RESUMEN

Endothelin is a potent vasoconstrictor peptide isolated from endothelial cells and it induces smooth muscle cell proliferation. Endothelin-1 secretion is increased in atheroma and induces deleterious effects such as vasospasm and atherosclerosis. Oxidized low-density lipoproteins (LDLs) induce atherosclerosis in the vascular wall, as well as endothelin-1 secretion in endothelial cells and are activators of both peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and PPAR-gamma. PPAR-alpha (fibric acids) and PPAR-gamma (glitazones) activators are used to treat dyslipoproteinemias and type 2 diabetes, respectively. Furthermore, these drugs induce numerous pleiotropic effects, such as inhibiting thrombin-induced endothelin-1 secretion in endothelial cells. This study shows that both PPAR-alpha (Wy 14643) and PPAR-gamma activation (rosiglitazone) partially inhibit oxidized LDL-induced protein kinase C activity and endothelin-1 secretion in endothelial cells at the transcriptional levels and suggests that synthetic PPAR activators are stronger PPAR activators than oxidized LDL. This study also suggests that fibrate and glitazone treatments should have beneficial effects on the vascular wall by reducing endothelin-1 secretion and the resulting vasospasm and atherosclerosis.

Asunto(s)

Endotelina-1/metabolismo , Endotelio Vascular/efectos de los fármacos , Lipoproteínas LDL/farmacología , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Lipoproteínas LDL/antagonistas & inhibidores , Oxidación-Reducción , Proteína Quinasa C/metabolismo

RESUMEN

Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses.

Asunto(s)

Células Dendríticas/fisiología , Monocitos/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Tiazolidinedionas , Factores de Transcripción/fisiología , Quimiocina CCL4 , Quimiocina CCL5/biosíntesis , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Humanos , Interleucina-12/biosíntesis , Proteínas Inflamatorias de Macrófagos/biosíntesis , Receptores Citoplasmáticos y Nucleares/análisis , Rosiglitazona , Células TH1/fisiología , Tiazoles/farmacología , Factores de Transcripción/análisis

RESUMEN

The nature of the interactions between the intravascular parasite Schistosoma mansoni and the host pulmonary vasculature is critical in determining the outcome of infection. In this report, we show that lung schistosomula selectively induce the synthesis of IL-6 mRNA and protein in cultured human and mouse lung microvascular endothelial cells (EC) and that parasite excretory/secretory lipophilic compounds, particularly prostaglandin E(2), are responsible for this effect. In vivo, a striking increase of IL-6 expression is observed in the pulmonary microvasculature of S. mansoni-infected C57BL/6 mice suggesting that, in vivo, parasites also induce the synthesis of IL-6 in lung EC. In infected mice, IL-6 deficiency results in an accelerated mobilization of eosinophils into the lung tissue and in a dramatic increased number of recruited leukocytes, particularly eosinophils, in the airway. This effect is associated with an enhanced production of eotaxin (CCL11) and IL-5 in the lungs of IL-6 knockout (KO) animals. Finally, compared to wild-type mice, we detect a dramatic increased level of parasite mortality in the lungs of IL-6 KO mice. Taken together, we suggest that parasite larvae activate EC to produce IL-6 to escape the inflammatory reaction that develops in the lungs of infected hosts. Finally, we show that the parasite-induced IL-6 synthesis is mediated by a protein kinase A-dependent pathway that principally targets the cAMP-response element and the nuclear factor-kappaB sites from the -256/+20 region of the IL-6 promoter.

Asunto(s)

Quimiocinas CC , Endotelio Vascular/inmunología , Interleucina-6/genética , Interleucina-6/fisiología , Eosinofilia Pulmonar/inmunología , Schistosoma mansoni/patogenicidad , Esquistosomiasis mansoni/inmunología , Animales , Capilares/citología , Capilares/inmunología , Células Cultivadas , Quimiocina CCL11 , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Citocinas/biosíntesis , Citocinas/genética , Dinoprostona/fisiología , Humanos , Interleucina-5/biosíntesis , Interleucina-5/genética , Interleucina-6/biosíntesis , Pulmón/citología , Pulmón/inmunología , Pulmón/parasitología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Eosinofilia Pulmonar/parasitología , ARN Mensajero/biosíntesis , Elementos de Respuesta , Esquistosomiasis mansoni/parasitología , Activación Transcripcional

