RESUMEN
BACKGROUND: Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. RESULTS: Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. CONCLUSION: MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals.
Asunto(s)
Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Células COS , Chlorocebus aethiops , Ratones , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas ras/químicaRESUMEN
Although peroxisome proliferator-activated receptor γ (PPARγ) has anti-inflammatory actions in macrophages, which macrophage populations express PPARγ in vivo and how it regulates tissue homeostasis in the steady state and during inflammation remains unclear. We now show that lung and spleen macrophages selectively expressed PPARγ among resting tissue macrophages. In addition, Ly-6C(hi) monocytes recruited to an inflammatory site induced PPARγ as they differentiated to macrophages. When PPARγ was absent in Ly-6C(hi)-derived inflammatory macrophages, initiation of the inflammatory response was unaffected, but full resolution of inflammation failed, leading to chronic leukocyte recruitment. Conversely, PPARγ activation favored resolution of inflammation in a macrophage PPARγ-dependent manner. In the steady state, PPARγ deficiency in red pulp macrophages did not induce overt inflammation in the spleen. By contrast, PPARγ deletion in lung macrophages induced mild pulmonary inflammation at the steady state and surprisingly precipitated mortality upon infection with Streptococcus pneumoniae. This accelerated mortality was associated with impaired bacterial clearance and inability to sustain macrophages locally. Overall, we uncovered critical roles for macrophage PPARγ in promoting resolution of inflammation and maintaining functionality in lung macrophages where it plays a pivotal role in supporting pulmonary host defense. In addition, this work identifies specific macrophage populations as potential targets for the anti-inflammatory actions of PPARγ agonists.
Asunto(s)
Resistencia a la Enfermedad/inmunología , Mediadores de Inflamación/fisiología , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , PPAR gamma/fisiología , Animales , Regulación de la Expresión Génica/inmunología , Inflamación/inmunología , Inflamación/microbiología , Inflamación/prevención & control , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/uso terapéutico , Pulmón/microbiología , Macrófagos Alveolares/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , PPAR gamma/biosíntesis , PPAR gamma/deficiencia , Streptococcus pneumoniae/inmunologíaRESUMEN
Development of osteoporosis severely complicates long-term glucocorticoid (GC) therapy. Using a Cre-transgenic mouse line, we now demonstrate that GCs are unable to repress bone formation in the absence of glucocorticoid receptor (GR) expression in osteoblasts as they become refractory to hormone-induced apoptosis, inhibition of proliferation, and differentiation. In contrast, GC treatment still reduces bone formation in mice carrying a mutation that only disrupts GR dimerization, resulting in bone loss in vivo, enhanced apoptosis, and suppressed differentiation in vitro. The inhibitory GC effects on osteoblasts can be explained by a mechanism involving suppression of cytokines, such as interleukin 11, via interaction of the monomeric GR with AP-1, but not NF-kappaB. Thus, GCs inhibit cytokines independent of GR dimerization and thereby attenuate osteoblast differentiation, which accounts, in part, for bone loss during GC therapy.
Asunto(s)
Glucocorticoides/toxicidad , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Animales , Apoptosis , Diferenciación Celular , Dimerización , Interleucina-11/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/efectos de los fármacos , Receptores de Glucocorticoides/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
OBJECTIVE: Mouse aorta smooth muscle cells (SMC) express tumor necrosis factor receptor superfamily member 1A (TNFR-1) and lymphotoxin beta-receptor (LTbetaR). Circumstantial evidence has linked the SMC LTbetaR to tertiary lymphoid organogenesis in hyperlipidemic mice. Here, we explored TNFR-1 and LTbetaR signaling in cultured SMC. METHODS AND RESULTS: TNFR-1 signaling activated the classical RelA NF-kappaB pathway, whereas LTbetaR signaling activated the classical RelA and alternative RelB NF-kappaB pathways, and both signaling pathways synergized to enhance p100 inhibitor processing to the p52 subunit of NF-kappaB. Microarrays showed that simultaneous TNFR-1/LTbetaR activation resulted in elevated mRNA encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Importantly, SMC acquired features of lymphoid tissue organizers, which control tertiary lymphoid organogenesis in autoimmune diseases through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. TNFR-1/LTbetaR cross-talk resulted in augmented secretion of lymphorganogenic chemokine proteins. Supernatants of TNFR-1/LTbetaR-activated SMC markedly supported migration of splenic T cells, B cells, and macrophages/dendritic cells. Experiments with ltbr(-/-) SMC indicated that LTbetaR-RelB activation was obligatory to generate the lymphoid tissue organizer phenotype. CONCLUSIONS: SMC may participate in the formation of tertiary lymphoid tissue in atherosclerosis by upregulation of lymphorganogenic chemokines involved in T-lymphocyte, B-lymphocyte, and macrophage/dendritic cell attraction.
