RESUMEN
Hidradenitis Suppurativa (HS) is a chronic, recurrent, inflammatory, follicular skin disease whose pathology is complex and not fully understood. The objective of this study was to elucidate the role of IL-17A in moderate-to-severe HS. Transcriptomic and histological analyses were conducted on ex vivo HS (n = 19; lesional and non-lesional) and healthy control (n = 8) skin biopsies. Further, a Phase II exploratory, randomized, double-blind, placebo-controlled study was carried out in moderate-to-severe HS patients. Patients were treated with either CJM112 300 mg (n = 33), a fully human anti-IL-17A IgG1/κ monoclonal antibody, or placebo (n = 33). The main outcome of the translational analyses was to identify IL-17A-producing cells and indications of IL-17A activity in HS lesional skin. The primary objective of the clinical study was to determine the efficacy of CJM112 in moderate-to-severe HS patients by HS-Physician Global Assessment (HS-PGA) responder rate at Week 16. Transcriptomic and histopathologic analyses revealed the presence of heterogeneous cell types in HS lesional skin; IL-17A gene signatures were increased in HS lesional vs non-lesional or healthy skin. High expression of IL-17A was localized to T cells, neutrophils, and mast cells, confirming the transcriptional data. Clinically, the proportion of Week 16 HS-PGA responders was significantly higher (p = 0.03) in the CJM112 group vs placebo (32.3% vs 12.5%). This study elucidated the role of the IL-17A pathway in HS pathogenesis and clinically validated the IL-17A pathway in moderate-to-severe HS patients in a proof-of-concept study using the anti-IL-17A-specific antibody CJM112.
Asunto(s)
Dermatitis , Hidradenitis Supurativa , Humanos , Anticuerpos Monoclonales/uso terapéutico , Dermatitis/metabolismo , Hidradenitis Supurativa/genética , Inmunoglobulina G/metabolismo , Piel/metabolismoRESUMEN
Fragile X syndrome (FXS) is a monogenic form of intellectual disability and autism spectrum disorder caused by the absence of the fragile X mental retardation protein (FMRP). In biological models for the disease, this leads to upregulated mRNA translation and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here, we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here, we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those in neurons we suggest the validity of this peripheral biomarker. Our study offers a potential explanation for the comprehensive drug development program undertaken thus far yielding negative results and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis.
Asunto(s)
Trastorno del Espectro Autista/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Adolescente , Adulto , Anciano , Animales , Trastorno del Espectro Autista/fisiopatología , Niño , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/biosíntesis , Síndrome del Cromosoma X Frágil/fisiopatología , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/patología , Adulto JovenRESUMEN
BACKGROUND: This phase 2 randomized, double-blind, placebo-controlled study evaluated the efficacy and safety of the nicotinic acetylcholine receptor α7 agonist AQW051 in patients with Parkinson's disease and levodopa-induced dyskinesia. METHODS: Patients with idiopathic Parkinson's disease and moderate to severe levodopa-induced dyskinesia were randomized to AQW051 10 mg (n = 24), AQW051 50 mg (n = 24), or placebo (n = 23) once daily for 28 days. Coprimary end points were change in Modified Abnormal Involuntary Movement Scale and Unified Parkinson's Disease Rating Scale part III scores. Secondary outcomes included pharmacokinetics. RESULTS: In total, 67 patients completed the study. AQW051-treated patients experienced no significant improvements in Modified Abnormal Involuntary Movement Scale or Unified Parkinson's Disease Rating Scale part III scores by day 28. AQW051 was well tolerated; the most common adverse events were dyskinesia, fatigue, nausea, and falls. CONCLUSIONS: AQW051 did not significantly reduce dyskinesia or parkinsonian severity. © 2016 International Parkinson and Movement Disorder Society.
