AngII-induced Atherosclerosis Research Articles

Vasohibin-2 Aggravates Development of Ascending Aortic Aneurysms but not Abdominal Aortic Aneurysms nor Atherosclerosis in ApoE-Deficient Mice.

Vasohibin-2 (VASH2) has been isolated as a homologue of vasohibin-1 (VASH1) that promotes angiogenesis counteracting with VASH1. Chronic angiotensin II (AngII) infusion promotes both ascending and abdominal aortic aneurysms (AAs) in mice. The present study aimed to investigate whether exogenous VASH2 influenced AngII-induced vascular pathology in apolipoprotein E-deficient (ApoE-/-) mice. Male, ApoE-/- mice (9-14 weeks old) were injected with Ad LacZ or Ad VASH2. After a week, saline or AngII (1,000 ng/kg/minute) was infused into the mice subcutaneously via mini-osmotic pumps for 3 weeks. Consequently, all these mice were divided into 4 groups: saline + LacZ (n = 5), saline + VASH2 (n = 5), AngII + LacZ (n = 18), and AngII + VASH2 (n = 17). Exogenous VASH2 had no significant effect on ex vivo maximal diameters of abdominal aortas (AngII + LacZ: 1.67 ± 0.17 mm, AngII + VASH2: 1.52 ± 0.16 mm, n.s.) or elastin fragmentation and accumulation of inflammatory cells. Conversely, exogenous VASH2 significantly increased intima areas of aortic arches (AngII + LacZ: 16.6 ± 0.27 mm2, AngII + VASH2: 18.6 ± 0.64 mm2, P = 0.006). VASH2 effect of AngII-induced ascending AAs was associated with increased cleaved caspase-3 abundance. AngII-induced atherosclerosis was not altered by VASH2. The present study demonstrated that augmented VASH2 expression had no effect of AngII-induced abdominal AAs or atherosclerosis, while increasing dilation in the ascending aorta.

Abstract 103: Vascular Smooth Muscle Cell PGC1alpha Deletion is Atheroprotective in vivo

Introduction: In vivo studies found H 2 O 2 leads to lipid oxidation in the arterial wall and atherosclerosis formation. Catalase overexpression was found to reduce the development of aortic atherosclerosis. In vitro studies demonstrated effective transcriptional control of catalase expression through a PPARgamma coactivator 1-alpha (PGC1α)-mediated signaling cascade in the setting of ANG II exposure. Here we sought to characterize in vivo the role of PGC1α as a regulator in atherosclerosis formation. Methods: All mice were homozygous for floxed PGC1α. In addition, littermates were either knockin for SM22-Cre (PGC1α SMC-/- ) or Cre-negative (PGC1α SMC+/+ ). A retro-orbital injection of rAAV8 vectors carrying gain-of-function PCSK9 was given to 10-week-old PGC1α SMC-/- (7 M) and PGC1α SMC+/+ (9 M) mice. Mice were fed with high-fat diet and received SQ ANG II at 0.75 mg/kg/day over 6 weeks. Descending thoracoabdominal and ascending thoracic aorta atherosclerotic lesions were assessed en face and with microscopy, respectively. Catalase expression was assessed by WB in isolated MASMs and IHC staining of aortic cross-sections. Statistical significance was determined by t test. Results: When comparing PGC1α SMC-/- and PGC1α SMC+/+ mice, we found no significant difference in baseline SBP, hypertensive response to Ang II and serum LDL levels. PGC1α SMC-/- mice had significantly less thoracoabdominal aorta atherosclerosis formation (Figure A and B). PGC1α SMC-/- MASMs showed increased catalase protein expression (Figure C) but no difference in ROS production at baseline. After ANG II stimulation, PGC1α SMC-/- MASMs were noted to have decreased ROS production. IHC confirmed increased catalase staining in the tunica media of PGC1α SMC-/- mice, which was more evident after ANG II infusion for 14 days (Figure D). Conclusion: Vascular SMC PGC1α deletion leads to increased expression of catalase with a subsequent decrease in ANG II-induced atherosclerosis formation in vivo.

Mas receptor deficiency augments angiotensin II-induced atherosclerosis and aortic aneurysm ruptures in hypercholesterolemic male mice

