(A) Neointima sizes, as quantified in Movat-stained sections of carotid arteries 4 weeks after wire-injury in Apoe^−/− mice depleted of neutrophils for indicated periods. Representative images show neointima formation in mice with intact white blood cell count (WBC, top) and in neutropenic mice (bottom). n=6–8. *p<0.05 versus control mice. Kruskal-Wallis-test with posthoc-Dunn-test. (B) Immunohistochemical analysis of neointima one week after wire-injury in mice with intact white blood cell count (WBC) or with neutropenia. Displayed are neointimal area (left), Mac-2⁺ macrophage content (middle), and luminal coverage with CD31⁺ endothelium (right), and representative images (lower panel). Green fluorescence stems from secondary FITC-conjugated antibody, red fluorescence derives from secondary Cy3-conjugated antibody. n=7–9. *p<0.05 versus intact WBC. Mann-Whitney test. (C,D) Neointima size (C) and endothelial coverage (D) in Apoe^−/−, beigeApoe^−/−, Dppi^−/−Apoe^−/−, WT→Apoe^−/−, and Cramp^−/− →Apoe^−/− mice one week after wire-injury. Experiments were performed in mice with intact WBC or neutropenia. Representative images show neointima formation and CD31 staining in WT→Apoe^−/−, and Cramp^−/−→Apoe^−/− mice one week after wire-injury. n=6–8. *p<0.05 versus Apoe^−/− or WT→Apoe^−/− with intact WBC. Mann-Whitney test (left), Kruskal-Wallis-test with posthoc-Dunn-test (right). Scale bars, 100 μm.

(A) Adhesion of neutrophils to injured carotid arteries of monocyte-depleted Lysm^egfp/egfpApoe^−/− mice. Recordings were taken at different time points after injury and quantifications are given as percent fluorescent of total arterial area. Representative images indicate neutrophil adhesion at indicated time points. n=4–5. (B) In vivo detection of CRAMP along injured carotid arteries of mice with intact white blood cell count (WBC) or neutropenia, as detected by protein G-coupled fluorescent beads conjugated with an anti-CRAMP antibody. The number of immobilized beads was quantified. n=4. *p<0.05 versus intact WBC. Mann-Whitney-test. (C) anti-CRAMP bead complexes were detected luminally using intravital 2-photon microscopy. Blue fluorescence indicating collagen in the arterial wall originates from second harmonic generation, green fluorescence derives from beads conjugated with antibodies to CRAMP. (D) Surface binding of LL-37 to human aortic endothelial cells (HAoECs) assessed by FACS analysis. Untreated HAoECs (ctrl), HAoECs pretreated with heparinase (Hep, 50U/ml), chondroitinase (Chon, 20U/ml), or both, activated with TNF (10ng/ml, 4h) or rendered apoptotic with cyclohexamide (CHX, 500ng/ml), as indicated, were incubated with LL-37 (1 μg/ml, 15min). MFI, mean fluorescence intensity. n=7. *p<0.05 versus ctrl. Kruskal-Wallis-test with posthoc-Dunn-test. (E) Interaction of LL-37 with extracellular matrix proteins. LL-37 (1 μg/ml) was reacted with indicated extracellular matrix proteins and FCS or FCS alone immobilized on cell culture dishes and detected by immunofluorescence. n=8. *p<0.05 versus FCS alone. Kruskal-Wallis-test with posthoc-Dunn-test. Scale bars, 100 μm.