Asunto(s)
Antiparkinsonianos/farmacología , Compuestos de Azabiciclo/farmacología , Dopaminérgicos/efectos adversos , Discinesia Inducida por Medicamentos/tratamiento farmacológico , Levodopa/efectos adversos , Evaluación de Resultado en la Atención de Salud , Enfermedad de Parkinson/tratamiento farmacológico , Piridinas/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Anciano , Antiparkinsonianos/administración & dosificación , Antiparkinsonianos/efectos adversos , Compuestos de Azabiciclo/administración & dosificación , Compuestos de Azabiciclo/efectos adversos , Método Doble Ciego , Discinesia Inducida por Medicamentos/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piridinas/administración & dosificación , Piridinas/efectos adversosRESUMEN
AIMS: Lectin-like oxLDL receptor-1 (LOX-1) mediates the uptake of oxidized low-density lipoprotein (oxLDL) in endothelial cells and macrophages. However, the different atherogenic potential of LOX-1-mediated endothelial and macrophage oxLDL uptake remains unclear. The present study was designed to investigate the in vivo role of endothelial LOX-1 in atherogenesis. METHODS AND RESULTS: Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). Oxidized low-density lipoprotein uptake was enhanced in cultured endothelial cells, but not in macrophages of LOX-1TG mice. Six-week-old male LOX-1TG and wild-type (WT) mice were fed a high-cholesterol diet (HCD) for 30 weeks. Increased reactive oxygen species production, impaired endothelial nitric oxide synthase activity and endothelial dysfunction were observed in LOX-1TG mice as compared with WT littermates. LOX-1 overexpression led to p38 phosphorylation, increased nuclear factor κB activity and subsequent up-regulation of vascular cell adhesion molecule-1, thereby favouring macrophage accumulation and aortic fatty streaks. Consistently, HCD-fed double-mutant LOX-1TG/ApoE(-/-) displayed oxidative stress and vascular inflammation with higher aortic plaques than ApoE(-/-) controls. Finally, bone marrow transplantation experiments showed that endothelial LOX-1 was sufficient for atherosclerosis development in vivo. CONCLUSIONS: Endothelial-specific LOX-1 overexpression enhanced aortic oxLDL levels, thereby favouring endothelial dysfunction, vascular inflammation and plaque formation. Thus, LOX-1 may serve as a novel therapeutic target for atherosclerosis.
Asunto(s)
Aterosclerosis/etiología , Endotelio Vascular/metabolismo , Receptores Depuradores de Clase E/metabolismo , Animales , Aorta Abdominal/metabolismo , Aorta Torácica/metabolismo , Aortitis/etiología , Apolipoproteínas E/metabolismo , Células Cultivadas , Colesterol en la Dieta/administración & dosificación , Masculino , Ratones Transgénicos , Placa Aterosclerótica/etiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Stress is known to correlate with the incidence of acute myocardial infarction. However, the molecular mechanisms underlying this correlation are not known. This study was designed to assess the effect of experimental stress on arterial thrombus formation, the key event in acute myocardial infarction. Mice exposed to 20 h of restraint stress displayed an increased arterial prothrombotic potential as assessed by photochemical injury-induced time to thrombotic occlusion. This increase was prevented by chemical sympathectomy performed through 6-hydroxydopamine (6-OHDA). Blood-born tissue factor (TF) activity was enhanced by stress and this increase could be prevented by 6-OHDA treatment. Vessel wall TF, platelet count, platelet aggregation, coagulation times (PT, aPTT), fibrinolytic system (t-PA and PAI-1) and tail bleeding time remained unaltered. Telemetric analysis revealed only minor hemodynamic changes throughout the stress protocol. Plasma catecholamines remained unaffected after restraint stress. Tumor necrosis factor alpha (TNF-α) plasma levels were unchanged and inhibition of TNF-α had no effect on stress-enhanced thrombosis. These results indicate that restraint stress enhances arterial thrombosis via the sympathetic nervous system. Blood-borne TF contributes, at least in part, to the observed effect whereas vessel wall TF, platelets, circulating coagulation factors, fibrinolysis and inflammation do not appear to play a role. These findings shed new light on the understanding of stress-induced cardiovascular events.
