Research ArticleAtherosclerosis

Neutrophil-Derived Cathelicidin Protects from Neointimal Hyperplasia

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Science Translational Medicine  05 Oct 2011:
Vol. 3, Issue 103, pp. 103ra98
DOI: 10.1126/scitranslmed.3002531

Abstract

Percutaneous transluminal angioplasty with stent implantation is used to dilate arteries narrowed by atherosclerotic plaques and to revascularize coronary arteries occluded by atherothrombosis in myocardial infarction. Commonly applied drug-eluting stents release antiproliferative or anti-inflammatory agents to reduce the incidence of in-stent stenosis. However, these stents may still lead to in-stent stenosis; they also show increased rates of late stent thrombosis, an obstacle to optimal revascularization possibly related to endothelial recovery. Here, we examined the contribution of neutrophils and neutrophilic granule proteins to arterial healing after injury. We found that neutrophil-borne cathelicidin (mouse CRAMP, human LL-37) promoted reendothelization and thereby limited neointima formation after stent implantation. We then translated these findings to an animal model using a neutrophil-instructing, biofunctionalized, miniaturized Nitinol stent coated with LL-37. This stent reduced in-stent stenosis in a mouse model of atherosclerosis, suggesting that LL-37 may promote vascular healing after interventional therapy.

Introduction

Progressive atherosclerosis causes luminal narrowing and obstruction that can be treated by mechanical revascularization; however, this procedure induces severe arterial damage and an inflammatory response, leading to restenosis. Implantation of drug-eluting stents reduces the incidence of restenosis with a favorable safety profile and lower cost than surgical procedures (1). However, clinical data implicate an increased risk of late stent thrombosis when using drug-eluting stents compared with bare-metal stents (2). This risk may partially be attributable to impaired endothelial recovery; however, the link between reendothelialization and late stent thrombosis is mostly based on associative evidence from autopsy and angioscopy studies. Coating stents with polymers such as poly(ethylene glycol) (PEG) can prevent adhesion of serum proteins and platelets to reduce in-stent thrombosis (3). Thus, novel concepts of biofunctionalization may further improve treatment of in-stent restenosis and thrombosis.

The initial inflammatory response to arterial injury is characterized by early influx of neutrophils (4). These immune cells contribute to the regulation of the inflammatory cascade by discharging preformed granule proteins, such as azurocidin, α-defensins, or LL-37, which are crucial mediators of neutrophil-dependent activities in inflammation (5, 6). These secretory proteins are potent activators of antigen-presenting cells, but they also activate endothelial cells (ECs), inducing permeability changes and neovascularization (7, 8).

After arterial damage, reendothelialization can regulate inflammation and smooth muscle cell (SMC) accumulation; thus, reendothelialization represents a crucial mechanism to limit neointima formation. Endothelial regeneration primarily relies on migration and proliferation of ECs from adjacent vascular segments. However, angiogenic early outgrowth cells (EOCs) can be recruited to sites of injury and accelerate reendothelialization (9, 10). Notably, infusion of EOCs can attenuate neointimal hyperplasia after arterial injury (11, 12), which has inspired EOC-based strategies to reduce restenosis. These strategies include elution of cyclic RGD peptides or CXCR2 ligands, such as NAP-2, which can recruit EOCs to the stent surface or sites of injury (13, 14).

Here, we examined the involvement of neutrophil-derived granule proteins in arterial healing. We identified a prominent role for neutrophil-borne cathelicidin (mouse CRAMP, human LL-37) in promoting reendothelialization and limiting neointima formation. Consequently, we translated these findings into a biofunctionalized stent coated with LL-37 and demonstrate that a neutrophil-instructing device can reduce in-stent stenosis in mice.

