Role of Adipokines and Perivascular Adipose Tissue in Abdominal Aortic Aneurysm: A Systematic Review and Meta-Analysis of Animal and Human Observational Studies

Improved understanding of abdominal aortic aneurysms (AAA) pathogenesis is required to identify treatment targets. This systematic review summarized evidence from animal studies and clinical research examining the role of adipokines and perivascular adipose tissue (PVAT) in AAA pathogenesis. Meta-analyses suggested that leptin (Standardized mean difference [SMD]: 0.50 [95% confidence interval (CI): −1.62, 2.61]) and adiponectin (SMD: −3.16 [95% CI: −7.59, 1.28]) upregulation did not significantly affect AAA severity within animal models. There were inconsistent findings and limited studies investigating the effect of resistin-like molecule-beta (RELMβ) and PVAT in animal models of AAA. Clinical studies suggested that circulating leptin (SMD: 0.32 [95% CI: 0.19, 0.45]) and resistin (SMD: 0.63 [95% CI 0.50, 0.76]) concentrations and PVAT to abdominal adipose tissue ratio (SMD: 0.56 [95% CI 0.33, 0.79]) were significantly greater in people diagnosed with AAA compared to controls. Serum adiponectin levels were not associated with AAA diagnosis (SMD: −0.62 [95% CI −1.76, 0.52]). One, eight, and one animal studies and two, two, and four human studies had low, moderate, and high risk-of-bias respectively. These findings suggest that AAA is associated with higher circulating concentrations of leptin and resistin and greater amounts of PVAT than controls but whether this plays a role in aneurysm pathogenesis is unclear.


INTRODUCTION
Screening studies suggest that abdominal aortic aneurysms (AAA) prevalence is approximately 3% in men and 1% in women aged over 65 years (1). The main complication of AAA is aortic rupture which is estimated to cause 200,000 deaths worldwide each year (2). Currently the management of AAA is limited to surgical repair (3). Randomized clinical trials have demonstrated that early elective surgical repair does not reduce mortality for people with small asymptomatic AAAs (4,5). Current guidelines therefore recommend simply monitoring small (<50mm in women and <55m in men) asymptomatic AAAs (6). Most small AAAs continue to expand and eventually reach a diameter when surgical treatment is indicated (6). Drug therapies which limit growth of small AAAs would be valuable in clinical practice. Previous small randomized controlled trials have however failed to identify any effective medications (7).
Adipocytes are fat depots that store excess energy as triglycerides. They also have an endocrine function by secreting adipokines, such as leptin, adiponectin, dipeptidyl peptidase-4 (DPP-4), resistin and resistin-like molecule beta (RELMb) (8). Leptin is an adipokine involved in regulating energy homeostasis and obesity. It is considered an important regulator of b cell mass and survival (9). In patients with leptin deficiency, recombinant leptin replacement therapy suppresses appetite and increases energy expenditure and leptin has been developed as a treatment for obesity (10). The adipokine adiponectin has insulin-sensitizing, anti-inflammatory and anti-apoptotic effects (11). Adiponectin and agonists of its receptor have also been suggested as a treatment for obesity and its complications (12). DPP4 release has been shown to strongly correlate with adipocyte size, potentially representing an important source of DPP4 in obesity (13). Resistin belongs to an RELM family and is believed to be an important link between obesity, insulin resistance, and diabetes (14). Increasing evidence suggests that resistin plays an important role in a variety of biological processes involved in cardiovascular diseases, autoimmune disease, and asthma (14). RELM-b is another member of RELM family that is mainly secreted from the adipose tissue in rodents (15) and from adipose-associated macrophages in humans (16). Adipose is deposited over multiple subcutaneous and visceral sites but when considering a role in aortic pathology, perivascular aortic adipose tissue (PVAT) is of particular interest. Adipose tissue residing in the vascular adventitia has been proposed to act like endocrine cells that respond to inflammatory stimuli by releasing adipokines and other signaling mediators to maintain vascular homeostasis and potentially play a role in aortic pathology (17,18). This review summarizes published animal and human studies that investigated the role of these adipokines and PVAT in AAA pathogenesis.

