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(Circulation Research. 2003;93:980.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Bone Marrow Monocyte Lineage Cells Adhere on Injured Endothelium in a Monocyte Chemoattractant Protein-1–Dependent Manner and Accelerate Reendothelialization as Endothelial Progenitor Cells

Soichiro Fujiyama, Katsuya Amano, Kazutaka Uehira, Masayuki Yoshida, Yasunobu Nishiwaki, Yoshihisa Nozawa, Denan Jin, Shinji Takai, Mizuo Miyazaki, Kensuke Egashira, Takayuki Imada, Toshiji Iwasaka, Hiroaki Matsubara

From the Department of Medicine II (S.F., K.A., T. Imada, T. Iwasaka) and Medicine I (K.U.), Kansai Medical University; Department of Medical Biochemistry (M.Y., Y. Nishiwaki), Tokyo Medical and Dental University; Pharmacobioregulation Research Laboratory (Y. Nozawa), Taiho Pharmaceutical Co Ltd; Department of Pharmacology (D.J., M.M.), Osaka Medical College; Department of Cardiovascular Medicine (K.E.), Kyushu University of Medicine; and Department of Cardiovascular Medicine (H.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan.

Correspondence to Hiroaki Matsubara, MD, Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan. E-mail matsubah{at}koto.kpu-m.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peripheral blood (PB)-derived CD14⁺ monocytes were shown to transdifferentiate into endothelial cell (EC) lineage cells and contribute to neovascularization. We investigated whether bone marrow (BM)- or PB-derived CD34^-/CD14⁺ cells are involved in reendothelialization after carotid balloon injury. Although neither hematopoietic nor mesenchymal stem cells were included in human BM-derived CD34^-/CD14⁺ monocyte lineage cells (BM-MLCs), they expressed EC-specific markers (Tie2, CD31, VE-cadherin, and endoglin) to an extent identical to mature ECs. When BM-MLCs were cultured with vascular endothelial growth factors, hematopoietic markers were drastically decreased and new EC-specific markers (Flk and CD34) were induced. BM-MLCs were intra-arterially transplanted into balloon-injured arteries of athymic nude rats. When BM-MLCs were activated by monocyte chemoattractant protein-1 (MCP-1) in vivo or in vitro, they adhered onto injured endothelium, differentiated into EC-like cells by losing hematopoietic markers, and inhibited neointimal hyperplasia. Ability to prevent neointimal hyperplasia was more efficient than that of BM-derived CD34⁺ cells. MCP-dependent adhesion was not observed in PB-derived CD34^-/CD14⁺ monocytes. Regenerated endothelium exhibited a cobblestone appearance, blocked extravasation of dye, and induced NO-dependent vasorelaxation. Basal adhesive activities on HUVECs under laminar flow and ß₁-integrin expression (basal and active forms) were significantly increased in BM-MLCs compared with PB-derived monocytes. MCP-1 markedly enhanced adhesive activity of BM-MLCs (2.8-fold) on HUVECs by activating ß₁-integrin conformation. Thus, BM-MLCs can function as EC progenitors that are more potent than CD34⁺ cells and acquire the ability to adhere on injured endothelium in a MCP-1–dependent manner, leading to reendothelialization associated with inhibition of intimal hyperplasia. This will open a novel window to MCP-1–mediated biological actions and vascular regeneration strategies by cell therapy.

Key Words: endothelium • angioplasty • endothelial progenitor cells • bone marrow • reendothelialization

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intimal thickening and smooth muscle cell (SMC) proliferation attributable to balloon catheter denudation of endothelial cells (ECs) remain major problems after vascular manipulations.^1,2 Effective regeneration of ECs via administration of vascular endothelial growth factor (VEGF) is one of the most potent inhibitors of SMC proliferation.^3–5 VEGF-mediated reendothelialization may be partly attributable to its ability to mobilize endothelial progenitor cells (EPCs)³ or augment NO release from the endothelium.⁶

EC migration and sprouting from locally residing endothelium and recruitment of circulating EPCs play an important role in reendothelialization in vascular repair. EPCs have been identified among human leukocytes enriched for CD34⁺ cells.^7–10 Although earlier studies reported that adult blood-derived CD34⁺VEGFR-2⁺ angioblasts differentiate into ECs,^7–10 recent accumulating findings have suggested that peripheral blood (PB)-derived mature CD14⁺ monocytes can also transdifferentiate into EC lineage cells under angiogenic conditions^11–14 and play a role in neovascularization via leukocyte-leukocyte interaction with CD34⁺ cells.¹³

