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Cardiovascular Research 2003 58(2):469-477; doi:10.1016/S0008-6363(03)00266-9
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Copyright © 2003, European Society of Cardiology

Vascular gene delivery of anticoagulants by transplantation of retrovirally-transduced endothelial progenitor cells

Daniel P. Griesea,*, Stefan Achatza,1, Christian A. Batzlspergera,1, Ulrike G. Strauchb, Bernhard Grumbecka, Joachim Weila and Günter A.J. Rieggera

aKlinik und Poliklinik für Innere Medizin II, Universität Regensburg, Franz-Josef-Strauß-Allee 11, 93042 Regensburg, Germany
bKlinik und Poliklinik für Innere Medizin I, Universität Regensburg, Franz-Josef-Strauß-Allee 11, 93042 Regensburg, Germany

daniel.griese{at}klinik.uni-regensburg.de

* Corresponding author. Tel.: +49-941-944-7211; fax: +49-941-944-7213.

Received 5 September 2002; accepted 29 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Recent studies have documented the presence of bone marrow-derived endothelial progenitor cells (EPC) in the circulation of several species. This study was designed to evaluate the use of engineered EPC for vascular gene delivery into angioplasty-induced arterial lesions. Methods and Results: EPC could easily be isolated from whole bone marrow and peripheral blood of adult rats. Differentiation was induced by culture on fibronectin in the presence of endothelial specific growth factors. Rat EPC shared several phenotypic and functional properties with mature endothelial cells. Recombinant retroviruses were generated encoding for the anticoagulants tissue-type plasminogen activator (tPA) and hirudin. Efficient (>90%) ex vivo gene transfer could be achieved resulting in high levels of transgene production. Engineered EPC were locally infused into freshly balloon-injured carotid arteries. Analysis of day 7 vessels showed 73±10% luminal coverage of the lesioned arterial bed with transduced EPC. Sustained secretion of both anticoagulants could be detected in organ cultures of explanted arteries. EPC seeding inhibited dilation of the injured arterial segment and prevented reduction of media thickness. However, rapid repopulation with EPC failed to attenuate neointima formation in this model. Conclusions: Peripheral blood and bone marrow can be used as source for endothelial lineage cells. Cultured EPC can be genetically engineered by retroviral gene transfer and serve as cellular vehicles for vascular gene and drug delivery of anticoagulants. Local transplantation of EPC attenuates reendothelialization of angioplasty-injured arteries but fails to inhibit neointima proliferation.

KEYWORDS Angioplasty; Anticoagulants; Cell isolation; Endothelial function; Gene therapy


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Recent studies have demonstrated a previously unrecognized degree of plasticity in postnatal stem cell (SC) populations [1]. The experimental data suggest that an expansion of the traditional view of stem cell biology is needed. Hematopoietic SC, originally known to reconstitute the hematopoietic lineages in the blood following lethal irradiation, have now been shown to give rise to cells of multiple organ systems like skeletal muscle [2,3], central nervous system [4,5], intestinum, lung [6], liver [7], heart [8,9], and vasculature [10–13]. It is therefore believed that SC from both bone marrow and other tissue compartments are capable of entering the blood stream and participate in processes of tissue growth and regeneration [14].

This evolving concept of a seemingly unrestricted potential of adult SC to (trans)differentiate into various tissue-specific cell types is likely to have enormous implications for clinical medicine [15]. There is an increasing number of experimental studies addressing the potential use of postnatal SC for cellular therapies of various organ damage. In cardiovascular medicine both vascular [16–18] and myocardial [8,19] disease models have been tested, and functional improvement was documented.

Despite advances in pharmacological therapies, generalized atherosclerosis and ischemic heart disease remain the principal cause of morbidity and mortality in industrialized countries. Patients with advanced vascular disease often require percutaneous angioplasty to restore sufficient organ perfusion. This interventional procedure, however, is plagued with relevant failure rates caused by adverse vascular remodelling. The inevitable injury of the intact endothelial monolayer during endovascular procedures is believed to be a significant factor to further trigger local activation of the coagulation cascade and the inflammatory burst within the distended vessel wall. Subsequently, acute thrombotic occlusions and progressing neointima formation can severely complicate this treatment modality.

