RESUMO
Recent evidence indicates that an imbalance between cardiomyocyte hypertrophy and blood vessel growth in the remote myocardium may contribute to heart failure in ischaemic heart disease. It remains, however, largely unknown which angiogenic factors are capable of stimulating vessel growth in the remote myocardium after myocardial infarction (MI) and whether systemic, rather than local, administration of such factors suffices to ameliorate post-MI cardiac recovery. We therefore analysed the effect of systemic placental growth factor (PlGF) delivery on myocardial recovery post-MI in mice. MI was induced by permanent ligation of the left anterior descending coronary (LAD) artery in C57Bl6/J mice, followed by systemic injection of a PlGF adenovirus, resulting in elevated circulating levels of PlGF for 4 weeks. Functional and morphological analysis revealed that PlGF treatment induced cardiomyocyte hypertrophy and improved cardiac recovery at day 28 post-MI. PlGF stimulated angiogenesis in the infarct border and vessel enlargement in the remote myocardium. In this mouse model, capillary-to-cardiomyocyte ratios in the remote myocardium were maintained post-MI, but PlGF increased the vascular perfusion area in balance with the cardiomyocyte hypertrophy. Overall, systemic delivery of PlGF improves cardiac performance and promotes adaptive remodelling of the post-MI heart.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Proteínas da Gravidez/genética , Análise de Variância , Animais , Vasos Coronários/patologia , Ecocardiografia , Feminino , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Infarto do Miocárdio/patologia , Miocárdio/patologia , Fator de Crescimento Placentário , Tempo , Transdução Genética/métodosRESUMO
Essentials HDL subclasses were studied in acute coronary syndrome (ACS). HDL2 from ACS patients have better antiplatelet potency than HDL from non ACS subjects. ACS remodels the antiplatelet properties of HDL subclasses. Oxidized polyunsaturated fatty acids content of HDL is modified by ACS. SUMMARY: Background Although HDLs have antithrombotic effects by reducing platelet activation, the relationship between HDL levels and the risk of acute coronary syndrome (ACS) is unclear, as HDL particles are heterogeneous in composition and biological properties. Objective To characterize the effects of HDL2 and HDL3 subclasses from ACS patients and non-coronary artery disease (CAD) subjects on platelet activation. Methods We measured platelet aggregation and ex vivo thrombus formation, analyzed signaling pathways by flow cytometry, and performed a targeted lipidomics analysis on HDL subclasses. Results Analysis of human platelet aggregation in suspension, adhesion on von Willebrand factor and thrombus formation on collagen under arterial shear demonstrated that HDL2 from ACS patients had higher antiplatelet potency than HDL3 from ACS patients and HDL from non-CAD subjects. HDL binding to scavenger receptor class B type I was essential for this effect. A lipidomics analysis revealed that HDL2 from ACS patients had more oxidized polyunsaturated fatty acids (PUFAs). An inverse correlation between the concentrations of 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), the eicosapentaenoic acid metabolite 18-hydroxyeicosapentaenoic acid (18-HEPE) and hydroxyeicosatetraenoic acid isomers in HDL2 and platelet aggregation was observed. This relationship was further demonstrated by the direct inhibitory effects of 18-HEPE, 9-HODE, 13-HODE, 17-hydroxydocosahexaenoic acid and 14-hydroxydocosahexaenoic acid on collagen-related peptide-induced platelet aggregation, indicating that oxidized PUFAs contribute to the antithrombotic effect of ACS HDL2. Conclusions Our data shed new light on the antiplatelet effects of HDL subclasses, and suggest physiological adaptation through the modulation of HDL properties in ACS patients that may limit their platelet-dependent thrombotic risk.
