International Journal of Cardiology 173 (2014) 80–91

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International Journal of Cardiology

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Distinct CD11b+-monocyte subsets accelerate endothelial cell recoveryafter acute and chronic endothelial cell damage

Ulrich M. Becher a, Lisa Möller a, Vedat Tiyerili a, Mariuca Vasa Nicotera a, Felix Hauptmann a,Katrin Zimmermann b, Alexander Pfeifer b, Georg Nickenig a, Sven Wassmann c, Nikos Werner a,⁎a Medizinische Klinik und Poliklinik II, Innere Medizin, Universitätsklinikum Bonn, Germanyb Institut für Pharmakologie und Toxikologie, Universitätsklinikum Bonn, Germanyc Kardiologische Abteilung, Innere Medizin, Isarklinik München, Germany

⁎ Corresponding author at: Medizinische Klinik und PoBonn, Sigmund Freud Str. 25, 53105 Bonn, Germanyfax: +49 228 287 16026.

E-mail address: nikos.werner@ukb.uni-bonn.de (N. W

http://dx.doi.org/10.1016/j.ijcard.2014.02.0040167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 3 August 2013Received in revised form 12 January 2014Accepted 8 February 2014Available online 20 February 2014

Keywords:Endothelial regenerationCD11b+-monocytesTransfusionCD11b-DTR-mice

Background: Endothelial cell recovery requires replenishment of primary cells from the endothelial lineage.However, recent evidence suggests that cells of the innate immune system enhance endothelial regeneration.Methods and results: Focusing on mature CD11b+-monocytes, we analyzed the fate and the effect of transfusedCD11b+-monocytes after endothelial injury in vivo. CD11b-diphtheria-toxin-receptor-mice – a mouse modelin which administration of diphtheria toxin selectively eliminates endogenous monocytes and macrophages –were treated with WT-derived CD11b+-monocytes from age-matched mice. CD11b+-monocytes improvedendothelium-dependent vasoreactivity after 7 days while transfusion of WT-derived CD11b−-cells had nobeneficial effect on endothelial function. In ApoE−/−-CD11b-DTR-mice with a hypercholesterolemia-inducedchronic endothelial injury transfusion of WT-derived CD11b+-monocytes stimulated by interferon-γ (IFNγ)decreased endothelial function, whereas interleukin-4-stimulated (IL4) monocytes had no detectable effect

on vascular function. Bioluminescent imaging revealed restriction of transfused CD11b+-monocytes to theendothelial injury site in CD11b-DTR-mice depleted of endogenous monocytes. In vitro co-culture experimentsrevealed significantly enhanced regeneration properties of human endothelial outgrowth cells (EOCs) whencultured with preconditioned-media (PCM) or monocytes of IL4-stimulated-subsets compared to the effects ofIFNγ-stimulated monocytes.Conclusion: CD11b+-monocytes play an important role in endothelial cell recovery after endothelial injury byhoming to the site of vascular injury, enhancing reendothelialization and improving endothelial function.In vitro experiments suggest that IL4-stimulated monocytes enhance EOC regeneration properties most likelyby paracrine induction of proliferation and cellular promotion of differentiation. These results underlinenovel insights in the biology of endothelial regeneration and provide additional information for the treatmentof vascular dysfunction.

© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Effective endothelial restoration after injury requires dynamicorchestration of diverse cell types. In addition to cells derived fromthe endothelial lineage, cells of the innate immune system displayendothelial-regenerating properties. Circulating monocytes are amongthe first in responding to endothelial damage and are recruited to sitesof vascular remodeling after angioplasty or stent implantation [1,2].Adhesion molecules expressed on the surface of activated endothelialcells (ECs) enable the recruitment of circulating monocytes to sites ofendothelial injury and inflammation, followed by migration towards

liklinik II, Universitätsklinikum. Tel.: +49 228 287 16025;

erner).

.

distinct cytokine and chemokine gradients [3]. In the inflammatorymilieu attracted monocytes mature into foamy macrophages uponingestion of oxidized LDL particles. Within foam cells production ofkey pro-inflammatory cytokines beyond necessary levels triggersprogression of vascular inflammation and atherogenesis, counteractingresolution of inflammation in later healing stages [4]. However, uponacute EC damage in physiological environment monocytes may shiftthe balance from chronic vascular deterioration towards induction andmaintenance of vascular regeneration and repair. Importantly, interac-tions between monocytes and ECs regulate endothelial cell survivaland proliferation essential for vascular recovery and repair [5,6].Distinct from the pro-angiogenic and growth promoting cytokinessecretion of differentiated macrophages, contact-dependent directcellular interactions of monocytes are specific to EC survival [5]. Thiscomplex interactions of contact-dependent and paracrine stimulatedcellular cross-talk early after injury is critical for the recovery and

81U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

maintenance of the endothelial monolayer and its function [5].Although monocyte plasticity is functionally restricted mainly toconversion into tissue macrophages, subsets with pluripotent plasticityexists andmay differentially interactwith ECs [7].More recentwork hasdemonstrated significant heterogeneity among monocytes and theirpotentially different roles in atherogenesis [8]. Pro-inflammatorymonocytes, identified by high expression of the surface marker Ly6C,are CCR2+ [9]. A second monocyte subset identified by low expressionof Ly6C is largely CCR2− and thought to be involved in the post-inflammatory reparative functions of monocytes [10]. Given the rangeof vascular modulating populations of circulating monocytes and thecontroversy effects on the role of monocytes in vascular regenerationand deterioration, we studied the effect of CD11b+-monocytes onvascular function and their destination after transfusion and inducedvascular injury in vivo. Furthermore, we analyzed the response ofimmature human endothelial outgrowth cells (EOCs) after interactionwith blood monocytes prior to and after stimulation with IFNγ or IL4in vitro.

To advance the understanding of monocytes in the pathophysiologyof vascular inflammation and regeneration we focused on remodelingand repair processes and explored the role of circulating CD11b+-monocytes in vascular regeneration of acute and chronic endothelialinjuries.We investigated a transgenic ApoE−/−-mouse-model of hyper-cholesterolemia induced endothelial dysfunction and a transgenicmouse (CD11b+-DTR-mouse) in which diphtheria toxin (DT) condi-tionally eliminates monocytes and macrophages [11]. Thus, cellspecific transgenic expression of human heparin-binding EGF-likegrowth factor confers DT sensitivity to murine CD11b+-monocytesin vivo. We correlated outcomes of endothelial regeneration afterCD11b+-cell depletion and transfusion in this mouse model andfound that CD11b+-monocytes display not only early placeholderfunction in damaged endothelium but also profoundly affect localendothelial cell recovery in vitro demonstrating that depending onthe prestimulation (IFNγ or IL4) and the mode of interaction(paracrine or cell–cell-interaction) monocytes affect proliferation,differentiation, apoptosis, and function of immature human EOCs,relevant in endothelial regeneration.

2. Methods

2.1. Mice

Male, 16-week-old wild-type-mice (WT, C57BL/6J background), ApoE−/−-mice(C57BL/6J background), CD11b+-DTR-mice (FVB/N background) and ApoE−/−-CD11b-DTR-mice (C57BL/6J × FVB/N background) were used for these studies. The animalswere maintained in a 22 °C room with a 12-hour light and dark cycle and receiveddrinking water ad libitum. All animal experiments were performed in accordancewith institutional guidelines and the German animal protection law. All surgicalprocedures were approved and in accordance with institutional guidelines.

