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 (AT 1a ) 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 AT 1a receptor deficiency ablated Ang II-induced ascending AAs (P<0.001). To determine the role of AT 1a receptors on leukocytes, LDL receptor ؊/؊ ؋AT 1a receptor ؉/؉ or AT 1a receptor ؊/؊ mice were irradiated and repopulated with bone marrow-derived cells isolated from either AT 1a receptor ؉/؉ or AT 1a receptor ؊/؊ mice. Deficiency of AT 1a receptors in bone marrow-derived cells had no effect on Ang II-induced ascending AAs. To determine the role of AT 1a receptors on vascular wall cells, we developed AT 1a receptor floxed mice with depletion on either smooth muscle or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT 1a receptor deletion in smooth muscle cells had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology. Key Words: ascending aneurysm Ⅲ angiotensin II Ⅲ AT 1a receptor Ⅲ Tek-cre A scending aortic aneurysm (AA) is an asymptomatic expansion of this restricted region in which rupture has catastrophic consequences. 1 It has now become apparent that ascending AAs have a significantly higher incidence than originally thought. Furthermore, studies and communities in which autopsies are routinely performed have demonstrated that the incidence of aortic aneurysms is increasing. 2 Recent human and experimental studies have inferred a role for angiotensin II (Ang II) in the development of ascending AAs. [3][4][5][6] Ascending AAs can be generated in transgenic mice that express a common mutation of fibrillin-1 present in patients with Marfan syndrome. There are also several other genes that have been associated with AAs. 4 The aortic dilation that is localized to the ascending region is prevented by the administration of the AT 1 receptor antagonist, losartan. 5 Retrospective analysis of pharmacological treatments given to individuals with Marfan syndrome demonstrated that administration of losartan attenuated dilation of the ascending aorta. 3 Prospective evaluations of angiotensin receptor antagonists are currently being performed in populations afflicted with Marfan syndrome. 7 Ang II has diverse effects that could be implicated in aneurysm formation in the ascending aorta. Ang II exerts its bioactive effects mainly via stimulation o...

Objective This study determined whether angiotensinogen (AGT) has angiotensin (Ang)II-independent effects using multiple genetic and pharmacological manipulations. Approach and Results All study mice were in LDL receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were > 90% lower compared to their wild type littermates. HypoAGT mice had lower SBP, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide (ASO) administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in LDL receptor -/- mice. In mice with established adiposity, administration of AGT ASO versus aliskiren led to equivalent reductions of SBP and atherosclerosis. AGT ASO administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat and human revealed 4 highly conserved sequences within the des(AngI)AGT domain. des(AngI)AGT, through adeno-associated viral infection in hepatocyte-specific AGT deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. Conclusions AGT contributes to body weight gain and liver steatosis through functions of the des(AngI)AGT domain, which are independent of AngII production.

Angiotensin II (Ang II) promotes development of ascending aortic aneurysms (AAs), but progression of this pathology is undefined. We evaluated factors potentially involved in progression, and determined the temporal sequence of tissue changes during development of Ang II-induced ascending AAs. Ang II infusion into C57BL/6J mice promoted rapid expansion of the ascending aorta, with significant increases within 5 days, as determined by both in vivo ultrasonography and ex vivo sequential acquisition of tissues. Rates of expansion were not significantly different in LDL receptor-null mice fed a saturated fat-enriched diet, demonstrating a lack of effect of hypercholesterolemia. Augmenting systolic blood pressure with norepinephrine infusion had no significant effect on ascending aortic expansion. Pathological changes observed within 5 days of Ang II infusion included increased medial thickness and intramural hemorrhage characterized by erythrocyte extravasation in outer lamellar layers of the media. Intramedial hemorrhage was not observed after prolonged Ang II infusion, although partial medial disruption was present. Elastin fragmentation and transmural medial breaks of the ascending aorta were observed with continued Ang II infusion, which were restricted to anterior aspects. CD45(+) cells accumulated in adventitia but were minimal in media. Similar pathology was observed in tissues obtained from patients with ascending AAs. In conclusion, Ang II promotes ascending AAs through region-specific changes that are independent of hypercholesterolemia or systolic blood pressure.