When monocytes were induced to infiltrate the heart by overexpression of monocyte chemoattractant protein-1 (MCP-1), the invading monocytes seemed to form erythrocyte-containing vascular-like tunnels.¹⁵ Considering that only 0.05% of PB leukocytes express CD34 and yet as many as 10% to 20% of ECs are blood-derived cells in the neovasculature in ischemic tisssues,¹⁶ such monocyte-derived EC lineage cells may also partly act as angioblasts to be incorporated into neovasculature. Although we^17,18 have demonstrated that bone marrow (BM)-derived mononuclear cells differentiate into neocapillaries in ischemic limbs or myocardium, the involvement of BM monocyte–derived EC lineage cells remains undefined. Considering that the administration of granulocyte-macrophage colony–stimulating factor mobilized angioblasts from BM¹⁹ and that statin treatment^20,21 accelerated reendothelialization after vascular injury by mobilizing BM-derived EC lineage cells, transdifferentiation of BM monocyte lineage cells (BM-MLCs) to EC-like cells may contribute to reendothelialization. We report a novel finding that MLCs in BM cells can adhere on the injured endothelium by a MCP-1–dependent mechanism to cause reendothelialization as EC progenitors more potent than BM-derived CD34⁺ cells, leading to inhibition of intimal hyperplasia.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
MCP-1 Gene Transfection and Arterial Injury
Bupivacaine was injected into two sites of thigh muscle (750 µg per site) of immunodeficient nude rats (F344/N rnu/rnu) to induce a higher transfection efficiency of MCP-1 plasmid. Human MCP-1 plasmid DNA (300 µg) encapsulated in hemagglutinating virus of Japan liposome was injected into the same sites (2 sites in each 150 µL) 3 days after bupivacaine.²² Three days after MCP-1 gene transfer, deendothelialization injury of left common carotid artery was produced by ballooning (3 times) with 2F Fogarty catheter. Immediately after surgery, we administered a cell solution (1 mL including 10⁷ of cells) from the left common carotid artery (BM-MLCs, BM-CD34⁺ cells, PB-derived monocytes, saline infusion control; n=12 each). Rats were killed 2 weeks after injury.

Isolation of BM-MLCs
Human BM cells were obtained with informed consent when patients with peripheral artery diseases received angiogenic cell therapy,²³ and BM mononuclear cells (BM-MNCs) were isolated by Percoll gradient centrifugation (Lymphoprep, NYCOMED).¹⁷ CD34^- fraction in BM-MNCs was negatively selected three times by CD34⁺ antibody-coated magnetic beads (Miltenyi Biotec). CD34⁺ cells were separated using CD34 magnetic beads. The purity of enriched CD34⁺ cells was 88±2.7% (n=5) by FACS analysis. CD14⁺ cells in CD34^- fraction were positively selected twice by CD14⁺ antibody-coated magnetic beads (Miltenyi Biotec). The purity of CD14⁺ cells was >99% by flow cytometry (n=10), and CD34⁺ cells were <1%. The CD34^-/CD14⁺ cells were labeled with green-fluorescence cell linker (PKH2-GL, Sigma).¹⁷

Cell Culture and Flow Cytometry
For FACS analysis, CD14-positive cells (2x10⁶ cells/dish) were plated on fibronectin-coated dishes (6-cm diameter), incubated with EGM-2 medium supplemented with VEGF (20 ng/mL), and detached with 1 mmol/L EDTA 14 days after plating. The suspended cells were treated with human IgG polyclonal antibody, washed with PBS, and then incubated with the following monoclonal antibodies: IgG1-FITC and IgG1-PE (Becton Dickinson), CD11b and CD68 (CALTAG Laboratories), CD19, CD15, CD14, CD31, and CD34 (PharMingen), CD45 (Immunotech), VE-cadherin (Chemicon), FLK-1 (Sigma), endoglin (Ancell), and polyclonal anti–Flt-1 and Tie2 antibodies (Santa Cruz). Anti-rabbit polyclonal IgG-FITC, anti-mouse monoclonal IgG-PE (DAKO), and anti-biotin PE (Miltenyi Biotec) were used as secondary antibodies. For immunohistochemical analysis, samples were snap frozen, cut with a cryostat, and incubated with anti–von Willebrand factor (anti-vWF) (DAKO), anti–endothelial NO synthase (anti-eNOS) (Calbiochem), anti–VE-cadherin (Santa Cruz), anti-CD45 (PharMingen), or anti-CD11b (PharMingen) antibody.¹⁶

Histological Analysis
To measure the reendothelialized area, animals were perfused in vivo with Evans Blue dye (Sigma) at time points immediately before death and the remaining denuded area was planimetrically determined. For the intima to media (I/M) ratio, serial cross sections of paraffin-embedded specimens were stained with elastic trichrome stain.

