University of Groningen

Stem cell-mediated regeneration of the infarcted heartVelde, Susanne van der

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Chapter 7

Monocytic CD14+ cells in patients with stable and unstable angina pectoris: a choice between macrophage and endothelial-like phenotype?

S. Vandervelde1, M.J.A. van Luyn¹, X. Gallego y van Seijen¹, R.A. Tio², F. Zijlstra², M.C. Harmsen¹

Departments of Pathology & Laboratory Medicine1 and Cardiology²,University Medical Center Groningen, the Netherlands

Submitted

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AbstractObjective. The destiny of CD14+ monocytes is to become macrophages, but they appear also to be capable to contribute to neovascularisation by becoming Endothelial Progenitor Cells (EPC). Here, we compared the phenotype of CD14+ monocytes cultured in EPC medium and macrophage medium. Moreover, we tested whether this differentiation potential of monocytes of patients with unstable angina pectoris (UAP) is altered compared to patients with stable angina pectoris (SAP). Methods. CD14+ monocytes were isolated from the peripheral blood by magnetic beads and were cultured on fibronectin-coated plates in either MF medium containing M-CSF or EC medium containing VEGF and bFGF. Results. CD14+ monocytes obtained a comparable phenotype after 7 days of culture in MF or EC medium, also on an ultrastructural level. Cultured monocytes did not express endothelial specific markers and had a comparable expression of macrophage markers regardless medium type. Both MF and EC cultured monocytes exhibited EPC features of LDL uptake, Lectin binding, and CFUs formation, but also phagocytozed large particles. MF cultured monocytes of patients with SAP had an even more pronounced differentiation towards macrophages compared to UAP, which effect could also be induced by addition of serum of SAP to culture of monocytes of healthy donors.Conclusion. Notwithstanding small phenotypical differences of monocytes cultured in EPC medium or macrophage medium, it seemed that CD14+ monocytes are predestined towards a macrophage-like phenotype. Nevertheless, regardless culture medium these cultured monocytes were transcriptionally active and contained vasculogenic factors, and may therefore be well-capable of inducing neovascularization.

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IntroductionInflammation is an important feature in cardiovascular disease, in particular in artherosclerotic vascular lesions. Atherosclerotic plaques are inflammatory microenvironments in which macrophages, i.e. derivates of circulating CD14+ monocytes, are determinative in the pathophysiology. Yet also endothelial cells play a central role. Although macrophages are well known for foam-like appearance in atherosclerotic plaques, they may also contribute to neovascularization and tissue repair under vasculopathologic conditions [1;2]. It is currently appreciated that macrophages and their circulating monocytic progenitors can functionally adapt their phenotype in response to changes in cytokine environment [3;4]. In recent publications it was demonstrated that monocytes, precursor of macrophages, can also differentiate into endothelial progenitor cells (EPCs), which have been shown to augment vascularisation and vascular repair. More specific, peripheral blood monocytes can differentiate into an endothelial-like phenotype under appropriate culture conditions containing VEGF and bFGF [5;6] and are able to improve neovascularization [7]. These cells are often referred to as endothelial progenitor cells (EPCs). However, the phenotype of EPCs is a matter of debate. At first, a certain subpopulation of hematopoietic stem cells (HSC) was considered as EPC. These EPC carried surface marker CD34, CD133 or KDR or a combination of these markers [8;9]. The scarcity of these cells has led to a more practical approach in which EPCs were obtained after culture of the total peripheral blood mononuclear cell fraction on fibronectin coated culture dishes in the presence of endothelial growth factors. The latter EPCs are phenotypically defined as adherent cells that form colony forming units (CFUs), take up Acetylated-LDL and bind to Lectins, and that have various endothelial progenitor-like markers on their surface after culture (such as CD31 and VE-Cadherin) [10;11]. Most likely, these cultured EPCs are derived from peripheral blood (CD14+) monocytes 5. Thus, purified CD14+ monocytic cells, which form a substantial subpopulation of the MNC fraction may indeed be able to differentiate into both macrophages and endothelial-like cells. It would appear that, in vivo, the environment the monocyte encounters determines its fate. In the first part of this study we compared the phenotype of CD14+ monocytes cultured in either endothelial cell medium (EC) containing VEGF-A and FGF2 or macrophage medium (MF) containing M-CSF. In the second part we tested whether this differentiation potential is altered in patients with unstable angina pectoris compared to stable angina pectoris. It is suggested that cardiovascular disease may shift the balance between cells that contribute to neovascularization and repair towards cells that might induce a harmful inflammatory reaction [12]. We hypothesize that the higher inflammatory status in unstable angina pectoris has an influence on the outcome of the differentiation of CD14+ monocytes.

