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Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier
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European Journal of Cell Biology 87 (2008) 81–90 www.elsevier.de/ejcb
Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier Fabienne Funckea,b, Heike Hoyerb, Florian Breniga, Caroline Steingena, Dennis Ladageb, Jochen Mu¨ller-Ehmsenb, Annette Schmidta, Klara Brixiusa, Wilhelm Blocha, a
Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Carl-Diem-Weg 6, D-50933 Cologne, Germany b Laboratory for Muscle Research and Molecular Cardiology, Department III for Internal Medicine, University of Cologne, Cologne, Germany Received 12 April 2007; received in revised form 8 August 2007; accepted 31 August 2007
Abstract In vitro cultured endothelial progenitor cells (cEPC) are used for intracoronary cell therapy in cardiac regeneration. The aim of this study was to investigate whether cEPC and circulating mononuclear cells (MNC), which include a small number of in vivo circulating EPC, are able to transmigrate through the endothelial barrier into the cardiac tissue. MNC and EPC were isolated from the peripheral blood from healthy male volunteers (n ¼ 13, 2576 years) and stained with a fluorescent marker. The cells were perfused in vitro through organs with endothelial layers of different phenotypes (rat aorta, human umbilical vein, isolated mouse heart). The endothelium and the basal lamina were then stained by immunofluorescence and the cryo-sections analysed using a confocal laser scanning microscope. After perfusion through the rat aorta, an adhesion/integration of MNC was observed at the endothelial layer and the basal lamina beneath endothelial cells. However, no migration of MNC over the endothelial barrier was found. This remained true even when the cell numbers were increased (from 0.5 to 10 million cells/h), when the time of perfusion was prolonged (1.5–4 h) and when the aorta was cultivated for 24 h. In the Langendorff-perfused mouse heart with intact endothelium, no migration of MNC (1 107) or cEPC (1 106) was observed after 0.5 and 2 h. In conclusion, MNC and cEPC do not possess any capacity to transmigrate the endothelial barrier. In the context of stem cell therapy, these cells may therefore serve as endothelial regenerators but not as cardiomyocyte substitutes. r 2007 Elsevier GmbH. All rights reserved. Keywords: Cell therapy; Endothelial barrier; Transmigration
Introduction The importance of stem and progenitor cells for the regeneration and adaptation of definite tissues, for Corresponding author. Tel.: +49 221 4982 5380; fax: +49 221 4982 8370. E-mail address: [email protected] (W. Bloch).
0171-9335/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2007.08.002
example skeletal muscle and cell lines such as haematopoietic cells, has been known for a long time. Over the past few years, several studies have shown that stem cells and progenitor cells play an important role in cardiac (Oh et al., 2003) and vascular regeneration (Dimmeler and Zeiher, 2004). The importance of the role of endothelial progenitor cells (EPC) in this context has become widely recognised.
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EPC are bone marrow-derived cells that circulate in the peripheral blood. A small number of them are found among the mononuclear cells (MNC). Their mobilisation from the bone marrow to the peripheral circulation, as well as their proliferation, occurs in response to stimuli such as growth factors and cytokines. In this context, vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1), both of which have been shown to be released by ischemic tissue, seem to play an important role in the mobilisation of EPC (Asahara et al., 1999; Askari et al., 2003; Ceradini et al., 2004). EPC are able to proliferate and differentiate into mature endothelial cells when grown under appropriate conditions in vitro. Asahara et al. (1997) showed that CD34-positive cells from human peripheral blood grew into cells with endothelial characteristics after being plated on a fibronectin-coated surface. Tso and et al. (2006) have shown that EPC adhere to endothelium in response to an inflammation in the thoracic aorta of mice and differentiate into mature endothelial cells. Endothelial health reflects a balance between endothelial injury and repair. The endothelium is at least partly regenerated by circulating adult endothelial cells. However, this pool is very limited, proliferates infrequently (Schwartz et al., 1976) and is not sufficiently active to be able to satisfy this regeneration process. It is well known that the amount of circulating EPC in the blood is decreased in patients with cardiac risk factors (Hill et al., 2003; Vasa et al., 2001) and is increased in patients with acute myocardial infarction and active ischemic heart disease (Massa et al., 2005; Shintani et al., 2001). Thus, it seems promising that EPC could be used to develop therapies for the treatment of cardiovascular diseases (e.g. after atherosclerotic or cardiac events). There is also evidence that EPC trans-differentiate into cells of the myocardial lineage (Murasawa et al., 2005) and may thereby contribute to myocardial repair. In this context, it may be assumed that EPC not only adhere to, but also transmigrate through the endothelial barrier. The aim of this study was to investigate the interaction between EPC and the endothelial barrier. For this purpose, co-culture and perfusion experiments were performed with MNC, cultivated EPC (cEPC) and endothelial cells of rat aorta, murine heart and umbilical vein.
(2576 years old). After Ficoll-density gradient centrifugation, these cells were washed twice with Dulbecco´s phosphate-buffered saline (DPBS; 9.5 mM phosphate without calcium and magnesium, Invitrogen, Paisley, UK) and prepared for further analysis.
Culturing of EPC Immediately after isolation of MNC, 1 107 cells were plated onto culture dishes coated with fibronectin and maintained in endothelial basal medium MV2 (PromoCell, Heidelberg, Germany) supplemented with 5% foetal calf serum (FCS; Invitrogen), epidermal growth factor, hydrocortisone, VEGF, basic fibroblast growth factor (hbFGF), IGF-1, ascorbic acid, gentamycin, and amphotericin B. After 3 days in culture (95% humidity, 5% CO2), non-adherent cells were removed by washing with DPBS and the remaining adherent cells (i.e. cEPC) were incubated in fresh medium for 24 h before perfusion through isolated organs (Urbich et al., 2005). For staining, cells were removed gently from the dishes with 3 ml accutases (PAA, Pasching) and washed with DPBS.
PKH cell staining Vital cell staining of MNC and cEPC was carried out using a MINI 67 staining kit with PKH-67 (SigmaAldrich, Steinheim, Germany), a green fluorescent cell tracking dye. Its long alkyl-chains rapidly partition into cell membranes and provide strong anchorage in the lipid bi-layer of living cells. The cells (MNC or cEPC) were resuspended in diluent C and PKH-67 diluted in diluent C was added. Staining was performed at 25 1C for 5 min in the dark and then stopped by adding FCS for 1 min. The cells were washed with MV2 medium and counted in a Neubauer chamber.
‘Static’ stem cell aorta model for co-culture
Materials and methods
Male Wistar rats (8–12 weeks old) were sacrificed by cervical dislocation. The aorta was removed immediately after death, cut into rings, opened and attached to 12-mm glass cover slips with the endothelium facing the top using a two-component fibrin sealant (Tissucol Kit, Baxter, Vienna, Austria). The aortas were transferred in well plates and incubated with 6 104 PKH67 vital stained MNC for 105 min in endothelial basal medium MV2. These co-culture experiments between MNC and endothelial cells of rat aorta were performed three times.
Isolation of mononuclear cells
MNC perfusion through rat aorta
MNC were isolated from the peripheral blood of thirteen healthy, male human, non-smoking volunteers
The aorta was removed from rats as described above and cannulated for perfusion. The stained MNC were
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resuspended in 50 ml MV2 medium containing 5% FCS. The aortas were perfused with 1.5 107 MNC for 1.5 and 4 h (0.55 and 0.21 ml/min). In order to investigate if the perfusion time was too short for the beginning of migration of MNC, the perfused aorta was cultured in tyrode solution (in mM: NaCl 119.8, KCl 5.4, MgCl2 1.05, CaCl2 1.8, NaHCO3 22.6, NaHPO4 0.42, glucose 5.05, ascorbic acid 0.28, disodium EDTA 0.05, pH 7.4, gassed with 100% O2, 37 1C) for 24 and 48 h (95% humidity, 5% CO2). The MNC perfusion through isolated rat aortic rings was repeated 21 times.