Adhesion Assay Under Laminar Flow Conditions
Adhesion assay under laminar flow was previously described.^24,25 HUVECs transfected with recombinant type 5 adenovirus E-selectin were positioned in the flow chamber (shear stresses of 1 dyne/cm²). The entire period was recorded on videotape, and captured images were transferred to a PC computer (10 randomly selected x20 microscopic fields for each experiment). Cells were considered to be adherent after 10 seconds of stable contact with the monolayer.

Acetylcholine-Induced Vasodilation
Resting tension of isolated cervical aorta was adjusted to 0.25 g optimal for maximal contraction as previously described.²⁶ Contractile response to 50 mmol/L KCl was confirmed and preincubated for equilibration with or without L-NAME (10 µmol/L) for 30 minutes. Constrictive response of aorta was induced by norepinephrine (30 nmol/L), and at the maximal constriction level, acetylcholine (10 µmol/L) was added to induce relaxation. After acetylcholine-mediated relaxation, papaverine (100 µmol/L) was added to induce maximal relaxation. Acetylcholine-mediated relaxation was assessed by percent relaxation relative to papaverine-mediated relaxation (100%).

Reverse Transcription–Polymerase Chain Reaction
Total RNA was prepared using the RNeasy kit (Quiagen). Polymerase chain reaction (PCR) protocol and primer design were performed on basis of previous studies.^11,12 PCR products for vWF, VE-cadherin, eNOS, and GAPDH were 434, 226, 836, and 301 bp, respectively.

Statistical Analysis
Statistical analyses were performed with one-way ANOVA followed by pairwise contrasts using the Dunnett’s test. Data (mean±SE) were considered significant when P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial Marker Expression in BM-MLCs
We characterized the cell population in CD34^-/CD14⁺ BM-MLCs by analyzing the expressions of CD3 (T cell), CD19 (B cell), CD15 (granulocytes), and CD68 (macrophage). FACS analysis (Figure 1) indicated that the expressions of these cell markers are <2.4%, whereas CD11b-positive (monocytes) or CD45-positive (monocytes/lymphocytes) cells were >99%. Endothelial-lineage precursor cells derived from hematopoietic stem cells are known to be AC133⁺/FLK⁺/CD34⁺ cells.⁹ Inclusion of such cell population was <1% (Figure 1). Additional analysis indicated that BM-MLCs abundantly expressed various EC markers (Tie2, 99%; VE-cadherin, 45%; endoglin, 36%) (Figure 2). Mesenchymal stem cells were defined to be endoglin positive and CD45 negative.²⁷ Because CD34^-/CD14⁺ BM-MLCs are CD45 positive (99.9%), it is unlikely that such mesenchymal stem cells are included in this study population. PB-derived CD34^-/CD14⁺ cells did not express such EC markers.

View larger version (56K): [in this window] [in a new window]   Figure 1. FACS analysis of fresh BM-MLCs. CD34-/CD14+ BM-MLCs were sorted from BM mononuclear cells and analyzed by FACS. Cells were incubated with direct-labeled monoclonal antibodies (CD14-FITC, CD3-FITC, CD19-FITC, CD15-FITC, CD68-FITC, and CD11b-FITC) or the corresponding isotype control. The quadrants were set based on IgG-FITC or IgG-PE/SSC profile. Bottom, Histogram representing cell numbers (y-axis) versus fluorescence intensity (x-axis). Similar results were obtained in 4 separate experiments. Numbers shown are the mean percent of cells for 4 separate experiments determined by comparison with the corresponding negative labeling.

