CLINICAL PERSPECTIVE

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(Circulation. 2007;115:553-561.)
© 2007 American Heart Association, Inc.

Coronary Heart Disease

Mobilization of CD34-Positive Bone Marrow–Derived Cells After Coronary Stent Implantation

Impact on Restenosis

Teruo Inoue, MD; Masataka Sata, MD; Yutaka Hikichi, MD; Ryoichi Sohma, BSc; Daiju Fukuda, MD; Toshihiko Uchida, MD; Minoru Shimizu, MD; Hiroshi Komoda, MSc; Koichi Node, MD

From the Department of Cardiovascular and Renal Medicine, Saga University Faculty of Medicine, Saga (T.I., Y.H., R.S., H.K., K.N.); Department of Cardiovascular Medicine, University of Tokyo, Tokyo (M. Sata, D.F.); and Department of Cardiology, Koshigaya Hospital, Dokkyo Medical University, Koshigaya (T.U., M. Shimizu), Japan.

Correspondence to Teruo Inoue, MD, Department of Cardiovascular and Renal Medicine, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan. E-mail inouete{at}med.saga-u.ac.jp

Received February 16, 2006; accepted November 21, 2006.

	Abstract

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Background— Recently, accumulating evidence has indicated that bone marrow–derived stem cells are capable of differentiating into vascular cells. It has been hypothesized that the inflammatory response after vascular injury triggers the mobilization of endothelial and smooth muscle progenitor cells from bone marrow.

Methods and Results— We measured circulating CD34-positive mononuclear cells, activation of integrin Mac-1 on the surface of neutrophils, and plasma granulocyte-colony stimulating factor levels in 40 patients undergoing coronary stenting. After bare-metal stenting, CD34-positive cells increased, reaching a maximum on day 7 after stenting. The maximum change compared with baseline before stenting was more striking in patients with restenosis than without restenosis (332±108% versus 148±49%; P<0.05). In contrast, CD34-positive cells decreased after sirolimus-eluting stenting (72±21% on day 7). The change in CD34-positive cells on day 7 relative to baseline was closely correlated with that in activated Mac-1 at 48 hours (R=0.52, P<0.01) and that in granulocyte-colony stimulating factor levels at 24 hours (R=0.42, P<0.05). Cell culture assay on day 7 showed that mononuclear cells differentiated into CD31-positive endothelium-like cells after bare-metal stenting. In patients with restenosis, mononuclear cells differentiating into -smooth muscle actin–positive smooth muscle–like cells also were observed. Implantation of sirolimus-eluting stents suppressed both types of differentiation.

Conclusions— Stent implantation may induce differentiation of bone marrow cells into endothelial or smooth muscle cells. Endothelial cells may participate in reendothelialization, a protective reaction against vascular injury, whereas smooth muscle cells may participate in neointimal thickening and restenosis. Sirolimus-eluting stents appear to inhibit the mobilization and differentiation of bone marrow cells.

Key Words: stem cells • inflammation • reendothelialization • restenosis • stents

	Introduction

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Stent placement is accompanied by stretch of the entire artery, deendothelialization, and compression of the plaque. This often results in dissection of the tunica media and occasionally dissection of the adventitia. These events induce a substantial local inflammatory reaction in the injured vessel wall that is followed by the proliferation of vascular components such as smooth muscle cells and extracellular matrix, leading to neointimal thickening and restenosis.^1–3 In contrast, reendothelialization of at least part of the injured vessel surface may occur at the stented site, protecting against early-stage thrombotic complications and possibly late restenosis.⁴

