https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Köln Klinik III für Innere Medizin - Experimentelle Kardiologie Köln, Deutschland; 2Herzzentrum der Universität zu Köln Klinik III für Innere Medizin Köln, Deutschland; 3Uniklinik Köln Experimentelle Kardiologie/ Innere Med. III Köln, Deutschland; 4LMU Klinikum der Universität München Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten München, Deutschland; 5Institut für Allgemeine Pathologie und Pathologische Anatomie Köln, Deutschland; 6Universitätsklinikum Düsseldorf Klinik für Gefäß- und Endovaskularchirurgie Düsseldorf, Deutschland; 7Universitätsklinikum Düsseldorf Institut für Molekulare Medizin III Düsseldorf, Deutschland; 8Augusta University Immunology Center of Georgia Augusta, GA, USA; 9Karolinska Institutet Department of Molecular Medicine and Surgery Stockholm, Schweden; 10Herzzentrum der Universität zu Köln Klinik für Kardiologie, Angiologie, Pneumologie und Internistische Intensivmedizin Köln, Deutschland
Background:
Abdominal aortic aneurysm (AAA) is a chronic, inflammatory disease characterized by dilation of the abdominal aorta above 3.5cm or 50% of its original diameter. Macrophage-mediated inflammation has been highlighted as a key contributor to maladaptive vascular remodeling in AAA. Yet, the exact mechanisms driving macrophage activation in AAA are unknown. The olfactory receptor 2 (Olfr2) is a G-protein coupled receptor involved in mediating the sense of smelling. Extra-nasal expression of Olfr2 has been identified on vascular macrophages, but its role in AAA is unknown.Methods & Results:
We performed in-silico micro array analysis of AAA tissue with and without intraluminal thrombus (ILT) of 76 patients as well as 13 controls. OR6A2 (the human ortholog of Olfr2) was significantly increased in AAA with ILT compared to control tissue. By immunofluorescence staining we detected OR6A2 on 30% of macrophages residing in human AAA.
To investigate the dynamics of Olfr2 expression on murine aortic leukocytes in AAA we induced AAA by infusion of porcine pancreatic elastase (PPE) into the infrarenal aorta of WT mice and applied spectral flow cytometry utilizing a 27-marker panel on aortic tissue at different stages of AAA development. Olfr2 was regulated by MHCII+CCR2low monocytes and macrophages peaking at day 7 post AAA induction. We next studied the functional role of Olfr2 in AAA by comparing Olfr2-deficient (KO) and WT control mice after aneurysm induction. Ultrasound analysis revealed marked protection from aneurysm formation in KO mice which coincided with preserved elastin structure and higher collagen content. Additionally, FACS analysis of AAA tissue at day 7 and day 28 post PPE revealed a significant reduction of monocytes and macrophages in KO aortae.
To evaluate the effects of pharmacological modulation of Olfr2 on AAA, we injected WT mice with the Olfr2 antagonist citral or the agonist octanal. Citral treated mice showed reduced aortic dilation and macrophage content compared to control mice whereas octanal induced the inverse phenotype.
We further assessed the recruitment of WT and KO monocytes in AAA, by analyzing chemokine receptor expression on Ly6Chigh monocytes in the blood. FACS analysis revealed a significant reduction of CX3CR1 on Olfr2-/- monocytes compared to WT. To determine whether reduced CX3CR1 expression affects the migratory response, we performed a chemotaxis assay. Isolated KO monocytes showed impaired migration towards the CX3CR1 ligand CX3CL1 compared to WT. Next, we tested the adhesion of monocytes to activated endothelium in an ex-vivo assay. Carotid arteries of WT mice were stimulated with TNFα and perfused with labeled WT and KO monocytes 1:1 in a perfusion chamber. Adherent monocytes were imaged under a 2-photon-microscope. We detected significantly less KO monocytes adhering to the endothelium.
To evaluate these findings in our in-vivo model, we adoptively transferred labeled WT and KO monocytes into WT mice at day 3 post PPE. FACS analysis 24h post injection revealed reduced aortic infiltration of injected KO monocytes compared to WT.
Finally, gene set enrichment analysis of bulk RNA sequencing data of sorted splenic Ly6Chigh monocytes at day 7 post PPE showed positive enrichment for TNFα signaling and Interferon-γ response pathways in WT monocytes compared to KO.
Conclusion:
We highlight a novel mechanism by which Olfr2 promotes AAA development by augmenting CX3CR1-mediated monocyte recruitment.