International Journal of Cardiology 173 (2014) 80–91

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Distinct CD11b+-monocyte subsets accelerate endothelial cell recovery after 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 b c

Medizinische Klinik und Poliklinik II, Innere Medizin, Universitätsklinikum Bonn, Germany Institut für Pharmakologie und Toxikologie, Universitätsklinikum Bonn, Germany Kardiologische Abteilung, Innere Medizin, Isarklinik München, Germany

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Article history: Received 3 August 2013 Received in revised form 12 January 2014 Accepted 8 February 2014 Available online 20 February 2014 Keywords: Endothelial regeneration CD11b+-monocytes Transfusion CD11b-DTR-mice

a b s t r a c t 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 transfused CD11b+-monocytes after endothelial injury in vivo. CD11b-diphtheria-toxin-receptor-mice – a mouse model in which administration of diphtheria toxin selectively eliminates endogenous monocytes and macrophages – were treated with WT-derived CD11b+-monocytes from age-matched mice. CD11b+-monocytes improved endothelium-dependent vasoreactivity after 7 days while transfusion of WT-derived CD11b−-cells had no beneficial effect on endothelial function. In ApoE−/−-CD11b-DTR-mice with a hypercholesterolemia-induced chronic 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 the endothelial injury site in CD11b-DTR-mice depleted of endogenous monocytes. In vitro co-culture experiments revealed significantly enhanced regeneration properties of human endothelial outgrowth cells (EOCs) when cultured with preconditioned-media (PCM) or monocytes of IL4-stimulated-subsets compared to the effects of IFNγ-stimulated monocytes. Conclusion: CD11b+-monocytes play an important role in endothelial cell recovery after endothelial injury by homing 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 likely by paracrine induction of proliferation and cellular promotion of differentiation. These results underline novel insights in the biology of endothelial regeneration and provide additional information for the treatment of vascular dysfunction. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Effective endothelial restoration after injury requires dynamic orchestration of diverse cell types. In addition to cells derived from the endothelial lineage, cells of the innate immune system display endothelial-regenerating properties. Circulating monocytes are among the first in responding to endothelial damage and are recruited to sites of vascular remodeling after angioplasty or stent implantation [1,2]. Adhesion molecules expressed on the surface of activated endothelial cells (ECs) enable the recruitment of circulating monocytes to sites of endothelial injury and inflammation, followed by migration towards ⁎ Corresponding author at: Medizinische Klinik und Poliklinik II, Universitätsklinikum Bonn, Sigmund Freud Str. 25, 53105 Bonn, Germany. Tel.: + 49 228 287 16025; fax: + 49 228 287 16026. E-mail address: [email protected] (N. Werner).

http://dx.doi.org/10.1016/j.ijcard.2014.02.004 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

distinct cytokine and chemokine gradients [3]. In the inflammatory milieu attracted monocytes mature into foamy macrophages upon ingestion of oxidized LDL particles. Within foam cells production of key pro-inflammatory cytokines beyond necessary levels triggers progression of vascular inflammation and atherogenesis, counteracting resolution of inflammation in later healing stages [4]. However, upon acute EC damage in physiological environment monocytes may shift the balance from chronic vascular deterioration towards induction and maintenance of vascular regeneration and repair. Importantly, interactions between monocytes and ECs regulate endothelial cell survival and proliferation essential for vascular recovery and repair [5,6]. Distinct from the pro-angiogenic and growth promoting cytokines secretion of differentiated macrophages, contact-dependent direct cellular interactions of monocytes are specific to EC survival [5]. This complex interactions of contact-dependent and paracrine stimulated cellular cross-talk early after injury is critical for the recovery and

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maintenance of the endothelial monolayer and its function [5]. Although monocyte plasticity is functionally restricted mainly to conversion into tissue macrophages, subsets with pluripotent plasticity exists and may differentially interact with ECs [7]. More recent work has demonstrated significant heterogeneity among monocytes and their potentially different roles in atherogenesis [8]. Pro-inflammatory monocytes, identified by high expression of the surface marker Ly6C, are CCR2+ [9]. A second monocyte subset identified by low expression of Ly6C is largely CCR2− and thought to be involved in the postinflammatory reparative functions of monocytes [10]. Given the range of vascular modulating populations of circulating monocytes and the controversy effects on the role of monocytes in vascular regeneration and deterioration, we studied the effect of CD11b+-monocytes on vascular function and their destination after transfusion and induced vascular injury in vivo. Furthermore, we analyzed the response of immature human endothelial outgrowth cells (EOCs) after interaction with blood monocytes prior to and after stimulation with IFNγ or IL4 in vitro. To advance the understanding of monocytes in the pathophysiology of vascular inflammation and regeneration we focused on remodeling and repair processes and explored the role of circulating CD11b+monocytes in vascular regeneration of acute and chronic endothelial injuries. We investigated a transgenic ApoE−/−-mouse-model of hypercholesterolemia induced endothelial dysfunction and a transgenic mouse (CD11b+-DTR-mouse) in which diphtheria toxin (DT) conditionally eliminates monocytes and macrophages [11]. Thus, cell specific transgenic expression of human heparin-binding EGF-like growth factor confers DT sensitivity to murine CD11b+-monocytes in vivo. We correlated outcomes of endothelial regeneration after CD11b+ -cell depletion and transfusion in this mouse model and found that CD11b+-monocytes display not only early placeholder function in damaged endothelium but also profoundly affect local endothelial cell recovery in vitro demonstrating that depending on the 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−/−-CD11bDTR-mice (C57BL/6J × FVB/N background) were used for these studies. The animals were maintained in a 22 °C room with a 12-hour light and dark cycle and received drinking water ad libitum. All animal experiments were performed in accordance with institutional guidelines and the German animal protection law. All surgical procedures 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 encapsulated 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) by reverse-phase evaporation technique. This encapsulation allows systemic distribution without adverse effects of the drug until the liposome is taken up by a monocyte or other phagocytic cells. Clodronate is a highly charged substance, not able to pass through the lipid bilayer of the liposome or the cell, so that the influence of clodronate on nonphagocytic cells e.g. endothelial cells is of no or only minor relevance. Clodronate liposomes (CLs) but not free clodronate significantly reduced the number and proliferation of viable cells that phagocytize and degrade the liposome in a dose dependent manner but did not affect vascular smooth muscle cells (VSMCs) or ECs viability and proliferation at concentrations up to 500 μmol/L. Perielectric carotid injury was performed in WT-mice as described previously. Briefly, the mice were anesthetized with 150 mg/kg body weight ketamine hydrochloride (Ketanest®, Pharmacia) and 0.1 mg/kg body weight xylazine hydrochloride (Rompun® 2%, Bayer). The common carotid artery (CCA) was exposed and submitted to an electric injury starting at the level of the bifurcation and continuing to the proximal part of the artery (in total 4 mm denudation).