Results

Neutrophil-derived cathelicidin limits neointima formation

To address a role of neutrophils in inflammatory processes after arterial injury, we studied neointima formation in atherosclerosis-prone Apoe−/− mice with intact white blood cell (WBC) count or neutropenia (Fig. 1A) 4 weeks after wire injury. Neutrophil depletion (table S1) significantly increased neointima formation, suggesting a beneficial function of neutrophils in arterial healing. To dissect stage-dependent effects, we depleted neutrophils during the first or the fourth week after injury. Whereas early neutropenia significantly increased the neointimal area, late depletion had no effect (Fig. 1A). Hence, we restricted subsequent analyses to 1 week after injury (Fig. 1B, fig. S1, and table S2). Herein, neutropenic mice exhibited a larger neointimal area than mice with intact WBCs. Paralleling a marked reduction of neutrophils in the neointima, the number of TUNEL+ (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling–positive) cells was reduced in neutropenic mice (fig. S1). Analyzing neointimal cell composition revealed that total macrophage content was reduced in neutropenic mice (Fig. 1B), in line with a role of neutrophils in inflammatory monocyte recruitment (15, 16). Conversely, neutrophil depletion increased neointimal collagen content but did not significantly affect CD3+ T lymphocyte numbers, SMC content (table S2), or the number of SMCs undergoing proliferation or apoptosis (table S3), nor did LL-37 alter SMC migration, proliferation, or apoptosis in vitro (fig. S2). As evident by CD31 staining, endothelial coverage was reduced by 48% in neutropenic mice, indicating diminished reendothelialization (Fig. 1B). Defective endothelial recovery in neutropenic mice was confirmed by Evans blue staining of en face–prepared carotid arteries (fig. S3).

Fig. 1

Neutrophil-derived cathelicidin limits neointima formation. (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 WBC count (top) and in neutropenic mice (bottom). n = 6 to 8. *P < 0.05 versus control mice, Kruskal-Wallis test with post hoc Dunn test. (B) Immunohistochemical analysis of neointima 1 week after wire injury in mice with intact WBC count 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 fluorescein isothiocyanate–conjugated antibody; red fluorescence derives from secondary Cy3-conjugated antibody. n = 7 to 9. *P < 0.05 versus intact WBCs, Mann-Whitney test. (C and D) Neointima size (C) and endothelial coverage (D) in Apoe−/−, beigeApoe−/−, Dppi−/−Apoe−/−, WT→Apoe−/−, and Cramp−/−Apoe−/− mice 1 week after wire injury. Experiments were performed in mice with intact WBCs or neutropenia. Representative images show neointima formation and CD31 staining in WT→Apoe−/− and Cramp−/−Apoe−/− mice 1 week after wire injury. n = 6 to 8. *P < 0.05 versus Apoe−/− or WT→Apoe−/− with intact WBCs, Mann-Whitney test (left) and Kruskal-Wallis test with post hoc Dunn test (right). Scale bars, 100 μm.

Neutrophils regulate acute inflammatory responses through discharge of preformed granule proteins (5, 6). To dissect the role of distinct groups of granule proteins, we used beigeApoe−/− and Dppi−/−Apoe−/− mice, which have reduced mobilization of primary neutrophil granule contents or lack active forms of neutrophil-derived serine proteases (proteinase-3, cathepsin G, and neutrophil elastase), respectively (17, 18). Neointima formation and luminal CD31+ cell coverage after injury in these mice did not differ from that of Apoe−/− mice with intact WBCs (Fig. 1, C and D). Neutropenic mice of all three strains behaved similarly, indicating that primary granule contents and neutrophil-derived proteases are not required for the neutrophil-mediated healing process. Hence, we examined the role of CRAMP, which attracts monocytes to promote wound healing or angiogenesis (8, 18), in neointima formation. Atherosclerotic mice transplanted with Cramp−/− bone marrow (BM) (Cramp−/−Apoe−/−) (19) displayed larger neointimal areas and reduced luminal CD31+ lining compared to mice receiving BM from wild-type mice (WT→Apoe−/−) (Fig. 1, C and D). Neutropenia in Cramp−/−Apoe−/− mice did not further aggravate this phenotype, which resembled that in neutropenic WT→Apoe−/− mice.

Cathelicidins are deposited at sites of endothelial injury

We suspected that CRAMP, a highly positively charged polypeptide, was deposited by adherent neutrophils at sites of injury. Monocyte-depleted Lysmegfp/egfpApoe−/− mice (table S4) carrying only fluorescent neutrophils (20) were used to monitor the time course of neutrophil adhesion. Although neutrophil adhesion did not occur in uninjured carotid arteries, neutrophils rapidly adhered after injury, peaking at 4 to 8 hours (Fig. 2A). Hence, we studied arterial deposition of CRAMP 4 hours after injury. Injection of fluorescent beads conjugated to an antibody to CRAMP allowed for in vivo detection of immobilized CRAMP (Fig. 2B), which was largely abolished in neutropenic mice. The luminal location of CRAMP was corroborated by two-photon microscopy (Fig. 2C). To characterize interactions of cathelicidins with ECs, we co-incubated LL-37 with human aortic ECs (HAoECs). LL-37 binding to HAoECs was reduced by pretreatment with heparinase but not chondroitinase (Fig. 2D). Activation with tumor necrosis factor (TNF) or induction of apoptosis with cycloheximide in HAoECs did not modulate LL-37 binding, but vitronectin, laminin, and fibrinogen strongly bound LL-37 when compared to serum proteins alone (Fig. 2E).

