Published online before print January 26, 2006, doi: 10.1161/01.ATV.0000205607.98538.9a
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© 2006 American Heart Association, Inc.
Vascular Biology |
Granulocyte Colony-Stimulating Factor–Mobilized Circulating c-Kit+/Flk-1+ Progenitor Cells Regenerate Endothelium and Inhibit Neointimal Hyperplasia After Vascular Injury
From the Department of Cardiovascular Medicine (M.T., M.O., N.U., K.T., T.N., K.Z., E.A., T. Tatsumi, T. Takahashi, H.M.), Kyoto Prefectural University School of Medicine, Japan; Department of Pharmacology (D.J., S.T., M.M.), Osaka Medical College, Takatsuki, Japan; Pharmacobioregulation Research Laboratory (Y.N.), Taiho Pharmaceutical Co. Ltd, Saitama, Japan; and Department of Pathology II (Y.A.), Kansai Medical University, Moriguchi, Japan.
Correspondence to Mitsuhiko Okigaki MD, Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan. E-mail okigakim{at}koto.kpu-m.ac.jp
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Abstract |
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Background— Granulocyte colony-stimulating factor (G-CSF) treatment was shown to inhibit neointimal formation of balloon-injured vessels, whereas neither the identification of progenitor cells involved in G-CSF–mediated endothelial regeneration with a bone marrow (BM) transplant experiment nor the functional properties of regenerated endothelium have been studied.
Methods and Results— Recombinant human G-CSF (100 µg/kg per day) was injected daily for 14 days starting 3 days before balloon injury in the rat carotid artery. Neointimal formation of denuded vessels on day 14 was markedly attenuated by G-CSF (39% versus the control; P<0.05). Endothelial cell–specific immunostaining revealed an enhancement of re-endothelialization (1.8-fold increase versus the control; P<0.05) and inhibition of extravasation of Evans Blue dye (47%; P=0.02). The regenerated endothelium exhibited acetylcholine-mediated vasodilatation in NO-dependent manner. G-CSF increased the circulating c-Kit+/Flk-1+ cells (9.1-fold; P<0.02), which showed endothelial properties in vitro (acetylated low-density lipoprotein uptake and lectin binding) and incorporated into the regenerated endothelium in vivo. A BM replacement experiment with green fluorescent protein (GFP)–overexpressing cells showed that BM-derived GFP+/CD31+ endothelial cells occupied 39% of the total luminal length in the G-CSF–mediated neo-endothelium (2% in the control).
Conclusion— The G-CSF–induced mobilization of BM-derived c-Kit+/Flk-1+ cells contributes to endothelial regeneration, and this cytokine therapy may be a feasible strategy for the promotion of re-endothelialization after angioplasty.
Subcutaneous injection of G-CSF increases in re-endothelialization of the denuded vessels, followed by inhibition of neointimal formation. The G-CSF–induced endothelium exhibited normal acetylcholine-mediated vasodilatation in NO-dependent manner. Bone marrow (BM) replacement by GFP-overexpressing cells showed that G-CSF–mobilized Lin-/c-Kit+/Flk-1+ cells from BM contributes to endothelial regeneration.
