Debra L. Rateri

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.

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.

ANG II infusion promotes abdominal aortic aneurysms independent of increased blood pressure in hypercholesterolemic mice

Infusion of ANG II in hyperlipidemic mice augments atherosclerosis and causes formation of abdominal aortic aneurysms (AAAs). The purpose of this study was to define the contribution of ANG II-induced hypertension to these vascular pathologies. Male apolipoprotein E (apoE)- and LDL receptor (LDLr)-deficient mice were infused with ANG II (1,000 ng.kg(-1).min(-1)) or norepinephrine (NE; 5.6 mg.kg(-1).day(-1)) for 28 days. Infusion of ANG II or NE increased mean arterial pressure (MAP; ANG II, 133 +/- 2.8; NE, 129 +/- 13 mmHg) to a similar extent compared with baseline blood pressures (MAP, 107 +/- 2 mmHg). Abdominal aortic width increased in both apoE-deficient (apoE(-/-)) or LDLr-deficient (LDLr(-/-)) mice infused with ANG II (apoE(-/-): 1.4 +/- 0.1; LDLr(-/-): 1.6 +/- 0.2 mm). In contrast, NE did not change diameters of abdominal aortas (apoE(-/-): 0.91 +/- 0.03; LDLr(-/-): 0.87 +/- 0.02 mm). Similarly, atherosclerotic lesions in aortic arches were much greater in mice infused with ANG II compared with NE. At a subpressor infusion rate of ANG II (500 ng.kg(-1).min(-1)), AAAs developed in 50% of apoE(-/-) mice. Alternatively, administration of hydralazine (250 mg/l) to ANG II-infused apoE(-/-) mice (1,000 ng.kg(-1).min(-1)) lowered systolic blood pressure (day 28: ANG II, 157 +/- 6; ANG II/hydralazine, 135 +/- 6 mmHg) but did not prevent AAA formation or atherosclerosis. These results demonstrate that infusion of ANG II to hyperlipidemic mice induces AAAs and augments atherosclerosis independent of increased blood pressure.

Bone Marrow Transplantation Reveals That Recipient AT1a Receptors Are Required to Initiate Angiotensin II-Induced Atherosclerosis and Aneurysms

Objective—Angiotensin II (AngII) infusion into hypercholesterolemic mice accelerates atherosclerosis and promotes formation of abdominal aortic aneurysms (AAAs). The purpose of this study was to define whether AngII interacts with receptors on infiltrating versus resident cells in promoting vascular pathologies. Methods and Results—Male LDL receptor−/− mice, that were either AT1a receptor +/+ or −/−, were fed a fat enriched diet and infused with either saline or AngII. AngII-induced augmentation of atherosclerosis and formation of AAAs was ablated in AT1a receptor−/− mice. Bone marrow transplantation studies were performed to determine the role of AT1a receptors expressed on infiltrating cells. AT1a receptor +/+ and −/− mice were irradiated and repopulated with bone marrow–derived stem cells of either genotype. These 4 groups of chimeric mice were infused with either saline or AngII. Repopulation of irradiated AT1a receptor +/+ mice with −/− bone marrow–derived cells resulted in modest reductions in AngII-induced atherosclerosis. Unexpectedly, AT1a receptor–deficient recipient mice were dramatically protected from AngII-induced vascular pathologies, irrespective of donor genotype. Conclusion—AngII promotes vascular pathology via AT1a receptors. AT1a receptors expressed on infiltrating cells exert modest regulation of AngII-induced atherosclerosis. However, the presence of this receptor in resident tissue is required for the initiation of AngII-induced atherosclerosis and AAAs.

Depletion of Natural Killer Cell Function Decreases Atherosclerosis in Low-Density Lipoprotein Receptor Null Mice

Objective—Natural killer (NK) cells are a key component of innate immunity. Despite being identified in human and mouse atherosclerotic lesions, the role of NK cells in the disease process in unknown. To determine this role, we created chimeric atherosclerosis-susceptible low-density lipoprotein (LDL) receptor null (ldl-r−/−) mice that were deficient in functional NK cells through expression of a transgene encoding for Ly49A. Methods and Results—Bone marrow cells from Ly49A transgenic and nontransgenic littermates were used to repopulate the hematopoietic system of lethally-irradiated female ldl-r−/− mice. After a recovery period to permit sufficient engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was no difference in either serum total cholesterol concentrations or lipoprotein cholesterol distribution in mice repopulated with nontransgenic versus Ly49A transgenic marrow cells. Using immunohistochemistry, we detected NK cells in atherosclerotic lesions of both groups of mice. However, deficiency of functional NK cells significantly reduced the size of atherosclerosis by 70% (P=0.0002) in cross-sectional analysis of the aortic root and by 38% (P=0.004) in en face analysis of the intimal surface of the aortic arch. Conclusion—These studies demonstrate that NK cells infiltrate the vessel wall and promote atherosclerotic lesion development.

Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE-/- mice.