Editorial p 548

Clinical Perspective p 561

Recent exciting research suggests that endothelial progenitor cells mobilized from bone marrow into peripheral blood contribute to endothelial cell regeneration and postnatal neovascularization.^5,6 In addition, regenerated endothelial cells differentiated from bone marrow–derived endothelial progenitor cells also may contribute to reendothelialization as part of the process of vascular repair.⁵ On the other hand, it has been hypothesized that after vascular injury smooth muscle progenitor cells also are mobilized from bone marrow, triggered by the inflammatory response. These progenitors migrate to the site of vascular damage, differentiate into smooth muscle cells, proliferate, and cause neointimal hyperplasia. Evidence in support of these hypotheses comes from a vascular injury model in bone marrow chimeric mice⁷ and human autopsy findings in sex-mismatched bone marrow transplantation subjects.⁸ Therefore, under stimulation by vascular injury such as stenting, bone marrow–derived cells are thought to have the potential to differentiate into both vascular endothelial cells that may lead to reendothelialization and vascular smooth muscle cells that may lead to neointimal thickening and restenosis. This study was designed to assess the mobilization of bone marrow–derived CD34-positive stem cells and to assess its linkage to inflammation in poststent local vascular injury, repair, and regeneration in human.

	Methods

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Study Design
The subjects were 40 patients with stable coronary artery disease who underwent elective single coronary stent implantation. Bare-metal stents were implanted in 30 patients; sirolimus-eluting stents were placed in the remaining 10. All patients were receiving standard daily oral medications for angina, including 81 mg aspirin, and were started on 200 mg oral ticlopidine at least 2 days before stenting. Ticlopidine therapy was continued for 1 month after stenting or longer if necessary. Intravenous heparin was administered to maintain an adequate activated clotting time during the procedure and for 48 hours after coronary stenting. Follow-up coronary angiography was recommended for all patients at 6 months after coronary stenting but was performed earlier if necessary on the basis of clinical indications. Before the stent procedure, a coronary sinus catheter was placed and left for 48 hours after stenting. Both coronary sinus blood and peripheral blood were collected before stenting and at 15 minutes, 24 hours, and 48 hours after stenting; peripheral blood alone was collected again on 7 and 14 days and 1 month after stenting. Samples were immediately collected into tubes containing acid citrate dextrose and tubes containing EDTA. Using peripheral blood samples, we measured the number of circulating CD34-positive bone marrow–derived stem cells before and at each time point from baseline until 1 month after stenting. We also assessed activation of integrin Mac-1 (CD11b/CD18) on the surface of neutrophils and plasma level of granulocyte-colony stimulating factor (G-CSF) in coronary sinus blood at each time point from baseline until 48 hours after stenting. Activation of integrin Mac-1 plays a key role in the inflammatory process in vascular injury, and G-CSF is involved in stem cell mobilization. In addition, we cultured the patients’ peripheral blood mononuclear cells and assessed their differentiation to vascular cells before stenting and on day 7 after stenting. Finally, using the coronary sinus blood sampled at 24 hours after stenting, we measured creatine kinase-MB activity to assess postprocedural myocardial injury. The local institutional review board approved the study protocol, and written informed consent was obtained from each patient.

Quantitative Coronary Angiographic Analysis
Coronary lesions were assessed by quantitative coronary angiography with a computer-based QUANTCOR system (Siemens, Munich, Germany). Lesion length, reference diameter, and minimal lumen diameter were measured, and late lumen loss (minimal lumen diameter after stenting minus minimal lumen diameter at follow-up angiography) was calculated as an index of neointimal thickening. Restenosis was defined as >50% reduction in the luminal diameter at the site of the initial stenosis.

Measurement of Circulating CD34-Positive Cells
Circulating CD34-positive mononuclear cells were measured using flow cytometry based on a previously described method⁹ with minor modifications. In brief, EDTA-treated peripheral blood (3 mL) was incubated with test reagent or control reagent. The reagent mixture consisted of a nucleic acid dye (SY-III-8, Molecular Probes, Eugene, Ore), the FITC-conjugated monoclonal antibody CD45 (HLE-1, Becton Dickinson Immunocytometry System, San Jose, Calif), and the phycoerythrin-conjugated monoclonal antibody CD34 (HPCA-2, clone 8G12, Becton Dickinson Immunocytometry System) in the test reagent or IgG1 control antibody (IgG1 isotype control, Becton Dickinson Immunocytometry System). The samples were incubated for 20 minutes at room temperature and after incubation were diluted with a fluorescent-activated cell sorter lysing solution (Becton Dickinson Immunocytometry System) for hemolysis. Flow cytometric analysis was then performed with the fluorescent-activated cell sorter Calibur laser flow cytometer. Each measurement consisted of 10⁶ events of all white blood cells, which exceeded a threshold set on SY-III-8 fluorescence (nucleated cells). After gating on the CD45-positive area, the absolute numbers of CD34-positive cells were quantified.

