Numerous publications have demonstrated that AngII infusion into mice leads to development of AAAs.16,17
More recently, it has been observed that AngII infusion into mice also leads to pronounced aortic dilation and dissections that are highly restricted to the ascending aortic region.6,18
Although AngII generates aortic aneurysms in both regions, the pathology of ascending AAs demonstrates many distinctions, including concentric dilation without local medial rupture and medial thickening that is most pronounced on the adventitial aspect.6,19
This ascending aortic pathology that evolves during AngII infusion bears many similarities to a model of Marfan’s syndrome in which mice express a mutant allele of fibrillin-1.5
In the current study, whole body deficiency of AT1a receptors ablated development of AngII-induced ascending AAs. To define the cell type expressing AT1a receptors that contributed to development of ascending AAs, a combination of approaches was taken using bone marrow transplantation and cell-specific conditional deficiencies. These approaches studied three major cell types in AngII-induced ascending AAs: leukocytes, SMCs, and endothelial cells. Using these approaches, we were unable to detect any effect of AT1a receptor deficiency in leukocytes or SMCs on development of AngII-induced ascending AAs. In contrast, depletion of AT1a receptors in endothelium attenuated development of AngII-induced ascending AAs.
Many of the physiological and pathological effects of AngII are mediated by stimulation of AT1 receptors.20
In rodents, chromosomal duplication has led to expression of two subtypes of AT1 receptors, termed a and b.21
Although AT1b receptor expression is more restricted compared to AT1a receptors, aortic tissue contains both subtypes with a predominance of the b subtype.22,23
Only the b subtype is involved in AngII-induced contractile activity that has been previously demonstrated to be restricted to the infrarenal region.24,25
Despite the presence of functional AT1b receptors in aorta, AT1a receptors plays an essential role in AngII-induced ascending AAs based on the ablation of the pathology in AT1a receptor deficient mice. It may be presumed that minor amino acid differences between AT1a and AT1b receptor subtypes lead to differential stimulation of signaling pathways that differentiate AngII-induced contractile versus pathological processes.26
Leukocytes are prominent components in both AngII-induced AAAs and ascending AAs.6,19
Evaluation for a role of AngII acting directly on leukocytes was performed in AAAs using bone marrow transplantation to create mice that are chimeric for AT1a receptors.8
While this approach did not distinguish the cells types that arise from the bone marrow, macrophages are the most dominant cell type from this origin in AngII-induced AAAs.18,19,27
Similarly, macrophages are the most abundant leukocytes in AngII-induced ascending AAs. Repopulation of irradiated mice demonstrated no effect of AT1a receptors on donor cells to development of AngII-induced AAAs.8
The current study also failed to demonstrate a role for AT1a receptors on donor cells in promoting AngII-induced ascending AAs. Furthermore, while functional AT1 receptors have been identified on mouse macrophages,28
we were unable to demonstrate a role for AT1a receptors on this cell type in directly influencing development of ascending AAs.
AngII is known to exert region-specific effects on aortic SMCs in physiological and pathological responses.14,29
We have demonstrated previously that the mechanism of AngII-induced SMC-rich medial thickening in vivo differs in the ascending aorta relative to all other regions.15
This difference may be a consequence of the different developmental origin of SMCs throughout the aortic tree.30
In the present study, we initially sought to demonstrate whether any regional differences in SMC responses to AngII can be detected ex vivo. Previous studies have demonstrated that contractile responses to AngII differ between aortic regions, with minimal contraction in the descending thoracic aorta compared to the infra-renal region.14,25
Responses may differ in the ascending aorta since SMCs in this region have a different developmental origin from the descending aorta, with the potential for different functional characteristics.31,32
However, similar to the descending aorta, we were also unable to detect any AngII-induced SMC contraction in rings isolated from the ascending aorta, despite this region contracting to KCl and 5-HT. Therefore, AngII does not have demonstrable effects on a physiological function of SMCs in this aortic area.
