Objective: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. Methods and Results:C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-␣. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10 ؊7 mol/L). Conclusions: Genetic

OBJECTIVE-The degradation of angiotensin (Ang) II by ACE2, leading to the formation of Ang 1-7, is an important step in the renin-angiotensin system (RAS) and one that is significantly altered in the diabetic kidney. This study examines the role of ACE2 in early renal changes associated with diabetes and the influence of ACE2 deficiency on ACE inhibitor-mediated renoprotection.RESEARCH DESIGN AND METHODS-Diabetes was induced by streptozotocin in male c57bl6 mice and ACE2 knockout (KO) mice. After 5 weeks of study, animals were randomized to receive the ACE inhibitor perindopril (2 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ). Wild-type mice were further randomized to receive the selective ACE2 inhibitor MLN-4760 (10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) and followed for an additional 5 weeks. Markers of renal function and injury were then assessed.RESULTS-Induction of diabetes in wild-type mice was associated with a reduction in renal ACE2 expression and decreased Ang 1-7. In diabetic mice receiving MLN-4760 and in ACE2 KO mice, diabetes-associated albuminuria was enhanced, associated with an increase in blood pressure. However, renal hypertrophy and fibrogenesis were reduced in diabetic mice with ACE2 deficiency, and hyperfiltration was attenuated. Diabetic wild-type mice treated with an ACE inhibitor experienced a reduction in albuminuria and blood pressure. These responses were attenuated in both diabetic ACE2 KO mice and diabetic mice receiving MLN-4760. However, other renoprotective and antifibrotic actions of ACE inhibition in diabetes were preserved in ACE2-deficient mice.CONCLUSIONS-The expression of ACE2 is significantly modified by diabetes, which impacts both pathogenesis of kidney disease and responsiveness to RAS blockade. These data indicate that ACE2 is a complex and site-specific modulator of diabetic kidney disease.

Aims/hypothesis Formation of AGEs is increased in the diabetic milieu, which contributes to accelerated atherogenesis. We studied whether delayed treatment with AGE-inhibiting compounds, alagebrium chloride and pyridoxamine dihydrochloride, affected established atherosclerosis in experimental diabetes in comparison with the angiotensin-converting enzyme inhibitor, quinapril. Methods Streptozotocin-induced diabetic male Apoe −/− mice (n=24 per group) received, by oral gavage, from week 10 to 20 of diabetes: no treatment; alagebrium (1 mg kg −1 day −1 ); pyridoxamine (1 g/l in drinking water); or quinapril (30 mg kg −1 day −1 ). Atherosclerotic lesion area (en face analysis) was evaluated for all groups. Results Delayed intervention with alagebrium decreased plaque area in the diabetic Apoe −/− mice compared with untreated mice (total plaque area: alagebrium 10.6±1.6%, untreated, 15.1±1.5%, p<0.05). This anti-atherosclerotic effect was comparable with that achieved with quinapril (quinapril 8.4±1.4%, vs untreated, p<0.05). Pyridoxamine also attenuated plaque development in diabetic mice (5.7± 1.2% vs untreated 11.9 ± 1.1%, p < 0.05). The antiatherosclerotic effect conferred by alagebrium and quinapril was associated with a significant reduction in vascular oxidative stress and circulating AGEs and methylglyoxal, although preformed AGEs were not removed from the vascular wall with either delayed intervention. Conclusions/interpretation Inhibition of AGE accumulation, using a delayed intervention with alagebrium or pyridoxamine, significantly attenuated the progression of established diabetes-associated atherosclerosis, similar to results obtained with quinapril. These findings provide further evidence that blockade of AGE-mediated pathways may present a novel therapy for the prevention of atherosclerosis in diabetes.

Aims/hypothesis We evaluated the anti-atherosclerotic effect of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, rosuvastatin, and the angiotensin II receptor blocker (ARB), candesartan, alone and in combination, in the streptozotocin-induced diabetic apolipoprotein Edeficient (Apoe −/− ) mouse. Methods Control and streptozotocin-induced diabetic Apoe −/− mice received rosuvastatin (5 mg kg −1 day −1 ), candesartan (2.5 mg kg −1 day −1 ), dual therapy or no treatment for 20 weeks. Aortic plaque deposition was assessed by Sudan IV staining and subsequent visual quantification. The abundance of proteins was measured using immunohistochemistry. Results Diabetes was associated with a fourfold increase in total plaque area. Rosuvastatin attenuated plaque area in diabetic mice in the absence of lipid-lowering effects. The anti-atherosclerotic effect of rosuvastatin was comparable to that observed with candesartan. A similar beneficial effect was seen with dual therapy, although it was not superior to monotherapy. Rosuvastatin treatment was associated with attenuated accumulation of AGE and AGE receptor (RAGE) in plaques. Similar beneficial effects on markers of oxidative stress were seen with the ARB and statin. Candesartan was more effective at reducing macrophage accumulation and collagen I abundance in plaques compared with rosuvastatin. The combined effect of candesartan and rosuvastatin was superior in reducing macrophage infiltration, monocyte chemoattractant protein-1 level, vascular AGE accumulation and RAGE abundance in the vascular wall. Furthermore, the combination tended to be more effective in reducing smooth muscle cell infiltration and connective tissue growth factor abundance in plaques. Conclusions/interpretation Rosuvastatin has direct antiatherosclerotic effects in diabetic macrovascular disease. These effects are independent of effects on lipids and comparable to the effects observed with candesartan.

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In the print version of the article listed above, Fig. 5C is incorrect. During the revision process, Fig. 5A was mistakenly duplicated into Fig. 5C. The correct Fig. 5C appears below.