El ratón deficiente en apolipoproteína E, un modelo traslacional para el estudio de la aterosclerosis / Apolipoprotein E–deficient mouse, a translational model for the study of atherosclerosi

INTRODUCCIÓN: La aterosclerosis es una de las principales causas de morbimortalidad en países desarrollados que presenta varias similitudes histopatológicas con la inflamación crónica. Los ratones deficientes en apolipoproteína E (apoE-/−) son ampliamente utilizados en el estudio de los mecanismos implicados en el inicio y el progreso de las lesiones ateroscleróticas. OBJETIVO: Evaluar el impacto en la formación de la placa de ateroma de una dieta aterogénica en el ratón apoE-/−. MATERIAL Y MÉTODOS: Ratones apoE-/− de 2 meses de edad fueron sometidos o no a una dieta hipercolesterolémica (10,8% de grasa, 0,75% en colesterol) durante 2 meses adicionales. Se determinó el perfil lipídico, la lesión y el contenido en macrófagos, linfocitos, colágeno, células de la musculatura lisa vascular (CMLV) y core necrótico por técnicas histológicas e inmunohistoquímicas. Cuantificación de las interacciones leucocito-endotelio por microscopia intravital en la microcirculación cremastérica. RESULTADOS: Los ratones apoE-/− sometidos a dieta hipercolesterolémica mostraron elevados niveles circulantes de colesterol total y triglicéridos frente aquellos sometidos a dieta control. Estos efectos fueron acompañados de un claro desarrollo de lesión aterosclerótica en la aorta caracterizada por un mayor contenido en macrófagos (Mac3+), linfocitos (CD3+), colágeno, core necrótico y CMLV. Paralelamente hubo una mayor adhesividad de los leucocitos al endotelio arteriolar en aquellos animales sometidos a dieta grasa. CONCLUSIÓN: El modelo de aterosclerosis que se desarrolla en el ratón apoE-/− sometido a dieta aterogénica presenta numerosas similitudes con la lesión humana, y constituye un adecuado modelo para la detección de nuevas dianas terapéuticas y ensayo de nuevos fármacos

INTRODUCTION: Atherosclerosis is one of the leading causes of morbidity and mortality in Western countries and bears several histopathological similarities to chronic inflammation. Mice deficient in apolipoprotein E (apoE-/−) are widely used in the study of the mechanisms involved in the onset and progression of the atherosclerotic lesion. OBJECTIVE: To evaluate the impact of an atherogenic diet in lesion formation in apoE-/− mice. MATERIAL AND METHODS: Two month-old apoE-/− mice were subjected, or not (controls), to a high fat/high cholesterol diet (10.8% fat, .75% cholesterol) for two months. Lipid profile, lesion formation, and macrophage, lymphocyte, collagen, vascular smooth muscle cells (VSMC), and necrotic core content, were determined within the lesion using histological and immunohistochemical techniques. Leukocyte-endothelial cell interactions were quantified by intravital microscopy in the cremaster microcirculation. RESULTS: apoE-/− mice subjected to a hypercholesterolemic diet showed increased circulating levels of total cholesterol and triglycerides compared to those subjected to a control diet. These effects were accompanied by a clear development of atherosclerotic lesion in the aorta, which was characterized by enhanced macrophage (Mac3+), lymphocyte (CD3+) collagen, VSMC and necrotic core content. In parallel, increased adhesiveness of leukocytes to the arteriolar endothelium in those animals subjected to an atherogenic diet was also detected. CONCLUSION: The atherosclerosis model in apoE-/− mice subjected to an atherogenic diet shares common features with the human atherosclerotic lesion, and constitutes an appropriate model to detect new therapeutic targets and evaluate novel developed drugs

Combined treatment with bexarotene and rosuvastatin reduces angiotensin-II-induced abdominal aortic aneurysm in apoE(-/-) mice and angiogenesis.