To directly determine the role of AngII stimulation of SMCs in the pathological process of ascending AAs development, we generated AT1a receptor floxed mice to develop cell-specific deficient mice. The floxed sites were located either side of exon 3 that is responsible for the entire translatable portion of the protein. AT1a receptor deficiency on SMCs was created by breeding mice that were hemizygous for Cre driven by the SM22 promoter.33
This Cre expression strategy has been used previously to determine the role of SMCs in experimental AAAs, but has not been used to determine ascending AAs.33–35
The normal mouse aorta not only has different developmental origins of SMCs, but the media may also contain other cell types, such as fibroblasts and myofibroblasts.36
Despite this potential heterogeneity, studies in which SM22 driven Cre was expressed in ROSA26 mice demonstrated the uniformity of gene depletion across the media that coincided with α-actin immunostaining. Although we were unable to validate spatial distribution of AT1a receptor protein expression, the combined evidence from the SM22-Cre-ROSA26 mice and the absence of AT1a receptor mRNA in aortic SMCs is consistent with an effective depletion. In mice with this depletion, we were unable to detect any difference in AngII-induced ascending AAs. This is consistent with the lack of direct effects of AngII on SMCs to initiate and propagate AAs.
Endothelial function has a profound effect on vascular physiology and pathology and endothelial cells express angiotensin II receptors.37
To determine a role of AngII stimulation on endothelial cells in the development of AngII-induced ascending AAs, we bred AT1a receptor floxed mice to those expressing Cre under the control of the Tek promoter. Use of the Tek promoter has the potential complication of deleting genes in cells of myeloid origin, in addition to endothelial. Given that myeloid and endothelial cells are derived from a common precursor cell of the hemangioblast, any genes expressed at this stage of development would have a broader spectrum of effects on these different cell types.38
However, since we were unable to demonstrate any effect of AT1a receptor expression on bone marrow-derived cells, the attenuation of AngII-induced ascending AAs in Tek-driven Cre mice was not confounded by deletion of the receptor in myeloid cells.
Although deficiency of AT1a receptors in endothelium attenuated the development of AngII-induced ascending AAs, the effect was not as profound as the complete ablation of disease in mice with whole body AT1a receptor deficiency. In contrast, deficiency of AT1a receptors in SMCs had no discernible effect on the development of aneurysms. Furthermore, bone marrow transplantation studies failed to reveal any effect of leukocyte AT1a receptor deficiency on aneurysm development. Therefore, of the three major cell types present in aneurysmal tissue, only depletion of AT1a receptors in the endothelium reduced aneurysmal disease, but not as dramatically as whole body deficiency. One potential explanation is a technical shortcoming that the activity of Cre was not sufficient to delete the floxed exon 3. It would have been preferable to demonstrate deletion by determination of changes in AT1a receptor protein. Unfortunately, we were unable to validate that commercially available antibodies authentically stained the AT1a receptor protein. We were also unable to develop antibodies that specifically recognize this protein. Therefore, the index of deletion was dependent on measurement of mRNA abundance. In agreement with previous studies using Cre under the control of SM22 Cre,33,34,39
we demonstrated a highly effective deletion of the floxed components of the gene. Given the inability to isolate mRNA from the endothelial cells harvested from the ascending aorta, we performed this analysis on endothelial cells isolated from lung. Findings in these cells were directly complemented by studies using Tek expressing mice in the ROSA26 background. Analyses of sections from ascending aortas of these mice demonstrated a uniform expression of β
-galactosidase in the endothelium. Therefore, expression of Cre in either endothelial or smooth muscle selective manner appears to have been effective in removing the exon that translates the entire functional protein.
Another explanation of the different extent of whole body versus cell-specific AT1a receptor deficiency on ascending AAs may be the involvement of other cell types. There is emerging evidence for a role of fibroblasts and myofibroblasts in the development of experimental aneurysms by exposure to calcium chloride or AngII infusion.18,36,40
These cell types have been detected in both the media and adventitia of mice. Future studies in mice expressing Cre under a fibroblast-specific promoter will provide insight into the role of this cell type.
Depletion of AT1a receptors in endothelial cells reduced the development of AngII-induced ascending AAs to a similar extent to that previously described in mice deficient in CCR2, the cognate receptor for monocyte chemoattractant protein-1 (MCP-1). With both deficiencies there was a decrease in lumen area and medial thickness. AngII can stimulate the release of MCP-1 from endothelial cells.41
Therefore, it is interesting to speculate that AngII stimulation of endothelial AT1a receptor promotes MCP-1 release that permeates the media due to blood pressure. This could promote leukocyte recruitment via the adventitial aspect of the aorta with the subsequent elaboration of elastolytic enzymes. The recent availability of MCP-1 floxed mice will permit the testing of this hypothesis.42
In summary, we demonstrate that endothelial-specific deficiency of AT1a receptors markedly attenuates the development of AngII-induced AAs. Future studies will define the nature of the mediator released from the endothelium that promotes this localized disease.