RESUMO
Ischemic retinopathies, such as retinopathy of prematurity and diabetic retinopathy are characterized by an initial microvascular degeneration, followed by an abnormal hypoxia-induced neovascularization. Oxygen-induced retinopathy (OIR) is a well-established in vivo model of ischemic retinopathies, which, although the triggering insult varies, all share a common end result of capillary loss. Understanding the mechanisms of normal retinal vascular development as well as the pathophysiological processes leading to the primary vascular loss is the key to develop treatments to prevent the sight-threatening neovascularization associated with human ischemic retinopathies. The importance of oxygen-dependent vascular endothelial growth factor in the pathophysiology of both phases of OIR has long been recognized. However, recent studies point out that OIR is a multifactorial disease, resulting from additive effects of an unbalanced expression of pro- and anti-angiogenic factors, interrelated with protective effects of nutritional factors and cytotoxic effects of oxidative and nitro-oxidative stress-dependent mediators. This review summarizes the most recent aspects of the research on OIR conducted in our laboratory and others, with a particular focus on the role of new mediators of nitro-oxidative stress, the trans-arachidonic acids, in microvascular degeneration, and on a novel pathway of metabolic signaling where hypoxia-driven succinate, via receptor GPR91, governs normal and pathological retinal angiogenesis.
Assuntos
Retinopatia Diabética/etiologia , Modelos Animais de Doenças , Isquemia/etiologia , Oxigênio/toxicidade , Retinopatia da Prematuridade/etiologia , Animais , Humanos , Recém-Nascido , Estresse Oxidativo , Ratos , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologiaRESUMO
OBJECTIVE: Angiogenesis is essential physiologically in growth and pathologically in tumor development, chronic inflammatory disorders, and proliferative retinopathies. Activation of protease-activated receptor 2 (PAR2) leads to a proangiogenic response, but its mechanisms have yet to be specifically described. Here, we investigated the mode of action of PAR2 in retinal angiogenesis. METHODS AND RESULTS: PAR2-activating peptide, SLIGRL, increased retinal angiogenesis associated with an induction of vascular endothelial growth factor and angiopoetin-2 and most notably tie2 in the retina in vivo as well as in cultured neuroretinal endothelial cells. SLIGRL also induced release of the proinflammatory and angiogenic mediator tumor necrosis factor-alpha (TNF-alpha) via the MEK/extracellular signal-regulated kinase (ERK) (MEK/ERK) pathway in these endothelial cells. TNF-alpha, in turn, elicited tie2 expression by activating the MEK/ERK pathway. PAR2-evoked tie2 expression, endothelium proliferation (in vitro), and retinal neovascularization (in vivo) were abrogated by selective TNF-alpha blockers (neutralizing antibody infliximab and soluble TNF-alpha receptor-Fc fusion protein etanercept) as well as the MEK inhibitor PD98059. CONCLUSIONS: The proangiogenic properties of PAR2 are intertwined with its proinflammatory effects, such that in retinal vasculature, they depend on TNF-alpha and subsequent induction of tie2 via the MEK/ERK pathway.
Assuntos
Neovascularização Fisiológica , Receptor PAR-2/fisiologia , Angiopoietina-2/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Endotélio Vascular/fisiologia , Oligopeptídeos/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor TIE-2/metabolismo , Retina/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Encéfalo/irrigação sanguínea , Endotélio Vascular/fisiologia , Óxido Nítrico Sintase/biossíntese , Receptores de Prostaglandina E/fisiologia , Animais , Células Cultivadas , Dinoprostona/farmacologia , Microcirculação/fisiologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Suínos , Regulação para Cima/efeitos dos fármacosRESUMO
Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Consequently, the retina constitutes an excellent model for testing the functional consequences of membrane lipid peroxidation. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane (TXA2) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA2 and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. Despite these advances there are still a number of important questions that remain to be answered before we can confidently target pathological signals. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.
