The identification of risk factors for AAA formation provides strategies for AAA prevention and therapy. In the past few decades, various factors including male gender, aging, cigarette smoking, and elevated blood cholesterol level, etc., have been found to be related to AAA initiation and progression (Figure 1) ^[6][14][15][6,14,15]. The incidence of AAA is directly proportional to an increase in age, and AAA is a prominent cause of death in older people from 65 years of age [5]. It is often seen in older men, and the ratio of male and female patients ranges 3.5–6:1 based on various screening studies ^[16][17][16,17]. Therefore, the men with an age of more than 65 years old are the high-risk population for AAA development.

Smoking has been recognized as a strong risk factor for AAA in various epidemiological studies ^[14][16][18][14,16,18]. Approximately 87% of the men with AAA were reported to be current or ex-smokers in a screening study of AAA among 65-year-old Swedish men with an odd ratio of 3.5 (95%CI: 2.4–5.1) [19]. The nicotine in plasma contributes to AAA progression, which has been confirmed in Apolipoprotein E deficient (ApoE−/−) mouse model and elastase-perfusion model of AAA ^[20][21][22][20,21,22]. The mechanisms underlying smoking induced AAA involve VSMC dysfunction and inflammatory cell function that have been discussed in Norman, P. E. and Curci, J. A. published review [20]. Hypercholesterolemia is a common facilitating cause of AAA, although there is limited knowledge as to its role in the development of AAA [15]. The abdominal aortic region is prone to the atherosclerotic lesion, which is found in virtually all cases of human AAA patients and that atherosclerosis is involved in the process of aortic dilation. Additional evidence to support the important role of hypercholesterolemia in aneurysm formation is the increased AAA formation in angiotensin II (Ang II) infused ApoE−/− mice (hypercholesterolemia mouse model) compared with wild-type (WT) C57BL/6 mice [23]. Genetic analyses of AAA from population screening and gene-associated studies show that different genetic risk factors are involved in the pathogenesis of AAA [24]. A heritability of 70% was reported in a large twin study by Jesper Swedenborg’s research group [25]. Several other studies have found that the risk of AAA in first-degree relatives of affected individuals was almost doubled ^[26][27][28][26,27,28]. Two loci for AAA have been identified and mapped on chromosome 19q13 and 4q31 ^[29][30][29,30]. The genome wide association studies (GWAS) have identified associations of single nucleotide polymorphisms (SNPs) with AAAs, such as CDKN2BAS1 rs10757274, the low-density lipoprotein receptor (LDLR) rs6511720, and DAB2IP rs10985349 ^[31][32][31,32]. In addition, other factors including DNA methylation, hypertension, and viral infections (e.g., cytomegalovirus, CMV) are also associated with AAA pathogenesis ^{[33][34][35][36][37]}[33,34,35,36,37].

The histopathology of AAA is characterized by aortic wall inflammation, EC alteration, SMC dysfunction, oxidative stress, and ECM degradation, which cause a progressive luminal dilation, and finally a rupture (Figure 2) ^{[6][13][38][39]}[6,13,38,39]. The aortic wall inflammation with the infiltration of inflammatory cells, including T-cells, B-cells, and macrophages, are essential features of AAA (Figure 2) ^[40][41][42][40,41,42]. In clinical and experimental AAA, a number of pro-inflammatory cytokines are increased, such as monocyte chemoattractant protein 1 (MCP-1), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Polymorphisms in inflammatory cytokines may affect the production of these cytokines and, therefore, influence the pathogenesis of AAA ^[23][43][44][23,43,44]. SNPs (rs1800795 and rs1800796) in the IL-6 promoter have been linked with the development of AAA ^[44][45][44,45]. These inflammatory mediators play a critical role in inflammatory cell infiltration, which promotes an inflammatory response and subsequent SMC dysfunction, ECM degradation (mainly through matrix metalloproteinases, MMPs), and eventual AAA formation ^{[42][46][47][48]}[42,46,47,48]. In addition, fragments of the aortic wall degradation serve as attracting agents for macrophage infiltration into the aortic wall to initiate the immune responses and AAA formation [49]. Dysregulation of EC function is another important factor implicated in AAA initiation and/or progression ^[50][51][50,51]. The increased EC expression of MCP-1 and vascular cell adhesion molecule 1 (Vcam-1) recruits macrophages into the aortic wall and leads to ECM degradation and, finally, aneurysm formation (Figure 2). In addition, EC apoptosis with the reduced expression of endothelial nitric oxide synthase (eNOS) also facilitates AAA formation by affecting the activity of NO, which is important in the stability of vascular tone, blood pressure, and SMC relaxation.

3. Application of Single-Cell RNA-Sequencing (scRNA Seq) in AAA Studies

scRNA seq emerges as a powerful approach to study transcriptome profile changes that are useful for identifying cellular clusters and exploring cellular responses in AAA. During AAA development, a myriad of cell types is involved, ranging from circulating immune cells to vascular resident cells (e.g., SMC). Recently, studies using scRNA seq have shed light on the heterogeneity and cellular responses of vascular cells in AAA progression. One recent study identified 17 clusters representing nine-cell lineages. Further Seurat clustering analysis identified four SMC subpopulations and five monocyte/macrophage subpopulations. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas a small subpopulation was increased with downregulated SMC contractile markers and increased pro-inflammatory genes ^[52][163], suggesting phenotypic changes. Interestingly, scRNA seq analysis of lesioned aortas has identified macrophage-derived Netrin-1 as a robust inducer of the intracellular calcium flux and MMP3 activity by VSMCs, thereby it mediates the dynamic crosstalk between inflammation and ECM remodeling in AAA ^[53][164]. Single-cell analysis of the clinical aortic specimens from Marfan syndrome patients also revealed defective TGF-β signaling, i.e., downregulated TGFBR2 and Smad in a subset of SMCs ^[54][165]. Furthermore, an altered subpopulation of dedifferentiated proliferative SMCs was noted in the aortic tissues from Marfan syndrome patients but not from control subjects. These studies underscore the importance of the selective targeting of subgroups of VSMCs based on their transcriptome profiles. The scRNA seq analysis of AAA tissues are useful for dissecting the heterogeneity of cell subpopulations, deregulated signaling pathways, and cellular responses, as well as their interactions during AAA development. It also holds the key for identifying disease-relevant transcriptional signatures in VSMC-lineage cells, which might provide clues for disease predication, diagnosis, and prevention.