Materials and MethodsStudy Subjects. We studied 14 unstable and 14 stable angina pectoris patients. Patients hospitalized for acute coronary syndrome except for ST-elevated myocardial infarction on electrocardiogram (unstable angina pectoris) were included within 24 hours of onset of clinical symptoms. 14 patients with stable angina matched for age and sex were selected randomly from the cardiology outpatient clinic (Table 1). Exclusion criteria were clinical or biological evidence for the presence of concomitant disease, such as (autoimmune) inflammatory disease, malignant disease, chronic renal insufficiency, thrombocytopenia

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or anemia and atrial fibrillation. A group of 14, non-smoking volunteers (average age of 24.9 ± 2.9 years) without medication use served as donors of control CD14+ monocytes. All subjects gave informed consent. This study was approved by the institutional review board of the University Medical Center Groningen, the Netherlands.

CD14+ cell count and isolation. Human peripheral blood (PB) (20 mL) was withdrawn by venapuncture into heparin-coated syringes. The mononuclear cell (MNC) fraction was isolated by density gradient method on LymphoprepTM (Axis-Shield PoC AS, Oslo, Norway). Residual erytrocytes were lysed on ice (10 min) with erylysis (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.4)). MNCs were recollected in degassed isolation buffer containing PBS (pH 7.2), 0.5% Fetal Calf Serum (FCS) (Cambrex Bio Science, Verviers, Belgium) and 2 mM EDTA (Merck, Darmstadt, Germany). Total MNCs per mL PB was counted and a small fraction of the isolated MNCs was stained with FITC-conjungated anti-human CD14 mononuclear antibody (IQProducts, Groningen, the Netherlands) according to the manufacturers protocol. The percentage CD14+ cells of total MNC fraction was determined by analysis on a FACS Calibur with Cellquest computer software (Becton-Dickinson, Franklin Lakes, NJ USA). In addition, the number of CD14+ cells per MNC number per mL PB was calculated. CD14+ cells were isolated from MNCs by magnetic activated cell sorting (MACS) using CD14 specific MACS beads (MACS, Miltenyi Biotec, Gladbach, Germany) and a magnetic cell sorting device according to manufacturers’ protocol (Miltenyi Biotec, Gladbach, Germany).

CD14+ cell culture. Purified CD14+ cells were cultured in either macrophage (MF) medium or endothelial cell (EC) medium. MF medium consisted of RPMI (Cambrex Bio Science), 10% FCS (Cambrex Bio Science) and 50 ng/mL recombinant human M-CSF (Peprotech, Rocky Hill, New Jersey), with addition of 2mM L-Glutamine (Cambrex Bio Science), 5 U/mL Heparin (Leo Pharmaceutical Products B.V., Weesp, the Netherlands), 100 IE/mL Penicillin (Yamanouchi Pharma BV, Leiderdorp, the Netherlands , 100 μg/mL Streptomicin (Radiumpharma, Italy). ECM medium contained DMEM (Cambrex Bio Science) supplemented with 1 ng/mL human recombinant VEGF (Peprotech),10 ng/mL bFGF (Peprotech), 50 μg/mL Endothelial Cell Growth Factor (isolate according to Maciag et al 13), 20% FCS (Cambrex Bio Science, Verviers, Belgium), together with 2mM L-Glutamine, 5 U/mL Heparin, 100 IE/mL Penicillin, 100 μg/mL Streptomycin. Culture dishes and cover slips were coated with 5 ug/mL fibronectin (Harbor Bio-Products, Norwood, MA) in 1% gelatin (Sigma Chemcial Co, St. Louis, USA). Purified CD14+ cells were plated in triplicate at a density of 0.1* 106 / cm² for MF 0.5* 106 / cm² for ECM-culture. At day 4 fresh medium was added. At day 7 cell clusters were counted per well and cells in culture dishes were detached with Accutase (Chemicon International, Temecula, CA, USA) according to manufactures’ protocol, and subsequently the cells were collected for further analysis. Pictures of cell cultures were taken with an inverted microscope (Leica DMIL, Leica Microsystems, Wetzlar, Germany).