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Immunofluorescence staining of the basal lamina Tissue slices were incubated in 5% bovine serum albumin (BSA) and in Tris-buffered saline (TBS) (1 h, room temperature) for blocking. The primary antibody rabbit anti-laminin (Sigma, St. Louis, MO, USA) was diluted 1:500 in 0.8% BSA and incubated overnight at 4 1C. As a secondary antibody, Cy5-conjugated affine pure goat anti-rabbit IgG (Jackson Immuno Research, Suffolk, UK) (1:500) was used.
Immunofluorescence staining of MNC and EPC Perfusion of cells through umbilical vein A human umbilical vein was clamped in the perfusion model as described above and 1.5 107 MNC suspended in 50 ml medium were perfused through it for 4 h (0.21 ml/min). The MNC perfusion through an umbilical vein was done once.
Perfusion of cells through the murine heart Wild-type mice (4–6 weeks old) were sacrificed by cervical translocation. The heart was quickly removed and the aorta cannulated for the initiation of a retrograde perfusion in a Langendorff-modified apparatus. The stained cells were resuspended in 50 ml MV2 medium containing 5% FCS. The hearts were perfused with 1 107 MNC and 1 106 cEPC for 0.5 and 2 h (1.67 and 0.42 ml/min). The MNC perfusion through murine hearts was repeated 16 times and the EPC perfusion 10 times.
Fixation of the tissue and cryo-sections Co-culture experiments: The tissue was immediately fixed with 4% formaldehyde (freshly prepared from paraformaldehyde, Merck, VWA, Darmstadt, Germany) for 20 min followed by three washes with DPBS. Perfusion experiments: The tissue was immediately fixed with 4% formaldehyde for 4–6 h while being cooled at 4 1C. After this time, the tissue was washed with DPBS, fixed again with 18% sucrose and then frozen in tissue-tecs(Sakura Finetek Europe, Zoeterwoude, NL) at 80 1C followed by a cryo-slicing to 10 mm thickness and immunofluorescence microscopy.
Visualising of endothelial cells Tissue slices were stained for 30 min with rhodamineGriffonia (Bandeireaea) simplicifolia lectin I (Vector Laboratories, Burlingame, USA). After washing with DPBS, the tissue was embedded in Aquapoly-mounts (Polysciences Inc., Warrington, USA).
For characterisation of MNC and EPC, immunofluorescence staining was performed as described above. The first antibody used was either CD34 (Santa Cruz, Biotechnology, Inc., Heidelberg, Germany) diluted 1:400 or rabbit polyclonal to VEGFR-2 (Abcam, Cambridge, UK) diluted 1:500 in 0.8% BSA. As secondary antibody, Cy5-conjugated affine pure goat anti-rabbit IgG (Jackson Immuno Research) (1:500) was used.
Microscopic assessment Microscopy was performed using a confocal laser scanning microscope (LSM 510 META, Zeiss, Go¨ttingen). For each experimental condition, at least 60 single cells were investigated.
Results Co-culture experiments between MNC and endothelial cells of rat aorta To investigate whether MNC interact with endothelial cells, a ‘static’ stem cell aorta model was established to perform co-culture experiments between isolated MNC and endothelial cells of rat aortas. A freshly isolated MNC before the experiment is shown in Fig. 1A. It appears in a completely spherical shape. Fig. 1B shows an MNC after being co-cultivated on the endothelial layer of the rat aorta. The spherical shape of the MNC has been lost, the cell has flattened and extensions have formed in close proximity to the endothelial cells. In Fig. 1C the aorta has been rotated 901 around a horizontal axis. The tissue was fixed on the right side on a glass coverslip (see arrow), so the endothelium faces to the left. It can clearly be seen that the MNC have attached to the endothelial layer, but did not form any extensions through it. These pictures clearly show that the MNC attempted to interact with the endothelium when being co-cultured. Morphological changes
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Fig. 1. Interaction between MNC and endothelium under co-culture conditions. (A) MNC before being in contact with endothelial cells. (B) Morphological change of MNC upon interaction with endothelial cells (105 min of co-culture on an endothelial layer of rat aorta). (C) Co-culture as in (B). 901 rotation around the horizontal axis of an MNC without any extension into the subendothelial layer (arrow: side of the glass coverslip). Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamineGriffonia simplicifolia lectin I.
Fig. 2. MNC perfusion through rat aorta and interaction with the tissue. (A) Adhesion of MNC at endothelial cells (A) and at endothelial-free regions (B) after a perfusion time of 4 h. (C) Morphological changes of MNC and integration into the endothelial layer. (D) Adhesion of MNC to lower tissues where the basal lamina was disrupted, but no migration into the vessel tissue. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I. White: basal lamina stained with anti-laminin and Cy5-conjugated secondary antibody. Arrow: basal lamina beneath the endothelial layer.
including the formation of extensions into the endothelial cells can be detected.
MNC perfusion through isolated rat aortic rings To investigate whether MNC also interact with endothelial cells under perfusion conditions, MNC were perfused through isolated rat aortas. Perfusion was performed with 1.5 107 MNC for 1.5 and 4 h (0.55 and 0.21 ml/min). Cross sections of the perfused rat aortas, examined by confocal microscopy, are shown in Fig. 2. The adhesion of MNC (green) to endothelial cells (red) is shown in Fig. 2A and the interaction of MNC with endothelial-free regions (i.e. with the basal lamina) in Fig. 2B. In Fig. 2C morphological changes of MNC can be seen when being attached to the endothelium. A flattening of the cell can be observed. This is a sign for direct interaction of the cell with the endothelium. The adhesion of MNC to lower tissue of the rat aorta is shown in Fig. 2D (white ¼ basal lamina) where the basal lamina was also disrupted. One hundred cells were investigated, but migration of MNC through the subendothelial tissue of the rat aorta was not detected in any case.
In order to see if the two perfusion times were too short for the beginning of migration of MNC, the aortas were cultivated for 24 and 48 h after the perfusion. The results are shown in Fig. 3. Again, no transmigration of MNC into subendothelial parts of the aortic wall was observed under these conditions.
MNC perfusion through isolated umbilical vein Since there is evidence for regional differences in the endothelial cell phenotype (Verhamme and Hoylaerts, 2006), it was investigated whether the transmigrational behaviour of MNC is dependent on the endothelial phenotype. MNC (1.5 107) were perfused through an umbilical vein for 4 h (0.21 ml/min). Cross sections through the umbilical vein are shown in Fig. 4. The adhesion of MNC to endothelial cells is shown in Fig. 4A and the adhesion to the basal lamina in Fig. 4B (endothelium-free tissue). As for the results with rat aorta, a morphological flattening of the cells was observed, but migration of MNC through the endothelial layer was not seen in any of the 50 cells investigated.
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Fig. 3. Cultivation of the rat aorta for 24 h (A) or 48 h (B) after perfusion with MNC. No migration into lower parts of the tissue can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I. White: basal lamina stained with anti-laminin and Cy5-conjugated secondary antibody.
Fig. 4. MNC perfusion through umbilical vein. Adhesion of MNC at endothelial cells (A) and endothelial-free regions (B). No migration into lower parts of the vessel tissue, but morphological changes can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I. White: basal lamina stained with anti-laminin and Cy5conjugated secondary antibody.