Evaluation of Activated Mac-1 on the Surface of Neutrophils and Serum G-CSF Level
Acid citrate dextrose–treated whole blood (3 mL) from the coronary sinus was used for flow cytometric analysis for activation of Mac-1 on the surface of neutrophils as described in our previous study.¹⁰ We used a purified monoclonal antibody, 8B2 (provided by Dr Thomas Edgington), with a high sensitivity and specificity for the recognition of the activation-dependent neoepitope of Mac-1.¹¹ Purified mouse IgG1 also was used as an isotype-negative control. The fluorescein-conjugated second-step reagents for indirect immunofluorescence were FITC-conjugated F(ab')2 fragments of anti-mouse IgG goat immunoglobulins (Dako Cytomation, Glostrup, Denmark). Indirect immunofluorescence labeling was performed on whole blood incubated with 8B2 (100 µg/mL). Then, the flow cytometric analysis for 8B2 binding (activated Mac-1) was performed with an EPICS XL flow cytometer (Coultronics, Sunnyvale, Calif). Mean channel fluorescence intensity was calculated as an index of activated Mac-1 on the surface of neutrophils.

EDTA-treated blood (2 mL) from the coronary sinus was centrifuged at 1500g for 15 minutes at room temperature for measurement of G-CSF. The plasma was frozen and stored at –80°C until analysis. G-CSF was measured by a solid-phase sandwich enzyme-linked immunosorbent assay with a monoclonal antibody specific for human G-CSF and a biotinylated monoclonal second antibody. The assay was performed with a commercially available kit (BioSource International, Camarillo, Calif) according to the manufacturer’s instructions.

Cell Culture Assay and Immunocytochemistry
Cell culture assay to assess the differentiation of mononuclear cells into vascular cells was performed using EDTA-treated blood (40 mL) according to previously described methods.¹² Briefly, the mononuclear cell layer (2x10⁷ cells) was isolated and incubated on the human fibronectin–coated 96-well plates with Medium 199 (Gibco, Grand Island, NY) including 20% FBS, 20 µg/mL bovine pituitary extract as a growth supplement, 10 U/mL heparin (Sigma, St Louis, Mo) as endothelial progenitor cell medium, and HuMedia SG2 (KURABO, Osaka, Japan), including 5% FBS, 10 ng/mL platelet-derived growth factor (Sigma), 0.5 ng/mL endothelial growth factor (KURABO), 10 ng/mL basic fibroblast growth factor (KURABO), 5 µg/mL insulin (KURABO), 50 µg/mL gentamicin (KURABO), and 50 ng/mL amphotericin B (KURABO) as smooth muscle progenitor cell medium. After 3 days, floating cells were removed, and the adhering cells were incubated for 1 week in endothelial progenitor cell medium or for 2 weeks in smooth muscle progenitor cell medium.

Immunocytochemistry was used to assay cultured cells for the expression of CD31 as an endothelial cell marker and for the expression of -smooth muscle actin as a smooth muscle cell marker. Cells were fixed with cold 4% paraformaldehyde, followed by incubation with Block Ace (Dainippon Pharma, Osaka, Japan) to block nonspecific antibody binding. To stain for -smooth muscle actin, cells were treated with 0.2% Triton X-100 before the blocking step because this marker exists in cytoplasm. Primary monoclonal antibodies against human CD31 (clone JC70A, IgG1, Dako Cytomation) and -smooth muscle actin (clone 1A4, IgG2a, Dako Cytomation) were then applied. Nonimmune mouse IgG2a and IgG1 (Dako Cytomation) were used as negative controls. After washing 5 times with Tris-buffered saline, an alkaline phosphatase-conjugated rabbit anti-mouse IgG was applied. Finally, to visualize the immunoreactive products, Fast Red Substrate System (Dako Cytomation) was used according to the manufacturer’s instructions.