ObjectivePrevious studies demonstrated that deficiency of angiotensin-converting enzyme 2 (ACE2) augmented angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. Effects of ACE2 deficiency could arise from increased concentrations of its substrate, AngII, or decreased concentrations of its product, angiotensin-(1-7) [Ang-(1-7)]. Infusion of Ang-(1-7), a Mas receptor (MasR) ligand, to hypercholesterolemic male mice reduced AngII-induced atherosclerosis, suggesting a protective role of the Ang-(1-7)/MasR axis. However, it is unclear whether endogenous Ang-(1-7) acts at MasR to influence AngII-induced vascular diseases. The purpose of this study was to define the role of MasR deficiency in AngII-induced atherosclerosis and AAA formation and severity in hypercholesterolemic male mice. MethodsMasR+/+ and MasR−/− male mice on a low-density lipoprotein receptor-deficient (Ldlr−/−) or apolipoprotein E-deficient (Apoe−/−) background were infused with AngII at either 600 or 1000 ng/kg/min by osmotic minipump for 28 days. Atherosclerosis was quantified at study end point as percentage lesion surface area of the aortic arch in Ldlr−/− mice. Abdominal aortic internal diameters were quantified by ultrasound, and maximal external AAA diameters were quantified at study end point. Blood pressure was quantified by radiotelemetry and a tail cuff-based technique. Serum cholesterol concentrations and vascular tissue characterization were examined at study end point. ResultsMasR deficiency did not influence body weight, systolic blood pressure at baseline and during AngII infusion, or serum cholesterol concentrations in either Apoe−/− or Ldlr−/− mice. MasR deficiency increased AngII-induced atherosclerosis in aortic arches of Ldlr−/− mice (P < .05), associated with increased oxidative stress and apoptosis in aortic root sections (P < .05). MasR deficiency also augmented internal and external AAA diameters and increased aortic ruptures of both Ldlr−/− and Apoe−/− mice (P < .05). These effects were associated with increased elastin breaks and T-lymphocyte and macrophage accumulation into abdominal aortas of AngII-infused MasR-deficient mice (P < .05). ConclusionsThese results demonstrate that MasR deficiency augmented AngII-induced atherosclerosis and AAA rupture through mechanisms involving increased oxidative stress, inflammation, and apoptosis, suggesting that MasR activation may provide therapeutic efficacy against vascular diseases.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Voluntary Exercise Stabilizes Established Angiotensin II-Dependent Atherosclerosis in Mice through Systemic Anti-Inflammatory Effects.

We have previously demonstrated that exercise training prevents the development of Angiotensin (Ang) II-induced atherosclerosis and vulnerable plaques in Apolipoprotein E-deficient (ApoE-/-) mice. In this report, we investigated whether exercise attenuates progression and promotes stability in pre-established vulnerable lesions. To this end, ApoE-/- mice with already established Ang II-mediated advanced and vulnerable lesions (2-kidney, 1-clip [2K1C] renovascular hypertension model), were subjected to sedentary (SED) or voluntary wheel running training (EXE) regimens for 4 weeks. Mean blood pressure and plasma renin activity did not significantly differ between the two groups, while total plasma cholesterol significantly decreased in 2K1C EXE mice. Aortic plaque size was significantly reduced by 63% in 2K1C EXE compared to SED mice. Plaque stability score was significantly higher in 2K1C EXE mice than in SED ones. Aortic ICAM-1 mRNA expression was significantly down-regulated following EXE. Moreover, EXE significantly down-regulated splenic pro-inflammatory cytokines IL-18, and IL-1β mRNA expression while increasing that of anti-inflammatory cytokine IL-4. Reduction in plasma IL-18 levels was also observed in response to EXE. There was no significant difference in aortic and splenic Th1/Th2 and M1/M2 polarization markers mRNA expression between the two groups. Our results indicate that voluntary EXE is effective in slowing progression and promoting stabilization of pre-existing Ang II-dependent vulnerable lesions by ameliorating systemic inflammatory state. Our findings support a therapeutic role for voluntary EXE in patients with established atherosclerosis.

Exogenous 17-β estradiol administration blunts progression of established angiotensin II-induced abdominal aortic aneurysms in female ovariectomized mice.

BackgroundAbdominal aortic aneurysms (AAAs) occur predominately in males. However, AAAs in females have rapid growth rates and rupture at smaller sizes. Mechanisms contributing to AAA progression in females are undefined. We defined effects of ovariectomy, with and without 17-β estradiol (E2), on progression of established angiotensin II (AngII)-induced AAAs in female mice.MethodsWe used neonatal testosterone exposures at 1 day of age to promote susceptibility to AngII-induced AAAs in adult female Ldlr−/− mice. Females were infused with AngII for 28 days to induce AAAs, and then stratified into groups that were sham, ovariectomized (Ovx, vehicle), or Ovx with E2 administration for 2 months of continued AngII infusions. Aortic lumen diameters were quantified by ultrasound and analyzed by linear mixed model, and maximal AAA diameters were analyzed by one-way ANOVA. Atherosclerosis was quantified en face in the aortic arch. AAA tissue sections were analyzed for cellular composition. We quantified effects of E2 on abdominal aortic smooth muscle cell (SMC) growth, α-actin and transforming growth factor-beta (TGF-β) production, and wound healing.ResultsSerum E2 concentrations were increased significantly by E2. Aortic lumen diameters increased over time in sham-operated and Ovx (vehicle) females, but not in Ovx females administered E2. At day 70, E2 administration decreased significantly aortic lumen diameters compared to Ovx vehicle and sham-operated females. Compared to Ovx females (vehicle), maximal AAA diameters were reduced significantly by E2. AAA tissue sections from Ovx females administered E2 exhibited significant increases in α-actin and decreases in neutrophils compared to Ovx females administered vehicle. In abdominal aortic SMCs, E2 resulted in a concentration-dependent increase in α-actin, elevated TGF-β, and more rapid wound healing. E2 administration to Ovx females also significantly reduced atherosclerotic lesions compared to sham-operated females. This effect was accompanied by significant reductions in serum cholesterol concentrations.ConclusionsE2 administration to Ovx females abolished progressive growth and decreased severity of AngII-induced AAAs. These effects were accompanied by increased SMC α-actin, elevated TGF-β, and reduced neutrophils. Similarly, E2 administration reduced AngII-induced atherosclerosis. These results suggest that loss of E2 in post-menopausal females may contribute to progressive growth of AAAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-015-0030-1) contains supplementary material, which is available to authorized users.