We and others have demonstrated the successful transplantation of endothelial cells (EC) into a denuded vascular bed or prosthetic graft [17,20,21]. The EC used, however, have been limited to a venous source or those derived from adipose tissue [22], neither of which seems feasible for broad clinical applications.

The aim of this study was to explore the potential for cell transplantation strategies for angioplasty-injured arteries based on the existence of endothelial progenitor cells (EPC) in the circulation and bone marrow. Following genetic modification to establish sustained secretion of anticoagulative agents, we sought to use EPC as vehicles to create an endothelial biosurface for local vascular drug delivery to overcome complicating factors of percutaneous angioplasty.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Whole bone marrow was isolated from both femurs and tibias of adult male Sprague–Dawley rats (Charles River, Sulzfeld, Germany), and heparinized peripheral blood (10 ml per animal) was collected by puncture of the right ventricle. Mononuclear cells were fractionated by density gradient centrifugation (Ficoll-PaqueTM PLUS, Amersham Biosciences, Freiburg, Germany). Subsequently, cells were cultured on dishes precoated with human fibronectin (Sigma) in EBM (PromoCell, Heidelberg, Germany) supplemented with 5% fetal calf serum, recombinant rat VEGF (10 ng/ml, R&D Systems, Wiesbaden, Germany), recombinant bovine bFGF (1 ng/ml, R&D Systems), recombinant murine IGF-1 (10 ng/ml, R&D Systems), human EGF (10 ng/ml, PromoCell), hydrocortisone (1 µg/ml, PromoCell), and antibiotics. Adherent cells were extensively washed on day 4 to remove unattached cells, and fresh growth media was added. All experiments were performed with first passage cells.

2.2 Cell characterization, quantification of transgene expression, and Western blot
For determination of active LDL-cholesterol uptake, cells were incubated with acLDL-Dil (10 µg/ml) for 4 h at 37°C. Lectin binding was analysed using FITC-conjugated UEA-1-lectin (Sigma, 10 µg/ml). For histochemistry and Western blot analysis, the following antibodies were used: von Willebrand factor (Sigma, dilution 1:200), VEGF-receptor 2 (Santa Cruz Biotechnology, clone A-3, 1:100), Tie-2 (Santa Cruz Biotechnology, clone C-20, 1:100), and eGFP (Santa Cruz Biotechnology, 1:100). Isotype controls and secondary antibodies were from Jackson Immunoresearch (distributed by Dianova, Hamburg, Germany). Vectashield including DAPI nuclear counterstain (Vector Laboratories, Burlingame, CA) was used as mounting media. For tube formation cells were mobilized and reseeded on MatrigelTM matrix (BD Biosciences). tPA- and hirudin-content was quantified using commercially available kits according to the manufacturers instructions (IMUBIND Hirudin ELISA, IMUBIND tPA ELISA; America Diagnostica, Greenwich, CT, USA). For Western blot analysis vessels were homogenized in 100 µl lysis buffer (25 mM Tris, 1 mM EDTA, 1 mM EGTA). Then 35 µg of protein per lane was separated on a 4–15% SDS–PAGE gradient, and blotted onto a PVDF membrane (Bio-Rad, Hercules, CA). Immunodetection was performed according to standard protocols.

2.3 Recombinant retroviruses and ex vivo gene transfer
The murine retroviral construct pC.MMP has been described elsewhere [23,24]. The human tPA cDNA (1670 bp), hirudin cDNA (278 bp), and eGFP cDNA (737 bp) were inserted into the Nco I and BamH I cut vector. For correct modification and secretion of the functional peptide, the hirudin cDNA is preceeded by the signalling peptide of the human growth hormone reading frame. The IRES-eGFP element was taken as Bgl II to BamH I fragment from pMSCV2.2-IRES-eGFP and cloned into the BamH I site. Constructs were confirmed by automated DNA sequencing. VSV-G pseudotyped retroviruses were generated as previously described. Briefly, vector construct (24 µg), pMD.M-MLV.gag.pol (18 µg), and pMD.M-VSV.G (6 µg) were co-transfected into subconfluent 293T cells in 100-mm dishes by standard calcium-phosphate-DNA coprecipitation. Retroviral supernatant (10 ml/plate) was harvested on day 2. Infectious titer was determined by eGFP expression on NIH/3T3 cells, and was usually ~1–5x106 IU/ml. EPC were infected by exposure to VSV-G pseudotypes on days 5 and 8 of culture for 8 h in the presence of polybrene (8 µg/ml). Analysis of eGFP expression was performed on an Epics Coulter FACS.

