Potent Inhibitory Effect of Sirolimus on Circulating Vascular Progenitor Cells
http://www.100md.com
循环学杂志 2005年第2期
the Department of Cardiovascular Medicine (D.F., M.S., K.T., R.N.), University of Tokyo Graduate School of Medicine, Tokyo, Japan
PRESTO (M.S.), Japan Science and Technology Agency Kawaguchi, Saitama, Japan
Abstract
Background— Neointimal hyperplasia is the major cause of in-stent restenosis (ISR). The sirolimus-eluting stent (SES) has emerged as a promising therapy to prevent ISR; however, the exact mechanism by which locally delivered sirolimus, an immunosuppressive agent, prevents ISR remains unknown. Recent evidence suggests that circulating progenitor cells may contribute to neointimal formation.
Methods and Results— Mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers. Smooth muscle (SM)–like cells outgrew from the culture of MNCs (1x106) in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas endothelial cell–like cells were obtained in the presence of vascular endothelial growth factor. Sirolimus potently inhibited SM-like cell outgrowth. The number of SM-like cells was significantly reduced at a concentration as low as 0.1 ng/mL (15.9±5.8% of control, P<0.001). Sirolimus also exerted an inhibitory effect on endothelial cell–like cells that originated from MNCs. Wire-mediated vascular injury was induced in femoral arteries of bone marrow chimeric mice. Either vehicle or sirolimus was administered locally to the perivascular area of the injured arteries. Sirolimus significantly reduced neointima hyperplasia at 4 weeks (intima/media ratio 2.0±0.3 versus 1.0±0.2, P<0.05) with a decreased number of bone marrow–derived SM-like cells and hematopoietic cells in the lesion. Reendothelialization was retarded in the arteries treated with sirolimus.
Conclusions— The potent inhibitory effects of sirolimus on circulating smooth muscle progenitor cells may mediate the clinical efficacy of SES, at least in part. Sirolimus potentially may affect reendothelialization after stent implantation.
Key Words: stents ; restenosis ; balloon ; atherosclerosis ; endothelium
Introduction
Implantation of coronary stents has been shown to reduce the risk of periprocedural complications and restenosis more than balloon angioplasty alone.1 However, in-stent restenosis (ISR) remains a significant clinical problem limiting the long-term success of percutaneous treatment.1 The principal cause of ISR is neointimal hyperplasia resulting from the excessive accumulation of smooth muscle cells (SMCs). Recently, the sirolimus-eluting stent (SES) has emerged as a promising strategy to prevent ISR.2,3 A randomized, double-blind trial demonstrated that the use of SES reduced the rates of restenosis and clinical events even in patients with complex lesions.3 Despite increasing clinical interest in SES, very little is known about the mechanism by which locally delivered sirolimus, a hydrophobic macrolide with potent immunosuppressive activity, prevents ISR.2
We and others suggested that bone marrow (BM) cells give rise to vascular progenitor cells that home in on injured arteries and differentiate into SMCs or endothelial cells (ECs), thereby contributing to vascular repair and lesion formation.4–9 Here, we investigated the effects of sirolimus on human vascular progenitor cells that may contribute to neointimal hyperplasia and reendothelialization after stent implantation. The in vivo effect of sirolimus was also evaluated in a model of neointimal hyperplasia with bone marrow chimeric mice. Results suggest that sirolimus has a potent inhibitory effect on both smooth muscle progenitor cell and endothelial progenitor cell incorporation at the sites of vascular lesions.
Methods
Cell Culture
Peripheral mononuclear cells (MNCs) were isolated from blood of healthy human volunteers by density gradient centrifugation with HISOPAQUE-1077 (Sigma). MNCs were cultured at a density of 1x106 cells per fibronectin-coated well in a 96-well dish in HuMedia-SG2 (KURABO) supplemented with 10 ng/mL platelet-derived growth factor (PDGF)-BB and 10 ng/mL basic fibroblast growth factor (smooth muscle progenitor cell medium). The same number of MNCs were cultured in EBM (Clonetics) supplemented with 1 μg/mL hydrocortisone, 3 μg/mL bovine brain extract, 10 ng/mL vascular endothelial growth factor, and 20% FBS (endothelial progenitor cell medium). Sirolimus (Wako Pure Chemical) was added to the culture medium at a concentration of 0.1 to 100 ng/mL. At 4, 8, and 12 days, the culture medium was changed, and nonadherent cells were removed. Jurkat cells and Sertoli cells were purchased from the American Type Culture Collection (Rockville, Md). Human aortic SMCs (HASMCs) were purchased from KURABO and cultured in HuMedia-SG2. Human umbilical vein ECs (HUVECs) were purchased from Clonetics and cultured in EGM-2 (Clonetics). HASMCs or HUVECs were seeded at a density of 5x103 cells per well in 24-well plates. After culture for 72 hours in the presence of sirolimus, cells were counted.
