Elevated Adiponectin Levels Suppress Perivascular and Aortic Inflammation and Prevent AngII-induced Advanced Abdominal Aortic Aneurysms

Abstract

Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by aortic dilation and rupture leading to sudden death. Currently, no non-surgical treatments are available and novel therapeutic targets are needed to prevent AAA. We investigated whether increasing plasma levels of adiponectin (APN), a pleiotropic adipokine, provides therapeutic benefit to prevent AngII-induced advanced AAA in a well-established preclinical model. In the AngII-infused hyperlipidemic low-density lipoprotein receptor-deficient mouse (LDLR^-/-) model, we induced plasma APN levels using a recombinant adenovirus expressing mouse APN (AdAPN) and as control, adenovirus expressing green florescent protein (AdGFP). APN expression produced sustained and significant elevation of total and high-molecular weight APN levels and enhanced APN localization in the artery wall. AngII infusion for 8 weeks induced advanced AAA development in AdGFP mice. Remarkably, APN inhibited the AAA development in AdAPN mice by suppressing aortic inflammatory cell infiltration, medial degeneration and elastin fragmentation. APN inhibited the angiotensin type-1 receptor (AT1R), inflammatory cytokine and mast cell protease expression, and induced lysyl oxidase (LOX) in the aortic wall, improved systemic cytokine profile and attenuated adipose inflammation. These studies strongly support APN therapeutic actions through multiple mechanisms inhibiting AngII-induced AAA and increasing plasma APN levels as a strategy to prevent advanced AAA.

Figure 1. Adiponectin expression and plasma adiponectin levels in AngII-infused LDLR^−/− mice fed high-fat diet and injected AdAPN or AdGFP adenovirus during 8 weeks.

(a) Plasma adiponectin levels at 8 weeks after injection measured by ELISA. (AdGFP n = 14 and AdAPN n = 10/group, *p < 0.001 Student’s t-test. (b) Western blot analysis of plasma APN levels at 8 weeks of adiponectin expression quantified by densitometry with NIH ImageJ software. (c) Distribution of circulating APN oligomeric forms quantified by gel electrophoresis, HMW, high molecular weight, MMW, medium molecular weight, LMW, low molecular weight. (d) AngII infusion suppressed adipose APN mRNA expression which was not affected by adenoviral APN expression in hyperlipidemic LDLR^−/− mice (n = 8/group), *p < 0.05 Student’s t-test (e) Immunolocalization of APN in abdominal aorta (red arrows) from AngII-infused AdGFP or AdAPN mice. Mice infused with PBS were used as control (n = 8). Abdominal aortic cross-sections represented in boxes are shown at higher magnification. Scale bar = 50 μm.

Figure 2. Adiponectin elevation inhibits AngII-induced AAA formation.

(a) Representative photographs showing macroscopic view of AAA induced by AngII infusion (red arrow). (b) Luminal diameter of abdominal aortas. Control n = 8, AdGFP n = 12, AdAPN n = 8, **p < 0.01 vs AdGFP and AdAPN, *p < 0.05 vs AdGFP, ANOVA (c) Outer aortic diameter of abdominal aortas. Control n = 8, AdGFP n = 12, AdAPN n = 8 **p < 0.01 vs AGFP and AdAPN, *p < 0.05 vs AdGFP, ANOVA. (d) Percent of AngII-infused LDLR^−/− mice injected AdAPN or Ad-GFP that developed advanced AAA. *p < 0.05 vs AdGFP (e) Sudan IV-stained en face aortic preparations showing abdominal atherosclerotic lesions and aneurysm. (f) Representative cross-sections of abdominal aortas from AngIIAdGFP and AngIIAdAPN showing atherosclerosis (magnification 10X).

Figure 3. Adiponectin expression inhibited AngII-induced elastin degradation, infiltration of inflammatory cells and preserved vascular smooth muscle cells in the abdominal aorta of hyperlipidemic LDLR^−/− mice.