MNC perfusion through isolated murine hearts In order to investigate the migrational activity of MNC and EPC in functional tissue with a complete endothelial layer, the cells we perfused through isolated murine hearts in which the endothelial layer of the capillaries was completely functional and not manually disrupted by dissection. To minimise the rate of inflammatory processes due to dissection, the perfusion with MNC and cEPC was started immediately after removing the heart. Fig. 5 shows transversal cross sections through murine hearts perfused with 1 107 MNC for 30 min or 120 min (1.67 and 0.42 ml/min). At least 60 cells were observed for each condition. At a perfusion time of 30 min (Fig. 5A), migration of MNC over the endothelial layer could not be observed in any case. In order to find out if 30 min were too short for MNC migration, the cells were perfused for 120 min. No migration could be detected (Fig. 5B) here either.
To investigate whether MNC adhere strongly to endothelial cells in the capillaries, the mice hearts were firstly perfused with the MNC suspension for 30 or 120 min (1.67 and 0.42 ml/min) and afterwards with 5 ml DPBS for 10 min (0.5 ml/min). Cross sections of that are shown in Fig. 6A. In this experiment no loosening of the MNC from the endothelial layer was seen. This showed that there was a strong contact and interaction between the perfused MNC and endothelial cells in the capillaries. The next idea was that cell perfusion, i.e. erythrocytes, could cause a loosening of the attached MNC when subsequently being perfused through the small capillaries of the mouse heart. So 5 ml of human EDTAblood was perfused through mice hearts after the perfusion of MNC for 30 and 120 min for 10 min (0.5 ml/min). The results show that no loosening of the cells occurred. A cross section is shown in Fig. 6B. This led to the conclusion that the MNC were strongly attached to the endothelial cells in the capillaries of the
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Fig. 5. MNC perfusion through isolated murine hearts for 30 min (A) or 120 min (B). No migration of cells over the endothelial barrier can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I.
Fig. 6. MNC perfusion through isolated murine hearts, subsequently washed for 10 min with DPBS (A) or EDTA-blood containing erythrocytes (B). No loosening of the cells from the endothelium can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I.
heart so that integration, but not transmigration, could take place. To obtain further information on whether it is possible to remove the cells from the endothelium we compared the number of MNC in isolated hearts that had been directly fixed in formaldehyde after MNC perfusion with the number of MNC in hearts that had been perfused with DPBS or blood after MNC perfusion. The comparison was made by counting the number of MNC in the microscope field of vision. We did not observe any significant differences in the number of MNC between the different conditions (direct fixation: 3.371.6 MNC per field of vision; perfusion with DPBS after MNC perfusion: 4.072.2 MNC per field of vision; blood perfusion after MNC perfusion: 3.771.9 MNC per field of vision). Overall it can be said that MNC, which contain circulating EPC, attach strongly to the endothelial cells of vessel walls when being perfused, but do not migrate
over the endothelial barrier into subendothelial parts of the vessel tissue.
EPC perfusion through isolated murine hearts The number of EPC in MNC is extremely low (Mancuso et al., 2001; Kim et al., 2003). In order to increase the number of EPC in the cell suspension, 1 107 MNC were cultured to obtain 1 106 cEPC. These cEPC were resuspended in 50 ml medium and perfused through mice hearts for 30 and 120 minutes (1.67 and 0.42 ml/min). In Fig. 7 the cross sections of the murine hearts perfused with cEPC are shown. This experiment conformed with the previous results obtained after MNC perfusion by showing no migration of cEPC over the endothelial barrier into subendothelial tissue after 30 min (Fig. 7A) and 120 min (Fig. 7B).
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Fig. 9. EPC are positive for CD34 and VEGFR-2. Green: MNC vital stained with PHK-67. White: Immunostaining for CD34 (A) or VEGFR-2 (B).
Discussion
Fig. 7. EPC perfusion through isolated murine hearts for 30 min (A) or 120 min (B). No migration of the cells over the endothelial barrier can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamineGriffonia simplicifolia lectin I.
Fig. 8. MNC are positive for CD34 and VEGFR-2. Green: MNC vital stained with PHK-67. White: Immunostaining for CD34 (A) or VEGFR-2 (B).
Characterisation of MNC and EPC by immunofluorescence staining MNC were characterised by their surface markers. A dual-staining for PKH and either the expression of the VEGF receptor 2 or CD34 was performed. The MNC used for the experiments were positive for CD34 (Fig. 8A) and the VEGF receptor 2 (Fig. 8B). EPC were also characterised by their surface markers. A dual-staining for PKH and either the expression of the VEGF receptor 2 or CD34 was performed. The EPC used for the experiments are positive for CD34 (Fig. 9A) and the VEGF receptor 2 (Fig. 9B).
The aim of this study was to investigate the interaction between circulating and in vitro-cultivated EPC and the endothelial barrier. The results demonstrated that MNC and cEPC interact with endothelial cells. Morphological changes in the shape of the progenitor cells were seen when MNC or cEPC were attached to the endothelium. They flattened and built up extensions when in contact with the endothelium. However, migration of MNC and cEPC through and over the endothelial barrier was not detected. These results explain the process of angiogenesis and vasculogenesis in terms of neovascularisation. Angiogenesis is the process of new vessel growth. Mature, differentiated endothelial cells break free from their basement membrane, and migrate and proliferate to form sprouts from parent vessels. Vasculogenesis involves the participation of EPC, which circulate to sites of neovascularisation where they differentiate in situ into mature endothelial cells (Isner and Asahara, 1999). Incorporated EPC may also release growth factors that promote angiogenesis by acting on mature endothelial cells (Fuchs, 2001). However in terms of cardiac regeneration, especially when infused intra-arterially, migration out of the vessel into the surrounding tissue is required (Dimmeler et al., 2005). In vivo this migration process involves multiple steps. The EPC have to leave their normal environment, the bone marrow, and reach the target tissue by migrating through the vessel wall. The present paper focuses on the second migratory process. This is divided into different steps which are currently of high interest in research: (1) the EPC must come into contact with the endothelial cells; (2) they have to dissociate the cell–cell contact of the endothelium; (3) they must integrate into the endothelial layer, and finally (4) they have to pass and leave this barrier (Schmidt et al., 2006). So far, the mechanism of cell mobilisation and homing is only rudimentarily understood. It is hypothesised that once the cells are mobilised from bone marrow
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as a result of a stimulus (VEGF, GM-SCF, SDF-1, angiopoietin-1, placental growth factor or erythropoietin via matrix metalloproteinase 9), the first adhesion to endothelium takes place. Here SDF-1 appears to play a pivotal role as an attractor for EPC to ischemic tissue. Also the high mobility group box protein 1, a chromatin-binding protein released by cell necrosis, may stimulate EPC attraction (Dimmeler et al., 2005). After cell attraction, the first weak interaction takes place by rolling the EPC over the endothelial cells, which is governed by L-selectins and their ligands (Biancone et al., 2004). Duan et al. (2006) recently showed that EPC express LFA-1 and VLA-4 and that their ligands ICAM-1 and VCAM-1 are expressed in vessel endothelium in ischemic tissues. The next step, a strong adhesion of the cells, is triggered by b2-integrins (Chavakis et al., 2005). At this point the transmigration of the cell through the endothelium and invasion into the target tissue could take place. It needs to be mentioned that MNC were deliberately perfused in this study because it was assumed that these cells represent the native cell fraction which is responsible for endothelial regeneration processes in the body and which contains a small number of EPC (o500/ml (Mancuso et al., 2001) or 40.2710.2/ml (Kim et al., 2003)). This was because no clear differentiation of EPC has yet been found and the authors used different methods for detection. Also, changes of any (surface) properties of native EPC were avoided in order to get representative results of how vessel regeneration takes place in vivo. For enhancement of the number of EPC in the perfused cell suspension, the MNC were kept in culture for 4 days under EPC-growing conditions, as done by other groups before (Urbich et al., 2005). This was done in order to perfuse mostly EPC rather than the large and undefined pool of MNC. Under culture conditions, however, the cells experienced a change in morphology, as can be seen by their change in size, and do not represent the natural cells circulating in the body. The problem is that by now no consensus has been reached on the characteristics of EPC, and published studies mostly characterise EPC differently. Another limitation of the study is that a xenograft model was used. It cannot be excluded that this influenced the results. No transmigration of cells into the subendothelial tissue was observed in this study. However, these results do not exclude the migrational activity of EPC. In the case of wound healing, for example, the cells are able to migrate along a gradient of growth factors released by the damaged tissue in order to reach the site where wound healing takes place (Suh et al., 2005). However, in wounds, the tissue is damaged and an intact endothelial barrier no longer exists. From the results obtained here it can only be said that EPC are not able to cross the endothelial barrier by transmigration.