Fig. 2

Neutrophils deposit cathelicidin at sites of injury. (A) Adhesion of neutrophils to injured carotid arteries of monocyte-depleted Lysmegfp/egfpApoe−/− mice. Recordings were taken at different time points after injury, and quantifications are presented as percent fluorescence of total arterial area. Representative images indicate neutrophil adhesion at indicated time points. n = 4 to 5. (B) In vivo detection of CRAMP along injured carotid arteries of mice with intact WBC count or neutropenia, as detected by G protein (heterotrimeric guanosine 5′-triphosphate–binding protein)–coupled fluorescent beads conjugated with an antibody to CRAMP. The number of immobilized beads was quantified. n = 4. *P < 0.05 versus intact WBCs, Mann-Whitney test. (C) Anti-CRAMP bead complexes were detected luminally using intravital two-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 HAoECs assessed by fluorescence-activated cell sorting (FACS) analysis. Untreated HAoECs [control (ctrl)] and HAoECs pretreated with heparinase (Hep, 50 U/ml), chondroitinase (Chon, 20 U/ml), or both, activated with TNF (10 ng/ml, 4 hours) or rendered apoptotic with cycloheximide (CHX, 500 ng/ml), as indicated, were incubated with LL-37 (1 μg/ml, 15 min). MFI, mean fluorescence intensity. n = 7. *P < 0.05 versus control, Kruskal-Wallis test with post hoc 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 post hoc Dunn test. Scale bars, 100 μm.

Cathelicidins mediate EOC recruitment

Neointima formation is governed by an ambiguous role of BM-derived cells. Whereas SMC progenitor cells contribute to neointimal hyperplasia, EOCs limit neointima formation (10). Because neutropenia and CRAMP deficiency affected reendothelialization rather than SMC accumulation, we investigated their role in lesional recruitment of BM-derived EOC-like cells in Apoe−/− mice reconstituted with gfp+ (green fluorescent protein–positive) BM and subjected to arterial wire injury. After 1 week, gfp+ cells staining for CD31 were quantified in the neointima of mice with neutropenia or intact WBCs (fig. S4). Neutrophil depletion reduced the number of BM-derived gfp+CD31+ EOC-like cells in the recovering endothelial lining.

Cathelicidins exert multiple immune-regulating functions via formyl peptide receptor 2 (FPR2) (5, 21). As evidenced by flow cytometry, FPR2 was expressed on human EOCs under basal conditions and up-regulated by VEGF (vascular endothelial growth factor), TNF, or hypoxia at the transcriptional level (Fig. 3A). In Ca2+ flux experiments, stimulation of EOCs with LL-37 resulted in rapid Ca2+ mobilization, a response abolished by the FPR antagonist boc-PLPLP (Fig. 3B).

Fig. 3

Cathelicidin promotes EOC recruitment. (A) Flow cytometry (left, middle) and real-time polymerase chain reaction (right) analyses of FPR2 expression in EOCs. EOCs were treated with VEGF (20 ng/ml) and TNF (50 ng/ml) or exposed to hypoxic conditions. Representative histograms for FPR2 expression (green) and isotype control (red) at baseline are shown. n = 4. *P < 0.05 versus control, Kruskal-Wallis test with post hoc Dunn test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Fluorescence intensity was recorded in EOCs loaded with the Ca2+-sensitive fluo4-AM for 90 s before and 180 s after stimulation with LL-37 in the 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 analysis of variance (ANOVA) followed by Tukey test. (C and 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 WBCs or into neutropenic mice 4 hours after wire injury. EOCs adherent to injured arteries were counted. Representative images in (C) show adherent EOC (arrows) in mice with intact WBC count or in neutropenic mice. n = 6. *P < 0.05 versus untreated EOCs injected into mice with intact WBCs, Kruskal-Wallis test with post hoc Dunn test. Scale bars, 100 μm. (E and F) Adhesion of EOCs to activated HAoECs and matrix proteins is enhanced by LL-37. LL-37 was deposited for 15 min on TNF-activated HAoECs. EOCs pretreated with or without boc-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 the presence or absence of LL-37. n = 4 to 6. *P < 0.05 versus controls and boc-PLPLP treatment, Kruskal-Wallis test with post hoc 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.