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2.3. Splenectomy Mice assigned to cell transfusion experiments were anesthetized with 150 mg/kg body weight ketamine hydrochloride (Ketanest, Pharmacia) and 0.1 mg/kg body weight xylazine hydrochloride (Rompun 2%, Bayer). The spleen was dissected through a lateral incision of the left abdomen. Vessels were carefully ligated using 6/0 silk. After removal of the spleen, the abdomen was closed with sutures using 6/0 silk. Animals were allowed to 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, and mononuclear 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 colloidal superparamagnetic microbeads conjugated to monoclonal rat anti-mouse-CD11bantibody (MACS MicroBeads, Miltenyi Biotec). After incubation and additional washing, magnetic cell separation was performed. The collected effluent contained the negative MNC fraction depleted of CD11b+-cells (Suppl. Fig. 1). Attached CD11b+-MNCs were collected in buffer. Separated subpopulations were resuspended in 200 μL of normal saline solution for intravenous injection. Bone marrow from WT-mice was explanted and mechanically minced. About 5 × 106 cells were obtained per mouse and cultured for 72 h in RPMI Medium 1640 (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. On day 4 of culture cells were stimulated with INFγ (100 ng/mL) or IL4 (10 ng/mL) for 24 h. On day 5 the monocytes were washed, collected in buffer and resuspended according to the protocol of the transfusion experiments in vivo. Using this standard method we routinely achieve 95% purity. This method allows by simple means the generation of high numbers of murine monocytes of classical 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 diet that 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 INFγor IL4-stimulated CD11b+-MNCs (ApoE−/−-CD11b-DTR-mice, Fig. 5) by intravenous tail vein injection on three consecutive days. Control animals received a corresponding amount of cell-free saline (Figs. 2, 3, 4, 5 and 6). On day 7 after i.v. treatment, endothelial function of aortic ring segments of the thoracic aorta was analyzed. Vasodilatation and vasoconstriction of isolated aortic ring preparations were determined in organ baths 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-wildtype mice as described previously [13]. Immediately, 24 and 48 h after induction of CCAI mice received either 1 × 106 spleen-derived MNCs or CD11b-depleted spleen-derived by intravenous tail vein injection (see Fig. 3). Control animals received a corresponding amount of normal saline (see Fig. 3). Animals were allowed to recover. Considerable time was spent to assure rigid standardization of all conditions and manual manipulations. One highly trained investigator adapted to the microsurgical approach carried out the carotid artery injury and performed and evaluated more than 100 operations. To evaluate neointima formation, perfusion-fixed carotid arteries were harvested on 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. For morphometric analyses Lucia Measurement Version 4.6 software was used to measure external elastic lamina, internal elastic lamina, and lumen circumference as well as medial and neointimal area of 25 sections per animal. An investigator blinded to the type of experimental 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 endothelial regeneration after CCAI, we used a transgenic mouse (CD11b-DTR-mice) in which application of DT conditionally ablates monocytes and macrophages. Using this system, DT treatment reduced the endogenous monocytes population by 81 ± 6% confirmed by FACS analysis (Suppl. Fig. 2). CD11b-DTR-mice treated with DT received either vehicle or DT to suppress endogenous monocyte population. CD11b-DTR-mice treated with DT (15 ng/g bw) received 1 × 106 spleen-derived luciferase expressing CD11b + -monocytes by intravenous tail vein injection immediately, 24, and 48 h after induction of CCAI (Fig. 6). Control animals received a corresponding amount of normal saline by intravenous tail vein injection. Luciferase expressing CD11b +cells were transduced with a HIV-derived lentiviral vector (LV) carrying a CMV promoter-driven luciferase expression cassette. The lentivector carrying the fireflyluciferase gene under control of the CMV promoter (CMV-Luc) was kindly provided by Inder Verma (Salk Institute, La Jolla, California 92037, USA) and the production

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

2.10. Statistical methods Data are presented as mean ± SEM. Statistical analysis was performed using the ANOVA test followed by the Newman–Keuls post hoc analysis. p b 0.05 indicates statistical significance.

3. Results 2.8. Reendothelialization in CD11b-DTR-mice For assessment of reendothelialization, common carotid arteries were submitted to a perielectric injury as described previously [15]. Vessel preparation was started at the bifurcation and continued to the proximal part of the artery. Care was taken in applying as little mechanical pressure to the vessel as possible; electrical pulses lasted 4 s with 2 W. Immediately, 24, and 48 h after induction of the perielectric CCAI vehicle and DT treated CD11bDTR-animals received either 1 × 106 spleen-derived CD11b+-monocytes by intravenous tail vein injection (Fig. 4). Control animals received a corresponding amount of normal saline by intravenous tail vein injection at corresponding time points (Fig. 4). The denuded area was determined on day 5 after intravenous injection of 50 μL Evans blue in an enface preparation of the vessel. Complete vessel area and Evans blue stained denuded areas were measured using AxioVision version 4.5.0 software. The percentage of reendothelialized area was quantified. Perielectric carotid artery injury was induced as described previously.