That EPC possess the ability to migrate was also shown in different in vitro experiments in Boyden chambers. A Boyden chamber consists of two compartments separated by a Millipore filter (3–8 mm) to test for chemotaxis. In most cases, EPC are placed in basal medium in the upper compartment of the Boyden chamber (Laufs et al., 2005). The lower chamber contains basal medium and the chemoattractant, mostly VEGF. This chamber is cultured for a certain period of time. Any EPC that migrate to the lower chamber can then be detected and counted. This showed that the EPC have certain migration ability in vitro. In contrast to EPC, Schmidt et al. (2006) have recently shown that mesenchymal stem cells (MSC) possess the ability to transmigrate over the endothelial barrier, making them a potentially useful tool for cellular therapy in injured tissue. MSC were able to split tight junctions between endothelial cells, became integrated into the endothelial wall of the capillary vessel and could finally fully pass the endothelial barrier (Schmidt et al., 2006). It needs to be pointed out that after application of EPC to patients with ischemic events, i.e. myocardial infarction or cardiomyopathies, a clear improvement of the ejection fraction could be measured (Britten et al., 2003). After ischemic events cardiac tissue is damaged in a way that is associated with a complete loss of vascular integrity and thus a loss of the function of the endothelial barrier. EPC do not have to cross this barrier for cardiac regeneration. In the case of acute damage the cells can ‘bleed’ freely into the cardiac tissue. However, our results show that in a healthy or chronically damaged heart where there is no acute damage of the endothelial barrier, non-endothelial cell replacement via EPC migration is unlikely to occur. Thus, under these conditions, there must be mechanisms other than the migration of EPC over the endothelial barrier that enable these cells to support the cardiac regeneration process without acting themselves as replacement cells, pointing this connection the paracrine effects of endothelial progenitor cells have been postulated. One possibility could be that the EPC themselves express growth factors which directly affect the surrounding tissue in order to start the regeneration process. Urbich et al. (2005) reported the release of soluble factors by EPC which might explain their incorporation into newly formed vessels, enhanced neovascularisation and improvement of cardiac function. These authors postulated that EPC promote neovascularisation and cardiac regeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilise tissue-residing progenitor cells. It was found that EPC express higher levels of VEGF-A, VEGF-B, SDF-1 and IDF-1 than human umbilical vein endothelial cells or human microvascular endothelial cells. So EPC exhibit a high expression of
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angiogenic growth factors that enhance migration of mature endothelial cells and tissue-resident cardiac progenitor cells. The fact that MNC adhere strongly to the vessel wall and cannot be removed by passing blood supports the view that they act in a paracrine manner on surrounding endothelial cells in order to support the cardiac regeneration process without acting themselves as replacement cells. It is possible that application of granulocyte-colony stimulating factor (G-CSF) prior to EPC isolation alters their migratory capacity. It has been shown that such treatment increases the expression of the CXCR4 receptor and may thus facilitate the homing of the EPC in patients with coronary artery disease (Powell et al., 2005). However, there are also reports indicating that G-CSF impairs the functional capacity of EPC, e.g. in an experimental mouse model of hind limb ischemia (Honold et al., 2006). Thus, the functional (migratory) capacity of EPC may be influenced by pharmacological treatment. This has to be investigated in further studies.
Conclusion The work presented here demonstrates that EPC possess the capability to interact with the endothelial layer of different organs in a way that causes morphological changes and strong adhesion to the tissue, but are not able to transmigrate over the endothelial barrier.
Acknowledgements This work was supported by the Novartis Foundation through a grant to W. Bloch, U. Mehlhorn and R.H.G. Schwinger. We thank the clinic of gynaecology and midwifery, University of Cologne that supplied an umbilical cord. We would also like to thank Anika Voss and Esra Koroglu¨ for their excellent technical assistance.
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Biancone, L., Cantaluppi, V., Duo`, D., Deregibus, M.C., Torre, C., Camussi, G., 2004. Role of L-selectin in the vascular homing of peripheral blood-derived endothelial progenitor cells. J. Immunol. 173, 5268–5274. Britten, M.B., Abolmaali, N.D., Assmus, B., Lehmann, R., Honold, J., Schmitt, J., Vogl, T.J., Martin, H., Scha¨chinger, V., Dimmeler, S., Zeiher, A.M., 2003. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 108, 2212–2218. Ceradini, D.J., Kulkarni, A.R., Callaghan, M.J., Tepper, O.M., Bastidas, N., Kleinman, M.E., 2004. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat. Med. 10, 858–864. Chavakis, E., Aicher, A., Heeschen, C., Sasaki, K., Kaiser, R., El Makhfi, N., Urbich, C., Peters, T., ScharffetterKochanek, K., Zeiher, A.M., Chavakis, T., Dimmeler, S., 2005. Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells. J. Exp. Med. 201, 63–72. Dimmeler, S., Zeiher, A.M., 2004. Vascular repair by circulating endothelial progenitor cells: the missing link in atherosclerosis? J. Mol. Med. 82, 671–677. Dimmeler, S., Zeiher, A.M., Schneider, M.D., 2005. Unchain my heart: the scientific foundations of cardiac repair. J. Clin. Invest. 115, 572–583. Duan, H., Cheng, L., Sun, X., Wu, Y., Hu, L., Wang, J., Zhao, H., Lu, G., 2006. LFA-1 and VLA-4 involved in human high proliferative potential-endothelial progenitor cells homing to ischemic tissue. Thromb. Haemost. 96, 807–815. Fuchs, S., 2001. Transendothelial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J. Am. Coll. Cardiol. 37, 1726–1732. Hill, J.M., Zalos, G., Halcox, J.P., Schenke, W.H., Waclawiw, M.A., Quyyumi, A.A., 2003. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med. 348, 593–600. Honold, J., Lehmann, R., Heeschen, C., Walter, D.H., Assmus, B., Sasaki, K., Martin, H., Haendeler, J., Zeiher, A.M., Dimmeler, S., 2006. Effects of granulocyte colony stimulating factor on functional activities of endothelial progenitor cells in patients with chronic ischemic heart disease. Arterioscler. Thromb. Vasc. Biol. 26, 2238–22343. Isner, J.M., Asahara, T., 1999. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J. Clin. Invest. 103, 1231–1236. Kim, H.K., Song, K.S., Kim, H.O., Chung, J.H., Lee, K.R., Lee, Y.J., Lee, D.H., Lee, E.S., Kim, H.K., Ryu, K.W., 2003. Circulating numbers of endothelial progenitor cells in patients with gastric and breast cancer. Cancer Lett. 198, 83–88. Laufs, U., Urhausen, A., Werner, N., Scharhag, J., Heitz, A., Kissner, G., Bo¨hm, M., Kindermann, W., Nickenig, G., 2005. Running exercise of different duration and intensity: effect on endothelial progenitor cells in healthy subjects. Eur. J. Cardiovasc. Prev. Rehabil. 12, 407–414. Mancuso, P., Burlini, A., Pruneri, G., Goldhirsch, A., Martinelli, G., Bertolini, F., 2001. Resting and activated
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endothelial cells are increased in the peripheral blood of cancer patients. Blood 97, 3658–3661. Massa, M., Rosti, V., Ferrario, M., Campanelli, R., Ramajoli, I., Rosso, R., 2005. Increased circulating hematopoietic and endothelial progenitor cells in the early phase of acute myocardial infarction. Blood 105, 199–206. Murasawa, S., Kawamoto, A., Horii, M., Nakamori, S., Asahara, T., 2005. Niche-dependent translineage commitment of endothelial progenitor cells, not cell fusion in general, into myocardial lineage cells. Arterioscler. Thromb. Vasc. Biol. 25, 1388–1394. Oh, H., Bradfute, S.B., Gallardo, T.D., Nakamura, T., Gaussin, V., Mishina, Y., Pocius, J., Michael, L.H., Behringer, R.R., Garry, D.J., Entman, M.L., Schneider, M.D., 2003. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc. Natl. Acad. Sci. USA 100, 12313–12318. Powell, T.M., Paul, J.D., Hill, J.M., Thompson, M., Benjamin, M., Rodrigo, M., McCoy, J.P., Read, E.J., Khuu, H.M., Leitman, S.F., Finkel, T., Cannon III, R.O., 2005. Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 25, 296–301. Schmidt, A., Ladage, D., Steingen, C., Brixius, K., Schinko¨the, T., Klinz, F.J., Schwinger, R.H., Mehlhorn, U., Bloch, W., 2006. Mesenchymal stem cells transmigrate over the endothelial barrier. Eur. J. Cell Biol. 85, 1179–1188.