2.2. Clodronate liposomes injection in WT-mice

Clodronate (Sifavitor) and rhodamine RE (Avanti Polar Lipids) were encapsulat-ed in liposomes composed of 50 μmol/L distearoyl phosphatidylglycerol (DSPG)(Avanti), 100 μmol/L cholesterol (Sigma-Aldrich, Taufkirchen, Germany Chemicals),and 150 μmol/L of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti) byreverse-phase evaporation technique. This encapsulation allows systemic distributionwithout adverse effects of the drug until the liposome is taken up by a monocyte orother phagocytic cells. Clodronate is a highly charged substance, not able to pass throughthe lipid bilayer of the liposome or the cell, so that the influence of clodronate on non-phagocytic cells e.g. endothelial cells is of no or only minor relevance. Clodronateliposomes (CLs) but not free clodronate significantly reduced the number andproliferationof viable cells that phagocytize and degrade the liposome in a dose dependentmanner butdid not affect vascular smooth muscle cells (VSMCs) or ECs viability and proliferation atconcentrations up to 500 μmol/L. Perielectric carotid injury was performed in WT-miceas described previously. Briefly, the mice were anesthetized with 150 mg/kg body weightketamine hydrochloride (Ketanest®, Pharmacia) and 0.1 mg/kg body weight xylazinehydrochloride (Rompun® 2%, Bayer). The common carotid artery (CCA) was exposedand submitted to an electric injury starting at the level of the bifurcation and continuingto the proximal part of the artery (in total 4 mm denudation).

2.3. Splenectomy

Mice assigned to cell transfusion experiments were anesthetized with 150 mg/kgbody weight ketamine hydrochloride (Ketanest, Pharmacia) and 0.1 mg/kg body weightxylazine hydrochloride (Rompun 2%, Bayer). The spleen was dissected through a lateralincision of the left abdomen. Vessels were carefully ligated using 6/0 silk. After removalof the spleen, the abdomen was closed with sutures using 6/0 silk. Animals were allowedto recover for 2 days before further treatment was performed.

2.4. Preparation of donor cells

Spleens from wild-type mice were explanted and mechanically minced, andmononuclear cells (MNCs) were isolated using a Ficoll gradient (Lympholyte-M,Cedarlane). For intravenous injection MNCs were separated into CD11b+- or CD11b−-cells (Supp. Fig. 1). In brief, MNCs were washed, resuspended, and mixed with colloidalsuperparamagnetic microbeads conjugated to monoclonal rat anti-mouse-CD11b-antibody (MACS MicroBeads, Miltenyi Biotec). After incubation and additional washing,magnetic cell separation was performed. The collected effluent contained the negativeMNC fraction depleted of CD11b+-cells (Suppl. Fig. 1). Attached CD11b+-MNCs werecollected in buffer. Separated subpopulationswere resuspended in200 μL of normal salinesolution for intravenous injection.

Bonemarrow fromWT-mice was explanted and mechanically minced. About 5 × 106

cells were obtained per mouse and cultured for 72 h in RPMI Medium 1640 (Gibco, LifeTechnologies, Paisley, Scotland) with M-CSF and 2 mM L-Glu (Invitrogen, Karlsruhe,Deutschland) at 37 °C, 21% O2, 5% CO2 and 95% H2O. On day 4 of culture cells werestimulated with INFγ (100 ng/mL) or IL4 (10 ng/mL) for 24 h. On day 5 the monocyteswerewashed, collected in buffer and resuspended according to the protocol of the transfu-sion experiments in vivo.Using this standardmethodwe routinely achieve 95% purity. Thismethod allows by simple means the generation of high numbers of murine monocytes ofclassical or alternative phenotype macrophages with very low contaminations.

2.5. Organ chamber experiments

ApoE−/−-mice and ApoE−/−-CD11b-DTR-mice were fed on a high fat cholesterol dietthat contained 21% fat, 19.5% casein, and 1.25% cholesterol (Ssniff, Germany) for 5 weeks.They received either 1 × 106 CD11b+- or CD11b−-MNCs (ApoE−/−-mice, Fig. 2) or INFγ-or IL4-stimulated CD11b+-MNCs (ApoE−/−-CD11b-DTR-mice, Fig. 5) by intravenous tailvein injection on three consecutive days. Control animals received a correspondingamount of cell-free saline (Figs. 2, 3, 4, 5 and 6). On day 7 after i.v. treatment, endothelialfunction of aortic ring segments of the thoracic aorta was analyzed. Vasodilatation andvasoconstriction of isolated aortic ring preparations were determined in organbaths filled with oxygenated modified Tyrode buffer (37 °C), as previously described [12].An investigator blinded to the type of experimental group performed the experiments.

2.6. Neointima formation

Curved wire common carotid artery injury (CCAI) was induced in C57BL/6J-wildtypemice as described previously [13]. Immediately, 24 and 48 h after induction of CCAI micereceived either 1 × 106 spleen-derived MNCs or CD11b-depleted spleen-derived byintravenous tail vein injection (see Fig. 3). Control animals received a correspondingamount of normal saline (see Fig. 3). Animals were allowed to recover. Considerabletimewas spent to assure rigid standardization of all conditions andmanualmanipulations.One highly trained investigator adapted to the microsurgical approach carried out thecarotid artery injury and performed and evaluated more than 100 operations.

To evaluate neointima formation, perfusion-fixed carotid arteries were harvestedon day 7 after wire injury, embedded in Tissue Tek OCT embedding medium (Miles),snap-frozen, and stored at −80 °C. Samples were sectioned on a Leica cryostat (7 μm)and placed on slides coated with poly-L-lysine (Sigma-Aldrich, Taufkirchen, Germany).For morphometric analyses H&E staining was performed according to standard protocols.Formorphometric analyses LuciaMeasurement Version 4.6 softwarewas used tomeasureexternal elastic lamina, internal elastic lamina, and lumen circumference aswell as medialand neointimal area of 25 sections per animal. An investigator blinded to the type ofexperimental group performed the experiments.

2.7. In vivo bioluminescence imaging in CD11b-DTR-mice

To examine the fate of CD11b-depleted mononuclear cells on early endothelialregeneration after CCAI, we used a transgenic mouse (CD11b-DTR-mice) in whichapplication of DT conditionally ablates monocytes and macrophages. Using thissystem, DT treatment reduced the endogenous monocytes population by 81 ± 6%confirmed by FACS analysis (Suppl. Fig. 2). CD11b-DTR-mice treatedwith DT receivedeither vehicle or DT to suppress endogenous monocyte population. CD11b-DTR-micetreated with DT (15 ng/g bw) received 1 × 106 spleen-derived luciferase expressingCD11b+-monocytes by intravenous tail vein injection immediately, 24, and 48 hafter induction of CCAI (Fig. 6). Control animals received a corresponding amountof normal saline by intravenous tail vein injection. Luciferase expressing CD11b+-cells were transduced with a HIV-derived lentiviral vector (LV) carrying a CMVpromoter-driven luciferase expression cassette. The lentivector carrying the firefly-luciferase gene under control of the CMV promoter (CMV-Luc) was kindly providedby Inder Verma (Salk Institute, La Jolla, California 92037, USA) and the production

82 U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

of lentivirus containing supernatants was done as described previously [14].Positioning of luciferase-expressing, transgenic monocytes was analyzed bynon-invasively in vivo imaging of bioluminescence after intraperitoneally applicationof luciferin. Bioluminescent images were collected immediately after transfusion usingthe IVIS® 100 Imaging System (Xenogen Corp., Alameda, CA). The bioluminescent andgray-scale images were overlaid using Living Image software and pseudocolor imagerepresents bioluminescence intensity (blue, least intense, and red, most intense).