Assuntos
Síndrome Coronariana Aguda/sangue , Plaquetas/metabolismo , Ácidos Graxos Insaturados/sangue , Lipoproteínas HDL/sangue , Agregação Plaquetária , Trombose/sangue , Síndrome Coronariana Aguda/diagnóstico , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Adesividade Plaquetária , Receptores Depuradores Classe B/sangue , Transdução de Sinais , Trombose/diagnóstico , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Periodontopathogens antibodies have been shown to be associated with primary myocardial events, but little is known regarding their impact on major adverse events after a prior acute myocardial infarction (AMI). The present prospective study evaluates the association between antibody levels of 4 periodontopathogens and the risk of all-cause death or non-fatal myocardial infarction (MI) at 1â year in 975 patients admitted for acute ST segment or non-ST segment elevation MI in French Registry of Acute ST-Elevation and Non-ST-Elevation Myocardial Infarction (FAST-MI), a nationwide French survey. METHODS: Multiserotype ELISAs were performed to assess levels of IgG and IgA against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Tannerella forsythia. RESULTS: Adjusted HRs indicate the lack of association between IgG-anti-Po. gingivalis levels (0.96 (0.78 to 1.18)), IgA-anti-Po. gingivalis levels (1.13 (0.90 to 1.42)) and the risk of all-cause death or non-fatal MI at 1â year. Additionally, no significant association was found between the occurence of an event at 1 year and immunoglobulins levels against the others periodontopathogens. CONCLUSIONS: The present data indicate that circulating levels of periodontopathogens antibodies are not associated with an increased risk of major adverse events in patients with a prior AMI. Studies dealing with bacterial and clinical data are needed to assess the role of oral health in comprehensive cardiac rehabilitation programmes.
RESUMO
The mechanism(s) by which exercise reduces atherogenic risk remains unknown. This study tested the hypothesis that sustained exercise-induced oxidative stress may increase antioxidant defense in the arterial wall. Acute exercise induced an increase in antibodies to oxidatively modified proteins and catalase in the aortic walls of normal mice compared with sedentary control mice. In male atherogenic diet-fed low density lipoprotein (LDL) receptor-deficient mice, exercise lowered plasma cholesterol (15%) and decreased atherosclerotic lesions by 40% compared with values in sedentary control mice, with a concomitant increase in arterial catalase and endothelial NO synthase. Because these mice lack the LDL receptor, the results indicate that the LDL receptor might not be responsible for the exercise-induced lowering of plasma cholesterol. Vitamin E supplementation to exercising LDL receptor-deficient mice did not reduce atherosclerotic lesion formation significantly as opposed to lesion formation in untreated exercised mice. Moreover, vitamin E counteracted the beneficial effects of exercise by preventing the induction of aortic catalase activity and endothelial NO synthase expression. These results might indicate that although vitamin E might have prevented the exercise-induced oxidative stress, its availability in the artery was insufficient to prevent the atherosclerotic process. These results indicate that exercise-induced plasma oxidative stress could be responsible for the prevention of atherosclerosis by stimulating arterial antioxidant response. Furthermore, vitamin E supplementation could be deleterious in exercisers by inhibiting antioxidant enzyme buildup in the arterial wall.
Assuntos
Artérias/enzimologia , Arteriosclerose/enzimologia , Estresse Oxidativo , Condicionamento Físico Animal , Animais , Antioxidantes/metabolismo , Artérias/efeitos dos fármacos , Arteriosclerose/imunologia , Arteriosclerose/patologia , Autoanticorpos/biossíntese , Catalase/biossíntese , Colesterol/sangue , Dieta Aterogênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Receptores de LDL/genética , Vitamina E/farmacologiaRESUMO
In this work, two biosensors were developed for the detection of High-Density Lipoproteins (HDL) particles, which are biomarkers inversely correlated with cardiovascular risk and which represent therapeutic targets for atherosclerosis. The electrochemical properties of the grafted antibody on interdigitated gold electrode were achieved by Impedance Spectroscopy (IS). The used deposition method was based on oriented antibody Anti-ApoA1 with an intermediate thin layer of protein G. The developed biosensor was able to detect both native plasma HDL and reconstituted HDL (rHDL) particles respectively with the detection limit of 50n g/mL and 1 ng/mL, respectively. Dynamic contact angle and atomic force microscopy were used. The developed biosensors are able to differentiate the HDL particles according to their differences in size and interactions with the immobilized antibody.
