Lisa A. Cassis

Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E–deficient mice

Increased plasma concentrations of angiotension II (Ang II) have been implicated in atherogenesis. To examine this relationship directly, we infused Ang II or vehicle for 1 month via osmotic minipumps into mature apoE(-/-) mice. These doses of Ang II did not alter arterial blood pressure, body weight, serum cholesterol concentrations, or distribution of lipoprotein cholesterol. However, Ang II infusions promoted an increased severity of aortic atherosclerotic lesions. These Ang II-induced lesions were predominantly lipid-laden macrophages and lymphocytes; moreover, Ang II promoted a marked increase in the number of macrophages present in the adventitial tissue underlying lesions. Unexpectedly, pronounced abdominal aortic aneurysms were present in apoE(-/-) mice infused with Ang II. Sequential sectioning of aneurysmal abdominal aorta revealed two major characteristics: an intact artery that is surrounded by a large remodeled adventitia, and a medial break with pronounced dilation and more modestly remodeled adventitial tissue. Although no atherosclerotic lesions were visible at the medial break point, the presence of hyperlipidemia was required because infusions of Ang II into apoE(+/+) mice failed to generate aneurysms. These results demonstrate that increased plasma concentrations of Ang II have profound and rapid effects on vascular pathology when combined with hyperlipidemia, in the absence of hemodynamic influences.

Mouse Models of Abdominal Aortic Aneurysms

Abstract—Many mouse models of abdominal aortic aneurysms have been developed that use a diverse array of methods for producing the disease, including genetic manipulation and chemical induction. These models could provide insight into potential mechanisms in the development of this disease. Although experimental studies on abdominal aortic aneurysms (AAAs) have used a variety of mammalian and avian approaches, there is an increasing reliance on the use of mice. The models recapitulate some facets of the human disease including medial degeneration, inflammation, thrombus formation, and rupture. Most of the mouse models of AAA are evoked either by genetically defined approaches or by chemical means. The genetic approaches are spontaneous and engineered mutations. These include defects in extracellular matrix maturation, increased degradation of elastin and collagen, aberrant cholesterol homeostasis, and enhanced production of angiotensin peptides. The chemical approaches include the intraluminal infusion of elastase, periaortic incubations of calcium chloride, and subcutaneous infusion of AngII. A common feature of these models is the reduction of AAA incidence and severity by the prophylactic administration of matrix metalloproteinase (MMP) inhibitors or genetically engineered deficiencies of specific members of this proteolytic protein family. The validation of mouse models of AAAs will provide insight into the mechanisms of progression of the human disease.

Aortic Dissection Precedes Formation of Aneurysms and Atherosclerosis in Angiotensin II-Infused, Apolipoprotein E-Deficient Mice

Objective—We sought to define the temporal characteristics of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and to provide mechanistic insight into the development of this vascular pathology in apolipoprotein E-deficient (apoE-/-) mice. Methods and Results—Male apoE-/- mice were infused with AngII for 1 to 56 days. Suprarenal arteries were sequentially sectioned, and cellular features were defined by histologic and immunocytochemical techniques. The initial identified event was medial accumulation of macrophages in regions of elastin degradation. Subsequent medial dissection was associated with luminal dilation and thrombus formation. Thrombi were usually constrained by adventitial tissue, although ≈10% of mice died due to rupture. Thrombi led to profound inflammation that was characterized by infiltration of macrophages and T and B lymphocytes. Remodeling of the tissues was associated with regeneration of elastin fibers and reendothelialization of the dilated luminal surface. Aneurysmal tissue underwent profound neovascularization. Atherosclerotic lesions were only detected after development of the aneurysms. Conclusions—The initial event in AngII-induced AAA is a focal dissection in the suprarenal region. The progression of AAA precedes the development of overt atherosclerotic lesions.

Osteopontin mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance in mice

Obesity is associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and macrophage infiltration into adipose tissue, which may contribute to the development of insulin resistance. During immune responses, tissue infiltration by macrophages is dependent on the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine that promotes monocyte chemotaxis and cell motility. In the present study, we used a murine model of diet-induced obesity to examine the role of osteopontin in the accumulation of adipose tissue macrophages and the development of insulin resistance during obesity. Mice exposed to a high-fat diet exhibited increased plasma osteopontin levels, with elevated expression in macrophages recruited into adipose tissue. Obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on diet-induced obesity, body composition, or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of inflammation, both in adipose tissue and systemically. Taken together, these results suggest that osteopontin may play a key role in linking obesity to the development of insulin resistance by promoting inflammation and the accumulation of macrophages in adipose tissue.