BACKGROUND AND PURPOSE: Abdominal aortic aneurysm (AAA) is a degenerative vascular disease associated with angiogenesis. Bexarotene is a retinoid X receptor (RXR) ligand with anti-angiogenic activity. Statins also exert anti-angiogenic activity and activate PPARs. Because RXR ligands form permissive heterodimers with PPARs and a single anti-angiogenic drug may not be sufficient to combat the wide array of angiogenic factors produced during AAA, we evaluated the effect of combined low doses of bexarotene and rosuvastatin in a mouse model of AAA. EXPERIMENTAL APPROACH: The effect of the combined treatment was investigated in a murine model of angiotensin II-induced AAA in apoE(-/-) mice. This combination therapy was also evaluated in in vivo (Matrigel plug assay) and in vitro (endothelial cell differentiation assay) models of angiogenesis as well as the underlying mechanisms involved. KEY RESULTS: Co-treatment with bexarotene plus rosuvastatin reduced aneurysm formation, inflammation and neovascularization compared with each single treatment. In HUVEC, the combination of suboptimal concentrations of bexarotene and rosuvastatin inhibited angiotensin II-induced morphogenesis, proliferation and migration. These effects were accompanied by diminished production of pro-angiogenic chemokines (CXCL1, CCL2 or CCL5) and VEGF, and seemed to be mediated by RXRα/PPARα and RXRα/PPARγ activation. This combined therapy reduced the activation of members of the downstream PI3K pathway (Akt/mTOR and p70S6K1) in vivo and in vitro. CONCLUSIONS AND IMPLICATIONS: The combination of RXR agonists with statins at low doses synergistically interferes with the signalling pathways that modulate inflammation and angiogenesis and may constitute a new and safer therapeutic treatment for the control of AAA.

PPARß activation restores the high glucose-induced impairment of insulin signalling in endothelial cells.

BACKGROUND AND PURPOSE: PPARß enhances insulin sensitivity in adipocytes and skeletal muscle cells, but its effects on insulin signalling in endothelial cells are not known. We analysed the effects of the PPARß/δ (PPARß) agonists, GW0742 and L165041, on impaired insulin signalling induced by high glucose in HUVECs and aortic and mesenteric arteries from diabetic rats. EXPERIMENTAL APPROACH: Insulin-stimulated NO production, Akt-Ser(473) and eNOS-Ser(1177) phosphorylation, and reactive oxygen species (ROS) production were studied in HUVECs incubated in low- or high-glucose medium. Insulin-stimulated relaxations and protein phosphorylation in vessels from streptozotocin (STZ)-induced diabetic rats were also analysed. KEY RESULTS: HUVECs incubated in high-glucose medium showed a significant reduction in insulin-stimulated production of NO. High glucose also reduced insulin-induced Akt-Ser(473) and eNOS-Ser(1177) phosphorylation, increased IRS-1-Ser(636) and ERK1/2-Thr(183) -Tyr(185) phosphorylation and increased ROS production. The co-incubation with the PPARß agonists GW0742 or L165041 prevented all these effects induced by high glucose. In turn, the effects induced by the agonists were suppressed when HUVEC were also incubated with the PPARß antagonist GSK0660, the pyruvate dehydrogenase kinase (PDK)4 inhibitor dichloroacetate or after knockdown of both PPARß and PDK4 with siRNA. The ERK1/2 inhibitor PD98059, ROS scavenger catalase, inhibitor of complex II thenoyltrifluoroacetone or uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone, also prevented glucose-induced insulin resistance. In STZ diabetic rats, oral GW0742 also improved insulin signalling and the impaired NO-mediated vascular relaxation. CONCLUSION AND IMPLICATIONS: PPARß activation in vitro and in vivo restores the endothelial function, preserving the insulin-Akt-eNOS pathway impaired by high glucose, at least in part, through PDK4 activation.

Taurine chloramine inhibits functional responses of human eosinophils in vitro.