Key Words: restenosis • endothelium • carotid artery • cytokines • vascular biology
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Introduction |
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Endothelial cells (ECs) cover the luminal surface of blood vessels and maintain multiple vascular functions. The disruption of endothelial coverage causes a decrease in the production of vasculoprotective mediators such as NO, leading to elevated vascular tonus, enhanced inflammation, and medial smooth muscle cell proliferation. The resultant neointimal hyperplasia causes restenosis after angioplasty.1
Bone marrow (BM)–derived endothelial progenitor cells (EPCs) was isolated from the mononuclear cell (MNC) population in the peripheral blood (PB).2,3 Transplantation of autologous circulating EPCs (CEPCs) to balloon-denuded arteries was reported to induce rapid re-endothelialization of the injured artery.4,5 Moreover, transfusion of spleen-derived EPCs or EPCs overexpressing endothelial NO synthase reduced neointimal formation after vascular injury.6,7 Delivery of cultured PB-MNCs to balloon-injured arteries accelerated re-endothelialization associated with endothelium-dependent vasoreactivity and reduced neointimal formation.7
Cytokines efficiently mobilize hematopoietic precursor cells from BM.8 Takahashi et al showed that exogenous granulocyte/macrophage colony-stimulating factor (CSF) mobilize CEPCs from BM and thereby contributes to neovascularization of ischemic tissues.9 Recently, granulocyte-CSF (G-CSF) was shown to recruit BM-derived EPCs10 and enhance the BM cell mobilization to brain, leading to angiogenesis and eventually a reduction in the volume of cerebral infarction.11 G-CSF was also reported to increase angiogenesis in the BM of G-CSF–treated patients.12 Treatment with G-CSF plus macrophage CSF accelerates neovascularization in limb ischemia.13 G-CSF enhances endothelialization of small-caliber prosthetic implanted grafts,14,15 and intracoronary infusion of G-CSF–mobilized PB-MNCs improved cardiac regional flow in patients with myocardial infarction.16 Kong et al reported that mobilization of CEPCs by exogenous G-CSF facilitates re-endothelialization and inhibits neointimal development,17 whereas the cell types of BM-derived cells contributing to G-CSF–mediated endothelial regeneration in the vascular repair model has not been defined in a BM transplant experiment, and neither the involvement of G-CSF–mediated outgrowth of resident ECs bordering the injured area nor the functional properties of the regenerated endothelium were studied in G-CSF–treated animals. To further elucidate these undetermined issues, the present study was designed, and it provided the additional novel findings that: (1) BM-derived c-Kit+/Flk-1+ progenitor cells mobilized by G-CSF directly contribute to the endothelial regeneration after EC-denuded injury and can differentiate to EC-like cells in vitro, (2) the contribution of the G-CSF–mediated outgrowth of resident ECs is negligible, and (3) the repaired artery showed NO-mediated arterial relaxation and inhibition of neointimal hyperplasia. These findings suggested that G-CSF therapy can be a feasible therapy to inhibit neointimal hyperplasia after angioplasty.
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Materials and Methods |
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Balloon Injury Model
A 2Fr Fogarty arterial embolectomy catheter (Edwards Lifesciences) was inserted into the right common carotid artery of Lewis rats (LEW/SsN Slc; 10 to 12 weeks of age) and inflated 3 times with 300 µL of air. Human G-CSF (Lenograstim; 10, 30, or 100 µg/kg per day) or vehicle (saline) was subcutaneously injected daily for 14 days from 3 days before injury. The lesion was harvested on day 14. Green fluorescent protein (GFP) transgenic mice were generously donated by Dr Okabe (Osaka University, Japan).18 All experimental procedures complied with the institutional guidelines for animal experiments.
Morphometric Analysis
The injured lesion was fixed with 4% paraformaldehyde, paraffin sectioned, and stained with hematoxylin and eosin or Elastica van Gieson. Three sections from each carotid artery at 300-µm intervals were analyzed with NIH image software. The absolute intimal area or relative ratio of intimal-to-medial area (I/M ratio) was evaluated. Evans blue dye (5%; Sigma) was transfused to rats 10 minutes before euthanasia to identify the remaining denuded area. Furthermore, to analyze the EC-recovered area, samples were incubated with horseradish peroxidase (HRP)–conjugated anti-FactorVIII antibody, followed by visualization with 3, 3'-diaminobenzidine (DAB). Slides were then counterstained with hematoxylin.
Functional Assay of Regenerated Endothelium
NO-mediated vasorelaxation of regenerated endothelium was evaluated as we described previously.5
Transfusion of G-CSF–Induced PB-MNCs
PB-MNCs were isolated by Percoll gradient centrifugation (Lymphoprep; NYCOMED) from 5-day G-CSF–treated donor rats and labeled with DiI19 and transfused to the recipient Lewis rat after arterial injury (1x107 cells). After a 5-day G-CSF treatment in GFP-overexpressing mice, PB-MNCs were prepared and incubated with phycoerythrin (PE)–Cy5-conjugated anti-mouse CD45 or anti-mouse c-Kit antibodies (BD Pharmingen), as well as PE-conjugated anti-mouse Flk-1 (Becton Dickinson). GFP+/c-Kit+/Flk-1+ and GFP+/c-Kit+/Flk-1– cells were sorted and transfused to the recipient nude rats (F344/N rnu/rnu) after arterial injury. On day 14, the lesion was frozen sectioned and incubated with anti-CD31 antibody (Santa Cruz Biotechnology), followed by rhodamine-conjugated secondary antibody (DAKO).