Assuntos
Técnicas Biossensoriais , Lipoproteínas HDL/análise , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Antígenos/imunologia , Apolipoproteína A-I/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Eletrodos , Ouro/química , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Lipoproteínas HDL/sangue , Lipoproteínas HDL/imunologia , Microscopia de Força Atômica , Soroalbumina Bovina/químicaRESUMO
1. Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2). 2. Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium-dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL. 3. Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration-dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3+/-2.1 micromol l[-1]) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4+/-3.2, 14.1+/-4.1, 20.4+/-4.4 and 72.6+/-9.2 micromol l(-1) for ferulic, protocatechuic, ellagic and p-coumaric acids, respectively). 4. The anti-apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity ('indirect' protective effect); (ii) a 'direct' cytoprotective effect, acting at the cellular level. 5. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 6. In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL-induced apoptosis.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Dieta , Endotélio Vascular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Fenóis/farmacologia , Antioxidantes/administração & dosagem , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Endotélio Vascular/citologia , Humanos , Metamioglobina/farmacologia , Fenóis/administração & dosagem , Raios UltravioletaRESUMO
The atherogenic oxidative modification of low-density lipoprotein is suggested to occur in the aortic intima. There is reasonable evidence to suggest that antioxidants might be beneficial in preventing or retarding the progression of atherosclerosis. Exercise, estrogens, and substitution of polyunsaturated fat for saturated fat are beneficial in the prevention of atherosclerosis. Yet, paradoxically, they are capable of inducing an oxidative stress. To reconcile with this paradox, we postulate that under certain conditions an oxidative stress might be beneficial by inducing antioxidant enzymes in arterial cells. However, those with genetic deficiency in antioxidant enzymes or those who poorly respond to oxidative stress or those with overwhelming plasma oxidative stress might need additional antioxidant protection.
Assuntos
Antioxidantes , Lipídeos/sangue , Lipoproteínas/sangue , Estresse Oxidativo , Animais , Artérias/metabolismo , Arteriosclerose , Gorduras Insaturadas na Dieta , Estrogênios , Exercício Físico , Humanos , Lipoproteínas LDL/sangue , Lipoproteínas LDL/metabolismoRESUMO
Iron deposits are observed in tissue of abdominal aortic aneurysm (AAA) patients, although the underlying mechanisms are not completely elucidated. Therefore we explored circulating markers of iron metabolism in AAA patients, and tested if they could serve as biomarkers of AAA. Increased red blood cell (RBC)-borne iron retention and transferrin, transferrin receptor and ferritin expression was observed in AAA tissue compared to control aorta (immunohistochemistry and western blot). In contrast, decreased circulating iron, transferrin, mean corpuscular haemoglobin concentration (MCHC) and haemoglobin concentration, along with circulating RBC count, were observed in AAA patients (aortic diameter >3 cm, n=114) compared to controls (aortic diameter <3 cm, n=88) (ELISA), whereas hepcidin concentrations were increased in AAA subjects (MS/MS assay). Moreover, iron, transferrin and haemoglobin levels were negatively, and hepcidin positively, correlated with aortic diameter in AAA patients. The association of low haemoglobin with AAA presence or aortic diameter was independent of specific risk factors. Moreover, MCHC negatively correlated with thrombus area in another cohort of AAA patients (aortic diameter 3-5 cm, n=357). We found that anaemia was significantly more prevalent in AAA patients (aortic diameter >5 cm, n=8,912) compared to those in patients with atherosclerotic aorto-iliac occlusive disease (n=17,737) [adjusted odds ratio=1.77 (95% confidence interval: 1.61;1.93)]. Finally, the mortality risk among AAA patients with anaemia was increased by almost 30% [adjusted hazard ratio: 1.29 (95% confidence interval: 1.16;1.44)] as compared to AAA subjects without anaemia. In conclusion, local iron retention and altered iron recycling associated to high hepcidin and low transferrin systemic concentrations could lead to reduced circulating haemoglobin levels in AAA patients. Low haemoglobin levels are independently associated to AAA presence and clinical outcome.