AngII (angiotensin II) induces atherosclerosis and AAAs (abdominal aortic aneurysms) through multiple proposed mechanisms, including chemotaxis. Therefore, we determined the effects of whole-body deficiency of the chemokine receptor CCR2 (CC chemokine receptor 2) on these diseases. To meet this objective, apoE (apolipoprotein E)−/− mice that were either CCR2+/+ or CCR2−/−, were infused with either saline or AngII (1000 ng·kg−1 of body weight·min−1) for 28 days via mini-osmotic pumps. Deficiency of CCR2 markedly attenuated both atherosclerosis and AAAs, unrelated to systolic blood pressure or plasma cholesterol concentrations. During the course of the present study, we also observed that AngII infusion led to large dilatations that were restricted to the ascending aortic region of apoE−/− mice. The aortic media in most of the dilated area was thickened. In regions of medial thickening, distinct elastin layers were discernable. There was an expansion of the distance between elastin layers in a gradient from the intimal to the adventitial aspect of the media. This pathology differed in a circumscribed area of the anterior region of ascending aortas in which elastin breaks were focal and almost transmural. All regions of the ascending aorta of AngII-infused mice had diffuse medial macrophage accumulation. Deficiency of CCR2 greatly attenuated the AngII-induced lumen dilatation in the ascending aorta. This new model of ascending aortic aneurysms has pathology that differs markedly from AngII-induced atherosclerosis or AAAs, but all vascular pathologies were attenuated by CCR2 deficiency.

Endothelial Cell–Specific Deficiency of Ang II Type 1a Receptors Attenuates Ang II–Induced Ascending Aortic Aneurysms in LDL Receptor−/− Mice

Rationale: Human studies and mouse models have provided evidence for angiotensin II (Ang II)–based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that Ang II infusion induces aneurysmal pathology in the ascending aorta. Objective: The aim of this study was to define the role of angiotensin II type 1a (AT1a) receptors and their cellular location in Ang II–induced ascending aortic aneurysms (AAs). Methods and Results: Male LDL receptor−/− mice were fed a saturated fat–enriched diet for 1 week before osmotic mini-pump implantation and infused with either saline or Ang II (1000 ng/kg per minute) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT1a receptor deficiency ablated Ang II–induced ascending AAs (P<0.001). To determine the role of AT1a receptors on leukocytes, LDL receptor−/−×AT1a receptor+/+ or AT1a receptor−/− mice were irradiated and repopulated with bone marrow–derived cells isolated from either AT1a receptor+/+ or AT1a receptor−/− mice. Deficiency of AT1a receptors in bone marrow–derived cells had no effect on Ang II–induced ascending AAs. To determine the role of AT1a receptors on vascular wall cells, we developed AT1a receptor floxed mice with depletion on either smooth muscle or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT1a receptor deletion in smooth muscle cells had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology. Conclusions: Ang II infusion promotes aneurysms in the ascending aorta via stimulation of AT1a receptors that are expressed on endothelial cells.

T Lymphocytes in Atherosclerosis The Yin-Yang of Th1 and Th2 Influence on Lesion Formation

The presence of activated T lymphocytes in all stages of human atherosclerotic lesion development implies their involvement in this vascular disease process.1 However, the specific role T lymphocytes play in atherogenesis remains unclear. It is not feasible to regulate the immune system in humans to determine its association with atherosclerotic-related diseases. Therefore, dissection of the role of T lymphocytes in lesion development will be dependent on animal models. Appropriate animal models need to mimic the cellular composition of human lesions, particularly in content of T lymphocytes. In this respect, the most commonly used mouse models of atherosclerosis, such as apolipoprotein E −/− and low-density lipoprotein (LDL) receptor −/− mice, contain T lymphocytes, although the number of cells is less than in human lesions.2 T-lymphocyte presence has functional consequences, because their complete absence reduces lesion formation during moderate hypercholesterolemia.3,4⇓ The major class of T lymphocytes present in atherosclerotic lesions is CD4+. In response to the local milieu of cytokines, CD4+ cells differentiate into the Th1 or Th2 lineage. Among the principal inducers of the Th1 and Th2 cells are interleukin (IL)-12 and IL-10, respectively. Activated T lymphocytes are functionally defined by the cytokines produced with interferon (IFN)-γ secreted from the Th1 cells and IL-4 from the Th2 cells. Much of the emphasis in atherosclerosis research in relation to T lymphocytes has focused on the role of Th1-type responses. The evidence for the role of Th1 cells …

Prolonged infusion of angiotensin II in apoE(-/-) mice promotes macrophage recruitment with continued expansion of abdominal aortic aneurysm.

Angiotensin II (AngII) infusion initiates abdominal aortic aneurysm (AAA) development due to medial disruption and results in luminal dilation and thrombus formation. The objective of this study was to determine whether AAA progressed during protracted AngII infusion. Male apoE(-/-) mice were infused with AngII using miniosmotic pumps. On day 27, suprarenal aortic luminal diameters were ultrasonically measured to identify mice exhibiting AAAs. Mice were designated to three groups with similar mean luminal dilation. Group 1 mice were sacrificed on day 28. Group 2 and 3 mice were subsequently infused with saline or AngII, respectively, for an additional 56 days. In Group 2, saline infusion-after the initial 28 days of AngII infusion-led to an immediate decrease in systolic blood pressure. Over the subsequent 56 days of saline infusion, there were no aneurysm-related deaths or significant changes in luminal diameter. In contrast, continuous AngII infusion in Group 3 maintained persistently increased systolic blood pressure, with aneurysmal rupture-associated deaths, increased luminal diameters, and tissue remodeling. Aortic aneurysmal segments that expanded during continuous AngII infusion exhibited macrophage accumulation in regions of medial disruption, predominantly on the adventitial aspect. Macrophages immunostained for CD206 more than for iNOS, consistent with an M2 phenotype. In conclusion, prolonged AngII infusion promotes AAA expansion, and is associated with enhanced rupture rates and increased macrophage infiltration.

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.