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Integrative Physiology |
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 |
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Key Words: endothelium angioplasty endothelial progenitor cells bone marrow reendothelialization
| Introduction |
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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.710 Although earlier studies reported that adult blood-derived CD34+VEGFR-2+ angioblasts differentiate into ECs,710 recent accumulating findings have suggested that peripheral blood (PB)-derived mature CD14+ monocytes can also transdifferentiate into EC lineage cells under angiogenic conditions1114 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 we17,18 have demonstrated that bone marrow (BM)-derived mononuclear cells differentiate into neocapillaries in ischemic limbs or myocardium, the involvement of BM monocytederived EC lineage cells remains undefined. Considering that the administration of granulocyte-macrophage colonystimulating factor mobilized angioblasts from BM19 and that statin treatment20,21 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-1dependent mechanism to cause reendothelialization as EC progenitors more potent than BM-derived CD34+ cells, leading to inhibition of intimal hyperplasia.
| Materials and Methods |
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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 antiFlt-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 antivon Willebrand factor (anti-vWF) (DAKO), antiendothelial NO synthase (anti-eNOS) (Calbiochem), antiVE-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 TranscriptionPolymerase 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 Dunnetts test. Data (mean±SE) were considered significant when P<0.05.
| Results |
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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.
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MCP-1Dependent 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-1activated 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-1activated cells but not in MCP-1untreated cells.
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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-1activated BM-MLCs or MCP-1activated 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.
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As shown in Figure 5B, EC-like cells regenerated by MCP-1activated 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-MLCderived reendothelialization is shown in Figure 4A.
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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-1Dependent 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-1dependent 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-1mediated effects on PB-derived CD34-/CD14+ monocytes were much weaker.
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Expression level of ß1-integrin and MCP-1mediated activation of ß1-integrin conformation have important implications in transendothelial chemotaxis of monocytes.28 Neutralizing antiß1-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-1mediated 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 |
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MCP-1 binding to its receptor CCR2 induces a conformational change in ß1-integrin via Gi
-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-integrindependent 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-1mediated 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 antiMCP-1 therapy significantly prevents intimal hyperplasia.35 Thus, migration of MCP-1activated PB monocytes into the vessel wall is a key step in the progression of intimal hyperplasia. In this study, we focused on MCP-1mediated 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-1mediated 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.710 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.1114 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 musclelike cells in aortic or cardiac transplant arteriopathy.36,37 In these allotransplant models, intimal hyperplastic lesions composed of smooth musclelike 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-MLCderived EC-like cells. These findings suggest that if BM-MLCs preferentially transdifferentiate to endothelial-like cells on injured endothelium, infiltration of BM-derived smooth musclelike cells into intimal lesions may be blocked and thus restenosis can be prevented. Alternatively, precursor cells for smooth musclelike cells may not be included in the BM-MLCs.
Our present study demonstrated that transfusion of MCP-1activated 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 musclelike cells. Werner et al39 recently reported that intravenous transfusion of spleen-derived EPC enhances reendothelialization after vascular injury when spleen is removed. MCP-1activated 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-1mediated 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 |
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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 |
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| References |
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