To explore a link of the FPR2-cathelicidin axis to reendothelialization, we compared the adhesion of human EOCs to injured carotid arteries in neutropenic mice and mice with intact WBCs. Depletion of neutrophils significantly reduced adhesion of injected EOCs (Fig. 3C). Similarly, pretreatment of EOCs with boc-PLPLP decreased adhesion to levels observed in neutropenic mice receiving untreated EOCs. Notably, boc-PLPLP did not further reduce adhesion of EOCs injected into neutropenic mice (Fig. 3C). Moreover, platelet-depleted mice showed reduced EOC adhesion (fig. S5), likely attributable to altered EOC interactions with platelets or platelet-derived microparticles (22) or secondary effects related to lower neutrophil adhesion in thrombocytopenic mice (20). As seen for monocytes, blocking CXCR2 on EOCs reduced their adhesion (fig. S6), an effect partially independent of neutrophils, implying a nonredundant role of this putative LL-37 receptor (23). Antagonists to P2X7 or TLR4 (Toll-like receptor 4), both involved in cathelicidin-dependent cell activation, failed to alter EOC adhesion (fig. S7A).

A potential interaction of cathelicidin with FPR2 underlying neutrophil-mediated EOC adhesion was addressed in atherosclerotic mice. The adhesion of EOCs was reduced by neutrophil depletion in WT→Apoe−/− mice but did not differ between neutropenic and normal Cramp−/−Apoe−/− mice (Fig. 3D). Moreover, EOCs treated with boc-PLPLP before injection into WT→Apoe−/− or Cramp−/−Apoe−/− mice showed similar adhesion as in neutropenic WT→Apoe−/− mice or in Cramp−/−Apoe−/− mice with intact WBCs or neutropenia, indicating that CRAMP mediates neutrophil-dependent EOC adhesion through FPRs. In vitro flow assays confirmed that deposition of LL-37 on TNF-activated HAoECs significantly enhanced EOC arrest, which was abrogated by boc-PLPLP (Fig. 3E). Moreover, LL-37 co-immobilized with vitronectin, laminin, or fibrinogen enhanced EOC adhesion, which was abolished by the FPR antagonist (Fig. 3F). Coordinated cell spreading is essential for effective adhesion, requiring cytoskeletal reorganization and actin polymerization. We therefore analyzed LL-37–dependent spreading of EOCs immobilized on fibrinogen. The presence of LL-37 rapidly decreased circularity (Fig. 3G), indicating prompt cellular activation and actin reorganization.

Cathelicidins promote survival of EOCs

Once immobilized, EOCs may facilitate reendothelialization by proliferation, maturation, or secretion of growth factors. We assessed the relevance of LL-37 in the repopulation of scratch wounds inflicted in EOC monolayers. Compared to controls, incubation with LL-37 promoted EOC regrowth into the wounded area (Fig. 4A). Mechanistically, we investigated the effects of LL-37 on EOC apoptosis, proliferation, and maturation. LL-37 prevented TNF-induced EOC apoptosis in an FPR-dependent manner (Fig. 4B) but did not affect proliferation (Fig. 4C) or maturation, as evidenced by unaltered expression of endothelial (Fig. 4D) and myeloid (Fig. 4E) markers. Moreover, LL-37 did not modulate expression of CXCR2 and CXCR4 (Fig. 4F), and serum levels of CXCL12 and mCXCL1, ligands of CXCR4 or CXCR2, respectively, were unaltered in neutropenic mice after wire injury (table S5).

Fig. 4

LL-37 promotes EOC survival. (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 ANOVA followed by Tukey test. (B) EOCs were treated with TNF (50 ng/ml) in the presence or absence of LL-37 or boc-PLPLP. Representative histograms and percentage of annexin V+ EOCs are displayed. n = 3. *P < 0.05 versus TNF and TNF + LL-37 + boc-PLPLP, Kruskal-Wallis test with post hoc Dunn test. (C) Cell cycle analysis based on propidium iodide incorporation of EOCs treated with or without LL-37. n = 3. (D to 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.