Asunto(s)
Diferenciación Celular/fisiología , Tejido Linfoide/citología , Receptor beta de Linfotoxina/fisiología , Miocitos del Músculo Liso/citología , FN-kappa B/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Transducción de Señal/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Aorta/citología , Aorta/efectos de los fármacos , Aorta/fisiología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Movimiento Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Tejido Linfoide/fisiología , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Blood of both humans and mice contains 2 main monocyte subsets. Here, we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation, some molecules were conversely expressed between the 2 species' subsets, including CD36, CD9, and TREM-1. Other differences included a prominent peroxisome proliferator-activated receptor gamma (PPARgamma) signature in mouse monocytes, which is absent in humans, and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus, whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized, important differences between the species deserve consideration when models of human disease are studied in mice.
Asunto(s)
Perfilación de la Expresión Génica , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Human B lymphocytes can produce leukotriene B4 but the biological function of the 5-lipoxygenase (5-LO) pathway in B cells is unclear. In order to better understand and define the role of 5-LO in B cells, we investigated the expression of 5-LO mRNA and protein in subsets of B cells from human tonsils and different types of B cell lymphoma. RESULTS: Based on RT-PCR and western blot/immunohistochemical staining, with a polyclonal antibody raised against 5-LO, high expression of 5-LO was found in mantle zone B cells from tonsils. By contrast, only a weak expression of 5-LO was detected in germinal centre cells and no expression in plasma cells from tonsils. This pattern of 5-LO expression was preserved in malignant lymphoma with high expression in mantle B cell lymphoma (MCL) and weak or no expression in follicular lymphoma. Primary leukemized MCL, so called B-prolymphocytic leukaemia cells, and MCL cell lines also expressed 5-LO and readily produced LTB4 after activation. CONCLUSION: The present report demonstrates the expression of 5-LO mainly in normal and malignant mantle zone B cells while the expression is low or absent in germinal centre B cells and plasma cells, indicating a role of the 5-LO pathway in B cells before the cells finally differentiate to plasma cells.
Asunto(s)
Araquidonato 5-Lipooxigenasa/biosíntesis , Subgrupos de Linfocitos B/enzimología , Linfocitos B/enzimología , Leucemia Prolinfocítica Tipo Células B/enzimología , Linfoma Folicular/enzimología , Linfoma de Células del Manto/enzimología , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/inmunología , Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Western Blotting , Diferenciación Celular , Línea Celular , Transformación Celular Neoplásica , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Inmunidad Celular , Memoria Inmunológica , Inmunofenotipificación , Leucemia Prolinfocítica Tipo Células B/inmunología , Leucotrieno B4/metabolismo , Activación de Linfocitos , Linfoma Folicular/inmunología , Linfoma de Células del Manto/inmunología , Microscopía Fluorescente , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Reacción en Cadena de la Polimerasa , Transducción de SeñalRESUMEN
Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.
Asunto(s)
Aorta Abdominal/crecimiento & desarrollo , Apolipoproteínas E/genética , Tejido Conectivo/crecimiento & desarrollo , Receptor beta de Linfotoxina/fisiología , Transducción de Señal/fisiología , Envejecimiento , Animales , Aorta Abdominal/metabolismo , Aterosclerosis/genética , Transporte Biológico , Células Cultivadas , Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Análisis por Conglomerados , Tejido Conectivo/metabolismo , Perfilación de la Expresión Génica , Hibridación in Situ , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/crecimiento & desarrollo , Tejido Linfoide/metabolismo , Receptor beta de Linfotoxina/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Organogénesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túnica Íntima/crecimiento & desarrollo , Túnica Íntima/metabolismo , Túnica Media/crecimiento & desarrollo , Túnica Media/metabolismoRESUMEN
Glucocorticoids (GCs) are widely used in the treatment of allergic skin conditions despite having numerous side effects. Here we use Cre/loxP-engineered tissue- and cell-specific and function-selective GC receptor (GR) mutant mice to identify responsive cell types and molecular mechanisms underlying the antiinflammatory activity of GCs in contact hypersensitivity (CHS). CHS was repressed by GCs only at the challenge phase, i.e., during reexposure to the hapten. Inactivation of the GR gene in keratinocytes or T cells of mutant mice did not attenuate the effects of GCs, but its ablation in macrophages and neutrophils abolished downregulation of the inflammatory response. Moreover, mice expressing a DNA binding-defective GR were also resistant to GC treatment. The persistent infiltration of macrophages and neutrophils in these mice is explained by an impaired repression of inflammatory cytokines and chemokines such as IL-1beta, monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and IFN-gamma-inducible protein 10. In contrast TNF-alpha repression remained intact. Consequently, injection of recombinant proteins of these cytokines and chemokines partially reversed suppression of CHS by GCs. These studies provide evidence that in contact allergy, therapeutic action of corticosteroids is in macrophages and neutrophils and that dimerization GR is required.
