Parsing Aortic Aneurysms: More Surprises

Summary

The progression of aortic aneurysms involves complex changes in wall structure and results from the interplay of multiple cell types including endothelial cells, SMCs, macrophages and adventitial cells. It is important to realize that these pathogenic steps play out on different response backgrounds in the ascending thoracic aorta compared to the abdominal aorta. Much like genetic modifiers at work in different inbred strains of mice that can sometimes produce dramatically different phenotypic manifestations of the same mutation on different genetic backgrounds, the SMC lineage diversity intrinsic to different aortic segments may play important modifier roles in the origins and progression of aneurysms in thoracic versus abdominal aortic segments in the same individual. Future work will focus on identification of factors produced by endothelial cells exposed to chronic infusions of AngII that initiate response cascades in SMCs and macrophages leading to aneurysm formation. It will also bring into greater focus the ways that advances in understanding basic pathways of vascular development provide important insights into mechanisms of disease pathogenesis in the adult vascular system.

Introduction

Aortic aneurysms are progressive enlargements of the aorta that can lead to life-threatening degenerative changes in the structure of the artery wall including medial dissections and wall rupture. The abdominal aorta is the most common site of aneurysm formation yet the ascending thoracic aorta can also exhibit aneurysmal expansion with potentially dire outcomes. Ascending aortic aneurysms are frequently associated with connective tissue diseases such as Marfan's syndrome, a disorder of the extracellular matrix protein fibrillin-1 ¹. Mouse models carrying hypomorphic mutations in fibrillin-1 also develop aortic dilations associated with macrophage infiltration, elastin fragmentation and enhanced TGFβ signaling in the artery wall ². Aortic aneurysms in fibrillin-1 hypomorphic mice can be prevented by treatment with either anti-TGFβ neutralizing antibodies or with the angiotensin II (AngII) type I receptor (AT1) antagonist losartan ³. Indeed, chronic infusion of AngII into apoE^-/- mice is an effective experimental model to induce aortic aneurysm formation and to study the molecular pathways underlying its pathogenesis ⁴.

The Role of AT1a Receptors in Ascending Aortic Aneurysms

AngII signals through two types of angiotensin receptors, AT1 and AT2 ⁵. AT2 receptors have been shown to elicit anti-proliferative responses in vascular smooth muscle cells (SMCs), possibly by antagonism of signaling through AT1 receptors ⁵. In mice, the AT1 receptor exists as two subtypes, AT1a and AT1b, and both receptor subtypes are expressed in the artery wall. The receptor that mediates angiotensin-induced contractile responses in vascular smooth muscle is predominantly the AT1b subtype ⁶. The AT1 receptor subtype that mediates aortic aneurysm formation has been unknown. In a study that appears in this issue of Circulation Research, Rateri et al utilized a series of mouse models to genetically dissect the process of AngII-induced aneurysm formation in the ascending portion of the thoracic aorta ⁷ (Figure). Using mice in which a null mutation of the AT1a receptor was placed on an LDL receptor (LDLR)-deficient background, the authors showed that increases in ascending aortic diameters (a measure of aneurysmal dilation) produced by chronic infusion of AngII via subcutaneous mini-osmotic pumps were substantially reduced compared to LDLR^-/- mice expressing a normal complement of AT1a receptors. These experiments demonstrated that AngII-induced ascending aortic aneurysm formation was mediated by the AT1a receptor.

(A) Normal appearance of thoracic aorta in an untreated adult mouse (-AngII). (B) Wall structure of the ascending thoracic aorta of normal adult mice. Lumen is to the left. Endothelial cells (EC) extend over medial smooth muscle cells (SMCs) that are distributed between five layers of elastin (dark lines). Resident tissue macrophages (MΦ) are found in the adventitia. (C) Pronounced ascending aorta dilation is produced by chronic infusion of angiotensin II (+AngII). (D) Wall structure of the dilated ascending aortic segment. Lumen is to the left. The aortic media is greatly distended, elastic fibers are fragmented, the media and adventitia are invaded by macrophages, and a weakened wall structure is produced. The dilated aortic segment is confined to the portion of thoracic aorta composed of neural crest-derived SMCs (A, C, black) and does not extend into the more distal aortic segment composed of somitederived SMCs (A, C, dark gray). The aortic root is composed of SMCs derived from second heart field progenitors (A, C, light gray) and is a frequent site of aortic coarctation in Williams-Beuren syndrome ¹⁴.

Cell Type-Specific Deletions of AT1a Receptor

Knowing that critical events downstream of the AT1a receptor mediate development of ascending aortic aneurysms, the next question to tackle was what cell type[s] in the ascending aorta were critical targets of AngII in this experimental model. One logical target is the macrophage since infiltration of the aortic wall with macrophages is a prominent feature of the dilated segment in both mouse and human aortic aneurysms. To address this possibility, the authors carried out a series of reciprocal bone marrow transplantion experiments with the somewhat surprising result that no significant contribution of AT1a receptors on bone marrow-derived cells, including myeloid cells, was evident by this analysis ⁷. These results argued that the critical sites of AT1a receptor expression for aneurysm formation were most likely to be found in resident artery wall cells themselves. Since the pathological manifestations of aneurysmal dilation are clearly seen in the medial layer of the ascending aorta, the obvious cell type of interest in this regard is the medial SMC. To test this possibility, Rateri et al generated mice carrying a floxed allele of the AT1a receptor in which Cre recombinase-mediated deletion of the floxed genomic sequence would remove exon 3 which contains the entire region of translation for the AT1a receptor thus producing a null allele. Floxed AT1a mice were bred to mice that express Cre recombinase under control of genetic regulatory elements from the smooth muscle differentiation marker gene SM22α (also called transgelin). Despite nearly extinguishing AT1a receptor expression at the mRNA level from SMCs in ascending aorta segments, there was no significant reduction in aneurysm formation after infusion of AngII in these mice ⁷. Taken together, the experiments argue that neither macrophages nor aortic SMCs, the two seemingly most likely targets, are the critical AngII-responsive cell type in this model of ascending aortic aneurysm formation.