Objective Gain-of-function mutations of proprotein convertase subtilisin/kexin type 9 (PCSK9) lead to hypercholesterolemia. This study was to determine whether infection of normocholesterolemic mice with an adeno-associated viral vector (AAV) expressing a gain-of-function mutation of mouse PCSK9 increased angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs). Approach and Results In an initial study, male C57BL/6 mice were injected intraperitoneally with either an empty vector or PCSK9 gain-of-function mutation (D377Y).AAV at three doses and fed a saturated fat-enriched diet for 6 weeks. Two weeks after AAV injection, mice were infused with AngII for 4 weeks. Plasma PCSK9 concentrations were increased dose-dependently in mice injected with AAV containing PCSK9D377Y mutation, and positively associated with elevations of plasma cholesterol concentrations. Infection with intermediate and high doses of PCSK9D377Y.AAV led to equivalent increases of maximal width of abdominal aortas in C57BL/6 mice infused with AngII. Therefore, the intermediate dose was used in subsequent experiments. We then determined effects of PCSK9D377Y.AAV infection on 5 normolipidemic mouse strains, demonstrating that C57BL/6 mice were the most susceptible to this AAV infection. PCSK9D377Y.AAV infected male C57BL/6 mice were also compared with age-matched male low-density lipoprotein (LDL) receptor −/− mice. Although plasma cholesterol concentrations were lower in mice infected with PCSK9D377Y.AAV, these mice had equivalent AAA formation, compared to LDL receptor −/− mice. In a separate study, reduced plasma PCSK9 concentrations by PCSK9 antisense oligonucleotides in male LDL receptor −/− mice did not influence AngII-induced AAAs. Conclusion AAV-mediated infection with a mouse PCSK9 gain-of-function mutation is a rapid, easy, and efficient approach for inducing hypercholesterolemia and promoting AAAs in C57BL/6 mice infused with AngII.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

Objective LRP1, a multifunctional protein involved in endocytosis and cell signaling pathways, leads to a range of vascular pathologies when deleted in vascular smooth muscle cells (SMCs). The purpose of this study was to determine whether LRP1 deletion in SMCs influenced AngII-induced arterial pathologies. Approach and Results LRP1 protein abundance was equivalent in selected arterial-regions, but SMC-specific LRP1 depletion had no effect on abdominal and ascending aortic diameters in young mice. To determine the effects of LRP1 deficiency on AngII vascular responses, SMC-specific LRP1 (smLRP1) +/+ and -/- mice were infused with saline, AngII, or norepinephrine (NE). Several smLRP-/- mice died of superior mesenteric arterial (SMA) rupture during AngII infusion. In surviving mice, AngII profoundly augmented SMA dilation in smLRP1-/- mice. SMA dilation was blood pressure-dependent as demonstrated by a similar response during NE infusion. SMA dilation was also associated with profound macrophage accumulation, but minimal elastin fragmentation. AngII infusion led to no significant differences in abdominal aorta diameters between smLRP1+/+ and -/- mice. In contrast, ascending aortic dilation was exacerbated markedly in AngII-infused smLRP1-/- mice, but NE had no significant effect on either aortic region. Ascending aortas of smLRP1-/- mice infused with AngII had minimal macrophage accumulation but significantly increased elastin fragmentation and mRNA abundance of several LRP1 ligands including MMP-2 and uPA. Conclusions smLRP1 deficiency had no effect on AngII-induced abdominal aortic aneurysm formation. Conversely, AngII infusion in smLRP1-/- mice exacerbated SMA and ascending aorta dilation. Dilation in these two regions had differential association with blood pressure and divergent pathologic characteristics.

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 -10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Objective Dyslipidemia is implicated in abdominal aortic aneurysms (AAAs) in humans and angiotensin (Ang)II-infused mice. This study determined effects of major lipoprotein classes on AngII-induced AAAs using multiple mouse strains with dietary and pharmacological manipulations. Approach and Results Western diet had minor effects on plasma cholesterol concentrations and the low incidence of AngII-induced AAAs in C57BL/6J mice. Low incidence of AAAs in this strain was not attributed to protection from HDL, since apolipoprotein (apo)AI deficiency did not increase AngII-induced AAAs. ApoAI deletion also failed to alter AAA occurrence in hypercholesterolemic mice. Low density lipoprotein (LDL) receptor−/− mice fed normal diet had low incidence of AngII-induced AAAs. Western diet feeding of this strain provoked pronounced hypercholesterolemia due to increased apoB-containing lipoproteins with attendant increases of atherosclerosis in both genders, but AAAs only in male mice. ApoE−/− mice fed normal diet were modestly hypercholesterolemic, whereas this strain fed Western diet was severely hypercholesterolemic due to increased apoB-containing lipoprotein concentrations. The latter augmented atherosclerosis, but did not change the high incidence of AAAs in this strain. To determine whether reductions in apoB-containing lipoproteins influenced AngII-induced AAAs, ezetimibe was administered at a dose that partially reduced plasma cholesterol concentrations to apoE−/− mice fed Western diet. This decreased atherosclerosis, but not AAAs. This ezetimibe dose in apoE−/− mice fed normal diet significantly decreased plasma apoB-containing lipoprotein concentrations and reduced AngII-induced AAAs. Conclusions ApoB-containing lipoproteins contribute to augmentation of AngII-induced AAA in male mice. However, unlike atherosclerosis, AAA occurrence was not correlated with increases in plasma apoB-containing lipoprotein concentrations.