RESUMEN
BACKGROUND: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain. RESULTS: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385-418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies. CONCLUSION: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.
Asunto(s)
Asparagina/química , Carbohidratos/inmunología , Células Dendríticas/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Pliegue de Proteína , Asparagina/metabolismo , Asparagina/fisiología , Sitios de Unión , Antígenos CD4/inmunología , Carbohidratos/química , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/química , Células HeLa , Humanos , Pruebas de NeutralizaciónRESUMEN
INTRODUCTION: Atherosclerotic plaque microvessels are associated with plaque hemorrhage and rupture. The mechanisms underlying plaque angiogenesis are largely unknown. Angiopoietin (Ang)-1 and -2 are ligands of the endothelial receptor Tie-2. Ang-1 induces formation of stable vessels, whereas Ang-2 destabilizes the interaction between endothelial cells and their support cells. We studied the expression patterns of Ang-1 and -2 in relation to plaque microvessels. METHODS AND RESULTS: Carotid endarterectomy specimens were studied (n = 100). Microvessel density (MVD) was correlated with the presence of macrophages and with a (fibro)atheromatous plaque phenotype. A negative correlation was observed between Ang-1 expression and MVD. A positive correlation was observed between the ratio of Ang-2/Ang-1 and MVD. Ang-2 expression was correlated with matrix metalloproteinase-2 (MMP-2) activity. Immunohistochemical staining of Ang-1 was observed in smooth muscle cells, whereas Ang-2 was detected in endothelial cells, smooth muscle cells and macrophages. CONCLUSIONS: In plaques with high MVD, the local balance between Ang-1 and Ang-2 is in favor of Ang-2. Plaque Ang-2 levels are associated with MMP-2 activity. Ang-2-induced MMP-2 activity might play a role in the development of (unstable) plaque microvessels.
Asunto(s)
Angiopoyetina 1/metabolismo , Angiopoyetina 2/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Vasos Sanguíneos/patología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Microcirculación , Coloración y EtiquetadoRESUMEN
BACKGROUND: Extra domain A (EDA), splice-variant of fibronectin, is a Toll-like receptor 4 (Tlr4) ligand. Recently, EDA has been demonstrated to enhance atherogenesis in mice but human data on the role of EDA in atherosclerotic disease are lacking. We hypothesized that EDA is associated with unstable plaque phenotypes and that plasma EDA could serve as biomarker for atherosclerosis. METHODS: EDA levels were assessed in carotid endarterectomy specimen (206 patients) and related with plaque phenotype. In a second patient cohort, systemic EDA levels in atherosclerotic patients (73 patients) were compared to risk-factor matched controls (68 patients). RESULTS: EDA plaque levels were associated with characteristics of stable plaques; more smooth muscle cells (P=0.003), more collagen (P=0.071) and less fat (P=0.023). Concomitantly, asymptomatic patients showed higher EDA values in the plaque compared to symptomatic patients (P=0.004). EDA plasma levels did not differ between atherosclerotic patients versus controls (P=0.134). CONCLUSION: EDA plaque levels are higher in asymptomatic patients and are associated with a stable plaque phenotype. EDA is not a plasma marker for atherosclerotic disease. These results suggest that local presence of endogenous Tlr4 ligand EDA is not associated with in an unstable plaque phenotype in humans.
Asunto(s)
Aterosclerosis/sangre , Aterosclerosis/metabolismo , Fibronectinas/química , Arterias/patología , Biomarcadores , Estudios de Cohortes , Regulación de la Expresión Génica , Humanos , Glándulas Mamarias Humanas/irrigación sanguínea , Metaloproteinasas de la Matriz/metabolismo , Fenotipo , Estudios Prospectivos , Estructura Terciaria de Proteína , Factores de Riesgo , Receptor Toll-Like 4/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Anti-inflammatory qualities are held partially responsible for the reduction of cardiovascular events after statin treatment. We examined the phenotype of carotid atherosclerotic plaques harvested during carotid endarterectomy in relation to the previous use of different statins prescribed in clinical practice. METHODS: Three hundred and seventy-eight patients were included. Atherosclerotic plaques were harvested, immunohistochemically stained and semiquantitively examined for the presence of macrophages (CD68), smooth muscle cells, collagen and fat. Adjacent atherosclerotic plaques were used to study protease activity and interleukin levels. Patients' demographics were recorded and blood samples were stored. RESULTS: Serum cholesterol, low-density lipoprotein, apolipoprotein B, and C-reactive protein levels were lower in patients treated with statins compared with patients without statin treatment. Atheromatous plaques were less prevalent in patients receiving statins compared with patients without statin therapy (29% versus 42%, P=0.04). An increase of CD68 positive cells was observed in patients receiving statins compared with nonstatin treatment (P=0.05). This effect was specifically related to atorvastatin treatment. In patients treated with atorvastatin, the increased amount of CD68 positive cells were not associated with increased protease activity. In contrast, a dose-dependent decrease in protease activity was shown in the atorvastatin group. Interleukin 6 expression was lower in plaques obtained from patients treated with statins (P=0.04). CONCLUSIONS: Statin use may exert pleiotropic effects on plaque phenotype. However, not the presence of macrophages but activation with subsequent protease and cytokine release may be attenuated by statin use.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Enfermedades de las Arterias Carótidas/patología , Endarterectomía Carotidea , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Inflamación/patología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/cirugía , Atorvastatina , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/cirugía , Citocinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Ácidos Heptanoicos/uso terapéutico , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inmunohistoquímica , Macrófagos/metabolismo , Macrófagos/patología , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Fenotipo , Pravastatina/uso terapéutico , Pirroles/uso terapéutico , Estudios Retrospectivos , Simvastatina/uso terapéuticoRESUMEN
