Olfactory receptor 2 signaling drives abdominal aortic aneurysm

Patrik Schelemei (Köln)1, F. S. R. Picard (Köln)1, H. Nemade (Köln)1, D. Mehrkens (Köln)2, S. Grimm (Köln)2, K. Tinaz (Köln)2, E. Wagner (Köln)3, W. Kreuzberg (Köln)1, M. Orecchioni (Augusta)4, M. Wagenhäuser (Düsseldorf)5, H. Schelzig (Düsseldorf)5, M. L. Liljeqvist (Solna)6, J. Roy (Solna)6, S. Baldus (Köln)7, M. Mollenhauer (Köln)2, H. Winkels (Köln)2

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 Köln, Deutschland; 4Augusta University Immunology Center of Georgia Augusta, USA; 5Universitätsklinikum Düsseldorf - Klinik für Gefäß- und Endovaskularchirurgie Düsseldorf, Deutschland; 6Karolinska Institutet Department of Molecular Medicine and Surgery Solna, Schweden; 7Herzzentrum der Universität zu Köln Klinik für Kardiologie, Angiologie, Pneumologie und Internistische Intensivmedizin Köln, Deutschland


Abdominal aortic aneurysm (AAA) is defined as localized enlargement of the aorta above 3cm or 50% of its original diameter. It is characterized by degradation of the extra cellular matrix driven by smooth muscle cell death, elastin fragmentation and fibrosis.  Current treatment options are only limited to open surgical or endovascular intervention, while pharmacological treatment to prevent its progression is lacking.
Particularly macrophage mediated inflammation of the vessel wall has been associated with disease progression, leading to extensive tissue remodeling, yet the underlying mechanisms are incompletely understood.
A recent study has shown extra-nasal expression of the olfactory receptor 2 (Olfr2) on vascular macrophages, in which activation induced a pro-atherogenic response.
The role of macrophage Olfr2 signaling in AAA, however, is unknown.

Methods & Results:
To determine whether the human Olfr2 ortholog OR6A2 is expressed in human AAA, we performed immunofluorescence stainings of human AAA sections. Co-localization of OR6A2 with the CD68 macrophage marker indicated expression on human AAA macrophages.  Additionally, in silico micro-array analysis of human AAA tissue has shown OR6A2 to be specifically upregulated in AAA tissue with thrombus formation compared to healthy control tissue.

We further investigated the dynamics of Olfr2 in AAA, by inducing AAA by porcine pancreas (PPE) infusion into the infrarenal aorta of wild type (WT) mice and performing spectral flow cytometry (FACS) utilizing a 27-marker panel at day 7, day 28 as well as sham conditions.
Olfr2 peaked at day 7 and was reduced to baseline levels at day 28 particularly on MHCII+CCR2low vascular monocytes and MHCII+CCR2low macrophages. Other monocyte and macrophage clusters did not regulate or express Olfr2.
To test for the functional relevance of OLFR2 deficiency on AAA, we further induced AAA in OLFR2 knock-out (KO) mice and WT controls. Olfr2 KO mice were widely protected from AAA progression measured via ultrasound. Furthermore, elastin degradation was reduced while collagen content, measured via second harmonics generation in the 2-photon-microscopy, was increased in the KO group. Additionally, immunofluorescence analysis showed significantly less macrophages while maintaining higher alpha-smooth muscle actin (ASMA) content in the KO mice compared to WT. Plasma cytokine levels, including Interferon-ɣ, IL-6 and TNF-⍺ were also reduced in Olfr2 KO mice at day 28 after aneurysm induction.

Transcriptomically, gene set enrichment analysis of bulk RNA sequencing data of KO aortae versus WT controls at day 7 after PPE showed highly upregulated pathways associated with vascular smooth muscle cell contractility while leukocyte activation was downregulated.
This finding was corroborated by FACS analysis of vascular leukocytes of Olfr2 WT and KO mice at day 7 and day 28 showing decreased overall leukocyte numbers at both time points in the KO group. Particularly macrophages, Ly6Chigh monocytes, dendritic cells, B cells and T cells where reduced in KO aortae whereas neutrophils were unaffected. Unexpectedly, Ly6Clow monocytes were shown to be increased in KO aortae at day 7 after PPE compared to WT.

We show that OLFR2 deficiency in mice protects from abdominal aortic aneurysm development and aortic remodeling by reducing overall vascular inflammation.

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