View larger version (43K): [in this window] [in a new window]   Figure 2. FACS and RT-PCR analyses of BM-MLCs after 14 days in culture with VEGF. Each panel is a histogram representing cell numbers (y-axis) versus fluorescence intensity (x-axis). BM-MLCs or PB monocytes (2x106cells/dish) were plated on fibronectin-coated dishes and incubated with EGM-2 medium supplemented with VEGF (20 ng/mL). The medium was exchanged every 2 days, and the cells were detached with 1 mmol/L EDTA 14 days after plating. Similar results were obtained in 4 separate experiments, and numbers shown are same as Figure 1. , , and indicate the decrease, increase, and unchanged levels in the expression of surface makers, respectively. RT-PCR analysis indicates the mRNA levels for vWF, VE-cadherin, and eNOS in PB monocytes incubated with EGM-2 medium supplemented with VEGF (20 ng/mL). The expression of their mRNAs was observed 14 days after incubation but not in fresh day-0 cells. HUVEC indicates positive control.

When BM-MLCs were cultured for 14 days with VEGF, hematopoietic markers (CD14, CD45, and CD11b) were drastically decreased (>98% to 2.7% to 16.8%), whereas the expressions of VE-cadherin (45.3% to 88.5%), Flk (0.88% to 24.4%), endoglin (36% to 99.8%), and CD34 (0.94% to 19.3%) were increased (Figure 2A). When PB-derived CD34^-/CD14⁺ monocytes were cultured with VEGF, marked decreases in hematopoietic markers and increase in one of EC markers (Tie2, from 11.3% to 54.7%) were observed (Figure 2B). Because other EC markers were not detected by FACS, we examined their expressions by reverse transcription (RT)-PCR. Although mRNA expressions for vWF, VE-cadherin, and eNOS were not observed in fresh day-0 PB monocytes, 28-day culture of cells with VEGF induced their mRNA expressions (Figure 2C). Thus, the present study demonstrates that characters of CD34^-/CD14⁺ BM-MLCs are obviously different from those of PB monocytes, suggesting that our study population is mostly derived from BM-MLCs and that the influence of contaminated PB monocytes is negligible.

Time-Dependent Detachment of Balloon-Injured Endothelium
We examined time-dependent detachment of endothelium after balloon-mediated endothelial injury (Figure 3). The endothelial layer was preserved 1 day after balloon injury, as indicated by vWF expression. Detachment of endothelium was observed from day 2, and apparent intimal hyperplasia was seen on day 14 (n=12, each time point). Extravasation of Evans Blue was observed in aortic samples obtained immediately after ballooning. These findings suggest that the endothelium already lost endothelial function immediately after balloon injury, although the injured endothelial cells seemed to be present on the inner wall 1 day after balloon injury. Therefore, BM-MLCs were injected immediately after balloon injury to examine their attachment on endothelium, and reendothelialization was studied on day 14.

View larger version (62K): [in this window] [in a new window]   Figure 3. Endothelial detachment after balloon-mediated angioplasty. Time-dependent detachment of the endothelial layer after balloon-mediated injury was shown by H&E staining and immunofluorescence analysis. Endothelial cells were immunostained with anti–vWF antibody followed by TRITC-conjugated secondary antisera (red inner layer indicated by arrowheads). Endothelial detachment (shown by arrows) was observed at day 2, and apparent intimal hyperplasia was seen at day 14.

MCP-1–Dependent Adhesion of BM-MLCs and Reendothelialization
Planimetric analysis of rat carotid artery specimens indicated that total areas of initial balloon injury were similar between all experimental groups (no cell infusion: control, 17±1.4; BM-MLCs, 18±1.5; PB monocytes, 17±1.4 mm²; n=12 each). Intra-arterial transfusion of BM-MLCs prelabeled with green fluorescence PKH2 showed no attachment of BM-MLCs on the injured endothelium. We hypothesized that MCP-1–activated BM-MLCs may acquire the ability to adhere on the injured endothelium by activation of ß₁-integrin.²⁸

We have reported that injection of naked MCP-1 plasmid into skeletal muscle causes a sustained increase (3 fold) in the circulating MCP-1 levels over 14 days, with a peak increase at day 4.²² Animals were pretreated with an intramuscular injection of the MCP-1 gene, and then balloon injury and intravenous infusion of BM-MLCs were performed. BM-MLCs were transfused immediately after balloon injury, because reendothelialization efficiency was much lower when cells were infused 2 days after injury (61±4% versus 92±2.7%, P<0.0001; infusion immediately after injury; n=10 each), suggesting that the preservation of endothelial layer plays a key role for the infused BM-MLCs to attach on the inner layer.