Role of Endothelial Cells in Aortic Aneurysm

Like the proverbial gorilla in the room, that leaves one major cell type, the endothelium, unaccounted for. At first glance, it is not obvious that endothelial cells should be the principal target of AngII infusion in this model of aneurysm formation. The increases in blood pressure produced by chronic infusion of AngII are relatively modest (~10-20mmHg) and the effects of losartan as an inhibitor of aneurysm formation are not mimicked by equal reductions in blood pressure produced by an unrelated antagonist, propranolol ³. Moreover, the cell type most responsive to increases in wall tension produced by elevated blood pressures would be medial SMCs and not endothelial cells. Furthermore, the most pronounced structural changes in aneurysm are in the media not in the intima. Yet when AT1a floxed mice were crossed with Tek (also called Tie2)-cre mice, significant reductions in outer curvature length, measured from the aortic root to the brachiocephalic origin, were found ⁷. In addition to endpoints of aortic dilation, cross-sectional measurements of medial thickness and the number of breaks in elastic lamellae were also significantly reduced following AngII infusion in Tek cre-expressing mice ⁷. Although Tek-driven cre activity is also found in bone marrow-derived cells, the reciprocal marrow transplantation experiments described above argue against an important role for AT1a receptors on bone marrow-derived cells in this model. These results argue strongly for a critical signal relay initiated in the endothelium and transmitted to medial SMCs resulting in aortic wall dilation, expansion of interlamellar spaces in the tunica media, recruitment of macrophages and fragmentation of elastin that is characteristic of the disease (Figure). Of course, endothelial cells are known to express angiotensin receptors and many responses of these cells to AngII have been described, including induction of monocyte chemoattractant protein-1 expression ^{5, 8}. It will clearly be of importance to further investigate what mediators are responsive to endothelial AT1a receptor stimulation in vivo and how these mediators direct underlying SMCs to undergo the degenerative wall remodeling responses that lead to aneurysm formation.

Arterial Segment-Specific Differences in Aneurysmal Dilation

Chronic AngII infusion produces dilation and aneurysm formation of both the ascending thoracic aorta and the abdominal aorta in the same animal 9. However, the structural and histological characteristics of aneurysms arising in the ascending thoracic aorta are distinctly different from those found in the abdominal aorta ^{4, 9}. Abdominal aortic aneurysms develop focal transmural disruption of elastin matrix structure, and focal macrophage accumulation in the presence of T- and B-lymphocytes ^{4, 9}. Thoracic aortic aneurysms develop with widespread rather than focal macrophage accumulation, elastin fragmentation that may be extensive but generally not transmural, and a greater bias toward medial changes near the adventitial side of the artery wall ⁹. These region-specific differences in aneurysm progression may be related to differences in the embryonic origin of medial SMCs in the ascending aorta, which arise from neural ectoderm-derived progenitors in the cardiac neural crest, compared to SMCs in the abdominal aorta, which originate from paraxial mesoderm-derived progenitors in somites ^{10, 11}. This possibility is made more intriguing by considering the important role of TGFβ signaling in aneurysm formation ³ since SMCs from these two embryonic origins exhibit lineage-specific differences in the ways that they respond to stimulation by TGFβ1 ¹². When compared under identical conditions ex vivo, DNA synthesis, cell proliferation and plasminogen activator inhibitor-1 (PAI-1) promoter activity were strongly stimulated by TGFβ1 (0.4 to 400 pmol/L) in neural crest-derived SMCs whereas somite-derived SMCs exhibited only minimal increases in PAI-1 promoter activity and were consistently growth inhibited by the same concentrations of TGFβ1 under identical conditions in culture ¹². It is of particular interest that the distal limit of the ascending aortic wall dilation responses to chronic AngII infusion in mice ⁹ maps very closely to the lineage boundary where SMCs of neural crest origin exhibit a sharply demarcated transition zone into aortic tunica media composed of SMCs that arise from somite-derived progenitors ^{10, 11, 13}. Likewise in William's syndrome ¹⁴ coarctation of the ascending aorta occurs in close proximity to a second SMC lineage boundary in the aortic root that is situated between neural crest-derived SMCs and aortic SMCs that arise from progenitors in the second heart field ¹⁵ (Figure). These observations lead to the speculation that when exposed to a common stimulus, in this case chronic infusion with AngII, different aortic segments follow distinct response pathways that are dictated by their different embryonic origins and lineage histories ¹⁰. This property may underlie the transfer of susceptibility to atherosclerosis in aortic homograft transplants reported many years ago in which atherosclerosis-susceptible abdominal aortic segments retained their propensity to develop severe intimal lesions even when transplanted to atherosclerosis-resistant positions in the thoracic aorta ¹⁶. It may also be relevant to the contrasting roles of TGFβ signaling reported by different investigators studying aneurysm formation in the thoracic compared to abdominal aortic segments ^17,18.