2.8. Reendothelialization in CD11b-DTR-mice

For assessment of reendothelialization, common carotid arteries were submitted to aperielectric injury as describedpreviously [15]. Vessel preparationwas started at the bifur-cation and continued to the proximal part of the artery. Carewas taken in applying as littlemechanical pressure to the vessel as possible; electrical pulses lasted 4 s with 2W. Imme-diately, 24, and 48 h after induction of the perielectric CCAI vehicle and DT treated CD11b-DTR-animals received either 1 × 106 spleen-derived CD11b+-monocytes by intravenoustail vein injection (Fig. 4). Control animals received a corresponding amount of normal sa-line by intravenous tail vein injection at corresponding time points (Fig. 4). The denudedarea was determined on day 5 after intravenous injection of 50 μL Evans blue in an enfacepreparation of the vessel. Complete vessel area and Evans blue stained denuded areaswere measured using AxioVision version 4.5.0 software. The percentage ofreendothelialized area was quantified. Perielectric carotid artery injury was inducedas described previously.

2.9. Cell cultures and co-culture experiments

Human endothelial outgrowth cells (EOCs) and human primary CD11b+-monocyteswere prepared from isolated mononuclear cells of human buffy coats using a Ficollgradient (Lympholyte-M, Cedarlane). EOCs were generated as described before [16].CD11b+-monocytes were further isolated by magnetic bead separation using conjugatedmonoclonal rat anti-mouse-CD11b-antibody (MACS MicroBeads, Miltenyi Biotec).Human primary CD11b+-monocytes were counted and grown in RPMI 1640 media(Gibco, Life Technologies, Paisley, Scotland) with M-CSF and 2 mM L-Glu (Invitrogen,Karlsruhe, Deutschland) at 37 °C, 21% O2, 5% CO2 and 95% H2O before initiation ofco-culture experiments.

To elucidate the findings of in vivo transfusion experiments with IFNγ- and IL4-stimulated monocytes, we evaluated the response of EOC regenerative cellular propertiesdepending on co-cultured monocyte phenotypes or their preconditioned media in vitro.EOC regenerative properties included proliferation, differentiation, apoptosis, and tubeformation as a functional test of angiogenesis in vitro. To gain monocyte phenotypescultured primary monocytes were further stimulated with INFγ (100 ng/mL) or IL4(10 ng/mL) for 24 h on day 4. On day 5 the monocytes were washed, collected in bufferand resuspended according to the protocol of the co-culture experiments in vitro. Forexperiments, EOCs were seeded into gelatine-coated 24-well plates and grown to 50%confluence.When initiating the co-cultures, a total of 0.5mL of EBM-2medium containing1 × 106 CD11b+-monocytes were added to EOCs and the cells were further cultured for48 h. To test paracrine effects preconditioned media (PCM) from supernatant ofCD11b+-cells were added to EOC and the EOCs were cultured for further 48 h. Volumesof 50 μL (low) and 100 μL (high) PCM were applied. Control wells containing separatelycultured cells were handled in the sameway. To evaluate proliferation and differentiationstatus of EOCs we performed anti-vWF- and anti-ki-67-immunohistochemistry. Rabbitanti-vWF-antibody (diluted 1:100, DakoCytomation, Glostrup, Denmark) and mouseanti-ki-67-antibody (1:100, DakoCytomation, Glostrup, Denmark) were used inco-culture experimentswith primary CD11b+-monocytes. Evaluation of EOC regenerativeproperties after co-culture with IFNγ- and IL4-stimulated CD11b+-monocytes or theirPCM included proliferation by anti-BrdU-immunohistochemistry, differentiation byanti-vWF-immunohistochemistry, apoptosis by anti-Annexin-V-immunohistochemistry,and tube formation by the tube formation assay (Ibidi, Martinsried, Germany). As second-ary antibodies, a Cy2-conjugated anti-rabbit-antibody and a Cy3-conjugated anti-mouse-antibody (all diluted 1:400, all from Dianova, Hamburg, Germany) were employed.Cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.2%Triton-X (Roth) and blocked with 5% donkey serum (Vector Laboratories). Primaryantibodies were incubated for 2 h; secondary antibodies were incubated for 1 h at roomtemperature. For nuclear staining, Hoechst-33258 (1 μg/mL; Sigma-Aldrich, Taufkirchen,Germany) was applied to the sections for 15 min. Stained samples were mounted withfluorescence mounting medium (Sigma-Aldrich, Taufkirchen, Germany) and studiedwith a Zeiss Axiovert 200 microscope (Carl Zeiss Jena, Germany) equipped with anApoTome and an AxioCam MRc5. Images were acquired with Zeiss AxioVision softwareand processedwith Corel Graphics Suite. Immunohistochemistrywas used to characterizeand quantify the different cell types in culture. All quantitative analyses were carried outby randomly taken photographs of several samples. For classification of differentiating orproliferating EOCs we used the same criteria as reported earlier: ki-67/BrdU-negativeand vWF-positive cells were considered as differentiating cells; ki-67/BrdU-positiveand vWF-negative cells represented proliferating cells. Percentages of proliferating,differentiating or apoptotic EOCs were calculated by dividing their number by thetotal number of Hoechst-positive nuclei. Quantification of tube formation was performedas recommended by the assay protocol. For each condition,five pictures (20×magnification)of three independent experiments were recorded and analyzed.

2.10. Statistical methods

Data are presented as mean ± SEM. Statistical analysis was performed using theANOVA test followed by the Newman–Keuls post hoc analysis. p b 0.05 indicatesstatistical significance.

3. Results

3.1. Clodronate-induced monocyte destruction deceleratesreendothelialization in WT mice

To evaluate the biological effect of endogenous monocytes onreendothelialization after perielectric vascular injury, WT-mice weretreated with liposome-encapsulated clodronate (15 mg/kg) in orderto deplete the pool of circulating monocytes. The reendothelializedareas in percent of the damaged endothelial area were determinedby Evans blue staining in a whole vessel preparation five days afterinduction of EC damage (Fig. 1). Evans blue staining of the injuredvessel revealed that clodronate induced depletion of endogenousmonocytes was associated with a significant decrease in reendothelial-ization comparedwith untreatedWT-mice (Fig. 1; 26± 3% vs. 40± 5%,p b 0.05, n = 5/group).