Assuntos
Anemia/diagnóstico , Aorta/metabolismo , Aneurisma da Aorta Abdominal/diagnóstico , Biomarcadores/metabolismo , Eritrócitos/fisiologia , Hemoglobinas/metabolismo , Ferro/metabolismo , Idoso , Anemia/complicações , Anemia/mortalidade , Aorta/patologia , Aneurisma da Aorta Abdominal/complicações , Aneurisma da Aorta Abdominal/mortalidade , Feminino , Ferritinas/metabolismo , Hepcidinas/metabolismo , Humanos , Masculino , Prognóstico , Receptores da Transferrina/metabolismo , Fatores de Risco , Análise de Sobrevida , Transferrina/metabolismoRESUMO
High-density lipoproteins (HDLs) represent a family of particles characterized by the presence of apolipoprotein A-I (apoA-I) and by their ability to transport cholesterol from peripheral tissues back to the liver. In addition to this function, HDLs display pleiotropic effects including antioxidant, anti-apoptotic, anti-inflammatory, anti-thrombotic or anti-proteolytic properties that account for their protective action on endothelial cells. Vasodilatation via production of nitric oxide is also a hallmark of HDL action on endothelial cells. Endothelial cells express receptors for apoA-I and HDLs that mediate intracellular signalling and potentially participate in the internalization of these particles. In this review, we will detail the different effects of HDLs on the endothelium in normal and pathological conditions with a particular focus on the potential use of HDL therapy to restore endothelial function and integrity.
Assuntos
Endotélio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Modelos Biológicos , Receptores de Lipoproteínas/metabolismo , Vasculite/metabolismo , Animais , Apoptose , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Angiopatias Diabéticas/imunologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Angiopatias Diabéticas/terapia , Sistemas de Liberação de Medicamentos , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/uso terapêutico , Lisofosfolipídeos , Esfingosina/análogos & derivados , Vasculite/imunologia , Vasculite/fisiopatologia , Vasculite/terapiaRESUMO
OBJECTIVE: Cytokines are important mediators of immune-inflammatory responses implicated in abdominal aortic aneurysm (AAA) pathogenesis. Our objective was to investigate the cytokine expression profile in plasma of AAA patients. METHODS: Cytokine protein expression was measured in plasma of 5 large AAA patients (aortic size >50mm) and 5 controls (aortic size <30 mm) using a 20-cytokine antibody-based protein array. IGFBP-1 plasma concentrations were analyzed by ELISA. IGFBP-1 protein levels were analyzed in AAA thrombus by immunohistochemistry and Western blot. Platelet aggregation was assessed by conventional optical aggregometry. RESULTS: Several proteins including MIP-3 alpha (CCL20), Eotaxin-2 and IGFBP-1 were increased in AAA patients compared to controls. Among them, IGFBP-1 concentrations were significantly higher in large AAA patients vs control subjects. These data were validated in plasma of patients with large AAA (n = 30) compared to matched controls (n = 30) [834(469-1628) vs 497(204-893) pg/ml, p<0.01]. Furthermore, the potential association of IGFBP-1 with AAA size was analyzed in a second independent group of subjects [large AAA (n = 59), small AAA patients (aortic size = 30-50mm, n = 54) and controls (n = 30)]. Interestingly, IGFBP-1 levels correlated with AAA size (r = 0.4, p<0.001), which remained significant after adjusting for traditional risk factors. IGFBP-1 was localized in the luminal part of AAA thrombus and IGFBP-1 levels were increased in AAA thrombus conditioned media compared to media layer and healthy media. Interestingly, IGFBP-1 abrogated the potentiation of ADP-induced platelet aggregation triggered by IGF-1. CONCLUSIONS: IGFBP-1 has been identified by a protein array approach as a potential novel biomarker of AAA. The biological role of IGFBP-1 in AAA pathogenesis could be related to the modulation on the effect of IGF-1 on platelet aggregation.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Análise Serial de Proteínas , Proteômica/métodos , Idoso , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/imunologia , Aortografia/métodos , Biomarcadores/sangue , Western Blotting , Estudos de Casos e Controles , Meios de Cultivo Condicionados/metabolismo , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Agregação Plaquetária , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Espanha , Técnicas de Cultura de Tecidos , Tomografia Computadorizada por Raios X , Regulação para CimaRESUMO
