RESUMO
Vascular smooth muscle cells (VSMC) are present in arterial intima before atherosclerotic plaques develop and are likely to be exposed to unmodified serum lipids as they enter the vessel wall. We examined the effects of sera from mice on the morphology and function of mouse VSMC. Incubation of a mouse VSMC line (MOVAS) with sera from normocholesterolemic (C57BL/6J) or hypercholesterolemic (APOE(-/-)) mice caused concentration-dependent increases in lipid accumulation as measured by AdipoRed, with the extent of lipid uptake significantly greater with the latter sera type. Inhibition of c-Jun N-terminal kinases (SP600125), Src kinases (AG1879), and clathrin-dependent endocytosis (monodansylcadaverine) to disrupt scavenger receptor-mediated uptake of lipids had no effect on serum-induced lipid accumulation by VSMC. By contrast, inhibition of macropinocytosis with antagonists of PI-3 kinase (LY294002) and actin (cytochalasin D) markedly reduced lipid accumulation. Serum exposure reduced the expression of the ATP-binding cassette transporter A1, consistent with impaired cholesterol efflux, but had no effect on the expression of markers of VSMC differentiation. Moreover, the expression of several inflammation and foam cell markers was unchanged (CCL2, CCL5, and CD68) by mouse sera. The accumulation of unmodified serum lipids by VSMC involves a macropinocytosis-like uptake pathway and is associated with the downregulation of the ATP-binding cassette transporter. We speculate that VSMC may play an atheroprotective role in arterial intima by acting as a "sink" for unmodified lipids.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Hipercolesterolemia/metabolismo , Lipídeos/sangue , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Pinocitose , Actinas/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Biomarcadores/sangue , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Mediadores da Inflamação/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Pinocitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fatores de TempoRESUMO
Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO) decreases reactive oxygen species production by endothelial Nox2. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO, also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angeli's salt and isopropylamine NONOate (IPA/NO), on Nox2-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1µmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral (MCA)) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angeli's salt (1µmol/L) or IPA/NO (1µmol/L). Similarly, phorbyl 12,13-dibutyrate (10µmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1µmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger l-cysteine (3mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100µmol/L; NO scavenger), ODQ (10µmol/L; soluble guanylyl cyclase (sGC) inhibitor), or Rp-8-pCPT-cGMPS (10µmol/L; cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2(-/y)) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1µmol/L) had no effect on superoxide levels in arteries from Nox2(-/y) mice. Finally, angiotensin II (1-1000µmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1µmol/L), whereas constrictor responses to either the thromboxane A2 mimetic U46619 (1-100 nmol/L) or high potassium (122.7mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC-cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2-dependent vascular dysfunction.
Assuntos
Hidrazinas/administração & dosagem , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Óxidos de Nitrogênio/metabolismo , Doenças Vasculares/enzimologia , Angiotensina II/metabolismo , Animais , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/metabolismo , Humanos , Hidrazinas/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Terapia de Alvo Molecular , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , Óxido Nítrico/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/patologiaRESUMO
The Nox family NADPH oxidases are reactive oxygen species (ROS)-generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved. Here we investigated the role of the Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE(-/-)) mice. Wild-type (C57Bl6/J), ApoE(-/-), and Nox2(-/y)/ApoE(-/-) mice were maintained on a high-fat (21%) diet from 5 wk of age until they were 12 or 19 wk old. Mice were euthanized and their aortas removed for measurement of Nox2 expression (Western blot analysis and immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (contractions to N(omega)-nitro-L-arginine), and atherosclerotic plaque development along the aorta and in the aortic sinus. Nox2 expression was upregulated in the aortic endothelium of ApoE(-/-) mice before the appearance of lesions, and this was associated with elevated ROS levels. Within developing plaques, macrophages were also a prominent source of Nox2. The absence of Nox2 in Nox2(-/y)/ApoE(-/-) double-knockout mice had minimal effects on plasma lipids or lesion development in the aortic sinus in animals up to 19 wk of age. However, an en face examination of the aorta from the arch to the iliac bifurcation revealed a 50% reduction in lesion area in Nox2(-/y)/ApoE(-/-) versus ApoE(-/-) mice, and this was associated with a marked decrease in aortic ROS production and an increased NO bioavailability. In conclusion, this is the first demonstration of a role for Nox2-NADPH oxidase in vascular ROS production, reduced NO bioavailability, and early lesion development in ApoE(-/-) mice, highlighting this Nox isoform as a potential target for future therapies for atherosclerosis.
Assuntos
Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Aterosclerose/enzimologia , Aterosclerose/patologia , Glicoproteínas de Membrana/fisiologia , NADPH Oxidases/fisiologia , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Disponibilidade Biológica , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Imuno-Histoquímica , Isoenzimas/genética , Lipídeos/sangue , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , NADPH Oxidase 2 , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , NG-Nitroarginina Metil Éster/farmacologia , Espécies Reativas de OxigênioRESUMO
A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.