Primary Culture of G-CSF–Induced PB-MNCs
PB-MNCs were prepared from 5-day G-CSF–treated rats and cultured on fibronectin-coated chamber slides (Becton Dickinson) for 7 days with 10% FBS-DMEM (GIBCO). Adherent cells were incubated with 2.4 µL/mL of DiI-labeled acetylated LDL (Molecular Probes) for 120 minutes, fixed with 2% paraformaldehyde, and stained with 10 ng/mL of fluorescein isothiocyanate (FITC)–conjugated Ulex europaeus agglutinin-1 (UEA-1) lectin (Sigma). The double fluorescent cells were counted in 4 randomly selected high-power fields.
Fluorescence-Activated Cell Sorter Analysis of G-CSF–Induced Mice PB-MNCs
PB-MNCs were prepared from 5-day G-CSF–treated C57BL/6 mice, incubated with PE-conjugated antibody against CD3, CD8, B220, CD11b, Ter119 or Gr-1, and FITC-conjugated anti-CD34 antibody as well as biotin-conjugated mouse antibody against Flk-1 or c-Kit, followed by activated protein C–conjugated secondary antibody (all from Pharmingen). Samples were analyzed with fluorescent-activated cell sorter (FACS) caliber using Cell Quest (Becton Dickinson).
BM Transplantation
1x107 BM cells from GFP-overexpressing mice were transplanted to nude rats (F344/N rnu/rnu) after 6 Gray irradiation. At week 4, arterial injury was conducted, and G-CSF (100 µg/kg per day) was injected daily for 14 days from 3 days before arterial injury. The lesions were frozen sectioned and stained with anti-CD31 antibody with rhodamine-conjugated secondary antibody (DAKO).
Immunohistochemistry
The injured lesions were paraffin sectioned at 14 days after balloon injury and immunostained with anti-rat CD45 (BD Pharmingen) and rat cross-reactive anti–interleukin-1ß (IL-1ß) antibodies (sc-7884; Santa Cruz Biotechnology) and HRP-conjugated secondary antibody, followed by visualization with DAB and counterstained with hematoxylin.
Statistical Analysis
Statistical analyses were performed with 1-way ANOVA followed by pairwise contrasts using Dunnett test. Data (means±SE) were considered statistically significant when P was <0.05.
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Results |
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G-CSF Inhibits Neointimal Hyperplasia After Arterial Injury
We determined whether G-CSF treatment inhibits neointimal hyperplasia after EC-denuded balloon injury of carotid artery. Neointimal lesions developed in the vehicle-treated vessels 2 weeks after injury, whereas G-CSF treatment markedly reduced the neointimal formation in a dose-dependent manner (10 to 100 µg/kg per day; Figure 1). Morphometric analysis revealed a remarkable decrease in the neointimal area of high-dose G-CSF (100 µg/kg per day)–treated rats compared with the vehicle-treated group (39±3% decrease; n=10; P<0.05). The I/M ratio in G-CSF–treated rats was less than that in the vehicle-treated group (46.0±7.6% versus 84.3±7.3%; n=10; P<0.05; Figure 1B). Thus, it is unlikely that G-CSF directly affects the outgrowth of resident smooth muscle cells in the injured artery in vivo, although G-CSF was reported to stimulate the growth of the cultured vascular smooth muscle cells.20
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We also studied whether G-CSF aggravated inflammatory cell infiltration and cytokine expression in the injured arteries by examining the infiltration of inflammatory cells (CD45+ cells) and the expression of inflammatory cytokine (IL-1ß) in the injured arteries. Figure I (available online at http://atvb.ahajournals.org) shows that the infiltration of CD45+ cells and the expression of IL-1ß are markedly inhibited in the day-14 neoendothelium of G-CSF–treated rats compared with those in the saline-treated controls, consistent with the previous observation that G-CSF pellet–induced angiogenic activity on the cornea occurred without any sign of inflammatory reactions.21
G-CSF treatment dose dependently (10, 30, and 100 µg/kg per day) elevated the number of white blood cells 14 days after treatment (14 500±2000, 19 660±430, 33 520±2171 cells; n=15 each), which were significantly higher compared with the vehicle-injected control (3640±153; n=15; P<0.01). Any dose of G-CSF (10, 30, or 100 µg/kg per day) did not affect the survival rate and body weight of the administrated rats and did not cause any macroanatomic change. Because 100 µg/kg per day of human G-CSF used here is considered to be similar to the human clinical dose based on the species difference between human and rodents,22 and therefore this dose was used in the following experiment.
