Second Heart Field-Derived Cells Contribute to Angiotensin II-Mediated Ascending Aortopathies.

BACKGROUND: The ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies. METHODS: Ascending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry-assisted proteomics and molecular features of SHF-derived cells were determined by single-cell transcriptomic analyses. Genetic deletion of either Lrp1 (low-density lipoprotein receptor-related protein 1) or Tgfbr2 (transforming growth factor-ß receptor type 2) in SHF-derived cells was conducted to examine the effect of SHF-derived cells on vascular integrity. RESULTS: Pathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas after AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion before overt pathology occurred evoked downregulation of smooth muscle cell proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single-cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single-cell transcriptomics results identified PAI1 (plasminogen activator inhibitor 1) as the most increased protein in SHF-derived smooth muscle cells and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies. CONCLUSIONS: SHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and transforming growth factor-ß signaling associated with increases of aortic PAI1.

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Smooth Muscle Cells Derived From Second Heart Field and Cardiac Neural Crest Reside in Spatially Distinct Domains in the Media of the Ascending Aorta-Brief Report.

OBJECTIVE: Smooth muscle cells (SMCs) of the proximal thoracic aorta are embryonically derived from the second heart field (SHF) and cardiac neural crest (CNC). However, distributions of these embryonic origins are not fully defined. The regional distribution of SMCs of different origins is speculated to cause region-specific aortopathies. Therefore, the aim of this study was to determine the distribution of SMCs of SHF and CNC origins in the proximal thoracic aorta. APPROACH AND RESULTS: Mice with repressed LacZ in the ROSA26 locus were bred to those expressing Cre controlled by either the Wnt1 or Mef2c (myocyte-specific enhancer factor 2c) promoter to trace CNC- and SHF-derived SMCs, respectively. Thoracic aortas were harvested, and activity of ß-galactosidase was determined. Aortas from Wnt1-Cre mice had ß-galactosidase-positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, ß-galactosidase-positive areas in Mef2c-Cre mice extended from the aortic root throughout the ascending aorta. This distribution occurred independent of sex and aging. Cross and sagittal aortic sections demonstrated that CNC-derived cells populated the inner medial aspect of the anterior region of the ascending aorta and transmurally in the media of the posterior region. Interestingly, outer medial cells throughout anterior and posterior ascending aortas were derived from the SHF. ß-Galactosidase-positive medial cells of both origins colocalized with an SMC marker, α-actin. CONCLUSIONS: Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta. The outer medial cells of the ascending aorta form a sleeve populated by SHF-derived SMCs.

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

OBJECTIVE: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Relaxin and Matrix Metalloproteinase-9 in Angiotensin II-Induced Abdominal Aortic Aneurysms.

BACKGROUND: This study determined whether relaxin or matrix metalloproteinase (MMP)-9 influences angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA).Methods and Results:Male C57BL/6 or apolipoprotein E-/-mice were infused with AngII with or without relaxin. Relaxin did not influence AngII-induced AAA in either mouse strain. Infusion of AngII reduced, but relaxin increased, MMP-9 mRNA in macrophages. We then determined the effects of MMP-9 deficiency on AAA in apolipoprotein E-/-mice. MMP-9 deficiency led to AAA formation in the absence of AngII, and augmented AngII-induced aortic rupture and AAA incidence. CONCLUSIONS: MMP-9 deficiency augmented AngII-induced AAA.

Fibroblast Angiotensin II Type 1a Receptors Contribute to Angiotensin II-Induced Medial Hyperplasia in the Ascending Aorta.

OBJECTIVE: Angiotensin II (Ang II) infusion causes aortic medial thickening via stimulation of angiotensin II type 1a (AT1a) receptors. The purpose of this study was to determine the cellular loci of AT1a receptors that mediate this Ang II-induced aortic pathology. APPROACH AND RESULTS: Saline or Ang II was infused into AT1a receptor floxed mice expressing Cre under control of cell-specific promoters. Initially, AT1a receptors were depleted in aortic smooth muscle cell and endothelium by expressing Cre under control of SM22 and Tie2 promoters, respectively. Deletion of AT1a receptors in either cell type had no effect on Ang II-induced medial thickening. To determine whether this effect was related to neural stimulation, AT1a receptors were depleted using an enolase 2-driven Cre. Depletion of AT1a receptors in neural cells attenuated Ang II-induced medial thickening of the ascending, but not descending aorta. Lineage tracking studies, using ROSA26-LacZ, demonstrated that enolase 2 was also expressed in adventitial cells adjacent to the region of attenuated thickening. To determine whether adventitial fibroblasts contributed to this attenuation, AT1a receptors in fibroblasts were depleted using S100A4 driven Cre. Similar to enolase 2-Cre, Ang II-induced medial thickening was attenuated in the ascending, but not the descending aorta. Lineage tracking demonstrated an increase of S100A4-LacZ positive cells in the media of the ascending region during Ang II infusion. CONCLUSIONS: AT1a receptor depletion in fibroblasts attenuates Ang II-induced medial hyperplasia in the ascending aorta.