Asunto(s)
Restricción Física , Estrés Psicológico , Sistema Nervioso Simpático/fisiología , Tromboplastina/fisiología , Trombosis/etiología , Animales , Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Arterias Carótidas/fisiología , Etanercept , Inmunoglobulina G/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidopamina/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral , Flujo Sanguíneo Regional , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
OBJECTIVE: To assess the effects of aging on arterial thrombus formation by comparing 2-year-old with 11-week-old C57Bl6 mice. METHODS AND RESULTS: Aging is a major risk factor for cardiovascular disease. In humans, assessing the direct effects of aging on vascular homeostasis is difficult because it occurs in the presence of other risk factors. Arterial thrombosis is the critical event in cardiovascular diseases; however, it is not known whether aging per se promotes its occurrence. Mice represent an interesting system to address this issue because they age without spontaneously developing other risk factors. Organ chamber experiments confirmed the advanced level of aging of old mice. As previously shown, old mice exhibited endothelial dysfunction; however, arterial thrombosis induced by photochemical injury was unchanged. Arterial tissue factor expression and activity; expressions of tissue factor pathway inhibitor, thrombomodulin, and plasminogen activator inhibitor 1; prothrombin time; partial thromboplastin time; thrombin-antithrombin complex; and platelet activation were comparable in both groups. CONCLUSIONS: Although these results cannot be directly extrapolated to humans, this study contributes novel important information on the direct effect of aging on arterial thrombosis and underscores the importance of controlling modifiable risk factors in aged individuals.
Asunto(s)
Envejecimiento/patología , Envejecimiento/fisiología , Trombosis de las Arterias Carótidas/fisiopatología , Endotelio Vascular/fisiopatología , Envejecimiento/genética , Animales , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/fisiopatología , Secuencia de Bases , Coagulación Sanguínea , Plaquetas/fisiología , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/patología , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Riesgo , Tromboplastina/genética , Tromboplastina/metabolismo , VasodilataciónRESUMEN
OBJECTIVE: Enhanced endothelial permeability leading to intimal accumulation of low-density lipoproteins (LDL) stimulates the formation of atherosclerotic lesions. Histamine is known to increase vascular permeability. Whether this affects the formation of atherosclerotic lesions, however, remains elusive. METHODS AND RESULTS: Apolipoprotein E-null (ApoE(-/-)) mice treated with a histamine H1 receptor but not an H2 receptor antagonist developed 40% fewer atherosclerotic lesions in the aorta than placebo-treated controls. Similarly, genetic deletion of the H1 but not the H2 receptor resulted in a 60% reduction of lesions compared with ApoE(-/-) controls. The H1 receptor enhanced LDL permeability and lipid accumulation in the aorta, whereas plasma lipoprotein levels remained unaltered. In contrast, the H1 receptor did not affect proliferation and migration of vascular smooth muscle cells. Bone marrow transplantation confirmed that the formation of atherosclerotic lesions depended on the H1 receptor in vascular cells, whereas its presence in bone marrow-derived cells was irrelevant for plaque development. Mice expressing the H1 receptor exhibited higher levels of the chemokine (C-C motif) ligand 5 and higher numbers of macrophages and T-helper lymphocytes in plaques, higher numbers of circulating lymphocytes, and larger spleens. CONCLUSION: These data indicate that H1 but not H2 receptor activation drives the formation of atherosclerotic lesions through an increased vascular permeability for LDL, which is associated with an enhanced secondary aortic and systemic inflammation. These data open novel perspectives for the prevention and treatment of atherosclerotic vascular disease.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Permeabilidad Capilar , Histamina/metabolismo , Lipoproteínas LDL/metabolismo , Receptores Histamínicos H1/metabolismo , Animales , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aortitis/inmunología , Aortitis/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Aterosclerosis/prevención & control , Trasplante de Médula Ósea , Permeabilidad Capilar/efectos de los fármacos , Proliferación Celular , Modelos Animales de Enfermedad , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Linfocitos/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Receptores Histamínicos H1/deficiencia , Receptores Histamínicos H1/efectos de los fármacos , Receptores Histamínicos H1/genética , Receptores Histamínicos H2/metabolismoRESUMEN