Abstract 587: Leukocyte Calpain Deficiency Reduces Angiotensin II-induced Inflammation and Atherosclerosis in Hypercholesterolemic Mice

Background and Objective: Chronic angiotensin II (AngII) infusion promotes atherosclerosis and abdominal aortic aneurysms (AAAs) in mice. Recently, we demonstrated that pharmacological inhibition of calpain-1 and -2 (a class of calcium-activated, neutral cysteine proteases) attenuated AngII-induced atherosclerosis, AAAs, and medial macrophage accumulation in mice. Using mice that overexpress calpastatin (CAST), the endogenous inhibitor of ubiquitous calpain-1 and -2 or mice with specific deficiency of calpain-1 or -2, the purpose of this study was to determine whether calpains in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and AAAs in hypercholesterolemic mice. Methods and Results: Irradiated male LDL receptor -/- mice were repopulated with BM-derived cells that were harvested from either wild type (WT) or CAST overexpression transgenic (Tg) mice. Four weeks after BM repopulation, recipient mice were fed a saturated fat-enriched diet and infused with either saline or AngII (1,000 ng/kg/min) for 4 weeks. AngII infusion increased systolic blood pressure equivalently in both genotypes. Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in WT and Tg mice (1.28 ± 0.11 versus 1.32 ± 0.08 mm, P = NS). Interestingly, overexpression of CAST in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches by en face measurement (Intimal lesions: WT = 19.9 ± 1.8 % vs Tg = 9.9 ± 1.0 %; n=14, P&lt;0.05). Using either BM cells-derived from calpain-1 deficient mice or mice with leukocyte-specific calpain-2 deficiency (Calpain-2f/f LysM Cre), further studies demonstrated that deficiency of either calpain-1 or -2 in leukocytes resulted in a modest, but significant, (P&lt;0.05) decrease in AngII-induced atherosclerosis (Calpain-1: WT- 15 ± 1.2 % vs KO- 10 ± 0.96 %; Calpain-2f/f: LysM Cre WT-16 ± 1.4 % vs KO-11 ± 1.2 %). CAST overexpression significantly attenuated AngII-induced inflammatory responses in macrophages in vivo, and suppressed macrophage migration towards MCP-1 and adhesion to endothelial cells in vitro. Conclusion: Calpain inhibition in BM-derived cells attenuated AngII-induced atherosclerosis by influencing macrophage function.

Abstract 454: An XX Sex Chromosome Complement, in the Presence of Testosterone, Markedly Promotes Angiotensin II-Induced Abdominal Aortic Aneurysms in Hypercholesterolemic Male Mice

Objective: Male sex is a strong risk factor for abdominal aortic aneurysms (AAAs). Similarly, angiotensin II (AngII)-induced AAAs exhibit a 4-fold higher prevalence in male compared to female hypercholesterolemic mice, which is abolished by removal of testosterone. We recently demonstrated that acute administration of testosterone to neonatal female mice markedly promoted their adult susceptibility to AngII-induced AAAs in the absence of a requirement for adult testosterone. In this study, we tested the hypothesis that testosterone acts through the sex chromosome complement (SCC) to promote AngII-induced AAAs. Methods and Results: Male transgenic mice with deletion of Sry from the Y-chromosome expressing Sry on autosomes (8-12 weeks of age) were bred to female Ldlr-/- mice to generate male mice with an XY or an XX SCC. Mice were fed a western diet and segregated into sham and orchiectomized (ORC) groups. Two weeks later, mice were implanted with osmotic micropumps to infuse AngII (1,000 ng/kg/min) for 28 days. Internal abdominal aortic lumen diameters were significantly larger in sham XX versus XY male mice at day 27 (XX, 1.77 ± 0.08; XY, 1.59 ± 0.05, p= 0.019). Castration decreased internal aortic lumen diameters in both genotypes, eliminating group differences (p=0.389). AAA external diameters were significantly increased in sham XX versus XY males (XX, 2.12 ± 0.17; XY, 1.55 ± 0.12, p=0.004), and this effect was abolished by castration. The presence of an additional X chromosome increased AAA incidence from 75% to 95% (p=0.136) and rupture rate from 11% to 30% (p=0.238). In contrast, an XY SCC promoted AngII-induced atherosclerosis (ORC: XY, 32.1 ± 6.5%; XX, 14.9 ± 1.6%, p=0.02), and this effect was reversed by castration (sham, XY: 19.4 ± 4.2, p=0.036). Conclusion: These results demonstrate that testosterone acts at an XX SCC to markedly promote the severity of AngII-induced AAAs in male mice. In contrast, testosterone acts at an XY SCC to protect against AngII-induced atherosclerosis in male mice. These results demonstrate, for the first time, that complex interactions between sex hormones and sex chromosomes diversely influence vascular diseases promoted by AngII.