Cathelicidins modulate secretory properties of EOCs

The low numbers of circulating EOCs and the contribution of endothelial regrowth to arterial healing led us to explore whether the cathelicidin-EOC axis exerts paracrine effects on ECs. As potent sources of growth factors (22, 24), EOCs exposed to LL-37 showed increased release of VEGF and EGF (epidermal growth factor), which was abolished by boc-PLPLP (Fig. 5A) but not antagonists to P2X7 or TLR4 (fig. S7B). To examine functional properties of EOC secretory products, we subjected confluent HAoECs to scratch injury and incubated them with conditioned medium harvested from LL-37–treated EOCs (LL-37/CM), untreated EOCs (CM), or control medium with 1% fetal calf serum (FCS). To exclude effects of LL-37 in CM, we immunodepleted LL-37, as confirmed by dot blot analysis (Fig. 5B). Moreover, LL-37 failed to induce or enhance proliferation of HAoECs (fig. S8), indicating that direct effects on local EC proliferation are unlikely. Injured HAoEC monolayers in control medium recovered to about 20% within 24 hours, and endothelial recovery was slightly improved by CM but markedly enhanced by LL-37/CM (Fig. 5C). We analyzed alterations of apoptosis, proliferation, or migration, possibly underlying improved recovery with LL-37/CM. Control medium and CM but not LL-37/CM induced apoptosis and reduced proliferation and migration in HAoECs compared to EC medium (Fig. 5, D to F).

Fig. 5

LL-37–treated EOCs exert paracrine proendothelial effects. (A) EOCs were treated with medium (ctrl) or LL-37 (1 μg/ml) in the presence or absence of boc-PLPLP. EGF and VEGF were measured in medium or supernatant of nonactivated EOCs (ctrl) or LL-37–treated EOCs. n = 4. *P < 0.05 versus all bars, Kruskal-Wallis test with post hoc Dunn test. (B) LL-37 was detected in conditioned medium harvested from untreated EOCs (CM), LL-37–treated EOCs before or after immunodepletion of LL-37, or synthetic LL-37 by dot blot. (C) 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–immunodepleted CM from LL-37–treated EOCs (LL-37 CM). n = 3. *P < 0.05 versus other groups, one-way ANOVA followed by Tukey test. (D to F) HAoEC apoptosis (D), proliferation (E), and migration (F) are 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 bars) served as control. n = 5. *P < 0.05 versus ctrl and CM treatment, Kruskal-Wallis test with post hoc Dunn test.

Cathelicidin biofunctionalization reduces in-stent stenosis

Because of its biocompatibility, corrosion resistance, and elasticity, Nitinol is frequently used for stents. The role of the LL-37–EOC axis in protecting against neointima formation prompted us to develop biofunctionalized stents exploiting this principle. We assessed the biofunctional properties of surface-modified Nitinol in EOC adhesion assays. First, Nitinol foils were aminosilanized with N-[3-trimethoxysilyl-propyl]ethylenediamine and coated with six-arm star-shaped PEG (star-PEG), creating an inert and antiadhesive surface, as evidenced by minimal unspecific background adhesion of EOCs, when compared to uncoated or fibronectin-coated plastic surfaces or untreated Nitinol foils (Fig. 6A). The aminosilanized, star-PEG–coated Nitinol foils were biofunctionalized by dip coating with recombinant P-selectin, RGD, and LL-37 individually or in combination. The adhesion of EOCs to Nitinol foils was unaffected by coating with any proteins alone but markedly enhanced by the combination of P-selectin, RGD, and LL-37 (Fig. 6B). This prompted us to explore the effectiveness of LL-37–coated stents in mice. Miniaturized stents with shape memory were braided from Nitinol wire, aminosilanized, and coated with star-PEG, and biofunctionalized by covalently binding P-selectin/RGD without or with LL-37, which was well retained on stent surfaces, as shown by two-photon microscopy and fluorescent LL-37 (fig. S9).

