Background
Abdominal aortic aneurysms (AAA) affect more than 1.1 million people in the United States and bear an 85% chance of death after rupture [
1]. Each year in the United States, more than 16,000 deaths result from rupture of abdominal aortic aneurysms, and 33,000 patients undergo repair of asymptomatic abdominal aortic aneurysms to prevent future rupture [
2]. Albeit the high morbidity and mortality associated with aneurysm formation, there are no validated or approved medical therapies for attenuating aneurysm growth and rupture. With increasing recognition of the devastating impact of aneurysmal rupture, ongoing research efforts aim to determine mechanisms of initiation and progression of aneurysms.
Medial infiltration of macrophages and the activation of inflammatory signaling represents the earliest histological change during the initiation of AAA formation [
3,
4]. Locally secreted inflammatory signals act on these infiltrating myeloid cells by inducing signal-stimulated transcription factors. Among these, the NR4A subgroup of orphan nuclear receptors represents immediate-early response genes, which are expressed rapidly in response to inflammatory stimulation and control essential cellular functions in all processes of pathological vascular remodeling [
5,
6]. The NR4A3 subfamily member NOR1 is highly expressed in human abdominal aortic aneurysm tissues [
7] and has been implicated previously in proliferation of myeloid progenitor cells [
8,
9], recruitment of monocytes to sites of inflammation [
10], transcription of inflammatory gene expression [
11,
12], and macrophage apoptosis [
13]. Based on this key function of NOR1 in the control of inflammation combined with the well-established contribution of macrophage-induced inflammatory signaling to AAA formation [
14,
15], we investigated whether NOR1 deletion affects development of AAA.
Discussion
NOR1 expression in macrophages regulates proliferation [
8,
9], inflammation [
11,
12], macrophage lipid-uptake [
12], and apoptosis [
13]. While these cellular functions have been implicated in initiation of AAA formation [
24], it remains currently unknown whether NOR1 contributes to their formation. Therefore, we depleted macrophages of NOR1 using bone marrow transplantation and analyzed whether macrophage NOR1 function affected AAA development in chimeric mice. Although deletion of the NOR1 locus attenuated expression of multiple inflammatory genes, NOR1 deficiency in wall-infiltrating cells derived from hematopoietic progenitors did not affect the initiation of angiotensin II-induced AAA formation.
NOR1 is expressed in all cell types of the vascular wall, including endothelial cells, smooth muscle cells, and infiltrating macrophages [
5]. While basal levels of expression are low, NOR1 is induced rapidly in response to mitogenic and inflammatory signaling underlying pathological vascular remodeling [
5]. Macrophage activation represents a major mechanism of vascular disease initiation, as inhibition of their infiltration and inflammatory signaling prevents all major vascular diseases, including atherosclerosis and AAA formation [
25,
26]. Several prior studies have identified a key role of inducible NOR1 expression in the regulation of macrophage function, including activation of pro-inflammatory [
11] but also anti-inflammatory gene regulation [
12,
27]. In contrast to these previous studies using viral NOR1 overexpression, our inflammation-biased profiling experiments employed loss-of-function using cells isolated from mice harboring genetic deletion of the NOR1 locus. While different in approach, we observed that 36 out of the 184 analyzed inflammatory genes were differentially expressed, although the magnitude of significant differences was overall mild. Among those regulated genes, the majority were pro-inflammatory, and their expression was decreased in NOR1-deficient cells, including for example IL-6 and TNFα. These loss-of-function studies are consistent with the observations of Pei et al. that NOR1 expression induces transcription of inflammatory genes and activates NF-κB-dependent gene regulation in macrophages [
11]. Furthermore, several of the differentially regulated genes revealed putative NBRE binding sites for NOR1, suggesting potentially direct mechanisms of gene regulation. However, in addition to direct gene regulation, NOR1 deletion induces the Ly6-C(hi) monocyte population [
8,
9] and is likely to affect myeloid lineage specification, which may further alter inflammatory signaling, particularly in vivo.
Considering the literature supporting an important function of NOR1 in macrophage gene regulation [
11,
12,
27], combined with our own observations discussed above, and further that infiltration of circulating monocytes represents one of the earliest mechanisms of AAA formation [
4], we tested whether NOR1 deletion in bone marrow-derived cells affected AAA formation and progression. Activation of the renin-angiotensin system constitutes a key mechanism of AAA formation, and infusion of angiotensin II induces AAA formation in hyperlipidemic mice [
20], which closely resembles many aspects of human pathology [
22]. Unexpectedly, bone marrow cell deletion of NOR1 did not affect formation or rupture incidence of AAA. Since infiltrating macrophages during AAA formation are primarily derived from the bone marrow [
28], NOR1 expression in these cells appeared not to be a major determinant of AAA formation, albeit the noted differential gene expression seen in NOR1-deficient macrophages.
There are several potential explanations for these seemingly discrepant observations. First, it is possible that those genes that were regulated by NOR1 are either not causally involved in AAA or that the magnitude of regulation by NOR1 is insufficient to affect the disease development. However, in our in vitro studies, IL-6 expression, for example, was decreased in NOR1-deficient macrophages, and inhibition of IL-6 has been demonstrated to prevent angiotensin II-induced aneurysm formation [
29]. Second, it is more likely that there is a balance between the regulation of pro- and anti-inflammatory genes by NOR1, and that the overall net effect on macrophage function does not modulate the initiation and severity of AAA formation. That this hypothesis could be conceivable is supported by the observation that IL-4, which polarizes macrophages towards the more anti-inflammatory M2 phenotype, was increased in NOR1-deficient cells. Third, potential anti-inflammatory effects of NOR1 deletion within locally infiltrating macrophages may become mitigated by an increased number of circulating monocytes. NR4A nuclear receptors have been identified previously as tumor suppressors for myeloid lineage, and deletion of NOR1 increases the Ly6-C(hi) monocyte population [
8,
9]. Therefore, NOR1 deficiency specifically in the hematopoietic lineage may increase the number of pro-inflammatory monocytes in the circulation, which could compensate for decreased inflammatory gene expression and ultimately increase AAA formation [
28]. Finally, it is possible, if not likely, that decreased inflammation of infiltrating NOR1-deficient macrophages during angiotensin II-induced vascular remodeling may be compensated by an increase in inflammatory gene expression or matrix remodeling enzymes in resident endothelial and vascular smooth muscle cells. Although we have not performed an extensive histological examination of tissues, this notion is supported by the recent findings that expression of NOR1 in endothelial cells increased monocyte adhesion through the regulation of VCAM-1 and ICAM-1 [
30], which constitutes an important early mechanism of AAA formation [
31]. However, dissecting the more cell-specific contribution of NOR1 in endothelial cells, smooth muscle cells, and mature macrophages will depend on genetic deletion using conditional, cell-specific gene targeting in future experiments.