Disturbed blood flow up-regulates vascular CCRL2 expression that promotes leucocyte adhesion and plaque formation. (A) CCRL2 mRNA in the aortic arch (AA) and descending aorta (DA) of C57BL/6J mice was assessed by RT–qPCR (n = 5 per group). **P < 0.01 by unpaired Student’s t-test. (B) CCRL2 protein levels in the aortic arch and descending aorta of WT mice were measured by IF staining with anti-CCRL2 and anti-CD31 antibodies. Bar = 20 μm. (C) CCRL2 protein expression in the GC and LC of the aortic arch in WT mice was detected by en face staining. (red, CCRL2; green, CD31; blue, DAPI). Bar = 100 μm. (D and E) HUVECs were treated for 24 h by OS or LS with a cone-and-plate shear stress device. The expression of CCRL2 was examined by RT–qPCR (D) and Western blotting (E) (n = 3 per group). Full blots can be found in the Supplementary material online, Figure S8B. **P < 0.01 by unpaired Student’s t-test. (F) To induce d-flow, the left carotid artery (LCA) of WT or CCRL2^−/− mice was partially ligated. Total carotid RNA before and 24 h after ligation was isolated and analysed for CCRL2 mRNA expression by RT–qPCR, normalized to GAPDH, and expressed as the ratio of ligated LCA to non-ligated (RCA) control (n = 5 per group). ***P < 0.001 by unpaired Student’s t-test. (G) Immunofluorescence staining was performed by en face staining of the carotid intima (red, CCRL2; green, CD31; blue, DAPI). Bar = 20 μm. (H and I) Chimeric mice were generated by transplanting CCRL2^+/+ GFP⁺ bone marrow into irradiated CCRL2^+/+ and CCRL2^−/− recipients. After 6–8 weeks to allow engraftment and haematopoietic reconstitution, d-flow was induced in the mouse LCA by PCL. After 2 days or 7 days, leucocyte adhesion to the vascular endothelium of the LCA was assessed by en face staining. Green, GFP; red, CD31; Bar = 20 μm. The number of adherent GFP⁺ leucocytes (H) or the area of adherent GFP⁺ leucocytes (I) on the vessel wall was quantified in 10–17 fields from 3–4 mice. ***P < 0.001, ****P < 0.0001 by unpaired Student’s t-test. (J) CCRL2^+/+ApoE^−/− and CCRL2^−/−ApoE^−/− mice were subjected to PCL. After 2 weeks on HFD, lipid deposition (in red) on mouse LCA was analysed by en face Sudan IV staining. Bar = 1 mm. The lesion surface area was quantified and displayed as the percentage area of the LCA. **P < 0.01 by unpaired Student’s t-test. n = 7 mice per group. (K) Male CCRL2^−/−ApoE^−/− or CCRL2^+/+ApoE^−/− mice were lethally irradiated and transplanted with CCRL2^+/+ApoE^−/− mouse bone marrow cells. Plaque formation in chimeric mice induced by PCL and HFD. *P < 0.05 by unpaired Student’s t-test. n = 6–7 mice per group.