-1 cells exhibited increased VCAM-1 levels and adhesion to THP-1 cells. Adiponectin abrogated these effects. From a mechanistic standpoint, the effects of adiponectin on MP release and molecular signaling occurred at least in part through the AMPK, Akt and NFκB pathways. CONCLUSION: Adiponectin exerts novel effects to limit the production and action of mono-MPs, underscoring yet another pleiotropic effect of this adipokine.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Adiponectina/genética , Aterosclerose/genética , Regulação da Expressão Gênica , NF-kappa B/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA/genética , Proteínas Quinases Ativadas por AMP/biossíntese , Adiponectina/biossíntese , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Humanos , Inflamassomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Monócitos/metabolismo , Monócitos/ultraestrutura , NF-kappa B/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Transdução de SinaisRESUMO
The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular (125) I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE(-/-) mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age-dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.
Assuntos
Autofagia/genética , Endotélio Vascular/metabolismo , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipoproteínas LDL/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Homeostase/genética , Metabolismo dos Lipídeos/genética , Lipoproteínas LDL/genética , Camundongos Knockout , Oxirredução , Epitélio Pigmentado da Retina/metabolismoRESUMO
Obesity is a multifactorial chronic disease characterized by an accumulation of visceral and subcutaneous fat, which leads to a predisposition toward cardiometabolic diseases. A plethora of mechanisms, including abnormalities in lipid metabolism, insulin resistance, inflammation, endothelial dysfunction, adipokine imbalance, and inflammasome activation have been suggested to underlie the relationship between obesity and atherosclerosis. More recent data point toward an emerging role of impaired autophagy and altered gut microbiome homeostasis as potentially contributing factors. This review provides an overview of this area.
Assuntos
Tecido Adiposo/metabolismo , Aterosclerose/etiologia , Metabolismo Energético , Obesidade/complicações , Aterosclerose/metabolismo , Homeostase , Humanos , Obesidade/metabolismoRESUMO
BACKGROUND: Angiotensin II and endothelin-1 are potent endothelium-derived contracting factors. The effects of acute endothelin antagonism on endothelial function in saphenous vein from patients treated with and without angiotensin-converting enzyme inhibitors were compared. METHODS: Vascular segments of saphenous vein were obtained perioperatively from 14 patients on angiotensin-converting enzyme inhibitors and 29 controls. In vitro endothelium-dependent and -independent responses to acetylcholine and sodium nitroprusside were assessed by constructing isometric dose-response curves in precontracted rings in the presence and absence of bosentan (endothelinA/B receptor antagonist) and BQ-123 (endothelinA antagonist) using isolated organ baths. Percent maximum relaxation and sensitivity were compared between interventions. RESULTS: Endothelium-dependent relaxation to acetylcholine was augmented in the angiotensin-converting enzyme inhibitor-treated group (p < 0.005). Both specific and mixed endothelin receptor blockade improved acetylcholine-mediated relaxation in the angiotensin-converting enzyme inhibitor-treated and untreated groups (p < 0.02). The effects of these antagonists were endothelium specific as endothelium-independent responses to sodium nitroprusside remain unaltered. CONCLUSIONS: These data demonstrate that (1) chronic angiotensin-converting enzyme inhibition improves endothelial function in saphenous veins, and (2) this effect can be further augmented by acute endothelin blockade. These data suggest that antagonism of both angiotensin II and endothelin may be important in attenuating saphenous vein arteriosclerosis.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antagonistas dos Receptores de Endotelina , Endotélio Vascular/fisiologia , Veia Safena/fisiologia , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Angiotensina II/fisiologia , Bosentana , Ponte de Artéria Coronária , Relação Dose-Resposta a Droga , Endotelina-1/fisiologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Nitroprussiato/farmacologia , Peptídeos Cíclicos/farmacologia , Veia Safena/efeitos dos fármacos , Sulfonamidas/farmacologia , Vasoconstrição/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
