RESUMEN
OBJECTIVES: To (i) determine whether sustained disease activity states, as measured by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score (ASDAS), impact function, and (ii) evaluate characteristics predicting sustained low functional impairment in a prospective axial spondyloarthritis (axSpA) cohort. METHODS: Biologic Treatment Registry Across Canada (BioTRAC) was a multi-center, prospective registry that collected real-world data on axSpA patients receiving infliximab or golimumab between 2006 and 2017. Generalized estimating equations (GEE) were used to test baseline characteristics, treatment, and duration (at 6 and 12 months vs. only at 6 or 12 months vs. neither) of low BASDAI (< 3), ASDAS-inactive disease (ID)(< 1.3), and ASDAS-low disease activity (LDA) in predicting sustained low Bath Ankylosing Spondylitis Functional Index (BASFI)(< 3) between 12 and 18 months. The adjusted impact of achieving low disease state at 6 and/or 12 months on BASFI at 18 months was analyzed by generalized linear models. RESULTS: Eight hundred ten patients were enrolled. 33.7%, 13.4%, and 24.7% achieved sustained low BASDAI, ASDAS-ID, and ASDAS-LDA, respectively. In univariable GEE of baseline variables, age and baseline BASDAI, BASFI, and ASDAS significantly predicted sustained low BASFI. In multivariable GEE, sustained low BASDAI (p < 0.001), low BASDAI only at 6 or 12 months (p = 0.001), and baseline BASFI (p < 0.001) were the only predictors of sustained low BASFI. Sustained ASDAS-ID (p = 0.040) and ASDAS-LDA (p < 0.001) were also predictors when forced into the model. Similar results were obtained when evaluating the BASFI score at 18 months. CONCLUSION: Sustained BASDAI < 3 may be a valid and feasible target for a treat-to-target strategy in axSpA having function as treatment goal.
Asunto(s)
Espondiloartritis , Espondilitis Anquilosante , Humanos , Espondilitis Anquilosante/diagnóstico , Espondilitis Anquilosante/tratamiento farmacológico , Infliximab , Canadá/epidemiología , Índice de Severidad de la Enfermedad , Espondiloartritis/diagnóstico , Espondiloartritis/tratamiento farmacológicoRESUMEN
Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific proinflammatory genes. The mechanism of action of these phospholipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G protein-coupled receptors (GPCRs). However, increasing evidence suggests the existence of a functional intracellular GPCR population. It has been suggested that immediate effects are mediated by cell surface receptors, whereas long-term responses are mediated by intracellular receptors. PAF and LPA(1) receptors localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pig, rat hepatocytes, and cells overexpressing each receptor, and stimulation of isolated nuclei reveal biological functions, including transcriptional regulation of major genes, namely cylooxygenase-2 and inducible nitric oxide synthase. This mini review focuses on the nuclear localization and signaling of GPCRs, recognizing PAF and LPA phospholipids as ligands. Theories on how nuclear PAF and LPA1 receptors activate gene transcription and nuclear localization pathways are discussed. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; moreover, intracellular GPCRs constitute a distinctive mode of action for gene regulation.
Asunto(s)
Sistema Cardiovascular/metabolismo , Regulación de la Expresión Génica/fisiología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/fisiología , Animales , Sistema Cardiovascular/citología , Humanos , Ratas , PorcinosRESUMEN
BACKGROUND: In the preterm newborn, a patent ductus arteriosus is in large part a result of the increased sensitivity of the immature ductus to prostaglandin E2 (PGE2). PGE2 acts through 3 G protein-coupled receptors (EP2, EP3, and EP4) that activate both adenyl cyclase and K(ATP) channels. We explored these pathways to identify the mechanisms responsible for the increased sensitivity of the immature ductus to PGE2. METHODS AND RESULTS: We measured EP receptor content (mRNA and protein), receptor binding, cAMP production, and isometric tension in rings of ductus taken from immature (65% gestation) and mature (95% gestation) sheep and baboon fetuses. Ductus relaxation and cAMP generation were augmented in response to selective EP receptor agonists in the immature ductus. 8-Br-cAMP, a stable cAMP analogue, produced greater relaxation in the immature ductus. In the presence of a selective protein kinase A inhibitor, Rp-8-CPT cAMPS, the developmental differences in sensitivity to PGE2 could no longer be demonstrated. EP2, EP3, and EP4 receptor densities were higher in immature ductus, despite similar receptor mRNA and protein contents at the 2 gestational ages. In contrast, forskolin and NaF, direct activators of adenyl cyclase and Gs, respectively, elicited comparable increases in cAMP in both age groups. KATP channel inhibition also had similar effects on PGE2-induced relaxation in both age groups. CONCLUSIONS: Two mechanisms explain the increased sensitivity of the immature ductus to PGE2: (1) increased cAMP production because of increased binding of PGE2 to the individual EP receptors and (2) increased potency of cAMP on protein kinase A-regulated pathways.