MCP-1 gene treatment alone (no cell infusion) did not significantly affect vascular remodeling (reendothelialized area or I/M ratio) compared with the control (no cell infusion, without MCP-1) (Figure 4A). Transfusion of BM-derived CD34⁺ cells (10⁷ cells same as BM-MLCs number) after balloon injury of nude rats caused moderate reendothelialization (64±4%), the extent of which was relatively weaker (P<0.001) than that of BM-MLCs (92±2.7%), suggesting a more efficient function of BM-MLCs as an endothelial progenitor compared with CD34⁺ fraction (Figure 4A). Reendothelialization by transplantation of CD34⁺ cells was observed only in MCP-1–activated cells but not in MCP-1–untreated cells.

View larger version (50K): [in this window] [in a new window]   Figure 4. MCP-1–mediated adhesion of BM-MLCs and inhibition of intimal hyperplasia. A, Quantification of reendothelialized area and I/M ratio in the aortic ring expressed as mean±SE. MCP-1 plasmid was injected into the thigh muscle of rats 3 days before balloon-mediated injury. CD34-/CD14+ BM-MLCs, PB-derived CD34-/CD14+ monocytes, or BM-derived CD34+ cells (107 cells, each) were intra-arterially transfused immediately after balloon-mediated injury. Aortic samples were examined 14 days after transfusion of cells. Reendothelialized area was evaluated by planimetric analysis (extravasation of Evans blue), and I/M ratio was determined by specimens stained by elastic trichrome stain. *P<0.001 vs no cell infusion (n=12 in each group). B, CD34-/CD14+ BM-MLCs or PB-derived CD34-/CD14+ monocytes (107 cells) were prelabeled with green fluorescence PKH2 and intra-arterially transfused immediately after injury. BM-MLCs attaching on the endothelial layer (green fluorescent indicated by white arrows) expressed endothelial marker vWF (red fluorescence indicated by white arrows) and inhibited neointimal hyperplasia 14 days after transplantation. In contrast, transfusion of PB monocyte had no effect on cell attachment or neointimal hyperplasia

Interestingly, the green-labeled cells adhered onto the endothelial layer (Figure 4B), where reendothelialization with vWF-expressing EC-like cells was observed and intimal hyperplasia was markedly inhibited; the reendothelialized area and I/M ratio were 93±2.7% and 1.1±0.2%, respectively (n=12, Figure 4A). In contrast, infusion of PB-derived monocytes failed to cause attachment of labeled cells (Figure 4B) and showed neither reendothelialization (23±2.4%) nor inhibition of intimal hyperplasia (2.9±0.7%, n=12).

Monocytes as well as endothelium were reported to express MCP-1 receptor CCR2.²⁸ We next tested whether MCP-1–activated BM-MLCs or MCP-1–activated endothelium plays a more dominant role in the attachment of BM-MLCs. BM-MLCs were exposed to MCP-1 in vitro and then transfused. As shown in Figure 5A, green-labeled BM-MLCs activated by MCP-1 in vitro firmly attached onto the injured endothelium, where EC-like cells expressing eNOS (Figure 5A) as well as CD31, vWF, or VE-cadherin (not shown) accompanied the inhibition of intimal hyperplasia.

View larger version (27K): [in this window] [in a new window]   Figure 5. Adhesion and transformation of MCP-1–activated BM-MLCs to EC-like cells. BM-MLCs were incubated with serum-free medium containing MCP-1 (50 ng/mL) for 1 hour, green labeled by PKH2, and intra-arterially transfused immediately after balloon-mediated injury. A, Confocal analysis (merge with PKH2+vWF) indicated that BM-MLCs attached on the endothelial layer (green-labeled inner wall indicated by arrows) and express eNOS (red-stained inner wall indicated by arrows). B, Immunofluorescent analysis using anti-CD31 (endothelial cell marker), anti-CD11b (monocyte marker), and anti-CD45 (monocyte/lymphocyte marker) antibodies. Most of the PKH2 green-labeled BM-MLCs express CD31, whereas the numbers of BM-MLCs expressing hematopoietic markers CD11b and CD45 are very few (red-stained cells indicated by arrows).

As shown in Figure 5B, EC-like cells regenerated by MCP-1–activated BM-MLCs were double-positive for PKH2 (green) and EC marker CD31 (red), whereas these transfused BM-MLCs lost the hematopoietic markers for monocytes (CD11b) or monocytes/lymphocytes (CD45), and only a few cells expressed such hematopoietic markers (indicated by arrows in Figure 5B).