2.4 Experimental angioplasty and cell transplantation
Balloon injury of the right common carotid artery was performed as previously described [25]. Briefly, rats (400–450 g) were anesthetized by an intraperitoneal injection of ketamin/xylazine, and the right carotid artery was exposed. Hemostatic controls were placed at the proximal common carotid and the internal carotid. A Fogarty 2F embolectomy catheter (Edwards Lifesciences, Unterschleissheim, Germany) was introduced into the external carotid, advanced to the common carotid, inflated, and withdrawn three times with rotation. EPC were mobilized with trypsin/EDTA, washed twice in PBS, and 1.0x106 per animal were resuspended in 200 µl EBM supplemented with 20% (v/v) rat serum and heparin (20 U/ml). Cell solution (mock infected EPC, n = 4; eGFP infected EPC, n = 6; hirudin-eGFP infected EPC, n = 6; tPA-eGFP infected EPC, n = 5) was instilled and incubated in the freshly injured arterial bed for 25–30 min. Unbound cells were removed by rinsing the isolated arterial segment with 1 ml of EBM/20% rat serum. Finally, the catheter was removed, and blood flow was restored. All animal procedures were performed in accordance with institutional guidelines and conformed with the Guide for the Care and Use of Laboratory Animals as published by the US NIH. On day 7 animals were euthanized and perfusion-fixed with 10% buffered formalin. Vessels were embedded in O.C.T. and frozen in liquid nitrogen. Cryosections were stained for eGFP and vWF as described above. For organ cultures carotids were excised and placed in 0.5 ml EBM growth media for 24 h. Hirudin and tPA was quantified in triplicate in organ culture supernatant and serum. For histomorphology (six animals per group, four sections/carotid artery) cross-sections were stained with hematoxylin/eosin and examined for vessel diameter, media area, and intima to media area ratio.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Endothelial progenitor cells can be isolated from bone marrow and peripheral blood of adult rats
Peripheral blood and whole bone marrow was isolated from adult rats. Mononuclear cells were separated by density gradient centrifugation. This enriched cell population was CD45 (leukocyte common antigen, LCA)-positive, indicating its hematopoietic origin (data not shown). Cells (2.0±0.4x108 per animal, 8.3±0.5x106 per animal) were cultured at high densities of 0.8–1.0x106 cells/cm2 on fibronectin-precoated dishes in the presence of the endothelial cell (EC) specific growth factors vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1). On day 4 media was changed for the first time and cultures were washed thoroughly to remove unattached cells. Colonies of adherent cells increased in size and resembled EC characteristic cobble-stone morphology (Fig. 1a,b). All further experiments were performed with bone marrow-derived first passage cells on days 11–13 of culture (yield 1.4±0.3x104 cells/cm2). Adherent cells took up LDL-cholesterol particles from the media and stained positive for UEA-1-lectin (Fig. 1c,d). These criteria are not exclusively specific for EC but are widely used to define EPC of hematopoietic origin. To confirm their differentiation into cells of the endothelial lineage, bone marrow-derived cells were further analyzed by immunohistochemistry. At day 11 of culture rat EPC showed expression of several EC-specific markers like von Willebrand factor, VEGF-receptor 2, and Tie-2 (Fig. 1e–g). When mobilized and reseeded onto matrigel, formation of primitive vascular tubes could be observed within 6–12 h (Fig. 1h). Additional FACS analysis of surface receptors revealed that rat EPC after 11–13 days of in vitro culture had lost expression of the hematopoietic marker CD45 (LCA). Our culture conditions resulted in high purity so that at the first passage less than 5% of contaminating CD45-positive cells were present. These contaminating cells further diminished when EPC were replated. Most of the EPC showed expression of CD90 (Thy 1.1), which is known as marker for hematopoietic stem and progenitor cells, and a subset of EC. Interestingly, first passage rat EPC had positive labeling for the intercellular adhesion molecule 1 (ICAM-1, CD54) which is found on activated EC and known to mediate adhesion of inflammatory cells to the EC surface (Fig. 2a–d).