OBJECTIVES: Diminished soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) concentrations are associated with cardiovascular diseases. We have analyzed sTWEAK levels and its relation with expansion rate in subjects with abdominal aortic aneurysm (AAA). METHODS: sTWEAK levels were measured by ELISA. RESULTS: sTWEAK concentrations were diminished in small AAA (≤ 5 cm; 353 ± 12 pg/mL; n = 25, p = 0.03) and large AAA (>5 cm; 315 ± 21 pg/mL; n = 18, p = 0.004) compared with healthy subjects (411 ± 22 pg/mL; n=27). Moreover, sTWEAK concentrations were negatively associated with AAA size (r = -0.4; p = 0.008). sTWEAK was also negatively associated with AAA expansion rate with 5 years of follow-up (n = 79, r = -0.263; p = 0.031). Multivariate regression analysis revealed that sTWEAK levels were independently associated with AAA growth rate (ß = -0.208; p = 0.046). CONCLUSIONS: sTWEAK plasma levels were decreased in subjects with AAA and were independently related with AAA expansion rate indicating that this protein could be a novel diagnostic and prognostic biomarker of AAA.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Fatores de Necrose Tumoral/sangue , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Biomarcadores/sangue , Estudos de Casos e Controles , Citocina TWEAK , Dinamarca , Progressão da Doença , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Modelos Logísticos , Prognóstico , Medição de Risco , Fatores de Risco , Espanha , Fatores de Tempo , UltrassonografiaRESUMO
OBJECTIVE: Oxidative stress is a main mechanism involved in vascular pathologies. Increased thioredoxin (TRX) levels have been observed in several oxidative stress-associated cardiovascular diseases. We aim to test the potential role of TRX as a biomarker of oxidative stress in abdominal aortic aneurysm (AAA). METHODS: TRX levels were analysed in both AAA intraluminal thrombus (ILT) tissue and in tissue-conditioned media by immunohistochemistry, Western blot and ELISA. Moreover, serum TRX levels were assessed in AAA Caucasian patients by ELISA. RESULTS: TRX was mainly localized in the luminal part of ILT in AAA. Compared with the abluminal layer, TRX release was increased in the luminal layer of the ILT of AAA (31+/-9 ng/ml vs. 9+/-3 ng/ml, p<0.05). The interest of this approach is that we can identify proteins potentially released into the blood compartment, which could serve as biomarkers of the pathology. In a training population, serum TRX levels were significantly increased in patients with AAA relative to healthy subjects (50+/-6 ng/ml vs. 26+/-3 ng/ml, p<0.05). These results were validated in a second independent group of patients. Moreover, a positive correlation between TRX and AAA size (rho=0.5, p<0.001) was observed. Finally, in AAA samples with follow-up, TRX was positively associated to aneurismal growth rate (rho=0.25, p=0.027). CONCLUSIONS: TRX release is increased in the luminal part of AAA and TRX serum levels are increased in AAA patients compared with healthy subjects. TRX levels correlates with AAA size and expansion, suggesting its potential role as a biomarker of AAA evolution.
Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Estresse Oxidativo , Tiorredoxinas/metabolismo , Idoso , Aorta Abdominal/patologia , Aorta Abdominal/cirurgia , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/cirurgia , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Meios de Cultivo Condicionados/metabolismo , Dinamarca , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Masculino , Espanha , Tiorredoxinas/sangue , Técnicas de Cultura de Tecidos , Regulação para CimaRESUMO
Apoptosis participates in every step of atherogenesis, but the process of clearance of apoptotic cells by phagocytosis has been underestimated. Rapid removal of apoptotic cells is critical for tissue homeostasis, in order to avoid accumulation of necrotic material and subsequent inflammation in the pathological vascular wall. We have demonstrated by RT-PCR, western blot and immunocytofluorescence that vascular smooth muscle cells (VSMCs) express the phosphatidylserine receptor (PSR). We then tested the involvement of PSR in the ability of VSMCs to bind and engulf apoptotic cells. We used a model of senescent erythrocytes, which expose PS after 4 days of culture (85% of cells relative to 8% in freshly isolated erythrocytes). The pseudo-peroxidase activity of haemoglobin contained within erythrocytes allowed us to quantify per se both binding and phagocytosis by VSMCs. We have also shown by light and confocal microscopy that VSMCs were able to ingest aged erythrocytes. Addition of a blocking antibody or transfection of VSMCs by a siRNA directed against PSR reduced the binding and engulfment of aged erythrocytes by more than 90%. These results suggest that PSR is involved in phagocytosis of PS-presenting cells. Incubation of aged erythrocytes with VSMCs also significantly increased the expression of PSR, suggesting that the tethering/ingestion of apoptotic cells triggers this process. Immunostaining for PSR in complicated atherosclerotic plaques shows positivity in the media and macrophage-rich areas. The mechanisms underlying phagocytosis and involving PSR in vivo, within the pathological arterial wall, deserve further investigation.