3.2. CD11b+-monocytes improve endothelial function in ApoE−/−-mice

Focusing on CD11b+-monocytes, we hypothesized that systemicapplication of exogenous CD11b+-monocytes potentially enhancesregeneration processes of the diseased endothelium and therebyconsequently limits abnormalities in vasoreactivity. We tested thishypothesis in an established model of endothelial dysfunctionusing ApoE−/−-mice which were fed on a 5-week cholesterol-richdiet. Endothelium-dependent vasodilatation was profoundly im-paired in hypercholesterolemic ApoE−/−-mice (Fig. 2) in contrastto WT-mice (maximal relaxation: ApoE−/−-mice 92.2 ± 3.7% versusWT-mice 25.5 ± 2.7%, p b 0.05). Endothelium-independent vasore-laxation induced by nitroglycerin was comparable between groups(data not shown). Next, ApoE−/−-mice received three repetitivetransfusions of either spleen-derived CD11b+-monocytes or CD11b−-mononuclear cells (MNCs). ControlWT-micewere treatedwith normalsaline solution without cells intravenously on three consecutive days.Endothelial function of aortic segments was assessed seven days afterthe last transfusion. Transfusion of CD11b+-cells, significantly im-proved endothelium-dependent vasodilatation after seven days,although complete recovery of endothelial function was not achieved(Fig. 2; maximal relaxation: ApoE−/−-mice transfused with CD11b+-monocytes 64.0 ± 1.9% vs. ApoE−/−-mice with control 92.2 ± 3.7%, pb 0.05). In contrast, no significant improvement in endothelial functionwas noted in ApoE−/−-mice transfused with CD11b−-MNCs or saline-treated ApoE−/−-mice (Fig. 2; maximal relaxation: ApoE−/−-micetransfused with CD11b−-MNCs 95.7 ± 2.3% vs ApoE−/−-mice withcontrol, 9.2.2 ± 3.7%). Endothelium-independent vasorelaxation wasnot altered between groups (data not shown).

3.3. CD11b-depleted MNCs do not reduce neointima formation after CCAI

Impaired reendothelialization is associated with enhanced neo-intima formation. Therefore, we evaluated neointima formationafter perielectric vascular CCAI and transfusion of unfractionatedspleen-derivedMNCs or CD11b-negativeMNCs.Morphometric analysisshowed increased neointima formation after transfusion of CD11b-depleted MNCswhile transfusion of unfractionatedMNCs containingCD11b+-cells resulted in a significant reduction in neointimaarea (Fig. 3; CD11bdepleted MNCs: 5 × 105 ± 0.9 × 105 μm2 vs. MNCs2.3 × 105 ± 0.4 × 105 μm2, p b 0.05).

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Fig. 1. Experimental setting of clodronate liposome (CL) injection inWT-mice. Liposomal clodronate (15 mg/kg)was injected on day−1 until day+5 every 24 h. Organswere harvestedon day 5. The percentage of reendothelialized area was quantified. Depletion of endogenous monocytes by CL results in delayed endothelial reendothelialization after denudation of theendothelium of the CCA compared to untreated control WT mice (WTCL: 26 ± 3 vs. WTuntreated: 40 ± 5%, p b 0.05, n = 5/group).

83U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

3.4. CD11b+-monocytes increase reendothelialization in CD11b-DTR-mice

Focusing on CD11b+-monocytes, we hypothesized that systemicdepletion of endogenous CD11b+-monocytes potentially inhibits thevascular repair process of the injured endothelium. To evaluate thesepostulated effects of CD11b-positive monocytes depletion on reendo-thelialization, we performed an endothelial common carotid arteryinjury (CCAI) in a CD11b-DTR-transgenic mouse model, wherebyadministration of DT selectively depletes endogenous monocytes andmacrophages [11]. Immediately, 24, and 48 h after CCAI, DT or vehicletreated animals received 1 × 106 CD11b-positive cells or cell free salinevia tail vein injection (Fig. 4). DT treatment resulted in an 81 ± 6%depletion of endogenous CD11+-monocytes after application of 15 ng

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Fig. 2. CD11b+-monocytes improve endothelial function in ApoE−/−-mice. Endothelium-depcompared to WT-mice (maximal relaxation: ApoE−/−control: 92.2 ± 3.7% vs. WT-mice 23.3 ±ApoE−/−-mice had beneficial effects on endothelial function demonstrated by improved eto ApoE−/−-mice treated with cell-free saline control (ApoE−/− + CD11b+: 64.0 ± 1.9% vsCD11b-depleted-cell-population had no beneficial effect on vasoreactivity in ApoE−/−-mice (m

DT i.p./g bodyweight (BW) (Suppl. Fig. 2). Rescue experiments showedthat systemic transfusion of WT-CD11b+-monocytes in CD11b-DTR-mice treated with either vehicle or DT resulted in a comparableenhancement of reendothelialization. Compared to control animals(Fig. 4; CD11b-DTR-mice+vehicle 44 ± 3% vs. CD11b-DTR-mice+DT 31 ±2%, p b 0.05) morphometric analysis of Evans blue staining showedsignificantly increased reendothelialization in CD11b+-monocyte trans-fused animals with no significant differences between the vehicle or DTtreated groups (Fig. 4; CD11b-DTR-mice+vehicle + WT-CD11b+-monocytes 69 ± 4% vs. CD11b-DTR+DT + WT-CD11b+-monocytes 71 ±2%, p N 0.05). Transfusion of mice with CD11b-depleted-WT-MNCstreated with either vehicle or DT resulted in no significant beneficialeffect on reendothelialization compared to controls but significant

* p < 0.05 vs. ApoE-/--mice control#p < 0.05 vs WT-mice control

n = 4/group

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endent vasodilatation was profoundly impaired in hypercholesterolemic ApoE−/−-mice2.7%, p b 0.05). Systemic application of mature CD11b+-monocytes in splenectomized

ndothelium-dependent vasodilation 7 days after the first cell transfusion compared. ApoE−/−control: 92.2 ± 3.7%, p b 0.05). Intravenous application of the WT-derivedaximal relaxation: ApoE−/− + CD11b−: 95.7 ± 2.3% vs. ApoE−/−control: 92.2 ± 3.7%).

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CD11bneg MNC Control

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Fig. 3. Neointima formation after CCAI. Experimental setting and transfusion regimen to evaluate the impact of CD11b+-monocytes on neointima formation. An endothelial CCAI wasperformed in a CD11b-DTR-transgenic mouse model. DT-treated CD11b+-DTR-mice received intravenously either spleen-derived wildtype mononuclear cells (MNCs) or wildtypeCD11b-depleted MNCs after injury of the CCA. Morphometric analysis showed increased neointima formation after transfusion of CD11b-depleted MNCs while transfusion ofunfractionated MNCs containing CD11b+-cells resulted in a substantial reduction in neointima area indicating decreased re-endothelialization potential without the CD11b-positivemonocyte subset (CD11bdepleted MNCs: 5 × 105 ± 0.9 × 105 μm2 vs. MNCs 2.3 × 105 ± 0.4 × 105 μm2, p b 0.05).