Immunocytochemistry
To identify smooth muscle (SM)–like cells, adherent cells were fixed at 14 days in 4% paraformaldehyde and permeabilized with 0.5% NP40. After blocking with 1% goat serum, cells were incubated with an alkaline phosphatase–conjugated anti--smooth muscle actin (-SMA) antibody (Sigma) and stained with a Vector red substrate kit (Vector). To identify EC-like cells, adherent cells were incubated with 10 μg/mL acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyamine perchlorate (DiI-Ac-LDL; Biomedical Technologies Inc) for 4 hours. Cells were washed in PBS, fixed with 4% formaldehyde, and counterstained with FITC-labeled lectin from Bandeiraea simplicifolia (FITC-BS lectin, Sigma). Cells that were positive for both DiI-Ac-LDL and FITC-BS lectin were deemed EC-like cells, as described previously.8
Reverse Transcription–Polymerase Chain Reaction
Total RNA was prepared with RNazol reagent (Tel-Test, Inc). Reverse transcription was performed with 1 μg of RNA, random hexamer primers, and MMLV reverse transcriptase (ReverTraAce-, TOYOBO). Polymerase chain reaction (PCR) primers were as follows: -SMA 5'-CCAGCTATGTGTGAAGAAGAG-3' and 5'-GTGATCTCCTTCTGCATTCGGT-3'; GAPDH 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. PCR reactions were performed for 30 cycles of 30-second denaturation at 94°C, 30 seconds of annealing at 60°C, and 1 minute of extension at 72°C followed by 10 minutes of final extension. PCR for FK506-binding protein 12 (FKBP12) was performed with a primer set of 5'-GAGAAAAGCCAGCATAAAGC-3' and 5'-TCTAGAACTTCGGTGGAAAG -3' for 30 cycles of denaturation (94°C, 15 seconds), annealing (48°C, 30 seconds), and polymerization (68°C, 45 seconds). After agarose gel (3%) electrophoresis in the presence of ethidium bromide, the PCR products were revealed by ultraviolet irradiation.
Bone Marrow Transplantation
LacZ mice, which are knock-in mice that express the LacZ gene in essentially all tissues (C57BL/6x129S background),10 were originally purchased from Jackson Laboratory (B6; 129S-Gtrosa26, stock No. 002073, Bar Harbor, Me). LacZ mice were maintained in our animal facility and intercrossed with C57BL/6J mice. The resulting littermates were used for the present study.4 The homozygotes and wild-type mice were screened by PCR analysis of tail DNA according to the protocol provided by Jackson Laboratory. GFP mice, which are transgenic mice (C57BL/6 background) that ubiquitously express enhanced green fluorescent protein (GFP), were a generous gift from Dr Masaru Okabe (Osaka University, Osaka, Japan). Bone marrow transplantation (BMT) was performed from LacZ mice to wild-type mice (BMTLacZWild) or GFP mice to wild-type mice (BMTGFPWild) as described previously.4,11 A total of 85% to 95% of peripheral leukocytes had been reconstituted as determined by in situ hybridization for Y chromosome (BMTMale LacZFemale Wild mice) or flow cytometry (BMTGFPWild mice). All procedures that involved experimental animals were performed in accordance with protocols approved by the institutional committee for animal research of the University of Tokyo and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).
Vascular Injury and Local Delivery of Sirolimus
Twenty weeks after BMT, both femoral arteries of BM chimeric mice were injured by inserting a straight spring wire (0.38 mm in diameter, No. C-SF-15-15, Cook) as described previously.12 Sirolimus was suspended in DMSO at a concentration of 25 μg/μL. After vascular injury, 25 μg (n=8) of sirolimus suspended in 50 μL of 20% Pluronic F-127 gel (Sigma) was administered to the perivascular area of the right femoral artery in BMTLacZWild mice, and the same volume of Pluronic gel containing only DMSO was administered around the left femoral artery.
X-Gal Staining and Morphometric Analysis
Four weeks after surgery, mice were euthanized with an overdose of pentobarbital and perfused at a constant pressure via the left ventricle with 0.9% sodium chloride solution. The injured arteries were excised and stained with X-gal (5-bromo-4-chloro-3-indolyl ;-D-galactopyranoside) as described previously.4,11 The arteries were embedded in paraffin, and cross sections were made as described previously.12 After counterstaining with hematoxylin, morphometric analysis was performed with image-analysis software (Image-Pro Plus version 4.5, Media Cybernetics, USA) as described previously.12 Intima/media ratio and the number of LacZ-positive cells were averaged on 3 different sections from each artery.
Immunofluorescence Staining
To study the effect of sirolimus on reendothelialization, cross sections were incubated with an anti-CD31 antibody (clone MEC13.3, BD Biosciences) and Cy3-conjugated anti--SMA antibody (Sigma), followed by incubation with an FITC-conjugated anti-rat Ig secondary antibody (Jackson ImmunoResearch). To characterize the BM-derived cells observed in the neointima of BMTLacZWild mice, X-gal–stained cross sections were incubated with a Cy3-conjugated anti--SMA antibody and an anti-CD45 antibody (BD Biosciences), followed by incubation with an FITC-conjugated anti-rat Ig secondary antibody. The femoral arteries harvested from BMTGFPWild mice were embedded in plastic resin (Technovit 8100, Heraeus Kulzer) as described previously.11 An anti-SM1 rat monoclonal antibody was kindly provided by Dr Kazuhide Hasegawa (Kyowa Hakko, Japan). Thin sections (4 μm) were incubated with Cy3-conjugated anti--SMA antibody or anti-SM1 antibody followed by Cy3-conjugated anti-rat Ig antibody. After immunofluorescence staining, nuclei were counterstained with Hoechst 33258 (Sigma). The sections were mounted with the Prolong Antifade Kit (Molecular Probes) and observed under a PROVIS AX80 microscope (Olympus) equipped with a mercury/halogen dual-illumination system and a charge-coupled device camera (DP50, Olympus). High-resolution fluorescence images were taken with a confocal laser scanning system (FLUOVIEW FV300, Olympus).
Statistical Analysis
Values are expressed as mean±SEM for continuous variables. Comparisons of multiple groups were made by 1-way ANOVA, followed by Scheffe multiple comparison test. A probability value <0.05 was considered statistically significant.