Figure 1
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  		Fig. 1 In vitro differentiation of rat EPC. (a) Bone marrow-derived EPC, day 10; (b) peripheral blood-derived EPC-colony, day 12 (original magnification x200); (c) uptake of acetylated LDL by BM-derived EPC. By immunohistochemistry rat EPC stain positive for: (d) UEA-1-lectin; (e) von Willebrand-factor (small frame: isotype control); (f) VEGF-receptor 2 (small frame: isotype control); and (g) Tie-2 (small frame: isotype control), nuclear blue staining with DAPI, original magnification x1000. (h) Rat EPC form primitive vascular tube-like structures when replated on matrigel.

 

Figure 2
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  		Fig. 2 Characterization of rat EPC. Representative FACS analysis of surface receptor expression on first-passage bone marrow-derived rat EPC. Histograms display fluorescence intensity (x-axis) versus relative cell number (y-axis). Black line, mouse IgG isotype-control-labeled cells; gray fill, rat EPC labeled with: (a) CD45 (leukocyte common antigen, LCA); (b) UEA-1-lectin; (c) CD90 (Thy1.1); and (d) CD54 (ICAM-1).

 
3.2 Recombinant murine retroviruses allow efficient genetic modification of rat endothelial progenitor cells
A series of replication-defective retroviruses was generated to genetically modify EPC during the differentiation and expansion period. The complimentary DNA encoding human tissue-type plasminogen activator (tPA) and the thrombin-inhibitor hirudin were inserted into the murine retroviral vector pC.MMP. By cloning of an internal ribosome entry site (IRES) element downstream of the first transgene, constructs were made bicistronic and allowed simultaneous expression of the enhanced green fluorescence protein eGFP (Fig. 3a,b). This simplified the determination of infectious titers and allowed follow up of transduced cells both in vitro and in vivo. Retroviral particles generated from pC.MMP-tPA-IRES-eGFP, pC.MMP-hirudin-IRES-eGFP, and pC.MMP-eGFP were pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelopes. Upon reverse transcription and integration, transcriptional activity for transgene expression is regulated by the retroviral long terminal repeat.


Figure 3
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  		Fig. 3 Retroviral gene transfer of rat EPC. (a) Simplified scheme of the retroviral vector constructs pC.MMP-tPA-IRES-eGFP, pC.MMP-hirudin-IRES-eGFP, and pC.MMP-eGFP (SD, splice donor; SA, splice acceptor; {psi}, encapsidation signal); (b) retrovirally-transduced EPC exhibit strong expression of eGFP; (c) representative FACS analysis of mock-infected EPC and MMP-eGFP-infected EPC showing >90% of transduced cells.

 
Stable ex vivo gene transfer was achieved by exposure of EPC cultures to VSV-G pseudotyped retroviruses (~2x106 infectious units each) on days 5 and 8. With the high proliferative rate during this culture period of differentiation and expansion, retroviral gene transfer could be optimized so that transduction efficiencies of 89±7% were reached (Fig. 3c).

Transduced cells could be identified by strong eGFP expression, and showed sustained production of both anticoagulants. As seen in Fig. 4, high levels of hirudin- and tPA-production were measured in supernatants from infected EPC and control NIH/3T3 cells (infected at a multiplicity of infection of 5).


Figure 4
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  		Fig. 4 Production of human tPA and hirudin by rat EPC in vitro. EPC and NIH/3T3-cells were infected with recombinant retroviruses as described above. Concentrations of human tPA and hirudin were quantified in supernatants using commercially available ELISA kits. Values are mean±S.D.

