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

Abstract 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 ADAMTS-7 risk allele represent the thus far only known gene-environment interaction in CAD. The molecular and cellular mechanisms, however, remain unknown. Methods and Results Our analysis of carotid plaques revealed significantly elevated expression of the extracellular matrix protease ADAMTS-7 in ever-smokers compared to never-smokers. Similarly, cigarette smoke (CS) exposure in vivo in C57BL/6 mice led to increased aortic ADAMTS-7 expression on both mRNA and protein levels compared to mice maintained in room air (RA). We then performed bulk RNA sequencing of lung tissues from these groups, identifying 20 downregulated and 173 upregulated transcripts associated with CS. Notably, among the upregulated transcripts was C-C motif chemokine ligand 17 (CCL17), which was also elevated in plasma, as confirmed by a cytokine profiler assay. To explore the vascular effects of CCL17 in vitro, we treated primary vascular smooth muscle cells (VSMCs) with recombinant CCL17 which led to a significant increase in ADAMTS-7 mRNA expression in these cells. We next employed RNA interference to silence the expression of CCR4, the bona fide receptor for CCL17. Silencing CCR4 abolished the effect of CCL17 on ADAMTS-7 expression, indicating that CCL17 upregulates ADAMTS-7 via CCR4. Then, we collected supernatants from ADAMTS-7 KD and scramble KD VSMCs treated with CCL17 and incubated them with endothelial cells. Supernatants from CCL17-stimulated ADAMTS-7 KD VSMCs reduced leukocyte adhesion to endothelial cells and the mRNA levels of key adhesion molecules, i.e., VCAM1, ICAM1, SELE, and CDH5 compared to the scramble control. We further evaluated vascular inflammation in proatherogenic Apoe-/-mice exposed to RA or CS and in Apoe-/-Adamts-7-/- mice exposed to CS, using flow cytometry of aortic cell suspensions. CS exposure increased numbers of neutrophils, Ly6Chi monocytes, and macrophages in Apoe-/- mice compared to RA controls. In contrast, Apoe-/- Adamts-7-/- mice exposed to CS showed leukocyte and macrophage levels comparable to Apoe-/- mice in RA, highlighting the role of ADAMTS-7 in mediating CS-induced vascular inflammation. Conclusion Our data provide a mechanistic explanation of the gene-environment interaction between ADAMTS-7 and smoking. Upregulation of the inflammatory cytokine CCL17 in lung tissue secondary to smoking contributes to enhanced vascular expression of ADAMTS-7 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.