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARalpha is highly expressed in liver, skeletal muscle, kidney, heart and the vascular wall. PPARgamma is predominantly detected in adipose tissue, intestine and macrophages. PPARs are activated by fatty-acid derivatives and pharmacological agents such as fibrates and glitazones which are specific for PPARalpha and PPARgamma respectively. PPARs regulate lipid and lipoprotein metabolism, glucose homeostasis, cell proliferation and differentiation, and apoptosis. PPARalpha controls intra- and extracellular lipid metabolisms whereas PPARgamma triggers adipocyte differentiation and promotes lipid storage. In addition, PPARs also modulate the inflammatory response. PPAR activators have been shown to exert anti-inflammatory activities in various cell types by inhibiting the expression of proinflammatory genes such as cytokines, metalloproteases and acute-phase proteins. PPARs negatively regulate the transcription of inflammatory response genes by antagonizing the AP-1, nuclear factor-kappaB (NF-kappaB), signal transducer and activator of transcription and nuclear factor of activated T-cells signalling pathways and by stimulating the catabolism of proinflammatory eicosanoids. These recent findings indicate a modulatory role for PPARs in inflammation with potential therapeutical applications in chronic inflammatory diseases.

Asunto(s)

Inflamación/fisiopatología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Transducción de Señal/fisiología

RESUMEN

Retinoid-related orphan receptor alpha (ROR alpha) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the ROR alpha gene, suffer from immune abnormalities, we generated an adenovirus encoding ROR alpha1 to investigate its potential role in control of the inflammatory response. We demonstrated that ROR alpha is expressed in human primary smooth-muscle cells and that ectopic expression of ROR alpha1 inhibits TNFalpha-induced IL-6, IL-8 and COX-2 expression in these cells. ROR alpha1 negatively interferes with the NF-kappaB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of ROR alpha1 on NF-kappaB is associated with the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signalling pathway, whose expression was found to be transcriptionally upregulated by ROR alpha1 via a ROR response element in the IkappaB alpha promoter. Taken together, these data identify ROR alpha1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Asunto(s)

Proteínas de Unión al Calcio , Proteínas I-kappa B , Inflamación/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Transactivadores/fisiología , Adenoviridae/genética , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Ciclooxigenasa 2 , ADN Complementario/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Isoenzimas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Músculo Liso/citología , Músculo Liso/metabolismo , Músculo Liso Vascular/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Prostaglandina-Endoperóxido Sintasas/metabolismo , Unión Proteica , ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sinaptotagmina I , Sinaptotagminas , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba

RESUMEN

Regulation of the expression of platelet-activating factor (PAF) receptor by atherogenic lipoproteins might contribute to atherogenesis. We show that progressive oxidation of low-density lipoprotein (LDL) gradually inhibits PAF receptor expression on the macrophage cell surface. We tested the effect of oxidized LDL (oxLDL) on PAF receptor expression in human monocytes that do not contain peroxisome-proliferator-activated receptor gamma (PPARgamma), a nuclear receptor activated by oxLDL. OxLDL decreased by 50% (P < or = 0.001) and by 29% (P < or = 0.05) the binding of PAF and the expression of PAF receptor mRNA respectively. Next we demonstrated that progressive oxidation of LDLs significantly activated PPARalpha-dependent transcription in transfected mouse aortic endothelial cells. Finally we demonstrated, in mature macrophages, that fenofibrate (20 microM), a specific PPARalpha agonist, but not the specific PPARgamma agonist BRL49653 (20 nM), significantly decreased both PAF binding and PAF receptor mRNA expression, by 65% and 40% (P < or = 0.001) respectively. Additionally, another PPARalpha agonist, Wy14,643, decreased PAF receptor promoter activity by 70% (P < or = 0.05) in transfected THP-1 cells, suggesting the involvement of the proximal promoter region (-980 to -500) containing a series of four nuclear factor (NF)-kappaB motifs. Thus PPARalpha might be involved in the down-regulation of PAF receptor gene expression by oxLDLs in human monocytes/macrophages. The oxidation of one or more lipid components of LDLs might result in the formation of natural activators of PPARalpha. It is hypothesized that such activators might modulate inflammation and apoptosis upon atherogenesis by decreasing the expression of PAF receptor.