INTRODUCTION: Atherosclerotic carotid artery disease is responsible for a variety of clinical presentations, ranging from asymptomatic to cerebral ischemic events. Considering the upcoming use of noninvasive imaging modalities, plaque characteristics could serve as a marker in the selection of patients eligible for carotid endarterectomy (CEA). This would be more likely if characteristics corresponded with clinical manifestations and were predictive of future events. In this study, we hypothesized that plaque characteristics correlate with the clinical presentation of carotid artery disease. METHODS: We included 404 patients undergoing a carotid endarterectomy (CEA). Ipsilateral clinical symptoms and duplex measurements were recorded. Patients could be asymptomatic (23.5%) or symptomatic with stroke (26.5%), transient ischemic attack (TIA) (36.1%), or amaurosis fugax (AFX) (13.9%). Plaques were stained and semi-quantitatively analyzed for the presence of macrophages, smooth muscle cells, collagen, calcifications, and thrombus. Plaques were categorized in three phenotypes by their overall presentation and the amount of fat. In addition, plaque matrix metalloproteinase (MMP) activity and cytokines expressions were measured. RESULTS: Fibrous, fibro-atheromatous, and atheromatous plaques were observed in 30.2%, 35.6%, and 34.2%, respectively. Atheromatous plaques were more prevalent in patients with stroke and TIA compared with asymptomatic patients or patients with AFX (P = .001). Collagen staining was less evident in patients with TIA and stroke compared with asymptomatic patients or patients with AFX (P < .001). Plaques of patients with TIA and stroke showed significantly higher activity levels of MMP-8 and MMP-9 and higher levels of interleukin-8 compared with asymptomatic and AFX patients. CONCLUSION: Plaque phenotype of patients with TIA is comparable to that of patients with stroke; whereas, the plaque phenotype of patients with AFX resembles the plaque phenotype of asymptomatic patients. Follow-up studies should be encouraged to determine whether plaque characteristics visualized by imaging techniques might help to identify patients most likely to benefit from CEA.
Asunto(s)
Amaurosis Fugax/etiología , Enfermedades de las Arterias Carótidas/complicaciones , Interleucina-8/metabolismo , Ataque Isquémico Transitorio/etiología , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Accidente Cerebrovascular/etiología , Anciano , Amaurosis Fugax/metabolismo , Angiografía , Biomarcadores/metabolismo , Enfermedades de las Arterias Carótidas/diagnóstico , Enfermedades de las Arterias Carótidas/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Ataque Isquémico Transitorio/metabolismo , Imagen por Resonancia Magnética , Masculino , Estudios Prospectivos , Factores de Riesgo , Índice de Severidad de la Enfermedad , Accidente Cerebrovascular/metabolismo , Tomografía Computarizada por Rayos X , Ultrasonografía DopplerRESUMEN
OBJECTIVE: The extent of atherosclerotic plaque burden and the incidence of atherosclerosis-related cardiovascular events accelerate with increasing age. The composition of the plaque is associated with plaque thrombosis and acute coronary occlusion. Surprisingly, however, the relation between advancing age and atherosclerotic plaque composition is still unclear. In the present study, we investigated the association between plaque characteristics and advancing age in a population of patients with haemodynamically significant carotid artery stenosis. METHODS: Patients (N=383), ages 39-89 years, underwent carotid endarterectomy (CEA). Morphometric analysis was performed on the dissected atherosclerotic plaques to study the prevalence of fibrous and atheromatous plaques. Picro sirius red, haematoxylin eosin, alfa actin and CD68 stainings were performed to investigate the extent of collagen, calcification, smooth muscle cells and macrophages in carotid plaques, respectively. The presence of metalloproteinases-2 and -9 was assessed by ELISA. RESULTS: With aging, a decrease in fibrous plaques and an increase in atheromatous plaques were observed. This was accompanied by an age-associated decrease in smooth muscle cell content in carotid plaques. Macrophage content slightly increased with age. In addition, total matrix metalloprotease (MMP)-2 was negatively and MMP-9 positively related with age. Differences in plaque phenotype were most prominent for the youngest age quartile compared with older age quartiles. CONCLUSIONS: With increasing age, the morphology of atherosclerotic plaques from patients with carotid artery stenosis changes. Plaques become more atheromatous and contain less smooth muscle cells with increasing age. Local inflammation and MMP-9 levels slightly increased with age in plaques obtained from patients suffering from haemodynamically significant advanced atherosclerotic lesions.