2.9. Cell cultures and co-culture experiments Human endothelial outgrowth cells (EOCs) and human primary CD11b+-monocytes were prepared from isolated mononuclear cells of human buffy coats using a Ficoll gradient (Lympholyte-M, Cedarlane). EOCs were generated as described before [16]. CD11b+-monocytes were further isolated by magnetic bead separation using conjugated monoclonal 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% H2 O before initiation of co-culture experiments. To elucidate the findings of in vivo transfusion experiments with IFNγ- and IL4stimulated monocytes, we evaluated the response of EOC regenerative cellular properties depending on co-cultured monocyte phenotypes or their preconditioned media in vitro. EOC regenerative properties included proliferation, differentiation, apoptosis, and tube formation as a functional test of angiogenesis in vitro. To gain monocyte phenotypes cultured 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 buffer and resuspended according to the protocol of the co-culture experiments in vitro. For experiments, EOCs were seeded into gelatine-coated 24-well plates and grown to 50% confluence. When initiating the co-cultures, a total of 0.5 mL of EBM-2 medium containing 1 × 106 CD11b+-monocytes were added to EOCs and the cells were further cultured for 48 h. To test paracrine effects preconditioned media (PCM) from supernatant of CD11b+-cells were added to EOC and the EOCs were cultured for further 48 h. Volumes of 50 μL (low) and 100 μL (high) PCM were applied. Control wells containing separately cultured cells were handled in the same way. To evaluate proliferation and differentiation status of EOCs we performed anti-vWF- and anti-ki-67-immunohistochemistry. Rabbit anti-vWF-antibody (diluted 1:100, DakoCytomation, Glostrup, Denmark) and mouse anti-ki-67-antibody (1:100, DakoCytomation, Glostrup, Denmark) were used in co-culture experiments with primary CD11b+-monocytes. Evaluation of EOC regenerative properties after co-culture with IFNγ- and IL4-stimulated CD11b+-monocytes or their PCM included proliferation by anti-BrdU-immunohistochemistry, differentiation by anti-vWF-immunohistochemistry, apoptosis by anti-Annexin-V-immunohistochemistry, and tube formation by the tube formation assay (Ibidi, Martinsried, Germany). As secondary antibodies, a Cy2-conjugated anti-rabbit-antibody and a Cy3-conjugated anti-mouseantibody (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). Primary antibodies were incubated for 2 h; secondary antibodies were incubated for 1 h at room temperature. For nuclear staining, Hoechst-33258 (1 μg/mL; Sigma-Aldrich, Taufkirchen, Germany) was applied to the sections for 15 min. Stained samples were mounted with fluorescence mounting medium (Sigma-Aldrich, Taufkirchen, Germany) and studied with a Zeiss Axiovert 200 microscope (Carl Zeiss Jena, Germany) equipped with an ApoTome and an AxioCam MRc5. Images were acquired with Zeiss AxioVision software and processed with Corel Graphics Suite. Immunohistochemistry was used to characterize and quantify the different cell types in culture. All quantitative analyses were carried out by randomly taken photographs of several samples. For classification of differentiating or proliferating EOCs we used the same criteria as reported earlier: ki-67/BrdU-negative and vWF-positive cells were considered as differentiating cells; ki-67/BrdU-positive and vWF-negative cells represented proliferating cells. Percentages of proliferating, differentiating or apoptotic EOCs were calculated by dividing their number by the total number of Hoechst-positive nuclei. Quantification of tube formation was performed as recommended by the assay protocol. For each condition, five pictures (20× magnification) of three independent experiments were recorded and analyzed.

3.1. Clodronate-induced monocyte destruction decelerates reendothelialization in WT mice To evaluate the biological effect of endogenous monocytes on reendothelialization after perielectric vascular injury, WT-mice were treated with liposome-encapsulated clodronate (15 mg/kg) in order to deplete the pool of circulating monocytes. The reendothelialized areas in percent of the damaged endothelial area were determined by Evans blue staining in a whole vessel preparation five days after induction of EC damage (Fig. 1). Evans blue staining of the injured vessel revealed that clodronate induced depletion of endogenous monocytes was associated with a significant decrease in reendothelialization compared with untreated WT-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 systemic application of exogenous CD11b+-monocytes potentially enhances regeneration processes of the diseased endothelium and thereby consequently limits abnormalities in vasoreactivity. We tested this hypothesis in an established model of endothelial dysfunction using ApoE−/−-mice which were fed on a 5-week cholesterol-rich diet. Endothelium-dependent vasodilatation was profoundly impaired in hypercholesterolemic ApoE−/−-mice (Fig. 2) in contrast to WT-mice (maximal relaxation: ApoE−/−-mice 92.2 ± 3.7% versus WT-mice 25.5 ± 2.7%, p b 0.05). Endothelium-independent vasorelaxation induced by nitroglycerin was comparable between groups (data not shown). Next, ApoE −/−-mice received three repetitive transfusions of either spleen-derived CD11b+-monocytes or CD11b−mononuclear cells (MNCs). Control WT-mice were treated with normal saline solution without cells intravenously on three consecutive days. Endothelial function of aortic segments was assessed seven days after the last transfusion. Transfusion of CD11b +-cells, significantly improved 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%, p b 0.05). In contrast, no significant improvement in endothelial function was noted in ApoE−/−-mice transfused with CD11b−-MNCs or salinetreated ApoE−/−-mice (Fig. 2; maximal relaxation: ApoE−/−-mice transfused with CD11b−-MNCs 95.7 ± 2.3% vs ApoE−/−-mice with control, 9.2.2 ± 3.7%). Endothelium-independent vasorelaxation was not altered between groups (data not shown).

3.3. CD11b-depleted MNCs do not reduce neointima formation after CCAI Impaired reendothelialization is associated with enhanced neointima formation. Therefore, we evaluated neointima formation after perielectric vascular CCAI and transfusion of unfractionated spleen-derived MNCs or CD11b-negative MNCs. Morphometric analysis showed increased neointima formation after transfusion of CD11bdepleted MNCs while transfusion of unfractionated MNCs containing CD11b+ -cells resulted in a significant reduction in neointima area (Fig. 3; CD11bdepleted MNCs: 5 × 105 ± 0.9 × 105 μm2 vs. MNCs 2.3 × 105 ± 0.4 × 105 μm2, p b 0.05).