Asunto(s)

Regulación hacia Abajo/fisiología , Lipoproteínas LDL/fisiología , Macrófagos/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Receptores de Superficie Celular , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores Acoplados a Proteínas G , Factores de Transcripción/fisiología , Células Cultivadas , Humanos , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. They are divided into three subtypes (alpha, beta or delta, and gamma) and are involved in lipid and glucose homeostasis and in the control of inflammation. In this study, we analyzed the expression of PPARs in murine dendritic cells (DCs), the most potent antigen presenting cells. We find that immature as well as mature spleen-derived DCs express PPARgamma, but not PPARalpha, mRNA and protein. We also show that the PPARgamma activator rosiglitazone does not interfere with the maturation of DCs in vitro nor modifies their ability to activate naive T lymphocytes in vivo. Finally, we present evidence that PPARgamma activators down-modulate the CD40-induced secretion of interleukin-12, a potent Th1-driving factor. These data suggest a possible role for PPARgamma in the regulation of immune responses.

Asunto(s)

Células Dendríticas/metabolismo , Interleucina-12/biosíntesis , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazolidinedionas , Factores de Transcripción/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos CD40/metabolismo , Antígenos CD40/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dimetilsulfóxido/farmacología , Interferón gamma/metabolismo , Interleucinas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , ARN Mensajero/biosíntesis , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Rosiglitazona , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tiazoles/farmacología , Factores de Transcripción/agonistas , Factores de Transcripción/genética

RESUMEN

Chronic inflammation is a hallmark of degenerative diseases such as atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily, which are expressed in the cells of the atherosclerosic lesion. PPARalpha ligands have been reported to exert anti-inflammatory activities in different cell types by antagonizing the transcriptional activity of NF-kappaB. In the present study, the influence of PPARalpha activators on the NF-kappaB signaling pathway was investigated. Our results show that fibrates, synthetic PPARalpha activators, induced the expression of the inhibitory protein IkappaBalpha in human aortic smooth muscle cells as well as in primary human hepatocytes, whereas neither IkappaB-kinase activity nor the degradation rate of IkappaBalpha were affected. Using PPARalpha-null mice, we demonstrated that fibrates induced IkappaBalpha in liver in vivo and that this action required PPARalpha. Furthermore, fibrate treatment induced IkappaBalpha protein expression in the cytoplasm and also enhanced IL-1beta-induced accumulation of IkappaBalpha protein in the nucleus. These actions of fibrates on IkappaBalpha expression were accompanied by a decrease in NF-kappaB DNA binding activity as demonstrated by electrophoretic mobility shift assays. Taken together, these data provide an additional molecular mechanism for the anti-inflammatory activity of PPARalpha agonists and reinforce their potential use in the treatment of inflammatory diseases.

Asunto(s)

Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Inflamación/fisiopatología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Transducción de Señal

RESUMEN

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a transcription factor belonging to the PPAR subfamily of nuclear receptors. Fatty acids and eicosanoids are natural PPARalpha ligands. Here, we show using transient transfection assays that oxidized (oxLDL) but not native low-density lipoproteins (LDL) dose-dependently activate PPARalpha in endothelial cells without affecting PPARalpha protein expression. Fractioning of oxLDL lipids followed by transactivation experiments demonstrated that the oxidized phospholipid component in oxLDL is responsible for PPARalpha activation. Using specific inhibitors, it is shown that oxLDL-mediated PPARalpha activation requires phospholipase A2 activity and that the oxidized fatty acids 9- and 13-HODE activate PPARalpha directly. Finally, we found that, similar to the synthetic PPARalpha ligand Wy-14643, oxLDL induced expression of the fatty acid transport protein-1 in human primary endothelial cells. Our findings define a novel group of PPARalpha activators and provide a molecular basis for certain effects of these biologically active phospholipids on gene transcription.

