Sean E. Thatcher

Obesity Promotes Inflammation in Periaortic Adipose Tissue and Angiotensin II-Induced Abdominal Aortic Aneurysm Formation

Objective—Obesity promotes macrophage infiltration into adipose tissue and is associated with increases in several cardiovascular diseases. Infusion of angiotensin II (AngII) to mice induces formation of abdominal aortic aneurysms (AAAs) with profound medial and adventitial macrophage infiltration. We sought to determine whether obesity promotes macrophage infiltration and proinflammatory cytokines in periaortic adipose tissue surrounding abdominal aortas and increases AngII-induced AAAs. Methods and Results—Hypertrophied white adipocytes surrounded abdominal aortas, whereas brown adipocytes surrounded thoracic aortas of obese mice. mRNA abundance of macrophage proinflammatory chemokines and their receptors were elevated with obesity to a greater extent in abdominal compared to thoracic periaortic adipose tissue. Periaortic adipose tissue explants surrounding abdominal aortas of obese mice released greater concentrations of MCP-1 and promoted more macrophage migration than explants from thoracic aortas. Male C57BL/6 mice were fed a high-fat (HF) diet for 1, 2, or 4 months and then infused with AngII (1000 ng/kg/min) for 28 days. AAA incidence increased progressively with the duration of HF feeding (18%, 36%,and 60%, respectively). Similarly, AngII-infused ob/ob mice exhibited increased AAAs compared to lean controls (76% compared to 32%, respectively, P<0.05). Infusion of AngII to obese mice promoted further macrophage infiltration into periaortic and visceral adipose tissue, and obese mice exhibiting AAAs had greater macrophage content in visceral adipose tissue than mice not developing AAAs. Conclusions—Increased macrophage accumulation in periaortic adipose tissue surrounding abdominal aortas of AngII-infused obese mice is associated with enhanced AAA formation.

ACE2 is expressed in mouse adipocytes and regulated by a high-fat diet

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low- (LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 +/- 253; HF: 6,829 +/- 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.

The adipose renin–angiotensin system: Role in cardiovascular disease

Several reviews have highlighted the importance of local tissue production of components of the renin-angiotensin system (RAS) [Bader, M., Ganten, D., 2008. Update on tissue renin-angiotensin systems. J. Mol. Med. 86, 615-621; Krop, M., Danser, A.H., 2008. Circulating versus tissue renin-angiotensin system: on the origin of (pro)renin. Curr. Hypertens. Rep. 10, 112-118; Paul, M., Poyan Mehr, A., Kreutz, R., 2006. Physiology of local renin-angiotensin systems. Physiol. Rev. 86, 747-803]. While the concept of tissue RAS is gaining more widespread acceptance, the concept of local angiotensin II (AngII) production, acting in coordinate or independently of the endocrine RAS, continues to be debated. The primary reasons that local AngII production has been studied by many investigators are that components of the RAS are expressed by multiple cell types, and that the endocrine RAS cannot fully explain all effects of AngII. Moreover, through the development and study of genetically altered models for over-expression or knockdown of individual RAS components within specific cell types, it is becoming increasingly more evident that local RAS contribute to effects of AngII in normal physiology and disease. The purpose of this review is to define the presence and physiological significance of a local RAS in adipose tissue in relation to cardiovascular disease.

Adipocyte Deficiency of Angiotensinogen Prevents Obesity-Induced Hypertension in Male Mice