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

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

Developed tension (% of max. phenylephrine-induced)

Focusing on CD11b+-monocytes, we hypothesized that systemic depletion of endogenous CD11b+-monocytes potentially inhibits the vascular repair process of the injured endothelium. To evaluate these postulated effects of CD11b-positive monocytes depletion on reendothelialization, we performed an endothelial common carotid artery injury (CCAI) in a CD11b-DTR-transgenic mouse model, whereby administration of DT selectively depletes endogenous monocytes and macrophages [11]. Immediately, 24, and 48 h after CCAI, DT or vehicle treated animals received 1 × 106 CD11b-positive cells or cell free saline via tail vein injection (Fig. 4). DT treatment resulted in an 81 ± 6% depletion of endogenous CD11+-monocytes after application of 15 ng

DT i.p./g bodyweight (BW) (Suppl. Fig. 2). Rescue experiments showed that systemic transfusion of WT-CD11b+-monocytes in CD11b-DTRmice treated with either vehicle or DT resulted in a comparable enhancement 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 showed significantly increased reendothelialization in CD11b+-monocyte transfused animals with no significant differences between the vehicle or DT treated 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-MNCs treated with either vehicle or DT resulted in no significant beneficial effect on reendothelialization compared to controls but significant

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Carbachol (log mol/l) * p < 0.05 vs. ApoE-/--mice control #p < 0.05 vs WT-mice control n = 4/group Fig. 2. CD11b+-monocytes improve endothelial function in ApoE−/−-mice. Endothelium-dependent vasodilatation was profoundly impaired in hypercholesterolemic ApoE−/−-mice compared to WT-mice (maximal relaxation: ApoE−/−control: 92.2 ± 3.7% vs. WT-mice 23.3 ± 2.7%, p b 0.05). Systemic application of mature CD11b+-monocytes in splenectomized ApoE−/−-mice had beneficial effects on endothelial function demonstrated by improved endothelium-dependent vasodilation 7 days after the first cell transfusion compared to ApoE−/−-mice treated with cell-free saline control (ApoE−/− + CD11b+: 64.0 ± 1.9% vs. ApoE−/−control: 92.2 ± 3.7%, p b 0.05). Intravenous application of the WT-derivedCD11b-depleted-cell-population had no beneficial effect on vasoreactivity in ApoE−/−-mice (maximal relaxation: ApoE−/− + CD11b−: 95.7 ± 2.3% vs. ApoE−/−control: 92.2 ± 3.7%).

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Neointima formation (µm2)

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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 was performed in a CD11b-DTR-transgenic mouse model. DT-treated CD11b+-DTR-mice received intravenously either spleen-derived wildtype mononuclear cells (MNCs) or wildtype CD11b-depleted MNCs after injury of the CCA. Morphometric analysis showed increased neointima formation after transfusion of CD11b-depleted MNCs while transfusion of unfractionated MNCs containing CD11b+-cells resulted in a substantial reduction in neointima area indicating decreased re-endothelialization potential without the CD11b-positive monocyte subset (CD11bdepleted MNCs: 5 × 105 ± 0.9 × 105 μm2 vs. MNCs 2.3 × 105 ± 0.4 × 105 μm2, p b 0.05).

less reendothelialization compared to transfusion of WT-CD11b+monocytes (Fig. 4; CD11b-DTR-mice+vehicle + CD11b-depleted-WTMNCs 53 ± 7% vs. CD11b-DTR+ DT + CD11b-depleted-WT-MNCs 39 ± 9%, p N 0.05). Therefore, transfusion lacking WT-CD11b +monocytes significantly reduced reendothelialization, especially when DT was applied to deplete endogenous CD11b+-monocytes. These results demonstrate that depletion of endogenous CD11b+-monocytes

significantly delays reendothelialization, which can be rescued by exogenous applied CD11b+-monocytes. 3.5. IFNγ-stimulated CD11b+-monocytes increase endothelial dysfunction Focusing on the subpopulations of CD11b+-monocytes, we evaluated the endothelial function of transfused IFNγ- and IL4-stimulated

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Fig. 4. CD11b+-monocytes increase reendothelialization in CD11b-DTR-mice. Experimental setting and transfusion regimen to further determine the effect of systemic transfusion of CD11b+-monocytes on the reendothelization process. Monocyte-depleted-CD11b+-DTR-animals were treated with CD11b+-monocytes after perielectrical vascular injury. Compared to control animals (CD11b-DTR-mice+vehicle 44 ± 3% vs. CD11b-DTR-mice+DT 31 ± 2%, p b 0.05) morphometric analysis of Evans blue staining showed significantly increased reendothelialization in WT-CD11b + -monocyte transfused animals with no significant differences between the vehicle or DT treated groups (CD11b-DTR-mice + vehicle + WTCD11b +-monocytes 69 ± 4% vs. CD11b-DTR + DT + WT-CD11b +-monocytes 71 ± 2%, p N 0.05). Transfusion of mice with CD11b-depleted-WT-MNCs treated with either vehicle or DT resulted in no significant beneficial effect on reendothelialization compared to controls but significant less reendothelialization compared to transfusion of WT-CD11b+monocytes (CD11b-DTR-mice+ vehicle + CD11b-depleted-WT-MNCs 53 ± 7% vs. CD11b-DTR + DT + CD11b-depleted-WT-MNCs 39 ± 9%, p N 0.05). Therefore, transfusion lacking WT-CD11b+-monocytes significantly reduced reendothelialization, especially when DT was applied to deplete endogenous CD11b+-monocytes.

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Carbachol (log mol/l) * p < 0.05 vs. IL4-stimulated and control n = 4/group Fig. 5. Transfusion of CD11b+-subpopulations to ApoE−/−-CD11b-DTR-mice. Systemic application of IFNγ- and IL4-stimulated CD11b-positive monocytes was performed after depletion of endogenous CD11b+-monocytes in splenectomized ApoE−/−-CD11b-DTR-mice. ApoE−/−-CD11b-DTR-mice were put on a 5-week cholesterol-rich diet leading to profoundly impaired endothelium-dependent vasodilatation. Administration of DT selectively depleted endogenous monocytes and macrophages. Depleted ApoE−/−-CD11b-DTR-mice received three repetitive 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 endotheliumdependent vasodilatation after seven days (maximal relaxation: ApoE−/−-CD11b-DTR-mice transfused with IFNγ-stimulated CD11b+-monocytes: 83.0 ± 1.9% vs. IL4-stimulated CD11b+-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 noted after IL4-stimulated CD11b+-cell transfusion to ApoE−/−-CD11b-DTR-mice compared to control (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 not altered between groups (data not shown).