Identification and Inclusion of Studies in This Review
Studies were identified from the PubMed database published until 5 th August 2020 using the following search terms ["adiposity" OR "adipose tissue" OR "adipokines" OR "adiponectin" OR "leptin" OR "resistin" OR "subcutaneous fat" OR "visceral fat" OR "peri aortic fat" OR "obesity"] AND ["Abdominal aortic aneurysm" OR "aortic occlusive disease" OR "aortic rupture"]. Animal and observational human studies that investigated the role of adipokines or PVAT in AAA were included. Outcomes included aortic diameter and adipokine concentrations or relative amount. Included articles were identified by one author (ST) and reviewed by another author (JG). A minimum of two studies reporting the aortic diameter in animal studies and adipokines levels or PVAT in human studies were required for meta-analyses to be performed. In studies where the effect of deficiency and inhibition of a specific adipokine or PVAT were reported, data were converted to an equivalent format in order to include in the same meta-analyses. Meta-analysis findings were presented as standardized mean difference (SMD) and 95% confidence intervals (CI). Metaanalyses were performed using random effects model and an inverse variance method was used to estimate the heterogeneity between studies. All analyses were conducted using the "meta" package of R software version 3.4.4. p value of ≤0.05 were considered significant.
The quality of the animal studies was assessed using a modified version of the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guideline criteria. The quality of the included human studies was assessed using the modified version of Newcastle-Ottawa Scale 10 which assessed the study design, sample size estimation, age and sex-matching of controls, whether imaging was performed to measure AAA size, method of AAA size assessment, adjustment for confounders, and whether the observers were blinded to group during analysis. Assessment scores with <50, 50-75, and >75% were considered to have high, moderate, and low risk of bias respectively.

RESULTS AND DISCUSSION
The initial search identified 383 studies. Based on the inclusion criteria, 321 studies were excluded due to not meeting the entry criteria. The full text of the remaining 62 studies (19 animal and 43 human) were reviewed of which ultimately 10 animal and 8 human studies were included (Figures 1 and 2).
Peri-aortic application of leptin was reported to promote a significant increase in AAA severity (diameter) in the Ang-II model (19). Leptin also promoted aortic and visceral adipose macrophage infiltration. A further study reported that perivascular implantation of white adipose tissue in obese or lean mice resulted in an increased AAA size over controls in the CaCl 2 model (20). Perivascular implantation of adipose tissue from leptin deficient mice abolished the increase in AAA diameter size that was demonstrated when adipose tissue from leptin sufficient mice was used, suggesting a role of leptin in adipose tissue induced AAA development (20).
In contrast to the above findings, germline leptin deficiency has been reported to promote the risk of AAA rupture in obese mice infused with Ang-II but AAA size was not reported in this study (21). In keeping with a role of leptin in inhibiting AAA pathogenesis, intraperitoneal injection of leptin (600 mg/kg daily) has been reported to reduce AAA diameter and risk of rupture in the Ang-II model (22). Leptin was reported to downregulate expression of the Th2 cytokine IL-4 and mRNA levels of GATA binding protein-3 (GATA-3), a key transcription factor for Th2 polarization. In addition, leptin also upregulated Th1 cytokine interferon-gamma (INF-g) and T-box binding transcription factor (T-bet), a key transcription factor for Th1 polarization (22). These results led the authors to suggest that leptin could be a potential treatment for the prevention of AAA formation.
A meta-analysis of three eligible studies suggested that leptin upregulation had no significant effect on AAA diameter (SMD: 0.50 [95% CI: −1.62, 2.61]) ( Figure 3). Overall the findings of these studies suggest that the effect of leptin depends on the method and site of up and downregulation. All four studies used the same mouse strain (C57BL/6) and experimental period but the method of leptin upregulation varied markedly which likely was the cause of the inconsistent findings. The studies reported that leptin promoted IL18 binding to inflammatory and vascular cells by upregulating the expression of IL18, the IL18-receptor, and the Na-Cl co-transporter in addition to augmenting MMP-9 synthesis (19,20,22).