Asunto(s)

Regulación de la Expresión Génica , Proteínas de Transporte de Membrana , Fosfolipasas A/metabolismo , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Proteínas Portadoras/genética , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteínas de Transporte de Ácidos Grasos , Humanos , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana/genética , Fosfolipasas A2 , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional

RESUMEN

Oncostatin M (OSM), a cytokine first identified from activated monocytes and T lymphocytes, is one of the most potent autocrine growth factor for AIDS and Kaposi's sarcoma. Little is known about the effects of OSM on normal vascular cells. We thus exposed human aortic smooth muscle cells (hASMCs) to OSM, examined cell proliferation and morphology, and determined interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) expression. OSM had a weak antiproliferative effect. After a 4-day incubation with 100 ng/mL OSM, cell count decreased to 69+/-3% of control. However, OSM induced striking changes in hASMC morphology, characterized by a polyclonal shape, in contrast to the spindle morphological feature of control hASMCs. OSM stimulated the release of IL-6 by hASMCs in a dose-dependent way; after a 48-hour exposure, values were 8.5+/-0.7, 29.7+/-3.5, 50.9+/-4.4, and 73.8+/-7.6x10(3) U/mL (n=6) at OSM concentrations of 0, 1, 10, and 100 ng/mL, respectively. OSM induced marked expression of COX-2 protein and mRNA. Leukemia inhibitory factor had no effect on hASMCs, indicating that OSM effects on hASMCs were mediated by the OSM type II receptor and not by the leukemia inhibitory factor receptor. OSM used the JAK/STAT signaling pathway, as demonstrated by rapid phosphorylation of JAK1 and specific activation of STAT1. Interestingly, OSM acted in synergy with IL-1beta on IL-6 production and COX-2 expression. In conclusion, OSM is a novel regulator of human smooth muscle cell functions, acting in concert with IL-1beta, and OSM may play a role in major vascular diseases such as atherosclerosis.

Asunto(s)

Inhibidores de Crecimiento/farmacología , Interleucina-1/farmacología , Interleucina-6/biosíntesis , Isoenzimas/biosíntesis , Músculo Liso Vascular/metabolismo , Péptidos/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Células Cultivadas , Ciclooxigenasa 2 , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana , Oncostatina M

RESUMEN

Interleukin-6 (IL-6) is a pleiotropic cytokine, whose plasma levels are elevated in inflammatory diseases such as atherosclerosis. We have previously reported that peroxisome proliferator-activated receptor alpha (PPARalpha) ligands (fibrates) lower elevated plasma concentrations of IL-6 in patients with atherosclerosis and inhibit IL-1-stimulated IL-6 secretion by human aortic smooth muscle cells (SMC). Here, we show that aortic explants isolated from PPARalpha-null mice display an exacerbated response to inflammatory stimuli, such as lipopolysaccharide (LPS), as demonstrated by increased IL-6 secretion. Furthermore, fibrate treatment represses IL-6 mRNA levels in LPS-stimulated aortas of PPARalpha wild-type, but not of PPARalpha-null mice, demonstrating a role for PPARalpha in this fibrate action. In human aortic SMC, fibrates inhibit IL-1-induced IL-6 gene expression. Furthermore, activation of PPARalpha represses both c-Jun- and p65-induced transcription of the human IL-6 promoter. Transcriptional interference between PPARalpha and both c-Jun and p65 occurs reciprocally, since c-Jun and p65 also inhibit PPARalpha-mediated activation of a PPAR response element-driven promoter. This transcriptional interference occurs independent of the promoter context as demonstrated by cotransfection experiments using PPARalpha, p65, and c-Jun Gal4 chimeras. Overexpression of the transcriptional coactivator cAMP-responsive element-binding protein-binding protein (CBP) does not relieve PPARalpha-mediated transcriptional repression of p65 and c-Jun. Finally, glutathione S-transferase pull-down experiments demonstrate that PPARalpha physically interacts with c-Jun, p65, and CBP. Altogether these data indicate that fibrates inhibit the vascular inflammatory response via PPARalpha by interfering with the NF-kappaB and AP-1 transactivation capacity involving direct protein-protein interaction with p65 and c-Jun.

