RESUMO
Experimental animals placed on a high-cholesterol diet for 2 or more weeks exhibit an inflammatory response in postcapillary venules. The aims of this study were to determine (1) whether superoxide mediates the hypercholesterolemia-induced inflammatory response and (2) whether leukocyte and/or vessel wall NAD(P)H oxidase contributes to this response. Intravital videomicroscopy was used to quantify leukocyte-endothelial cell adhesion in cremasteric postcapillary venules of wild-type (WT) mice, CuZn-superoxide dismutase transgenic (SOD TgN) mice, and mice heterozygous (p47(phox)+/-) or homozygous (p47(phox)-/-) for NAD(P)H oxidase placed on either a normal diet or high-cholesterol diet (HCD) for 2 weeks. The number of adherent and emigrated leukocytes in postcapillary venules of WT HCD mice was significantly higher than that detected in venules of their normal-diet counterparts. However, the HCD-induced recruitment of adherent and emigrated leukocytes was not observed in SOD TgN mice. Whereas hypercholesterolemic p47(phox)+/- and WT mice exhibited similar inflammatory responses, p47(phox)-/- mice did not. Bone marrow chimeras were developed to selectively delete p47(phox) from either the vessel wall or circulating leukocytes. Whereas WT marrow transplanted into WT mice produced a normal inflammatory response of venules to HCD, chimeric mice with p47(phox) deficiency in either the vessel wall or leukocytes exhibited an attenuated inflammatory response to HCD that was comparable with that observed in p47(phox)-/- HCD mice. Our findings indicate that enhanced superoxide production is a critical event that initiates the leukocyte-endothelial cell adhesion in postcapillary venules of HCD mice. NAD(P)H oxidase appears to be an important source of this superoxide.
Assuntos
Adesão Celular/fisiologia , Endotélio Vascular/citologia , Hipercolesterolemia/fisiopatologia , Leucócitos/citologia , NADH NADPH Oxirredutases/metabolismo , Superóxidos/metabolismo , Animais , Movimento Celular/fisiologia , Colesterol/sangue , Feminino , Genótipo , Hipercolesterolemia/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NADPH Oxidases , Fosfoproteínas/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Thermotolerance describes the process in which hyperthermia induces a transient resistance of the stressed cells to subsequent episodes of oxidative stress. The aims of this study were first, to assess the effect of ischemia-reperfusion (IR) injury on renal function and the expression of the ICAM-1 receptor and MHC antigens, and second, to evaluate the protective effects of thermotolerance on IR induced renal injury and its potential for decreasing allograft rejection, by decreasing alloantigen expression. Sprague-Dawley rats were randomized into three groups: control, IR, and hyperthermia + IR (HIR) (n=8 per group). Thermotolerance was induced 18 hr prior to IR by increasing the core body temperature to 41 degrees C+/-0.5 degrees C for 15 min. After left uninephrectomy, IR was induced by clamping the right renal pedicle for 45 min, followed by 2 hr reperfusion. Myeloperoxidase (MPO) activity was used as an indicator of renal neutrophil influx. Kidney edema was assessed using the weight difference between left and right kidneys. Renal function was evaluated by measuring serum creatinine and urea 2 hr following clamp removal. Immunocytochemistry was used to measure expression of ICAM-1 and MHC antigen. Renal function was significantly impaired by IR with serum creatinine and urea levels of 131.5+/-5.01 microM and 11.2+/-0.71 mM, respectively, compared with controls of 67.9+/-5.11 microM and 8.1+/-0.36 mM, P<0.005 in both cases. Renal function was preserved in the HIR group, serum creatinine (84.8+/-8.58 microM) and urea (9.0+/-0.52 mM) were comparable to that of controls. Renal endothelium was activated in the IR group compared with controls, with increased ICAM-1, and tubular epithelium showed increased class II MHC expression. This up-regulation was prevented by prior induction of thermotolerance. Endothelial permeability was increased in the IR group with MPO activity of 0.8+/-0.08 units/g tissue--twice that of control levels P<0.05--and a marked increase in organ edema. Thermotolerance preserved endothelial barrier function. Thermotolerance may prevent IR injury by preventing endothelium activation and has the potential to modify allograft rejection by decreasing expression of ICAM-1, an important T cell receptor, and class II MHC.
Assuntos
Aclimatação/fisiologia , Temperatura Alta , Molécula 1 de Adesão Intercelular/fisiologia , Rim/irrigação sanguínea , Traumatismo por Reperfusão/fisiopatologia , Animais , Permeabilidade da Membrana Celular/efeitos da radiação , Creatinina/sangue , Antígenos de Histocompatibilidade/fisiologia , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe II/análise , Imuno-Histoquímica , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Ureia/sangueRESUMO
Reperfusion of ischaemic tissues often leads to microvascular dysfunction that is manifested as impaired endothelium-dependent dilation of arterioles, enhanced fluid filtration and leucocyte plugging in capillaries, and the trafficking of leucocytes and plasma protein extravasation in postcapillary venules. Efforts to define the mechanisms that underlie these microvascular responses to ischaemia and reperfusion have largely relied on pharmacological agents and monoclonal antibodies. Gene-targeting technology has been applied to the production of transgenic and knockout mice that are rapidly gaining acceptance as tools for mechanistic studies of ischaemia-reperfusion (I/R) injury that obviate some of the concerns (e.g. specificity) raised about previously employed experimental strategies. This review summarizes some of our efforts to apply gene-targeted mice to the study of I/R injury in the splanchnic vascular bed. A role for endothelial cell adhesion molecules (CAMs) and reactive oxygen metabolites is supported by results from mutant mice. Low density lipoprotein receptor mice also reveal that the microvascular and inflammatory responses to I/R are greatly exaggerated during chronic hypercholesterolaemia. The wide variety of mutant mice that have been produced for inflammation-related research makes this experimental strategy particularly promising for mechanistic investigations of the tissue responses to I/R.