Schwartz, S.M., Benditt, E.P., 1976. Clustering of replicated cells in aortic endothelium. Proc. Natl. Acad. Sci. USA 73, 651–653. Shintani, S., Murohara, T., Ikeda, H., Ueno, T., Honma, T., Katoh, A., 2001. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation 103, 2776–2779. Suh, W., Kim, K.L., Kim, J.M., Shin, I.S., Lee, Y.S., Lee, J.Y., Jang, H.S., Lee, J.S., Byun, J., Choi, J.H., Jeon, E.S., Kim, D.K., 2005. Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization. Stem Cells 23, 1571–1578. Tso, C., Martinic, G., Fan, W.H., Rogers, C., Rye, K.A., Barter, P.J., 2006. High-density lipoproteins enhance progenitor-mediated endothelium repair in mice. Arterioscler. Thromb. Vasc. Biol. 26, 1144–1149. Urbich, C., Aicher, A., Heeschen, C., Dernbach, E., Hofmann, W.K., Zeiher, A.M., Dimmeler, S., 2005. Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J. Mol. Cell. Cardiol. 39, 733–742. Vasa, M., Fichtlscherer, S., Aicher, A., Adler, K., Urbich, C., Martin, H., 2001. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ. Res. 89, E1–E7. Verhamme, P., Hoylaerts, M.F., 2006. The pivotal role of the endothelium in haemostasis and thrombosis. Act. Clin. Belg. 61, 213–219.
European Journal of Cell Biology 87 (2008) 81–90 www.elsevier.de/ejcb
Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier Fabienne Funckea,b, Heike Hoyerb, Florian Breniga, Caroline Steingena, Dennis Ladageb, Jochen Mu¨ller-Ehmsenb, Annette Schmidta, Klara Brixiusa, Wilhelm Blocha, a
Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Carl-Diem-Weg 6, D-50933 Cologne, Germany b Laboratory for Muscle Research and Molecular Cardiology, Department III for Internal Medicine, University of Cologne, Cologne, Germany Received 12 April 2007; received in revised form 8 August 2007; accepted 31 August 2007
Abstract In vitro cultured endothelial progenitor cells (cEPC) are used for intracoronary cell therapy in cardiac regeneration. The aim of this study was to investigate whether cEPC and circulating mononuclear cells (MNC), which include a small number of in vivo circulating EPC, are able to transmigrate through the endothelial barrier into the cardiac tissue. MNC and EPC were isolated from the peripheral blood from healthy male volunteers (n ¼ 13, 2576 years) and stained with a fluorescent marker. The cells were perfused in vitro through organs with endothelial layers of different phenotypes (rat aorta, human umbilical vein, isolated mouse heart). The endothelium and the basal lamina were then stained by immunofluorescence and the cryo-sections analysed using a confocal laser scanning microscope. After perfusion through the rat aorta, an adhesion/integration of MNC was observed at the endothelial layer and the basal lamina beneath endothelial cells. However, no migration of MNC over the endothelial barrier was found. This remained true even when the cell numbers were increased (from 0.5 to 10 million cells/h), when the time of perfusion was prolonged (1.5–4 h) and when the aorta was cultivated for 24 h. In the Langendorff-perfused mouse heart with intact endothelium, no migration of MNC (1 107) or cEPC (1 106) was observed after 0.5 and 2 h. In conclusion, MNC and cEPC do not possess any capacity to transmigrate the endothelial barrier. In the context of stem cell therapy, these cells may therefore serve as endothelial regenerators but not as cardiomyocyte substitutes. r 2007 Elsevier GmbH. All rights reserved. Keywords: Cell therapy; Endothelial barrier; Transmigration
Introduction The importance of stem and progenitor cells for the regeneration and adaptation of definite tissues, for Corresponding author. Tel.: +49 221 4982 5380; fax: +49 221 4982 8370. E-mail address: [email protected] (W. Bloch).
0171-9335/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2007.08.002
example skeletal muscle and cell lines such as haematopoietic cells, has been known for a long time. Over the past few years, several studies have shown that stem cells and progenitor cells play an important role in cardiac (Oh et al., 2003) and vascular regeneration (Dimmeler and Zeiher, 2004). The importance of the role of endothelial progenitor cells (EPC) in this context has become widely recognised.
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EPC are bone marrow-derived cells that circulate in the peripheral blood. A small number of them are found among the mononuclear cells (MNC). Their mobilisation from the bone marrow to the peripheral circulation, as well as their proliferation, occurs in response to stimuli such as growth factors and cytokines. In this context, vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1), both of which have been shown to be released by ischemic tissue, seem to play an important role in the mobilisation of EPC (Asahara et al., 1999; Askari et al., 2003; Ceradini et al., 2004). EPC are able to proliferate and differentiate into mature endothelial cells when grown under appropriate conditions in vitro. Asahara et al. (1997) showed that CD34-positive cells from human peripheral blood grew into cells with endothelial characteristics after being plated on a fibronectin-coated surface. Tso and et al. (2006) have shown that EPC adhere to endothelium in response to an inflammation in the thoracic aorta of mice and differentiate into mature endothelial cells. Endothelial health reflects a balance between endothelial injury and repair. The endothelium is at least partly regenerated by circulating adult endothelial cells. However, this pool is very limited, proliferates infrequently (Schwartz et al., 1976) and is not sufficiently active to be able to satisfy this regeneration process. It is well known that the amount of circulating EPC in the blood is decreased in patients with cardiac risk factors (Hill et al., 2003; Vasa et al., 2001) and is increased in patients with acute myocardial infarction and active ischemic heart disease (Massa et al., 2005; Shintani et al., 2001). Thus, it seems promising that EPC could be used to develop therapies for the treatment of cardiovascular diseases (e.g. after atherosclerotic or cardiac events). There is also evidence that EPC trans-differentiate into cells of the myocardial lineage (Murasawa et al., 2005) and may thereby contribute to myocardial repair. In this context, it may be assumed that EPC not only adhere to, but also transmigrate through the endothelial barrier. The aim of this study was to investigate the interaction between EPC and the endothelial barrier. For this purpose, co-culture and perfusion experiments were performed with MNC, cultivated EPC (cEPC) and endothelial cells of rat aorta, murine heart and umbilical vein.