Assuntos
Isquemia/imunologia , Lipídeos/imunologia , Retina , Doenças Retinianas/imunologia , Vasos Retinianos/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Peroxidação de Lipídeos , Estresse Oxidativo , Oxigênio/metabolismo , Fosfolipases A/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Prostaglandinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fluxo Sanguíneo Regional , Retina/imunologia , Retina/patologia , Doenças Retinianas/patologia , Vasos Retinianos/citologia , Vasos Retinianos/patologia , Transdução de Sinais/fisiologia , Tromboxano A2/metabolismoRESUMO
BACKGROUND AND PURPOSE: Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension. METHODS: Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 micro m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and K(Ca) channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining. RESULTS: Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156+/-2 and 155+/-1 mm Hg, respectively) than that of control offspring (137+/-1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A(2) mimetic and dilation to carba-prostaglandin I(2) did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of K(Ca) (another important mediator of NO action) and relaxation to the K(Ca) opener NS1619 were unchanged by antenatal diet. CONCLUSIONS: Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.
Assuntos
GMP Cíclico/análogos & derivados , Endotélio Vascular/fisiopatologia , Hipertensão/etiologia , Hipertensão/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal , Sistema Vasomotor/fisiopatologia , Animais , Doença Crônica , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas Alimentares/farmacologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Guanilato Ciclase , Técnicas In Vitro , Microcirculação/efeitos dos fármacos , Microcirculação/fisiopatologia , Neurotransmissores/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Pia-Máter/irrigação sanguínea , Gravidez , Deficiência de Proteína/fisiopatologia , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Sistema Vasomotor/efeitos dos fármacosRESUMO
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.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/irrigação sanguínea , Dinoprosta/análogos & derivados , Dinoprostona/análogos & derivados , Endotélio Vascular/efeitos dos fármacos , F2-Isoprostanos/farmacologia , Microcirculação/efeitos dos fármacos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Injeções Intraventriculares , Isoprostanos/farmacologia , L-Lactato Desidrogenase/metabolismo , Microcirculação/citologia , Microcirculação/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Necrose , Ratos , Ratos Sprague-Dawley , Suínos , Tromboxano A2/metabolismo , Tromboxano-A Sintase/antagonistas & inibidoresRESUMO
Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.
Assuntos
Óxido Nítrico/fisiologia , Estresse Oxidativo , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Vasos Retinianos/patologia , Tirosina/análogos & derivados , Animais , Animais Recém-Nascidos , Antioxidantes/farmacologia , Glutationa/análise , Isoenzimas/análise , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Malondialdeído/análise , Metaloporfirinas/farmacologia , Microcirculação/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitroarginina/farmacologia , Oxirredução , Oxigênio/toxicidade , Ratos , Ratos Sprague-Dawley , Retina/química , Retina/efeitos dos fármacos , Retina/patologia , Doenças Retinianas/induzido quimicamente , Vasos Retinianos/efeitos dos fármacos , Salicilatos/farmacologia , Tirosina/análise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Because prostaglandins (PGs) are implicated in acute hypercapnia-induced hyperemia, this study was conducted to test the hypothesis that prolonged hypercapnia may cause a sustained increase in retinal blood flow (RBF) through a PG-dependent induction of endothelial nitric oxide synthase (eNOS). METHODS: Time-dependent RBF (microsphere technique), PGE(2), nitrite (NO(2)(-)), and NOS protein (reduced nicotinamide adenine dinucleotide phosphate [NADPH]-diaphorase staining) production were measured in hypercapnia (6% CO(2))-treated piglets. From the same species, PGE(2), eNOS mRNA, NOS protein, and vasomotor responses were measured in eyecup preparations, as were Ca(2+) transients in neuroretinovascular endothelial cells. RESULTS: Hypercapnia caused biphasic (at 0.5 hours and 6-8 hours) increases in RBF that were abolished with normalization of the pH. The early phase (0.5 hour) was associated with an increase in PGE(2) levels and the latter phase (6-8 hours) with an increase in NO(2)(-) and NOS protein. Inhibition of cyclooxygenase by diclofenac prevented the early and late increase in RBF. NOS inhibitor L-nitro-arginine prevented only the latter. Hypercapnic acidosis increased retinal PGE(2) levels and eNOS-dependent vasorelaxation ex vivo. The ex vivo time course of eNOS mRNA expression corresponded with the late-phase increase in RBF and was blocked by the transcription inhibitor actinomycin D and the receptor-operated Ca(2+) channel blocker SK&F96365. In neuroretinovascular cells, acidosis increased Ca(2+) transients, which were inhibited by SK&F96365, but not diclofenac. CONCLUSIONS: This study discloses a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induced expression of eNOS mediated by PGE(2).