CD11b+-monocytes after depletion of endogenous CD11b+-monocytes in an animal model of ApoE−/−-CD11b-DTR-mice. ApoE−/−-CD11b-DTR-mice were fed on a 5-week cholesterol-rich diet. Administration of DT selectively depleted endogenous monocytes and macrophages. Endothelium-dependent vasodilatation was profoundly impaired in depleted hypercholesterolemic ApoE−/−-CD11bDTR-mice (Fig. 5). Depleted ApoE−/−-CD11b-DTR-mice received three repetitive 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 endotheliumdependent 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 function was 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 not altered between groups (data not shown). These results demonstrated that WT-derived CD11b +-monocytes stimulated by IFNγ decreased endothelial function, whereas IL4-stimulated monocytes had no detectable effect on vascular function within this experimental setting. 3.6. CD11b+-monocytes are located to the injury site in CD11b+-DTR-mice Using bioluminescent imaging, transfused CD11b+-monocytes specifically expressing the luciferase gene were tracked in vivo permitting analysis of monocyte dynamics in living CD11b-DTR-mice in realtime (Fig. 6). Transfused CD11b+-monocytes were restricted to the injury site at the CCA and application side after tail vein injection in monocyte-depleted-CD11b+-DTR-mice (Fig. 6F–J). While bioluminescence at the application side was detectable up to three days after application (Fig. 6I), bioluminescence at the CCA injury side was found up to five days after injury (Fig. 6J, arrow), demonstrating

that transfused circulating CD11b+-monocytes reach and stay at the area of endothelial repair under physiological flow conditions in living mice. 3.7. Mode of interaction and monocyte phenotype differentially affect regeneration properties of EOCs in vitro Co-culture experiments were used to examine the influence of primary CD11b+-monocytes on endothelial outgrowth cell proliferation and differentiation (Fig. 7). Cell proliferation marker ki-67 and differentiation marker vWF supporting maturation were examined. While incubation of an immature starting population of EOCs with preconditioned medium (PCM) of CD11b+-monocytes showed a significant increase of proliferation pointing towards a paracrine stimulation, co-culture of EOC in the presence of CD11b +-monocytes resulted in a significant higher number of differentiating EOCs than in untreated control cells. In vitro these co-culture experiments revealed increasing differentiation potential of EOCs co-cultured with CD11b+-monocytes demonstrating that circulating CD11b+monocytes might contribute to endothelial cell regeneration and restoration associated with an improved vascular function. To elucidate the findings of in vivo transfusion experiments with IFNγ- and IL4-stimulated monocytes, we evaluated the response of EOC regenerative cellular properties depending on co-cultured monocyte phenotypes or their preconditioned media in vitro. Evaluation of EOC regenerative properties after co-culture with IFNγ- and IL4-stimulated monocytes or their PCM included proliferation by anti-BrdU-immunohistochemistry, differentiation by anti-vWFimmunohistochemistry, apoptosis by anti-Annexin-V-immunohistochemistry, and tube formation by the tube formation assay. Upon co-culture the IFNγ-induced CD11b+-monocyte phenotype showed increased numbers of apoptotic EOCs independent of direct cellular (Fig. 8A, black column) or paracrine interactions (Fig. 8B, black columns), whereas no effect on EOC-apoptosis was detected by IL4-stimulated monocytes (Fig. 8A, gray column) or its PCM (Fig. 8B, gray columns). In line with increased apoptotic EOCs the proliferation rate was significantly decreased upon co-culture with prior IFNγ-stimulated monocytes (Fig. 8C, black column) or their PCM (Fig. 8D, black columns). However, PCM of IL-4-stimulated

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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 transfused CD11b+-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 of monocytes 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 protein expression 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 of luciferase-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 mice received 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 image representing the spatial distribution of detected photons (panels F–O). Our studies revealed that transfused CD11b + -monocytes were restricted to the injury site in monocyte-depleted-CD11b-DTR-mice demonstrating that circulating CD11b+-monocytes contribute to early endothelial regeneration (panels F–J, arrow, pseudo-color images represent luciferase activity). Bar: panel A 100 μm and panel B 50 μm.

monocytes induced EOC proliferation in a dose dependent manner (Fig. 8D, gray columns). Tube formation resembling the ability of EOCs to build up vessel like structures was found significantly increased on direct cellular co-culture of IL-4-stimulated monocytes with EOCs (Fig. 8E, gray column), while co-culture of IFNγinduced CD11b +-monocytes (Fig. 8E, black column) or PCM (Fig. 8F, black column) reduced this capacity significantly. Moreover, differentiation measured by EOC vWF-expression was significantly reduced upon co-culture with IFNγ-stimulated monocytes (Fig. 8G, black column) or PCM (Fig. 8H, black columns). Conclusively, depending on the prestimulation CD11b +-monocyte (IFNγ or IL4) and the mode of monocyte–EOC-interaction (paracrine or cell–cell-interaction) we found differentially affected regeneration properties of immature EOCs. To investigate the elevated proliferation rates of EOCs cultured in PCM of IL4-stimulated monocytes we measured the concentration of monocyte-derived vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), both known to effect EC proliferation and survival. We found significant higher VEGF concentrations in PCM of IL4-stimulated monocytes compared to IFNγ-stimulated monocytes (Suppl. Fig. 3A), but significant 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 paracrine effects in EOCs without direct cell–cell-contact. 4. Discussion Although the importance of monocytes in atherogenesis is reasonably well established, at least in rodent models potential roles of monocytes in the setting of vascular regeneration and resolution of inflammation have been less-well explored. Here we could demonstrate that CD11b+-monocytes play an important role in endothelial cell recovery after acute and chronic endothelial injury. Monocytes home to the site of vascular injury enhance reendothelialization and improve endothelial function, most likely by inducing paracrine and cell-differentiation processes. Apparently monocytes have divergent roles in the vascular compartment [2,3,8]. During atherogenesis confounding factors such as dyslipidemia, hypertension or proinflammatory mediators cause activation of the endothelium leading to increased adhesion and migration of circulating monocytes. By uptake of oxidized lipoproteins they mature into pro-inflammatory macrophages and convert to foam cells maintaining chronic vascular inflammation. Studies in mice provided evidence on the important role of endothelial activation on endothelial- and monocyte–cell-interactions

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20% vWF Fig. 7. Endothelial outgrowth cells (EOCs) and CD11b+-monocyte co-culture. Co-culture experiments were used to examine the influence of primary CD11b+-monocytes or preconditioned 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 B and 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) resulted in a significant higher number of differentiating EOCs than in untreated control cells (panel F). In vitro these experiments revealed increasing differentiation potential of EOCs cocultured 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 paracrine stimulation of proliferation (panel E). Bar: 100 μm.