Smooth muscle cell deletion of low-density lipoprotein receptor-related protein 1 augments angiotensin II-induced superior mesenteric arterial and ascending aortic aneurysms.

OBJECTIVE: Low-density lipoprotein receptor-related protein 1 (LRP1), a multifunctional protein involved in endocytosis and cell signaling pathways, leads to several vascular pathologies when deleted in vascular smooth muscle cells (SMCs). The purpose of this study was to determine whether LRP1 deletion in SMCs influenced angiotensin II-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 angiotensin II vascular responses, SMC-specific LRP1 (smLRP1(+/+)) and smLRP1-deficient (smLRP1(-/-)) mice were infused with saline, angiotensin II, or norepinephrine. Several smLRP(-/-) mice died of superior mesenteric arterial (SMA) rupture during angiotensin II infusion. In surviving mice, angiotensin II profoundly augmented SMA dilation in smLRP1(-/-) mice. SMA dilation was blood pressure dependent as demonstrated by a similar response during norepinephrine infusion. SMA dilation was also associated with profound macrophage accumulation, but minimal elastin fragmentation. Angiotensin II infusion led to no significant differences in abdominal aorta diameters between smLRP1(+/+) and smLRP1(-/-) mice. In contrast, ascending aortic dilation was exacerbated markedly in angiotensin II-infused smLRP1(-/-) mice, but norepinephrine had no significant effect on either aortic region. Ascending aortas of smLRP1(-/-) mice infused with angiotensin II had minimal macrophage accumulation but significantly increased elastin fragmentation and mRNA abundance of several LRP1 ligands including MMP-2 (matrix metalloproteinase-2) and uPA (urokinase plasminogen activator). CONCLUSIONS: smLRP1 deficiency had no effect on angiotensin II-induced abdominal aortic aneurysm formation. Conversely, angiotensin II infusion in smLRP1(-/-) mice exacerbated SMA and ascending aorta dilation. Dilation in these 2 regions had differential association with blood pressure and divergent pathological characteristics.

Deficiency of Endogenous Acute-Phase Serum Amyloid A Protects apoE-/- Mice From Angiotensin II-Induced Abdominal Aortic Aneurysm Formation.

OBJECTIVE: Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. APPROACH AND RESULTS: Plasma SAA increased ≈60-fold in apoE(-/-) mice 24 hours after intraperitoneal Ang II injection (100 µg/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE(-/-) mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE(-/-) mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II-infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. CONCLUSIONS: Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.

Angiotensin II induces region-specific medial disruption during evolution of ascending aortic aneurysms.

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.

Abdominal aortic aneurysm: novel mechanisms and therapies.

PURPOSE OF REVIEW: Abdominal aortic aneurysm (AAA) is a pathological condition of permanent dilation that portends the potentially fatal consequence of aortic rupture. This review emphasizes recent advances in mechanistic insight into aneurysm pathogenesis and potential pharmacologic therapies that are on the horizon for AAAs. RECENT FINDINGS: An increasing body of evidence demonstrates that genetic factors, including 3p12.3, DAB2IP, LDLr, LRP1, matrix metalloproteinase (MMP)-3, TGFBR2, and SORT1 loci, are associated with AAA development. Current human studies and animal models have shown that many leukocytes and inflammatory mediators, such as IL-1, IL-17, TGF-ß, and angiotensin II, are involved in the pathogenesis of AAAs. Leukocytic infiltration into aortic media leads to smooth muscle cell depletion, generation of reactive oxygen species, and extracellular matrix fragmentation. Preclinical investigations into pharmacological therapies for AAAs have provided intriguing insight into the roles of microRNAs in regulating many pathological pathways in AAA development. Several large clinical trials are ongoing, seeking to translate preclinical findings into therapeutic options. SUMMARY: Recent studies have identified many potential mechanisms involved in AAA pathogenesis that provide insight into the development of a medical treatment for this disease.