Fig. 6

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

Stents were implanted into the left carotid artery of Apoe−/− mice through insertion into a silicon tube, with careful forward feeding of the stent while retracting the tube to allow for shape memory–based expansion (fig. S9). After 1 and 4 weeks, we performed histological analyses to assess lumen and neointima by Giemsa staining. Whereas neointima size was only reduced in mice receiving LL-37–coated stents compared to bare-metal stents and aminosilanized, star-PEG–coated, and P-selectin/RGD–coated stents without LL-37 (fig. S10), the luminal area was markedly increased in LL-37–coated stents (Fig. 6, C and D), revealing protective effects against in-stent stenosis. Improved endothelial coverage of the luminal lining with CD31+ ECs was observed by immunofluorescence staining in carotid arteries that had received LL-37–coated stents, compared to control stents, and an abundance of CD31+ ECs within the stented area, but no significant thrombosis was detected in carotid arteries with LL-37–coated stents (fig. S11). As seen in other models of arterial injury, for example, wire-induced denudation, in mice, a period of 4 weeks is commonly sufficient to obtain a maximal degree of neointimal hyperplasia. Hence, it is conceivable that long-term efficiency and patency of LL-37–coated stents would persist beyond 4 weeks. To corroborate that CRAMP limits in-stent stenosis, we implanted uncoated Nitinol stents in Apoe−/− mice reconstituted with WT or Cramp−/− BM. Mice lacking leukocyte CRAMP displayed larger neointima but reduced in-stent lumen size (fig. S12).

Discussion

Neutrophils are the first line of immune cells recruited to sites of injury after percutaneous transluminal angioplasty, and the rapid release of their granule proteins critically shapes the vascular inflammatory response. Here, we identified cathelicidins as important mediators of neutrophil-dependent repair after arterial injury. The activity of these effectors was based on their EOC-activating capacities: They enhanced EOC recruitment and released regenerative growth factors. Both mechanisms cooperated to promote reendothelialization and to reduce the extent of neointima formation. We translated this knowledge by designing an LL-37–coated stent, which was effective in limiting in-stent stenosis.

Neutrophils have been underestimated players in arterial disease. Recent studies revealed an exacerbating effect of neutrophils in atherosclerosis, which was promoted by neutrophilia but attenuated in neutropenic mice (20). In contrast, we showed that neutrophils exert protective effects during acute arterial injury. Previous studies revealed that a lack of β2 integrins (25, 26) or P-selectin (27), both expressed by neutrophils, resulted in smaller neointima size. Because these molecules are not specific for neutrophils and are also involved in recruiting monocytes, platelets, and progenitor cells, we believe that these studies identify molecular rather than cellular targets. Moreover, the contribution of P-selectin to neointima formation seems to be restricted to platelets (28). In contrast, blocking mCXCL1, which can target neutrophil recruitment, increased neointima formation while decreasing endothelial recovery (29). Conversely, elevated neutrophil counts after granulocyte colony-stimulating factor (G-CSF) application were found to reduce neointima size and promote reendothelialization (30). Hence, neutrophils may exert protective effects during early phases of arterial injury.

Healing after vascular injury is currently thought to be differentially affected by recruitment of BM-derived progenitor cells: Whereas EOCs are thought to limit neointima formation by accelerating reendothelialization, SMC progenitor cells contribute to neointimal hyperplasia (10). Our analysis revealed that neutropenia affected endothelial recovery but not SMC content in neointimal lesions. Thus, we focused on the interrelation between neutrophils and EOCs. Two major mechanisms are thought to account for beneficial effects of EOCs on endothelial recovery: recruitment and release of growth factors promoting EC regrowth (31).

The CXCR4-CXCL12 axis is a major determinant of EOC mobilization and homing to sites of arterial injury. Platelet-derived microparticles amplify vasoregenerative functions of EOCs by transferring CXCR4 (22). Moreover, the CXCR2-CXCL1 axis plays a pivotal role in EOC recruitment and angiogenesis, as evidenced by CXCR2−/− mice or neutralizing CXCR2 and its ligands (14, 32, 33). Neutrophils, however, seem to modulate neither axis, because CXCR4 and CXCR2 expression on EOCs and CXCL12 and CXCL1 serum levels were unaltered by LL-37 or neutropenia, respectively.

In addition, LL-37 acts through FPR2 rather than CXCR4 in EOC-related monocytes (15, 21), whereas some functional ligand activity for CXCR2 was observed in neutrophils (23). Indeed, LL-37–FPR interactions are important in mediating neutrophil-dependent EOC recruitment; neutrophil- and LL-37–mediated EOC adhesion was abrogated by antagonizing FPRs. Notably, adherent neutrophils could seed LL-37 at sites of injury. Based on its cationic nature, LL-37 interacts with negatively charged proteins of the basement membrane and the EC glycocalyx. In a similar sequence of events, azurocidin deposited by emigrating neutrophils could induce monocyte adhesion (34), and activated platelets could deposit chemokines from intracellular stores to trigger monocyte arrest (35). The FPR2–LL-37 interaction is important during EC activation and proliferation leading to angiogenesis (8). Activation of embryonic endothelial progenitor cells with LL-37 ex vivo has been found to enhance nuclear factor κB (NF-κB) activity and to promote adhesion of these cells at sites of ischemia (36). Moreover, LL-37 has been implicated in regulating cell survival, with high concentrations being cytotoxic, whereas lower concentrations may prevent apoptosis (37, 38). Here, we show that LL-37 protected EOCs against apoptosis in an FPR2-dependent manner, providing an adjuvant mechanism for EOC accumulation under inflammatory conditions. However, unlike in ECs (8), LL-37 did not stimulate EOC proliferation.