(a) Elastin staining using Verhoeff’s van Geison stain in representative abdominal aorta cross-sections from control (PBS) and AngII-infused and AdGFP or AdAPN injected mice after 8 weeks. (b) Quantification of elastin degradation in abdominal aorta of control (PBS-infused, n = 8) and AngII-infused and AdGFP (n = 12) or AdAPN (n = 8) injected mice after 8 weeks. Results are presented in box plot using median (line) with the 25th and 75th percentiles. T-bars indicate outliers 1.5 times the box height and the circles 1.5 times the interquartile range. **p < 0.01 vs AdGFP and Control, *p < 0.05 vs AdAPN, ANOVA. (c) Representative immunostained images of vascular smooth muscle cells (SMC22α) localization in the abdominal aortic cross-sections from control (PBS-infused) and AngII-infused and AdGFP or AdAPN injected mice after 8 weeks. Immunolocatization of macrophages (MOMA2) (d) and T-lymphocytes (CD3e) (e) in abdominal aorta from AngII-infused and AdGFP or AdAPN injected mice after 8 weeks. Infiltration of inflammatory cells is detected only in AngII-infused mice and not in PBS-infused mice. Red arrows represent macrophage-rich (d) and T-lymphocyte-positive (e) areas. Areas of abdominal aortic cross-sections (a,c,d,e) represented in boxes are shown at higher magnification. Scale bar = 50 μm.

Figure 4. Adiponectin regulates AngII-mediated inflammatory and extracellular matrix gene expression in the aneurysmal wall.

Gene expression analysis of angiotensin type 1a receptor (AT1aR), MMP9, mast cell chymase, mast cell tryptase, smooth muscle cell marker (SM22α), T-lymphocyte marker (CD3e), collagen, α-actin and lysyl oxidase (LOX) in abdominal aorta of control (PBS-infused) (n = 8) and AngII-infused AdGFP (n = 12) or AdAPN (n = 8)-injected mice after 8 weeks after AngII infusion. AT1aR analyzed with n = 5/group. RNA samples from abdominal aorta were analyzed by QRT-PCR and normalized to TBP. *p < 0.05 vs Control, **p < 0.01 vs AdGFP and Control, ANOVA.

Figure 5. Adiponectin expression improves systemic inflammatory cytokine/chemkine profile.

Circulating cytokine/chemokine and growth factor levels in AngII-infused hyperlipidemic LDLR^−/− mice, 8 weeks after injection of AdGFP or AdAPN. Plasma samples pooled from individual mice from AdGFP (n = 9) and AdAPN (n = 8) group were analyzed by multiplex ELISA immunoassay. *p < 0.05 vs AdGFP, Student’s t-test.

Figure 6. Adiponectin inhibits perivascular (abdominal) and visceral adipose inflammatory cell infiltration, renin-angiotensin system component and inflammatory cytokine/chemokine expression.

Gene expression analysis of adiponectin (APN), angiotensin type 1a receptor (AT1aR), angiotensin-converting enzyme (ACE), macrophage marker (CD68), T-cell marker (CD4), Tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2) in perivascular adipose tissue (surrounding abdominal aorta) of AngII-infused mice 8 weeks after injection of AdGFP or AdAPN (n = 8/group). RNA samples from abdominal perivascular adipose tissue aorta were analyzed by QRT-PCR and normalized to GAPDH. *p < 0.05 vs AdGFP, Student’s t-test.

Figure 7. Schematic showing potential mechanisms by which APN inhibits AngII-induced advanced AAA development.

This study shows that the protective APN actions are mediated by multiple mechanisms including inhibition of the RAS components, inflammatory cell infiltration, vascular SMC degeneration and elastic fragmentation in the aneurysmal wall. In addition, APN suppressed perivascular and visceral adipose inflammation. (Mφ, macrophage, ↑ increase, ↓ decrease).