Animal Studies Investigating the Effect of Adiponectin on AAA
Two studies including 64 animals investigated the effect of adiponectin on AAA development in mouse models (23,24) ( Table 1B). In high-fat fed mice with LDL-r deficiency, intravenous injection of a recombinant adenoviral vector encoding adiponectin was reported to inhibit AAA development in the Ang-II infusion model (23). Adiponectin was reported to inhibit AAA development by suppressing aortic inflammatory cell infiltration, medial degeneration, and elastin fragmentation (23). Adiponectin was also shown to inhibit the angiotensin type-1 receptor (AT1R), downregulate expression of inflammatory cytokines, mast cell protease, and adipose inflammation, and upregulate lysyl oxidase (LOX) expression in the aortic wall (23). In keeping with this, germline deletion of adiponectin has been reported to promote AAA development in the Ang-II infusion model (24). Adiponectin-deficiency augmented the early infiltration of macrophages and increased

Animal Studies Investigating the Effect of RELMb on AAA
Two studies including 60 mice in one study and an unreported number in one study investigated the role of RELMb in AAA development (25,26) (Table 1C). Both studies used the same mouse strain (ApoE −/− ) and experimental period. Germline deletion of RELMb resulted in significantly reduced AAA diameter within the Ang-II infusion model (25). This was associated with reduced macrophage accumulation and decreased expression of proinflammatory cytokines (monocyte chemoattractant protein 1 and IL6) and MMP2 and 9 in the aortic wall. Further, it has also been reported that RELMb expression was upregulated within the aortas of mice with Ang-II induced AAA compared to controls (26). These limited investigations suggest that RELMb could have a role in promoting AAA formation via induction of pro-inflammatory cytokines. Meta-analysis was not possible due to the lack of available studies.

Animal Studies Investigating the Effect of PVAT on AAA
Two studies reporting the effect of PVAT in AAA development in 54 animals were identified (27,28) (Table 1D). In mice deficient in osteopontin, angiotensin II type 1a (AT1a) deficiency of PVAT significantly attenuated the size of AAA developing, macrophage infiltration, and gelatinolytic activity in the Ang-II model (27). Another study investigated the effect of AAA development following the removal of PVAT in a hypoperfusion induced AAA rat model (28). In this model, the authors intended to isolate the oxygen supply and induce hypoxia in the infra-renal aortic region. First, the PVAT was removed from around the infra-renal aorta to prevent oxygen supply via the adventitial vasa vasorum (VV). Following this, a polyurethane catheter was inserted via a small incision in the infra-renal aorta to separate the lumen blood flow from the aortic wall, thus isolating the oxygen supply to the aortic wall from the blood directly. These two steps reduced the oxygen supply and induced hypoxia in the infra-renal aortic region without affecting the systemic circulation, promoting AAA formation. It was reported that 28 days after the removal of the PVAT the number of CD44+ and CD90+ cells and adipocytes in the AAA wall was significantly less when PVAT had been removed. Mice that has PVAT removed were reported to have significantly smaller AAA diameter than controls (28). Evidence from other animal studies has suggested that PVAT contributes to intimal hyperplasia via leptin signaling (29). Samples from

Quality of Prior Animal Studies Investigating Adipokines and PVAT in AAA
All studies reported an ethics approval statement, AAA model type and included relevant control groups ( Table 2). None of the studies reported sample size estimates. Two studies reported that the assessors were blinded during the aortic diameter and/or adipokine assessments (20,21). One study reported the reproducibility of aortic diameter assessment (20). Three studies did not report aortic diameter following the assessment period (21,26,28). One study did not report either the age or weight of the animals used in the study (26). One study did not report the number of animals included in each group (26).