Results
Potent Inhibitory Effect of Sirolimus on SM Progenitor Cells
Human peripheral MNCs were cultured in fibronectin-coated wells in the presence of PDGF-BB and basic fibroblast growth factor (SM progenitor cell medium). When the medium was changed at 4 days, there were adherent cells that consisted of heterogeneous cell types. Some cells remained round, whereas others showed a spindlelike shape and were displayed as a monolayer. At 14 days, cells with a polygonal and stellate shape became dominant. Consistent with a previous report,5 mRNA for -SMA was detected in adherent cells (Figure 1A). Immunocytochemistry revealed that >90% of adherent cells at 14 days were positive for -SMA (Figure 1B). Sirolimus had little effect on the number of adherent cells at 4 days; however, the number of -SMA–positive cells at 14 days was dramatically decreased by sirolimus (Figure 1B and 1C). At a concentration as low as 0.1 ng/mL, sirolimus significantly reduced the number of -SMA–positive cells to 15.9±5.8% of control (P<0.0001).
Effects of Sirolimus on Endothelial Progenitors, ECs, and SMCs
Sirolimus also inhibited the proliferation of HASMCs and HUVECs in a dose-dependent manner (data not shown); however, its inhibitory effect on these cells was relatively mild (Figure 2A). There was no significant reduction in cell number by sirolimus at 0.1 ng/mL, whereas at higher concentrations (1 ng/mL), sirolimus inhibited the proliferation of HASMCs (47.0±4.8% of control, P<0.001) and HUVECs (40.7±5.9% of control, P<0.05). Sirolimus had no obvious effect on the morphology of HASMCs and HUVECs. Human MNCs were also plated on fibronectin-coated 96-well plates and cultured in EBM with bovine brain extract, vascular endothelial growth factor, and 20% FBS (endothelial progenitor cell medium). Under these conditions, attached cells became spindle-shaped and were doubly stained by DiI-Ac-LDL and FITC-BS lectin.8 Sirolimus potently reduced the number of EC-like cells at 14 days in a dose-dependent manner. At a concentration as low as 0.1 ng/mL, sirolimus significantly reduced the number of EC-like cells to 36.7±1.7% of control (P<0.01). To obtain an insight into the mechanism by which sirolimus selectively inhibits differentiation of MNCs into SM-like or EC-like cells, we investigated the expression of FKBP12, which is known to be a receptor of sirolimus.2,13 Consistent with previous reports,14 HASMCs isolated from the aortic medial layer expressed little FKBP12 (Figure 2B). On the other hand, FKBP12 was abundantly expressed in human MNCs.
Effects of Locally Delivered Sirolimus on Neointimal Formation and Reendothelialization After Vascular Injury
The in vivo effects of sirolimus on vascular progenitor cells were studied in BM chimeric mice with a mouse model of vascular injury that induces reproducible neointima hyperplasia.11,12 At 4 weeks after injury, there was neointima hyperplasia that contained BM-derived LacZ-positive cells in the BMTLacZWild mice. Local delivery of sirolimus significantly reduced neointimal formation (intima/media ratio 2.0±0.3 versus 1.0±0.2, P=0.03; Figure 3A and 3B). The reduction in neointimal hyperplasia was associated with a decrease in the number of LacZ-positive cells in the neointima (number of LacZ-positive cells/neointima 20.2±4.1 versus 7.3±2.4, P=0.02). In the lesion treated with vehicle, 55.1±26.1% of the luminal side was coated with the regenerated endothelium as determined by anti-CD31 immunostaining. On the other hand, endothelium was detected only in 7.7±3.4% of the luminal side of the sirolimus-treated arteries. Taken together, these results indicate that locally delivered sirolimus possibly affects the reendothelialization process after severe vascular injury, although neointima hyperplasia is markedly attenuated (Figure 3C).
Characterization of BM-Derived Neointimal Cells
BM-derived cells observed in neointima were characterized by confocal immunofluorescence study. The wire injury induced vascular lesions that were predominantly composed of -SMA–positive cells.12 In BMTGFPWild mice, we readily detected GFP-positive intimal cells that were positive for -SMA (Figure 4A). On the other hand, there were few neointimal cells that expressed both GFP and SM1, a marker for differentiated SMCs (Figure 4B).15 These results suggest that BM-derived -SMA–positive cells do not become highly differentiated SMCs, at least at 4 weeks after wire injury.11 In BMTLacZWild mice, most of the BM-derived neointimal cells expressed -SMA. There were BM-derived cells that expressed both -SMA and CD45, a pan-hematopoietic marker (Table).4 Local administration of sirolimus potently reduced the number of LacZ+/-SMA+ cells in the neointima (Figure 4C and 4D). Sirolimus also reduced the number of LacZ+/CD45+ cells. Reduction in the number of LacZ+ cells in neointima was associated with a reduction in the total number of -SMA–positive neointimal cells (Figure 4E).
Expression of -SMA and CD45 by LacZ-Positive Cells in Neointima
Discussion
It is assumed that sirolimus locally delivered from SES prevents neointimal hyperplasia by inhibiting migration and proliferation of SMCs that originate from the adjacent media.2,13 However, it was reported that medial SMCs express little FKBP12,16 which sirolimus binds to prevent cell-cycle progression through the upregulation of the cyclin-dependent kinase inhibitor p27kip1.2,17 Thus, it is likely that sirolimus may also target cell types other than medial SMCs. Recent reports suggested that circulating progenitor cells may contribute to neointimal hyperplasia in models of transplant-associated arteriosclerosis, hyperlipidemia-induced atherosclerosis, and postangioplasty restenosis.4–7 It has been consistently reported that SM-like cells with specific growth, adhesion, and integrin profiles could grow from human peripheral MNCs.5 Moreover, a significant part of SMCs throughout the atherosclerotic vessel wall was shown to derive from donor BM in gender-mismatched BMT patients.6 Thus, it is possible that circulating cells may participate in the pathogenesis of human ISR.
