Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells

Original article

Soluble factors released by endothelial progenitor cells promote migrationof endothelial cells and cardiac resident progenitor cells

Carmen Urbich a, Alexandra Aicher a, Christopher Heeschen a, Elisabeth Dernbach a,Wolf K. Hofmann b, Andreas M. Zeiher a, Stefanie Dimmeler a,*

a Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germanyb Department of Hematology and Oncology, Internal Medicine II, University of Frankfurt, Theodor-Stern-Kai 7, Frankfurt, Germany

Received 12 October 2004; received in revised form 7 June 2005; accepted 11 July 2005

Available online 29 September 2005

Abstract

Circulating endothelial progenitor cells (EPC) are incorporated into newly formed capillaries, enhance neovascularization after hind limbischemia and improve cardiac function after ischemic injury. Incorporated progenitor cells may also promote neovascularization and cardiacregeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilize tissue residing progenitor cells.Therefore, we analyzed the expression profile of cytokines in human peripheral blood-derived EPC as opposed to human umbilical veinendothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and CD14+ monocytes by microarray technology. A gene treeanalysis revealed a distinct expression pattern of angiogenic growth factors in EPC, mature endothelial cells, and CD14+ monocytes. VEGF-A,VEGF-B, SDF-1, and IGF-1 mRNA levels were higher in EPC as compared to HUVEC or HMVEC. The enhanced mRNA expression wasparalleled by a significant release of VEGF, SDF-1, and IGF-1 protein into the cell culture supernatant of EPC. Moreover, immunohistologicalanalysis of ischemic limbs from nude rats revealed that VEGF is also released from recruited human EPC in vivo. As a functional conse-quence, conditioned medium of EPC induced a strong migratory response of mature endothelial cells, which was significantly inhibited byVEGF and SDF-1 neutralizing antibodies. Finally, conditioned medium of EPC significantly stimulated the migration of cardiac residentc-kit+ progenitor cells in vitro. Taken together, EPC exhibit a high expression of angiogenic growth factors, which enhanced migration ofmature endothelial cells and tissue resident cardiac progenitor cells. In addition to the physical contribution of EPC to newly formed vessels,the enhanced expression of cytokines may be a supportive mechanism to improve blood vessel formation and cardiac regeneration after celltherapy.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Growth factors; Endothelial progenitor cells; Angiogenesis

1. Introduction

Postnatal neovascularization is an important process to res-cue tissue from critical ischemia [1]. Circulating bonemarrow-derived endothelial progenitor cells (EPC) signifi-cantly contribute to adult blood vessel formation [2–4]. Theseendothelial progenitor cells are positive for endothelial markerproteins such as VEGF receptor 2 (KDR), von Willebrand

factor (vWF), VE-cadherin, eNOS, take-up of diacetylatedLDL, and bind lectin [5–7]. Injected EPC were shown to inte-grate into blood vessels and improve neovascularization ofischemic hind limbs and ischemic hearts in animal models[8–11]. In accordance, initial clinical trials indicate that bonemarrow-derived or circulating blood-derived progenitor cellsmay be therapeutically useful to improve blood flow ofischemic tissue and improve heart function [12,13].At presentit is unclear, whether the improved heart function seen inpatients after progenitor cell therapy is exclusively mediatedby an improvement of neovascularization or also may reflectregeneration of cardiac tissue. Indeed, EPC have been shownto acquire a cardiac phenotype in vitro after co-cultivation

* Corresponding author. Tel.: +49 69 6301 7440/5789; fax: +49 69 63017113/6374.

E-mail address: [email protected] (S. Dimmeler).

Journal of Molecular and Cellular Cardiology 39 (2005) 733–742

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with neonatal rat cardiac myocytes [14]. However, cardiacregeneration may also be mediated by mobilization of endog-enous tissue residing cardiac progenitor cells [15–17].

EPC are incorporated to a significant extent into the cap-illaries after ischemia and, thereby, physically contribute tothe formation of new capillaries in the ischemic tissue. How-ever, the lower number of incorporated cells with an endot-helial phenotype in some of the experimental studies may notentirely explain the strong therapeutic effect observed aftercell therapy in experimental models [7,9,18]. Thus, the effi-ciency of EPC-induced neovascularization may not solely bedue to the incorporation of EPC into newly formed vessels,but may also be influenced by the release of pro-angiogenicfactors in a paracrine manner. In line with this hypothesis,the deletion of Tie-2-positive bone marrow-derived cellsthrough activation of a suicide gene blocked tumor angiogen-esis, even though in this study, cells were not found to beintegrated into the endothelial lining of the tumor vessels butrather located adjacent to the vessel wall [19]. Moreover,recent studies suggest that monocytic cells as well as mesen-chymal stem cells augment arteriogenesis via the release ofangiogenic growth factors [20–22]. The local release ofgrowth factors by progenitor cells may additionally protectcardiac myocytes from apoptosis and potentially mobilize car-diac progenitor cells [23–25]. For example, HGF and IGF-1 were shown to exert a potent anti-apoptotic activity andmobilize cardiac progenitor cells [24–27].

In the present study, we analyzed the expression of poten-tial angiogenic growth factors in EPC as opposed to humanumbilical vein endothelial cells, human microvascular endot-helial cells, and CD14+ monocytes. Our results indicate thatEPC express higher levels of growth factors such as the vas-cular endothelial growth factor (VEGF) A, stromal cell-derived factor-1 (SDF-1), insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF) as compared tomature endothelial cells. All factors have been described aspro-angiogenic growth factors (for review see [28]).As a func-tional consequence, conditioned medium of EPC promotedmigration of mature endothelial cells and cardiac resident pro-genitor cells. The local EPC-mediated release of growth fac-tors may contribute to neovascularization and cardiac regen-eration after ischemia.

2. Material and methods

2.1. EPC culture assay

Mononuclear cells (MNCs) were isolated by density gra-dient centrifugation with Ficoll from peripheral blood ofhealthy human volunteers as previously described [6]. Imme-diately after isolation, 8 × 106 MNC/ml were plated on cul-ture dishes coated with human fibronectin (Sigma) and main-tained in endothelial basal medium (EBM; Cambrex,Verviers,Belgium) supplemented with hydrocortisone, bovine brain

extract, gentamicin, amphotericin B, epidermal growth fac-tor and 20% fetal calf serum (FCS). After 3 days in culture,nonadherent cells were removed by thorough washing withPBS and adherent cells were incubated in fresh medium for24 h before starting the experiments. EPC were characterizedby dual-staining for 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-labeled acetylated low-density lipopro-tein (DiLDL) and lectin and by the expression of endothelialmarker proteins KDR, VE-cadherin, eNOS and von Will-ebrand factor [9,29].

2.2. Cell culture

Pooled human umbilical venous endothelial cells (HU-VEC) were purchased from Cambrex and cultured in endot-helial basal medium (Cambrex) supplemented with hydrocor-tisone, bovine brain extract, gentamicin, amphotericin B,epidermal growth factor and 10% fetal calf serum until thethird passage. After detachment with trypsin, cells (4.0 × 105

cells) were grown on 6 cm cell culture dishes for at least 18 h.Human dermal microvascular endothelial cells (HMVEC)were purchased from Cambrex and cultured according to theinstructions of the manufacturer in endothelial basalmedium-2 (EBM-2; Cambrex) supplemented with epidermalgrowth factor, hydrocortisone, fibroblast growth factor, vas-cular endothelial growth factor, insulin-like growth factor,ascorbic acid, gentamicin, amphotericin-B and 5% FCS untilthe fifth passage.

2.3. Isolation of CD14+ monocytes

Human mononuclear cells were obtained from peripheralblood of healthy human volunteers by density gradient cen-trifugation using Ficoll separating solution (density 1.077;Biochrom, Berlin, Germany). CD14+ monocytes were puri-fied from mononuclear cells by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Ger-many) using a magnetic cell sorter device (Miltenyi Biotec).Purity assessed by FACS analysis was > 95%.

2.4. Oligonucleotide microarray analysis

Total RNA was isolated with the RNeasy Kit according toQiagen (Hilden, Germany). Ten micrograms of total RNAwas hybridized to the HG-U95Av2 microarray (Affymetrix,Santa Clara, CA, USA). The standard protocol used for samplepreparation and microarray processing is available fromAffymetrix. Expression data were analyzed using Microar-ray Suite version 5.0 (Affymetrix) and the software Gene-Spring version 4.2 (Silicon Genetics, San Carlos, CA, USA)as previously described [30].

2.5. Western blot analysis

Cells were incubated with lysis buffer (20 mmol/l Tris(pH 7.4), 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA,

734 C. Urbich et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 733–742

1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/lb-glycerolphosphate, 1 mmol/l Na3VO4, 1 µg/ml leupeptinand 1 mmol/l phenylmethylsulfonyl fluoride) for 15 min onice. After centrifugation for 15 min at 20,000 × g (4 °C), theprotein content of the samples was determined according toBradford. Proteins (50 µg per lane) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes.Western blots were performed using antibodies directedagainst VEGF (R&D systems). Enhanced chemilumines-cence was performed according to the instructions of themanufacturer (Amersham, Germany). Blots were reprobedwith actin (1:2000; Sigma). The autoradiographies werescanned and semiquantitatively analyzed.

2.6. Hind limb ischemia model in athymic rats

For transplantation of human EPC into a xenogeneic ratmodel, immunodeficient athymic rnu:rnu rats (5–7 week oldfemales; Harlan, Borchen, Germany) were required. Thesuperficial femoral artery was ligated. CM-Dil (MolecularProbes; Göttingen, Germany)-labeled cells (2 × 106 cells perrat) were intravenously injected 24 h after induction of uni-lateral hind limb ischemia.

2.7. Immunohistochemistry

In nude rats, the gastrocnemius muscles were harvested48 h after injection of human CM-Dil-labeled EPC. Frozentissue sections (5 µm) were then cut and fixed with ice coldacetone. Incorporated CM-Dil-labeled EPC were detected bythe red fluorescence of the cell tracker. Myocytes were stainedwith laminin (Abcam) followed by goat-anti-rabbit AlexaFluor 647 (Molecular Probes). VEGF was detected using anantibody against VEGF (R&D systems, Wiesbaden, Ger-many), which reacts with human VEGF and, to a lesser extent,with rat VEGF, followed by goat-anti-mouse Alexa Fluor 488(Molecular Probes). Nuclei were stained with Sytox (Molecu-lar Probes). Tissue sections were examined using confocalmicroscopy (LSM 510, Zeiss, Germany).

2.8. Preparation of conditioned media and ELISA

EPC and HUVEC were incubated with equal amounts ofbasal EBM medium without supplements and FCS for 24 h.The medium was collected and 10 × concentrated by centrifu-gation for 20 min at 5000 × g at 4 °C using Ultrafree-4 centrifugal filter tubes with Biomax-5 membrane (Milli-pore). Release of VEGF, SDF-1, HGF, and IGF-1 wasmeasured in concentrated (10×) cell culture supernatants byELISA according to the instructions of the manufacturer(R&D systems).

2.9. Cell migration

Migration of HUVEC was detected using a “scratchedwound assay”. Therefore, in vitro “scratched” wounds were

created by scraping cell monolayers [31,32]. Cells were grownon 6 cm wells, which were previously labeled with a tracedline. After injury, the cells were gently washed with PBS andincubated with the concentrated conditioned media of EPCor HUVEC in the presence or absence of a neutralizing anti-body against VEGF (R&D systems) or SDF-1 (R&D sys-tems). EC migration from the edge of the injured monolayerwas quantified by measuring the distance between the woundedges at the time of injury and after 24 h of incubation usinga computer-assisted microscope (Zeiss, Jena, Germany) at fivedistinct positions (every 5 mm).

2.10. Isolation of cardiac progenitor cells

Cardiac progenitor cells were isolated from femaleC57BL/6 mice at 2 months of age. Mice hearts were mincedand incubated with a mixture of 0.1% dispase (Gibco,Karlsruhe, Germany) and 0.2% pronase E (Merck, Darms-tadt, Germany) in PBS for 20 min at 37 °C, then minced again,and mixed with pure fetal calf serum to stop the digestion.After centrifugation, cells were washed with PBS and counted.c-kit (CD117)+ cells were purified using positive selectionwith anti-CD117-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity of sorted cells was determinedby FACS.

2.11. Statistical analysis

Data are expressed as mean ± S.E.M. from at least threeindependent experiments. Statistical analysis was performedby t-test. ANOVA was performed for serial analyses.

3. Results

3.1. Expression of pro-angiogenic factors in EPC

The endothelial phenotype of human peripheral blood-derived EPC was confirmed by expression of KDR, von Will-ebrand factor, CD146, VE-cadherin, and eNOS in previousstudies [9,29]. A gene tree analysis revealed a distinct geneexpression pattern of growth factors (99 different oligonucle-otides) in EPC as compared to other cell types. As shown inFig. 1, the expression of growth factors in EPC was differentas compared to HUVEC and HMVEC, and was also distin-guishable from those of CD14+ monocytes. The expressionpattern of HUVEC resembled that of HMVEC (Fig. 1). Indetail, VEGF A and VEGF B are highly expressed in EPC ascompared to HUVEC and HMVEC (Figs. 1 and 2A). In con-trast, VEGF C mRNA was exclusively expressed in HUVECand HMVEC (Figs. 1 and 2B). VEGF D was similarlyexpressed in EPC, HUVEC, and CD14+ monocytes and acidicFGF mRNA was similar in EPC, HMVEC, and CD14+ mono-cytes (Fig. 2B, C). Basic FGF revealed an enhanced expres-sion in HUVEC and HMVEC (Fig. 2C). The chemokinesSDF-1, IGF-1, HGF, and to a minor extent granulocyte colony

735C. Urbich et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 733–742

stimulating factor (G-CSF) were increased in EPC as com-pared to HUVEC (Fig. 2D–G). In contrast, no differenceswere observed for the isoforms SDF-2 and IGF-2(Figs. 2D,2E).

3.2. EPC-mediated release of growth factors in vitroand in vivo

Growth factors are secreted to stimulate neighboring cells.Therefore, we measured the release of different factors intocell culture supernatants. As shown in Fig. 3, the release of

VEGF, SDF-1, IGF-1, and HGF was profoundly increased inEPC cell culture supernatants as compared to HUVEC.

Because VEGF is a predominant growth factor involvedin angiogenesis as well as vasculogenesis, we focused on theexpression and function of VEGF in EPC. Consistent withthe mRNA expression and the release into supernatants, theprotein expression ofVEGFA was also significantly increasedin EPC as compared to HUVEC and HMVEC (Figs. 4A, B).In contrast to the mRNA expression, the protein expressionof VEGF was lower in CD14+ monocytes as compared toEPC. However, the release of VEGF from cell culture super-

Fig. 1. Gene expression analysis of growth factors in EPC, HMVEC, HUVEC and CD14+ monocytes. Total RNA of EPC, HMVEC, HUVEC and CD14+

monocytes (each N = 3) were isolated and gene expression profiles were assessed with the Affymetrix gene chip expression assay. A gene tree analysis ofgrowth factors is shown. The blue color indicates low expression, the red color indicates high expression. The brightness indicates the reliability of hybridiza-tion signal. The expression of prominent clusters is marked with boxes and the corresponding genes are listed. Of note, some genes (e.g. VEGF A) arerepresented by two or more different oligonucleotides within the clusters, emphasizing the reproducibility of the assay.


natants of CD14+ monocytes was similar to EPC (data notshown), suggesting a higher turnover and secretion of VEGFprotein in CD14+ monocytes as compared to EPC.

To measure the expression of VEGF in incorporated EPCin vivo, we injected CM-Dil-labeled human EPC in athymicrats 24 h after induction of hind limb ischemia. After 48 h,

Fig. 2. mRNA expression of various growth factors. The mRNA expression of various growth factors in EPC, HMVEC, HUVEC, and CD14+ monocytes issummarized. Data are normalized mean fluorescence intensity ± S.E.M. (N = 3). In case of two or more different oligonucleotides representing one gene themean is shown. The mRNA expression of VEGF A and VEGF B (A), VEGF C and VEGF D (B), acidic and basic FGF (C), SDF-1 and SDF-2 (D), IGF-1 andIGF-2 (E), G-CSF (F), and HGF (G) is shown.


immunohistochemistry of muscle sections of the ischemichind limb was performed. The co-localization of VEGF wasassessed with an antibody against VEGF, which reacts withhumanVEGF and, to a lesser extent, with ratVEGF.As shownin Fig. 4C, VEGF co-localized with incorporated human EPCin the ischemic tissue. However, VEGF is also expressed inthe ischemic area without EPC incorporation, which is inaccordance to previous findings [33].

3.3. Conditioned medium of EPC stimulates migrationof mature endothelial cells and cardiac progenitor cells

To test the functional activity of the growth factor releaseof EPC, we used the concentrated conditioned media of EPCand HUVEC, respectively, for stimulation of endothelial cellmigration in a scratched wound assay. Incubation with EPC-derived conditioned medium induced endothelial cell migra-tion to a greater extent than HUVEC-derived conditionedmedium (Fig. 5A). Co-incubation with a neutralizing anti-body directed against VEGF or SDF-1 inhibited the pro-migratory effect of the EPC-conditioned medium (Fig. 5A).The combined inhibition of VEGF and SDF-1 further slightlyreduced endothelial cell migration (Fig. 5A).

To determine whether EPC-conditioned medium alsoattracts tissue residing cardiac progenitor cells, we tested thepro-migratory effect of EPC-conditioned medium on iso-lated c-kit+ cardiac progenitor cells. Cardiac progenitor cellswere isolated by means of magnetic beads from mice heartsaccording to a previous publication [16].As shown in Fig. 5B,EPC-conditioned medium significantly increased the migra-tion of cardiac progenitor cells as compared to control mediumor HUVEC-conditioned medium.

4. Discussion

The data of the present study demonstrate that humanperipheral blood-derived EPC express and secrete a varietyof growth factors, which stimulate the migration of matureendothelial cells. Moreover, conditioned medium of EPC pro-moted the migration of cardiac progenitor cells.

One of the most important pro-angiogenic factors is VEGF.The endothelial cell-specific mitogen and survival factorVEGF also induces increased vascular permeability, prolif-eration, migration and recruitment of EPC from the bone mar-row (reviewed by [34–36]). The initially discovered factor

Fig. 3. Release of the pro-angiogenic factors VEGF, SDF-1, IGF-1, and HGF. HUVEC and EPC were incubated with equal amounts of fresh medium for 24 h.The medium was collected and 10× concentrated by centrifugation. The release of VEGF (A), SDF-1 (B), IGF-1 (C), and HGF (D) was measured in theconcentrated conditioned media of EPC and HUVEC by ELISA, N ≥ 5, P < 0.05.


VEGF A consists of five splice variants with 121, 145, 165,189, 206 amino acids, whereby VEGF 165 is the most pre-dominant protein (reviewed by [37]). Meanwhile, severalother members of the VEGF family have been identifiedincluding VEGF B, C and D, which differ in the activation ofVEGF receptor subtypes [37]. The biological effects ofVEGFare mediated by two tyrosine kinase receptors, namely Flt-1(VEGFR-1) and KDR (VEGFR-2). Interestingly, membersof the VEGF gene family were differentially regulated in thedifferent cell types. Whereas VEGF D mRNA expression was

similar in EPC, HUVEC, and CD14+ monocytes, VEGF CmRNA was highest in mature endothelial cells. In contrast,the most predominant VEGF isoform, VEGF A, was signifi-cantly higher expressed in EPC as compared to mature endot-helial cells and was secreted and produced in vitro and invivo by EPC. However, monocytes showed a fourfold highermRNA expression level of VEGF A as compared to EPC.This is consistent with previous findings demonstrating thepreferential expression of VEGF A in monocytes [38,39].Interestingly, despite the high expression of VEGF A in

Fig. 4. Increased protein expression of VEGF in EPC as compared to mature endothelial cells. EPC, HMVEC, HUVEC, and CD14+ cells were lyzed and theexpression of VEGF A was analyzed by Western blot. Actin serves as loading control. A) A representative blot out of 3 independent experiments is shown. B)Blots were scanned and protein expression was quantified by densitometric analysis. The ratio for VEGF A/loading is shown. Data are mean ± S.E.M., N ≥ 3;*P < 0.05 versus HUVEC. C) Human CM-Dil-labeled EPC were intravenously injected into rats 24 h after induction of hind limb ischemia. Histologicalsections were obtained 48 h after injection of EPC. Human EPC were identified by CM-Dil (red fluorescence). VEGF was stained with an antibody againsthuman and rat VEGF (green fluorescence). Laminin staining is given in white. Nuclei were stained with Sytox (blue fluorescence). Representative confocalmicrographs are shown.


CD14+ monocytic cells, the infusion of these cells did notaugment neovascularization after hind limb ischemia [9]. Thismight be explained by a lower protein expression as deter-mined by Western blot (Fig. 4). However, one may also specu-late that the release of pro-angiogenic factors may not be theonly prerequisite to improve neovascularization. This assump-tion is also supported by a recent publication of Hur andco-workers, who compared two different types of EPC, earlyspindle-shaped and late cobblestone-shaped EPC. Althoughlate EPC produced significantly lower levels of pro-angiogenic factors (6–10-fold) as compared to early EPC, thevasculogenic potential of early and late EPC in vivo was simi-lar [40]. Thus, although the release of pro-angiogenic factorsmay play a crucial role for augmenting neovascularization[20–22,41], distinct mechanisms may underlie the observedimprovement in blood flow after ischemia for different pro-genitor cell subsets. Supplementary to the improvement ofneovascularization, injection of EPC also significantlyimproved cardiac function in animal models [10,11]. Consis-tently, initial clinical trials indicate that bone marrow-derived or circulating blood-derived progenitor cells are thera-peutically useful to improve heart function [13,42]. However,the relative contributions of different potential mechanisms

(differentiation versus paracrine effects) are difficult to dis-sect in vivo, and, remain to be elucidated.

Other factors such as IGF-1, HGF and SDF-1 were alsohighly expressed in EPC as compared to mature endothelialcells or monocytes. IGF-1 and HGF are potent anti-apoptoticproteins and promote angiogenesis in different models[43,44]. In addition, IGF-1 plays a crucial role in muscleregeneration by promoting the proliferation and differentia-tion of satellite cells in the muscle, enabling them to fuse toexisting muscle fibers and repair damaged regions and sub-sequently to enhance muscle regenerative capacity [45,46].Recent studies provide further evidence that HGF contrib-utes to cardiac regeneration by affecting tissue resident pro-genitor cells. HGF stimulated the mobilization of cardiac pro-genitor cells from their niche and improved cardiacregeneration and heart function [26]. Interestingly, the super-natant of cultured EPC increased the migration of isolatedcardiac progenitor cells. SDF-1 represents an additional cytok-ine involved in the attraction of hematopoietic and endothe-lial progenitor cells. Together with VEGF, SDF-1 not onlystimulates the migration of mature endothelial cells but alsoacts as a chemoattractant to promote migration and tissue inva-sion of endothelial and hematopoietic progenitor cells [18,47].

Fig. 5. Conditioned medium of EPC enhances migration of endothelial cells and cardiac progenitor cells. A) HUVEC and EPC were incubated with equalamounts of fresh medium for 24 h. The medium was collected and 10× concentrated by centrifugation. The concentrated conditioned media of EPC andHUVEC was used to incubate endothelial cells to measure cell migration by the use of a scratched wound assay in the presence or absence of neutralizingantibodies against VEGF (1 µg/ml) and/or SDF-1 (100 µg/ml). IgG isotype antibody was used as control. Data are mean ± S.E.M., N = 4; *P < 0.05 versusconditioned medium of EPC. B) c-kit+ cardiac progenitor cells were isolated from minced mice hearts. Migration of c-kit+ cells was analyzed using conditionedmedia of HUVEC or EPC as stimulus in a modified Boyden chamber. Data are mean ± S.E.M. (% of control medium), N ≥ 3; *P < 0.05 vs. no cells, #P < 0.05 vs.EPC.


Consistently, overexpression of SDF-1 promoted neovascu-larization of ischemic tissues [18]. Thus, SDF-1 and HGFreleased from EPC that have already been recruited into theischemic tissue may further promote the mobilization andrecruitment of circulating and tissue residing progenitor cellsinto the ischemic tissue and, thereby, further accelerate therevascularization and regeneration process [36,47,48].

Interestingly, other members of the IGF and SDF family,namely IGF-2 and SDF-2, whose functions are not welldefined yet, are not differentially expressed in EPC as com-pared to other cell types studied. Moreover, the pro-angiogenicprotein bFGF was even higher in microvascular endothelialcells as compared to EPC. Obviously, the release of factorsfrom EPC involved in neovascularization is a dynamic pro-cess and it is very likely that the expression pattern of angio-genic factors in EPC may change during homing and in vivoincorporation in the ischemic tissue.

Taken together, the present study demonstrates that endot-helial progenitor cells express and release a variety of growthfactors, which in turn can support the survival and functionof tissue residing cells (e.g. mature endothelial cells or car-diac progenitors) in a paracrine manner, thereby, accelerat-ing the process of new blood vessel formation and regenera-tion of ischemic tissues.

Acknowledgments

We would like to thank Melanie Näher, Andrea Knau andMarion Muhly-Reinholz for their excellent technical help.This study was supported by the Deutsche Forschungsge-meinschaft (FOR501 Di600/6-1 and He 3044/2-1).

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Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells

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