Daniel E. Venegas-Pino

Hyperglycaemia is associated with impaired vasa vasorum neovascularization and accelerated atherosclerosis in apolipoprotein-E deficient mice

OBJECTIVE A direct correlation between blood glucose levels and the microvascular complications of diabetes is well established. However, the effects of hyperglycaemia on the vasa vasorum, a microvascular network which surrounds and supplies the walls of large arteries, is not known. The objective of this study is to investigate the effects of hyperglycaemia on the vasa vasorum and to examine correlations between these effects and the development of atherosclerosis in a mouse model. METHODS The micro- and macrovascular effects of hyperglycaemia were examined in streptozotocin-injected apolipoprotein-E deficient (ApoE(-/-)) mice. Retina and aortic sinus were isolated from hyperglycaemic mice and normoglycaemic controls at 5-20 weeks of age. Retinal and vasa vasorum microvessel densities were quantified and correlated to atherosclerotic lesion development. The expression levels of pro-angiogenic factors including vascular endothelial growth factor (VEGF) and VEGF receptor 2 were examined. RESULTS In normoglycaemic ApoE(-/-) mice atherogenesis is associated with vasa vasorum expansion, which likely corresponds to the increasing blood supply demands of the thickening artery wall. In hyperglycaemic ApoE(-/-) mice there is no significant neovascularization of the vasa vasorum, despite the fact that lesions are significantly larger. This defect may result from a localized deficiency in VEGF. CONCLUSIONS These findings are the first evidence that hyperglycaemia alters the structure of the vasa vasorum. Such microvascular changes directly correlate, and may contribute to, the development and progression of atherosclerosis in hyperglycaemic ApoE-deficient mice.

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

Sex-Specific Differences in an ApoE(-/-):Ins2(+/Akita) Mouse Model of Accelerated Atherosclerosis.

Diabetic patients have a twofold to fourfold increased risk of cardiovascular disease. Despite a vast amount of research, the underlying mechanisms that predispose individuals with diabetes to the development of cardiovascular disease are unclear. To further our understanding of how diabetes promotes atherosclerosis, we have established, characterized, and manipulated a new model of hyperglycemia-induced atherosclerosis: the apolipoprotein E-deficient (ApoE(-/-)):Ins2(+/Akita) mouse. All mice were fed a standard chow diet. Male ApoE(-/-):Ins2(+/Akita) mice developed chronic hyperglycemia, which significantly accelerated atherosclerosis. Female ApoE(-/-):Ins2(+/Akita) mice presented hyperglycemia that normalized by 15 weeks of age. Despite the transient hyperglycemia, advanced atherosclerosis was observed at 15 weeks of age compared with ApoE(-/-) females. To better understand these differences, subsets of mice were castrated or ovariectomized at 5 weeks of age. Castrated ApoE(-/-):Ins2(+/Akita) mice showed a reduction in blood glucose levels that correlated with the amelioration of atherosclerosis. Interestingly, castrated normoglycemic ApoE(-/-) mice developed larger atherosclerotic lesions than sham-operated on controls. Ovariectomized ApoE(-/-):Ins2(+/Akita) mice presented chronic hyperglycemia, and atherosclerosis appeared to be advanced. We have characterized the distinctive sex-specific phenotypes exhibited by the ApoE(-/-):Ins2(+/Akita) mouse model and present evidence for the action of sex hormones on pancreatic β-cell function and the vasculature that affect the regulation of blood glucose levels and the development of atherosclerosis. This model will provide a test bed to further delineate these effects.

Evidence of extensive atherosclerosis, coronary artery disease and myocardial infarction in the ApoE−/−:Ins2+/Akita mouse fed a western diet

BACKGROUND AND AIMS Diabetic patients with no history of cardiac infarction have a prevalence of coronary atherosclerosis and a risk of heart attack equivalent to euglycemic patients who have coronary atherosclerosis and have suffered a prior myocardial infarction. Although several murine models of diabetes have been established, none of these show indications of cardiac events. In an attempt to establish a diabetic mouse model with coronary atherosclerosis and myocardial injury, we have fed hyperglycemic ApoE-/-:Ins2+/Akita mice a western diet to enhance the dyslipidemic phenotype. METHODS Five-week-old ApoE-/-:Ins2+/Akita mice and ApoE-/- controls were fed a diet, 0.15% cholesterol and 21% anhydrous milk lipids, until 25 weeks of age. Changes in lifespan, clinical and metabolic parameters were evaluated as well as atherosclerosis and heart injury. RESULTS In comparison to male ApoE-/-, male ApoE-/-:Ins2+/Akita mice presented with chronic hyperglycemia (30.8 ± 1.2 mM vs. 9.3 ± 0.5 mM) accompanied by extremely high levels of total plasma cholesterol (49.3 ± 6.3 mM vs. 30.1 ± 1.5 mM) and triglycerides (11.6 ± 1.7 mM vs. 2.36 ± 0.18 mM). These mice have atherosclerosis at multiple vascular sites, including aortic sinus, ascending and descending aorta, brachiocephalic artery and coronary arteries. In addition, myocardial infarcts and a significant reduction of the lifespan (close to 20% of survival vs. other groups) were observed. Distinctively, both strains of female mice presented a parallel increase in plasma lipids, atherosclerosis, and no effects on mortality. CONCLUSIONS We have established a diabetic mouse model, the western-diet-fed male ApoE-/-:Ins2+/Akita mouse, with profound cardiovascular disease involving extensive atherosclerosis, coronary artery disease and myocardial infarct resulting in shortened lifespan.