In the present study, sirolimus potently inhibited MNC differentiation into SM-like cells. The inhibitory effect was achieved at a concentration as low as 0.1 mg/mL, at which sirolimus had no significant effect on the proliferation of HASMCs that expressed low FKBP12. In fact, in our mouse model of severe vascular injury, locally derived sirolimus efficiently decreased the number of BM-derived cells in the neointima and attenuated neointima hyperplasia. These results suggest that the clinical efficacy of SES against restenosis might be achieved, at least in part, through its inhibitory effect on circulating SM progenitors.
In the present study, local administration of sirolimus also potently reduced the numbers of BM-derived CD45-positive hematopoietic cells in the neointima, which was associated with a significant reduction in the total number of -SMA–positive cells in the neointima. It is possible that the inhibitory effect of sirolimus on neointima hyperplasia was also mediated by inhibition of the local inflammatory response after severe vascular injury. Consistent with this interpretation, it was reported that BM-derived immune cells mediate vascular repair and regeneration by the proliferation of hematopoietic and nonhematopoietic cells.9
Sirolimus potently inhibited differentiation of MNCs into EC-like cells at a concentration as low as 0.1 ng/mL. Sirolimus also inhibited the proliferation of human vascular ECs at 1.0 ng/mL or higher concentrations. Furthermore, locally delivered sirolimus potentially affected reendothelialization after wire-mediated vascular injury. It was reported that a decrease in circulating endothelial progenitor cells is associated with a high incidence of cardiovascular disease.18,19 Thus, inhibition of endothelial progenitor cells may result in delayed reendothelialization and wound healing after stent implantation, which may lead to fatal late thrombosis.20 In fact, it was observed that oral administration of everolimus, a macrolide of the same family as sirolimus, results in delayed endothelial coverage over the stent surface with loose EC junction, although in-stent neointimal growth was suppressed.21 Moreover, there have been autopsy case reports that revealed late stent thrombosis22 or delayed reendothelialization23 more than 18 months after SES deployment.
There are controversies about the potential of BM cells to differentiate into other lineages.4,24,25 In the present study, BM-derived cells substantially contributed to neointimal formation after vascular injury, as reported by us and others.4,9,11 Many BM-derived cells in neointima expressed -SMA as detected by an antibody (clone 1A4); however, we seldom detected BM-derived neointimal cells that were positive for SM1.15 There were -SMA–positive cells that expressed CD45, the pan-hematopoietic marker.25 Thus, it might be a rare property of BM cells to transdifferentiate into fully differentiated SMCs in vascular lesions, at least at 4 weeks after injury, although a significant portion of the neointima could be formed by BM-derived SMC-like cells.4,9,11 Consistent with this notion, numerous reports have documented that neointimal SMCs are quite distinct from medial cells in morphology and gene expression.16,26 It was reported that putative circulating fibrocytes can express -SMA after 3 weeks in culture.27
In conclusion, our results suggest that inhibitory effects on SM progenitor cells and on inflammatory cells may mediate the clinical efficacy of SES. We should be cautious about the potential deleterious effects of SES on reendothelialization.
Acknowledgments
This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (15659180, 15390241, 15039213, 16046204, and 16659194 to Dr Sata).
References
Jacobs AK. Coronary stents: Have they fulfilled their promise; N Engl J Med. 1999; 341: 2005–2006.
Marks AR. Sirolimus for the prevention of in-stent restenosis in a coronary artery. N Engl J Med. 2003; 349: 1307–1309.
Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med. 2003; 349: 1315–1323.
Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002; 8: 403–409.
Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation. 2002; 106: 1199–1204.
Caplice NM, Bunch TJ, Stalboerger PG, Wang S, Simper D, Miller DV, Russell SJ, Litzow MR, Edwards WD. Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc Natl Acad Sci U S A. 2003; 100: 4754–4759.
Fukuda D, Shimada K, Tanaka A, Kawarabayashi T, Yoshiyama M, Yoshikawa J. Circulating monocytes and in-stent neointima after coronary stent implantation. J Am Coll Cardiol. 2004; 43: 18–23.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
Boehm M, Olive M, True AL, Crook MF, San H, Qu X, Nabel EG. Bone marrow–derived immune cells regulate vascular disease through a p27(Kip1)-dependent mechanism. J Clin Invest. 2004; 114: 419–426.
Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A. 1997; 94: 3789–3794.
Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res. 2003; 93: 783–790.
Sata M, Maejima Y, Adachi F, Fukino K, Saiura A, Sugiura S, Aoyagi T, Imai Y, Kurihara H, Kimura K, Omata M, Makuuchi M, Hirata Y, Nagai R. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J Mol Cell Cardiol. 2000; 32: 2097–2104.
Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. Circ Res. 1995; 76: 412–417.
Zohlnhofer D, Klein CA, Richter T, Brandl R, Murr A, Nuhrenberg T, Schomig A, Baeuerle PA, Neumann FJ. Gene expression profiling of human stent-induced neointima by cDNA array analysis of microscopic specimens retrieved by helix cutter atherectomy: detection of FK506-binding protein 12 upregulation. Circulation. 2001; 103: 1396–1402.