Electromicroscopic and Functional Analysis
Electromicroscopic analysis revealed characteristic features of regenerated endothelium derived from BM-MLCs. The surface of the normal endothelium was smooth and covered with a monolayer coat, whereas the regenerated endothelium appeared rougher and showed a cobblestone-like appearance (Figure 6A). We next examined the function of regenerated endothelium by testing extravasation of Evans Blue. Evans Blue leaked into the medial layer in the balloon-injured endothelium, whereas there was no blue staining in the opposite normal artery with the intact endothelium (Figure 6B, top). In contrast, much less blue staining was observed in the endothelium of the balloon-injured artery treated by transfusion of BM-MLCs (Figure 6B, bottom). Quantitative analysis of BM-MLC–derived reendothelialization is shown in Figure 4A.

View larger version (88K): [in this window] [in a new window]   Figure 6. Electron-microscopic appearance and functional analysis of regenerated endothelium. A, Electron microscopy showing a cobblestone appearance of the regenerated endothelium by transfusion of BM-MLCs in contrast with normal endothelium covered with a monolayer coat. B, Inhibition of extravasation of Evans blue by the regenerated endothelium. LCCA indicates left common carotid artery; RCCA, right common carotid artery. C, Acetylcholine (10 µmol/L)-mediated relaxation of the carotid artery constricted by noradrenaline (30 nmol/L) with or without L-NAME treatment. D, Immunohistological analysis of aortic samples (4 and 14 days after balloon injury) using anti-VEGF, anti-VEGF receptor-2, and anti-bFGF antibodies (brown-stained neointima between arrow and arrowheads indicates immunopositive lesions). Intimal lesions abundantly express VEGF, VEGFR-2, and bFGF.

We additionally studied NO production from regenerated endothelium by testing acetylcholine-mediated relaxation of the carotid artery constricted by norepinephrine. Acetylcholine induced a relaxation response (40±1.9% versus papaverine-induced maximal relaxation, n=6) in the balloon-injured artery treated by transfusion of BM-MLCs, whereas L-NAME pretreatment abolished such an acetylcholine-mediated response (Figure 6C, top). This ratio of endothelial NO-dependent relaxation was comparable with that of the normal carotid artery (38±1.4%, n=6, Figure 6C, bottom).

We show in Figure 2 that hematopoietic markers on BM-MLCs were markedly decreased and in contrast EC markers were increased when BM-MLCs were cultured with VEGF. Therefore, we examined whether such VEGF system is locally activated in balloon-injured arteries. The expressions of VEGF, VEGF receptor-2, and bFGF were apparently detected in intimal lesions at day 4 after balloon injury (n=8, Figure 6D), whereas their immunostaining was not consistently observed in the earlier phase after injury (days 1 through 3, n=8, data not shown), suggesting that balloon-mediated denudation locally causes angiogenic conditions that enable EC differentiation of BM-MLCs.

MCP-1–Dependent Adhesion of BM-MLCs
We have shown in Figures 3 through 5 that BM-MLCs transfused immediately after balloon injury adhere on the endothelium in a MCP-1–dependent manner. We next investigated whether the adhesive activity of BM-MLCs on the endothelium is actually modulated by MCP-1 under flow conditions. Cellular rolling and adhesion was quantified using HUVEC monolayers perfused under laminar flow.^24,25 As shown in Figures 7A and 7B, basal rolling and adhesion activities of BM-MLCs were much higher (4.3-fold and 2.9-fold, respectively) compared with those of PB-derived CD34^-/CD14⁺ monocytes. Interestingly, MCP-1 treatment markedly stimulated the rolling and adhesion activities of BM-MLCs (2.7-fold and 2.2-fold, respectively) relative to their basal levels, whereas MCP-1–mediated effects on PB-derived CD34^-/CD14⁺ monocytes were much weaker.