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 vascular inflammation and its resolution as well as its transition into chronic inflammation. These contradictory roles of monocytes might be explained by the involvement of different subsets of monocytes. In our study, we focused on the role of CD11b-positive monocytes and their pro- or anti-inflammatory function on vascular reendothelialization after acute (focal vascular injury) and chronic (disseminated vascular disease) endothelial injury. In the present study, we could demonstrate that intravenous application of CD11b-positive cells improved endothelium-dependent vasodilation suggesting that this cell-based treatment can limit abnormalities in vasoreactivity associated with atherosclerotic plaque development. Here we induced elimination of CD11b+-monocytes and macrophages via the administration of DT to transgenic CD11b+-DTRmice expressing the diphtheria toxin receptor under the control of the CD11b-promoter to analyze their role in vascular repair and recovery of the endothelial layer [11]. Depletion of CD11b+-cells resulted in substantial disruption of the endothelial repair process and reduction of vascular healing. Our experimental design of the mice models gave further evidence on the contribution of CD11b +-monocytes/ macrophages to the vascular healing process. We administered DT around the time of an induced CCAI to deplete endogenous monocytes and macrophages during the initial inflammatory response to injury. CD11b +-cell depletion during the early phase after CCAI resulted in decreased endothelial reendothelialization. Transfusion of WT-CD11b+-monocytes, not vulnerable to DT, was associated 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 permitting analysis of monocyte origin and dynamics in living monocyte depleted CD11b+-DTR-mice in realtime. Our studies revealed that the transfused CD11b+-monocytes were restricted to the injury site five days after acute injury in monocyte-depleted-CD11b+-DTR-mice demonstrating that beneficial effects on endothelial regeneration are potentially mediated by local CD11b +-monocyte actions. Evidence on the importance of monocytes/macrophages to the recovery of vascular injuries has been obtained from other experimental studies in macrophage-deficient mice [18–20]. These studies found reduced and abnormal vascularization in macrophage-depleted wounds. Using in vitro experiments we analyzed proliferation and differentiation characteristics of immature human endothelial precursor cells (EOCs) which were incubated with precondition CD11b+-monocyte medium or co-cultured in the presence of CD11b+-monocytes. It has been demonstrated that monocytes and ECs interact in vascular repair and that the one cell type can induce proliferative effects, survival, and phenotypic changes in the other [21]. Indeed, EOCs cultured in CD11b+-monocyte conditioned medium displayed approximately 2-fold increased proliferation measured by ki-67 expression and reduced vWF expression. Increased differentiation of EOCs measured by vWF expression was observed after direct cell–cell interaction indicating a differential role for direct cellular and indirect paracrine interaction between CD11b+-monocytes and EOCs in vascular repair. To elucidate the findings of in vivo transfusion experiments with IFNγ- and IL4-stimulated monocytes, we evaluated the response of EOC regenerative cellular properties depending on co-cultured monocyte phenotypes or their preconditioned media in vitro. Evaluation of EOC regenerative properties after co-culture with IFNγ- and IL4-stimulated monocytes or their PCM included proliferation by anti-BrdU-immunohistochemistry, differentiation by

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anti-vWF-immunohistochemistry, apoptosis by anti-Annexin-Vimmunohistochemistry, and tube formation by the tube formation assay. Depending on the pre-stimulation of CD11b +-monocyte (IFNγ or IL4) and the mode of monocyte–EOC-interaction (paracrine or cell–cell-interaction) we found differentially affected regeneration properties of immature EOCs. Our findings suggest that depending on the monocyte phenotype factors involved in EOC proliferation and function modulate EC survival and function. One of the EOC protective paracrine factors might be VEGF, which we found to be highly expressed in IL4-stimulated CD11b +-monocytes but not in IFNγstimulated CD11b +-monocytes. However, the influence of other unidentified or unknown paracrine molecules underlying the proangiogenic effects has not been proven and one must admit that many of 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–cellcontact but also mediate contact-dependent effects on EOCs. In an early phase after endothelial denudation initializing of endothelial recovery coincides with adhesion of monocytes to the denuded arterial surface [22]. By secretion of soluble growth factors monocytes augment EC proliferation, spreading and migration preparing the occurrence of a second phase of vascular reendothelization in which replenished ECs

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directly interact with monocytes leading to contact-dependent inhibition of proliferation and EC differentiation. The indicated potent effect of not only primary but also distinct monocyte phenotypes on EC wound closure mediated by increased proliferation and migration of stimulated ECs into the denuded area is supported by several other studies which demonstrated that the soluble factor VEGF secreted by activated monocytes enhanced EC proliferation and wound closure in injured EC monolayers [23]. On the molecular level members of the family of Tie-2 ligands such as angiopoietin-1 and -2 have been recently shown to be involved in the monocyte–EC-interaction during vessel remodeling and regeneration [6]. It has been reported that on direct physical interaction of unactivated monocytes with EC, monocytes secreted high levels of Ang-1 leading to a transient activation of endothelial Tie2, contributing to the regulation of EC survival. The selective secretion of Ang-1 or Ang-2 may be regulated by the monocyte phenotype, as preactivation of monocytes with INFγ abolished the secretion of Ang-1 and increased the secretion of Ang-2. Moreover, a previous study in which soluble Tie2 receptor served as a neutralizing competitive antagonist for Ang-1 the protective effect of Ang-1 on EC was reversed, further supporting the role of monocytes/Ang-1 and Ang-2 in the regulation of EC survival [6].