84 U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

less reendothelialization compared to transfusion of WT-CD11b+-monocytes (Fig. 4; CD11b-DTR-mice+vehicle + CD11b-depleted-WT-MNCs 53 ± 7% vs. CD11b-DTR+DT + CD11b-depleted-WT-MNCs39 ± 9%, p N 0.05). Therefore, transfusion lacking WT-CD11b+-monocytes significantly reduced reendothelialization, especially whenDT was applied to deplete endogenous CD11b+-monocytes. Theseresults demonstrate that depletion of endogenous CD11b+-monocytes

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Fig. 4. CD11b+-monocytes increase reendothelialization in CD11b-DTR-mice. Experimental sCD11b+-monocytes on the reendothelization process. Monocyte-depleted-CD11b+-DTR-animacontrol animals (CD11b-DTR-mice+vehicle 44 ± 3% vs. CD11b-DTR-mice+DT 31 ± 2%, p b 0.05) mization in WT-CD11b+-monocyte transfused animals with no significant differenceCD11b+-monocytes 69 ± 4% vs. CD11b-DTR+DT + WT-CD11b+-monocytes 71 ± 2%, pvehicle or DT resulted in no significant beneficial effect on reendothelialization compared to cmonocytes (CD11b-DTR-mice+vehicle + CD11b-depleted-WT-MNCs 53 ± 7% vs. CD11blacking WT-CD11b+-monocytes significantly reduced reendothelialization, especially wh

significantly delays reendothelialization, which can be rescued byexogenous applied CD11b+-monocytes.

3.5. IFNγ-stimulated CD11b+-monocytes increase endothelial dysfunction

Focusing on the subpopulations of CD11b+-monocytes, we evaluat-ed the endothelial function of transfused IFNγ- and IL4-stimulated

Vehicle DT

+ CD11bdepleted

WT-MNC+ CD11b+

WT-MNC

Vehicle DT Vehicle DT

*

*p < 0.05n = 5/group

**

**

etting and transfusion regimen to further determine the effect of systemic transfusion ofls were treated with CD11b+-monocytes after perielectrical vascular injury. Compared toorphometric analysis of Evans blue staining showed significantly increased reendothelial-s between the vehicle or DT treated groups (CD11b-DTR-mice+vehicle + WT-N 0.05). Transfusion of mice with CD11b-depleted-WT-MNCs treated with either

ontrols but significant less reendothelialization compared to transfusion of WT-CD11b+--DTR+DT + CD11b-depleted-WT-MNCs 39 ± 9%, p N 0.05). Therefore, transfusionen DT was applied to deplete endogenous CD11b+-monocytes.

Transfusion of INF -stimulated CD11b+-WT-monocytes

Transfusion of IL4-stimulated CD11b+-WT-monocytes

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Fig. 5. Transfusion of CD11b+-subpopulations to ApoE−/−-CD11b-DTR-mice. Systemic application of IFNγ- and IL4-stimulated CD11b-positive monocytes was performed after depletionof endogenous CD11b+-monocytes in splenectomized ApoE−/−-CD11b-DTR-mice. ApoE−/−-CD11b-DTR-micewere put on a 5-week cholesterol-rich diet leading to profoundly impairedendothelium-dependent vasodilatation. Administration of DT selectively depleted endogenous monocytes and macrophages. Depleted ApoE−/−-CD11b-DTR-mice received threerepetitive transfusions of IFNγ-stimulated or IL4-stimulated CD11b+-monocytes. Control animals were treated with cell-free saline solution intravenously on three consecutive days.Endothelial function of aortic segments was assessed seven days after the last transfusion. Transfusion of IFNγ-stimulated CD11b+-cells significantly decreased endothelium-dependent vasodilatation after seven days (maximal relaxation: ApoE−/−-CD11b-DTR-mice transfused with IFNγ-stimulated CD11b+-monocytes: 83.0 ± 1.9% vs. IL4-stimulatedCD11b+-cell transfusion: 67.7 ± 1.3% or ApoE−/−-CD11b-DTR-mice controls: 69.2 ± 1.7%, p b 0.05). In contrast, no significant influence on endothelial function was notedafter IL4-stimulated CD11b+-cell transfusion to ApoE−/−-CD11b-DTR-mice compared to control (maximal relaxation: ApoE−/−-CD11b-DTR-mice transfused with IL4-stimulatedCD11b+-cell: 67.7± 1.3% vs ApoE−/−-CD11b-DTR-mice with cell-free saline: 69.2± 1.7%). Endothelium-independent vasorelaxationwas not altered between groups (data not shown).

85U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

CD11b+-monocytes after depletion of endogenous CD11b+-monocytesin an animal model of ApoE−/−-CD11b-DTR-mice.

ApoE−/−-CD11b-DTR-mice were fed on a 5-week cholesterol-richdiet. Administration of DT selectively depleted endogenous monocytesand macrophages. Endothelium-dependent vasodilatation was pro-foundly impaired in depleted hypercholesterolemic ApoE−/−-CD11b-DTR-mice (Fig. 5). Depleted ApoE−/−-CD11b-DTR-mice received threerepetitive transfusions of IFNγ-stimulated or IL4-stimulated CD11b+-monocytes. Control animals were treated with cell-free saline solutionintravenously on three consecutive days. Endothelial function of aorticsegmentswas assessed seven days after the last transfusion. Transfusionof IFNγ-stimulated CD11b+-cells significantly decreased endothelium-dependent vasodilatation after seven days (Fig. 5; maximal relaxation:ApoE−/−-CD11b-DTR-mice transfused with IFNγ-stimulated CD11b+-monocytes 87.0 ± 1.9% vs. IL4-stimulated CD11b+-cell 67.7 ± 1.3%or ApoE−/−-CD11b-DTR-mice with cell-free saline, 69.2 ± 1.7%,p b 0.05). In contrast, no significant influence on endothelial functionwas noted after IL4-stimulated CD11b+-cell transfusion to ApoE−/−-CD11b-DTR-mice compared to control (Fig. 5; maximal relaxation:ApoE−/−-CD11b-DTR-mice transfused with IL4-stimulated CD11b+-cell 67.7 ± 1.3% vs ApoE−/−-CD11b-DTR-mice with cell-free saline,69.2 ± 1.7%). Endothelium-independent vasorelaxation was notaltered between groups (data not shown). These results demon-strated that WT-derived CD11b+-monocytes stimulated by IFNγdecreased endothelial function, whereas IL4-stimulated mono-cytes had no detectable effect on vascular function within thisexperimental setting.

3.6. CD11b+-monocytes are located to the injury site in CD11b+-DTR-mice

Using bioluminescent imaging, transfused CD11b+-monocytesspecifically expressing the luciferase gene were tracked in vivo per-mitting analysis of monocyte dynamics in living CD11b-DTR-micein realtime (Fig. 6). Transfused CD11b+-monocytes were restrictedto the injury site at the CCA and application side after tail veininjection in monocyte-depleted-CD11b+-DTR-mice (Fig. 6F–J). Whilebioluminescence at the application side was detectable up to threedays after application (Fig. 6I), bioluminescence at the CCA injury sidewas found up to five days after injury (Fig. 6J, arrow), demonstrating

that transfused circulating CD11b+-monocytes reach and stay atthe area of endothelial repair under physiological flow conditionsin living mice.

3.7. Mode of interaction and monocyte phenotype differentially affectregeneration properties of EOCs in vitro

Co-culture experiments were used to examine the influence ofprimary CD11b+-monocytes on endothelial outgrowth cell prolifer-ation and differentiation (Fig. 7). Cell proliferation marker ki-67 anddifferentiation marker vWF supporting maturation were examined.While incubation of an immature starting population of EOCs withpreconditionedmedium (PCM) of CD11b+-monocytes showed a sig-nificant increase of proliferation pointing towards a paracrine stimu-lation, co-culture of EOC in the presence of CD11b+-monocytesresulted in a significant higher number of differentiating EOCs thanin untreated control cells. In vitro these co-culture experimentsrevealed increasing differentiation potential of EOCs co-culturedwith CD11b+-monocytes demonstrating that circulating CD11b+-monocytes might contribute to endothelial cell regeneration andrestoration associated with an improved vascular function.