Assuntos
Artérias Carótidas/patologia , Estenose das Carótidas/patologia , Miócitos de Músculo Liso/fisiologia , Fagocitose/fisiologia , Receptores de Superfície Celular/metabolismo , Anticorpos Monoclonais/imunologia , Apoptose , Western Blotting , Artérias Carótidas/metabolismo , Estenose das Carótidas/metabolismo , Células Cultivadas , Senescência Celular , Envelhecimento Eritrocítico , Imunofluorescência , Humanos , Microscopia Confocal , Interferência de RNA , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Development and progression of acquired abdominal aortic aneurysms (AAAs) involve proteolytic activity. In the present study, we investigate the distribution of fibrinolytic system components within mural thrombi of human AAAs. 20 mural thrombi and the remaining AAA walls were dissected. The luminal, intermediate and abluminal thrombus layers, and media and adventitia were separately incubated in cell culture medium. Conditioned media were then analysed for plasminogen activators (PAs), plasminogen activator inhibitor-1 (PAI-1), free-plasmin, plasmin alpha(2)-antiplasmin complexes (PAPs) and D-dimers release. In parallel, PA and PAI-1 mRNA expression analysis was performed by RT-PCR. The study was completed by immunohistochemical localization of these components in AAA, ex vivo functional imaging using (99m)Tc-aprotinin as a ligand and measurement of PAP and D-dimer plasma levels. All fibrinolytic system components were present in each aneurysmal layer. However, the mural thrombus was the main source of active serine-protease release. Interestingly, the luminal layer of the thrombus released greater amounts of PAPs and D-dimers. This paralleled the preferential immunolocalization of plasminogen and PAs, and the (99m)Tc-aprotinin scintigraphic signal observed in the luminal pole of the thrombus. In contrast, mRNA expression analysis showed an exclusive synthesis of tPA and PAI-1 within the wall, whereas uPA mRNA was also expressed within the thrombus. Taken together, these results suggest that the increased plasma concentrations of PAPs and D-dimers found in AAA patients are related to mural thrombus proteolytic activity, thus explaining their known link with AAA progression. Components of the fibrinolytic system could also represent a target for functional imaging of thrombus activities in AAA.
Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Fibrinolíticos/análise , Trombose/metabolismo , Idoso , Idoso de 80 Anos ou mais , Aorta Abdominal/química , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aprotinina/metabolismo , Ensaio de Imunoadsorção Enzimática , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Produtos de Degradação da Fibrina e do Fibrinogênio/genética , Fibrinolisina/análise , Fibrinolisina/genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Plasminogênio/análise , Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/análise , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/análise , Cintilografia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombose/diagnóstico por imagem , Ativador de Plasminogênio Tecidual/análise , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tipo Uroquinase/análise , Ativador de Plasminogênio Tipo Uroquinase/genética , alfa 2-Antiplasmina/análise , alfa 2-Antiplasmina/genéticaRESUMO
Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H(2)O(2)). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H(2)O(2) after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress.