Statistical Analysis
Values of circulating CD34-positive cells, activated Mac-1 on the surface of neutrophils, and serum G-CSF levels at each time point after stenting were expressed as percent changes from the baseline (percent baseline). The serial changes in the percent baseline values were assessed by repeated-measures ANOVA, followed by post hoc Turkey test for intragroup and intergroup comparisons. The CD31-positive cell clusters and -smooth muscle actin–positive cells was not normally distributed. Thus, the intergroup comparison of the number between 2 time points was assessed with the Wilcoxon rank-sum test. Correlations between 2 variables were assessed with Spearman rank correlation coefficient. Multiple regression analysis was performed to predict the late lumen loss using variables that could possibly affect restenosis. Normality of the distribution of variables was assessed with the Kolmogorov-Smirnov test with Lilliefors’ correlation. Values are expressed as mean±SD for parametric data and median values and interquartile ranges for nonparametric data. Values of P<0.05 were considered significant.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

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Circulating CD34-Positive Cells in the Peripheral Blood
By 6 months of follow-up, angiographic restenosis was seen in 7 of 30 patients (23%) treated with bare-metal stents and in 0 of 10 patients with sirolimus-eluting stents. Baseline characteristics were similar among bare-metal stent patients who experienced restenosis (late lumen loss, 1.44±0.40 mm), those who did not (0.49±0.40 mm), and sirolimus-eluting stent patients (0.02±0.16 mm) (Table 1). Compared with baseline before stenting, the number of CD34-positive mononuclear cells did not change during 48 hours after stent implantation. However, it increased on day 7 (maximum) and day 14 after bare-metal stent implantation, and the change (percent baseline) was more striking in patients with restenosis than without restenosis (332±108% versus 148±49%, P<0.05 on day 7; 205±58% versus 124±14%, P<0.05 on day 14). In contrast, the number of CD34-positive cells decreased after sirolimus-eluting stenting (72±21% on day 7, P<0.05 versus bare metal stent without restenosis group) (Figure 1).

View this table: [in this window] [in a new window]   TABLE 1. Baseline Characteristics in Patients Treated With Bare-Metal Stents With or Without Restenosis and Sirolimus-Eluting Stents

View larger version (19K): [in this window] [in a new window]   Figure 1. Serial changes (percent baseline) in circulating CD34-positive mononuclear cells in peripheral blood. The CD34-positive mononuclear cells increased on day 7 (maximum) and day 14 after bare-metal stenting. The change was more striking in patients with than without restenosis and was suppressed after sirolimus-eluting stenting.

Activation of Mac-1 and Plasma G-CSF Level in the Coronary Sinus
Activation of Mac-1 on the surface of neutrophils was accelerated 24 to 48 hours after bare-metal stenting with a maximum at 48 hours. The percent baseline was more striking in patients with restenosis than without restenosis (164±34% versus 122±15%; P<0.05 at 48 hours). In contrast, the activation of Mac-1 was suppressed after sirolimus-eluting stenting (84±15% at 48 hours, P<0.05 versus bare metal stent without restenosis group) (Figure 2, left). Plasma G-CSF level also increased 24 to 48 hours after bare-metal stenting with a maximum at 24 hours. The change (percent baseline) was more striking in patients with restenosis than without restenosis (194±62% versus 136±46%; P<0.05 at 24 hours). The G-CSF level did not change after sirolimus-eluting stenting (Figure 2, right). The change in number of CD34-positive cells on day 7 was closely correlated with the change in activated Mac-1 at 48 hours (R=0.52, P<0.01) and the change in G-CSF levels at 24 hours (R=0.42, P<0.05) (Figure 3) but not with balloon inflation times (R=0.13, P=NS), total duration of balloon inflation (R=0.18, P=NS), or creatine kinase-MB activity at 24 hours after stenting (R=0.21, P=NS).