Previous studies demonstrated that diet-induced obesity increased plasma angiotensin II concentrations and elevated systolic blood pressures in male mice. Adipocytes express angiotensinogen and secrete angiotensin peptides. We hypothesize that adipocyte-derived angiotensin II mediates obesity-induced increases in systolic blood pressure in male high fat-fed C57BL/6 mice. Systolic blood pressure was measured by radiotelemetry during week 16 of low-fat or high-fat feeding in Agtfl/fl and adipocyte angiotensinogen-deficient mice (AgtaP2). Adipocyte angiotensinogen deficiency had no effect on diet-induced obesity. Basal 24-hour systolic blood pressure was not different in low fat-fed Agtfl/fl compared with AgtaP2 mice (124±3 versus 128±3 mm Hg, respectively). In Agtfl/fl mice, high-fat feeding significantly increased systolic blood pressure (24 hours; 134±2 mm Hg; P<0.05). In contrast, high fat-fed AgtaP2 mice did not exhibit an increase in systolic blood pressure (126±2 mm Hg). Plasma angiotensin II concentrations were increased by high-fat feeding in Agtfl/fl mice (low fat, 32±14; high fat, 219±58 pg/mL; P<0.05). In contrast, high fat-fed AgtaP2 mice did not exhibit elevated plasma angiotensin II concentrations (high fat, 18±7 pg/mL). Similarly, adipose tissue concentrations of angiotensin II were significantly decreased in low fat- and high fat-fed AgtaP2 mice compared with controls. In conclusion, adipocyte angiotensinogen deficiency prevented high fat-induced elevations in plasma angiotensin II concentrations and systolic blood pressure. These results suggest that adipose tissue serves as a major source of angiotensin II in the development of obesity hypertension.

Angiotensin Converting Enzyme 2 Contributes to Sex Differences in the Development of Obesity Hypertension in C57bl/6 Mice

Objectives—Obesity promotes hypertension, but it is unclear if sex differences exist in obesity-related hypertension. Angiotensin converting enzyme 2 (ACE2) converts angiotensin II (AngII) to angiotensin-(1–7) (Ang-[1–7]), controlling peptide balance. We hypothesized that tissue-specific regulation of ACE2 by high-fat (HF) feeding and sex hormones contributes to sex differences in obesity-hypertension. Methods and Results—HF-fed females gained more body weight and fat mass than males. HF-fed males exhibiting reduced kidney ACE2 activity had increased plasma angiotensin II levels and decreased plasma Ang-(1–7) levels. In contrast, HF-fed females exhibiting elevated adipose ACE2 activity had increased plasma Ang-(1–7) levels. HF-fed males had elevated systolic and diastolic blood pressure that were abolished by losartan. In contrast, HF-fed females did not exhibit increased systolic blood pressure until females were administered the Ang-(1–7) receptor antagonist, D-Ala-Ang-(1–7). Deficiency of ACE2 increased systolic blood pressure in HF-fed males and females, which was abolished by losartan. Ovariectomy of HF-fed female mice reduced adipose ACE2 activity and plasma Ang-(1–7) levels, and promoted obesity-hypertension. Finally, estrogen, but not other sex hormones, increased adipocyte ACE2 mRNA abundance. Conclusions—These results demonstrate that tissue-specific regulation of ACE2 by diet and sex hormones contributes to sex differences in obesity-hypertension.

Angiotensin-converting enzyme 2 deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor-/- mice.

Objective—The renin-angiotensin system contributes to atherosclerotic lesion formation. Angiotensin-converting enzyme 2 (ACE2) catabolizes angiotensin II (Ang II) to angiotensin 1–7 (Ang-(1–7)) to limit effects of the renin-angiotensin system. The purpose of this study was to define the role of ACE2 in atherosclerosis. Methods and Results—Male Ace2−/y mice in an low-density lipoprotein receptor–deficient background were fed a high-fat diet for 3 months. ACE2 deficiency increased atherosclerotic area (Ace2+/y, 17±1; Ace2−/y, 23±2 mm2, P<0.002). This increase was blunted by losartan. To determine whether leukocytic ACE2 influenced atherosclerosis, irradiated low-density lipoprotein receptor–deficient male mice were repopulated with bone marrow–derived cells from Ace2+/y or Ace2−/y mice and fed a high-fat diet for 3 months. ACE2 deficiency in bone marrow–derived cells increased atherosclerotic area (Ace2+/y, 1.6±0.3; Ace2−/y, 2.8±0.3 mm2; P<0.05). Macrophages from Ace2−/y mice exhibited increased Ang II secretion and elevated expression of inflammatory cytokines. Conditioned media from mouse peritoneal macrophages of Ace2−/y mice increased monocyte adhesion to human umbilical vein endothelial cells. Incubation of human umbilical vein endothelial cells with Ang II promoted monocyte adhesion, which was blocked by Ang-(1–7). Coinfusion of Ang-(1–7) with Ang II reduced atherosclerosis. Conclusion—These results demonstrate that ACE2 deficiency in bone marrow–derived cells promotes atherosclerosis through regulation of Ang II/Ang-(1–7) peptides.