Consequently, we identified effectors by which LL-37–triggered EOC function may contribute to reendothelialization, namely, LL-37 stimulated release of the proangiogenic factors VEGF and EGF. A similar effect was identified for porcine cathelicidin PR-39, which inhibits ubiquitin proteasome–dependent degradation of hypoxia-inducible factor–1α (HIF-1α) to increase VEGF expression (39). In our experiments, LL-37–treated EOC secretion exhibited a potent paracrine activity on ECs, resulting in enhanced migration and proliferation but reduced apoptosis. Considering the limited number of EOCs directly incorporating into the endothelial lining, this activity may stand out as a major mechanism underlying neutrophil-dependent reendothelialization. Hence, LL-37 promoted reendothelialization by a variety of mechanisms including EOC recruitment and survival as well as endothelial recovery by paracrine effects.

Recent meta-analyses confirmed drug-eluting stents as a preferred option for coronary revascularization (1, 40). Nevertheless, concerns regarding delayed reendothelialization and in-stent thrombosis associated with drug-eluting stents have been raised (41, 42). As a result, current treatment regimens after stent implantation have adopted a dual antiplatelet therapy for at least 1 year. We have therefore designed an LL-37–coated stent, which—likely through instructing the cathelicidin-EOC axis—limits in-stent stenosis in mice. LL-37 is known to harbor not only both pro- and anti-inflammatory properties, for example, monocyte recruitment and activation (5, 16), stimulation of wound healing, and angiogenesis, but also deactivation of macrophages (8, 43, 44). The limited in-stent neointima in LL-37–coated stents suggests that the anti-inflammatory effects of LL-37 prevailed in this model. Furthermore, LL-37–coated surfaces could exert potent antimicrobial effects (45) but little proinflammatory activity in terms of platelet or complement activation (46). Although our study was limited to 4 weeks after implantation, no thrombus formation was observed in these stents. Thus, stents exploiting proendothelial properties of LL-37 may also be suitable to circumvent problems of late thrombosis in drug-eluting stents after longer periods of clinical use. The maximum degree of neointima formation in mice is commonly obtained within 4 weeks after arterial injury. It is well established that covalent coatings with antimicrobial polypeptides are highly stable, withstanding extreme environmental conditions such as severe changes in pH or temperature (47). Therefore, we have no reason to believe that the long-term efficiency and patency would be markedly different beyond 4 weeks in our model. Previously, novel stent developments have been primarily tested and validated in large animal models (14, 48). Our approach of implanting miniaturized and biofunctionalized stents in mouse carotid arteries allows for versatile analysis of novel stent coatings in a disease-relevant context of genetically altered mice.

Whereas the overall rate of stent thrombosis may be equivalent between drug-eluting and bare-metal stents at 5 years, its temporal incidence may differ, with higher rates of very late stent thrombosis in drug-eluting stents (49). The underlying mechanisms have been differentially related to hypersensitivity or excessive fibrin in first-generation drug-eluting stents (50). Clinical follow-up at 3 years, however, indicates that an everolimus-eluting stent may have more favorable rates of stent thrombosis, possibly extending to very late stent thrombosis (51). Although a link of reendothelialization to late stent thrombosis is not firmly established, differences in endothelialization of drug-eluting and bare-metal stents in autopsy and angioscopy studies and comparison of drug-eluting stents in rabbit model showed a lesser reduction in neointima size despite better healing and reendothelialization in zotarolimus-eluting stents (52). This difference may not be generalized to our purely proendothelial polymer stent concept without confounding effects of other drug eluents. Its beneficial effects may provide evidence for a link between endothelial recovery and reduced in-stent stenosis.