Asunto(s)
Envejecimiento , Arteriosclerosis/patología , Estenosis Carotídea/patología , Actinas/metabolismo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Biomarcadores/metabolismo , Estenosis Carotídea/complicaciones , Estenosis Carotídea/metabolismo , Estenosis Carotídea/cirugía , Endarterectomía Carotidea , Femenino , Humanos , Inmunohistoquímica , Macrófagos/patología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
The HIV-1 envelope glycoprotein complex (Env) can be stabilized by the introduction of a disulfide bond between the gp120 and gp41 subunits. The resulting protein is monomeric, but trimerization can be improved by the introduction of a single helix-breaking residue at the conserved Ile559 site in the N-terminal heptad repeat region of gp41. To provide more insight into how such a substitution in gp41 affects Env structure and function, we evaluated the effect on the wild-type Env in the context of replicating virus. The Ile559Gly and Ile559Pro mutations adversely affect Env biosynthesis and Env incorporation into virions. Biophysical studies show that the Ile559Pro mutation essentially disrupts the folding of a recombinant gp41 ectodomain core into a six-helix bundle structure. Viruses containing the Ile559Gly and Ile559Pro substitutions replicate poorly, but an evolutionary route is described that restores replication competence. In the escape virus, which contains a Pro559Leu first-site pseudoreversion, the local helical structure and, as a consequence, Env biosynthesis and function are restored.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , Sustitución de Aminoácidos , Línea Celular , Línea Celular Tumoral , Dicroismo Circular , Cartilla de ADN , Femenino , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Conformación Proteica , Linfocitos T , Neoplasias del Cuello Uterino , Virión/fisiologíaRESUMEN
BACKGROUND: We previously described the construction of an HIV-1 envelope glycoprotein complex (Env) that is stabilized by an engineered intermolecular disulfide bond (SOS) between gp120 and gp41. The modified Env protein antigenically mimics the functional wild-type Env complex. Here, we explore the effects of the covalent gp120 - gp41 interaction on virus replication and evolution. RESULTS: An HIV-1 molecular clone containing the SOS Env gene was only minimally replication competent, suggesting that the engineered disulfide bond substantially impaired Env function. However, virus evolution occurred in cell culture infections, and it eventually always led to elimination of the intermolecular disulfide bond. In the course of these evolution studies, we identified additional and unusual second-site reversions within gp41. CONCLUSIONS: These evolution paths highlight residues that play an important role in the interaction between gp120 and gp41. Furthermore, our results suggest that a covalent gp120 - gp41 interaction is incompatible with HIV-1 Env function, probably because this impedes conformational changes that are necessary for fusion to occur, which may involve the complete dissociation of gp120 from gp41.
Asunto(s)
Evolución Molecular , Productos del Gen env/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/fisiología , Sustitución de Aminoácidos , Línea Celular Tumoral , Disulfuros/análisis , Humanos , Transfección , Replicación ViralRESUMEN
In clinical practice, biological markers are not available to routinely assess the progression of atherosclerotic disease or the development of restenosis following endarterectomy or catheter based interventions. Endarterectomy procedures provide an opportunity to study mechanisms of restenosis and progression of atherosclerotic disease since atherosclerotic tissue is obtained. Athero-Express is an ongoing prospective study, initiated in 2002, with the objective to investigate the etiological value of plaque characteristics for long term outcome. Patients are included who undergo an endarterectomy of the carotid artery. At baseline blood is withdrawn, patients fill in an extensive questionnaire and diagnostic examinations are performed. Atherosclerotic plaques are freshly harvested, immunohistochemically stained and examined for the presence of macrophages, smooth muscle cells, collagen and fat. Parts of the atherosclerotic plaques are freshly frozen to study protease activity and protein and RNA expressions. Patients undergo a duplex follow up to assess procedural restenosis (primary endpoint) at 3 months, 1 year and 2 years. Secondary endpoints encompass major adverse cardiovascular events. In the future, the creation of this biobank with atherosclerotic specimen will allow the design of cross-sectional and follow up studies with the objective to investigate the expression of newly discovered genes and proteins and their interaction with patients and plaque characteristics in the progression of atherosclerotic disease. Objective is to include 1000-1200 patients in 5 years. In January 2004, 289 patients had been included. It is expected that 250 patients will be included yearly.