Deficiency of endogenous acute phase serum amyloid A does not affect atherosclerotic lesions in apolipoprotein E-deficient mice.

OBJECTIVE: Although elevated plasma concentrations of serum amyloid A (SAA) are associated strongly with increased risk for atherosclerotic cardiovascular disease in humans, the role of SAA in the pathogenesis of lesion formation remains obscure. Our goal was to determine the impact of SAA deficiency on atherosclerosis in hypercholesterolemic mice. APPROACH AND RESULTS: Apolipoprotein E-deficient (apoE(-/-)) mice, either wild type or deficient in both major acute phase SAA isoforms, SAA1.1 and SAA2.1, were fed a normal rodent diet for 50 weeks. Female mice, but not male apoE-/- mice deficient in SAA1.1 and SAA2.1, had a modest increase (22%; P≤0.05) in plasma cholesterol concentrations and a 53% increase in adipose mass compared with apoE-/- mice expressing SAA1.1 and SAA2.1 that did not affect the plasma cytokine levels or the expression of adipose tissue inflammatory markers. SAA deficiency did not affect lipoprotein cholesterol distributions or plasma triglyceride concentrations in either male or female mice. Atherosclerotic lesion areas measured on the intimal surfaces of the arch, thoracic, and abdominal regions were not significantly different between apoE-/- mice deficient in SAA1.1 and SAA2.1 and apoE-/- mice expressing SAA1.1 and SAA2.1 in either sex. To accelerate lesion formation, mice were fed a Western diet for 12 weeks. SAA deficiency had effect neither on diet-induced alterations in plasma cholesterol, triglyceride, or cytokine concentrations nor on aortic atherosclerotic lesion areas in either male or female mice. In addition, SAA deficiency in male mice had no effect on lesion areas or macrophage accumulation in the aortic roots. CONCLUSIONS: The absence of endogenous SAA1.1 and 2.1 does not affect atherosclerotic lipid deposition in apolipoprotein E-deficient mice fed either normal or Western diets.

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.

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 (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. CONCLUSIONS: Ang II infusion promotes aneurysms in the ascending aorta via stimulation of AT(1a) receptors that are expressed on endothelial cells.

Protein kinase C-delta mediates adventitial cell migration through regulation of monocyte chemoattractant protein-1 expression in a rat angioplasty model.

OBJECTIVE: The adventitia is increasingly recognized as an important player during the development of intimal hyperplasia. However, the mechanism of adventitial cell recruitment to the subintimal space remains largely undefined. We have shown previously that gene transfer of protein kinase C-delta (PKCδ) increases apoptosis of smooth muscle cells following balloon injury. In the current study, we investigated a potential role of PKCδ in regulating the recruitment of adventitial cells. METHODS AND RESULTS: Conditioned media from PKCδ-overexpressing smooth muscle cells stimulated migration and CCR2 expression of adventitial fibroblasts through a MCP-1 dependent mechanism. Following balloon injury of rat carotid arteries, overexpression of PKCδ in smooth muscle cells significantly increased MCP-1 and CCR2 expression and the number of adventitia-originated cells detected in the neointima. Administration of an anti-MCP-1 antibody markedly diminished the recruitment of adventitial cells. Combined PKCδ overexpression and anti-MCP-1 inhibited intimal hyperplasia more effectively than either approach alone. CONCLUSIONS: Our data suggest that PKCδ regulates recruitment of adventitial cells to the neointima via a mechanism involving upregulation of the MCP-1/CCR2 signaling axis in injured arteries. Blockage of MCP-1 while enhancing apoptosis may serve as a potential therapeutic strategy to attenuate intimal hyperplasia.

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-specific deficiency of angiotensinogen decreases plasma angiotensinogen concentration and systolic blood pressure in mice.

Previous studies demonstrated that overexpression of angiotensinogen (AGT) in adipose tissue increased blood pressure. However, the contribution of endogenous AGT in adipocytes to the systemic renin-angiotensin system (RAS) and blood pressure control is undefined. To define a role of adipocyte-derived AGT, mice with loxP sites flanking exon 2 of the AGT gene (Agt(fl/fl)) were bred to transgenic mice expressing Cre recombinase under the control of an adipocyte fatty acid-binding protein 4 promoter (aP2) promoter to generate mice with adipocyte AGT deficiency (Agt(aP2)). AGT mRNA abundance in adipose tissue and AGT secretion from adipocytes were reduced markedly in adipose tissues of Agt(aP2) mice. To determine the contribution of adipocyte-derived AGT to the systemic RAS and blood pressure control, mice were fed normal laboratory diet for 2 or 12 mo. In males and females of each genotype, body weight and fat mass increased with age. However, there was no effect of adipocyte AGT deficiency on body weight, fat mass, or adipocyte size. At 2 and 12 mo of age, mice with deficiency of AGT in adipocytes had reduced plasma concentrations of AGT (by 24-28%) compared with controls. Moreover, mice lacking AGT in adipocytes exhibited reduced systolic blood pressures compared with controls (Agt(fl/fl), 117 ± 2; Agt(aP2), 110 ± 2 mmHg; P < 0.05). These results demonstrate that adipocyte-derived AGT contributes to the systemic RAS and blood pressure control.