Transient Exposure of Neonatal Female Mice to Testosterone Abrogates the Sexual Dimorphism of Abdominal Aortic Aneurysms

Rationale: Abdominal aortic aneurysms (AAAs) exhibit marked sexual dimorphism with higher prevalence in men. Similarly, AAAs induced by angiotensin II (AngII) infusion into mice exhibit a higher prevalence in males. Testosterone promotes AAA pathology in adult male mice through regulation of angiotensin type 1A receptors (AT1aR) in abdominal aortas. However, mechanisms for sexual dimorphism of regional aortic angiotensin receptor expression and AAA formation are unknown. Objective: To define the role of developmental testosterone exposures in sexual dimorphism of AAAs, we determined if exposure of neonatal female mice to testosterone confers adult susceptibility to AngII-induced AAAs. Methods and Results: One-day–old female hypercholesterolemic mice were administered a single dose of either vehicle or testosterone. Neonatal testosterone administration increased abdominal aortic AT1aR mRNA abundance and promoted a striking increase in AngII-induced AAAs in adult females exhibiting low serum testosterone concentrations. AngII-induced atherosclerosis and ascending aortic aneurysms were also increased by testosterone administration to neonatal females. In contrast, neonatal testosterone administration in males had no effect on AngII-induced vascular pathologies. Deficiency of AT1aR in smooth muscle cells reduced effects of neonatal testosterone to promote AAAs in adult females but did not alter atherosclerosis or ascending aortic aneurysms. Testosterone increased AT1aR mRNA abundance and hydrogen peroxide generation in cultured abdominal aortic SMCs. Increased AT1aR mRNA abundance was maintained during progressive passaging of female smooth muscle cells. Conclusions: These data reveal an unrecognized role of transient sex hormone exposures during neonatal development as long-lasting mediators of regional aortic AT1aR expression and sexual dimorphism of AAAs.

Angiotensin-Converting Enzyme 2 Decreases Formation and Severity of Angiotensin II–Induced Abdominal Aortic Aneurysms

Objective— Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (AngII) to form angiotensin-(1–7) (Ang-(1–7)), which generally opposes effects of AngII. AngII infusion into hypercholesterolemic male mice induces formation of abdominal aortic aneurysms (AAAs). This study tests the hypothesis that deficiency of ACE2 promotes AngII-induced AAAs, whereas ACE2 activation suppresses aneurysm formation. Approach and Results— ACE2 protein was detectable by immunostaining in mice and human AAAs. Whole-body deficiency of ACE2 significantly increased aortic lumen diameters and external diameters of suprarenal aortas from AngII-infused mice. Conversely, ACE2 deficiency in bone marrow–derived cells had no effect on AngII-induced AAAs. In contrast to AngII-induced AAAs, ACE2 deficiency had no significant effect on external aortic diameters of elastase-induced AAAs. Because ACE2 deficiency promoted AAA formation in AngII-infused mice, we determined whether ACE2 activation suppressed AAAs. ACE2 activation by administration of diminazene aceturate (30 mg/kg per day) to Ldlr -/- mice increased kidney ACE2 mRNA abundance and activity and elevated plasma Ang-(1–7) concentrations. Unexpectedly, administration of diminazene aceturate significantly reduced total sera cholesterol and very low-density lipoprotein-cholesterol concentrations. Notably, diminazene aceturate significantly decreased aortic lumen diameters and aortic external diameters of AngII-infused mice resulting in a marked reduction in AAA incidence (from 73% to 29%). None of these effects of diminazene aceturate were observed in the Ace2 -/y mice. Conclusions— These results demonstrate that ACE2 exerts a modulatory role in AngII-induced AAA formation, and that therapeutic stimulation of ACE2 could be a benefit to reduce AAA expansion and rupture in patients with an activated renin–angiotensin system.

Weight loss in obese C57BL/6 mice limits adventitial expansion of established angiotensin II-induced abdominal aortic aneurysms.