Differential Effects of Doxycycline, a Broad-Spectrum Matrix Metalloproteinase Inhibitor, on Angiotensin II–Induced Atherosclerosis and Abdominal Aortic Aneurysms

Objective—Angiotensin II (AngII) infusion into hyperlipidemic mice leads to the rapid formation of atherosclerotic lesions and abdominal aortic aneurysms (AAAs). To define the role of matrix metalloproteinases (MMPs) in the development of these vascular pathologies, we administered the broad-spectrum MMP inhibitor doxycycline to saline- and AngII-infused LDL receptor−/− mice. Methods and Results—Mice were placed on a high-fat diet for 1 week before infusion with either saline or AngII (1000 ng · kg−1 · min−1) via osmotic pumps for 28 days. Doxycycline (30 mg · kg−1 · d−1) was administered in the drinking water to both saline- and AngII-infused mice. Administration of doxycycline did not significantly influence systolic blood pressure, serum cholesterol concentrations, or lipoprotein-cholesterol distribution. Doxycycline had no effect on the extent of atherosclerosis in saline- or AngII-infused mice. In contrast, doxycycline markedly reduced the incidence of AAA formation (86% vs 35%, AngII vs AngII+doxycycline, respectively;P <0.05), in addition to reducing aneurysm severity. Conclusions—These data do not imply a role for MMPs in AngII-induced atherosclerosis but provide evidence consistent with a role in AngII-induced AAA formation.

Hypercholesterolemia Stimulates Angiotensin Peptide Synthesis and Contributes to Atherosclerosis Through the AT1A Receptor

Background—Hypercholesterolemia-induced atherosclerosis is attenuated by either pharmacological antagonism of AT1 receptors or AT1A receptor deficiency. However, the mechanism underlying the pronounced responses to angiotensin II (Ang II) antagonism has not been determined. We hypothesized that hypercholesterolemia stimulates the production of angiotensin peptides to provide a rationale for the profound effect of AT1A receptor deficiency on atherogenesis. Methods and Results—Atherosclerotic lesions were analyzed in LDL receptor–deficient mice. Immunocytochemical analysis demonstrated that atherosclerotic lesions contained all the components of the conventional pathway for Ang II synthesis. AT1A receptor deficiency caused a marked decrease in atherosclerotic lesion size in both the aortic root and arch of male and female mice, without a discernible effect on composition. AT1A receptor deficiency–induced reductions in atherosclerosis were independent of systolic blood pressure and measurements of oxidation and chemoattractants. Aortic AT2 receptor mRNA expression was not altered in AT1A receptor–deficient mice, and AT2 receptor deficiency had no effect on lesion area or cellular composition. Hypercholesterolemia greatly augmented the systemic renin-angiotensin system, as demonstrated by large increases in plasma concentrations of angiotensinogen and angiotensin peptides (Ang II, III, IV, and 4–8). These increases were ablated in hypercholesterolemic AT1A receptor–deficient mice. Conclusions—AT1A receptor deficiency had a striking effect in reducing hypercholesterolemia-induced atherosclerosis in LDL receptor–negative mice. Hypercholesterolemia was associated with increased systemic angiotensinogen and angiotensin peptides, which were reduced in AT1A receptor–deficient mice. These results demonstrate that hypercholesterolemia-induced stimulation of angiotensin peptide production provides a basis for the marked effect of AT1A receptor deficiency in reducing atherosclerosis.

Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.

We have recently demonstrated that chronic infusion of Angiotensin II into apoE−/− mice promotes the development of abdominal aortic aneurysms. To determine the involvement of specific Angiotensin II receptors in this response, we co‐infused Angiotensin II (1000 ng kg−1 min−1 for 28 days) with losartan (30 mg kg−1 day−1) or PD123319 (3 mg kg−1 day−1) to antagonize AT1 and AT2 receptors, respectively. Infusion of Angiotensin II promoted the development of abdominal aortic aneurysms in 70% of mature female apoE−/− mice. The formation of aortic aneurysms was totally inhibited by co‐infusion of Angiotensin II with losartan (30 mg kg−1 day−1; P=0.003). In contrast, the co‐infusion of Angiotensin II with PD123319 resulted in a marked increase in the incidence and severity of aortic aneurysms. To determine whether AT2 antagonism also promoted Angiotensin II‐induced atherosclerosis, Angiotensin II was infused into young female apoE−/− mice that had little spontaneous atherosclerosis. In these mice, co‐infusion of PD123319 led to a dramatic increase in the extent of atherosclerosis. This increase was associated with no change in plasma lipid concentrations and only transient and modest increases in blood pressure during co‐infusion with PD123319. While antagonism of AT1 receptors totally prevented the formation of aneurysms, antagonism of AT2 receptors promoted a large increase in the severity of Angiotensin II‐induced vascular pathology.

Influence of perivascular adipose tissue on rat aortic smooth muscle responsiveness.