(A) Flow cytometry (left, middle) and real-time PCR (right) analyses of FPR2 expression in early outgrowth cells (EOCs). EOCs were treated with VEGF (20ng/ml), TNF (50ng/ml) or exposed to hypoxic conditions. Representative histograms for FRP2 expression (green) and isotype control (red) at baseline are shown. MFI, mean fluorescence intensity. n=4. *p<0.05 versus control. Kruskal-Wallis-test with posthoc-Dunn-test. (B) Fluorescence intensity was recorded in EOCs loaded with the Ca²⁺-sensitive fluo4-AM for 90s before and 180s after stimulation with LL-37 in presence or absence of boc-PLPLP (1 μM). Shown is the graph of one representative experiment. n=6. *p<0.05 versus ctrl or LL-37+boc-PLPLP. One-way analyses of variance followed by Tukey test. (C,D) Neutrophil-mediated adhesion of human EOCs to injured carotid arteries involves CRAMP-FPR. Calcein-labeled EOCs pretreated with boc-PLPLP were injected into Apoe^−/− mice (C) or Apoe^−/− mice transplanted with WT or Cramp^−/− BM (D) with intact WBC or into neutropenic mice 4h after wire-injury. EOCs adherent to injured arteries were counted. Representative images in C show adherent EOC (arrows) in mice with intact white blood cell PLPLP were perfused over HAoEC monolayers (E) or plates coated with matrix proteins and LL-37 (F) and adherent cells were counted. Representative images in E show EOC adhesion (arrows) to TNF-activated HAoECs in presence or absence of LL-37. n=4–6. *p<0.05 versus controls and boc-PLPLP treatment. Kruskal-Wallis-test with posthoc-Dunn-test. Scale bars, 20 μm. (G) Analysis of EOC spreading. EOCs were seeded onto fibrinogen coated with or without (ctrl) LL-37. Circularity was assessed at indicated time points (right). Representative images of F-actin-stained EOCs (left). n=4. *p<0.05 versus control. Mann-Whitney test. Scale bars, 10 μm.

(A) EOC monolayers were subjected to scratch injury and the recovered wound area is expressed as percentage of the initial wound area. Representative photomicrographs are displayed. n=4. *p<0.05 versus ctrl. One-way analyses of variance followed by Tukey test. (B) EOCs were treated with TNF (50ng/ml) in presence or absence of LL-37 or boc-PLPLP and reacted with Representative histograms and percentage of annexin-V⁺ EOCs are displayed. n=3. *p<0.05 versus TNF and TNF+LL-37+bocPLPLP. Kruskal-Wallis-test with posthoc-Dunn-test. (C) Cell cycle analysis based on propidium iodide incorporation of EOCs treated with or without LL-37. n=3. (D–F) FACS analysis of endothelial (D) and myeloid (E) markers, and chemokine receptors (F). Baseline expression was set to 100% (black bars), and expression after LL-37 stimulation (white bars) is shown relative to baseline. n=3.

(A) EOCs were treated with medium (ctrl) or LL-37 (1 μg/ml) in presence or absence of boc-PLPLP. EGF and VEGF were measured in medium or supernatant of non-activated EOCs (ctrl) or LL-37-treated EOCs. n=4. *p<0.05 versus all bars. Kruskal-Wallis-test with posthoc-Dunn-test. (B) LL-37 was detected in conditioned medium harvested from untreated EOCs (CM), LL-37-treated EOCs before (LL-37 CM, before) or after (LL-37 CM, after) immuno-depletion of LL-37, or synthetic LL-37 by dot blot. (C) Human aortic endothelial cell (HAoEC) monolayers were wounded linearly and the recovered area is expressed as percentage of the initial wound area. Monolayers were treated with medium containing 1% FCS (ctrl), CM from untreated EOCs or LL-37-immuno-depleted CM from LL-37-treated EOCs (LL-37 CM). n=3. *p<0.05 versus other groups. One-way analyses of variance followed by Tukey test. (D–F) HAoEC apoptosis (D), proliferation (E), and migration (F) is promoted by supernatant of LL-37-treated EOCs. Apoptosis is expressed as percentage of annexin-V⁺ HAoECs. Proliferation was assessed by cell cycle analysis using propidium iodide. The migration distance was measured after time-lapse tracking of HAoEC movement. HAoECs were treated as in (C). EC growth medium (solid bar) served as control. n=5. *p<0.05 versus ctrl and CM-treatment. Kruskal-Wallis-test with posthoc-Dunn-test.

(A,B) EOCs were seeded onto Nitinol foils coated as indicated for 15min and the number of adherent EOCs was quantified. *p<0.05 versus aminosilanized, star-PEG-coated foils. n=4. Kruskal-Wallis-test with posthoc-Dunn-test. (C,D) RGD/P-selectin- or RGD/P-selectin/LL-37-coated stents were implanted into Apoe^−/− mice and after 1 (C) or 4 weeks (D) luminal areas were analyzed by Giemsa staining. Representative images are displayed. Scale bars, 100 μm. n=9. *p<0.05. Mann-Whitney test.