(2576 years old). After Ficoll-density gradient centrifugation, these cells were washed twice with Dulbecco´s phosphate-buffered saline (DPBS; 9.5 mM phosphate without calcium and magnesium, Invitrogen, Paisley, UK) and prepared for further analysis.
Culturing of EPC Immediately after isolation of MNC, 1 107 cells were plated onto culture dishes coated with fibronectin and maintained in endothelial basal medium MV2 (PromoCell, Heidelberg, Germany) supplemented with 5% foetal calf serum (FCS; Invitrogen), epidermal growth factor, hydrocortisone, VEGF, basic fibroblast growth factor (hbFGF), IGF-1, ascorbic acid, gentamycin, and amphotericin B. After 3 days in culture (95% humidity, 5% CO2), non-adherent cells were removed by washing with DPBS and the remaining adherent cells (i.e. cEPC) were incubated in fresh medium for 24 h before perfusion through isolated organs (Urbich et al., 2005). For staining, cells were removed gently from the dishes with 3 ml accutases (PAA, Pasching) and washed with DPBS.
PKH cell staining Vital cell staining of MNC and cEPC was carried out using a MINI 67 staining kit with PKH-67 (SigmaAldrich, Steinheim, Germany), a green fluorescent cell tracking dye. Its long alkyl-chains rapidly partition into cell membranes and provide strong anchorage in the lipid bi-layer of living cells. The cells (MNC or cEPC) were resuspended in diluent C and PKH-67 diluted in diluent C was added. Staining was performed at 25 1C for 5 min in the dark and then stopped by adding FCS for 1 min. The cells were washed with MV2 medium and counted in a Neubauer chamber.
‘Static’ stem cell aorta model for co-culture
Materials and methods
Male Wistar rats (8–12 weeks old) were sacrificed by cervical dislocation. The aorta was removed immediately after death, cut into rings, opened and attached to 12-mm glass cover slips with the endothelium facing the top using a two-component fibrin sealant (Tissucol Kit, Baxter, Vienna, Austria). The aortas were transferred in well plates and incubated with 6 104 PKH67 vital stained MNC for 105 min in endothelial basal medium MV2. These co-culture experiments between MNC and endothelial cells of rat aorta were performed three times.
Isolation of mononuclear cells
MNC perfusion through rat aorta
MNC were isolated from the peripheral blood of thirteen healthy, male human, non-smoking volunteers
The aorta was removed from rats as described above and cannulated for perfusion. The stained MNC were
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resuspended in 50 ml MV2 medium containing 5% FCS. The aortas were perfused with 1.5 107 MNC for 1.5 and 4 h (0.55 and 0.21 ml/min). In order to investigate if the perfusion time was too short for the beginning of migration of MNC, the perfused aorta was cultured in tyrode solution (in mM: NaCl 119.8, KCl 5.4, MgCl2 1.05, CaCl2 1.8, NaHCO3 22.6, NaHPO4 0.42, glucose 5.05, ascorbic acid 0.28, disodium EDTA 0.05, pH 7.4, gassed with 100% O2, 37 1C) for 24 and 48 h (95% humidity, 5% CO2). The MNC perfusion through isolated rat aortic rings was repeated 21 times.
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Immunofluorescence staining of the basal lamina Tissue slices were incubated in 5% bovine serum albumin (BSA) and in Tris-buffered saline (TBS) (1 h, room temperature) for blocking. The primary antibody rabbit anti-laminin (Sigma, St. Louis, MO, USA) was diluted 1:500 in 0.8% BSA and incubated overnight at 4 1C. As a secondary antibody, Cy5-conjugated affine pure goat anti-rabbit IgG (Jackson Immuno Research, Suffolk, UK) (1:500) was used.
Immunofluorescence staining of MNC and EPC Perfusion of cells through umbilical vein A human umbilical vein was clamped in the perfusion model as described above and 1.5 107 MNC suspended in 50 ml medium were perfused through it for 4 h (0.21 ml/min). The MNC perfusion through an umbilical vein was done once.
Perfusion of cells through the murine heart Wild-type mice (4–6 weeks old) were sacrificed by cervical translocation. The heart was quickly removed and the aorta cannulated for the initiation of a retrograde perfusion in a Langendorff-modified apparatus. The stained cells were resuspended in 50 ml MV2 medium containing 5% FCS. The hearts were perfused with 1 107 MNC and 1 106 cEPC for 0.5 and 2 h (1.67 and 0.42 ml/min). The MNC perfusion through murine hearts was repeated 16 times and the EPC perfusion 10 times.
Fixation of the tissue and cryo-sections Co-culture experiments: The tissue was immediately fixed with 4% formaldehyde (freshly prepared from paraformaldehyde, Merck, VWA, Darmstadt, Germany) for 20 min followed by three washes with DPBS. Perfusion experiments: The tissue was immediately fixed with 4% formaldehyde for 4–6 h while being cooled at 4 1C. After this time, the tissue was washed with DPBS, fixed again with 18% sucrose and then frozen in tissue-tecs(Sakura Finetek Europe, Zoeterwoude, NL) at 80 1C followed by a cryo-slicing to 10 mm thickness and immunofluorescence microscopy.
Visualising of endothelial cells Tissue slices were stained for 30 min with rhodamineGriffonia (Bandeireaea) simplicifolia lectin I (Vector Laboratories, Burlingame, USA). After washing with DPBS, the tissue was embedded in Aquapoly-mounts (Polysciences Inc., Warrington, USA).
For characterisation of MNC and EPC, immunofluorescence staining was performed as described above. The first antibody used was either CD34 (Santa Cruz, Biotechnology, Inc., Heidelberg, Germany) diluted 1:400 or rabbit polyclonal to VEGFR-2 (Abcam, Cambridge, UK) diluted 1:500 in 0.8% BSA. As secondary antibody, Cy5-conjugated affine pure goat anti-rabbit IgG (Jackson Immuno Research) (1:500) was used.
Microscopic assessment Microscopy was performed using a confocal laser scanning microscope (LSM 510 META, Zeiss, Go¨ttingen). For each experimental condition, at least 60 single cells were investigated.
Results Co-culture experiments between MNC and endothelial cells of rat aorta To investigate whether MNC interact with endothelial cells, a ‘static’ stem cell aorta model was established to perform co-culture experiments between isolated MNC and endothelial cells of rat aortas. A freshly isolated MNC before the experiment is shown in Fig. 1A. It appears in a completely spherical shape. Fig. 1B shows an MNC after being co-cultivated on the endothelial layer of the rat aorta. The spherical shape of the MNC has been lost, the cell has flattened and extensions have formed in close proximity to the endothelial cells. In Fig. 1C the aorta has been rotated 901 around a horizontal axis. The tissue was fixed on the right side on a glass coverslip (see arrow), so the endothelium faces to the left. It can clearly be seen that the MNC have attached to the endothelial layer, but did not form any extensions through it. These pictures clearly show that the MNC attempted to interact with the endothelium when being co-cultured. Morphological changes
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Fig. 1. Interaction between MNC and endothelium under co-culture conditions. (A) MNC before being in contact with endothelial cells. (B) Morphological change of MNC upon interaction with endothelial cells (105 min of co-culture on an endothelial layer of rat aorta). (C) Co-culture as in (B). 901 rotation around the horizontal axis of an MNC without any extension into the subendothelial layer (arrow: side of the glass coverslip). Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamineGriffonia simplicifolia lectin I.