Abstract 249: Male Mice with XX Sex Chromosome Complement Exhibit Reduced Angiotensin II-Induced Hypertension and Atherosclerosis

Objective: Hypertension and atherosclerosis exhibit sexual dimorphism, partially ascribed to effects of sex hormones. However, recent studies suggest that sex chromosome complement may also contribute to sexual dimorphism of these diseases. The renin-angiotensin system has been suggested to contribute to sexual dimorphism of hypertension and atherosclerosis. However, the relative contribution of sex hormones, versus chromosomes, in the regulation of angiotensin II (AngII)-mediated responses is unknown. We hypothesized that the presence of an XX chromosome complement in male hyperlipidemic mice would reduce AngII-induced hypertension and atherosclerosis. Methods and Results: Male Ldlr-/- mice with an Sry mutation but with an Sry transgene on autosomes were bred to Ldlr-/- females to generate XY or XX males. Male XY or XX mice were segregated to sham-operated or orchiectomized (ORC) groups and fed a high fat diet (42% kcal from fat). Two weeks later, mice were infused with AngII (1,000 ng/kg/min) for 4 weeks. In the presence of sex hormones (sham groups), systolic blood pressure (SBP) was significantly lower in AngII-infused XX than XY males (XY, 136 ± 4; XX, 119 ± 4 mmHg, p=0.024). In XY males, ORC had no significant effect on SBP compared to sham (ORC XY, 129 ± 6 mmHg; p=0.48). However, following castration of XX males, SBP significantly increased (ORC XX 135 ± 3 mmHg; p =0.002). In XY males, ORC resulted in a significant increase in atherosclerotic lesion surface area in the aortic arch (sham, 19 ± 4; ORC, 32 ± 7%; p=0.04). In contrast, ORC had no significant effect on lesion surface area in XX males (sham, 17 ± 3; ORC, 15 ± 2%; p=0.53). Moreover, in the absence of sex hormones, lesion surface area was markedly increased in XY compared to XX males (XY, 32 ± 7; XX, 15 ± 2%; p=0.002). Conclusions: These results demonstrate that effects of testosterone to regulate AngII-induced hypertension and atherosclerosis are dependent on sex chromosome complement. In XY males, testosterone protects against AngII-induced atherosclerosis. In XX males, testosterone protects against AngII-induced hypertension. Moreover, there is pronounced effect of XY chromosome complement to promote AngII-induced atherosclerosis in males.

Voluntary exercise slows the progression of established angiotensin II-dependent advanced atherosclerotic lesions

Objectives: We have previously demonstrated that exercise training prevents the development of Angiotensin (Ang) II-induced atherosclerosis and vulnerable plaques in ApoE-/- mice. In the present study, we investigated whether exercise attenuates the progression of established Ang II-dependent atherosclerosis.

Abstract 495: Calpain Inhibition in Bone Marrow--Derived Cells Attenuates Atherosclerosis but Not Aortic Aneurysms in Hypercholesterolemic Mice Infused With Angiotensin II

Background and Objective: Calpains are calcium-dependent neutral cysteine proteases that are essential for multiple cellular functions such as cytoskeletal remodeling, cell cycle, and apoptosis. Angiotensin II (AngII)-infusion into mice profoundly increased both aortic protein and activity of calpains. In addition, pharmacological inhibition of calpain attenuated AngII-induced aortic medial macrophage accumulation, atherosclerosis, ascending and abdominal aortic aneurysm (AAs) in mice. Using mice that overexpress calpastatin, the endogenous inhibitor of calpains, the purpose of this study was to determine whether calpains in bone marrow-derived cells contribute to AngII-induced atherosclerosis and AAs in hypercholesterolemic mice. Methods and Results: Bone marrow cells were harvested from either calpastatin (CAST) overexpression transgenic (Tg) or wild type (WT) mice and injected (7.5 x 10 6 cells/mouse) into age-matched male LDL receptor -/- recipient mice that had been irradiated with a total of 900 Rads from a cesium source. Four weeks after bone marrow repopulation, recipient mice were fed a saturated fat-enriched diet and infused with either saline or AngII (1,000 ng/kg/min) for 4 weeks. The CAST Tg or WT genotypes used for repopulation had no effect on body weight, white blood cell counts and plasma cholesterol concentrations during AngII infusion. Baseline systolic blood pressure was not different, and AngII infusion increased systolic blood pressure equivalently in both genotypes. Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in WT and Tg mice (1.28 ± 0.11 versus 1.32 ± 0.08 mm, P = NS). Calpain inhibition showed no discernible effects on AngII-induced ascending AA development (11.7 ± 0.47 versus 11.1 ± 0.54 mm 2 , P = NS). Interestingly, overexpression of CAST in bone marrow-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches by en face measurement (Intimal lesions: WT = 19.9 ± 1.8 % versus Tg = 11.0 ± 1.2 %; n=14, P&lt;0.001). Conclusion: Calpain inhibition in bone marrow-derived cells attenuates AngII-induced atherosclerosis but does not influence formation of ascending and abdominal AAs in hypercholesterolemic mice.

Abstract 277: Diminazene Aceturate, an ACE2 Activator, Decreases AngII-Induced Atherosclerosis in Low-Density Lipoprotein Receptor--Deficient Mice

Objectives: Diminazene aceturate (DIZE) activates angiotensin converting enzyme 2 (ACE2) both in vitro and in vivo. ACE2 limits angiotensin II (AngII)/angiotensin type 1 receptor (AT1R) signaling through cleavage of AngII to form angiotensin-(1-7) (Ang-(1-7)). Previous studies in our laboratory demonstrated that whole body ACE2 deficiency increased diet-induced atherosclerosis in low density lipoprotein receptor (LDLr) deficient mice. The purpose of this study was to determine if administration of DIZE to LDLr-/- mice activates ACE2 to decrease AngII-induced atherosclerosis. Materials and Methods: Initial dose ranging studies determined the effects of 7.5, 15, or 30 mg/kg/day of DIZE infused subcutaneously by osmotic minipump for 7 days into male LDLr-/- mice. DIZE dose-dependently increased plasma Ang-(1-7) concentrations with significant elevations at 30 mg/kg/day (vehicle, 5.9 ± 0.6; DIZE, 7.9 ± 0.5 ng/mL, p&lt;0.05). Moreover, kidney ACE2 activity increased in mice administered DIZE (30 mg/kg/day) compared to vehicle-injected controls (vehicle, 208 ± 87; DIZE, 1716 ± 424 fmol/mg/min, p&lt;0.05). For optimal dosing, ACE2 wildtype (Ace2+/y) and deficient (Ace2-/y) LDLr-/- mice were administered either vehicle or DIZE by intramuscular injection for a total of 5 weeks. Seven days after initiation of DIZE administration, mice were infused with AngII (1,000 ng/kg/min for 28 days) to induce atherosclerosis. Compared to vehicle, administration of DIZE resulted in significant reductions in atherosclerotic lesion formation in aortic arches of AngII-infused Ace2+/y , but not Ace2-/y mice (vehicle Ace2+/y, 9.8 ± 1.2; DIZE Ace2+/y, 5.8 ± 1.2; DIZE Ace2-/y, 9.2 ± 1.8, p&lt;0.05). Interestingly, compared to vehicle, DIZE lowered serum cholesterol concentrations in Ace2+/y but not Ace2-/y mice (vehicle Ace2+/y, 837 ± 69; DIZE Ace2+/y, 571 ± 41; DIZE Ace2-/y, 774 ± 113 mg/dl, p&lt;0.05). Conclusions: These studies demonstrate that DIZE decreased AngII-induced atherosclerosis through an ACE2-dependent mechanism. Mechanisms for DIZE-induced reductions in AngII-induced atherosclerosis may include reductions in serum cholesterol concentrations.

Abstract 289: Sex Hormones Influence Progression of Angiotensin II-Induced Abdominal Aortic Aneurysms in Hypercholesterolemic Mice