To elucidate the findings of in vivo transfusion experimentswith IFNγ- and IL4-stimulated monocytes, we evaluated the responseof EOC regenerative cellular properties depending on co-culturedmonocyte phenotypes or their preconditioned media in vitro. Evalu-ation of EOC regenerative properties after co-culture with IFNγ- andIL4-stimulated monocytes or their PCM included proliferationby anti-BrdU-immunohistochemistry, differentiation by anti-vWF-immunohistochemistry, apoptosis by anti-Annexin-V-immunohis-tochemistry, and tube formation by the tube formation assay.Upon co-culture the IFNγ-induced CD11b+-monocyte phenotypeshowed increased numbers of apoptotic EOCs independent of directcellular (Fig. 8A, black column) or paracrine interactions (Fig. 8B,black columns), whereas no effect on EOC-apoptosis was detectedby IL4-stimulated monocytes (Fig. 8A, gray column) or its PCM(Fig. 8B, gray columns). In line with increased apoptotic EOCs theproliferation rate was significantly decreased upon co-culture withprior IFNγ-stimulated monocytes (Fig. 8C, black column) or theirPCM (Fig. 8D, black columns). However, PCM of IL-4-stimulated

A B

C

F

K L M N O

G H I J

D E

Fig. 6. Transfused CD11b+-monocytes are located to the injury site. The flow chart of the experimental setting and transfusion regime using bioluminescent imaging to track transfusedCD11b+-monocytes specifically expressing the firefly-luciferase gene in vivo is depicted. Analysis of monocyte dynamics was performed in living CD11b+-DTR-mice in realtime.Bioluminescence images were obtained on day 0, 1, 2, 3, and 5 post-transplantation. CD11b+-monocytes (panel A: cultured monocytes, bright field; panel B: immunohistochemistry ofmonocytes using anti-CD11b-antibody) were incubated for 20 min at room temperature. Thereafter, cells were washed with PBS and cultured for another 2 days. Luciferase proteinexpression was monitored 24 h and 48 h after transduction (panels C, D and E served as negative control) following interaction with luciferin substrate. Bioluminescent image ofluciferase-positive CD11b+-monocytes post-perielectric injury isolated from WT-mice was transplanted to a syngeneic mouse via tail vein infusion (panels F–J, n = 5). Control micereceived cell-free normal saline (panels K–O, n = 5, images show representative animal). A gray-scale body image was collected and overlaid by a pseudo-color imagerepresenting the spatial distribution of detected photons (panels F–O). Our studies revealed that transfused CD11b+-monocytes were restricted to the injury site inmonocyte-depleted-CD11b-DTR-mice demonstrating that circulating CD11b+-monocytes contribute to early endothelial regeneration (panels F–J, arrow, pseudo-color imagesrepresent luciferase activity). Bar: panel A 100 μm and panel B 50 μm.

86 U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

monocytes induced EOC proliferation in a dose dependent manner(Fig. 8D, gray columns). Tube formation resembling the ability ofEOCs to build up vessel like structures was found significantly in-creased on direct cellular co-culture of IL-4-stimulated monocyteswith EOCs (Fig. 8E, gray column), while co-culture of IFNγ-induced CD11b+-monocytes (Fig. 8E, black column) or PCM(Fig. 8F, black column) reduced this capacity significantly. More-over, differentiation measured by EOC vWF-expression was signifi-cantly reduced upon co-culture with IFNγ-stimulated monocytes(Fig. 8G, black column) or PCM (Fig. 8H, black columns). Conclusive-ly, depending on the prestimulation CD11b+-monocyte (IFNγ orIL4) and the mode of monocyte–EOC-interaction (paracrine orcell–cell-interaction) we found differentially affected regenerationproperties of immature EOCs. To investigate the elevated prolifera-tion rates of EOCs cultured in PCM of IL4-stimulated monocytes wemeasured the concentration of monocyte-derived vascular endothe-lial growth factor (VEGF) and hepatocyte growth factor (HGF), bothknown to effect EC proliferation and survival. We found significanthigher VEGF concentrations in PCM of IL4-stimulated monocytescompared to IFNγ-stimulated monocytes (Suppl. Fig. 3A), but signif-icant changes in HGF concentrations were not found (Suppl. Fig. 3B).These findings suggest that IL4-stimulated monocytes do release

soluble VEGF when cultured in vitro, mediating independent paracrineeffects in EOCs without direct cell–cell-contact.

4. Discussion

Although the importance of monocytes in atherogenesis isreasonably well established, at least in rodent models potentialroles of monocytes in the setting of vascular regeneration and resolu-tion of inflammation have been less-well explored. Herewe could dem-onstrate that CD11b+-monocytes play an important role in endothelialcell recovery after acute and chronic endothelial injury. Monocyteshome to the site of vascular injury enhance reendothelialization andimprove endothelial function, most likely by inducing paracrine andcell-differentiation processes. Apparently monocytes have divergentroles in the vascular compartment [2,3,8]. During atherogenesisconfounding factors such as dyslipidemia, hypertension or pro-inflammatory mediators cause activation of the endothelium leadingto increased adhesion and migration of circulating monocytes. By up-take of oxidized lipoproteins they mature into pro-inflammatorymacrophages and convert to foamcellsmaintaining chronic vascular in-flammation. Studies inmice provided evidence on the important role ofendothelial activation on endothelial- and monocyte–cell-interactions

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Fig. 7. Endothelial outgrowth cells (EOCs) and CD11b+-monocyte co-culture. Co-culture experiments were used to examine the influence of primary CD11b+-monocytes orpreconditioned medium (PCM) of primary CD11b+-monocyte on immature endothelial outgrowth cell (EOC) proliferation and differentiation. Cell proliferation marker ki-67(panels A–D, green) and differentiation marker vWF (panels A–D red, nuclei Hoechst blue) were examined. While incubation of EOCs with PCM of CD11b+-monocytes (panels Band C) showed a significantly increase of proliferation (panel E) pointing towards a paracrine stimulation, co-culture of EOCs in the presence of CD11b+-monocytes (panel D) resultedin a significant higher number of differentiating EOCs than in untreated control cells (panel F). In vitro these experiments revealed increasing differentiation potential of EOCs co-cultured in the presence of CD11b+-monocytes (panel F). Culturing of EOCs in CD11b+-cell-preconditioned medium increased the rate of proliferation in EOCs pointing towards a para-crine stimulation of proliferation (panel E). Bar: 100 μm.

87U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

in the development of endothelial dysfunction and atherosclerosis[17]. Thus, the interaction between endothelium and the monocyte–macrophage-system (MMS) plays an important role in acute vascularinflammation and its resolution as well as its transition into chronicinflammation. These contradictory roles of monocytes might be ex-plained by the involvement of different subsets of monocytes. In ourstudy, we focused on the role of CD11b-positive monocytes and theirpro- or anti-inflammatory function on vascular reendothelializationafter acute (focal vascular injury) and chronic (disseminated vasculardisease) endothelial injury.

