Cigarette smoking upregulates vascular expression of the novel atherosclerosis risk factor ADAMTS-7 via CCL17-CCR4 signaling

C. Abrahamian (München)¹, A. Sharifi (München)¹, J. Riechel (München)¹, H. Winter (München)², T. A. Dang (München)¹, S. Kraut (Gießen)³, S. Hadzic (Gießen)³, C. Nöcker (München)¹, J. Hinterdobler (München)¹, J. Werner (München)¹, N. Panyam (München)¹, C. Gräßer (München)¹, M. Gredic (Gießen)³, C.-Y. Wu (Gießen)³, Z. Aherrahrou (Lübeck)⁴, Y. Döring (Bern)⁵, L. Maegdefessel (München)⁶, N. Weißmann (Gießen)⁷, H. Schunkert (München)¹, H. Sager (München)¹, T. Keßler (München)^1
1Deutsches Herzzentrum München Klinik für Herz- und Kreislauferkrankungen München, Deutschland; ²Technische Universität München (TUM) Klinik und Poliklinik für vaskuläre und endovaskuläre Chirurgie München, Deutschland; ³Universitätsklinikum Gießen und Marburg GmbH Medizinische Klinik II - Pneumologie Gießen, Deutschland; ⁴Universitätsklinikum Schleswig-Holstein Institut für Kardiogenetik Lübeck, Deutschland; ⁵Inselspital - Universitätsspital Bern Universitätsklinik für Angiologie Bern, Schweiz; ⁶Klinikum rechts der Isar der Technischen Universität München Klinik für Vaskuläre und Endovaskuläre Chirurgie München, Deutschland; ⁷Justus-Liebig-Universität Giessen Medizinische Klinik II Gießen, Deutschland

Background: Cigarette smoking is an established risk factor for coronary artery disease (CAD) and myocardial infarction. The gene encoding the extracellular matrix protease ADAMTS-7 was recently identified as a genetic risk factor for CAD. Cigarette smoking and the ADAMTS7 risk allele represent the thus far only known gene-environment interaction in CAD. The molecular and cellular mechanisms, however, remain unknown.
 
Methods and Results: We used an in vivo cigarette smoking (CS) model. Secondary to CS, wild type (WT) mice displayed an upregulation of aortic ADAMTS7 expression which was also found in carotid plaques from ever-smokers undergoing carotid endarterectomy, as compared to WT mice exposed to room air (RA) or never-smokers, respectively. We next performed bulk RNA sequencing of lung tissues from WT mice exposed to CS or RA. CS was associated with a downregulation of 20 and an upregulation of 173 transcripts. Among the latter, we found C-C motif chemokine ligand 17 (CCL17) to also be detected at higher levels in plasma using a cytokine profiler. We then investigated the influence of CCL17 on primary vascular smooth muscle (VSMC) cells in vitro. Recombinant CCL17 upregulated ADAMTS7 expression in primary VSMC as compared to vehicle. Using RNA interference, we silenced the expression of CCL17’s bona fide receptor, i.e., C-C Motif Chemokine Receptor 4 (CCR4). As compared to a scrambled small interfering RNA, after downregulating CCR4 ADAMTS7 mRNA levels remained unchanged secondary to incubation of primary VSMC with CCL17, indicating that upregulation of ADAMTS7 expression by CCL17 is mediated via CCR4. We further evaluated vascular inflammation in proatherogenic Apoe-/- mice exposed to RA or CS and Apoe-/-Adamts7-/- mice exposed to CS using flow cytometry of aortic cell suspensions. Secondary to exposure to CS, we found more numerous neutrophils, Ly6Chi monocytes, and macrophages in aortic cell suspensions of Apoe-/- mice exposed to CS as compared to RA. In contrast, numbers of leukocytes in Apoe-/-Adamts7-/- mice exposed to CS were comparable to those of Apoe-/- mice exposed to RA.
 
Conclusions: Our data provide a mechanistic explanation of the gene-environment interaction between ADAMTS7 and smoking. Upregulation of the inflammatory cytokine CCL17 in lung tissue secondary to smoking contributes to enhanced vascular expression of ADAMTS7 by canonical CCR4 signaling. While cessation of CS represents an effective and important strategy in primary and secondary prevention, inhibition of ADAMTS-7 might be a promising therapeutic option in both smokers and non-smokers.