Asunto(s)
Adenosina/análogos & derivados , Alprostadil/análogos & derivados , AMP Cíclico/análogos & derivados , Dinoprostona/farmacología , Conducto Arterial/efectos de los fármacos , Receptores de Prostaglandina E/efectos de los fármacos , Sistema Vasomotor/efectos de los fármacos , 16,16-Dimetilprostaglandina E2/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenilil Ciclasas/metabolismo , Alprostadil/farmacología , Animales , Compuestos de Bifenilo/farmacología , Colforsina/farmacología , AMP Cíclico/biosíntesis , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Activación Enzimática/efectos de los fármacos , Femenino , Edad Gestacional , Gliburida/farmacología , Indometacina/farmacología , Contracción Isométrica , NG-Nitroarginina Metil Éster/farmacología , Nitroprusiato/farmacología , Papio , Canales de Potasio/efectos de los fármacos , Embarazo , Receptores de Prostaglandina E/fisiología , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Ovinos , Transducción de Señal , Fluoruro de Sodio/farmacología , Tionucleótidos/farmacología , Sistema Vasomotor/fisiologíaRESUMEN
Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.
Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Acoplados a Proteínas G , Androstadienos/farmacología , Animales , Western Blotting , Calcio/metabolismo , Caveolina 1 , Caveolinas/metabolismo , Sistema Libre de Células/metabolismo , Células Cultivadas , Quelantes/farmacología , Clatrina/metabolismo , Ácido Egtácico/farmacología , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Immunoblotting , Hígado/metabolismo , Microcirculación , Microscopía Electrónica , Microscopía Fluorescente , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Toxina del Pertussis/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Pruebas de Precipitina , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptores del Ácido Lisofosfatídico , Fracciones Subcelulares/metabolismo , Porcinos , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , WortmaninaRESUMEN
Prostaglandins are ubiquitous lipid mediators that play pivotal roles in cardiovascular homeostasis, reproduction, and inflammation, as well as in many important cellular processes including gene expression and cell proliferation. The mechanism of action of these lipid messengers is thought to be primarily dependent on their interaction with specific cell surface receptors that belong to the heptahelical transmembrane spanning G protein-coupled receptor superfamily. Accumulating evidence suggests that these receptors may co-localize at the cell nucleus where they can modulate gene expression through a series of biochemical events. In this context, we have recently demonstrated that prostaglandin E2-EP3 receptors display an atypical nuclear compartmentalization in cerebral microvascular endothelial cells. Stimulation of these nuclear EP3 receptors leads to an increase of eNOS RNA in a cell-free isolated nuclear system. This review will emphasize these findings and describe how nuclear prostaglandin receptors, notably EP3 receptors, may affect gene expression, specifically of eNOS, by identifying putative transducing elements located within this organelle. The potential sources of lipid ligand activators for these intracellular sites will also be addressed. The expressional control of G-protein-coupled receptors located at the perinuclear envelope constitutes a novel and distinctive mode of gene regulation.
Asunto(s)
Reguladores de Proteínas de Unión al GTP/fisiología , Expresión Génica/fisiología , Membrana Nuclear/fisiología , Prostaglandinas/genética , Prostaglandinas/fisiología , Receptores de Prostaglandina E/genética , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Animales , Reguladores de Proteínas de Unión al GTP/genética , Expresión Génica/genética , Membrana Nuclear/genética , Membrana Nuclear/ultraestructura , Receptores de Prostaglandina E/fisiología , Transducción de Señal/genética , Transcripción Genética/genéticaRESUMEN
BACKGROUND AND PURPOSE: Free radical-induced peroxidation is an important factor in the genesis of hypoxic-ischemic encephalopathy, including that of the preterm infant. Isoprostanes are major peroxidation products. Since microvascular dysfunction seems to contribute to ischemic encephalopathies, we studied the cytotoxicity of 8-iso-prostaglandin F2alpha (PGF2alpha) on cerebral microvascular cells. METHODS: Microvascular endothelial, astroglial, and smooth muscle cells from newborn brain were cultured. The cytotoxicity of 8-iso-PGF2alpha on these cells was determined by MTT assays and lactate dehydrogenase (LDH) release, propidium iodide incorporation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]). In addition, effects of intraventricular injections of 8-iso-PGF2alpha and possible involvement of thromboxane in 8-iso-PGF2alpha-induced cytotoxicity were determined. RESULTS: 8-Iso-PGF2alpha induced time- and concentration-dependent endothelial cell death (EC50=0.1 nmol/L) but exerted little effect on smooth muscle and astroglial cells; endothelial cell death seemed mostly of oncotic nature (propidium iodide incorporation and LDH release). Cell death was associated with increased endothelial thromboxane A2 (TXA2) formation and was prevented by TXA2 synthase inhibitors (CGS12970 and U63557A); TXA2 mimetics U46619 and I-BOP also caused endothelial cell death. Intraventricular injection of 8-iso-PGF2alpha induced periventricular damage, which was attenuated by CGS12970 pretreatment. CONCLUSIONS: These data disclose a novel action of 8-iso-PGF2alpha involving TXA2 in oxidant stress-induced cerebral microvascular injury and brain damage.