View larger version (21K): [in this window] [in a new window]   Figure 2. Serial changes (percent baseline) in activated Mac-1 on the surface of neutrophils (left) and plasma G-CSF levels (right) in the coronary sinus. Activation of Mac-1 (maximum at 48 hours) was accelerated and G-CSF levels (maximum at 24 hours) increased time dependently after bare-metal stenting and was more striking in patients with restenosis. These changes were suppressed after sirolimus-eluting stenting.

View larger version (24K): [in this window] [in a new window]   Figure 3. Positive correlation between the number of CD34-positive cells (percent baseline) on day 7 and activated neutrophil Mac-1 at 48 hours (left) and G-CSF levels (right) at 24 hours after stenting.

Prediction of Late Lumen Loss
Angiographic late lumen loss was correlated with the change in CD34-positive cell numbers on day 7 (R=0.48, P<0.01) (Figure 4). Multiple regression analysis showed that late lumen loss was not associated with any other variables possibly relating to restenosis but independently with the change in CD34-positive cell numbers on day 7 (R=0.43, P<0.01) and the change in neutrophil Mac-1 activation at 48 hours (R=0.39, P<0.01). The change in CD34-positive cell number was the most powerful predictor of late lumen loss (Table 2).

View larger version (25K): [in this window] [in a new window]   Figure 4. Positive correlation between angiographic late lumen loss and the number of CD34-positive cells (percent baseline) on day 7.

View this table: [in this window] [in a new window]   TABLE 2. Multiple Regression Analysis for Prediction of Angiographic Late Lumen Loss

Cell Culture Assay
Cell culture assay for peripheral blood mononuclear cells was performed in 21 patients. A large amount of mononuclear cells cultured in endothelial progenitor cell medium differentiated into CD31-positive spindle-shaped cells and formed clusters. We could not count such a large number of cells; instead, we assessed the number of cell clusters per 4-well plates. In 10 patients treated with bare-metal stents without restenosis, the number of CD31-positive cell clusters increased on day 7 after stenting compared with baseline before stenting. In 5 patients who developed restenosis, these changes were less striking. On the other hand, in 6 patients treated with sirolimus-eluting stents, the number of clusters decreased significantly (Figure 5).

View larger version (65K): [in this window] [in a new window]   Figure 5. A, Peripheral blood mononuclear cells on day 7 after stenting cultured in endothelial progenitor cell medium and immunostained with CD31. These cells differentiated into spindle-shaped cells in which CD31-positive cell clusters formed on day 7 after stenting in patients treated with bare-metal stents who did not develop restenosis (left). In patients treated with bare-metal stents who developed restenosis, cluster formation was less (middle). In patients implanted with sirolimus-eluting stents, not only cluster formation but also CD31-positive cell numbers were reduced. B, The number of CD31-positive cell clusters per 4-culture well plates. Compared with before stenting, cell clusters significantly increased on day 7 in patients treated with bare-metal stents who developed restenosis. In patients treated with bare-metal stents without restenosis, the change was less striking. In contrast, the cluster number significantly decreased in the patients treated with sirolimus-eluting stents. Solid line represents median value; box, interquartile range; and error bar, 95% confidence interval.

Some mononuclear cells cultured in smooth muscle progenitor cell medium differentiated into polygonal and stellate-shaped cells. In these cells, -smooth muscle actin–positive cells were assessed, counting cell number per total of 100 cells. Compared with baseline before stenting, the cell number did not change on day 7 after stenting in bare-metal stent patients without restenosis and in sirolimus-eluting stent patients. However, in patients treated with bare-metal stents who developed restenosis, the number of -smooth muscle actin–positive cells increased on day 7 (Figure 6).