Asunto(s)

FN-kappa B/fisiología , Flebitis/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/fisiología , Animales , Células COS , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación hacia Abajo , Humanos , Interleucina-1/farmacología , Interleucina-6/genética , Lipopolisacáridos/farmacología , Masculino , Ratones , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética , Transfección

RESUMEN

The recruitment of immune cells into the lungs is a key step in protection against murine schistosomiasis. In this phenomenon, pulmonary (micro)vascular endothelial cells (EC) probably play a central role, by expressing specific adhesion molecules on their surface. Recently, we have shown that Schistosoma mansoni schistosomula, the parasitic stage which resides in the lungs, could activate microvascular EC to acquire an anti-inflammatory phenotype. In the present study, we tested the hypothesis that schistosomula could also regulate the expression of adhesion molecules in vitro by human lung microvascular EC (HMVEC-l) in the present of the pro-inflammatory cytokine TNF-alpha. We found that lipophilic substance(s) present in the excretory/secretory products from schistosomula selectively reduce the TNF-alpha-induced synthesis of E-selectin and VCAM-1 mRNA and proteins without affecting ICAM-1. This inhibitory effect appears to be mediated by a cyclic AMP/protein kinase A (cAMP/PKA) pathway that probably interferes with the NF-kappaB pathway induced by TNF-alpha at the level of the E-selectin promoter, whereas a cAMP-independent pathway appears to operate in VCAM-1 down-modulation. Finally, schistosomula also significantly reduce the VLA-4/VCAM-1-dependent adherence of leukocytes to TNF-alpha-stimulated HMVEC-l. We speculate that this mechanism could represent a new stratagem that parasites may use to escape the immune system by controlling leukocyte recruitment to the lungs.

Asunto(s)

Selectina E/biosíntesis , Endotelio Vascular/inmunología , Pulmón/inmunología , FN-kappa B/inmunología , Schistosoma mansoni/inmunología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Animales , Adhesión Celular , Células Cultivadas , AMP Cíclico/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico , Selectina E/genética , Endotelio Vascular/citología , Expresión Génica , Células HL-60 , Humanos , Integrina alfa4beta1 , Integrinas/inmunología , Pulmón/irrigación sanguínea , Fosforilación , Proteínas Serina-Treonina Quinasas/inmunología , ARN Mensajero , Receptores Mensajeros de Linfocitos/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/genética

RESUMEN

Endothelin-1 (ET-1), a 21-amino acid vasoactive peptide mainly produced by vascular endothelial cells, is involved in the regulation of vascular tone and smooth muscle cell proliferation. Peroxisome proliferator-activated receptors (PPARs), key players in lipid and glucose metabolism, have been implicated in metabolic disorders that are predisposing to atherosclerosis. Because of the potential role of ET-1 in vascular disorders such as hypertension and atherosclerosis, we investigated the regulation of ET-1 expression by PPAR activators. Western blot and reverse transcription-polymerase chain reaction analyses demonstrated that both PPARalpha and PPARgamma are expressed in human coronary artery endothelial cells as well as in endothelial cell lines such as HMEC-1 and ECV304. In bovine aortic endothelial cells and HMEC-1 cells, both PPARalpha and PPARgamma ligands inhibited thrombin-induced ET-1 secretion, whereas basal ET-1 secretion was only slightly suppressed. Reverse transcription-polymerase chain reaction experiments showed that this inhibition of ET-1 production occurs at the gene expression level. Using transient transfection assays, we demonstrated that PPARs downregulate thrombin-activated transcription of the human ET-1 promoter. Transactivation studies with c-Jun and c-Fos expression plasmids indicated that PPARs negatively interfere with the activator protein-1 signaling pathway, which mediates thrombin activation of ET-1 gene transcription. Furthermore, electrophoretic mobility shift assays demonstrated that PPAR activators reduce the thrombin-stimulated binding activity of bovine aortic endothelial cell nuclear extracts as well as c-Jun binding to an activator protein-1 consensus site. Taken together, these data indicate that (1) both PPARalpha and PPARgamma are expressed in human vascular endothelial cells and (2) PPAR activators inhibit thrombin-induced ET-1 biosynthesis, indicating a novel role for PPARs in vascular endothelial function.