G-CSF Promotes Re-Endothelialization
To evaluate re-endothelialization, Evans Blue dye was administered premortem to stain-remaining nonendothelialized areas. Re-endothelialized areas appear white (Figure 2A) and were significantly larger in G-CSF–treated rats than in the vehicle-injected group (63.0±6.8% versus 35.7±6.6%; n=10; P<0.01). Immunostaining revealed that the ratio of FactorVIII+ endothelial layer relative to the total luminal surface was significantly greater in G-CSF–treated rats than in the control (67.4±7.9% versus 40.2±6.9%; n=10; P<0.01; Figure 2B), indicating that G-CSF promoted re-endothelialization, leading to the inhibition of neointimal hyperplasia.
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Functional Analysis of Regenerated Endothelium
NO production in the regenerated endothelium was measured by acetylcholine (Ach)-mediated relaxation of carotid artery preconstricted by norepinephrine. Ach caused a relaxation response in the normal carotid artery (30.9±1.8%, n=6) versus papaverine-induced maximal relaxation in an NO-dependent manner (as shown by NG-nitro-L-arginine methyl ester [L-NAME] inhibition), whereas in the EC-injured artery, this response was markedly abolished (2.8±0.9%, n=6). In contrast, in the G-CSF–treated injured artery, relaxation was restored to a level comparable to that of normal carotid artery (43.8±4.5%; n=6), whereas L-NAME pretreatment completely inhibited such an Ach-mediated response (Figure 3), suggesting that the regenerated endothelium exerts an NO-mediated vasorelaxation response.
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Endothelial Regeneration by G-CSF–Mobilized CEPCs
Because G-CSF was reported to increase CEPCs,10,17 we next examined whether the G-CSF–mobilized CEPCs actually contributed to endothelial regeneration after EC-denuded injury. PB-MNCs (1x107 cells) were prepared from 5-day G-CSF–treated or vehicle-injected donor rats, DiI-labeled, and transfused into the recipient rats after vascular injury. In the day-14 samples, DiI+ cells were incorporated into the neo-endothelium and double immunofluorescence with anti-CD31 antibody disclosed that DiI+ PB-MNCs derived from G-CSF–treated donor rats contributed to neoendothelium formation greater than the vehicle-injected rats (DiI+/CD31+ area 54.3±6.1% versus 6.3±1.2% to total luminal surface length; n=5; P<0.01; Figure 4A).
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PB-MNCs from G-CSF–treated or vehicle-injected rats were primarily cultured for 7 days. EPC-like cells were identified by their binding ability to FITC–UEA1-lectin and uptake of DiI–AcLDL. The ratio of UAE-1+/AcLDL+ cells to total adherent cells from G-CSF–treated rats was 4-fold higher than that from vehicle-injected rats (39.2±9.8% versus 9.5±5.0% to total cultured cells; n=6; P<0.05; Figure 4B).
Endothelial Regeneration by G-CSF–Mobilized c-Kit+/Flk-1+ Cells
We studied the cell type responsible for endothelial regeneration induced by G-CSF. Because the anti-rat Flk-1 antibody for FACS sorting was not available, we analyzed the PB from "mice." Hematopoietic lineage negative (Lin-)/c-Kit+ cells were sorted from the mice PB and further analyzed for the expression of endothelial lineage markers Flk-1 and CD34. G-CSF treatment markedly elevated the ratio of Lin-/c-Kit+ cells to total PB-MNCs (8.1±0.5-fold; n=5; P<0.01), whereas the CD34+/Flk-1+ population included in the Lin-/c-Kit+ cells was increased 9.8±0.8-fold (n=5; P<0.01; Figure 5A).