Studies Investigating the Association of Adipokines With AAA Diagnosis
Five studies were identified studying the association of circulating adipokines, including DPP-4, leptin, resistin, and adiponectin, with AAA in 1146 cases compared with 837 control participants (30-34) ( Table 3). The design of the studies included retrospective cohort, case control, and prospective cohort. Two studies matched cases and controls for age (33,34) and sex (32,34). Three studies reported that controls were healthy (30,32,34). A case-control study reported that plasma DPP-4 enzyme activity was significantly increased in both large and small AAA patients compared to controls and positively correlated with AAA diameter (32) ( Table 3A). However, circulating DPP-4 plasma levels were similar between control and small AAA participants and significantly lower in people with large AAA (32). This study suggested that increased DPP-4 activity may play a role in AAA pathogenesis but larger studies are needed.
Three studies including 1,034 AAA patients and 808 controls investigated the association of circulating leptin concentrations with AAA (31,33,34) (Table 3B). A cohort sample of 701 patients from the Atherosclerotic Risk in Communities (ARIC) study suggested that plasma leptin levels were inversely associated with AAA diagnosis after adjusting for confounders including age, sex, race, smoking status, pack-years of smoking, height, hypertension, high and low density lipoproteins (HDL and LDL), total cholesterol, and peripheral artery disease, but the study did not report aortic diameter (31). A prospective cohort study showed that serum levels of leptin were similar in AAA and control participants after adjusting for age, smoking, body mass index (BMI), carotid artery stenosis, diabetes, hypertension, lipids, C-reactive protein (CRP), and statin prescription,   WHR, Waist to hip ratio; *Adipokine levels were either mentioned as mean ± S.D. or median (25 th -75 th percentile range); **Adipokine levels data extracted from graph; ᵟSub-analysis data assessed adipokines from a larger cohort; # Aortic diameter measured using ultrasound (US); $ Aortic diameter measured using Computed tomography (CT).
suggesting that leptin was not associated with AAA (33). Another population based study reported leptin levels in 952 men screened for AAA, 318 of whom had an AAA diagnosed. Results from this study suggested that leptin levels were not independently associated with AAA after adjusting for confounders including age, smoking, BMI, carotid artery stenosis, diabetes, hypertension, HDL, LDL, total cholesterol, triglycerides, CRP, and statins (34). A meta-analysis suggested that overall circulating leptin levels were higher in people diagnosed with an AAA than controls (SMD: 0.32 [95% CI: 0.19, 0.45]) ( Figure 5). It was not possible to adjust this metaanalysis for confounding risk factors. Two studies including 333 AAA patients and 808 controls investigated the association of serum resistin levels with AAA (33, 34) ( Table 3C). A prospective cohort study showed a trend toward higher resistin levels in participants with AAA compared to controls (33). Multivariate analyses showed that serum resistin levels were independently associated with AAA after adjusting for age, smoking, BMI, carotid artery stenosis, diabetes, hypertension, HDL, LDL, cholesterol, triglycerides, adiponectin, leptin, CRP, and statin medication (33). A population-based study in men found that serum resistin concentrations were independently associated with AAA after adjusting for age, dyslipidemia, hypertension, smoking, CHD, and diabetes mellitus, waist-hip ratio, and serum glucose (34). Meta-analysis of these studies suggested that serum resistin levels were significantly higher in people diagnosed with AAA compared to controls (SMD: 0.63 [95% CI 0.50, 0.76]) ( Figure 6).
Three studies including 352 AAA patients and 817 controls investigated the association of circulating adiponectin with AAA (30,33,34) (Table 3D). A prospective cohort study showed no independent association of serum levels of adiponectin with AAA after adjusting for age, smoking, BMI, carotid artery stenosis, diabetes, hypertension, lipids, CRP, and statin prescription (33). A population-based study found that serum levels of adiponectin were associated with AAA ≥30 mm, but not AAA ≥40 mm, suggesting that adiponectin may play a role in the early stages of AAA development (34). A case-control study reported that serum levels of adiponectin were significantly lower in AAA patients compared to healthy control organ donors, however, adiponectin was significantly increased in PVAT surrounding the AAA, suggesting that adipocytes surrounding the aorta may be a source of inflammatory leukocytes that are attracted by adipocytes undergoing necrosis and by proinflammatory ceramides (30). Meta-analysis suggested that serum adiponectin levels were not significantly associated with AAA diagnosis (SMD: −0.62 [95% CI −1.76, 0.52]) (Figure 7). Phenotypic changes in PVAT resulting from vascular injury have been reported to promote neointimal formation via release of adipocytokines which in turn regulate inflammation, vascular smooth muscle cell proliferation, endothelial dysfunction, fibroblast activation and migration and neovascularization (35). Furthermore, high fat diet or smoking have also been suggested to promote vascular inflammation, reactive oxygen species production, and matrix degradation that augments AAA formation (36).