BACKGROUND: The haematological benefit of standard-of-care anti-C5 treatment for haemolytic paroxysmal nocturnal haemoglobinuria is limited by residual intravascular haemolysis or emerging C3-mediated extravascular haemolysis. Therefore, the aim of this phase 2 study was to assess the safety, tolerability, pharmacokinetics and pharmacodynamics, and activity of the new complement factor B inhibitor, iptacopan, in patients with paroxysmal nocturnal haemoglobinuria who have active haemolysis despite anti-C5 therapy. METHODS: In this multicentre, open-label, single-arm, phase 2 trial, we enrolled adult patients (aged 18-80 years) with paroxysmal nocturnal haemoglobinuria who showed signs of active haemolysis despite receiving eculizumab treatment. Patients were enrolled at Federico II University Hospital (Naples, Italy), Hôpital Saint-Louis (Paris, France), and University Hospital Essen (Essen, Germany). For enrolment, patients were required to show lactate dehydrogenase more than 1·5-times the upper limit of normal and a paroxysmal nocturnal haemoglobinuria type 3 erythrocyte or granulocyte clone size of 10% or greater. Patients with bone marrow failure, on systemic steroid or immunosuppressive drugs, or with severe comorbidities were excluded from the study. Iptacopan was given orally as an add-on therapy at a dose of 200 mg twice daily. The primary endpoint was the effect of iptacopan on the reduction of chronic residual intravascular haemolysis measured as change in lactate dehydrogenase from baseline value to week 13. At 13 weeks, patients could opt into a long-term study extension (ongoing), allowing for modifications of standard treatment. This trial is registered at ClinicialTrials.gov, NCT03439839. FINDINGS: Between May 31, 2018, and April 9, 2019, ten patients had twice daily 200 mg iptacopan. Iptacopan resulted in marked reduction of lactate dehydrogenase from baseline versus at week 13 (mean 539 IU/L [SD 263] vs 235 IU/L [44], change from baseline -309·2 IU/L [SD 265·5], 90% CI -473·77 to -144·68, p=0·0081), associated with significant improvement of haemoglobin concentrations (mean 97·7 g/L [SD 10·5] vs 129·5 g/L [18·3] change from baseline 31·9 g/L [14·5], 90% CI 23·42-40·28, p<0·0001). All biomarkers of haemolysis improved on iptacopan treatment. Observed haematological benefits were maintained longer than the 13-week study period, throughout the study extension, including seven patients who stopped concomitant standard-of-care treatment and continued iptacopan as monotherapy. There were no deaths or treatment-related serious adverse events during the study period. Of three non-related serious adverse events, two occurred in the same patient (one during run-in and before exposure to iptacopan). INTERPRETATION: Iptacopan at a chronic dose of 200 mg twice daily was well tolerated without any major drug-related safety findings and shows lactate dehydrogenase reduction and haemoglobin normalisation in most patients with paroxysmal nocturnal haemoglobinuria at week 13 and beyond, even in monotherapy. On the basis of these data, iptacopan will be tested as monotherapy in pivotal trials investigating its haematological benefit in a broader paroxysmal nocturnal haemoglobinuria population. FUNDING: Novartis Institutes for Biomedical Research.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Factor B del Complemento/antagonistas & inhibidores , Inactivadores del Complemento/uso terapéutico , Hemoglobinuria Paroxística/tratamiento farmacológico , Hemólisis , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/farmacología , Biomarcadores/sangre , Factor B del Complemento/metabolismo , Inactivadores del Complemento/farmacología , Quimioterapia Combinada , Eritrocitos/citología , Eritrocitos/metabolismo , Femenino , Hemoglobinas/análisis , Hemólisis/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. RESULTS: We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. CONCLUSIONS: We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Silenciador del Gen , Adolescente , Adulto , Niño , Inmunoprecipitación de Cromatina , Epigénesis Genética/genética , Epigenómica , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Marcadores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Interferencia de ARN , Adulto JovenRESUMEN
Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel(-/-)) animals were crossed with mice lacking Apolipoprotein E (ApoE(-/-)). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE(-/-)L-sel(-/-)) mice and the corresponding ApoE(-/-) controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE(-/-)L-sel(-/-) mice as compared to ApoE(-/-) controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE(-/-)L-sel(-/-) animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE(-/-)L-sel(-/-) and ApoE(-/-) mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE(-/-)L-sel(-/-) as compared to ApoE(-/-) mice. Leukocyte rolling on lesions in the aorta was similar in ApoE(-/-)L-sel(-/-) and ApoE(-/-) animals. ApoE(-/-)L-sel(-/-) mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE(-/-) controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Eliminación de Gen , Selectina L/metabolismo , Animales , Apolipoproteínas E/metabolismo , Aterosclerosis/inmunología , Aterosclerosis/patología , Quimiocina CCL2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Rodamiento de Leucocito , Recuento de Linfocitos , Ratones , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/patología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Cyclophilin A (CyPA) is a cytoplasmic protein secreted under inflammatory conditions. Extracellular CyPA is detected in atherosclerotic plaques and has been observed to activate endothelial cells as well as monocytes. METHODS AND RESULTS: Commercially available recombinant CyPA-induced expression of tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAEC). However, CyPA from commercial sources contained lipopolysaccharide at concentrations up to 18.9 ng/ml; moreover, it exhibited low purity as determined by protein spectrum analysis and low activity as assessed by peptidyl prolyl cis-trans isomerase (PPIase) assay. An in-house preparation of pure, active, and uncontaminated CyPA failed to induce endothelial TF or VCAM-1 expression; moreover, it was not chemotactic for HAEC. In contrast, such CyPA exhibited potent chemotactic activity on monocytic THP-1 cells, with a maximal effect on migration occurring at a concentration of 5.5 x 10(-9)mol/l. Pretreatment of CyPA with cyclosporine A prevented its effect on THP-1 cell migration; similarly, PPIase-deficient mutant CyPA protein did not induce migration of these cells. In-house prepared CyPA induced the release of Il-6, but not TNF-alpha, from THP-1 cells. CONCLUSIONS: Commercially available CyPA exhibits low purity and activity and may be contaminated by endotoxin. Pure, active, and uncontaminated CyPA does not induce endothelial TF or VCAM-1 expression; instead, it acts as a potent monocyte chemoattractant and induces monocyte Il-6 release, implying a role for extracellular CyPA in the pathogenesis of atherosclerosis via activation of monocytes rather than endothelial cells.
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Ciclofilina A/química , Ciclofilina A/fisiología , Contaminación de Medicamentos , Células Endoteliales/fisiología , Monocitos/fisiología , Aorta/citología , Células Cultivadas , Factores Quimiotácticos/fisiología , Ciclofilina A/biosíntesis , Células Endoteliales/efectos de los fármacos , Humanos , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Tromboplastina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
OBJECTIVE: Nitric oxide (NO) inhibits thrombus formation, vascular contraction, and smooth muscle cell proliferation. We investigated whether NO release is enhanced after endothelial NO synthase (eNOS) gene transfer in atherosclerotic human carotid artery ex vivo. METHODS AND RESULTS: Western blotting and immunohistochemistry revealed that transduction enhanced eNOS expression; however, neither nitrite production nor NO release measured by porphyrinic microsensor was altered. In contrast, transduction enhanced NO production in non-atherosclerotic rat aorta and human internal mammary artery. In transduced carotid artery, calcium-dependent eNOS activity was minimal and did not differ from control conditions. Vascular tetrahydrobiopterin concentrations did not differ between the experimental groups. Treatment of transduced carotid artery with FAD, FMN, NADPH, L-arginine, and either sepiapterin or tetrahydrobiopterin did not alter NO release. Superoxide formation was similar in transduced carotid artery and control. Treatment of transduced carotid artery with superoxide dismutase (SOD), PEG-SOD, PEG-catalase did not affect NO release. CONCLUSIONS: eNOS transduction in atherosclerotic human carotid artery results in high expression without any measurable activity of the recombinant protein. The defect in the atherosclerotic vessels is neither caused by cofactor deficiency nor enhanced NO breakdown. Since angioplasty is performed in atherosclerotic arteries,eNOS gene therapy is unlikely to provide clinical benefit.