Objectives: Similar to humans, angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA) exhibit marked sexual dimorphism with a greater prevalence in male than female mice. We demonstrated that castration of male mice reduced formation of AngII-induced AAAs, while castration of females had no effect. However, the role of sex hormones (testosterone or estrogen) on the progression of an established AAA is undefined. We tested the hypothesis that sex hormones influence the progression of AngII-induced AAAs. Methods/Results: Male ApoE-/- male mice were infused with AngII (1,000 ng/kg/min) for 28 days to induce AAAs, stratified to sham-operated or castration groups, and subsequently infused with AngII for 56 days. Aortic lumen diameters were decreased significantly in castrated compared to sham controls (1.9 ± 0.1 vs 1.6 ± 0.1 mm, P&lt;0.05). While external AAA diameters were not different between groups, 3D image analysis revealed increased wall/lumen ratios of AAAs from castrated compared to sham controls (9.5 ± 2.0 vs 4.8 ± 0.9 ratio; P&lt;0.05). Increased AAA wall thickness was associated with more pronounced α-actin immunostaining and increased matrix deposition. In contrast, castration had no effect on AngII-induced atherosclerosis. LDLr-/- female mice that received a single dose of testosterone at 1 day of age to promote adult AAA susceptibility were infused with AngII. Females with an established AAA were stratified to sham-operated, castrated, and castrated+estrogen (E 2 , 36 μg/ml) groups for 56 days of AngII infusions. Castration of females significantly increased aortic lumen diameters compared to castrated females administered E 2 (vehicle, 2.2 ±0.2; E 2 , 1.7 ± 0.1 mm, P&lt;0.05). Moreover, E 2 administration significantly decreased external aortic diameters (castrated, 2.6 ± 0.3; castrated+E 2 , 1.7 ± 0.1 mm, P&lt;0.05). Finally, castrated females administered E 2 had significantly decreased atherosclerosis compared to sham controls (38.9 ± 3.6 vs 49.6 ± 2.3 % lesion surface area; P&lt;0.05). Conclusions: These results indicate that testosterone contributes to AAA progression in males whereas E 2 administration lowered AAA progression in female mice.

Amlodipine Reduces AngII-Induced Aortic Aneurysms and Atherosclerosis in Hypercholesterolemic Mice

BackgroundThe purpose of this study was to determine effects of amlodipine, a dihydropyridine calcium channel blocker, on development of angiotensin II (AngII)-induced vascular pathologies.Methods and ResultsMale LDL receptor -/- mice were infused with vehicle, amlodipine (5 mg/kg/d), AngII (1,000 ng/kg/min), or AngII + amlodipine for 4 weeks through osmotic pumps (n=10/group). Mice were fed a saturated fat-enriched diet for 1 week prior to pump implantation and during 4 weeks of infusion. Infusion of amlodipine resulted in plasma concentrations of 32 ± 2 ng/ml and 27 ± 2 ng/ml for mice in saline + amlodipine and AngII + amlodipine groups, respectively. This infusion rate of amlodipine did not affect AngII-induced increases in systolic blood pressure. Three of 10 (30%) mice infused with AngII died of aortic rupture, while aortic rupture did not occur in mice co-infused with AngII + amlodipine. Suprarenal aortic width and intimal area of ascending aortas were measured to define aortic aneurysms. In the absence of AngII infusion, amlodipine did not change suprarenal aortic width and ascending aortic area. Infusion of AngII led to profound increases of suprarenal aortic width (saline + vehicle versus AngII + vehicle: 0.86 ± 0.02 versus 1.72 ± 0.26 mm; P=0.0006), whereas co-infusion of AngII and amlodipine diminished abdominal dilation (1.02 ± 0.14 mm; P=0.003). As expected, AngII infusion increased mean intimal area of ascending aortas (saline + vehicle versus AngII + vehicle: 8.5 ± 0.3 versus 12.5 ± 1.1 mm2; P=0.001), while co-infusion of AngII and amlodipine ablated dilation of the ascending aorta (8.6 ± 0.2 mm2; P=0.03). Co-administration of amlodipine also significantly attenuated AngII-induced atherosclerosis in the thoracic region as quantified by percent lesion area (AngII + vehicle versus AngII + amlodipine: 5.8 ± 2.1 % versus 0.3 ± 0.1%; P=0.05).ConclusionsAmlodipine inhibited AngII-induced aortic aneurysms in both the abdominal and ascending regions, and atherosclerosis in hypercholesterolemic mice.

The Effect of Soluble RAGE on Inhibition of Angiotensin II-Mediated Atherosclerosis in Apolipoprotein E Deficient Mice