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Fig. 8. Mode of interaction and monocyte phenotype differentially affect regeneration properties of EOCs in vitro. Evaluation of EOC regenerative properties after co-culture with IFNγ- and IL4-stimulated monocytes or their PCM included proliferation by anti-BrdU-immunohistochemistry, differentiation by anti-vWF-immunohistochemistry, apoptosis by anti-Annexin-V-immunohistochemistry, and tube formation by the tube formation assay. Upon co-culture the IFNγ-induced CD11b + -monocyte phenotype showed increased numbers of apoptotic EOCs, independent of direct cellular (panel A, black column) or paracrine interactions (panel B, black columns), while no effect on EOC-apoptosis was detected by IL4-stimulated monocytes (panel A, gray column) or its PCM (panel B, gray columns). In line with increased apoptotic EOCs the proliferation rate was significantly decreased upon co-culture with prior IFNγ-stimulated monocytes (panel C, black column) or their PCM (panel D, black columns). However, PCM of IL-4-stimulated monocytes induced EOC proliferation in a dose dependent manner (panel D, gray columns). Tube formation resembling the ability of EOCs to build up vessel like structures was found significantly increased on direct cellular co-culture of IL-4-stimulated monocytes with EOCs (panel E, gray column), while co-culture of IFNγ-induced CD11b+-monocytes (panel E, black column) or PCM (panel F, black column) reduced this capacity significantly. Moreover, differentiation measured by EOC vWF-expression was significantly reduced upon co-culture with IFNγ-stimulated monocytes (panel G, black column) or PCM (panel H, black columns).

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Fig. 8 (continued).

Another concept of interaction with increasing evidence is that the withdrawal of positive effectors such as VEGF is sufficient to result in blood vessel regression in various in vivo systems including tumors and developing organs [24]. These studies demonstrate the presence of an inverse correlation between VEGF expression and the levels of vessel recovery, implicating VEGF as an important survival factor for endothelial cells. A number of different growth factors have been shown to rapidly activate Akt dependent intracellular signaling mechanisms via PI3-kinase activation, thereby promoting survival of ECs and other cell types [24]. In ECs VEGF survival signals are known to be mediated by the Flk-1/KDR receptor through the PI3-kinase/Akt signal transduction pathway [24]. Additionally, it was reported that secretion of hepatocyte growth factor (HGF) of primary unactivated monocytes may also contribute to the protective effect on EC [25]. The ability to activate Akt has been described not only for VEGF but also for different other growth factors, such as platelet derived growth factor, epidermal growth factor, bFGF, insulin, and insulin-like growth factor 1 [24], yielding attractive targets for pharmacological therapy aimed to optimize endothelial cell regeneration. Moreover, an anti-apoptotic contact dependent effect of monocytes has been reported by the upregulation of bcl-2 homologue A1 in EC [6]. We and others demonstrated the key role of monocyte-induced proliferation and differentiation of EC in the reendothelization process of the endothelial monolayer. It is reasonable that in case of acute endothelial injury mature monocyte subsets dominate in early acute vascular reconstitution and may subsequently provide paracrine signals to initiate proliferation of immature endothelial and endothelial

precursor cells followed by cellular induction of endothelial cell differentiation restoring vascular homeostasis while limiting potential luminal obstruction and intimal hyperplasia. Our findings provide evidence that VEGF survival signals in immature endothelial cells are mediated by IL4-stimulated monocyte phenotype enhancing EOC proliferation by paracrine mechanisms and EOC function by direct cellular contacts, while INFγ-stimulated monocytes induce paracrine and cellular interactions leading to endothelial cell apoptosis and regression of tube formation in vitro and endothelial function in vivo. It is generally accepted that the tissue microenvironment determines macrophage phenotypic polarization. Here we found that IL4-induced polarization of primary monocytes and co-cultivation with subconfluent endothelial cells resulted in transient secretion of VEGF from monocytes and the activation of endothelial survival genes. This effect was abolished by coculturing monocytes after IFNγ-induced polarization. Although primary monocytes had some effects on endothelial regenerative properties, both polarization states showed more intense interactions with EOCs. In conclusion, monocyte subsets differentially affect endothelial cell survival and play a crucial role in vascular remodeling and homeostasis. 4.1. Study limitations An intrinsic problem of mouse models in the cardiovascular context is their divergence from the human organism, both with regard to response to inflammatory stimuli [26], and to the different activation and paracrine functions of monocyte subpopulations and macrophage phenotypes between both species. An additional difficulty is