 
3.3 Genetically-engineered EPC can serve as cell vehicles for treatment of balloon-injured rat carotid arteries
An experimental angioplasty procedure was performed in the common carotid artery of adult rats. Immediately following the intervention, transduced EPC (1x106 per animal) were locally infused into the freshly injured arterial bed. After an incubation period of 25–30 min unbound cells were aspirated, the isolated vessel segment was rinsed, and the regular blood flow was restored. Control animals received mock-infected or MMP-eGFP-infected EPC. Vessels were analyzed on day 7.

Transplanted vessels showed no sign of thrombosis. A high degree of repopulation was seen so that 73±10% of the lesion was covered with eGFP-positive cells (Fig. 5a,b). Histological examination revealed that transplanted EPC had formed a monolayer on the luminal surface. The EC phenotype in vivo was confirmed by counterstaining with an antibody specific for von Willebrand factor (Fig. 5c–f). Expression of the eGFP marker could also be detected by Western blot analysis using protein lysates of vessels that had received retrovirally-transduced EPC (Fig. 5g).


Figure 5
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  		Fig. 5 EPC transplantation into balloon-injured carotid arteries. Suspended EPC were locally seeded into the isolated carotid for 25–30 min. Transplanted cells covered large areas of the lesion on day 7 after the procedure. En face photograph of a representative vessel showing eGFP-positive EPC on (a) the luminal surface, original magnificationx40, and (b) transitional zone, original magnificationx320. Histology of EPC-seeded vessel showing: (c–e) GFP-positive EPC that form a monolayer on the luminal surface (non-specific green fluorescence in the vessel wall); (e) negative staining on control carotids without EPC seeding; and (f) EC phenotype of transplanted EPC confirmed by Texas Red-vWF staining. Nuclear blue staining with DAPI. Original magnification x1000. (g) Western blot analysis for eGFP expression in vessels transplanted with media control (lane C), mock-infected EPC (lane E), and MMP-eGFP-infected EPC (lane EG).

 
To confirm the sustained secretion of transferred anticoagulants, arterial segments were excised and placed into organ culture for 24 h. Both hirudin and tPA could be detected in significantly higher amounts in supernatants from vessels that had been transplanted with hirudin-eGFP or tPA-eGPF transduced EPC as compared to mock- or eGFP-infected cells (Fig. 6). Systemic concentrations of hirudin and tPA were not elevated and had identical levels as compared to serum taken from the control animals (data not shown).


Figure 6
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  		Fig. 6 In vivo vascular delivery of anticoagulants by local EPC transplantation. Injured carotid arteries were harvested 7 days after experimental angioplasty and EPC seeding. Arterial segments were placed in organ culture (0.5 ml EC growth media) for 24 h. Hirudin and tPA production was quantified in supernatants by ELISA. Values are mean±S.D.

 
3.4 Rapid reendothelialization with endothelial progenitor cells failed to inhibit neointima proliferation following balloon injury of rat carotid arteries
Cross-sections of injured arterial segments were examined on day 7 post EPC transplantation for histomorphological adaptations (Fig. 7). The balloon-injury procedure in the rat common carotid artery resulted in dilation of the lesioned arterial segment so that the vessel diameter was significantly increased from 0.748±0.040 mm (mean±S.E.M.) in uninjured control arteries to 0.822±0.023 mm in injured vessels that were incubated with media alone. This widening was not found in carotids that had received transplantation of transduced EPC irrespective of transgene expression (Fig. 8a). Vessel distension in injured arteries was associated with reduction in cross-sectional media area from 0.203±0.020 to 0.105±0.049 mm2 as a result of early smooth muscle cell death in the vessel wall. Thinning out of media area was not seen in EPC transplanted arteries (Fig. 8b). Again, secretion of tPA or hirudin had no additional impact on cross-sectional media area when compared to eGFP controls. However, accelerated reendothelialization mediated by EPC seeding did not result in reduced neointima formation in carotids on day 7 post injury. Indeed, intimal to media area ratio was significantly higher in EPC transplanted vessel segments (I/M ratio: media control 0.51±0.05, eGFP-transduced EPC 1.03±0.16; mean±S.E.M.). EPC transduced to secrete tPA seemed to aggravate intimal hyperplasia so that I/M ratio was further elevated to 1.50±0.27. Sustained production of the thrombin-inhibitor hirudin by transplanted EPC resulted in an I/M ratio of 1.01±0.14 in arteries on day 7 post injury (Fig. 8c).