Asunto(s)

Endotelina-1/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Proliferadores de Peroxisomas/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Transducción de Señal/efectos de los fármacos , Trombina/antagonistas & inhibidores , Factor de Transcripción AP-1/antagonistas & inhibidores , Factores de Transcripción/fisiología , Animales , Aorta/citología , Arteriosclerosis/fisiopatología , Capilares/citología , Bovinos , Vasos Coronarios/citología , ADN/genética , ADN/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/fisiología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombina/farmacología , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/efectos de los fármacos , Transcripción Genética , Transfección

RESUMEN

The purpose of the present investigation was to determine whether the beneficial effects of polyunsaturated fatty acids (PUFA) may influence ischemia-reperfusion-induced alterations of myocardial alpha- and beta-adrenoceptor (alpha-AR, beta-AR) responsiveness. This study was carried out using monolayer cultures of neonatal rat ventricular myocytes in a substrate-free, hypoxia-reoxygenation model of ischemia. The cardiomyocytes (CM) were incubated during 4 days in media enriched either with n-6 PUFA (arachidonic acid, AA) or with n-3 PUFA (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA). The n-6/n-3 ratio in n-3 CM was close to 1.2, compared to 20.1 in n-6 CM. The contractile parameters of n-6 CM and n-3 CM were similar in basal conditions as well as during hypoxia and reoxygenation. In basal conditions, the phospholipid (PL) enrichment with long chain n-3 PUFA resulted in an increased chronotropic response to isoproterenol (ISO) and to phenylephrine (PHE). After posthypoxic reoxygenation, the chronotropic response to beta-AR activation in n-6 CM was significantly enhanced as compared with the control response in normoxia. In opposition, the ISO-induced rise in frequency in n-3 CM in control normoxia and after reoxygenation was similar. In these n-3 CM, the changes in contractile parameters, which accompanied the chronotropic response, were also similar in reoxygenation and in normoxic periods, although the rise in shortening velocity was slightly increased after reoxygenation. In response to PHE addition, only the chronotropic effect of n-6 CM appeared significantly enhanced after hypoxic treatment. These results suggested that increasing n-3 PUFA in PL reduced the increase in alpha- and beta-AR functional responses observed after hypoxia-reoxygenation. This effect may partly account for the assumed cardiac protective effect of n-3 PUFA, through the attenuation of the functional response to catecholamines in the ischemic myocardium.

Asunto(s)

Hipoxia de la Célula , Ácidos Grasos Insaturados/metabolismo , Lípidos de la Membrana/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Células Cultivadas , Contracción Miocárdica , Isquemia Miocárdica/metabolismo , Miocardio/citología , Oxígeno/metabolismo , Fosfolípidos/metabolismo , Ratas , Ratas Wistar

RESUMEN

The polyunsaturated fatty acids (PUFAs) of the omega 3 series are known to modulate adrenergic functions in ventricular myocytes. This study evaluated the influence of hypoxia duration and PUFA composition on the ability of cultured rat cardiomyocytes in producing alpha- and beta-adrenergic messengers (IPs and cAMP). After hypoxia (1.5, 2.5 or 3.5 h) followed by reoxygenation (1h). IP and cAMP production was induced by phenylephrine or isoproterenol stimulation, respectively. Hypoxia did not affect the basal level of messenger production in unstimulated cells, but decreased the cAMP production elicited by isoproterenol stimulation (up to 50%). The decrease in IP production after phenylephrine stimulation was observed only after long-term hypoxia duration close to irreversible cellular damages. The use of modified culture media supplemented with either arachidonic acid (AA) or docosahexaenoic acid (DHA) induced cardiomyocytes displaying either an arachidonic acid membrane profile (35% AA and 2% DHA in the phospholipids) or a docosahexaenoic acid membrane profile (15% AA and 20% DHA). These modifications did not alter the basal level of either messenger production in unstimulated cells nor the IP released after alpha-adrenergic stimulation. Conversely, the decrease in cAMP production was significantly more pronounced in docosahexaenoic acid-enriched cells than in arachidonic acid-enriched cells. This study suggests that hypoxia alters the beta-adrenergic messenger production, and that the alpha-system may balance the depression of the beta-system. The depression of the beta-adrenergic function induced by the incorporation of docosahexaenoic acid in membrane phospholipids may contribute to the beneficial effect of this fatty acid in the reperfused heart.