BACKGROUND: Eosinophils are prominent effectors of allergic inflammation. Taurine-chloramine (TauCl), a derivative of the amino acid taurine, shows antioxidant properties in different cell systems but its effects on eosinophils have not been reported. OBJECTIVE: To study the effects of TauCl and taurine on functional responses of isolated human eosinophils activated by different stimuli. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of TauCl and taurine (0.1-1 mM) were investigated on the generation of superoxide anion (ferricytochrome-c reduction microassay), calcium signal (fluorimetry), p47phox-p67phox translocation (Western blot), leukotriene C4 (LTC4) production (enzymeimmunoassay), eosinophil peroxidase (EPO) release (spectrophotometry), eosinophil cationic protein (ECP) release (radioimmunoassay), apoptosis (flow cytometry with annexin V-propidium iodide), and nuclear factor-kappaB (NF-kappaB) activation (Western blot). RESULTS: TauCl inhibited superoxide anion generation triggered by N-formyl-Met-Leu-Phe (fMLP; 30 nM), phorbol myristate acetate (1 nM) and serum opsonized zymosan (0.5 mg/mL) with similar potency (IC50 approximately 200 microM) for the three stimuli, while taurine (0.1-1 mM) was scarcely effective. TauCl but not taurine inhibited p47phox-p67phox translocation. TauCl (200 microM) and taurine (1 mM) did not modify the [Ca2+]i responses to fMLP. TauCl inhibited the release of EPO (IC50 approximately 200 microM) and reduced ECP and LTC4 production from fMLP-activated eosinophils while taurine was without significant effects. TauCl (1 mM) did not change constitutive apoptosis but significantly attenuated the ability of granulocyte-monocyte colony-stimulating factor (GM-CSF) and IL-5 to prevent apoptosis. The activation of eosinophil NF-kappaB induced by GM-CSF and IL-5 was suppressed by TauCl. CONCLUSION: Taurine is without significant in vitro effects on human eosinophil functions but its derivative TauCl inhibits oxidative burst and generation of inflammatory mediators, and reverses the survival effect produced by inflammatory cytokines. Therefore, endogenous TauCl may help to suppress excessive inflammatory response in eosinophils at inflammatory sites.

Gastric hypersecretion associated to iodoacetamide-induced mild gastritis in mice.

Disturbances of gastric motor, secretory and/or sensory functions are frequently associated with gastritis. The aim of this study was to characterize motor and secretory alterations associated to chemically-induced gastritis in mice. Mild gastritis was induced with 0.1% iodoacetamide administered intragastrically and added to the drinking water for a 6 days period. A significant loss of body weight and a reduction in food and water intake was observed in iodoacetamide-treated animals compared with those receiving vehicle. At the end of the treatment period, no macroscopic alterations were observed in the gastric mucosa of iodoacetamide-treated mice. However, histological sections revealed a mixed inflammatory infiltrate, with a predominance of mast cells in the submucosa; suggesting a mild gastritis. Gastric emptying rate of a nutrient solid meal was not modified in mice with gastritis compared with normal controls. In animals with gastritis, basal gastric acid secretion was increased compared with normal controls. Basal gastric acid secretion was not modified by either indomethacin or compound 48/80. Secretory response to secretagogues (pentagastrin and histamine) was enhanced during gastritis. Hypersecretory responses to both gastrin and histamine in iodoacetamide-treated mice were blocked by the mast cell stabilizer sodium cromoglycate, and enhanced by indomethacin, without affecting the secretory response in normal mice. These results suggest that mild gastritis alters gastric acid secretory responses through a mechanism related, at least partially, to mast cells activation. Moreover, prostaglandins also modulate secretory responses during mild inflammation. This animal model of gastritis might be useful to characterize pathophysiological changes and potential therapeutic targets in secretory-related gastric pathologies.

Cyclic AMP elevating agents and nitric oxide modulate angiotensin II-induced leukocyte-endothelial cell interactions in vivo.

Angiotensin (Ang-II) is a key molecule in the development of cardiac ischaemic disorders and displays proinflammatory activity in vivo. Since intracellular cyclic nucleotides elevating agents have proved to be effective modulators of leukocyte recruitment, we have evaluated their effect on Ang-II-induced leukocyte-endothelial cell interactions in vivo using intravital microscopy within the rat mesenteric microcirculation. Pretreatment with iloprost significantly inhibited (1 nM) Ang-II-induced increase in leukocyte rolling flux, adhesion and emigration at 60 min by 96, 92 and 90% respectively, and returned leukocyte rolling velocity to basal levels. Pretreatment with salbutamol or co-superfusion with forskolin exerted similar effects. When theophylline was administered, leukocyte rolling flux, adhesion and emigration elicited by Ang-II were significantly attenuated by 81, 89 and 71% respectively. Rolipram administration caused similar reduction of Ang-II-induced leukocyte responses. Co-superfusion of Ang-II with the NO-donor, spermine-NO, or 8-Br-cyclic GMP, or pretreatment with a transdermal nytroglycerin patch, resulted in a significant reduction of the leukocyte-endothelial cell interactions elicited by Ang-II. Salbutamol preadministration did not modify leukocyte-endothelial cell interactions elicited by either L-NAME or L-NAME+Ang-II, indicating that the inhibitory leukocyte effects caused by cyclic AMP-elevating agents are mediated through NO release. In conclusion, we have provided evidence that cyclic AMP elevating agents and NO donors, are potent inhibitors of Ang-II-induced leukocyte-endothelial cell interactions. Thus, they could constitute a powerful therapeutical tool in the control of the leukocyte recruitment characteristic of the vascular lesions that occur in cardiovascular disease states where Ang-II plays a critical role.