View larger version (57K): [in this window] [in a new window]   Figure 6. A, Peripheral blood mononuclear cells on day 7 after stenting cultured in smooth muscle progenitor cell medium and immunostained with -smooth muscle actin. These cells differentiated into polygonal and stellate-shaped cells in which -smooth muscle actin–positive cells were present on day 7 after stenting in patients treated with bare-metal stents who experienced restenosis (middle, arrow). In patients treated with bare-metal stents who did not develop restenosis (right) and patients treated with sirolimus-eluting stents (left), -smooth muscle actin–positive cells were rarely present. B, The number of -smooth muscle actin–positive cells per 100 cells. Compared with before stenting, -smooth muscle actin–positive cells did not change on day 7 after stenting in patients treated with bare-metal stents who did not develop restenosis and in patients treated with sirolimus-eluting stents. However, in patients treated with bare-metal stents who experienced restenosis, -smooth muscle actin–positive cells significantly increased. Solid line represents median value; box, interquartile range; and error bar, 95% confidence interval.

	Discussion

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Vessel Injury and Bone Marrow–Derived Stem Cells
In this study, we demonstrated that the number of circulating CD34-positive mononuclear cells increased from day 7 to day 14 after bare-metal stent deployment. The change was more striking in patients who developed restenosis than in patients who did not develop restenosis. In contrast, the change was suppressed after sirolimus-eluting stenting. In addition, multiple regression analysis showed that the increase in CD34-positive cells on day 7 was a powerful independent predictor of angiographic late lumen loss. These results suggest the possibility that bare-metal stents induce whereas sirolimus-eluting stents suppress mobilization of bone marrow–derived stem cells in association with restenosis.

CD34 is a membrane surface marker for bone marrow–derived stem cells, including endothelial and smooth muscle progenitor cells. An increase in circulating CD34-positive cells after stenting in patients has been observed by other investigators. Shintani et al¹³ suggested that an increase in CD34-positive cells was the mechanism responsible for vasculogenesis in the ischemic myocardium. Schober et al¹⁴ demonstrated a relationship between an increase in CD34-positive cells and the risk of restenosis, similar to the results of the present study. To further elucidate the role of CD34-positive cells after coronary artery stenting with both bare-metal and sirolimus-eluting stents, we simultaneously performed cell culture assay to assess differentiation of bone marrow–derived cells into vascular cells after stent deployment. As a result, after bare-metal stenting, circulating mononuclear cells differentiated into CD31-positive endothelium-like cells that formed more clustering on day 7 after stenting, corresponding to the time when the CD34-positive cells maximally increased compared with baseline before stenting. The clustering formation of endothelium-like cells was less striking in patients with than without restenosis. Conversely, the mononuclear cells of patients with restenosis differentiated into more -smooth muscle actin–positive cells on day 7 than in patients without restenosis. Our results suggest that recruitment of bone marrow–derived stem cells and differentiation into vascular cells may contribute to reendothelialization and restenosis and that differentiation into more smooth muscle cells with less endothelial cells may lead to restenosis. Interestingly, after sirolimus-eluting stenting, both CD31-positive cells with cluster formation and -smooth muscle actin–positive cells were reduced in the cell culture assay on day 7. These results suggest that sirolimus-eluting stents may inhibit the differentiation of bone marrow cells into both smooth muscle–like cells and endothelium-like cells, possibly leading to impairment of reendothelialization. Accordingly, from our results, we can envision that the excessive mobilization of stem cells may lead to restenosis, whereas its absence may impair reendothelialization.

We have already demonstrated that sirolimus inhibited outgrowth of both smooth muscle–like cells and endothelium-like cells that originate from cultured mononuclear cells and that sirolimus reduced both neointimal hyperplasia with a decreased number of bone marrow–derived smooth muscle–like cells and reendothelialization using the bone marrow chimeric mouse vascular injury model.¹² The present study provides clinical data supporting the findings in these experimental models. However, it is not clear why the local delivery of sirolimus from a drug-eluting stent has a systemic effect to suppress the differentiation of bone marrow cells.