CD14+ cross serum culture. 1 ml of heparinized peripheral blood was separated before MNC isolation procedure and was centrifuged 14.000 rpm at 4°C for 15 minutes. Supernatant was collected and was centrifuged again at 14.000 rpm at 4°C for 15 minutes, to obtain platelet free serum. Serum was stored at -80°C until use. For cross serum experiments,

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serum of unstable (n=10), of stable angina patients(n=10) and of healthy controls (n=10) was pooled per group. CD14+ cells of healthy controls (n=5) were cultured as described above with ECM or MF medium 1) without human pooled serum (SF) or 2) with 10% healthy pooled serum (con), 3) with 10% pooled serum of SAP (SA) or 4) with 10% pooled serum of UAP (UA).

CD14+ characterization. Purified freshly isolated CD14+ cells and 7 day cultured CD14+ cells were collected in isolation buffer containing PBS, 0.5% FCS and 2 mM EDTA, and were characterized using directly conjugated monoclonal antibodies (mAbs) against human CD14, CD31 (IQProducts, Groningen, the Netherlands), CD163, CD68 (Santa Cruz Biotechnology, Santa Cruz, Calafornia), Ve-Cadherin and Tie-2 (R&D Systems, Minneapolis, USA). For determination of monocytic and macrophage lineages we used CD163 and CD68, whereas for characterization of endothelial features we used CD144 (Ve-Cadherin), Tie-2 and CD31 (PECAM-1). Directly conjungated isotype-matched antibodies served as controls. In short, cells were detached with Accutase (Chemicon International, Temecula, CA, USA) according to manufactures’ protocol. After rinsing, cells were incubated for 30 minutes on ice with the antibody using a dilution according to the manufacturers protocols. Next the cells were washed twice with isolation buffer and stored on ice until mAbs detection (maximum of 1 hour). For intracellular staining of CD68, cells were fixed in 2% paraformaldehyde (5 min) and then permeabilized with 0.1% saponin in PBS (5 min). mAb was added in isolation buffer containing 0.1% saponin and after incubation cells were first washed with 0.1% saponin, followed by a wash with isolation buffer. Cytometric analysis wereperformed using a FACS Calibur with Cellquest computer software (Becton-Dickinson). For measurement of LDL uptake and Lectin binding , cultured cells were incubated with 1 μg DiI-Ac-LDL (Harbor Bio-Products) in fresh medium overnight. After rinsing, cells were detached with Accutase (Chemicon International, Temecula, CA, USA) according to manufactures’ protocol, and cells were fixed in 2% paraformaldehyde (5 min). Following, cells were incubated with 4 μg lectin from Ulex europaeus (Sigma) in 400 μL fresh medum on ice for 1 hour. Immunohistochemical staining for VEGF-A (polyclonal rabbit anti-human VEGF-A-20, Santa Cruz Biotechnology, Santa Cruz, USA) was performed on CD14+ cells cultured on cover slips. Cells were fixed and permeabilized with aceton (10 min). Endogenous biotin and avidin were blocked with avidin/biotin blocking kit according to the manufacturer`s instructions (Vector Laboratories, Burlingame, USA). Endogenous peroxidase was blocked by incubation with 0,1% H2O2 in PBS for 10 min. Colour development in immunoperoxidase stainings was performed with 3-amino-9-ethyl-carbazole (AEC; Sigma, Steinheim, Germany).

Phagocytosis assay. To assess phagocytotic capacity 10 * 106 Fluoresbrite YG Microspheres with a diameter of 2,0 μm (Polysciences) were added to 7 days cultured cells for 0.5, 1 and 24 hours.

