Nitrated fatty acids as a promising pharmacological treatment for abdominal aortic aneurysm

P. Arkenberg (Köln)¹, M. Stei (Bonn)², T. Uebing (Düsseldorf)³, D. Mehrkens (Köln)⁴, A. Busch (Dresden)⁵, M. Adam (Köln)⁴, V. Hoerr (Bonn)⁶, G. Sengle ( Cologne)⁷, T. Riffelmacher (Köln)⁴, H. Winkels (Köln)⁴, G. Nickenig (Bonn)⁶, M. Kelm (Düsseldorf)⁸, S. Baldus (Köln)⁹, M. Wagenhäuser (Düsseldorf)¹⁰, M. Mollenhauer (Köln)^4
1Universitätsklinikum Köln Klinik III für Innere Medizin - Experimentelle Kardiologie Köln, Deutschland; ²Universitätsklinikum Bonn Molekulare Kardiologie // Geb. 370 Bonn, Deutschland; ³Universitätsklinikum Düsseldorf Klinik für Gefäß- und Endovaskularchirurgie Düsseldorf, Deutschland; ⁴Herzzentrum der Universität zu Köln Klinik III für Innere Medizin Köln, Deutschland; ⁵Viszeral-, Thorax- und Gefäßchirurgie Gefäß- und Endovaskuläre Chirurgie Dresden, Deutschland; ⁶Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; ⁷Faculty of Medicine and University Hospital of Cologne, University of Cologne Centre for Biochemistry Cologne, Deutschland; ⁸Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ⁹Herzzentrum der Universität zu Köln Klinik für Kardiologie, Angiologie, Pneumologie und Internistische Intensivmedizin Köln, Deutschland; ¹⁰Universitätsklinikum Düsseldorf Düsseldorf, Deutschland

Background: The abdominal aortic aneurysm (AAA) is a multifactorial disease with a high prevalence in individuals over 65 years of age, associated with significant mortality and morbidity. There is no specific pharmacological approach that directly targets AAA, and surgery remains the only treatment option. The nitrated fatty acid OANO2 is formed endogenously and has no known side effects. OANO2 is a highly pleiotropic compound that exhibits anti-inflammatory, anti-oxidative and anti-fibrotic effects through direct and indirect interactions with various signaling pathways. In this study, we explored OANO2 as a potential pharmacological treatment for AAA in mice and additionally elucidated the signal pathway modulated by OANO2. Methods: AAA was induced in wild-type (wt) mice via surgery. The abdominal aorta was ligated, blood flow interrupted and filled with porcine-pancreas-elastase (PPE) for five minutes. Mice were treated with OANO2 for 6 days to assess acute inflammation or 4 weeks for tissue remodeling and fibrosis. A control group was treated with polytethylen glycol (PEG). Treatment was administered via Alzet mini osmotic pumps, starting 3 days before (prophylactic) or after (therapeutic) AAA induction. Ultrasounds were performed to monitor surgery effects, aneurysm induction and growth. Freshly isolated aortas were used for flow cytometric analysis. Snap-frozen aortas were used for proteome and kinome analysis or embedded for histological analysis. In cell culture, the mRNA expression of proinflammatory cytokines in HL-60 cells was assessed in both groups, and a migration assay was performed. Results: In both the prophylactic and therapeutic settings, the AAA diameter in OANO2-treated mice was significantly smaller after 28 days of treatment (p=0.042; p<0.001) compared to the control group. In the prophylactic setting, elastin grading (p=0.023) and the number of smooth muscle cells (p=0.046) in OANO2-treated mice were significantly higher as compared to PEG-treated animals. In the therapeutic setting, OANO2-treated mice showed significantly lower immune cell recruitment, as indicated by CD45 (p=0.018) and CD68 (p=0.028) staining. Flow cytometric analysis demonstrated a significant reduction in myeloid cells (p=0.015), monocyte-derived macrophages (p=0.008), monocytes (p=0.027), tissue macrophages (p=0.04) and neutrophils (p=0.006) within the aortic tissue following three days of OANO2 treatment, as compared to the untreated controls. Cell culture experiments revealed significant downregulation of IL1b (p=0.001) and RANTES (p=0.015) mRNA expression as well as a significant lower migration of HL-60 cells (p<0.0001) after OANO2 treatment. Proteomic and GO-term analysis identified the downregulation of proteins involved in immune response, acute phase response and immune cell response in OANO2-treated mice. Kinome analysis showed a downregulation of tyrosine kinases after OANO2 treatment, while further analysis showed a high influence of OANO2 in the Pi3K/Akt-Pathway (p<0.001) as the underlying mechanism of the anti-inflammatory effect. Conclusions: We have shown that OANO2 is a promising pharmacological treatment option in both prophylactic and therapeutic AAA mouse models as it maintains aortic wall stability and reduces immune cell recruitment. Moreover, we identified the novel and remarkable finding that OANO2 modulates the Pi3K/Akt-Pathway, thereby mediating its anti-inflammatory activity.