Inflammatory Response Triggers Recruitment of Stem Cells
It has been suggested that tissue ischemia is the primary mechanism for recruitment of endothelial progenitor cells from bone marrow into peripheral blood that contributes to endothelial cell regeneration and neovascularization.^5,15 On the other hand, the recruitment of bone marrow–derived stem cells also is hypothesized to occur as part of the inflammatory process.⁷ Although myocardial ischemia induced by stent deployment might lead to an increase in CD34-positive cells, we found no association between the change in CD34-positive cells and balloon inflation times, total duration of balloon inflation, or creatine phosphokinase levels 24 hour after stenting. In contrast, an early-stage inflammatory reaction was observed after stenting and was related to stem cell mobilization. Before an increase in CD34-positive cells, activation of Mac-1 on the surface of neutrophils was accelerated, and plasma G-CSF levels increased time-dependently in the coronary sinus blood of patients with bare-metal stent implantation. More striking changes were evident in the patients who developed restenosis. Interestingly, these changes were substantially suppressed by the deployment of sirolimus-eluting stents. In addition, the change in CD34-positive cells on day 7 was correlated with the change in Mac-1 activation at 48 hours and the change in G-CSF levels at 24 hours.

In the early stage of the inflammatory process after vascular injury, activated leukocytes, neutrophils, and monocytes interact with platelets adhered at sites of injured vessel wall.^3,16,17 Mac-1 orchestrates the recruitment of leukocytes by promoting firm adhesion to, and transmigration across, a layer of platelets adhered at sites of injured vessel wall via binding to platelet ligands such as glycoprotein Ib.^18,19 Mac-1 is thought to be one of the important signaling molecules in the mechanism of restenosis. Monoclonal antibody blockade²⁰ and the absence of Mac-1²¹ reduce neointimal thickening after experimental angioplasty and stenting. Previous clinical studies also have implicated stent-induced activation and upregulation of Mac-1 in restenosis.^10,22–24 The relationship between CD34-positive cells and Mac-1 activation in this study suggests a causal relation between stem cell mobilization and neutrophil Mac-1 signaling. It has been demonstrated that neutrophil-released matrix metalloproteinase-9 induces stem cell mobilization²⁵ and that recruitment of stem and progenitor cells from the bone marrow niche requires metalloproteinase-9–mediated release of Kit ligand.²⁶ In addition, it has been shown that stimulatory signals from Mac-1 induce release of metalloproteinase-9 from human neutrophils.²⁷ We have observed that binding of 8B2, which we used in this study as the antibody recognizing the activation-dependent neoepitope of Mac-1, also stimulated neutrophil metalloproteinase-9 release (unpublished data). These experimental results suggest that Mac-1–dependent activated neutrophils may contribute in part to recruitment of bone marrow–derived stem cells.

G-CSF is a potent hematopoietic cytokine that mobilizes bone marrow–derived stem cells into the peripheral circulation. Because of its ability for neovascularization and cardiac regeneration in the ischemic myocardium, G-CSF therapy has been applied after acute myocardial infarction to improve cardiac function.²⁸ However, a study using G-CSF therapy with intracoronary infusion of mobilized peripheral CD34-positive cells unexpectedly increased the rate of restenosis.²⁹ It has been demonstrated that the expression of G-CSF is induced in the local arterial wall by injury.³⁰ Thus, endogenous G-CSF production by stent-induced injury might also contribute to mobilization of CD34-positive cells and restenosis.

Although a positive association of CD34-positive cell mobilization with Mac-1 activation and G-CSF levels was observed, the mechanism of this association remains speculative. However, the observation that these inflammatory responses (at 24 to 48 hours) preceded CD34-positive cell mobilization (on day 7 to 14) suggests that the inflammatory response is a major trigger for mobilization of bone marrow–derived stem cells after coronary stenting. Considering that the inflammatory mediators circulate systemically, it is possible that modulation of local inflammation by a drug-eluting stent may have a systemic effect to lower stem cell stimulation.