Asunto(s)
Dermatitis Alérgica por Contacto/tratamiento farmacológico , Dermatitis Alérgica por Contacto/inmunología , Dexametasona/administración & dosificación , Inmunosupresores/uso terapéutico , Macrófagos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Animales , Antiinflamatorios/uso terapéutico , Células Cultivadas , Dermatitis Alérgica por Contacto/patología , Dexametasona/uso terapéutico , Sistemas de Liberación de Medicamentos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/inmunología , Neutrófilos/patología , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genéticaRESUMEN
5-Lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes (LTs), which are implicated in immune reactions. Recently, it was shown that FITC-triggered epidermal Langerhans cell (LC) emigration to draining lymph nodes (LNs) is impaired in LTC4 export pump (multidrug resistance-associated protein 1)-deficient mice. Here, we sought genetic evidence for a role of endogenous LTs in dendritic cell function through the study of 5-LO-deficient mice. Though DC numbers in skin, spleen, and peripheral LNs were similar in both 5-LO-deficient and wild-type (WT) mice, DC homing from skin to draining LNs induced by FITC was reduced by 75% in 5-LO-deficient mice. Moreover, in WT mice, all epidermal LCs, dermal langerin+ LCs, and subsets of dermal macrophages and langerin+ LCs in T-cell areas of skin-draining LNs markedly expressed 5-LO. However, the enzyme was noticeably absent in all DC subsets of the dermis, thymus, spleen, Peyer's patches, mesenteric LNs, and mucosal surfaces of lung and intestine. As all epidermal cells other than LCs lacked 5-LO and because differentiation and activation of DCs generated from 5-LO-deficient mice in vitro were normal, these data support a selective role of endogenous LTs in DC homing following skin sensitization.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Movimiento Celular/fisiología , Células Dendríticas/fisiología , Células de Langerhans/enzimología , Leucotrienos/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/genética , Diferenciación Celular , Células Dendríticas/citología , Fluoresceína-5-Isotiocianato , Regulación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Intestinos/enzimología , Células de Langerhans/citología , Células de Langerhans/inmunología , Leucotrienos/genética , Pulmón/enzimología , Tejido Linfoide/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/enzimología , Timo/enzimologíaRESUMEN
Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(-)CX3CR1(++)Ly-6C(lo) monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X(3)-C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE(-/-) mice and skewed toward an increased frequency of CCR2(+)Ly-6C(hi) monocytes in apoE(-/-) mice fed a high-fat diet. CCR2(+)Ly-6C(hi) monocytes efficiently accumulated in plaques, whereas CCR2(-)Ly-6C(lo) monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2(-) monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE(-/-) mice. By comparison, CCR2(+)Ly-6C(hi) monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2(+) monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.
Asunto(s)
Aterosclerosis/sangre , Monocitos/fisiología , Receptores CCR5/fisiología , Receptores de Quimiocina/fisiología , Animales , Receptor 1 de Quimiocinas CX3C , Ratones , Modelos Animales , Monocitos/clasificación , Receptores CCR2 , Receptores CCR5/deficiencia , Receptores CCR5/genéticaRESUMEN
Activation of the 5-lipoxygenase (5-LO) pathway leads to the biosynthesis of proinflammatory leukotriene lipid mediators. Genetic studies have associated 5-LO and its accessory protein, 5-LO-activating protein, with cardiovascular disease, myocardial infarction and stroke. Here we show that 5-LO-positive macrophages localize to the adventitia of diseased mouse and human arteries in areas of neoangiogenesis and that these cells constitute a main component of aortic aneurysms induced by an atherogenic diet containing cholate in mice deficient in apolipoprotein E. 5-LO deficiency markedly attenuates the formation of these aneurysms and is associated with reduced matrix metalloproteinase-2 activity and diminished plasma macrophage inflammatory protein-1alpha (MIP-1alpha; also called CCL3), but only minimally affects the formation of lipid-rich lesions. The leukotriene LTD(4) strongly stimulates expression of MIP-1alpha in macrophages and MIP-2 (also called CXCL2) in endothelial cells. These data link the 5-LO pathway to hyperlipidemia-dependent inflammation of the arterial wall and to pathogenesis of aortic aneurysms through a potential chemokine intermediary route.