Transmission electron microscopy. After 7 days of culture in either MF or EC medium, adherent cells were washed with PBS. Next, the cells were fixed with 2% glutaraldehyde (GA, TAAB Laboratories, Aldermastron, UK) at 4°C for 24 hours. Then, cells were washed with PBS and 6.8% sucrose (pH 7.4), and postfixed at 4°C in 1% osmiumtetroxide

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(OsO4) dissolved in 1.5% potassium hexacyanoferrate(II)trihydrate (Merck, Darmstadt, Germany) in 0.1 M PBS. Thereafter, cells were washed with distilled water, dehydrated through a grades series of ethanol, and embedded in EPON 812 (Serva Feinbiochemica, Heidelberg, Germany). Ultra thin sections of approximately 80 nm were cut on a Sorvall microtome (Sorvall, Newton, CT) and contrasted with uranyl acetate and lead citrate. Sections were evaluated in a Philips 201 TEM (Philips, Eindhoven, The Netherlands) operated at 60 kV.

Statistical Analysis. FACS and culture data passed normal distribution tests and were evaluated using student’s t-test (two sided, unpaired). Results for continuous variables are expressed as mean ± SEM. Probability values of P<0.05 were considered statistically significant. Clinical variables were compared between groups using the Fishers’ exact test. Data were analyzed using statistical software (GraphPad Prism 4.0, Graphpad Software Inc.)

ResultsDifferentiation capacity of human CD14+ cells The purity of CD14+ magnetic bead isolation from the mononuclear cell fraction was 97.2% ± 2.21. In figure 1 representative pictures of CD14+ monocytes after 7 days of culture in either EC medium or MF medium are shown. After 7 days of culture in endothelial differentiation medium or macrophage differentiation medium, the MACS-sorted CD14+ cells had adhered and formed rounded as well as spindle shaped elongated cells in both types of medium, of which the latter could be observed in higher quantity in the macrophage differentiation medium (Fig 1A-B). On an ultrastructural level we found that rounded cells in both types of culture medium had an active nucleus, while the cytoplasm was almost entirely filled with vesicles containing proteins (Fig 1C-D,F). These cells also showed an extensive and active Golgi network (Fig 1F). Also spindle-shaped cells in both types of culture media had a similar organization as the rounded cells with many filled vesicles containing proteins (Fig 1F). The only difference at an ultra structural level between both culture media, was that cells in macrophage stimulating medium had consistently more pseudopodia (Fig 1D-E). FACS analysis of 7 day cultured CD14+ monocytes revealed that there was no expression of endothelial-specific markers Tie-2 or Ve-Cadherin on CD14+ cells in both EC and MF differentiation culture medium (Fig 2). For CD31, CD163 and CD68, the expression was comparable between in both EC and MF differentiation culture medium. Only CD14 expression was significantly higher after culture in MF differentiating medium than in EC differentiating medium (Fig 2B).

Figure 1. In this figure we show representative pictures of 7 days cultured CD14+ monocytes in either EC medium (left side of figure) and MF medium (right side of figure). In the first pictures showing an overview of cells in culture (A, original magnification 100x, B, original magnification 400x)), more spindle-shaped and elongated cells can be observed in MF medium. Pictures C-F show cultured CD14+ monocytes at an ultra structural level using transmission electron microscopy (original magnifaction range from 2000x- 7000x). All cells have a very active nucleus (*), an extensive golgi apparatus (G) and contain many large vesicles containing proteins (V), indicating that the cells are transcriptionally active. The presence of pseudopodia (P) on MF cultured CD14+ monocytes, was observed as the only difference between the two culture medium. (m = mitochondrion).

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EC MF

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Figure 2. Flow cytometric analysis of cultured CD14+ monocytes demonstrate that the cells did not express Ve-Cadherin and Tie-2, but did express CD31, CD68 and CD163 in comparable amounts in MF and EC culture medium (B, n=8). Representative flow cytometry histograms of CD14+ monocytes after 7 days of culture in either EC medium or MF medium show comparable expression (A). Para-meters for specific antibody staining (tick line) is set according to isotype controls (gray area). Human umbilical vein endothelial cells (HUVECs), which served as controls, expressed Ve-cadherin as expected, but did also express CD68, which is presumed to be a macrophage marker. However, CD163, which is only specifically expressed on monocytes and macrophages, is not expressed on HUVECs (A).