In conclusion, neutrophils play an important role during the early healing process after arterial injury by depositing cathelicidins as regulators of endothelial recovery. The FPR2-cathelicidin axis promoted adhesion and function of EOCs, improving reendothelialization and limiting neointima formation. Accordingly, biofunctionalized LL-37–coated stents showed effectiveness in a mouse model. These findings warrant investigation of the applicability of this strategy in large animal models and its translation into clinical use.

Materials and Methods

Arterial wire injury

All animal studies were approved by local authorities. Apoe−/− mice were transplanted with BM from Cramp−/− mice (19), CAGgfp/gfp mice (The Jackson Laboratory), or WT controls by intravenous injection 24 hours after ablative whole-body irradiation. Mice were fed an atherogenic diet containing 21% fat for 1 week before and up to 4 weeks after injury. Wire injury was performed and analyzed as described (22). Neutrophils were depleted by intraperitoneal injection of monoclonal antibody (mAb) 1A8 (20). Platelets were depleted by intraperitoneal injection of antiplatelet serum (20). Control animals were treated with immunoglobulin G2a (IgG2a) isotype control antibody or control serum. The braiding, coating, implantation, and processing of biofunctionalized stents are detailed in the Supplementary Material.

Cell culture

Isolation and culture of EOCs were as described (22). Briefly, peripheral blood mononuclear cells were separated by density gradient centrifugation and seeded on fibronectin-coated plates in MV2 endothelial growth medium (PromoCell) for 7 days.

Statistics

Data are expressed as means ± SD. Statistical calculations were performed with GraphPad Prism 5. Statistical tests are specified in the figure legends. P values of <0.05 were considered significant.

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/3/103/103ra98/DC1

Materials and Methods

Fig. S1. Neointimal cell composition.

Fig. S2. LL-37 does not affect migration, apoptosis, and proliferation of smooth muscle cells (SMCs).

Fig. S3. Neutropenia impairs endothelial recovery.

Fig. S4. Neutropenia reduces incorporation of bone marrow–derived cells into the endothelial lining.

Fig. S5. Platelet depletion reduces adhesion of EOCs.

Fig. S6. Adhesion of adoptively transferred monocytes and EOCs partially depends on CXCR2.

Fig. S7. Role of P2 receptors and TLR4 in LL-37–mediated EOC activation.

Fig. S8. LL-37 does not induce endothelial cell (EC) proliferation.

Fig. S9. Development of an LL-37–coated Nitinol stent.

Fig. S10. Neointima sizes are reduced in mice receiving LL-37–coated stents.

Fig. S11. Reendothelialization is improved in arteries with LL-37–coated stents.

Fig. S12. CRAMP prevents in-stent stenosis.

Table S1. Differential leukocyte counts in mice with intact WBCs or neutropenia.

Table S2. Neointimal cell composition in mice with intact WBCs or neutropenia.

Table S3. Immunohistochemical analysis of SMC homeostasis 1 week after wire injury.

Table S4. Differential leukocyte counts in mice depleted of monocytes.

Table S5. mCXCL1 and CXCL12 serum levels in mice with intact WBCs or neutropenia.

Footnotes

  • * These authors contributed equally to this work.

References and Notes

  1. Acknowledgments: We thank Y. Jansen and S. Roubrocks for technical assistance. Funding: This study was supported by Deutsche Forschungsgemeinschaft (SO 876/3-1, WE 1913/11-2, ZE 827/4-1), German Heart Foundation, IZKF Aachen, and RWTH Aachen. Author contributions: O.S. designed the study, performed intravital microscopy, analyzed the data, wrote the manuscript, and provided funding. S. Wantha performed in vitro assays. S.S. acquired stent data. Y.D. performed in vitro experiments. R.T.A.M. performed two-photon microscopy. S.F.M. performed enzyme-linked immunosorbent assay measurements. M.D. performed wire injuries. R.S. and S. Weinandy contributed to acquisition of stent data. F.S. and T.G. braided stents. S.V. contributed in vitro data. M.A.M.J.v.Z. supervised two-photon microscopy. B.A. provided reagents. C.T.P. and R.L.G. provided mouse strains. T.M.H. synthesized fluorescent LL-37. E.A.L. performed wire injuries and stent implantations, and acquired and analyzed stent data. A.Z. designed the study, developed and performed stent implantation, and provided funding. D.K. supervised stent development. C.W. designed the study, wrote the manuscript, and provided funding. Competing interests: The authors declare that they have no competing interests.
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