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Isquemia Encefálica/metabolismo , Encéfalo/irrigación sanguínea , Dinoprost/análogos & derivados , Dinoprostona/análogos & derivados , Endotelio Vascular/efectos de los fármacos , F2-Isoprostanos/farmacología , Microcirculación/efectos de los fármacos , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/patología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Inyecciones Intraventriculares , Isoprostanos/farmacología , L-Lactato Deshidrogenasa/metabolismo , Microcirculación/citología , Microcirculación/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Necrosis , Ratas , Ratas Sprague-Dawley , Porcinos , Tromboxano A2/metabolismo , Tromboxano-A Sintasa/antagonistas & inhibidoresRESUMEN
Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on periventricular (PV) microvessels of fetal ( approximately 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 +/- 2.6%) and newborn (22.14 +/- 3.2%) pigs but was ineffective in adults (<2.5%). Specific [(3)H]PAF binding was greater in fetus and newborn than in adults; a concordant developmental PAF-induced inositol phosphate formation was observed. PAF-induced vasoconstriction was abrogated by thromboxane A(2) (TXA(2)) synthase and receptor inhibitors, calcium channel blockers, and by removal of endothelium; vasoconstriction to TXA(2) mimetic U-46619 did not differ with age. Immunoreactive TXA(2) synthase expression and PAF-evoked TXA(2) formation revealed a fetus> newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TXA(2) formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Feto/efectos de los fármacos , Feto/fisiología , Fosfatos de Inositol/metabolismo , Modelos Logísticos , Porcinos/fisiología , Tromboxano B2/metabolismoRESUMEN
PURPOSE: To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death. METHODS: Retinovascular density was calculated in rat pups exposed to 80% O(2) from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media. RESULTS: PAF levels in retina were markedly increased by exposure of isolated rat retinas to H(2)O(2) (1 micro M) and of rat pups placed in 80% O(2). Exposure to 80% O(2) induced retinal vasoobliteration, which was equally significantly inhibited ( approximately 60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time- and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF-induced cell death was preceded by an increase in TXB(2) levels and was prevented by TXA(2) synthase inhibition (with CGS12970). CONCLUSIONS: The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA(2). These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity.
Asunto(s)
Oxígeno , Factor de Activación Plaquetaria/uso terapéutico , Receptores Acoplados a Proteínas G , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/tratamiento farmacológico , Vasos Retinianos/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Humanos , Hiperoxia/complicaciones , Técnicas In Vitro , Inyecciones , Microcirculación/efectos de los fármacos , Estrés Oxidativo , Pericitos/efectos de los fármacos , Factor de Activación Plaquetaria/análogos & derivados , Factor de Activación Plaquetaria/antagonistas & inhibidores , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/antagonistas & inhibidores , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología , Porcinos , Tromboxano A2/fisiología , Cuerpo VítreoRESUMEN
We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE(2) in tissues and presence of perinuclear PGE(2) receptors (EP). We presently studied mechanisms by which PGE(2) induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE(2) and selective EP(3) receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP(3) immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP(3) receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear K(Ca) channels were detected, and their functionality corroborated by NS1619-induced Ca(2+) signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca(2+) chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as K(Ca) channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-kappaB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca(2+) transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE(2) through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP(3) receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K(Ca) channels, protein kinases, and NF-kappaB; the roles for nuclear EP(3) receptors seem different from those on plasma membrane.