Figure 7
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  		Fig. 7 EPC transplantation and vascular remodeling in balloon-injured rat carotid arteries. Representative photomicrographs of balloon-injured, perfusion-fixed, hematoxylin-eosin-stained carotid arteries 7 days after angioplasty. Original magnification x40. Cross-sectional view of: (a) uninjured carotid artery; (b) injured carotid artery with media-control incubation; (c) injured carotid artery transplanted with EPC secreting hirudin; and (d) injured carotid artery transplanted with EPC secreting tPA.

 

Figure 8
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  		Fig. 8 EPC transplantation fails to inhibit neointima proliferation in balloon-injured rat carotid arteries. Vessels were perfusion-fixed with 10% buffered formalin solution 7 days after endovascular injury and EPC seeding. A total of four hematoxylin-eosin-stained cross-sections per lesioned arterial segment were analysed: (a) vessel diameter; (b) media area; and (c) (neo)intima area/media area ratio was determined by computer-assisted histomorphometry. Values are mean±S.E.M. *P<0.05 (unpaired Student's t-test).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The unique functional properties of endothelial cells render them favorable candidates for tissue engineering and cell-based vascular therapies. However, protocols involving EC transplantation have not found wide clinical applicability. This is in part due to the technical challenges needed to obtain sufficient quantities of autologous EC. Previously, EC cultures were initiated after enzymatic digestion of venous tissue. In addition, adipose tissue has been suggested as an alternative source for microvascular EC [22]. Both isolation methods require an invasive procedure, and bear a significant risk for fibroblast and smooth muscle cell contamination during further expansion of harvested EC.

It has been described that both peripheral blood and bone marrow can be used as a source for endothelial precursor cells [10,16,26]. This population has the potential to mature into functional EC under specific culture conditions.

This study was therefore designed to evaluate the use of EPC for cell and gene therapies of arterial injury in a rat angioplasty model. We show that EPC can easily be isolated from peripheral blood and bone marrow of adult rats. EPC adhered to fibronectin-coated dishes and were expanded in the presence of VEGF, bFGF, and IGF-1. As reported from other species, rat EPC actively take up LDL-cholesterol and stain positive for the Ulex europaeus lectin. They share other phenotypic characteristics with endothelial lineage cells, as shown by expression of the EC restricted markers vWF, VEGF-receptor, and Tie-2. Rat EPC were found to be CD45(LCA)-negative but kept expression of CD90 which is found not only on certain EC but also characterizes hematopoietic stem and progenitor cells. It is known that systemic administration of hematopoietic and vascular growth factors can effectively mobilize the progenitor cell pool from the bone marrow into the circulation [26]. One could thereby argue that EPC harvest from the peripheral blood could be optimized for clinical applications.

With the use of recombinant retrovirusus we could establish a stable and very efficient gene transfer method. Ex vivo modified EPC revealed high levels of transgene expression in vitro and following transplantation in vivo as shown for both anticoagulants used in our study.

This study is the first to demonstrate the use of EPC for local transplantation into denuded arterial beds in rats. Several genetic approaches to improve vascular remodelling following angioplasty have been shown to be effective in animal models, including overexpression of antioxidant enzymes [25,27], and transient inhibition of cell cycle progression [28]. However, most of these therapeutic strategies depended on direct in vivo gene transfer with recombinant adenoviral vectors to achieve untargeted transduction of various cell types within the vessel wall.

EPC seeding onto the freshly injured arterial bed resulted in a rapid repopulation of the lesion and cells covered most parts of the luminal surface. The degree of reendothelialization seen with our approach on day 7 is usually reached 4 weeks later in this model [29].