Fig. 2. MNC perfusion through rat aorta and interaction with the tissue. (A) Adhesion of MNC at endothelial cells (A) and at endothelial-free regions (B) after a perfusion time of 4 h. (C) Morphological changes of MNC and integration into the endothelial layer. (D) Adhesion of MNC to lower tissues where the basal lamina was disrupted, but no migration into the vessel tissue. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I. White: basal lamina stained with anti-laminin and Cy5-conjugated secondary antibody. Arrow: basal lamina beneath the endothelial layer.
including the formation of extensions into the endothelial cells can be detected.
MNC perfusion through isolated rat aortic rings To investigate whether MNC also interact with endothelial cells under perfusion conditions, MNC were perfused through isolated rat aortas. Perfusion was performed with 1.5 107 MNC for 1.5 and 4 h (0.55 and 0.21 ml/min). Cross sections of the perfused rat aortas, examined by confocal microscopy, are shown in Fig. 2. The adhesion of MNC (green) to endothelial cells (red) is shown in Fig. 2A and the interaction of MNC with endothelial-free regions (i.e. with the basal lamina) in Fig. 2B. In Fig. 2C morphological changes of MNC can be seen when being attached to the endothelium. A flattening of the cell can be observed. This is a sign for direct interaction of the cell with the endothelium. The adhesion of MNC to lower tissue of the rat aorta is shown in Fig. 2D (white ¼ basal lamina) where the basal lamina was also disrupted. One hundred cells were investigated, but migration of MNC through the subendothelial tissue of the rat aorta was not detected in any case.
In order to see if the two perfusion times were too short for the beginning of migration of MNC, the aortas were cultivated for 24 and 48 h after the perfusion. The results are shown in Fig. 3. Again, no transmigration of MNC into subendothelial parts of the aortic wall was observed under these conditions.
MNC perfusion through isolated umbilical vein Since there is evidence for regional differences in the endothelial cell phenotype (Verhamme and Hoylaerts, 2006), it was investigated whether the transmigrational behaviour of MNC is dependent on the endothelial phenotype. MNC (1.5 107) were perfused through an umbilical vein for 4 h (0.21 ml/min). Cross sections through the umbilical vein are shown in Fig. 4. The adhesion of MNC to endothelial cells is shown in Fig. 4A and the adhesion to the basal lamina in Fig. 4B (endothelium-free tissue). As for the results with rat aorta, a morphological flattening of the cells was observed, but migration of MNC through the endothelial layer was not seen in any of the 50 cells investigated.
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Fig. 3. Cultivation of the rat aorta for 24 h (A) or 48 h (B) after perfusion with MNC. No migration into lower parts of the tissue can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I. White: basal lamina stained with anti-laminin and Cy5-conjugated secondary antibody.
Fig. 4. MNC perfusion through umbilical vein. Adhesion of MNC at endothelial cells (A) and endothelial-free regions (B). No migration into lower parts of the vessel tissue, but morphological changes can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I. White: basal lamina stained with anti-laminin and Cy5conjugated secondary antibody.
MNC perfusion through isolated murine hearts In order to investigate the migrational activity of MNC and EPC in functional tissue with a complete endothelial layer, the cells we perfused through isolated murine hearts in which the endothelial layer of the capillaries was completely functional and not manually disrupted by dissection. To minimise the rate of inflammatory processes due to dissection, the perfusion with MNC and cEPC was started immediately after removing the heart. Fig. 5 shows transversal cross sections through murine hearts perfused with 1 107 MNC for 30 min or 120 min (1.67 and 0.42 ml/min). At least 60 cells were observed for each condition. At a perfusion time of 30 min (Fig. 5A), migration of MNC over the endothelial layer could not be observed in any case. In order to find out if 30 min were too short for MNC migration, the cells were perfused for 120 min. No migration could be detected (Fig. 5B) here either.
To investigate whether MNC adhere strongly to endothelial cells in the capillaries, the mice hearts were firstly perfused with the MNC suspension for 30 or 120 min (1.67 and 0.42 ml/min) and afterwards with 5 ml DPBS for 10 min (0.5 ml/min). Cross sections of that are shown in Fig. 6A. In this experiment no loosening of the MNC from the endothelial layer was seen. This showed that there was a strong contact and interaction between the perfused MNC and endothelial cells in the capillaries. The next idea was that cell perfusion, i.e. erythrocytes, could cause a loosening of the attached MNC when subsequently being perfused through the small capillaries of the mouse heart. So 5 ml of human EDTAblood was perfused through mice hearts after the perfusion of MNC for 30 and 120 min for 10 min (0.5 ml/min). The results show that no loosening of the cells occurred. A cross section is shown in Fig. 6B. This led to the conclusion that the MNC were strongly attached to the endothelial cells in the capillaries of the
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Fig. 5. MNC perfusion through isolated murine hearts for 30 min (A) or 120 min (B). No migration of cells over the endothelial barrier can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I.
Fig. 6. MNC perfusion through isolated murine hearts, subsequently washed for 10 min with DPBS (A) or EDTA-blood containing erythrocytes (B). No loosening of the cells from the endothelium can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamine-Griffonia simplicifolia lectin I.
heart so that integration, but not transmigration, could take place. To obtain further information on whether it is possible to remove the cells from the endothelium we compared the number of MNC in isolated hearts that had been directly fixed in formaldehyde after MNC perfusion with the number of MNC in hearts that had been perfused with DPBS or blood after MNC perfusion. The comparison was made by counting the number of MNC in the microscope field of vision. We did not observe any significant differences in the number of MNC between the different conditions (direct fixation: 3.371.6 MNC per field of vision; perfusion with DPBS after MNC perfusion: 4.072.2 MNC per field of vision; blood perfusion after MNC perfusion: 3.771.9 MNC per field of vision). Overall it can be said that MNC, which contain circulating EPC, attach strongly to the endothelial cells of vessel walls when being perfused, but do not migrate
over the endothelial barrier into subendothelial parts of the vessel tissue.
EPC perfusion through isolated murine hearts The number of EPC in MNC is extremely low (Mancuso et al., 2001; Kim et al., 2003). In order to increase the number of EPC in the cell suspension, 1 107 MNC were cultured to obtain 1 106 cEPC. These cEPC were resuspended in 50 ml medium and perfused through mice hearts for 30 and 120 minutes (1.67 and 0.42 ml/min). In Fig. 7 the cross sections of the murine hearts perfused with cEPC are shown. This experiment conformed with the previous results obtained after MNC perfusion by showing no migration of cEPC over the endothelial barrier into subendothelial tissue after 30 min (Fig. 7A) and 120 min (Fig. 7B).
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Fig. 9. EPC are positive for CD34 and VEGFR-2. Green: MNC vital stained with PHK-67. White: Immunostaining for CD34 (A) or VEGFR-2 (B).
Discussion
Fig. 7. EPC perfusion through isolated murine hearts for 30 min (A) or 120 min (B). No migration of the cells over the endothelial barrier can be observed. Green: MNC vital stained with PKH-67. Red: endothelium stained with rhodamineGriffonia simplicifolia lectin I.
Fig. 8. MNC are positive for CD34 and VEGFR-2. Green: MNC vital stained with PHK-67. White: Immunostaining for CD34 (A) or VEGFR-2 (B).
Characterisation of MNC and EPC by immunofluorescence staining MNC were characterised by their surface markers. A dual-staining for PKH and either the expression of the VEGF receptor 2 or CD34 was performed. The MNC used for the experiments were positive for CD34 (Fig. 8A) and the VEGF receptor 2 (Fig. 8B). EPC were also characterised by their surface markers. A dual-staining for PKH and either the expression of the VEGF receptor 2 or CD34 was performed. The EPC used for the experiments are positive for CD34 (Fig. 9A) and the VEGF receptor 2 (Fig. 9B).