Potential Limitations
Our study included too few subjects to conclude that there were no definitive differences in the baseline characteristics among groups. In addition, a causal relationship between mobilization of bone marrow–derived stem cells and an inflammatory response and its role in restenosis should be addressed in a larger cohort. In this study, we measured CD34-positive cells as bone marrow–derived stem cells to assess the potential for differentiation of these cells into both endothelial cells and smooth muscle cells. Future studies using more specific markers of endothelial or smooth muscle progenitors would strengthen our findings. Although no specific marker for smooth muscle progenitors has been identified, recent studies have demonstrated the detection of endothelial progenitor cells using kinase insert domain receptor (vascular endothelial growth factor receptor-2) and/or CD133 in addition to CD34.³¹ However, smooth muscle outgrowth cells from the progenitors also have been shown to express kinase insert domain receptor.³² Therefore, established methods to isolate each progenitor are pending.

Clinical Implications and Conclusions
Drug-eluting stents such as sirolimus- or paclitaxel-eluting stents have drastically reduced angiographic and clinical restenosis across broad lesions and patient subsets because of their pharmacological inhibition of vascular cell proliferation. However, excessive inhibition of vascular cell proliferation by current drug-eluting stents produces rare but serious complications such as late incomplete apposition or aneurysm formation.³³ In addition, impaired reendothelialization and wound healing may result in fatal late thrombosis.³⁴ These unfavorable effects of drug-eluting stents may be derived in part from excessive inhibition of inflammation and stem cell recruitment.

Recently, the use of antibodies against membrane receptors of circulating endothelial progenitor cells to capture these cells at the site of vascular injury has been proposed, and anti-CD34 antibody–coated stents have been developed.³⁵ These antibody-coated stents have been implanted in human coronary arteries in a multicenter pilot study.³⁶ However, the anti-CD34 antibody recognizes not only endothelial progenitor cells but also broad categories of bone marrow–derived cells, including smooth muscle progenitor cells. Therefore, we should also consider the possibility that the captured cells differentiate simultaneously into smooth muscle cells, leading to restenosis.

In this study, we demonstrated that coronary stenting induces recruitment of bone marrow–derived stem cells that potentially differentiate into vascular cells, in association with early inflammatory responses. These cellular responses, which were significantly suppressed by sirolimus-eluting stents, may lead to the development of restenosis and reendothelialization. From our results, we propose a novel pathophysiological mechanism of restenosis and point out the potential deleterious effects of drug-eluting stents.

	Acknowledgments

 
We acknowledge the technical support services of Toshiyasu Miyazaki, PhD, HJL Japan, Co, Kawasaki, Japan, for flow cytometric analysis. We also thank Thomas S. Edgington, MD, PhD, Scripps Research Institute, La Jolla, Calif, for generously providing the monoclonal antibody 8B2.

Sources of Funding

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a grant from Kimura Foundation, and by a research grant from the Japan Foundation of Cardiovascular Research.

Disclosures

None.

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CLINICAL PERSPECTIVE

In this study, we show that circulating CD34-positive bone marrow–derived stem cells increase 7 to 14 days after bare-metal stenting and that these increases are more striking in patients who subsequently develop restenosis. In addition, these increases were associated with early local inflammatory response as demonstrated by activation of integrin Mac-1 on the surface of neutrophils. Cultured circulating mononuclear cells sampled on day 7 after percutaneous coronary intervention differentiated into CD31-positive endothelium-like cells and into -smooth muscle actin–positive smooth muscle–like cells. Interestingly, all of these responses were strikingly suppressed in patients implanted with sirolimus-eluting stents. The results suggest that stent vascular injury induces mobilization and differentiation of bone marrow cells into vascular cells in the vascular wound healing process. Suppression of endothelial precursor-like cell recruitment by sirolimus-eluting stents may have important clinical implications.

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