View larger version (58K): [in this window] [in a new window]   Figure 7. MCP-1–dependent adhesion of BM-MLCs and increased expressions of ß1-integrin. A, Basal rolling and adhesive activities of BM-MLCs (n=10) or PB-derived monocytes (n=8) were quantified on adeno–E-selectin transduced HUVEC monolayer under laminar flow. Adhesive and rolling cells are indicated by yellow and red arrows, respectively. Cells were considered to be adherent after 10 seconds of stable contact with the monolayer. B, Activation by MCP-1 was performed by incubating BM-MLCs or PB monocytes with MCP-1 (50 ng/mL) for 1 hour with (n=8 each) or without (n=7 each) pretreatment by anti–ß1-integrin antibody. *P<0.01, **P<0.001 vs MCP-1–untreated controls; P<0.001 vs BM-MLCs pretreated with MCP-1 but not anti–ß1-integrin antibody. C, Cellular extracts from BM-MLCs were immunostained with antibodies specific for integrin ß1 chain (PharMingen), active conformational form of ß1-integrin (Chemicon), or CCR2 (Santa Cruz). Western blotting shows that the expressions of both basal and active forms of ß1-integrin are higher (3.4±0.3-fold and 1.8±0.2-fold, P<0.001, respectively) in BM-MLCs (n=6) relative to those in PB monocytes (n=6). MCP-1 treatment significantly (P<0.001) upregulated their expression levels in BM-MLCs (4.8±0.3-fold and 3.4±0.2-fold, respectively, P<0.001, n=8 each) but not in PB monocytes (1.3±0.2-fold and 1.2±0.2-fold, respectively, P<0.001, n=8 each). Signal densities determined by densitometry were arbitrarily calculated as values relative to the untreated PB monocytes (value=1), and the mean value is described in the Figure. D, Each panel is a histogram representing cell numbers (y-axis) versus fluorescence intensity (x-axis). Surface expression of basal or active forms of ß1-integrin was analyzed by FACS. Similar results were obtained in 4 separate experiments. Numbers shown are the mean percent of cells for 4 separate experiments determined by comparison with the corresponding negative labeling.

Expression level of ß₁-integrin and MCP-1–mediated activation of ß₁-integrin conformation have important implications in transendothelial chemotaxis of monocytes.²⁸ Neutralizing anti–ß₁-integrin antibody significantly (P<0.001, n=8) reduced the MCP-1 effects on rolling and adhesion of BM-MLCs (Figure 7B, right). Western blotting shows that both basal expression of ß₁-integrin and its active conformational change are markedly higher (3.4±0.3-fold and 1.8±0.2-fold, respectively, P<0.001) in BM-MLCs (n=6) relative to those in PB monocytes (n=6). MCP-1 treatment significantly (P<0.001) upregulated their expression levels in BM-MLCs (4.8±0.3-fold and 3.4±0.2-fold versus basal levels, respectively, P<0.001, n=8 each) but not in PB monocytes (1.3±0.2-fold and 1.2±0.2-fold, P<0.001, respectively, n=8 each). In contrast, the expression of CCR2 (MCP-1 receptor) did not differ between BM-MLCs and PB-derived monocytes (Figure 7C).

MCP-1–mediated changes in ß₁-integrin expressions were additionally supported by FACS analysis (Figure 7D), which disclosed the higher expression of ß₁-integrin in BM-MLCs and the more enhanced change to its active conformational form by MCP-1 compared with those in PB monocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Restenosis has remained a major clinical problem after coronary and peripheral artery angioplasty.²⁹ The present study demonstrates a novel strategy for vascular reendothelialization using BM-MLCs as EC progenitors and an unexpected biological action of MCP-1; BM-MLCs firmly adhered onto the injured endothelium via MCP-1–dependent ß₁-integrin activation, where they transdifferentiated into functional EC-like cells and markedly inhibited intimal hyperplasia. Attached BM-MLC–derived EC-like cells have the ability to cause acetylcholine-mediated vasorelaxation in a NO-dependent manner, indicating that they have functions and characteristics similar to native ECs. Recent studies reported that PB-derived monocytes can transform into EC-like cells in the presence of VEGF.^11–14 Although the present study also showed that PB-derived monocytes have the ability to transdifferentiate into EC-like cells, the potency was much lower than BM-MLCs, and they could not adhere onto the injured endothelium even after MCP-1 stimulation.

MCP-1 binding to its receptor CCR2 induces a conformational change in ß₁-integrin via G_i-mediated signaling, leading to firm adhesion on the injured endothelium.^24,28 Earlier studies have demonstrated that MCP-1 is involved in angiogenesis by promoting the migration of ECs³⁰ and that MCP-1 enhances capillary sprouting in ischemic limbs.³¹ In the heart overexpressing MCP-1, the invading monocytes formed erythrocyte-containing vascular-like tunnels.¹⁵ In the present study, we found that BM-MLCs require to be activated by MCP-1 in a ß₁-integrin–dependent mechanism to cause a firm adhesion onto the endothelium under laminar flow. Walter et al²⁰ reported that statin therapy accelerates reendothelialization by increasing ß₁-integrin expression on EPCs. Together, these findings suggest that the expression level of ß₁-integrin and its MCP-1–mediated activation play a crucial role for the firm adhesion of BM-MLCs on injured endothelium under flow.