BackgroundThe cross talk between RAGE and angiotensin II (AngII) activation may be important in the development of atherosclerosis. Soluble RAGE (sRAGE), a truncated soluble form of the receptor, acts as a decoy and prevents the inflammatory response mediated by RAGE activation. In this study, we sought to determine the effect of sRAGE in inhibiting AngII-induced atherosclerosis in apolipoprotein E knockout mice (Apo E KO).Methods and Results9 week old Apo E KO mice were infused subcutaneously with AngII (1 µg/min/kg) and saline for 4 weeks using osmotic mini-pumps. The mice were divided into 4 groups 1. saline infusion and saline injection; 2. saline infusion and sRAGE injection; 3. AngII infusion and saline injection; 4. AngII infusion and sRAGE injection. Saline or 0.5 µg, 1 µg, to 2 µg/day/mouse of sRAGE were injected intraperitoneally daily for 28 days. We showed that atherosclerotic plaque areas in the AngII-infused Apo E KO mice and markers of inflammation such as RAGE, ICAM-1, VCAM-1, and MCP-1 were increased in aorta compared to that of the Apo E KO mice. However, the treatment of 0.5 µg, 1 µg, and 2 µg of sRAGE in AngII group resulted in the dose-dependent decrease in atherosclerotic plaque area. We also demonstrated that sRAGE decreased RAGE expression level as well as inflammatory cytokines and cell adhesion molecules in AngII or HMGB1 treated-rat aorta vascular smooth muscle cells.ConclusionThe results demonstrated that partical blockade of RAGE activation by sRAGE prevent AngII -induced atherosclerosis. Therefore these results suggested that first, RAGE activation may be important in mediating AngII-induced atherogenesis, and second, AngII activation is a major pathway in the development of atherosclerosis. Taken together, results from this study may provide the basis for future anti- atherosclerotic drug development mediated through RAGE activation.

Prevention of TGFβ induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr−/− mice

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFβ is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFβ neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFβ1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFβ attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFβ-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.

Abstract 517: Effect of Hepatocyte-specific Depletion of AT1a Receptors on Atherosclerosis and Aneurysms

Background and Objective Whole body deficiency of AT1a receptors (AT1aRs) ablates angiotensin II (AngII) induced vascular pathologies that include aortic aneurysms and atherosclerosis. We created AT1aR floxed mice to determine the role of specific cell types expressing this receptor on these vascular pathologies. Unexpectedly, these mice demonstrated a lack of effect of smooth muscle specific AT1aR deficiency on the development of atherosclerosis or aneurysms. Also, endothelial-specific AT1aR deficiency had no effect in atherosclerosis and minimal effect on aneurysms. The purpose of this study was to determined if AngII infusion was releasing substances from the liver that influence vascular pathology. Methods and Results Hepatocyte depletion of AT1aRs was accomplished using two methods of introducing Cre into AT1aR floxed mice. One approach was injection of adenovirus Cre (AdCre) and the other by breeding to mice expressing Cre under the control of the albumin promoter. To induce atherosclerosis, groups of wild type and hepatocyte-specific AT1aR depleted male (N=15-21/group) and female (N=21/group) mice were fed a saturated fat enriched diet for 12 weeks. Hepatocyte-specific depletion of AT1aRs was confirmed using detection of Cre and absence of AT1aRs by PCR and RT-PCR, respectively. Body weights, serum cholesterol concentrations, and systemic blood pressures were not different between wild type and hepatocyte-specific AT1aR depleted mice grouped by gender and genotype. Atherosclerotic burden was similar in all groups. To examine AngII-induced atherosclerosis, male wild type and hepatocyte-specific AT1aR depleted mice (N=11-16/group) were fed a fat enriched diet for 5 weeks, and AngII (1,000 mg/kg/min) was infused using osmotic mini-pumps for 28 days. AngII infusion increased systolic blood pressure, promoted atherosclerosis and induced aneurysms to a similar degree in both genotypes. Conclusion Deletion of hepatocyte-specific AT1aRs did not affect dietary or AngII-induced vascular pathologies.

PD123319 Augments Angiotensin II-Induced Abdominal Aortic Aneurysms through an AT2 Receptor-Independent Mechanism

BackgroundAT2 receptors have an unclear function on development of abdominal aortic aneurysms (AAAs), although a pharmacological approach using the AT2 receptor antagonist PD123319 has implicated a role. The purpose of the present study was to determine the role of AT2 receptors in AngII-induced AAAs using a combination of genetic and pharmacological approaches. We also defined effects of AT2 receptors in AngII-induced atherosclerosis and thoracic aortic aneurysms.Methods and ResultsMale AT2 receptor wild type (AT2 +/y) and deficient (AT2 -/y) mice in an LDL receptor −/− background were fed a saturated-fat enriched diet, and infused with either saline or AngII (500 ng/kg/min). AT2 receptor deficiency had no significant effect on systolic blood pressure during AngII-infusion. While AngII infusion induced AAAs, AT2 receptor deficiency did not significantly affect either maximal width of the suprarenal aorta or incidence of AAAs. The AT2 receptor antagonist PD123319 (3 mg/kg/day) and AngII were co-infused into male LDL receptor −/− mice that were either AT2 +/y or −/y. PD123319 had no significant effect on systolic blood pressure in either wild type or AT2 receptor deficient mice. Consistent with our previous findings, PD123319 increased AngII-induced AAAs. However, this effect of PD123319 occurred irrespective of AT2 receptor genotype. Neither AT2 receptor deficiency nor PD123319 had any significant effect on AngII-induced thoracic aortic aneurysms or atherosclerosis.ConclusionsAT2 receptor deficiency does not affect AngII-induced AAAs, thoracic aortic aneurysms and atherosclerosis. PD123319 augments AngII-induced AAAs through an AT2 receptor-independent mechanism.