In the present study, we could demonstrate that intravenousapplication of CD11b-positive cells improved endothelium-dependentvasodilation suggesting that this cell-based treatment can limit abnor-malities in vasoreactivity associated with atherosclerotic plaque devel-opment. Here we induced elimination of CD11b+-monocytes andmacrophages via the administration of DT to transgenic CD11b+-DTR-mice expressing the diphtheria toxin receptor under the control of theCD11b-promoter to analyze their role in vascular repair and recoveryof the endothelial layer [11]. Depletion of CD11b+-cells resulted insubstantial disruption of the endothelial repair process and reductionof vascular healing. Our experimental design of the mice modelsgave further evidence on the contribution of CD11b+-monocytes/macrophages to the vascular healing process. We administeredDT around the time of an induced CCAI to deplete endogenousmonocytes and macrophages during the initial inflammatory re-sponse to injury. CD11b+-cell depletion during the early phaseafter CCAI resulted in decreased endothelial reendothelialization.Transfusion of WT-CD11b+-monocytes, not vulnerable to DT, wasassociated by increased reendothelialization of the induced injury.

To examine the fate of intravenously transfused CD11b+-monocytes, we performed in vivo tracking of transgenic luciferase

gene expressing CD11b+-monocytes specifically, thereby permittinganalysis of monocyte origin and dynamics in living monocyte depletedCD11b+-DTR-mice in realtime. Our studies revealed that the transfusedCD11b+-monocytes were restricted to the injury site five days afteracute injury in monocyte-depleted-CD11b+-DTR-mice demonstratingthat beneficial effects on endothelial regeneration are potentiallymediated by local CD11b+-monocyte actions. Evidence on theimportance of monocytes/macrophages to the recovery of vascularinjuries has been obtained from other experimental studies inmacrophage-deficient mice [18–20]. These studies found reducedand abnormal vascularization in macrophage-depleted wounds.

Using in vitro experiments we analyzed proliferation and differenti-ation characteristics of immature human endothelial precursor cells(EOCs) which were incubated with precondition CD11b+-monocytemedium or co-cultured in the presence of CD11b+-monocytes. It hasbeen demonstrated that monocytes and ECs interact in vascular repairand that the one cell type can induce proliferative effects, survival,and phenotypic changes in the other [21]. Indeed, EOCs cultured inCD11b+-monocyte conditioned medium displayed approximately2-fold increased proliferation measured by ki-67 expression andreduced vWF expression. Increased differentiation of EOCs measuredby vWF expression was observed after direct cell–cell interactionindicating a differential role for direct cellular and indirect paracrineinteraction between CD11b+-monocytes and EOCs in vascular repair.

To elucidate the findings of in vivo transfusion experimentswith IFNγ- and IL4-stimulated monocytes, we evaluated the responseof EOC regenerative cellular properties depending on co-culturedmonocyte phenotypes or their preconditioned media in vitro.Evaluation of EOC regenerative properties after co-culture withIFNγ- and IL4-stimulated monocytes or their PCM included prolif-eration by anti-BrdU-immunohistochemistry, differentiation by

88 U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

anti-vWF-immunohistochemistry, apoptosis by anti-Annexin-V-immunohistochemistry, and tube formation by the tube formationassay. Depending on the pre-stimulation of CD11b+-monocyte(IFNγ or IL4) and the mode of monocyte–EOC-interaction (paracrineor cell–cell-interaction) we found differentially affected regenerationproperties of immature EOCs. Our findings suggest that depending onthe monocyte phenotype factors involved in EOC proliferation andfunction modulate EC survival and function. One of the EOC protec-tive paracrine factors might be VEGF, which we found to be highlyexpressed in IL4-stimulated CD11b+-monocytes but not in IFNγ-stimulated CD11b+-monocytes. However, the influence of otherunidentified or unknown paracrine molecules underlying the pro-angiogenic effects has not been proven and one must admit that manyof these properties are also shared by other growth factors or cytokines.

Monocytes do release soluble growth inducing factors (e.g. VEGF)when cultured mediating independent effects without direct cell–cell-contact but also mediate contact-dependent effects on EOCs. In anearly phase after endothelial denudation initializing of endothelialrecovery coincides with adhesion of monocytes to the denuded arterialsurface [22]. By secretion of soluble growth factors monocytes augmentEC proliferation, spreading and migration preparing the occurrence of asecond phase of vascular reendothelization in which replenished ECs

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Fig. 8.Mode of interaction and monocyte phenotype differentially affect regeneration propIFNγ- and IL4-stimulated monocytes or their PCM included proliferation by anti-BrdU-imby anti-Annexin-V-immunohistochemistry, and tube formation by the tube formationincreased numbers of apoptotic EOCs, independent of direct cellular (panel A, black column)detected by IL4-stimulatedmonocytes (panel A, gray column) or its PCM (panel B, gray columnupon co-culture with prior IFNγ-stimulated monocytes (panel C, black column) or their PCM (peration in a dose dependent manner (panel D, gray columns). Tube formation resembling the acellular co-culture of IL-4-stimulatedmonocytes with EOCs (panel E, gray column), while co-cucolumn) reduced this capacity significantly. Moreover, differentiation measured by EOC vWF(panel G, black column) or PCM (panel H, black columns).

directly interact with monocytes leading to contact-dependent inhibi-tion of proliferation and EC differentiation. The indicated potent effectof not only primary but also distinct monocyte phenotypes on ECwound closure mediated by increased proliferation and migration ofstimulated ECs into the denuded area is supported by several otherstudies which demonstrated that the soluble factor VEGF secreted byactivated monocytes enhanced EC proliferation and wound closure ininjured EC monolayers [23].

On the molecular level members of the family of Tie-2 ligandssuch as angiopoietin-1 and -2 have been recently shown to beinvolved in the monocyte–EC-interaction during vessel remodelingand regeneration [6]. It has been reported that on direct physical in-teraction of unactivated monocytes with EC, monocytes secretedhigh levels of Ang-1 leading to a transient activation of endothelialTie2, contributing to the regulation of EC survival. The selectivesecretion of Ang-1 or Ang-2 may be regulated by the monocytephenotype, as preactivation of monocytes with INFγ abolished thesecretion of Ang-1 and increased the secretion of Ang-2. Moreover,a previous study in which soluble Tie2 receptor served as a neutralizingcompetitive antagonist for Ang-1 the protective effect of Ang-1 on ECwas reversed, further supporting the role of monocytes/Ang-1 andAng-2 in the regulation of EC survival [6].

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erties of EOCs in vitro. Evaluation of EOC regenerative properties after co-culture withmunohistochemistry, differentiation by anti-vWF-immunohistochemistry, apoptosisassay. Upon co-culture the IFNγ-induced CD11b+-monocyte phenotype showed

or paracrine interactions (panel B, black columns), while no effect on EOC-apoptosis wass). In line with increased apoptotic EOCs the proliferation rate was significantly decreasedanel D, black columns). However, PCM of IL-4-stimulated monocytes induced EOC prolif-bility of EOCs to build up vessel like structures was found significantly increased on directlture of IFNγ-induced CD11b+-monocytes (panel E, black column) or PCM (panel F, black-expression was significantly reduced upon co-culture with IFNγ-stimulated monocytes

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Fig. 8 (continued).