Involvement of the renin-angiotensin system in abdominal and thoracic aortic aneurysms.

Aortic aneurysms are relatively common maladies that may lead to the devastating consequence of aortic rupture. AAAs (abdominal aortic aneurysms) and TAAs (thoracic aortic aneurysms) are two common forms of aneurysmal diseases in humans that appear to have distinct pathologies and mechanisms. Despite this divergence, there are numerous and consistent demonstrations that overactivation of the RAS (renin-angiotensin system) promotes both AAAs and TAAs in animal models. For example, in mice, both AAAs and TAAs are formed during infusion of AngII (angiotensin II), the major bioactive peptide in the RAS. There are many proposed mechanisms by which the RAS initiates and perpetuates aortic aneurysms, including effects of AngII on a diverse array of cell types and mediators. These experimental findings are complemented in humans by genetic association studies and retrospective analyses of clinical data that generally support a role of the RAS in both AAAs and TAAs. Given the lack of a validated pharmacological therapy for any form of aortic aneurysm, there is a pressing need to determine whether the consistent findings on the role of the RAS in animal models are translatable to humans afflicted with these diseases. The present review compiles the recent literature that has shown the RAS as a critical component in the pathogenesis of aortic aneurysms.

Novel mechanisms of abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are a common but asymptomatic disease that has high susceptibility to rupture. Current therapeutic options are limited to surgical procedures because no pharmacological approaches have been proven to decrease either expansion or rupture of human AAAs. The current dearth of effective medical treatment is attributed to insufficient understanding of the mechanisms underlying the initiation, propagation and rupture of AAAs. This review will emphasize recent advances in mechanistic studies that may provide insights into potential pharmacological treatments for this disease. While we primarily focus on recent salient findings, we also discuss mechanisms that continue to be controversial depending on models under study. Despite the progress on exploring mechanisms of experimental AAAs, ultimate validation of mechanisms will require completion of prospective double-blinded clinical trials. In addition, we advocate increased emphasis of collaborative studies using animal models and human tissues for determination of mechanisms that explore expansion and rupture of existing AAAs.

MyD88 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation independent of signaling through Toll-like receptors 2 and 4.

OBJECTIVE: The purpose of this study was to determine whether myeloid differentiation factor 88 (MyD88) and its related Toll-like receptors (TLRs) 2 and 4 contributed to the development of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and atherosclerosis. METHODS AND RESULTS: AngII was infused into either apoE(-/-) or LDL receptor (LDLR)(-/-) male mice that were either MyD88(+/+) or (-/-). MyD88 deficiency profoundly reduced AngII-induced AAAs and atherosclerosis in both strains. To define whether deficiency of specific TLRs had similar effects, AngII was infused into LDLR(-/-) mice that were also deficient in either TLR2 or TLR4. TLR2 deficiency had no effect on AAA development but inhibited atherosclerosis. In contrast, TLR4 deficiency attenuated both AAAs and atherosclerosis. To resolve whether MyD88 and TLR4 exerted their effects through cells of hematopoietic lineage, LDLR(-/-) mice were lethally irradiated and repopulated with bone marrow-derived cells from either MyD88 or TLR4 strains. MyD88 deficiency in bone marrow-derived cells profoundly reduced both AngII-induced AAAs and atherosclerosis. However, TLR4 deficiency in bone marrow-derived cells had no effect on either pathology. CONCLUSIONS: These studies demonstrate that MyD88 deficiency in leukocytes profoundly reduces AngII-induced AAAs and atherosclerosis via mechanisms independent of either TLR2 or TLR4.

Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity.

Continuous exposure of polymorphonuclear leukocytes (PMNLs) to circulatory hemodynamics points to fluid flow as a biophysical regulator of their activity. Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membrane-bound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R(2) = 0.96; P < 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R(2) = 0.5; P < 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shear-responses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis).