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(Circulation Research. 2003;93:e17.)
© 2003 American Heart Association, Inc.

UltraRapid Communications

Intravenous Transfusion of Endothelial Progenitor Cells Reduces Neointima Formation After Vascular Injury

Nikos Werner, Stefan Junk, Ulrich Laufs, Andreas Link, Katrin Walenta, Michael Böhm, Georg Nickenig

From the Klinik und Poliklinik, Innere Medizin III, Universität des Saarlandes, Homburg/Saar, Germany.

Correspondence to Georg Nickenig, MD, Klinik und Poliklinik, Innere Medizin III, Universität des Saarlandes, 66421 Homburg/Saar, Germany. E-mail nickenig{at}med-in.uni-sb.de

	Abstract

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Endothelial cell damage is one important pathophysiological step of atherosclerosis and restenosis after angioplasty. Accelerated reendothelialization impairs neointima formation. We evaluated the role of intravenously transfused endothelial progenitor cells (EPCs) on reendothelialization and neointima formation in a mouse model of arterial injury. Spleen-derived mouse mononuclear cells (MNCs) were cultured in endothelial basal medium. A total of 91.8±3.2% of adherent cells showed uptake of acetylated low-density lipoprotein (Dil-Ac-LDL) and lectin binding after 4 days. Immunostaining and long-term cultures confirmed the endothelial progenitor phenotype. To determine the effect of stem cell transfusion on reendothelialization, mice received either fluorescent-labeled spleen-derived MNCs or in vitro differentiated EPCs intravenously after endothelial injury of the carotid artery. Transfused cells were strictly restricted to the injury site, and lectin binding confirmed the endothelial phenotype. Homing of transfused cells to the site of injury was only detectable in splenectomized mice. Cell transfusion caused enhanced reendothelialization associated with a reduction of neointima formation. Systemically applied spleen-derived MNCs and EPCs home to the site of vascular injury, resulting in an enhanced reendothelialization associated with decreased neointima formation. These results allow novel insights in stem cell biology and provide additional information for the treatment of vascular dysfunction and prevention of restenosis after angioplasty. The full text of this article is available online at http://www.circresaha.org.

Key Words: atherosclerosis • spleen • endothelial progenitor cells • transfusion • neointima

	Introduction

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Emerging evidence suggests that circulating bone marrow (BM)–derived stem and progenitor cells play an important role in tissue regeneration throughout the cardiovascular system.^1–3 Several studies demonstrated the beneficial effect of mobilized BM-derived cells⁴ or transplanted stem cells^5–9 on remodeling after myocardial infarction. Special attention has been directed to endothelial progenitor cells (EPCs), a subset of BM-derived progenitor cells, which have been shown to play an important role in (patho-)physiological neoangiogenesis^1,10–15 and vascular regeneration after myocardial infarction.^8,13,16–18

The role of stem cells and BM-derived progenitor cells in atherosclerosis, the underlying reason for myocardial infarction and tissue damage, remains unclear. Atherosclerotic lesion formation is initiated by endothelial cell damage leading to endothelial dysfunction. Apoptosis of endothelial cells, macrophage adhesion and invasion, and smooth muscle cell migration and growth finally lead to stenosis of the targeted vessel.¹⁹ Accelerated reendothelialization effectively impairs smooth muscle cell proliferation and neointima formation^20,21 and is therefore of special interest with regard to prevention of the early stages of atherosclerosis and restenosis after angioplasty.²²

It has been shown that the number of circulating EPCs inversely correlates with risk factors for atherosclerosis^23,24 and that risk factors such as diabetes impair the migratory capacity of EPCs.²⁵ Furthermore, reduced levels of EPCs are associated with endothelial dysfunction as determined by flow-mediated brachial-artery reactivity.²⁴ We and others have recently demonstrated that BM-derived progenitor cells significantly contribute to reendothelialization after endothelial cell injury. Mobilization of EPCs to the circulation by statins is associated with an enhanced reendothelialization and decreased neointima formation, resulting in a reduction of restenosis.^26,27 However, it could not be excluded that the demonstrated beneficial effects were exerted by statin therapy rather than EPC mobilization. To provide evidence that EPCs are involved in vascular remodeling, we evaluated the possible impact of intravenously transfused EPCs on reendothelialization and neointima formation in a mouse model of arterial injury.

	Materials and Methods

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Preparation of Mononuclear Cells
Spleens from C57bl6 mice were explanted and mechanically minced, and mononuclear cells (MNCs) were isolated using a Ficoll gradient (Lympholite-M, Cedarlane). MNCs were stained with PKH-26, a fluorescent dye with long aliphatic tails for incorporation in the cell membrane using the PKH26-GL Red Fluorescent Cell Linker Kit (Sigma) according to the manufacturer’s instructions. Labeled cells were counted, and 1x10⁶ cells were resuspended in 500 µL normal saline for intravenous injection.

Spleen-Derived EPCs
Spleen-derived MNCs 4x10⁶ were seeded on 24-well plates coated with fibronectin (Sigma) in 0.5 mL endothelial basal medium (CellSystems) supplemented with 1 µg/mL hydrocortisone, 3 µg/mL bovine brain extract, 30 µg/mL gentamicin, 50 µg/mL amphotericin B, 10 µg/mL human endothelial growth factor, and 20% fetal calf serum (FCS). After 4 days in culture, cells were extensively washed with normal saline and resuspended. Adherent cells were incubated with 2.4 µg/mL 1,1'-dioctadecyl-3,3,3',3'-tertamethylindocarbocyanine–labeled acetylated low-density lipoprotein (DiI-Ac-LDL, CellSystems) for 1 hour. For transfusion experiments, 1x10⁶ Dil-Ac-LDL–labeled cells were resuspended in 500 µL normal saline for intravenous injection. For long-term cultures, 4x10⁶ spleen-derived MNCs were cultured for 14 days in endothelial basal medium (CellSystems) with change of medium every second day.

Immunocytochemistry
Immunocytochemistry was performed according to standard protocols. After 4 days in culture, spleen-derived EPCs were extensively washed. Staining with DiI-Ac-LDL (CellSystems) and lectin (BS-1, Sigma) was performed according to the manufacturer’s instructions. Cells double positive for DiI-Ac-LDL and lectin staining were judged EPCs and counted. For characterization of surface markers, spleen-derived MNCs were plated on fibronectin-coated dishes and cultured for 4 days. After fixation with ice-cold methanol/acetone 1:1 for 10 minutes at -20°C, immunocytochemistry was performed using phycoerythrin (PE)-labeled antibodies against c-kit, Sca-1, vascular endothelial growth factor receptor 2 (VEGFR-2), and fluorescein (FITC)-labeled CD34 (all BD). Isotype-specific antibodies (BD) were used as negative controls. After additional nuclear staining with DAPI (4',6-diamidino-2-phenylindole, Linaris), cells were mounted for fluorescent microscopic analysis (Nikon E600 microscope).

Surgical Procedures
All animal experiments were approved and in accordance with institutional guidelines.

Splenectomy
Mice were anesthetized with 150 mg/kg body weight (bw) ketaminehydrochloride (Ketanest, Pharmacia) and 0.1 mg/kg bw xylazinehydrochloride (Rompun 2%, Bayer). The spleen was dissected through a lateral incision of the left abdomen. Vessels were carefully ligated using 6/0 silk. After removal of the spleen, the abdomen was closed layer by layer with single sutures using 6-0 silk. Animals were allowed to recover for 7 days before arterial injury was performed.

Carotid Artery Injury
Carotid artery injury was induced as described previously.²⁶ Briefly, the mice were anesthetized with 150 mg/kg bw ketaminehydrochloride (Ketanest, Pharmacia) and 0.1 mg/kg bw xylazinehydrochloride (Rompun 2%, Bayer). Using a dissecting microscope (MZ6; Leica), the bifurcation of the left carotid artery was exposed via a midline incision of the ventral side of the neck. Two ligatures were placed proximally and distally around the external carotid artery. The distal ligature was then tied off. After temporary occlusion of the internal and common carotid artery, a transverse arteriotomy was performed between the ligatures of the external carotid artery to introduce a curved flexible wire (0.13 mm in diameter) that is slightly curved (30 degrees) at the tip and completely fills out the vessel. The wire was passed along the common carotid artery in a rotating manner for three times. After removal of the wire, the proximal ligature of the external carotid artery was tied off. Normal blood flow was reassured, and the skin was closed with single sutures using 6/0 silk. Animals were allowed to recover, and carotid arteries were harvested at various time points.

Considerable time was spent to assure rigid standardization of all conditions and manual manipulations. Modifications of the original protocol of Lindner et al²⁶ were incorporated. Two highly trained investigators adapted to the microsurgical approach performed the carotid artery injury and performed and evaluated more than 200 operations until now. Injury was always induced by the same flexible wire. Because of standardization of the protocol, neointima formation after endothelial cell damage can be observed in virtually all animals. Figure 1C demonstrates neointima formation in the last 40 subsequent, unselected wild-type mice we studied.

View larger version (69K): [in this window] [in a new window]   Figure 1. Carotid artery injury model. Fourteen days after carotid artery injury, vessels were subjected to histological analysis. A, Representative H&E staining of neointima formation (arrow) after endothelial cell damage. B, Uninjured control carotid artery. C, Neointima formation in the last 40 subsequent, unselected wild-type mice, demonstrating highly reproducible neointima formation. Magnification x10. Scale bar=100 µm.

Transfusion Regimen
Mice received either 1x10⁶ PKH-26–stained spleen-derived MNCs or 1x10⁶ Dil-Ac-LDL–labeled spleen-derived EPCs by intravenous tail vein injection directly after induction of arterial injury and again 24 hours later. Control animals received a corresponding amount of normal saline. Thirty minutes before euthanasia, mice received 25 mg/kg body weight FITC-labeled lectin (BS-1, Sigma) intravenously. To evaluate the reendothelialization process, a subset of these animals received an injection of Evans blue dye (Sigma) 10 minutes before tissue harvesting.

En Face Microscopy and Immunohistochemistry
Perfusion-fixed carotid arteries were embedded in Tissue Tek OCT embedding medium (Miles), snap-frozen, and stored at -80°C. Samples were sectioned on a Leica cryostat (7 µm) and placed on slides coated with poly-L-lysine (Sigma) for immunohistochemical analysis. A subset of perfusion-fixed carotid arteries was opened longitudinally for en face microscopy, embedded in mounting medium (Dako), and directly assessed for Dil-Ac-LDL or PKH-26–positive cells using a Nikon TS100 microscope. For immunohistochemistry, tissue cryosections were postfixed in 4% formaldehyde for 2 minutes. Slides were preincubated with 0.5% Igpal (Sigma) and 5% normal goat serum (Sigma) for 30 minutes each. Sections were washed, stained with DAPI (Linaris), and mounted with fluorescent mounting medium (Dako) for fluorescent microscopic analysis. Embedded sections were assessed for Dil-Ac-LDL or PKH-26–positive cells and lectin expression. For morphometric analyses, H&E staining was performed according to standard protocols. All sections were examined under a Nikon E600 microscope. For morphometric analyses, Lucia Measurement Version 4.6 software was used to measure external elastic lamina, internal elastic lamina, and lumen circumference as well as medial and neointimal area of 25 sections per animal.

Statistical Analysis
Results are presented as mean±SEM. Significance between two measurements was determined by unpaired Student’s t test and in multiple comparisons was evaluated by the Newman-Keuls multiple comparison test. Values of P<0.05 were considered significant.

	Results

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Carotid Artery Injury Model
Fourteen days after carotid artery injury, vessels were harvested and subjected to histological analysis. Figure 1A shows a representative H&E staining of a highly reproducible neointima formation after endothelial cell damage compared with an uninjured carotid artery (Figure 1B). Figure 1C demonstrates the reproducible neointima formation after carotid artery injury in the last 40 subsequent, unselected wild-type mice.

The Spleen Is a Major Reservoir for EPCs in the Mouse
Because EPCs are a scarce cell population and are only found in low frequency in the peripheral blood, we evaluated the role of the spleen as a putative source of EPCs in the mouse. Isolated spleen-derived MNCs were cultured in vitro as described. After 4 days in endothelial cell selection medium, 91.8±3.2% of attached cells showed uptake of acetylated LDL and lectin binding, demonstrating endothelial cell characteristics (n=8 experiments, three high-power fields per section) (Figure 2A). Long-term cultures of isolated spleen-derived MNCs revealed that after 14 days, spleen-derived MNCs form tubular-like clusters comprising spindle-shaped cells at the periphery (Figure 2B). Additional immunostaining of spleen-derived MNCs was performed after 4 days of in vitro differentiation. Cultured spleen-derived MNCs showed expression of the mouse stem-cell marker Sca-1 (85.0±4.8%) as well as CD34 (88.6±3.1%) and c-kit (67.3±5.4%) in addition to the endothelial cell lineage antigen VEGFR-2 (82.9±5.2%) (Figure 2C), confirming that spleen-derived MNCs show typical characteristics of EPCs after in vitro culture with endothelial basal medium. Cell integrity was documented with corresponding nuclear staining (DAPI, Figure 2C) (n=5 experiments, three high-power fields per section).

View larger version (56K): [in this window] [in a new window]   Figure 2. Spleen-derived MNCs differentiate into cells with endothelial progenitor cell characteristics after in vitro expansion. A, Spleen-derived MNCs show uptake of acetylated LDL (left) and bind FITC-labeled lectin (middle) after 4 days in culture. Most adherent cells (91.8±3.2%; n=8, 3 high-power fields per section) are lectin-positive and Dil-Ac-LDL–positive (right). Magnification x10. B, Long-term (14-day) cultivated spleen-derived MNCs form tubular-like clusters comprising spindle-shaped cells at the periphery (arrows; left, magnification x20; right, magnification x100). C, Immunocytochemical analysis of spleen-derived MNCs after 4 days in culture revealed the expression of the mouse stem cell marker Sca-1, CD34, and c-kit as well as VEGFR-2 (arrows, top). All antibodies are directly labeled with phycoerythrin (PE) except CD34, which is FITC labeled. Cell integrity was documented with corresponding nuclear staining with DAPI (middle). Double staining of cells is shown in the bottom panel. n=8 experiments, 3 high power fields per section. Magnification x10. Scale bars=100 µm.

Bone Marrow– and Spleen-Derived MNCs and EPCs Home to Their Host Organ and not to the Vascular Injury Site After Intravenous Transfusion
To evaluate the effect of stem cell transfusion on reendothelialization after vascular injury, mice received either 1x10⁶ PKH-26–labeled spleen-derived MNCs or 1x10⁶ Dil-Ac-LDL–labeled spleen-derived EPCs intravenously directly after carotid artery injury and again 24 hours postoperatively. En face microscopy (Figure 3A) and immunohistochemistry (data not shown) revealed that only few, isolated cells were detected at the site of injury. Additional histological analysis of liver, spleen, and lung demonstrated that most spleen-derived cells could be detected in the spleen (Figure 3B).

View larger version (21K): [in this window] [in a new window]   Figure 3. Spleen-derived EPCs home to the spleen. To evaluate the role of transfused progenitor cells on reendothelialization after vascular injury, 1x106 PKH-26–labeled spleen-derived MNCs or cultured Dil-Ac-LDL–labeled EPCs were intravenously infused directly after carotid artery injury and again 24 hours postoperatively. A, Microscopic analysis showed only very few, isolated cells at the site of injury (arrow). B, Most spleen-derived cells were detected in the spleen. C, Corresponding nuclear staining of the spleen with DAPI. Magnification x10. Scale bar=100 µm.

Splenectomy Allows Homing of Transfused Spleen-Derived MNCs and EPCs to the Vascular Injury Site
To increase homing of the transfused cells to the injury site, mice were splenectomized and allowed to recover for 7 days before induction of arterial injury. Spleen-derived MNCs or EPCs 1x10⁶ were intravenously transfused directly after induction of injury and 24 hours later. En face microscopy 14 days after induction of injury and infusion of MNCs or EPCs revealed that compared with nonsplenectomized mice, a significant number of cells attached at the injury site, forming islets of transplanted cells within the deendothelialized area (Figure 4). Interestingly, the number of attaching cells was higher in the group treated with MNCs (204.3±15.4 mm²) (Figures 4A and 4E) compared with the group treated with EPCs (88.6±8.6 mm²) (Figures 4B and 4E). Homing of transfused cells was strictly restricted to the injury site, with no detectable cells in uninjured parts or in the contralateral vessel (Figures 4C and 4D). To demonstrate that the attached transfused cells at the site of injury represent endothelial cells, mice received a single dose of FITC-labeled lectin 30 minutes before tissue harvesting. Immunohistochemical analysis revealed that transfused MNCs (data not shown) and EPCs were predominantly found at the injury site, lining the intraluminal margin of the neointima (Figure 5A and, for higher magnification, Figure 5G). Lectin staining revealed that the attaching cells at the site of endothelial injury were lectin-positive (Figure 5B and, for higher magnification, Figure 5H). Nuclear staining with DAPI and overlay experiments (Figure 5C) clearly demonstrated the endothelial phenotype of transfused cells. In the placebo group (Figure 5D and, for higher magnification, Figure 5I), only few endogenous (lectin-positive, Dil-Ac-LDL–negative) endothelial cells were present, and the endothelial cell lining of the neointima appeared disrupted at day 14 after induction of injury (Figure 5E and, for higher magnification, Figure 5J) compared with the treatment group. Additional nuclear staining (DAPI) and overlay experiments confirmed these findings (Figure 5F).

View larger version (13K): [in this window] [in a new window]   Figure 4. EPCs home to the vascular injury site after splenectomy. To prevent homing of transfused cells to the spleen, mice were splenectomized 7 days before induction of arterial injury and transfusion of spleen-derived, PKH-26–labeled MNCs or Dil-Ac-LDL–labeled EPCs. A and B, En face microscopy revealed that after splenectomy, a significant number of spleen-derived MNCs (A) and EPCs (B) attached at the injury site. C and D, Homing of transfused cells was strictly restricted to the deendothelialized area with no detectable cells in uninjured parts or in the contralateral vessel. Scale bar=100 µm. E, Quantitative analysis showed that the number of attaching cells was significantly higher in mice treated with MNCs compared with EPC treatment. *P<0.05. n=3.

View larger version (45K): [in this window] [in a new window]   Figure 5. Transfused EPCs contribute to reendothelialization after endothelial cell damage. To demonstrate that the attached transfused cells at the injury site represent endothelial cells, mice received FITC-labeled lectin 30 minutes before tissue harvesting. A, Immunohistochemical analysis 14 days after the injury revealed that transfused Dil-Ac-LDL–labeled EPCs were predominantly found at the injury site, lining the intraluminal site of the neointima (arrows; for higher magnification, see panel G). B, Attaching cells at the site of endothelial injury were lectin-positive and therefore represent endothelial cells (arrows; for higher magnification, see panel H). C, Corresponding overlay with additional nuclear staining with DAPI. D and E, In the placebo group (D, for higher magnification, see panel I), only few (lectin-positive, Dil-Ac-LDL–negative) endothelial cells were lining the neointima (arrows, E; for higher magnification, see panel J), representing insufficient reendothelialization 14 days after induction of injury. F, Corresponding overlay with additional nuclear staining with DAPI. A through F, Magnification x10, scale bars=100 µm. G through J, Magnification x100, scale bars=50 µm.

Intravenously Transfused Spleen-Derived MNCs and EPCs Decrease Neointima Formation
To evaluate the biological effect of transfused spleen-derived PKH-26–labeled MNCs and EPCs on reendothelialization and neointima formation in transfused and placebo-treated mice, the deendothelialized areas were determined by Evans blue staining in a whole vessel preparation 14 days after induction of endothelial cell damage (n=5). Evans blue staining of the injured vessel revealed that transfusion of EPCs was associated with a significant increase in reendothelialization (86.4%±5.0%) compared with the placebo group (71.3%±6.0%) (Figure 6A). Computer-based morphometric analysis of 25 H&E-stained cryosections per animal (n=5 to 8) showed that the accelerated reendothelialization after transfusion of MNCs and EPCs was associated with a significant reduction of neointima formation (18 854±2639 µm² and 30 592±1028 µm², respectively) compared with the placebo group (37 724±2719 µm²) (Figure 6B). In contrast, medial area remained nearly unchanged after transfusion of PKH-26–labeled MNCs (20 904±4013 µm²) and DiI-Ac-LDL EPCs (24 286±1305 µm²) compared with placebo (20 075±1343 µm²). The corresponding intima to media ratio revealed a ratio of 0.9±0.15, 1.26±0.06, and 1.88±0.26, respectively.

View larger version (32K): [in this window] [in a new window]   Figure 6. Reendothelialization and neointima formation. To evaluate the effect of transfused spleen-derived EPCs on reendothelialization, the whole injured vessel of transfused and placebo-treated mice was stained with Evans blue to unmask deendothelialized areas. A, Computer-based morphometric analysis revealed that transfusion of EPCs resulted in a significant increase in reendothelialization compared with the placebo group, n=5. B, Additional morphometric analysis of hematoxylin-stained cryosections showed that the accelerated reendothelialization after transfusion of EPCs and MNCs was associated with a significant reduction of neointima formation compared with the placebo group. *P<0.05; **P<0.0001; n=5 to 8 mice, 25 sections per animal. C, In contrast, morphometric analysis of hematoxylin-stained cryosections (25 per animal, n=5 to 8) revealed that medial area remained constant in each treatment group. D, Neointima to media ratio, n=5 to 8, 25 sections per animal. Tx indicates transfusion.

	Discussion

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Spleen-derived MNCs can transdifferentiate into cells with an endothelial phenotype after stimulation with endothelial growth factors in vitro. Intravenous transfusion of spleen-derived MNCs or in vitro cultured EPCs resulted in a significantly enhanced reendothelialization associated with a diminished neointima formation after induction of endothelial cell damage.

Regeneration and proliferation of endothelial cells after tissue damage, during neoangiogenesis, or after vascular injury was long believed to be a local process managed by the adjacent endothelial cells within the intact parts of the intima.²⁸ This dogma was challenged by recent evidence demonstrating that circulating stem cells and BM-derived progenitor cells transit through the circulation, enter different organs, and differentiate into cells of the host organ or contribute to the pool of existing stem cells.²⁹ Several studies have evaluated the role of BM-derived EPCs after tissue damage and during angiogenesis, demonstrating that after profound injury (eg, myocardial infarction, coronary artery bypass grafting, burns), the number of circulating EPCs significantly increases.^30,31 Furthermore, the number of circulating EPCs is significantly reduced and cells are functionally impaired in patients with cardiovascular risk factors, suggesting an important role of EPCs in cardiovascular disease.^23–25 Consequently, it could be shown that application of EPCs enhances angiogenesis as well as myocardial regeneration after myocardial infarction.^13,16–18

However, the potentially protective role of BM-derived stem and progenitor cells in endothelial dysfunction and in the early stages of atherosclerosis, the disease that predominantly precedes tissue damage, infarction, and neoangiogenesis, has not been clarified yet. We and others demonstrated that BM-derived EPCs participate in reendothelialization processes and vascular repair after arterial injury of the adult blood vessel.^26,27 In these animal studies, the circulating number of EPCs was increased by statin treatment, which correlated with decreased neointima formation.^26,27 In any event, the experimental approach did not allow us to dissect whether other statin properties such as direct inhibition of smooth muscle cell proliferation or the described mobilization of EPCs accounted for the beneficial vascular effect. The herein-presented data indicate that enhancement of the circulating number of EPCs by transfusion specifically results in an enhanced reendothelialization, which in turn is known to be associated with inhibition of smooth muscle cell proliferation,²⁰ resulting in the observed diminished neointima formation. Thus, EPCs as well as spleen-derived MNCs are capable of accelerating repair of endothelial cell damage in adult blood vessels. It remains unclear why the circulating EPCs are obviously insufficient to overcome exogenously applied tissue or vessel damage. On one hand, this could be a problem of quantities because of inappropriate mobilization. On the other hand, it could be the result of an insufficient homing and adhesion process. It cannot be excluded that isolation and in vitro expansion procedures lead to the conditioning of EPCs and MNCs, which enhance homing to the injury site.

Interestingly, neointima reduction was more prominent after intravenous transfusion of spleen-derived MNCs compared with EPCs derived from a 4-day spleen MNC culture. This may be attributable to an increased transdifferentiation rate of MNCs to endothelial cells in the blood compared with the in vitro assay. Furthermore, Rehman et al³² recently demonstrated that in humans most cultured blood-derived EPCs are derived from macrophages and monocytes. The high proportion of monocytes and macrophages in spleen-derived MNCs with differentiation potential into endothelial cells³² may potentially explain the increased reendothelialization and enhanced neointima suppression after intravenous transfusion of spleen-derived MNCs. In addition, transfusion experiments with ex vivo expanded hematopoietic progenitors and BM-derived mesenchymal stem cells have revealed that the homing efficiency of these cultured cells to the BM after intravenous transfusion is reduced compared with freshly isolated cells.^33,34 This has been associated with a downregulation of integrins during ex vivo expansion.³⁴ Additional experiments will have to determine the exact differences in surface markers and transdifferentiation status between freshly isolated MNCs and cultured EPCs.

Homing signals for circulating stem or progenitor cells mostly result from local injury and guide the circulating stem or progenitor cell presumably to the target tissue.^2,4 In our study, intravenously transfused cells were exclusively found at the injury site when homing to the host organ was prevented by splenectomy. This observation is in accordance with reports demonstrating that intravenously transfused hematopoietic progenitor cells preferentially home to the BM and spleen.³⁵ It is intriguing to speculate that splenectomy prolonged the circulating time of EPCs in the peripheral circulation, resulting in a change of surface markers because of the homing signals of the injury site. The interaction of EPCs with growth and differentiation factors finally may lead to the activation of tissue-specific genes, resulting in a cell phenotype of the host organ.^29,36

Our data indicate that regeneration of endothelial cells either by endogenous or transfused progenitor cells may be an important and novel therapeutic approach for the therapy of vascular injury in the early stages of atherosclerosis. In addition, therapeutic transfusion of EPCs after angioplasty, which triggers a cascade partially comparable to atherogenesis,²² may be an important option in the prevention of restenosis of the targeted vessel. The knowledge that stem and progenitor cells from multiple sources can regenerate endothelial cells increases the feasibility and applicability of stem cell–based therapeutic approaches. Additional investigation will have to focus on the identification of surface markers and homing signals to identify the critical markers and cytokines that give the alley for engraftment. Despite the attraction to the homing site, strategies for enhancing the differentiation of engrafted progenitor cells to ensure that BM-derived cells give rise to functional organ-specific cells are fundamental to develop. The confirmation and extension of these findings will have important implications for the understanding of stem cell biology and stem cell–based therapeutic approaches in cardiovascular disease.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft. The authors thank Simone Jäger and Sybille Karl for excellent technical assistance.

	Footnotes

 
Original received April 10, 2003; revision received June 16, 2003; accepted June 18, 2003.

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