Angiotensin II is involved in nitric oxide synthase and cyclo-oxygenase inhibition-induced leukocyte-endothelial cell interactions in vivo.

1. Chronic inhibition of nitric oxide synthase (NOS) provokes a hypertensive state which has been shown to be angiotensin II (Ang-II) dependent. In addition to raising blood pressure, NOS inhibition also causes leukocyte adhesion. The present study was designed to define the role of Ang-II in hypertension and in the leukocyte-endothelial cell interactions induced by acute NOS or cyclo-oxygenase (COX) inhibition using intravital microscopy within the rat mesenteric microcirculation. 2. While pretreatment with an Ang-II AT(1) receptor antagonist (losartan) reversed the prompt increase in mean arterial blood pressure (MABP) caused by indomethacin, it had no effect on the increase evoked by systemic L-NAME administration. 3. Pretreatment with losartan inhibited the leukocyte rolling flux, adhesion and emigration which occurs after 60 min NOS inhibition by 83, 80 and 70% respectively, and returned leukocyte rolling velocity to basal levels. 4. Losartan significantly reduced the leukocyte-endothelial cell interaction elicited by COX inhibition. In contrast, leukocyte recruitment induced by acute mast cell activation was not inhibited by losartan. 5. AT(1) receptor blockade also prevented the drop in haemodynamic parameters such as mean red blood cell velocity (V(mean)) and shear rate caused by NOS and COX inhibition. 6. In this study, we have demonstrated a clear role for Ang-II in the leukocyte-endothelial cell interactions and haemodynamic changes which arise in the absence of NO or prostacyclin (PGI(2)). This is of interest since leukocyte recruitment, which culminates in the vascular lesions that occur in hypertension, atherosclerosis and myocardial ischemia-reperfusion injury, might be prevented using AT(1) Ang-II receptor antagonists.

Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT(1) and AT(2) receptor-mediated P-selectin upregulation.

BACKGROUND: Angiotensin II (Ang II) plays a critical role in the development of vascular lesions in hypertension, atherosclerosis, and several renal diseases. Because Ang II may contribute to the leukocyte recruitment associated with these pathological states, the aim of the present study was to assess the role of Ang II in leukocyte-endothelial cell interactions in vivo. METHODS AND RESULTS: Intravital microscopy of the rat mesenteric postcapillary venules was used. Sixty minutes of superfusion with 1 nmol/L Ang II induced a significant increase in leukocyte rolling flux (83.8+/-20. 7 versus 16.4+/-3.1 cells/min), adhesion (11.4+/-1.0 versus 0.8+/-0. 5 cells/100 microm), and emigration (4.0+/-0.7 versus 0.2+/-0.2 cells/field) without any vasoconstrictor activity. These effects were not mediated by mast cell activation. Intravenous pretreatment with AT(1) (losartan) or AT(2) (PD123,319) receptor antagonists significantly reduced Ang II-induced responses. A combination of both receptor antagonists inhibited the leukocyte rolling flux, adhesion, and extravasation elicited by Ang II at 60 minutes. Pretreatment of animals with fucoidin or an adhesion-blocking anti-rat P-selectin monoclonal antibody abolished Ang II-induced leukocyte responses. Furthermore, rat platelet P-selectin expression was not affected by Ang II stimulation. CONCLUSIONS: -Ang II induces significant leukocyte rolling, adhesion, and emigration, which may contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of this peptide are elevated.

Every optometrist's concern--patient's access to low vision services.

Persons with low vision are increasing in numbers and searching for access to quality low vision services. Working with these patients is an important part of all optometric service models. Issues that should be considered and services that should be provided to patients with low vision are discussed.