In addition we were able to show that recombinant retroviral vectors offer a safe possibility to genetically engineer the EPC vehicles prior to their transplantation in vivo. This might further improve the therapeutic potential of vascular EPC therapies. As proof of principle, we demonstrate in this study that EPC modified to produce anticoagulants reendothelialize balloon-injured carotid arteries in rats. Both tPA and hirudin have proven their therapeutic benefit in acute thromboembolic disorders. Systemic administration of these anticoagulants however bears the risk of significant hemorrhagic complications. Therefore targeted and local application would be favorable. This was achieved in our in vivo model. Sustained transgene secretion in vivo was documented by organ culture experiments of explanted vessels. This implies that significant quantities of the secreted anticoagulants were delivered locally and to the vasculature distal to the site of transplantation. It has been shown that adenoviral gene transfer of tPA prevents arterial thrombus formation and promotes local thromboresistance [30]. In addition to the anticoagulative properties of hirudin, it has been shown that adenoviral gene transfer of the thrombin inhibitor reduces neointima formation following balloon injury in the rat [31].

Endothelial dysfunction and discontinuity are known to play a pivotal role in degenerative vascular disease. For endovascular interventions it has been postulated that injury of the intact endothelial monolayer is a key factor in the evolution of subsequent adverse vascular remodelling. It has recently been shown that systemic administration of statins increases the frequency of circulating endothelial progenitor cells. Accelerated reendothelialization of denuded vascular lesions was associated with reduced intimal thickening in this model [12,29]. We therefore hypothesized that immediate repopulation with EPC after experimental angioplasty may restore regular vascular anatomy and thereby stabilize vessel wall function. Seeding of cultured jugular vein endothelial cells had failed to reduce neointima proliferation in a rabbit model of vascular injury [32].

In our study, local EPC transplantation inhibited dilation of the injured arterial segment. Cross-sectional media area was significantly reduced on day 7 in injured control arteries compared to their uninjured counterparts. This is caused by smooth muscle cell death in the media during the early phase following balloon injury. Cross-sectional media area was comparable to uninjured vessels in EPC seeded carotids irrespective of transgene expression. Interestingly, immediate reendothelialization was associated with a significant increase of intima/media ratio compared to unseeded controls. It can only be speculated at this time as to the reasons for this observation. It appears that immediate restoration of the endothelial layer partly inhibited the dramatic smooth muscle cell loss usually seen in this model. Transplanted EPC may provide costimulatory factors that further promote proliferation and migration of smooth muscle cells. We have shown that our culture conditions resulted in surface expression of ICAM-1. This may reflect an activated state of the EPC at the timepoint of transplantation. ICAM-1 is known to be involved in leukocyte adhesion. This may have resulted in enhanced migration of inflammatory cells into the vessel wall to indirectly support intimal hyperplasia. As seen by others, local overexpression of tPA by adenoviral gene therapy resulted in further enhancement of neointima formation [33,34]. This seems to be mediated by the mitogenic effect of tPA on smooth muscle cells. In contrast to the beneficial effects of hirudin on vascular remodelling reported by others, targeted delivery of the thrombin inhibitor failed to reverse the effect on neointima proliferation caused by EPC seeding in our model [31,35].

In summary, our studies demonstrate that endothelial progenitor cells can easily be isolated from bone marrow and peripheral blood of adult rats. Differentiation and expansion can be induced by specific culture conditions. Recombinant murine retroviruses offer a safe vector system for stable gene transfer into rat EPC ex vivo. Seeding of bone marrow-derived endothelial lineage cells results in rapid repopulation of the denuded artery with transplanted cells. Genetically engineered EPC can serve as cellular vehicles for targeted vascular gene transfer. Interventional reendothelialization with EPC restores vessel diameter and media thickness but accelerates neointima proliferation in balloon-injured rat carotid arteries. Further studies will be needed to clarify if EPC transplantation may be used as therapeutic strategy for the treatment of vascular disease.

The authors wish to thank J. Simon for excellent technical assistance. D.P.G. is recipient of the Heinz Meise-award 2001 by the Deutsche Herzstiftung. This work was supported by the Deutsche Forschungsgemeinschaft (Gr 1581/1-1) and the University of Regensburg (ReForM A grant to D.P.G. and U.G.S.).

Time for primary review 26 days.


   Notes
 
1 Stefan Achatz and Christian A. Batzlsperger contributed equally to this work. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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