Asunto(s)

Ácidos Grasos Insaturados/metabolismo , Miocardio/citología , Oxígeno , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Hipoxia de la Célula , Células Cultivadas , Líquido Intracelular/metabolismo , Fosfolípidos/metabolismo , Ratas , Ratas Wistar

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) have been implicated in metabolic diseases, such as obesity, diabetes, and atherosclerosis, due to their activity in liver and adipose tissue on genes involved in lipid and glucose homeostasis. Here, we show that the PPARalpha and PPARgamma forms are expressed in differentiated human monocyte-derived macrophages, which participate in inflammation control and atherosclerotic plaque formation. Whereas PPARalpha is already present in undifferentiated monocytes, PPARgamma expression is induced upon differentiation into macrophages. Immunocytochemistry analysis demonstrates that PPARalpha resides constitutively in the cytoplasm, whereas PPARgamma is predominantly nuclear localized. Transient transfection experiments indicate that PPARalpha and PPARgamma are transcriptionally active after ligand stimulation. Ligand activation of PPARgamma, but not of PPARalpha, results in apoptosis induction of unactivated differentiated macrophages as measured by the TUNEL assay and the appearance of the active proteolytic subunits of the cell death protease caspase-3. However, both PPARalpha and PPARgamma ligands induce apoptosis of macrophages activated with tumor necrosis factor alpha/interferon gamma. Finally, PPARgamma inhibits the transcriptional activity of the NFkappaB p65/RelA subunit, suggesting that PPAR activators induce macrophage apoptosis by negatively interfering with the anti-apoptotic NFkappaB signaling pathway. These data demonstrate a novel function of PPAR in human macrophages with likely consequences in inflammation and atherosclerosis.

Asunto(s)

Apoptosis/fisiología , Macrófagos/citología , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazolidinedionas , Factores de Transcripción/metabolismo , Arteriosclerosis/fisiopatología , Caspasa 3 , Caspasas/metabolismo , Diferenciación Celular/fisiología , Humanos , Inmunohistoquímica , Inflamación/fisiopatología , Interferón gamma/farmacología , Ligandos , FN-kappa B/metabolismo , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Rosiglitazona , Transducción de Señal/fisiología , Tiazoles/farmacología , Transcripción Genética/genética , Transfección/genética , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) are key players in lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as dyslipidaemia and diabetes. Whereas PPARgamma promotes lipid storage by regulating adipocyte differentiation, PPARalpha stimulates the beta-oxidative degradation of fatty acids. PPARalpha-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARalpha is also a modulator of inflammation. Hypolipidaemic fibrate drugs are PPARalpha ligands that inhibit the progressive formation of atherosclerotic lesions, which involves chronic inflammatory processes, even in the absence of their atherogenic lipoprotein-lowering effect. Here we show that PPARalpha is expressed in human aortic smooth-muscle cells, which participate in plaque formation and post-angioplasty re-stenosis. In these smooth-muscle cells, we find that PPARalpha ligands, and not PPARgamma ligands, inhibit interleukin-1-induced production of interleukin-6 and prostaglandin and expression of cyclooxygenase-2. This inhibition of cyclooxygenase-2 induction occurs transcriptionally as a result of PPARalpha repression of NF-kappaB signalling. In hyperlipidaemic patients, fenofibrate treatment decreases the plasma concentrations of interleukin-6, fibrinogen and C-reactive protein. We conclude that activators of PPARalpha inhibit the inflammatory response of aortic smooth-muscle cells and decrease the concentration of plasma acute-phase proteins, indicating that PPARalpha in the vascular wall may influence the process of atherosclerosis and re-stenosis.

Asunto(s)

Aorta , Músculo Liso Vascular/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Tiazolidinedionas , Factores de Transcripción/fisiología , Proteínas de Fase Aguda/metabolismo , Animales , Antiinflamatorios/farmacología , Aorta/citología , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/patología , Células COS , Enfermedad Coronaria/sangre , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Fenofibrato/farmacología , Gemfibrozilo/farmacología , Regulación Enzimológica de la Expresión Génica , Humanos , Hiperlipidemias/sangre , Hipolipemiantes/farmacología , Inflamación/enzimología , Inflamación/metabolismo , Interleucina-1/metabolismo , Interleucina-6/biosíntesis , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas de la Membrana , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , FN-kappa B/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandinas/biosíntesis , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/química , Rosiglitazona , Tiazoles/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Transcripción Genética