Virtually every blood vessel in the body is surrounded to some degree by adipose tissue. A potential role for perivascular adipose tissue as a neurohumoral regulator of vascular responsiveness was studied. Thoracic aortae obtained from male Sprague-Dawley rats were cut into rings for use in a standard in vitro smooth muscle bath set-up. The vessels were either cleaned of surrounding adipose tissue or left intact. Contractile responses to KCl and phenylephrine as well as relaxation responses to acetylcholine, isoproterenol and sodium nitroprusside were not different between cleaned and intact tissues. However, a significant decrease in the sensitivity to norepinephrine was observed in intact vessels. This altered response was corrected by prior treatment with desipramine plus deoxycorticosterone. Contractile responses of aortic ring preparations to tyramine and a potassium-free physiological solution were significantly greater in intact tissues. Electrical stimulation resulted in no response in cleaned tissues, however, a frequency-dependent contraction was elicited in intact vessels. Phentolamine blocked the contractile responses generated by these manipulations which activate the sympathetic neuroeffector system. Responses evoked by electrical stimulation in intact vascular preparations were significantly attenuated by prior exposure to the selective angiotensin II (AII) antagonist SarI-Ile8-AII. These observations demonstrate that perivascular adipose tissue significantly influences vascular responsiveness in the in vitro setting.

Polychlorinated Biphenyl-77 Induces Adipocyte Differentiation and Proinflammatory Adipokines and Promotes Obesity and Atherosclerosis

Background Obesity, an inflammatory condition linked to cardiovascular disease, is associated with expansion of adipose tissue. Highly prevalent coplanar polychlorinated biphenyls (PCBs) such as 3,3′,4,4′-tetrachlorobiphenyl (PCB-77) accumulate in adipose tissue because of their lipophilicity and increase with obesity. However, the effects of PCBs on adipocytes, obesity, and obesity-associated cardiovascular disease are unknown. Objectives In this study we examined in vitro and in vivo effects of PCB-77 on adipocyte differentiation, proinflammatory adipokines, adipocyte morphology, body weight, serum lipids, and atherosclerosis. Methods PCB-77 or 2,2′,4,4,5,5′-hexachlorobiphenyl (PCB-153) was incubated with 3T3-L1 adipocytes either during differentiation or in mature adipocytes. Concentration-dependent effects of PCB-77 were contrasted with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For in vivo studies, we treated C57BL/6 wild-type (WT) or aryl hydrocarbon receptor (AhR)−/− mice with vehicle or PCB-77 (49 mg/kg, by intraperitoneal injection) and examined body weight gain. In separate studies, we injected ApoE−/− mice with vehicle or PCB-77 over a 6-week period and examined body weight, adipocyte size, serum lipids, and atherosclerosis. Results Low concentrations of PCB-77 or TCDD increased adipocyte differentiation, glycerol–3-phosphate dehydrogenase activity, and expression of peroxisome proliferator–activated receptor γ, whereas higher concentrations inhibited adipocyte differentiation. Effects of PCB-77 were abolished by the AhR antagonist α-naphthoflavone. PCB-77 promoted the expression and release of various proinflammatory cytokines from 3T3-L1 adipocytes. Administration of PCB-77 increased body weight gain in WT but not AhR−/− mice. ApoE−/− mice injected with PCB-77 exhibited greater body weight, adipocyte hypertrophy, serum dyslipidemia, and augmented atherosclerosis. Conclusions Our findings suggest that PCB-77 may contribute to the development of obesity and obesity-associated atherosclerosis.

Renin inhibition reduces hypercholesterolemia-induced atherosclerosis in mice

The role of the renin angiotensin system (RAS) in atherosclerosis is complex because of the involvement of multiple peptides and receptors. Renin is the rate-limiting enzyme in the production of all angiotensin peptides. To determine the effects of renin inhibition on atherosclerosis, we administered the novel renin inhibitor aliskiren over a broad dose range to fat-fed LDL receptor-deficient (Ldlr(-/-)) mice. Renin inhibition resulted in striking reductions of atherosclerotic lesion size in both the aortic arch and the root. Subsequent studies demonstrated that cultured macrophages expressed all components of the RAS. To determine the role of macrophage-derived angiotensin in the development of atherosclerosis, we transplanted renin-deficient bone marrow to irradiated Ldlr(-/-) mice and observed a profound decrease in the size of atherosclerotic lesions. In similar experiments, transplantation of bone marrow deficient for angiotensin II type 1a receptors failed to influence lesion development. We conclude that renin-dependent angiotensin production in macrophages does not act in an autocrine/paracrine manner. Furthermore, in vitro studies demonstrated that coculture with renin-expressing macrophages augmented monocyte adhesion to endothelial cells. Therefore, although previous work suggests that angiotensin peptides have conflicting effects on atherogenesis, we found that renin inhibition profoundly decreased lesion development in mice.