Assuntos
Catalase/genética , Endotélio Vascular/enzimologia , Expressão Gênica/efeitos dos fármacos , Peróxidos Lipídicos/farmacologia , Músculo Liso Vascular/enzimologia , Oxidantes/farmacologia , Animais , Catalase/metabolismo , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Ácidos Graxos/farmacologia , Artéria Femoral , Humanos , Peróxido de Hidrogênio/farmacologia , Ácido Linoleico/metabolismo , Ácidos Linoleicos/farmacologia , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Oxirredução , RNA Mensageiro/metabolismo , Coelhos , Transfecção , Veias UmbilicaisRESUMO
Oxidized low density lipoprotein (Ox-LDL) has a plethora of components that are not present in native LDL. Their presence and quantity depends on the nature, type, and extent of oxidation. Lipids esterified to oxidized fatty acids are the major components formed during the early phase of oxidation and these show a number of proatherogenic properties in in vitro cell culture systems. Recently, evidence has been forthcoming to suggest that some of these oxidized lipids also could elicit "antioxidant;-antiatherogenic" responses from cells. Moreover, some of the cellular effects of Ox-LDL that were previously interpreted as atherogenic could also be reinterpreted to suggest an antiatherogenic cellular response. In addition to the above, the antioxidants that are carried in lipoproteins could have anomalous behavior attributable to their metabolism, ability to be internalized by arterial cells, and the presence of oxidative systems that could render them prooxidants. In conclusion, there are numerous contributing factors that need to be studied and understood before antioxidant therapy becomes an option for the treatment for cardiovascular diseases.
Assuntos
Antioxidantes/uso terapêutico , Arteriosclerose/prevenção & controle , Oxidantes/farmacologia , Animais , Antioxidantes/metabolismo , Arteriosclerose/metabolismo , Humanos , Lipoproteínas LDL/efeitos adversos , Lipoproteínas LDL/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Oxidantes/metabolismoRESUMO
The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.
Assuntos
Angiotensina II/biossíntese , Angiotensina I/metabolismo , Músculo Liso Vascular/enzimologia , Peptidil Dipeptidase A/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Compartimento Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Dexametasona/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Glucocorticoides/farmacologia , Humanos , Radioisótopos do Iodo , Músculo Liso Vascular/citologia , Ratos , Fosfolipases Tipo C/metabolismoRESUMO
Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed.
Assuntos
Apoptose , Morte Celular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Necrose , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Apoptose/efeitos dos fármacos , Calcimicina/farmacologia , Cálcio/metabolismo , Regulação para Baixo , Células HL-60 , Humanos , Ionóforos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Oxirredução , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Human endothelial cells (ECs) grown under standard conditions are able to generate a basal level of oxygen free radicals and induce progressive oxidation of LDLs. Inhibition of cell-mediated LDL oxidation by superoxide dismutase, EDTA, or desferrioxamine implicates a role for superoxide anion and/or transition metals in this process. The potential role of the mitochondrion was investigated by inducing mitochondrial deenergization by selective photosensitization or the addition of inhibitors of the mitochondrial respiratory chain. Mitochondria of human cultured ECs were selectively damaged by photosensitization of cells labeled with the mitochondrion-selective fluorescent dye 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide under conditions that induced only low levels of toxicity during the time of the experiment. Photosensitized ECs exhibited severe mitochondrial dysfunction, as suggested by the defect in mitochondrial uptake of the mitochondrion-selective fluorescent dyes [rhodamine 123 and 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide] and morphological alterations as shown by transmission electron microscopy. In mitochondria-photosensitized cells, superoxide anion generation was strongly decreased, as was LDL oxidation and the subsequent cytotoxicity. When ECs were incubated with the mitochondrial respiratory-chain inhibitors antimycin A or rotenone or with the carbonylcyanide-m-chlorophenylhydrazone uncoupler rhodamine 123, uptake and subcellular distribution were altered, and concomitantly superoxide anion production and LDL oxidation were strongly decreased. In conclusion, these data suggest that mitochondrial function is required, directly or indirectly, for the production of superoxide anion and the subsequent LDL oxidation by human vascular ECs.
Assuntos
LDL-Colesterol/metabolismo , Endotélio/citologia , Endotélio/metabolismo , Mitocôndrias/fisiologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Linhagem Celular , Desferroxamina/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Humanos , Ionóforos/farmacologia , Luz , Manitol/farmacologia , Mitocôndrias/efeitos da radiação , NG-Nitroarginina Metil Éster/farmacologia , Oxirredução , Superóxido Dismutase/antagonistas & inibidores , Superóxidos/metabolismo , ômega-N-Metilarginina/farmacologiaRESUMO
Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.