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Aterosclerosis/metabolismo , Arterias Carótidas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Adenoviridae/genética , Anciano , Animales , Aorta/enzimología , Aorta/metabolismo , Aterosclerosis/enzimología , Aterosclerosis/genética , Arterias Carótidas/enzimología , Línea Celular , Femenino , Vectores Genéticos , Humanos , Masculino , Arterias Mamarias/enzimología , Arterias Mamarias/metabolismo , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/genética , Ratas , Superóxidos/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Transducción GenéticaRESUMEN
Local strategies directed against vascular smooth muscle cell (VSMC) proliferation such as drug-eluting stents reduce the occurrence of restenosis. However, these approaches may also inhibit endothelial cell (EC) proliferation and, thus, impair reendothelialization. We compared the effects of tacrolimus on human VSMC and EC proliferation and migration to sirolimus, a compound with similar molecular structure. Thymidine incorporation was determined in growth factor-stimulated VSMC and EC. The drug concentration at which maximal VSMC proliferation was inhibited by 50% (IC50) was about 10-fold higher for tacrolimus (3.8 x 10 M) than for sirolimus (4.1 x 10 M; P = 0.055). It is interesting that the molar IC50 value in EC was around 10-fold higher for tacrolimus (2.3 x 10 M) than for sirolimus (7.1 x 10 M; P < 0.01). The profile of these drugs on VSMC and EC migration was similar to the one found in the proliferation assays. Inhibition of VSMC proliferation by both tacrolimus and sirolimus was associated with upregulation of the cell-cycle inhibitor p27. Thus, tacrolimus is less potent than sirolimus for inhibiting VSMC proliferation or migration. However, tacrolimus exerts markedly less antiproliferative effects on EC compared with sirolimus. In combination with its potent antiinflammatory effects, tacrolimus may represent a promising compound for the use in drug-eluting stents.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Sirolimus/farmacología , Tacrolimus/farmacología , Western Blotting , Recuento de Células/métodos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , ADN/antagonistas & inhibidores , ADN/biosíntesis , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Inmunosupresores/farmacología , Concentración 50 Inhibidora , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Timidina/metabolismo , TritioRESUMEN
In vitro studies suggest a role for c-Jun N-terminal kinases (JNKs) in proatherogenic cellular processes. We show that atherosclerosis-prone ApoE-/- mice simultaneously lacking JNK2 (ApoE-/- JNK2-/- mice), but not ApoE-/- JNK1-/- mice, developed less atherosclerosis than do ApoE-/- mice. Pharmacological inhibition of JNK activity efficiently reduced plaque formation. Macrophages lacking JNK2 displayed suppressed foam cell formation caused by defective uptake and degradation of modified lipoproteins and showed increased amounts of the modified lipoprotein-binding and -internalizing scavenger receptor A (SR-A), whose phosphorylation was markedly decreased. Macrophage-restricted deletion of JNK2 was sufficient to decrease atherogenesis. Thus, JNK2-dependent phosphorylation of SR-A promotes uptake of lipids in macrophages, thereby regulating foam cell formation, a critical step in atherogenesis.