The aim of this study was to investigate the interaction between circulating and in vitro-cultivated EPC and the endothelial barrier. The results demonstrated that MNC and cEPC interact with endothelial cells. Morphological changes in the shape of the progenitor cells were seen when MNC or cEPC were attached to the endothelium. They flattened and built up extensions when in contact with the endothelium. However, migration of MNC and cEPC through and over the endothelial barrier was not detected. These results explain the process of angiogenesis and vasculogenesis in terms of neovascularisation. Angiogenesis is the process of new vessel growth. Mature, differentiated endothelial cells break free from their basement membrane, and migrate and proliferate to form sprouts from parent vessels. Vasculogenesis involves the participation of EPC, which circulate to sites of neovascularisation where they differentiate in situ into mature endothelial cells (Isner and Asahara, 1999). Incorporated EPC may also release growth factors that promote angiogenesis by acting on mature endothelial cells (Fuchs, 2001). However in terms of cardiac regeneration, especially when infused intra-arterially, migration out of the vessel into the surrounding tissue is required (Dimmeler et al., 2005). In vivo this migration process involves multiple steps. The EPC have to leave their normal environment, the bone marrow, and reach the target tissue by migrating through the vessel wall. The present paper focuses on the second migratory process. This is divided into different steps which are currently of high interest in research: (1) the EPC must come into contact with the endothelial cells; (2) they have to dissociate the cell–cell contact of the endothelium; (3) they must integrate into the endothelial layer, and finally (4) they have to pass and leave this barrier (Schmidt et al., 2006). So far, the mechanism of cell mobilisation and homing is only rudimentarily understood. It is hypothesised that once the cells are mobilised from bone marrow
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as a result of a stimulus (VEGF, GM-SCF, SDF-1, angiopoietin-1, placental growth factor or erythropoietin via matrix metalloproteinase 9), the first adhesion to endothelium takes place. Here SDF-1 appears to play a pivotal role as an attractor for EPC to ischemic tissue. Also the high mobility group box protein 1, a chromatin-binding protein released by cell necrosis, may stimulate EPC attraction (Dimmeler et al., 2005). After cell attraction, the first weak interaction takes place by rolling the EPC over the endothelial cells, which is governed by L-selectins and their ligands (Biancone et al., 2004). Duan et al. (2006) recently showed that EPC express LFA-1 and VLA-4 and that their ligands ICAM-1 and VCAM-1 are expressed in vessel endothelium in ischemic tissues. The next step, a strong adhesion of the cells, is triggered by b2-integrins (Chavakis et al., 2005). At this point the transmigration of the cell through the endothelium and invasion into the target tissue could take place. It needs to be mentioned that MNC were deliberately perfused in this study because it was assumed that these cells represent the native cell fraction which is responsible for endothelial regeneration processes in the body and which contains a small number of EPC (o500/ml (Mancuso et al., 2001) or 40.2710.2/ml (Kim et al., 2003)). This was because no clear differentiation of EPC has yet been found and the authors used different methods for detection. Also, changes of any (surface) properties of native EPC were avoided in order to get representative results of how vessel regeneration takes place in vivo. For enhancement of the number of EPC in the perfused cell suspension, the MNC were kept in culture for 4 days under EPC-growing conditions, as done by other groups before (Urbich et al., 2005). This was done in order to perfuse mostly EPC rather than the large and undefined pool of MNC. Under culture conditions, however, the cells experienced a change in morphology, as can be seen by their change in size, and do not represent the natural cells circulating in the body. The problem is that by now no consensus has been reached on the characteristics of EPC, and published studies mostly characterise EPC differently. Another limitation of the study is that a xenograft model was used. It cannot be excluded that this influenced the results. No transmigration of cells into the subendothelial tissue was observed in this study. However, these results do not exclude the migrational activity of EPC. In the case of wound healing, for example, the cells are able to migrate along a gradient of growth factors released by the damaged tissue in order to reach the site where wound healing takes place (Suh et al., 2005). However, in wounds, the tissue is damaged and an intact endothelial barrier no longer exists. From the results obtained here it can only be said that EPC are not able to cross the endothelial barrier by transmigration.
That EPC possess the ability to migrate was also shown in different in vitro experiments in Boyden chambers. A Boyden chamber consists of two compartments separated by a Millipore filter (3–8 mm) to test for chemotaxis. In most cases, EPC are placed in basal medium in the upper compartment of the Boyden chamber (Laufs et al., 2005). The lower chamber contains basal medium and the chemoattractant, mostly VEGF. This chamber is cultured for a certain period of time. Any EPC that migrate to the lower chamber can then be detected and counted. This showed that the EPC have certain migration ability in vitro. In contrast to EPC, Schmidt et al. (2006) have recently shown that mesenchymal stem cells (MSC) possess the ability to transmigrate over the endothelial barrier, making them a potentially useful tool for cellular therapy in injured tissue. MSC were able to split tight junctions between endothelial cells, became integrated into the endothelial wall of the capillary vessel and could finally fully pass the endothelial barrier (Schmidt et al., 2006). It needs to be pointed out that after application of EPC to patients with ischemic events, i.e. myocardial infarction or cardiomyopathies, a clear improvement of the ejection fraction could be measured (Britten et al., 2003). After ischemic events cardiac tissue is damaged in a way that is associated with a complete loss of vascular integrity and thus a loss of the function of the endothelial barrier. EPC do not have to cross this barrier for cardiac regeneration. In the case of acute damage the cells can ‘bleed’ freely into the cardiac tissue. However, our results show that in a healthy or chronically damaged heart where there is no acute damage of the endothelial barrier, non-endothelial cell replacement via EPC migration is unlikely to occur. Thus, under these conditions, there must be mechanisms other than the migration of EPC over the endothelial barrier that enable these cells to support the cardiac regeneration process without acting themselves as replacement cells, pointing this connection the paracrine effects of endothelial progenitor cells have been postulated. One possibility could be that the EPC themselves express growth factors which directly affect the surrounding tissue in order to start the regeneration process. Urbich et al. (2005) reported the release of soluble factors by EPC which might explain their incorporation into newly formed vessels, enhanced neovascularisation and improvement of cardiac function. These authors postulated that EPC promote neovascularisation and cardiac regeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilise tissue-residing progenitor cells. It was found that EPC express higher levels of VEGF-A, VEGF-B, SDF-1 and IDF-1 than human umbilical vein endothelial cells or human microvascular endothelial cells. So EPC exhibit a high expression of
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angiogenic growth factors that enhance migration of mature endothelial cells and tissue-resident cardiac progenitor cells. The fact that MNC adhere strongly to the vessel wall and cannot be removed by passing blood supports the view that they act in a paracrine manner on surrounding endothelial cells in order to support the cardiac regeneration process without acting themselves as replacement cells. It is possible that application of granulocyte-colony stimulating factor (G-CSF) prior to EPC isolation alters their migratory capacity. It has been shown that such treatment increases the expression of the CXCR4 receptor and may thus facilitate the homing of the EPC in patients with coronary artery disease (Powell et al., 2005). However, there are also reports indicating that G-CSF impairs the functional capacity of EPC, e.g. in an experimental mouse model of hind limb ischemia (Honold et al., 2006). Thus, the functional (migratory) capacity of EPC may be influenced by pharmacological treatment. This has to be investigated in further studies.
Conclusion The work presented here demonstrates that EPC possess the capability to interact with the endothelial layer of different organs in a way that causes morphological changes and strong adhesion to the tissue, but are not able to transmigrate over the endothelial barrier.
Acknowledgements This work was supported by the Novartis Foundation through a grant to W. Bloch, U. Mehlhorn and R.H.G. Schwinger. We thank the clinic of gynaecology and midwifery, University of Cologne that supplied an umbilical cord. We would also like to thank Anika Voss and Esra Koroglu¨ for their excellent technical assistance.
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