Asunto(s)
Aneurisma de la Aorta Abdominal/etiología , Araquidonato 5-Lipooxigenasa/metabolismo , Regulación de la Expresión Génica , Hiperlipidemias/complicaciones , Leucotrienos/biosíntesis , Macrófagos/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Análisis de Varianza , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Quimiocina CCL2/sangre , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CXCL1 , Quimiocinas CXC/metabolismo , Colatos , Tejido Conectivo/metabolismo , Citocinas/sangre , Cartilla de ADN , Dieta Aterogénica , Técnicas Histológicas , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucotrieno D4/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Developing drugs to treat atherosclerosis is a daunting task. However, recent studies of advanced human atherosclerotic lesions have yielded new information on potential mechanisms of inflammation and immune responses in late-stage human atherosclerosis. As leukotrienes (LTs) are among the most powerful inflammatory mediators known and because the 5-lipoxygenase pathway is expressed in diseased arteries, the roles of LTs in atherogenesis merit consideration. It is also of interest to consider pharmacological strategies to develop anti-LT drugs in atherogenesis.
Asunto(s)
Arteriosclerosis/tratamiento farmacológico , Diseño de Fármacos , Antagonistas de Leucotrieno/uso terapéutico , Araquidonato 5-Lipooxigenasa/biosíntesis , Araquidonato 5-Lipooxigenasa/genética , Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Predicción , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Antagonistas de Leucotrieno/farmacología , Inhibidores de la LipooxigenasaRESUMEN
The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 microM, TFRGAP increased [3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.
Asunto(s)
Músculo Liso Vascular/citología , Miocitos del Músculo Liso/química , Receptores de Trombina/análisis , Señalización del Calcio , Replicación del ADN/efectos de los fármacos , Humanos , Cinética , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/química , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Receptores de Trombina/genética , Receptores de Trombina/fisiología , Vena Safena , Trombina/farmacologíaRESUMEN
OBJECTIVE: Inflammatory infiltrates and atherosclerotic lesions emerge when monocytes adhere to endothelial cells (ECs), migrate into the subendothelial space, and become macrophages (MPhi(s)). Leukotrienes (LTs), products of 5-lipoxygenase, are powerful inflammatory mediators. 5-lipoxygenase+ MPhi(s) have been shown to increase during atherogenesis, and LT receptor (LT-R) transcripts were identified in diseased arteries. To investigate LT-Rs in cells involved in inflammation and atherogenesis, we used the in vitro models of human umbilical vein ECs (HUVECs) and monocyte-derived MPhi(s). METHODS AND RESULTS: HUVECs primarily expressed transcripts of the cysteinyl (cys) LT2-R, which was strongly upregulated by interleukin-4. By contrast, MPhi(s) predominantly expressed transcripts of the cysLT1-R. Calcium responses toward LTs revealed differential cysLT-R utilization by both cell types: HUVECs responded to both cysLTs, whereas MPhi(s) preferentially responded to LTD4; HUVECs, but not MPhi(s), were resistant toward a cysLT1-R antagonist, montelukast; cysLTs generated regular calcium oscillations in HUVECs that lasted >60 minutes, resulting in >500 oscillations per cell. By contrast, calcium elevations in MPhi(s) returned to baseline within seconds and were nonoscillatory. CONCLUSIONS: Our data raise the possibility that MPhi-derived LTs differentially activate cysLT2-Rs via paracrine stimulation and cysLT1-Rs via autocrine and paracrine stimulation during inflammation and atherogenesis.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Arteriosclerosis/metabolismo , Calcio/metabolismo , Endotelio Vascular/metabolismo , Macrófagos/metabolismo , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Arteriosclerosis/etiología , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Inflamación/complicaciones , Inflamación/fisiopatología , Regulación hacia ArribaRESUMEN
Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.