OBJECTIVE: Autophagy is an evolutionary conserved adaptive response that is believed to promote cell survival in response to stressful stimuli via recycling of precursors derived from the degradation of endogenous cellular components. The autophagic molecular machinery is controlled by a large family of autophagy-related genes (ATGs) and downstream regulators. We sought to define the autophagy gene fingerprint associated with human ischemia and reperfusion (IR) injury using an intraoperative model developed by Sellke and colleagues. METHODS: Right atrial appendages, collected from human hearts before and after cardioplegic arrest and after reperfusion, were submitted for polymerase chain reaction (PCR) array, quantitative real-time PCR, and immunoblot analysis for autophagy proteins and their associated upstream regulators. RESULTS: Perioperative IR significantly upregulated 11 (13.1%) and downregulated 3 (3.6%) of 84 ATGs. Specifically, there were increases in the autophagy machinery components ATG4A, ATG4C, and ATG4D; tumor necrosis factor-related apoptosis-inducing ligand, MAPK8 and BCL2L1; and chaperone-mediated autophagy activity with increased heat shock protein (HSP) A8, HSP90AA1, and a-synuclein. Autophagy activity was confirmed through observations of higher LC3-I levels and an increase in the LC3-II/LC3-I ratio. Autophagy activation coincided with increased AMPK activation and decreased protein levels of the mammalian target of rapamycin, the latter a key negative regulator of autophagy. CONCLUSIONS: We provide the first human cardiac fingerprint of autophagy gene expression in response to IR. These findings may inform on appropriate cell- and gene-based therapeutic approaches to limit aberrant cardiac injury.
Assuntos
Apêndice Atrial/patologia , Autofagia/genética , Perfilação da Expressão Gênica , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Apoptose/genética , Apêndice Atrial/metabolismo , Apêndice Atrial/cirurgia , Ponte Cardiopulmonar , Ponte de Artéria Coronária , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Parada Cardíaca Induzida , Humanos , Masculino , Pessoa de Meia-Idade , Traumatismo por Reperfusão Miocárdica/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Excessive production of reactive oxygen species (ROS), in part via upregulation of DNA damage pathways, is a central mechanism governing pathologic activation of vascular smooth muscle cells (VSMCs). We hypothesized that the breast cancer 1, early onset (BRCA1) gene that is involved in cellular resistance to DNA damage limits ROS production and oxidative stress in VSMCs. METHODS: We evaluated basal and H2O2-stimulated expression of BRCA1 in human aortic smooth muscle cells (HASMCs). In vitro gain-of-function experiments were performed in BRCA1 adenovirus (Ad-BRCA1)-transfected HASMCs. ROS production and expression of Nox1 and its key regulatory subunit p47phox, key components of the ROS-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, were evaluated. In vivo gain-of-function experiments were performed in spontaneously hypertensive (SHR) rats treated with Ad-BRCA1 (5 × 10(10) IU/rat). Blood pressure, vascular ROS generation, Nox1, and p47phox expression were measured. RESULTS: BRCA1 was constitutively expressed in murine, rat, and human smooth muscle cells (SMCs). H2O2 significantly reduced BRCA1 expression with a resultant increase in ROS generation. BRCA1-overexpressing HASMCs were protected against H2O2-induced ROS generation, in part, via downregulation of the ROS-producing NADPH oxidase subunits Nox1 and p47phox. Ad-BRCA1 treatment in SHR rats was associated with a sustained increase in aortic BRCA1 expression, lower aortic ROS production, reduced γH2A.X levels, greater RAD51 foci, and decreases in blood pressure. CONCLUSIONS: BRCA1 is a novel and previously unrecognized target that may shield VSMCs from oxidative stress by inhibiting NADPH Nox1-dependent ROS production. Gene- and/or cell-based approaches that improve BRCA1 bioavailability may represent a new approach in the treatment of diverse vascular diseases associated with an aberrant VSMC phenotype.