Arakawa E, Hasegawa K, Irie J, Ide S, Ushiki J, Yamaguchi K, Oda S, Matsuda Y. L-ascorbic acid stimulates expression of smooth muscle–specific markers in smooth muscle cells both in vitro and in vivo. J Cardiovasc Pharmacol. 2003; 42: 745–751.
Zohlnhofer D, Klein CA, Richter T, Brandl R, Murr A, Nuhrenberg T, Schomig A, Baeuerle PA, Neumann FJ. Gene expression profiling of human stent-induced neointima by cDNA array analysis of microscopic specimens retrieved by helix cutter atherectomy: detection of FK506-binding protein 12 upregulation. Circulation. 2001; 103: 1396–1402.
Marx SO, Marks AR. Bench to bedside: the development of rapamycin and its application to stent restenosis. Circulation. 2001; 104: 852–855.
Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003; 348: 593–600.
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1–E7.
Farb A, Burke AP, Kolodgie FD, Virmani R. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation. 2003; 108: 1701–1706.
Farb A, John M, Acampado E, Kolodgie FD, Prescott MF, Virmani R. Oral everolimus inhibits in-stent neointimal growth. Circulation. 2002; 106: 2379–2384.
Virmani R, Guagliumi G, Farb A, Musumeci G, Grieco N, Motta T, Mihalcsik L, Tespili M, Valsecchi O, Kolodgie FD. Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious; Circulation. 2004; 109: 701–705.
Guagliumi G, Virmani R, Musumeci G, Motta T, Valsecchi O, Bonaldi G, Saino A, Tespili M, Greco N, Farb A. Drug-eluting versus bare metal coronary stents: long-term human pathology: findings from different coronary arteries in the same patient. Ital Heart J. 2003; 4: 713–720.
Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R, Helisch A, Schaper W. Bone marrow–derived cells do not incorporate into the adult growing vasculature. Circ Res. 2004; 94: 230–238.
Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002; 297: 2256–2259.
Walsh K, Perlman H. Molecular strategies to inhibit restenosis: modulation of the vascular myocyte phenotype. Semin Interv Cardiol. 1996; 1: 173–179.
Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 2004; 114: 438–446.(Daiju Fukuda, MD; Masatak)
PRESTO (M.S.), Japan Science and Technology Agency Kawaguchi, Saitama, Japan
Abstract
Background— Neointimal hyperplasia is the major cause of in-stent restenosis (ISR). The sirolimus-eluting stent (SES) has emerged as a promising therapy to prevent ISR; however, the exact mechanism by which locally delivered sirolimus, an immunosuppressive agent, prevents ISR remains unknown. Recent evidence suggests that circulating progenitor cells may contribute to neointimal formation.
Methods and Results— Mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers. Smooth muscle (SM)–like cells outgrew from the culture of MNCs (1x106) in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas endothelial cell–like cells were obtained in the presence of vascular endothelial growth factor. Sirolimus potently inhibited SM-like cell outgrowth. The number of SM-like cells was significantly reduced at a concentration as low as 0.1 ng/mL (15.9±5.8% of control, P<0.001). Sirolimus also exerted an inhibitory effect on endothelial cell–like cells that originated from MNCs. Wire-mediated vascular injury was induced in femoral arteries of bone marrow chimeric mice. Either vehicle or sirolimus was administered locally to the perivascular area of the injured arteries. Sirolimus significantly reduced neointima hyperplasia at 4 weeks (intima/media ratio 2.0±0.3 versus 1.0±0.2, P<0.05) with a decreased number of bone marrow–derived SM-like cells and hematopoietic cells in the lesion. Reendothelialization was retarded in the arteries treated with sirolimus.
Conclusions— The potent inhibitory effects of sirolimus on circulating smooth muscle progenitor cells may mediate the clinical efficacy of SES, at least in part. Sirolimus potentially may affect reendothelialization after stent implantation.
Key Words: stents ; restenosis ; balloon ; atherosclerosis ; endothelium
Introduction
Implantation of coronary stents has been shown to reduce the risk of periprocedural complications and restenosis more than balloon angioplasty alone.1 However, in-stent restenosis (ISR) remains a significant clinical problem limiting the long-term success of percutaneous treatment.1 The principal cause of ISR is neointimal hyperplasia resulting from the excessive accumulation of smooth muscle cells (SMCs). Recently, the sirolimus-eluting stent (SES) has emerged as a promising strategy to prevent ISR.2,3 A randomized, double-blind trial demonstrated that the use of SES reduced the rates of restenosis and clinical events even in patients with complex lesions.3 Despite increasing clinical interest in SES, very little is known about the mechanism by which locally delivered sirolimus, a hydrophobic macrolide with potent immunosuppressive activity, prevents ISR.2
We and others suggested that bone marrow (BM) cells give rise to vascular progenitor cells that home in on injured arteries and differentiate into SMCs or endothelial cells (ECs), thereby contributing to vascular repair and lesion formation.4–9 Here, we investigated the effects of sirolimus on human vascular progenitor cells that may contribute to neointimal hyperplasia and reendothelialization after stent implantation. The in vivo effect of sirolimus was also evaluated in a model of neointimal hyperplasia with bone marrow chimeric mice. Results suggest that sirolimus has a potent inhibitory effect on both smooth muscle progenitor cell and endothelial progenitor cell incorporation at the sites of vascular lesions.