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represented by the inherent high phenotypic flexibility of myeloid cells, representing a wide spectrum rather than two extreme states. Therefore, interpretation of data generated from these experiments has to be done with caution: The ex vivo stimulation of monocytes with IL-4 vs. IFNγ, although routinely used for M1- vs. M2-like macrophage differentiation ex vivo, might harbor the risk of generating cell culture artifacts in addition to the possibility of further phenotypic alterations after injection. In this respect it was shown that M2-like macrophages may exert both anti- and pro-inflammatory actions [27]. Moreover, mature monocyte and macrophage subsets may possess the ability to reconstitute vascular damage by transdifferentiation that has been described in a subset of CD14+/KDR+ myeloid cells previously [28,29]. Two major ways of interaction seem to drive endothelial cellmonocyte cross talk: first, a direct cellular contact-mediated mechanism of binding mediated through adhesion molecules of cell–cellinteraction expressed on the surface of activated ECs and circulating monocytes contributing to EC differentiation and second, secretion of soluble paracrine factors by activated monocytes and macrophages driving proliferation and migration of EC suggesting a complex signaling network required for normal recovery of vascular homeostasis [30]. Monocytes and macrophages synthesize and release a vast array of regulatory molecules relevant for regulatory interactions with other cellular populations at the side of injury temporally changing the cellular assembly at the time and side of injury and disassembled when repair is completed [31]. Regulation of vascular regeneration by growth factors and cytokines encompasses several families of signaling molecules associated with different aspects of repair. Multiple individual molecules are found in each family with broadly overlapping activities. Given that multiple monocyte–macrophage-subsets are involved in vascular regeneration and are capable of specifically producing different relevant cytokines and growth factors for interaction with ECs adds profound complexity to the physiological process of vascular recovery. Thus, pro-angiogenic activity of monocyte subsets is probably related to secretion of angiogenic cytokines. Vice versa, monocytes may activate surface receptors on ECs by secreting anti-apoptotic factors thus supporting endothelial cell survival [6]. Cellular orchestration in tissue recovery is controlled by a variety of cytokines and growth factors. Members of the VEGF family and their receptors have been shown to be prominently expressed in damaged vascular tissue and data outlined secretion of VEGF mainly of activated proangiogenic monocytes [31]. The therapeutic infusion of freshly isolated CD11b+-monocytes for accelerating endothelial regeneration after vascular injury has been controversially discussed [32,33]. As monocytes are abundant and easy to separate, their local therapeutic use in regeneration could be of interest rather in pathological settings such as acute endothelial damage after vascular intervention as well as chronic vascular deterioration in diabetes mellitus, where the function of circulating progenitor cells is overall compromised [34]. Several published studies have reported that cultured angiogenic cells are reliable tools in regenerative cardiology [35–37]. These were usually cultured out from peripheral blood mononuclear cells and displayed myeloid (CD45+, CD14+, CD16+, CD11b+) along with endothelial (VEGFR2+, CD31+, Tie2+) features, which partially correspond to classical and intermediate monocytes [35]. Strategies to modulate their number and to augment their regenerative function are under investigation [38,39]. The detailed differential mechanisms of interaction between primary CD11b +-monocytes or their subsets and EOCs to gain EC function and improve endothelial regeneration remain not to be yet clear. Most probably, there is an additive cross talk between different monocyte/macrophage subsets interplaying in vascular tissue repair and further efforts are intended to characterize and identify angiogenic monocyte populations with high pro-angiogenic capacity. The nature of this monocyte polarization into different phenotypes deserves our attention because it may provide a key to prophylactic transcriptional control of macrophage differentiation.

5. Conclusion Our data underline that monocytes not only promote formation, growth and complication of atherosclerotic plaques and are associated with plaque vulnerability but also conversely contribute to tissue regeneration 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. References [1] Welt FG, Tso C, Edelman ER, et al. Leukocyte recruitment and expression of chemokines following different forms of vascular injury. Vasc Med 2003;8:1–7. [2] Osterud B, Bjorklid E. Role of monocytes in atherogenesis. Physiol Rev 2003;83:1069–112. [3] Auffray C, Sieweke MH, Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol 2009;27:669–92. [4] Nahrendorf M, Majmudar M, Keliher E, et al. Monocyte-directed RNAi improves infarct healing in atherosclerosis-prone mice. Circulation 2013;127. [5] Schubert SY, Benarroch A, Ostvang J, Edelman ER. Regulation of endothelial cell proliferation by primary monocytes. Arterioscler Thromb Vasc Biol 2008;28:97–104. [6] Schubert SY, Benarroch A, Monter-Solans J, Edelman ER. Primary monocytes regulate endothelial cell survival through secretion of angiopoietin-1 and activation of endothelial Tie2. Arterioscler Thromb Vasc Biol 2011;31:870–5. [7] Zhao Y, Glesne D, Huberman E. A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci U S A 2003;100:2426–31. [8] Ley K, Miller Y, Hedrick C. Monocyte and macrophage dynamics during atherogenesis. Arterioscler Thromb Vasc Biol 2011;31:1506–16. [9] Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol 2005;5:953–64. [10] Carlin L, Stamatiades E, Auffray C, et al. Nr4a1-dependent Ly6c(low) monocytes monitor endothelial cells and orchestrate their disposal. Cell 2013;153:362–75. [11] Stoneman V, Braganza D, Figg N, et al. Monocyte/macrophage suppression in CD11b diphtheria toxin receptor transgenic mice differentially affects atherogenesis and established plaques. Circ Res 2007;100:884–93. [12] Wassmann S, Stumpf M, Strehlow K, et al. Interleukin-6 induces oxidative stress and endothelial dysfunction by overexpression of the angiotensin II type 1 receptor. Circ Res 2004;94:534–41. [13] Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circ Res 1993;73:792–6. [14] Pfeifer Alexander, Hofmann Andreas. Lentiviral transgenesis. Gene knockout protocols. Methods Mol Biol 2009;530:391–405. [15] Carmeliet P, Moons L, Stassen JM, et al. Vascular wound healing and neointima formation induced by perivascular electric injury in mice. Am J Pathol Feb 1997;150(2):761–76. [16] Becher MU, Nickenig G, Werner N. Regeneration of the vascular compartment. Herz 2010;35:342–51 [Review]. [17] Maruyama K, Ii M, Cursiefen C, et al. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest 2005;115:2363–72. [18] Goren I, Allmann N, Yogev N, et al. A transgenic mouse model of inducible macrophage depletion: effects of diphtheria toxin-driven lysozyme M-specific cell lineage ablation on wound inflammatory, angiogenic, and contractive processes. Am J Pathol 2009;175:132–47. [19] Mirza R, DiPietro LA, Koh TJ. Selective and specific macrophage ablation is detrimental to wound healing in mice. Am J Pathol 2009;175:2454–62. [20] Lucas T, Waisman A, Ranjan R, et al. Differential roles of macrophages in diverse phases of skin repair. J Immunol 2010;184:3964–77. [21] Antonov AS, Munn DH, Kolodgie FD, Virmani R, Gerrity RG. Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/ p27(Kip1) regulatory checkpoint. J Clin Invest 1997;99:2867–76. [22] Rogers C, Welt FG, Karnovsky MJ, Edelman ER. Monocyte recruitment and neointimal hyperplasia in rabbits. Coupled inhibitory effects of heparin. Arterioscler Thromb Vasc Biol 1996;16:1312–8. [23] Tomita N, Morishita R, Taniyama Y, et al. Angiogenic property of hepatocyte growth factor is dependent on upregulation of essential transcription factor for angiogenesis, ets-1. Circulation 2003;107:1411–7. [24] Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem Nov 13 1998;273(46):30336–43.

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