Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1β and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.

The specialized pro-resolving lipid mediator maresin 1 (MaR1) is involved in the resolution phase of tissue inflammation. It was hypothesized that exogenous administration of MaR1 would attenuate abdominal aortic aneurysm (AAA) growth in a cytokine-dependent manner via LGR6 receptor signaling and macrophage-dependent efferocytosis of smooth muscle cells (SMCs). AAAs were induced in C57BL/6 wildtype (WT) mice and smooth muscle cell specific TGF-β2 receptor knockout (SMC-TGFβr2 −/− ) mice using a topical elastase AAA model. MaR1 treatment significantly attenuated AAA growth as well as increased aortic SMC α-actin and TGF-β2 expressions in WT mice, but not SMC-TGFβr2 −/− mice, compared to vehicle-treated mice. In vivo inhibition of LGR6 receptors obliterated MaR1-dependent protection in AAA formation and SMC α-actin expression. Furthermore, MaR1 upregulated macrophage-dependent efferocytosis of apoptotic SMCs in murine aortic tissue during AAA formation. In vitro studies demonstrate that MaR1-LGR6 interaction upregulates TGF-β2 expression and decreases MMP2 activity during crosstalk of macrophage-apoptotic SMCs. In summary, these results demonstrate that MaR1 activates LGR6 receptors to upregulate macrophage-dependent efferocytosis, increases TGF-β expression, preserves aortic wall remodeling and attenuate AAA formation.Therefore, this study demonstrates the potential of MaR1-LGR6-mediated mitigation of vascular remodeling through increased efferocytosis of apoptotic SMCs via TGF-β2 to attenuate AAA formation. K E Y W O R D Saorta, aneurysm, efferocytosis, macrophage, maresin, smooth muscle cells, transforming growth factor beta 2 2 of 13 | ELDER Et aL.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. Resolvin D1 (RvD1) is derived from ω-3 polyunsaturated fatty acids and is involved in the resolution phase of chronic inflammatory diseases. The aim of this study was to decipher the protective role of RvD1 via formyl peptide receptor 2 (FPR2) receptor signaling in attenuating abdominal aortic aneurysms (AAA). The elastase-treatment model of AAA in C57BL/6 (WT) mice and human AAA tissue was used to confirm our hypotheses. Elastase-treated FPR2 −/− mice had a significant increase in aortic diameter, proinflammatory cytokine production, immune cell infiltration (macrophages and neutrophils), elastic fiber disruption, and decrease in smooth muscle cell αactin expression compared to elastase-treated WT mice. RvD1 treatment attenuated AAA formation, aortic inflammation, and vascular remodeling in WT mice, but not in FPR2 −/− mice. Importantly, human AAA tissue demonstrated significantly decreased FPR2 mRNA expression compared to non-aneurysm human aortas. Mechanistically, RvD1/FPR2 signaling mitigated p47 phox phosphorylation and prevented hallmarks of ferroptosis, such as lipid peroxidation and Nrf2 translocation, thereby attenuating HMGB1 secretion. Collectively, this study demonstrates RvD1-mediated immunomodulation of FPR2 signaling on macrophages to mitigate ferroptosis and HMGB1 release, leading to resolution of aortic inflammation and remodeling during AAA pathogenesis.

Background: Our previous studies showed that neutrophil infiltration and activation plays an important role in the pathogenesis of abdominal aortic aneurysms (AAA). However, there is a lack of noninvasive, inflammatory cell-specific molecular imaging methods to provide early diagnosis of AAA formation. Formyl peptide receptor 1 (FPR1) is rapidly upregulated on neutrophils during inflammation. Therefore, it is hypothesized that the use of cinnamoyl-F-(D)L-F-(D)L-F-K (cFLFLF), a PEGylated peptide ligand that binds FPR1 on activated neutrophils, would permit accurate and noninvasive diagnosis of AAA via singlephoton emission computed tomography (SPECT) imaging.Materials and methods: Male C57BL/6 (wild-type) mice were treated with topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat-inactivated elastase (control), and aortic diameter was measured by video micrometry. Comparative histology was performed on Day 14 to assess neutrophil infiltration in aortic tissue. We performed near-infrared fluorescence imaging using c-FLFLF-Cy7 probe on Days 7 and 14 postelastase treatment and measured fluorescence intensity ex vivo in excised aortic tissue. A separate group of animals were injected with 99m Tc-c-FLFLF 2 h before SPECT imaging on Day 14 using a SPECT/computed tomography/positron emission tomography trimodal scanner. Coexpression of neutrophils with c-FLFLF was also performed on aortic tissue by immunostaining on Day 14.Results: Aortic diameter was significantly increased in the elastase group compared with controls on Days 7 and 14. Simultaneously, a marked increase in neutrophil infiltration and elastin degradation as well as decrease in smooth muscle integrity were observed in aortic tissue after elastase treatment compared with controls. Moreover, a significant increase in fluorescence intensity of c-FLFLF-Cy7 imaging probe was also observed in elastase-treated mice on Day 7 (approximately twofold increase) and Day 14 (approximately 2.5-fold increase) compared with respective controls. SPECT imaging demonstrated

Introduction: Resolvin D1 (RvD1) is derived from a ω-3 polyunsaturated fatty acid and is involved in the resolution phase of inflammation during abdominal aortic aneurysm (AAA) formation. Hypothesis: The aim of this study was to decipher the protective role of RvD1 via formyl peptide receptor 2 (FPR2) receptor signaling in attenuating AAA formation. Methods: C57BL/6 (WT) and FPR2 -/- mice (8-12 week old) underwent treatment with topical elastase or heat-inactivated elastase (control). Separate groups of mice were administered RvD1 (100ng/kg) or saline i.p. daily from day(d)1 to d13 and aortic diameter was measured by video micrometry on d14. mRNA quantification of FPR2 expression in aortic tissue was performed by RT-PCR on d7, 14 and 21. Human aortic tissue from AAA patients and controls (organ transplant donors) were analyzed for quantification of FPR2 expression. Data is presented as mean ± SEM and statistical analysis was performed by using Mann-Whitney test for phenotype or Wilcoxon Signed Rank Test for gene expression data. Results: Elastase-treated WT mice had significantly increased aortic diameter compared to controls (127±8% vs. 1± 0.6%; p<0.0001). Importantly, elastase-treated FPR2 -/- had a significant increase in aortic diameter compared to elastase-treated WT mice (165±16% vs. 127± 8%; p=0.03). RvD1 treatment attenuated AAA formation in WT mice, but not FPR2 -/- mice, on d14 (56± 10% vs.121± 8%; p<0.0001). Immunohistology documented decreased elastin and smooth muscle cell α-actin expression, as well as increased neutrophil and macrophage infiltration in FPR2 -/- mice compared to WT mice, after respective treatments with RvD1. Elastase-treated WT mice had significantly increased FPR2 receptor mRNA expression compared to respective controls on d7, d14, and d21. Human AAA tissue demonstrated significantly decreased FPR2 mRNA expression compared to controls (p=0.02). Conclusions: This data identifies the role of FPR2 in dysregulation of inflammation-resolution during AAA pathogenesis. Immunomodulation of FPR2 signaling by exogenous RvD1 identifies a novel mechanism in resolution of aortic inflammation and vascular remodeling during AAA pathogenesis.