Methods
Cell Culture
Peripheral mononuclear cells (MNCs) were isolated from blood of healthy human volunteers by density gradient centrifugation with HISOPAQUE-1077 (Sigma). MNCs were cultured at a density of 1x106 cells per fibronectin-coated well in a 96-well dish in HuMedia-SG2 (KURABO) supplemented with 10 ng/mL platelet-derived growth factor (PDGF)-BB and 10 ng/mL basic fibroblast growth factor (smooth muscle progenitor cell medium). The same number of MNCs were cultured in EBM (Clonetics) supplemented with 1 μg/mL hydrocortisone, 3 μg/mL bovine brain extract, 10 ng/mL vascular endothelial growth factor, and 20% FBS (endothelial progenitor cell medium). Sirolimus (Wako Pure Chemical) was added to the culture medium at a concentration of 0.1 to 100 ng/mL. At 4, 8, and 12 days, the culture medium was changed, and nonadherent cells were removed. Jurkat cells and Sertoli cells were purchased from the American Type Culture Collection (Rockville, Md). Human aortic SMCs (HASMCs) were purchased from KURABO and cultured in HuMedia-SG2. Human umbilical vein ECs (HUVECs) were purchased from Clonetics and cultured in EGM-2 (Clonetics). HASMCs or HUVECs were seeded at a density of 5x103 cells per well in 24-well plates. After culture for 72 hours in the presence of sirolimus, cells were counted.
Immunocytochemistry
To identify smooth muscle (SM)–like cells, adherent cells were fixed at 14 days in 4% paraformaldehyde and permeabilized with 0.5% NP40. After blocking with 1% goat serum, cells were incubated with an alkaline phosphatase–conjugated anti--smooth muscle actin (-SMA) antibody (Sigma) and stained with a Vector red substrate kit (Vector). To identify EC-like cells, adherent cells were incubated with 10 μg/mL acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyamine perchlorate (DiI-Ac-LDL; Biomedical Technologies Inc) for 4 hours. Cells were washed in PBS, fixed with 4% formaldehyde, and counterstained with FITC-labeled lectin from Bandeiraea simplicifolia (FITC-BS lectin, Sigma). Cells that were positive for both DiI-Ac-LDL and FITC-BS lectin were deemed EC-like cells, as described previously.8
Reverse Transcription–Polymerase Chain Reaction
Total RNA was prepared with RNazol reagent (Tel-Test, Inc). Reverse transcription was performed with 1 μg of RNA, random hexamer primers, and MMLV reverse transcriptase (ReverTraAce-, TOYOBO). Polymerase chain reaction (PCR) primers were as follows: -SMA 5'-CCAGCTATGTGTGAAGAAGAG-3' and 5'-GTGATCTCCTTCTGCATTCGGT-3'; GAPDH 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. PCR reactions were performed for 30 cycles of 30-second denaturation at 94°C, 30 seconds of annealing at 60°C, and 1 minute of extension at 72°C followed by 10 minutes of final extension. PCR for FK506-binding protein 12 (FKBP12) was performed with a primer set of 5'-GAGAAAAGCCAGCATAAAGC-3' and 5'-TCTAGAACTTCGGTGGAAAG -3' for 30 cycles of denaturation (94°C, 15 seconds), annealing (48°C, 30 seconds), and polymerization (68°C, 45 seconds). After agarose gel (3%) electrophoresis in the presence of ethidium bromide, the PCR products were revealed by ultraviolet irradiation.
Bone Marrow Transplantation
LacZ mice, which are knock-in mice that express the LacZ gene in essentially all tissues (C57BL/6x129S background),10 were originally purchased from Jackson Laboratory (B6; 129S-Gtrosa26, stock No. 002073, Bar Harbor, Me). LacZ mice were maintained in our animal facility and intercrossed with C57BL/6J mice. The resulting littermates were used for the present study.4 The homozygotes and wild-type mice were screened by PCR analysis of tail DNA according to the protocol provided by Jackson Laboratory. GFP mice, which are transgenic mice (C57BL/6 background) that ubiquitously express enhanced green fluorescent protein (GFP), were a generous gift from Dr Masaru Okabe (Osaka University, Osaka, Japan). Bone marrow transplantation (BMT) was performed from LacZ mice to wild-type mice (BMTLacZWild) or GFP mice to wild-type mice (BMTGFPWild) as described previously.4,11 A total of 85% to 95% of peripheral leukocytes had been reconstituted as determined by in situ hybridization for Y chromosome (BMTMale LacZFemale Wild mice) or flow cytometry (BMTGFPWild mice). All procedures that involved experimental animals were performed in accordance with protocols approved by the institutional committee for animal research of the University of Tokyo and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).
Vascular Injury and Local Delivery of Sirolimus
Twenty weeks after BMT, both femoral arteries of BM chimeric mice were injured by inserting a straight spring wire (0.38 mm in diameter, No. C-SF-15-15, Cook) as described previously.12 Sirolimus was suspended in DMSO at a concentration of 25 μg/μL. After vascular injury, 25 μg (n=8) of sirolimus suspended in 50 μL of 20% Pluronic F-127 gel (Sigma) was administered to the perivascular area of the right femoral artery in BMTLacZWild mice, and the same volume of Pluronic gel containing only DMSO was administered around the left femoral artery.
X-Gal Staining and Morphometric Analysis
Four weeks after surgery, mice were euthanized with an overdose of pentobarbital and perfused at a constant pressure via the left ventricle with 0.9% sodium chloride solution. The injured arteries were excised and stained with X-gal (5-bromo-4-chloro-3-indolyl ;-D-galactopyranoside) as described previously.4,11 The arteries were embedded in paraffin, and cross sections were made as described previously.12 After counterstaining with hematoxylin, morphometric analysis was performed with image-analysis software (Image-Pro Plus version 4.5, Media Cybernetics, USA) as described previously.12 Intima/media ratio and the number of LacZ-positive cells were averaged on 3 different sections from each artery.