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To further elucidate the contribution of circulating c-Kit+/Flk-1+ cells to endothelial regeneration, 5x104 c-Kit+/Flk-1+/GFP+ or 1x106 c-Kit+/Flk-1-/GFP+ cells were isolated from enhanced GFP (EGFP) transgenic mice and injected into the nude rats after balloon injury. GFP mRNA transcription in the transgenic mice is driven by the chicken ß-actin promoter and cytomegalovirus enhancer, indicating that the transcript expression in the hematopoietic and EC lineages is strong enough to trace them in the recipient mouse organ. In fact, GFP-positive ECs can be detected with a strong signal in the neo-endothelium. Figure 5B shows that GFP+ cells in the day-14 samples were detected on the regenerated endothelium of c-Kit+/Flk-1+ cell–transfused nude rats, and that these GFP+ cells were positive for the EC marker CD31 (Figure 5B, top, arrowheads), whereas the GFP+ cells were barely detectable in c-Kit+/Flk-1– cell–transfused nude rats (Figure 5B, bottom), suggesting that c-Kit+/Flk-1+ cells contain the cell population that can differentiate into CD31+ EC-like cells in the EC-denuded lesion.
Analysis by BM Replacement Model
To examine whether G-CSF–mobilized EPCs were originated from the BM, BM cells from EGFP transgenic mice were transplanted into the nude rats of which marrow cells had been ablated with whole body irradiation. Six weeks after transplantation, 86±2% of PB-MNCs were replaced (FACS; data not shown). The nude rats that received arterial injury and mobilization of BM-derived GFP+ cells to the neo-endothelium was examined on day 14. GFP+ cells were detected in the endothelial layer, and the immunostaining disclosed that CD31+/GFP+ EC-like cells were detected only in the G-CSF–treated group but not in the vehicle-injected group (39.2±5.8% versus 2.2±1.5% to total luminal surface length; n=5 each; P<0.005; Figure 6), indicating that 37.0% of the total luminal area (39.2% G-CSF group)–(2.2% saline group) was derived from G-CSF–mobilized BM cells (GFP+/CD31+ area). Considering that G-CSF–promoted neo-endothelium was 37.2% of the total luminal area (67.4% G-CSF group)–(40.2% saline group), these findings suggest that BM-derived cells are mainly involved in G-CSF–promoted neoendothelial formation, and the contribution of the G-CSF–mediated outgrowth of resident ECs is negligible.
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Discussion |
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Because the natural regenerative process after EC-denuded injury is slow, it cannot prevent the onset of the neointimal lesions.1 A novel approach that promotes re-endothelialization is required to accelerate this process. Several reports have shown that EPCs can be harvested from PB, and the intravenous injection of EPCs into EC-denuded vessels accelerates the recovery of endothelial integrity, resulting in the inhibition of neointimal hyperplasia and the restoration of vasodilatation activity.4–7 The EPCs were shown to enhance the endothelialization of small-caliber prosthetic grafts.14,15 G-CSF treatment increases the circulating CD34+ cells expressing endothelial markers,10 and administration of G-CSF to patients with coronary artery disease increased the circulating MNCs with EPC properties.23 Furthermore, G-CSF inhibited the neointimal formation in balloon-injured vessels,17 whereas it remains undetermined what cell types of marrow-derived cells are directly involved in G-CSF–mediated endothelial regeneration. Furthermore, neither the functional properties of the regenerated endothelium were studied nor analysis using BM transplant experiments undertaken in G-CSF–treated animals. Our present study performed BM replacement experiments and clearly showed that G-CSF treatment increases the number of BM-derived c-Kit+/Flk-1+ EPCs that actually contribute to re-endothelialization of the balloon-injured arteries, leading to marked inhibition of neointimal formation. Furthermore, we found that the regenerated endothelium exerts Ach-mediated vasodilatory action in an NO-dependent manner. Interestingly, the injured arteries of G-CSF–treated rats have a better vasodilatory capacity compared with normal arteries. The data were analyzed in the 6 different animals with the reproducibility. Although we cannot sufficiently explain the molecular mechanism responsible for the better vasodilatory capacity, the neo-endothelium regenerated by G-CSF–mobilized BM cells or G-CSF–mediated direct action to the endothelial regeneration process might have the enhanced NO-mediated vasodilatory effect. Further studies will be required to clarify the underlying mechanism. Together, this evidence leads us to consider a more aggressive clinical use of G-CSF to mobilize CEPCs and promote vascular repair.