Colony Forming Units (CFUs) are a hallmark for Endothelial Progenitor Cells (EPCs) in culture. In figure 3A we show representative micrographs of CFUs in EC and MF culture, in which the EC cultured colonies had more attached rounded cells than MF cultured cells that comprised predominantly elongated cells. CD14+ monocytes were formed more colonies or clusters in EC medium than in MF medium (Fig 3B, P<0.05). Another hallmark for cultured EPCs is their uptake of LDL, together with the surface

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binding of Lectin. All cultured cells in this study took up DiI-labeled acetylated LDL, which was to be expected because also macrophages harbor LDL recepters. However, irrespective of the type of medium in which the cells were cultured, they specifically bound in same amounts to Ulex Europeus Lectin (Fig. 3C).

Figure 3. Representative pictures of colony forming units (CFUs) after 7 days of culture of CD14+ monocytes in either EC (left) or MF (right) medium show that more spindle shaped cells were attached to CFUs in MF medium (A). The number of CFUs after 7 days of culture in EC medium was higher than in MF medium (B, P<0.05). by transmission electron microscopy (E). Representative flow cytometry histograms of CD14+ monocytes after 7 days of culture in either EC medium or MF medium show comparable uptake of LDL and binding to Lectin (C). Parameters for specific antibody staining

(thick line) is set according to isotype controls (gray area). Representative pictures of 7 days cultured CD14+ monocytes after addition of 10 million 2 µm latex (particles to culture in EC left) or MF (right) medium at 1 hour (D) and 24 hours (E). One hour after addition more particles were attached to MF cultured cells compared to EC cultured cells, however, after 24 hours all particles were phagocytized regardless medium type. Phagocytozis was confirmed by transmission electron microscopy (E).

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We functionally characterized macrophages by their capacity to phagocytose large particles. Ten million latex particles with a diameter of 2.0 μm were added to the wells of culture of both EC and MF differentiating medium on day 7. More latex beads attached to or were phagocytosed by cells cultured in MF differentiating medium in 1 hour compared to cells cultured in EC differentiating medium (Fig 3D), but after 24 hours most latex beads were phagocytosed irrespective of culture medium (Fig 3E). Electron Microscopy revealed that latex bead particles were actually phagocytosed and were located inside cell (Fig 3E).Immunohistochemical staining of CD14+ cells cultured on cover slips revealed that all cells contained VEGF-A irrespective of culture medium (Fig. 4A,B). Moreover, VEGF-a staining had a patch-like appearance (Fig 4C, D), which suggests there is co-localization with the vesicles observed by transmission electron microscopy. Secretion of VEGF-A in a 4 hours time period was found in EC culture (17.25 pg/ml ± 6.95), but was not found at detectable levels in MF culture (data not shown).

Figure 4. Immunohistological staining of CD14+ monocytes after 7 days of culture on cover slips revealed that all cells contained VEGF-A in both EC and MF medium (representative pictures of isotype control (A) and VEGF-A staining (B) original magnification 100x). Original magnication of 1000x revealed that high density of VEGF-a has a patch-like appearance, which seem to co-localize with the vesicles containing protein as demonstrated by transmission electron microscopy pictures shown in figure 1C-F. Representative pictures of VEGF-A staining in EC medium (C) or MF medium (D) demonstrate a comparable pattern.

CD14+ cells of stable and unstable angina pectoris patients In table 1 we show patient characteristics. Risk factors and medication use of UAP and SAP were comparable. There was a higher prevalence of pre-existing coronary artery disease in SAP (P<0.05).