Immunofluorescence Staining
To study the effect of sirolimus on reendothelialization, cross sections were incubated with an anti-CD31 antibody (clone MEC13.3, BD Biosciences) and Cy3-conjugated anti--SMA antibody (Sigma), followed by incubation with an FITC-conjugated anti-rat Ig secondary antibody (Jackson ImmunoResearch). To characterize the BM-derived cells observed in the neointima of BMTLacZWild mice, X-gal–stained cross sections were incubated with a Cy3-conjugated anti--SMA antibody and an anti-CD45 antibody (BD Biosciences), followed by incubation with an FITC-conjugated anti-rat Ig secondary antibody. The femoral arteries harvested from BMTGFPWild mice were embedded in plastic resin (Technovit 8100, Heraeus Kulzer) as described previously.11 An anti-SM1 rat monoclonal antibody was kindly provided by Dr Kazuhide Hasegawa (Kyowa Hakko, Japan). Thin sections (4 μm) were incubated with Cy3-conjugated anti--SMA antibody or anti-SM1 antibody followed by Cy3-conjugated anti-rat Ig antibody. After immunofluorescence staining, nuclei were counterstained with Hoechst 33258 (Sigma). The sections were mounted with the Prolong Antifade Kit (Molecular Probes) and observed under a PROVIS AX80 microscope (Olympus) equipped with a mercury/halogen dual-illumination system and a charge-coupled device camera (DP50, Olympus). High-resolution fluorescence images were taken with a confocal laser scanning system (FLUOVIEW FV300, Olympus).
Statistical Analysis
Values are expressed as mean±SEM for continuous variables. Comparisons of multiple groups were made by 1-way ANOVA, followed by Scheffe multiple comparison test. A probability value <0.05 was considered statistically significant.
Results
Potent Inhibitory Effect of Sirolimus on SM Progenitor Cells
Human peripheral MNCs were cultured in fibronectin-coated wells in the presence of PDGF-BB and basic fibroblast growth factor (SM progenitor cell medium). When the medium was changed at 4 days, there were adherent cells that consisted of heterogeneous cell types. Some cells remained round, whereas others showed a spindlelike shape and were displayed as a monolayer. At 14 days, cells with a polygonal and stellate shape became dominant. Consistent with a previous report,5 mRNA for -SMA was detected in adherent cells (Figure 1A). Immunocytochemistry revealed that >90% of adherent cells at 14 days were positive for -SMA (Figure 1B). Sirolimus had little effect on the number of adherent cells at 4 days; however, the number of -SMA–positive cells at 14 days was dramatically decreased by sirolimus (Figure 1B and 1C). At a concentration as low as 0.1 ng/mL, sirolimus significantly reduced the number of -SMA–positive cells to 15.9±5.8% of control (P<0.0001).
Effects of Sirolimus on Endothelial Progenitors, ECs, and SMCs
Sirolimus also inhibited the proliferation of HASMCs and HUVECs in a dose-dependent manner (data not shown); however, its inhibitory effect on these cells was relatively mild (Figure 2A). There was no significant reduction in cell number by sirolimus at 0.1 ng/mL, whereas at higher concentrations (1 ng/mL), sirolimus inhibited the proliferation of HASMCs (47.0±4.8% of control, P<0.001) and HUVECs (40.7±5.9% of control, P<0.05). Sirolimus had no obvious effect on the morphology of HASMCs and HUVECs. Human MNCs were also plated on fibronectin-coated 96-well plates and cultured in EBM with bovine brain extract, vascular endothelial growth factor, and 20% FBS (endothelial progenitor cell medium). Under these conditions, attached cells became spindle-shaped and were doubly stained by DiI-Ac-LDL and FITC-BS lectin.8 Sirolimus potently reduced the number of EC-like cells at 14 days in a dose-dependent manner. At a concentration as low as 0.1 ng/mL, sirolimus significantly reduced the number of EC-like cells to 36.7±1.7% of control (P<0.01). To obtain an insight into the mechanism by which sirolimus selectively inhibits differentiation of MNCs into SM-like or EC-like cells, we investigated the expression of FKBP12, which is known to be a receptor of sirolimus.2,13 Consistent with previous reports,14 HASMCs isolated from the aortic medial layer expressed little FKBP12 (Figure 2B). On the other hand, FKBP12 was abundantly expressed in human MNCs.
Effects of Locally Delivered Sirolimus on Neointimal Formation and Reendothelialization After Vascular Injury
The in vivo effects of sirolimus on vascular progenitor cells were studied in BM chimeric mice with a mouse model of vascular injury that induces reproducible neointima hyperplasia.11,12 At 4 weeks after injury, there was neointima hyperplasia that contained BM-derived LacZ-positive cells in the BMTLacZWild mice. Local delivery of sirolimus significantly reduced neointimal formation (intima/media ratio 2.0±0.3 versus 1.0±0.2, P=0.03; Figure 3A and 3B). The reduction in neointimal hyperplasia was associated with a decrease in the number of LacZ-positive cells in the neointima (number of LacZ-positive cells/neointima 20.2±4.1 versus 7.3±2.4, P=0.02). In the lesion treated with vehicle, 55.1±26.1% of the luminal side was coated with the regenerated endothelium as determined by anti-CD31 immunostaining. On the other hand, endothelium was detected only in 7.7±3.4% of the luminal side of the sirolimus-treated arteries. Taken together, these results indicate that locally delivered sirolimus possibly affects the reendothelialization process after severe vascular injury, although neointima hyperplasia is markedly attenuated (Figure 3C).