It appears that the safety and feasibility of G-CSF treatment focusing on the induction of vascular occlusion in atherosclerotic lesions has not yet been established.24 In angina patients, the administration of G-CSF was associated with the onset of acute myocardial infarction (AMI).25 A high rate of restenosis after intracoronary infusion of G-CSF–mobilized PB-MNCs was reported in AMI patients.16 There are articles reporting the induction of AMI and cerebral infarction in G-CSF–treated BM transplantation patients.26,27 Differentiation of G-CSF–mobilized progenitor cells into smooth muscle cells within the stented segment as well as the induction of angiogenesis within the atherosclerotic lesion and the aggregation of mobilized inflammatory cells within the plaque may be a plausible explanation.24 Furthermore, G-SCF–mobilized neutrophils may cause EC damage by superoxide production.28–30 We have previously shown that polymorphonuclear cells inhibited the ischemia-induced recovery of blood perfusion in the hindlimb ischemia model.31 Furthermore, G-CSF induces the expression of adhesion molecules on ECs, leading to leukocyte adhesion and its activation32 or to a hypercoagulability state.33 Thus, because G-CSF induces non-EPC populations, including neutrophil or smooth muscle progenitor cells, the enrichment of the EPC population and their application are required to inhibit such harmful effects. Indeed, the injection of G-CSF–induced circulating CD34+EPCs into ischemic limb muscle resulted in satisfactory clinical improvement.34 Transplantation of an enriched CD34+MNC population re-established endothelial integrity in injured vessels, thereby inhibiting neointimal hyperplasia.4 We here described that a small number (5x104) of enriched population c-Kit+/Flk-1+ or CD45-/Flk-1+ EPCs regenerated endothelium much more efficiently than did a large number (1x106) of c-Kit+/Flk-1– or CD45-/Flk-1– cells. Consistent with our observation, in the hindlimb ischemia model of mice, merely 1x103 CD34+/Flk-1+ cells improved limb salvage and hemodynamic recovery better than 1x104 CD34+/Flk-1– cells.35 Thus, the application of an enriched EPC population may be feasible to improve the safety and efficiency of G-CSF therapy.
Increasing evidence suggests that BM-derived EPCs home to the ischemic region for the formation of new blood vessels.2 EPCs were reportedly derived from more differentiated CD34+ or immature CD133+ hematopoietic stem cells, as well as from mature PB-MNCs or CD14+ monocytes.5,36 They express endothelial markers, including Flk-1, FactorVIII, and endothelial NO synthase.2,36 Intravenous infusion of BM-derived EPCs enhanced neovascularization in vivo.37 Application of either BM-derived or PB-derived EPCs into the infarct artery beneficially affects postinfarction remodeling.38,39 Consistent with these previous reports, we presented the data indicating that the c-Kit+/Flk-1+ cells were actually mobilized by G-CSF administration (Figure 5A), and the infusion experiment of the sorted c-Kit+/Flk-1+ EGFP cells (mobilized by G-CSF) revealed their direct involvement in the G-CSF–promoted endothelial regeneration process in the vascular repair. Thus, the stem cells expressing the Flk-1 marker are mobilized in response to G-CSF and then exert the property as an endothelial progenitor, suggesting that c-Kit+/Flk-1+ cells are the cell type responsible for G-CSF–mediated endothelial regeneration.
In conclusion, we characterized the cell type responsible for G-CSF–mediated endothelial regeneration leading to an inhibition of neointimal hyperplasia and showed that the vascular repair was mainly attributable to the G-CSF–mobilized BM-derived cells rather than G-CSF–mediated outgrowth of resident ECs, and that the repaired artery responded well to NO-mediated vasodilatory stimulus. These findings suggest that the treatment with G-CSF might be a feasible and suitable supplement therapy for the prevention of restenosis after the revascularization procedures. Although the very recent clinical study has reported that administration of G-CSF to patients with AMI improves cardiac function without any adverse events during 6-month observation,40 G-CSF–mediated proatherogenic effects, such as the induction of angiogenesis within the atherosclerotic lesion and the aggregation of mobilized inflammatory cells within the atheromatous plaque, are the limitation of the present study and remain to be determined. Further basic and clinical studies focusing on these issues will be required.
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Acknowledgments |
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This work was supported in part by a grant from the Ministry of Education, Culture, Science and Technology of Japan. We appreciate Dr Takeshi Todo for his great help in BM replacement experiment.
Received June 6, 2005; accepted December 15, 2005.
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References |
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