89U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

Another concept of interaction with increasing evidence is that thewithdrawal of positive effectors such as VEGF is sufficient to result inblood vessel regression in various in vivo systems including tumorsand developing organs [24]. These studies demonstrate the presenceof an inverse correlation between VEGF expression and the levels ofvessel recovery, implicating VEGF as an important survival factor forendothelial cells. A number of different growth factors have beenshown to rapidly activate Akt dependent intracellular signaling mecha-nisms via PI3-kinase activation, thereby promoting survival of ECs andother cell types [24]. In ECs VEGF survival signals are known to be me-diated by the Flk-1/KDR receptor through the PI3-kinase/Akt signaltransduction pathway [24]. Additionally, it was reported that secretionof hepatocyte growth factor (HGF) of primary unactivated monocytesmay also contribute to the protective effect on EC [25]. The ability toactivate Akt has been described not only for VEGF but also for differentother growth factors, such as platelet derived growth factor, epidermalgrowth factor, bFGF, insulin, and insulin-like growth factor 1 [24],yielding attractive targets for pharmacological therapy aimed tooptimize endothelial cell regeneration. Moreover, an anti-apoptoticcontact dependent effect of monocytes has been reported by theupregulation of bcl-2 homologue A1 in EC [6].

We and others demonstrated the key role of monocyte-inducedproliferation and differentiation of EC in the reendothelization processof the endothelial monolayer. It is reasonable that in case of acuteendothelial injury mature monocyte subsets dominate in earlyacute vascular reconstitution and may subsequently provide paracrinesignals to initiate proliferation of immature endothelial and endothelial

precursor cells followed by cellular induction of endothelial cell differ-entiation restoring vascular homeostasis while limiting potentialluminal obstruction and intimal hyperplasia. Our findings provideevidence that VEGF survival signals in immature endothelial cells aremediated by IL4-stimulated monocyte phenotype enhancing EOC pro-liferation by paracrine mechanisms and EOC function by direct cellularcontacts, while INFγ-stimulated monocytes induce paracrine and cellu-lar interactions leading to endothelial cell apoptosis and regression oftube formation in vitro and endothelial function in vivo. It is generallyaccepted that the tissue microenvironment determines macrophagephenotypic polarization. Here we found that IL4-induced polarizationof primarymonocytes and co-cultivationwith subconfluent endothelialcells resulted in transient secretion of VEGF frommonocytes and the ac-tivation of endothelial survival genes. This effect was abolished by co-culturing monocytes after IFNγ-induced polarization. Although primarymonocytes had some effects on endothelial regenerative properties,both polarization states showed more intense interactions with EOCs. Inconclusion,monocyte subsets differentially affect endothelial cell survivaland play a crucial role in vascular remodeling and homeostasis.

4.1. Study limitations

An intrinsic problem of mouse models in the cardiovascular contextis their divergence from the human organism, both with regard toresponse to inflammatory stimuli [26], and to the different activationand paracrine functions of monocyte subpopulations and macro-phage phenotypes between both species. An additional difficulty is

90 U.M. Becher et al. / International Journal of Cardiology 173 (2014) 80–91

represented by the inherent high phenotypic flexibility of myeloidcells, representing a wide spectrum rather than two extreme states.Therefore, interpretation of data generated from these experimentshas to be done with caution: The ex vivo stimulation of monocyteswith IL-4 vs. IFNγ, although routinely used for M1- vs. M2-likemacrophage differentiation ex vivo, might harbor the risk of generatingcell culture artifacts in addition to the possibility of further phenotypicalterations after injection. In this respect it was shown that M2-likemacrophages may exert both anti- and pro-inflammatory actions [27].Moreover, mature monocyte and macrophage subsets may possessthe ability to reconstitute vascular damage by transdifferentiation thathas been described in a subset of CD14+/KDR+myeloid cells previously[28,29]. Two major ways of interaction seem to drive endothelial cell-monocyte cross talk: first, a direct cellular contact-mediated mecha-nism of binding mediated through adhesion molecules of cell–cell-interaction expressed on the surface of activated ECs and circulatingmonocytes contributing to EC differentiation and second, secretion ofsoluble paracrine factors by activated monocytes and macrophagesdriving proliferation and migration of EC suggesting a complex signal-ing network required for normal recovery of vascular homeostasis[30]. Monocytes and macrophages synthesize and release a vast arrayof regulatory molecules relevant for regulatory interactions with othercellular populations at the side of injury temporally changing the cellu-lar assembly at the time and side of injury and disassembled whenrepair is completed [31]. Regulation of vascular regeneration by growthfactors and cytokines encompasses several families of signaling mole-cules associated with different aspects of repair. Multiple individualmolecules are found in each family with broadly overlapping activities.Given that multiple monocyte–macrophage-subsets are involved invascular regeneration and are capable of specifically producingdifferentrelevant cytokines and growth factors for interaction with ECs addsprofound complexity to the physiological process of vascular recov-ery. Thus, pro-angiogenic activity of monocyte subsets is probablyrelated to secretion of angiogenic cytokines. Vice versa, monocytesmay activate surface receptors on ECs by secreting anti-apoptoticfactors thus supporting endothelial cell survival [6]. Cellular orches-tration in tissue recovery is controlled by a variety of cytokines andgrowth factors. Members of the VEGF family and their receptorshave been shown to be prominently expressed in damaged vasculartissue and data outlined secretion of VEGF mainly of activated pro-angiogenic monocytes [31].

The therapeutic infusion of freshly isolated CD11b+-monocytes foraccelerating endothelial regeneration after vascular injury has beencontroversially discussed [32,33]. As monocytes are abundant andeasy to separate, their local therapeutic use in regeneration could beof interest rather in pathological settings such as acute endothelial dam-age after vascular intervention as well as chronic vascular deteriorationin diabetes mellitus, where the function of circulating progenitor cells isoverall compromised [34]. Several published studies have reported thatcultured angiogenic cells are reliable tools in regenerative cardiology[35–37]. These were usually cultured out from peripheral blood mono-nuclear cells and displayed myeloid (CD45+, CD14+, CD16+, CD11b+)along with endothelial (VEGFR2+, CD31+, Tie2+) features, whichpartially correspond to classical and intermediate monocytes [35].Strategies to modulate their number and to augment their regenerativefunction are under investigation [38,39].

The detailed differential mechanisms of interaction betweenprimary CD11b+-monocytes or their subsets and EOCs to gain ECfunction and improve endothelial regeneration remain not to beyet clear. Most probably, there is an additive cross talk betweendifferent monocyte/macrophage subsets interplaying in vasculartissue repair and further efforts are intended to characterize andidentify angiogenic monocyte populations with high pro-angiogeniccapacity. The nature of this monocyte polarization into differentphenotypes deserves our attention because it may provide a key toprophylactic transcriptional control of macrophage differentiation.

5. Conclusion

Our data underline that monocytes not only promote formation,growth and complication of atherosclerotic plaques and are associatedwith plaque vulnerability but also conversely contribute to tissueregeneration by substantial input of distinct CD11b+-monocyte subsets.

Acknowledgments of grant support

U. M. Becher was supported by BONFOR (O-109.0028/O-109.0040).V. Tiyerili was supported by BONFOR (O-109.0033).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ijcard.2014.02.004.

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