Assuntos
Dinoprostona/farmacologia , Hipercapnia/enzimologia , Hiperemia/enzimologia , Óxido Nítrico Sintase/biossíntese , Vasos Retinianos/efeitos dos fármacos , Animais , Velocidade do Fluxo Sanguíneo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dactinomicina/farmacologia , Indução Enzimática/efeitos dos fármacos , Hipercapnia/fisiopatologia , Hiperemia/fisiopatologia , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Nitritos/metabolismo , RNA Mensageiro/metabolismo , Radioimunoensaio , Vasos Retinianos/enzimologia , Vasos Retinianos/fisiopatologia , Suínos , VasodilataçãoRESUMO
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.
Assuntos
Oxigênio , Fator de Ativação de Plaquetas/uso terapêutico , Receptores Acoplados a Proteínas G , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/tratamento farmacológico , Vasos Retinianos/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Humanos , Hiperóxia/complicações , Técnicas In Vitro , Injeções , Microcirculação/efeitos dos fármacos , Estresse Oxidativo , Pericitos/efeitos dos fármacos , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/antagonistas & inibidores , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologia , Suínos , Tromboxano A2/fisiologia , Corpo VítreoRESUMO
IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.
Assuntos
Anti-Inflamatórios , Inflamação/tratamento farmacológico , Proteína Antagonista do Receptor de Interleucina 1 , Peptídeos/química , Peptídeos/farmacologia , Receptores de Interleucina-1/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Western Blotting , Dermatite/tratamento farmacológico , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peptídeos/síntese química , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-DawleyRESUMO
Thromboxane A(2) (TXA(2)) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA(2)-evoked neural microvascular endothelial cell death. Stable TXA(2) mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1alpha,4alpha,5beta(Z),6alpha,(1E,3S)]]-9,11-dedioxy-9alpha,11alpha-methanolpoxy (U-46619) [as well as [1S-[1alpha,2alpha(Z),3beta(1E,3S(*)),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA(2) dependence of these effects was corroborated by antagonism using the selective TXA(2) receptor blocker (-)-6,8-difluoro-9-p-methyl-sulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA(2) (mimetics) augmented generation of known active m-calpain (but not mu-calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of alpha-spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17- and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA(2)-induced neurovascular endothelial cell death.
Assuntos
Calpaína/fisiologia , Endotélio Vascular/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Degeneração Retiniana/metabolismo , Vasos Retinianos/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Calpaína/metabolismo , Carbazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Fosfatos de Inositol/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Microcirculação/metabolismo , Microcirculação/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologiaRESUMO
Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane A(2) (TXA(2)) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA(2) and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.
Assuntos
Inflamação/fisiopatologia , Isquemia/fisiopatologia , Doenças Retinianas/fisiopatologia , Animais , Humanos , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos , Peroxidação de Lipídeos , Oxigênio/efeitos adversos , Neovascularização Retiniana/patologia , Vasos Retinianos/fisiopatologiaRESUMO
The synthesis of PGE(2), the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE(2) synthases (PGES). The factors implicated in increased PGE(2) synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A(2) (cPLA(2)) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE(2) in the DA of NB were approximately 7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA(2) in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE(2) in fetal DA to levels approaching those of the NB; cPGES, cPLA(2), and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE(2) levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE(2) levels in the perinate.
Assuntos
Dinoprostona/metabolismo , Canal Arterial/enzimologia , Oxirredutases Intramoleculares/metabolismo , Animais , Animais Recém-Nascidos , Ciclo-Oxigenase 2 , Citosol/enzimologia , Canal Arterial/embriologia , Técnicas In Vitro , Isoenzimas/metabolismo , Fosfolipases A/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases/metabolismo , SuínosRESUMO
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.