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Table 1. Clinical profile of investigated patients. UAP= unstable angina pectoris patients, SAP= stable angina pectoris patients, BMI= body mass index, DM = diabetes mellitus, CAD= coronary artery disease. * (P<0.05)

SAP had more circulating CD14+ monocytes than UAP (P<0.05), whereas healthy controls had an intermediate number of circulating monocytes in the peripheral blood (Fig 5A). FACS analysis of freshly isolated CD14+ cells (Fig. 5B) revealed that there was no expression of Ve-Cadherin on freshly isolated CD14+ monocytes of patients or controls. The expression of Tie-2 was very low on freshly isolated CD14+ cells, albeit two times higher on SAP than on controls (P<0.05). In UAP Tie-2 expression was also elevated, but not significantly different from controls (P=0.061). The expression of CD31 on freshly isolated CD14+ cells was higher on UAP than on SAP (P>0.05). There was no difference in expression of CD163 or CD68 between patients and controls. CD14 expression was higher on freshly isolated CD14+ cells of UAP and SAP in comparison to CD14+ cells of controls (P<0.05).

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Figure 5. The number of circulating CD14+ monocytes in peripheral blood is higher in SAP than in UAP (*P<0.05, n=14). The number of circulating CD14+ monocytes of healthy donors (con) were intermediate compared to both patient groups (A). Flow cytometric analysis of freshly isolated CD14+ monocytes of SAP, UAP and con revealed that both patient groups had higher expression of Tie-2 and of CD14 compared to healthy controls and CD31 expression on freshly isolated CD14+ monocytes of UAP was higher than on CD14+ monocytes of SAP (B, *P<0.05). MFI= mean fluorescent intensity.

Cultured CD14+ cells in EC medium revealed that there was no differences in endothelial and macrophage marker expression between UAP and SAP (Fig 6A). Surprisingly, the expression of CD163, a marker restricted to monocyte and macrophages [14] was high in this EC medium. Endothelial specific Tie-2-receptor or Ve-Cadherin were never expressed at detectable levels, irrespective of culture medium. Cultured CD14+ cells in MF medium had a similar surface expression of CD31 in both types of patients. However, cultured CD14+ cells of SAP had a higher expression of CD163, CD68 and CD14 than UAP (P<0.05), indicating a more pronounced macrophage-like marker profile. Similar to cultures of healthy control CD14+ cells, the number of CFUs was again higher in EC medium than in MF medium for both SAP and UAP (Fig 6C). Nevertheless, CD14+ cells from both patient groups were less prone to form CFUs than healthy control CD14+ cells in both types of medium (Fig. 3B, P<0.05). No statistically significant difference in number of CFUs formed by either CD14+ cells of SAP or CD14+ cells of UAP could be observed.

Figure 6. Flow cytometric analysis of CD14+ monocytes of UAP and SAP cultured in EC medium (left) revealed that expression of all investigated markers was comparable (A). Flow cytometric analysis of CD14+ monocytes of UAP and SAP cultured in MF medium (right) demonstrated that the expression of monocyte and macrophage markers CD163, CD68 and CD14 was increased in SAP compared to UAP (A). Number of CFUs were higher in EC medium compared to MF medium, but no differences between the two patient groups were observed (B). However, overall the number of CFUs formed by patient CD14+ monocytes were lower than the number of CFUs formed by healthy donor CD14+ monocytes (* p<0.05). MFI= mean fluorescent intensity.

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Cross serum cultures of controls. Addition of serum, either of controls or patients, to cultures of CD14+ cells in MF medium appeared to increase especially the expression of CD163 in culture with MF medium compared to EC medium (P<0.05, Fig 7). Serum from SAP tended to induce a stronger upregulation of especially monocyte/macrophage surface markers CD68, CD14 and C163 than serum from UAP or healthy controls (P<0.05). Moreover, expression of surface markers on CD14+ monocytes cultured in EC medium supplemented with serum did not increase, except for the expression of CD68, which is especially increased with addition of serum of SAP (P<0.05).

Figure 7. Flow cytometric analysis of CD14+ monocytes of healthy donors (n=5) cultured in EC medium or MF medium without serum addition (SF) or supplemented with pooled human serum of healthy donors (con), with pooled serum of SAP (SA) or with pooled serum of UAP (UA). Serum from SAP increased expression of especially monocyte/macrophage surface markers CD68, CD14 and C163 compared to serum from UAP or healthy controls (P<0.05).