Quality of Human Studies Investigating the Association of Adipokines With AAA
All studies measured adipokines in AAA cases and a comparator group ( Table 3) (30)(31)(32)(33)(34). Three studies reported the results after  adjusting for confounders (31,33,34). Three studies reported the methods used for aortic diameter imaging (30,32,34). Two studies reported the methods used for aortic diameter assessment (32,34). One study imaged both AAA cases and the comparator groups (34). One study reported the sample size estimates used (31).

Studies Examining the Association of PVAT With AAA
Three studies were identified investigating peri-aortic and visceral adipose tissue in 3,337 AAA cases compared with 382 control participants (37-39) ( Table 4). The design of the studies included retrospective, prospective, and longitudinal cohort and case-control studies. All three studies matched the AAA cases and comparators for age and sex (37)(38)(39). In a multicenter retrospective case-control study, comparison of participants with asymptomatic AAA, aortoiliac occlusive disease (AIOD), and healthy controls suggested that AAA was associated with higher PVAT density and the visceral to subcutaneous adipose tissue (VAT/SAT) ratio was significantly greater in AAA patients (37). This association persisted after adjustment for cardiovascular risk factors and other diseases. Another casecontrol study compared AAA and intermittent claudication control subjects and also reported that the visceral to total abdominal adipose volume ratio was significantly greater in participants diagnosed with AAA, but the association was abolished after adjusted for age, heart disease, diabetes, smoking, sex, and hypertension (38). In a Framingham study including 3,001 individuals, the authors reported a significant association between peri-aortic fat deposition and larger aortic dimensions after adjusting for age, sex, cardiometabolic risk factors, and BMI, further supporting the notion that fat depots present around the aorta may contribute to its remodeling (39). Meta-analysis suggested that PVAT to abdominal adipose tissue ratio was significantly associated with AAA diagnosis (SMD: 0.56 [95% CI 0.33, 0.79]) ( Figure 8).

Quality of Human Studies Investigating the Association of PVAT With AAA
All studies reported the methods used to image the aorta, methods used for aortic diameter assessment and adjusted for potential confounders during analysis ( Table 4) (37)(38)(39). Two studies reported the sample size estimate used (37,38). Only one study blinded the observer during analysis of experimental data (37).

CONCLUSIONS
Overall this systematic review suggests inconsistent effects of adipokines and PVAT on AAA development within rodent AAA models. Circulating levels of leptin and resistin and PVAT to abdominal adipose tissue ratio, but not adiponectin, were found to be significantly greater in people diagnosed with AAA compared to controls. Given the limited number of eligible animal and human studies, more evidence is needed before any robust conclusions about the role of adipokines and PVAT in AAA pathogenesis can be made. The quality assessments suggested that this past research had a moderate risk of bias.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.