Assuntos
Proteína BRCA1/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteína BRCA1/genética , Pressão Sanguínea , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Terapia Genética/métodos , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertensão/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidases/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/metabolismo , Rad51 Recombinase/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , TransfecçãoRESUMO
OBJECTIVE: Neointimal hyperplasia secondary to vascular smooth muscle cell (VSMC) activation limits the long-term patency of saphenous vein grafts (SVGs). We compared markers of vascular injury and VSMC activation in SVGs harvested using the pedicled 'no-touch' (NT) vs the conventional (CON) technique. METHODS: Patients undergoing coronary artery bypass surgery were enrolled in the PATENT SVG trial (clinicaltrials.gov NCT01488084). Patients were randomly allocated to have SVGs harvested with the NT technique from one leg and the CON method from the other. SVG segments underwent morphometry, histological and electron microscopy assessments and transcript measurements of VSMC activation and differentiation markers. Leg wound functional recovery and harvest site complications were assessed using a quality-of-life questionnaire. RESULTS: A total of 17 patients (65.3 ± 7.3 years) were enrolled. SVGs harvested using the NT vs CON technique exhibited preserved intimal, medial and adventitial architecture. CON harvest was associated with greater medial Kruppel-like factor 4 transcript levels (0.26 ± 0.05 vs 0.11 ± 0.02, P < 0.05). CON samples had significantly lower medial serum response factor (0.53 ± 0.11 vs 1.44 ± 0.50, P < 0.05) and myocardin (0.59 ± 0.08 vs 1.33 ± 0.33, P < 0.05) transcript levels. MicroRNA-145, an inhibitor of VSMC activation and differentiation, was higher in the NT vs CON samples (1.84 ± 1.03 vs 0.50 ± 0.19, P < 0.05). Leg assessment scores were worse in the NT legs at 3 months, but similar to CON scores at 12 months. CONCLUSIONS: SVGs harvested using the 'NT' technique exhibit an early molecular and morphological pattern consistent with decreased VSMC activation compared with CON harvesting. Functional leg recovery was similar in both groups at 12 months. Larger studies are required to corroborate these findings.
Assuntos
Ponte de Artéria Coronária/métodos , Músculo Liso Vascular/patologia , Veia Safena/patologia , Veia Safena/transplante , Coleta de Tecidos e Órgãos/métodos , Idoso , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Veia Safena/química , Veia Safena/citologia , Retalhos Cirúrgicos/patologiaRESUMO
OBJECTIVE: Alterations in cardiac energy and substrate metabolism play a critical role in the development and clinical course of heart failure. We hypothesized that the cardioprotective role of the breast cancer 1, early onset (BRCA1) gene might be mediated in part by alterations in cardiac bioenergetics. METHODS: We generated cardiomyocyte-specific BRCA1 homozygous and heterozygous knockout mice using the Cre-loxP technology and evaluated the key molecules and pathways involved in glucose metabolism, fatty acid metabolism, and mitochondrial bioenergetics. RESULTS: Cardiomyocyte-specific BRCA1-deficient mice showed reduced cardiac expression of glucose and fatty acid transporters, reduced acetyl-coenzyme A carboxylase 2 and malonyl-coenzyme A decarboxylase (key enzymes that control malonyl coenzyme A, which in turn controls fatty acid oxidation), and reduced carnitine palmitoyltransferase I, a rate-limiting enzyme for mitochondrial fatty acid uptake. Peroxisome proliferator-activated receptor α and γ and carnitine palmitoyltransferase I levels were also downregulated in these hearts. Rates of glucose and fatty acid oxidation were reduced in the hearts of heterozygous cardiomyocyte-restricted BRCA1-deficient mice, resulting in a decrease in the rate of adenosine triphosphate production. This decrease in metabolism and adenosine triphosphate production occurred despite an increase in 5'-adenosine monophosphate-activated protein kinase and AKT activation in the heart. CONCLUSIONS: Cardiomyocyte-specific loss of BRCA1 alters critical pathways of fatty acid and glucose metabolism, leading to an energy starved heart. BRCA1-based cell or gene therapy might serve as a novel target to improve cardiac bioenergetics in patients with heart failure.
Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Reparo do DNA , Metabolismo Energético , Miócitos Cardíacos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Carboxiliases/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Heterozigoto , Homozigoto , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: BRCA1, a tumor suppressor gene implicated in breast and ovarian cancers, exerts multiple effects on DNA repair and affords resistance against cellular stress responses. We hypothesized that BRCA1 limits endothelial cell apoptosis and dysfunction, and via this mechanism attenuates atherosclerosis. METHODS: Loss and gain of function were achieved in cultured endothelial cells by silencing and overexpressing BRCA1, respectively. In vivo loss and gain of function were performed by generating endothelial cell-specific knockout (EC-BRCA1(-/-)) mice and administering a BRCA1 adenovirus. Well-established cell and animal models of angiogenesis and atherosclerosis were used. RESULTS: BRCA1 is basally expressed in endothelial cells. BRCA1 overexpression protected and BRCA1 silencing exaggerated inflammation- and doxorubicin-induced endothelial cell apoptosis. Key indices of endothelial function were modulated in a manner consistent with an effect of BRCA1 to limit endothelial cell apoptosis and improve endothelial function. BRCA1 overexpression strongly attenuated the production of reactive oxygen species and upregulated endothelial nitric oxide synthase, phosphorylated endothelial nitric oxide synthase, phosphorylated Akt, and vascular endothelial growth factor-a expression. BRCA1 overexpression also improved capillary density and promoted blood flow restoration in mice subjected to hind-limb ischemia. BRCA1-overexpressing ApoE(-/-) mice fed a Western diet developed significantly less aortic plaque lesions, exhibited reduced macrophage infiltration, and generated less reactive oxygen species. Lung sections and aortic segments from EC-BRCA1(-/-) mice demonstrated greater inflammation-associated apoptosis and impaired endothelial function, respectively. BRCA1 expression was attenuated in the plaque region of human atherosclerotic carotid artery samples compared with the adjacent plaque-free area. CONCLUSIONS: These data collectively highlight a previously unrecognized role of BRCA1 as a gatekeeper of inflammation-induced endothelial cell function and a target to limit atherosclerosis. Translational studies evaluating endothelial function and atherosclerosis in individuals with BRCA1 mutations are suggested.
Assuntos
Aterosclerose/prevenção & controle , Proteína BRCA1/metabolismo , Células Endoteliais/metabolismo , Terapia Genética/métodos , Isquemia/terapia , Músculo Esquelético/irrigação sanguínea , Adenoviridae/genética , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apoptose , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Proteína BRCA1/genética , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Vetores Genéticos , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Bone morphogenetic protein-2 (BMP-2) is a major regulator of aortic valve calcification. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression and miRNA-141 is a known repressor of BMP-2-mediated osteogenesis. We hypothesized that miRNA-141 is a key regulator of aortic valve calcification. METHODS: Porcine valvular interstitial cells were isolated, transfected with miRNA-141 or control, and stimulated with transforming growth factor-ß. The BMP-2, extracellular signal-regulated kinase 1/2, and runt-related transcription factor 2 levels were determined by immunoblotting and reverse transcriptase polymerase chain reaction. To determine the role of miRNA-141 in bicuspid aortic valve disease, human bicuspid (n = 19) and tricuspid (n = 17) aortic valve leaflets obtained intraoperatively were submitted for GenoExplorer human microRNA array, immunoblotting, and histologic and immunohistochemical analyses. RESULTS: Stimulation of porcine aortic valvular interstitial cells with transforming growth factor-ß induced morphologic alterations consistent with myofibroblastic transformation, BMP-2 signaling, and calcification. Transfection with miRNA-141 restored transforming growth factor-ß-induced valvular interstitial cell activation, BMP-2 signaling, and alkaline phosphatase activity (3.55 ± 0.18 vs 4.01 ± 0.21, P < .05), suggesting upstream regulation by miRNA-141. miRNA microarray demonstrated differential expression of 35 of 1583 miRNA sequences in the bicuspid versus tricuspid aortic valve leaflets, with a 14.5-fold decrease in miRNA-141 in the bicuspid versus tricuspid leaflets (P < .05). This was associated with significantly increased BMP-2 protein expression in bicuspid aortic valve compared with the tricuspid aortic valve leaflets (P < .001). CONCLUSIONS: We report a completely novel role of miRNA-141 as a regulator of BMP-2-dependent aortic valvular calcification and demonstrate marked attenuation of miRNA-141 expression in patients with bicuspid aortic valve-associated aortic stenosis. Therapeutic targeting of miRNA-141 could serve as a novel strategy to limit progressive calcification in aortic stenosis.