Elevated levels of MCP-1 were found in patients with restenosis after coronary angioplasty³² in atherosclerotic lesions or areas of endothelial denudation.^33,34 It was reported that monocytes/macrophages migrate into the intima and media of carotid artery after balloon-mediated injury of rats and anti–MCP-1 therapy significantly prevents intimal hyperplasia.³⁵ Thus, migration of MCP-1–activated PB monocytes into the vessel wall is a key step in the progression of intimal hyperplasia. In this study, we focused on MCP-1–mediated action on BM-MLCs and found that this cell population has an EC-committed property. MCP-1 stimulated their adhesive activity on endothelial layer, where they preferentially transdifferentiated into endothelial-like cell rather than migration into medial layer. The MCP-1–mediated adhesive ability of BM-MLCs on the endothelium was apparently higher than that of PB monocytes. Thus, our present study demonstrates the opposite action of MCP-1 on BM-MLCs and PB monocytes; MCP-1 enhances reendothelialization by BM-MLCs associated with decreased intimal hyperplasia, whereas MCP-1 action on PB monocytes accelerates vascular remodeling. Transplantation of BM-MLCs may cause reendothelialization in vascular repair after angioplasty in situations such as atherosclerotic lesions, where systemic and local MCP-1 levels are elevated.

Blood-derived cells represented 10% of ECs in the neovasculature in hindlimb ischemia, and EC progenitors have been identified among leukocytes enriched for CD34-expressing cells.^7–10 Only 0.05% of PB leukocytes express CD34, and yet as many as 10% of ECs in the neovasculature are blood-derived cells.^8,13,15 We questioned whether such a small population of EC progenitors could have such a profound effect on neovascularization. Recent in vitro and in vivo studies reported that PB monocytes can function as EC-like cells and play a major role in new vessel formation in ischemic limbs.^11–14 In fact, we found in this study that BM-MLCs more efficiently regenerate the endothelium of injured artery compared with BM-derived CD34⁺ cells, suggesting a more efficient function of BM-MLCs as an endothelial progenitor than CD34⁺ fraction. It is possible that both EC progenitors and BM-MLCs mobilized from bone marrow in response to tissue ischemia are collaboratively functioning in neovascularization, to which the contribution of BM-MLCs may play a more important role.

BM-derived cells were demonstrated to differentiate into both EC-like cells and intimal smooth muscle–like cells in aortic or cardiac transplant arteriopathy.^36,37 In these allotransplant models, intimal hyperplastic lesions composed of smooth muscle–like cells and EC-like cells are barely detectable on the endothelial layer. Noishiki et al³⁸ established that pretreatment of vascular grafts with BM cells completely inhibited thrombus formation by reendothelialization of the inner lumen. Figure 5 showed that no transfused labeled BM-MLCs migrated into medial layer of injured artery, whereas the endothelial layer was covered with BM-MLC–derived EC-like cells. These findings suggest that if BM-MLCs preferentially transdifferentiate to endothelial-like cells on injured endothelium, infiltration of BM-derived smooth muscle–like cells into intimal lesions may be blocked and thus restenosis can be prevented. Alternatively, precursor cells for smooth muscle–like cells may not be included in the BM-MLCs.

Our present study demonstrated that transfusion of MCP-1–activated BM-MLCs markedly prevented intimal formation by transdifferentiation into functional EC-like cells on the injured endothelium, resulting in the inhibition of growth of smooth muscle–like cells. Werner et al³⁹ recently reported that intravenous transfusion of spleen-derived EPC enhances reendothelialization after vascular injury when spleen is removed. MCP-1–activated BM-MLCs or spleen-derived EPCs would be available for a cell-based reendothelialization therapy. The present study will open a novel window to not only MCP-1–mediated biological actions but also more effective cell therapy strategies for vascular regeneration, because a much larger population of BM-MLCs (10%) is present in BM cells compared with CD34⁺AC133⁺FLK-1⁺ EC progenitors (0.01%).

	Acknowledgments

 
Acknowledgments

This study was supported in part by research grants from the Ministry of Education, Science, Sports and Culture, Japan, the Study Group of Molecular Cardiology, the Japan Medical Association, Japan Smoking Foundation, Japan Society for the Promotion of Science, Uehara Memorial Foundation, and the Japan Heart Foundation.

	Footnotes

 
Original received May 28, 2003; resubmission received August 25, 2003; revised resubmission received September 23, 2003; accepted September 24, 2003.

	References

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