Previous studies demonstrated that obesity increases inflammation in periaortic adipose tissue and promotes angiotensin II (ANG II)-induced abdominal aortic aneurysms (AAAs). We sought to determine whether weight loss of obese C57BL/6 mice would influence the progression of established AAAs. Male C57BL/6 mice were fed a high-fat diet (HF) for 4 mo and then infused with either saline or ANG II (1,000 ng x kg(-1) x min(-1)) for 3 mo. Mice with dilated suprarenal aortas at 28 days of ANG II infusion were designated to groups fed the HF (HF/HF) or a low-fat diet (LF; 10% kcal as fat; HF/LF) to induce weight loss for the last 2 mo of infusions. Suprarenal aortic lumen diameters of obese mice were increased by ANG II infusion at day 28 (day 0: 1.03 + or - 0.02; day 28: 1.86 + or - 0.14 mm; P < 0.05), but did not progress with continued infusion in HF/HF mice. Moreover, aortic lumen diameters were not different between groups (HF/HF: 1.89 + or - 0.15; HF/LF: 1.79 + or - 0.18 mm). However, maximal diameters of excised AAAs were decreased with weight loss (HF/HF: 2.00 + or - 0.11; HF/LF: 1.55 + or - 0.13 mm; P < 0.05) and had reduced adventitial areas (HF/HF: 1.18 + or - 0.10; HF/LF: 0.54 + or - 0.02 mm(2); P < 0.05). Neovascularization of aortic adventitias was strikingly decreased in HF/LF mice (HF/HF: 43 + or - 5; HF/LF: 12 + or - 2 endothelial cells/adventitial area; P < 0.05). ANG II-induced elevations in adipose mRNA abundance of CD105, an adipose-derived stem cell marker, were abolished with weight loss. These results demonstrate that weight loss limits adventitial expansion of ANG II-induced AAAs. Reduced neovascularization from weight loss may limit progression of AAAs.

Castration of male mice prevents the progression of established angiotensin II-induced abdominal aortic aneurysms

OBJECTIVE Male sex is a nonmodifiable risk factor for abdominal aortic aneurysm (AAA) development. Similar to humans, male mice are more susceptible to angiotensin II (AngII)-induced AAAs than female mice. Previous studies demonstrated that castration of males markedly reduced the formation of AngII-induced AAAs. Progression of AAA size is associated with increased risk of aneurysm rupture. In this study, we hypothesized that castration of male mice would reduce the progression of established AngII-induced AAAs. METHODS Male apolipoprotein E-deficient mice were infused with AngII for 1 month to induce AAA formation. Aortic diameters were measured by ultrasound imaging, and mice were stratified into two groups that underwent a sham operation or castration. AngII infusions were continued for a further 2 months. Ultrasound imaging was used to quantify lumen diameters, and excised aortas were processed for quantification of AAA size, volume, and tissue characteristics. RESULTS Sham-operated mice exhibited progressive dilation of suprarenal aortic lumen diameters during the continued AngII infusion. Aortic lumen diameters were significantly decreased in castrated mice (n = 17) compared with sham-operated mice (n = 15) at study end point (1.63 ± 0.04 vs 1.88 ± 0.05 mm; P < .05). However, maximal external AAA diameters were not significantly different between sham-operated and castrated mice. The vascular volume/lumen volume ratio of excised AAAs imaged by ultrasound was significantly increased by castration (9.5% ± 2.0%) vs sham operation (4.8% ± 0.9%; n = 11 per group; P < .05). Moreover, compared with the thin-walled AAAs of sham-operated mice, aneurysm sections from castrated mice exhibited increased smooth muscle α-actin and collagen. CONCLUSIONS Removal of endogenous male hormones by castration selectively reduces aortic lumen expansion while not altering the external AAA dimensions. CLINICAL RELEVANCE There are no therapeutics that slow the progression of abdominal aortic aneurysms (AAAs), and as the size of an AAA increases, so does the risk of rupture and death. Male sex is a nonmodifiable risk factor for AAA development, but whether male sex hormones have a similar effect on AAA progression is unclear. Removal of male sex hormones in an established mouse model of angiotensin II-induced AAAs resulted in reduced progressive lumen dilation while not altering external AAA dimensions. Therapies that limit androgen action may provide benefit against AAA progression. Alternatively, supplemental testosterone may be contraindicated in men diagnosed with an AAA.