Abstract 402: Calpain-1 Deficiency Does Not Affect Angiotensin II-Induced Atherosclerosis or Abdominal Aortic Aneurysms in Male LDL Receptor--Deficient Mice

Background and Objective: Chronic infusion of angiotensin II (AngII) augments the development of atherosclerosis and induces abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Recently, we demonstrated that pharmacological inhibition of calpains, a class of calcium-activated, neutral cysteine proteases, attenuated AngII-induced atherosclerosis and AAAs in mice. The purpose of this study was to determine the contribution of calpain-1 in the development of AngII-induced atherosclerosis and AAA using genetically modified mice. Methods and Results: Male LDL receptor -/- mice (10-12 weeks old) that were either calpain-1 +/+ or -/- (n=20 per group) were fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for 5 weeks. After 1 week of feeding a saturated fat enriched diet, mice were infused subcutaneously with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. AngII increased systolic blood pressure similarly in both groups (pre-infusion- calpain-1 +/+: 122 ± 4; calpain-1 -/-: 123 ± 6 mmHg; post-infusion calpain-1 +/+: 156 ± 5; calpain-1 -/-: 153 ± 6 mmHg). Deficiency of calpain-1 had no effect on plasma cholesterol concentrations (calpain-1 +/+: 1589 ± 39; calpain-1 -/-: 1531 ± 70 mg/dL) or lipoprotein-cholesterol distributions during AngII infusion. AngII infusion led to development of AAA formation in 76% of calpain-1 +/+ mice and 65% in calpain-1 -/- mice. This difference was not statistically significant. AngII infusion greatly augmented the development of aortic arch atherosclerosis that was not attenuated in calpain-1 -/- mice, (calpain-1 +/+: 6.9 ± 1.0 %; calpain-1 -/- : 5.1 ± 0.8 % of arch area intimal lesions; P = not significant). Conclusion: These findings suggest that calpain-1 does not contribute to the development of angiotensin II-induced atherosclerosis or AAAs.

Abstract 399: Deficiency of Angiotensin Type 1a Receptors in Adipocytes Increases Fat Mass in Angiotensin II-Infused LDLR-Deficient Mice but Has No Effect on Vascular Pathologies

Objectives: Angiotensin II (AngII) promotes atherosclerosis and abdominal aortic aneurysms (AAA) through the angiotensin type 1a receptor (AT1aR). However, the cell type mediating these effects has not been defined. Adipose tissue surrounding the aorta may influence the inflammatory milieu within the vessel wall and thus contribute to disease progression. The purpose of this study was to determine the effect of adipocyte-AT1aR deficiency on development of AngII and diet-induced vascular pathologies in low density lipoprotein receptor deficient (LDLR-/-) mice. Methods and results: LDLR -/- mice with adipocyte-AT1aR deficiency (AT1aRaP2, n = 21) and littermates (AT1aRfl/fl, n=17) were fed a high-fat diet (HF, 42% kcal fat; 0.15 cholesterol) for 1 week prior to, and throughout, 28 days of AngII infusion (1,000 ng/kg/min). Adipocyte-AT1aR deficiency had no effect on AAA incidence (external diameter of aorta; AT1aRfl/fl 1.4±0.1 vs. AT1aRaP2 1.5±0.1 mm, p=0.41) and atherosclerotic lesion areas in the aortic arch (AT1aRfl/fl 7.1±0.8 vs. AT1aRaP2 8.3+0.8% lesion area, p=0.49). Serum cholesterol (AT1aRfl/fl 1981±109 vs. AT1aRaP2 2044±85 mg/dL, p=0.65) and triglyceride (AT1aRfl/fl 450±31 vs. AT1aRaP2 496±33 mg/dL, p=0.32) concentrations were also unaffected by adipocyte-AT1aR deficiency. Interestingly, body weight was significantly increased in AngII-infused AT1aRaP2 mice (AT1aRfl/fl 26.8±0.5 vs. AT1aRaP2 28.0±0.5 g, p=0.013 for interaction between genotype and time), due to increases in adipose mass (retroperitoneal fat; AT1aRfl/fl 0.26±0.03 vs. AT1aRaP2 0.36±0.02 g, p=0.017). Mice fed the HF diet for 3 months without AngII infusion did not exhibit any changes in atherosclerotic lesion area in the aortic arch (AT1aRfl/fl 14.3±1% vs. AT1aRaP2 11.7±1%, p=0.14). Conclusions: Adipocyte-AT1aR deficiency has no effect on development of diet- or AngII-induced atherosclerosis or AAAs in hypercholesterolemic mice. Interestingly, body weight and fat mass were increased in AngII-infused adipocyte AT1aR deficient mice. Future studies will elucidate the mechanisms through which AngII regulates body weight and fat mass under these conditions.