DiscussionIn this study we compared the phenotype of CD14+ monocytes cultured in either EPC medium or macrophage medium. Furthermore, we evaluated whether the differentiation potential is changed in CD14+ monocytes of patients with unstable angina pectoris (UAP) compared to CD14+ monocytes of stable angina pectoris (SAP), since the fate of monocytes might be altered by environmental factors released after atherosclerotic plaque rupture. In the first part, we showed that human CD14+ monocytes from healthy donors obtain a similar phenotype in either macrophage differentiating medium or endothelial cell differentiating medium after 7 days of culture, also on an ultra structural level. In both conditions cultured CD14+ cells demonstrated EPC-like features such as LDL uptake, binding to Lectin and colony formation (CFUs). Nevertheless, CD14+ monocytes cultured in EC medium formed more CFUs than CD14+ monocytes cultured in MF medium.

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The cell-specific marker profile of both MF and EC cultured monocytes showed no expression of the endothelial-cell specific markers Ve-Cadherin and Tie-2 and comparable expression of CD31, CD14 and macrophage-markers like CD68 and CD163, indicating a more macrophage-like phenotype. This is also indicated by the capacity of cultured CD14+ monocytes to phagocytose large particles, albeit that M-CSF stimulated CD14+ cells phagocytized the particles faster. However, cultured CD14+ monocytes appeared transcriptionally active on an ultra structural level and all cultured CD14+ monocytes contain VEGF-a regardless culture medium. Therefore, cultured CD14+ monocytes may be capable of inducing neovascularization, despite its macrophage-like phenotype. In the second part, we showed that CD14+ monocytes of SAP, but not of UAP cultured in MF medium had a more pronounced differentiation towards macrophages, demonstrated by their higher expression of CD163, CD68 and CD14. Concluding from our cross serum experiments, serum of SAP added to MF culture of CD14+ monocytes of healthy donors also induced stronger differentiation towards a macrophage phenotype. Altogether, it seemed that the CD14+ monocyte is predestined towards a macrophage-like phenotype, which could not be altered by culture conditions promoting endothelial differentiation. Moreover, CD14+ monocytes of SAP and healthy CD14+ monocytes supplemented with serum of SAP in MF culture displayed a more pronounced macrophage-like phenotype. However, because they are transcriptionally active and have vesicles containing proteins under which VEGF-a, they may support neovascularization by paracrine effects instead of changing phenotypically into endothelial-like cell. Future experiments will focus on the comparison of the functional capacity and contribution to neovascularization of MF and EC cultured CD14+ cells in ischemic animal models, such as myocardial infarction [15] or hind limb ischemia. Although phenotypically we did not observe great differences between MF and EC cultured CD14+ monocytes of healthy donors, there were functional differences, such as less CFU formation in MF cultured CD14+ cells and slower phagocytosis in EC cultured CD14+ cells. However, the consequences of these differences remain unclear.In contrast to EC culture of CD14+ cells by others [7;16] , we did not observe expression of endothelial specific markers Tie-2 and Ve-cadherin. Urbrich et al. found expression of endothelial markers KDR, CD105, vWF but also of CD45 on CD14+ cells after 4 days of EC-like culture. In our study, 7-days cultured CD14+ cells expressed CD31, but did not express Ve-Cadherin and Tie-2. We did not evaluate expression of KDR, CD105 and vWF. Sharpe et al. did observe Ve-cadherin co-expression on CD14+ cells 7 days of culture in a slightly different medium. Presumably, discrepancies can be explained by different culture techniques, i.e. supplemented factors, percentage of FCS or time of measurement.We have found a higher number of CD14+ monocytes in SAP compared to UAP. A possible explanation might be that, as it was shown in animal models, monocytes migrate to ischemic myocardium within the first hours of reperfusion as occurs in UAP [17]. Moreover, the more macrophage-prone monocytes observed in SAP might be more susceptible to migrate towards the ischemic myocardium, however future investigations are needed to elucidate this. In conclusion, CD14+ cells cultured in either macrophage medium containing M-CSF or endothelial cell medium containing VEGF and bFGF acquire a comparable macrophage-like phenotype after 7 days of culture, which is exaggerated in stable angina pectoris patients. Nevertheless, cultured CD14+ monocytes appeared transcriptionally active

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Chapter 7

and contain VEGF-a. Therefore, cultured CD14+ monocytes may be capable of inducing neovascularization, despite its macrophage-like phenotype.

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