Characterization of BM-Derived Neointimal Cells
BM-derived cells observed in neointima were characterized by confocal immunofluorescence study. The wire injury induced vascular lesions that were predominantly composed of -SMA–positive cells.12 In BMTGFPWild mice, we readily detected GFP-positive intimal cells that were positive for -SMA (Figure 4A). On the other hand, there were few neointimal cells that expressed both GFP and SM1, a marker for differentiated SMCs (Figure 4B).15 These results suggest that BM-derived -SMA–positive cells do not become highly differentiated SMCs, at least at 4 weeks after wire injury.11 In BMTLacZWild mice, most of the BM-derived neointimal cells expressed -SMA. There were BM-derived cells that expressed both -SMA and CD45, a pan-hematopoietic marker (Table).4 Local administration of sirolimus potently reduced the number of LacZ+/-SMA+ cells in the neointima (Figure 4C and 4D). Sirolimus also reduced the number of LacZ+/CD45+ cells. Reduction in the number of LacZ+ cells in neointima was associated with a reduction in the total number of -SMA–positive neointimal cells (Figure 4E).
Expression of -SMA and CD45 by LacZ-Positive Cells in Neointima
Discussion
It is assumed that sirolimus locally delivered from SES prevents neointimal hyperplasia by inhibiting migration and proliferation of SMCs that originate from the adjacent media.2,13 However, it was reported that medial SMCs express little FKBP12,16 which sirolimus binds to prevent cell-cycle progression through the upregulation of the cyclin-dependent kinase inhibitor p27kip1.2,17 Thus, it is likely that sirolimus may also target cell types other than medial SMCs. Recent reports suggested that circulating progenitor cells may contribute to neointimal hyperplasia in models of transplant-associated arteriosclerosis, hyperlipidemia-induced atherosclerosis, and postangioplasty restenosis.4–7 It has been consistently reported that SM-like cells with specific growth, adhesion, and integrin profiles could grow from human peripheral MNCs.5 Moreover, a significant part of SMCs throughout the atherosclerotic vessel wall was shown to derive from donor BM in gender-mismatched BMT patients.6 Thus, it is possible that circulating cells may participate in the pathogenesis of human ISR.
In the present study, sirolimus potently inhibited MNC differentiation into SM-like cells. The inhibitory effect was achieved at a concentration as low as 0.1 mg/mL, at which sirolimus had no significant effect on the proliferation of HASMCs that expressed low FKBP12. In fact, in our mouse model of severe vascular injury, locally derived sirolimus efficiently decreased the number of BM-derived cells in the neointima and attenuated neointima hyperplasia. These results suggest that the clinical efficacy of SES against restenosis might be achieved, at least in part, through its inhibitory effect on circulating SM progenitors.
In the present study, local administration of sirolimus also potently reduced the numbers of BM-derived CD45-positive hematopoietic cells in the neointima, which was associated with a significant reduction in the total number of -SMA–positive cells in the neointima. It is possible that the inhibitory effect of sirolimus on neointima hyperplasia was also mediated by inhibition of the local inflammatory response after severe vascular injury. Consistent with this interpretation, it was reported that BM-derived immune cells mediate vascular repair and regeneration by the proliferation of hematopoietic and nonhematopoietic cells.9
Sirolimus potently inhibited differentiation of MNCs into EC-like cells at a concentration as low as 0.1 ng/mL. Sirolimus also inhibited the proliferation of human vascular ECs at 1.0 ng/mL or higher concentrations. Furthermore, locally delivered sirolimus potentially affected reendothelialization after wire-mediated vascular injury. It was reported that a decrease in circulating endothelial progenitor cells is associated with a high incidence of cardiovascular disease.18,19 Thus, inhibition of endothelial progenitor cells may result in delayed reendothelialization and wound healing after stent implantation, which may lead to fatal late thrombosis.20 In fact, it was observed that oral administration of everolimus, a macrolide of the same family as sirolimus, results in delayed endothelial coverage over the stent surface with loose EC junction, although in-stent neointimal growth was suppressed.21 Moreover, there have been autopsy case reports that revealed late stent thrombosis22 or delayed reendothelialization23 more than 18 months after SES deployment.
There are controversies about the potential of BM cells to differentiate into other lineages.4,24,25 In the present study, BM-derived cells substantially contributed to neointimal formation after vascular injury, as reported by us and others.4,9,11 Many BM-derived cells in neointima expressed -SMA as detected by an antibody (clone 1A4); however, we seldom detected BM-derived neointimal cells that were positive for SM1.15 There were -SMA–positive cells that expressed CD45, the pan-hematopoietic marker.25 Thus, it might be a rare property of BM cells to transdifferentiate into fully differentiated SMCs in vascular lesions, at least at 4 weeks after injury, although a significant portion of the neointima could be formed by BM-derived SMC-like cells.4,9,11 Consistent with this notion, numerous reports have documented that neointimal SMCs are quite distinct from medial cells in morphology and gene expression.16,26 It was reported that putative circulating fibrocytes can express -SMA after 3 weeks in culture.27
In conclusion, our results suggest that inhibitory effects on SM progenitor cells and on inflammatory cells may mediate the clinical efficacy of SES. We should be cautious about the potential deleterious effects of SES on reendothelialization.
Acknowledgments
This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (15659180, 15390241, 15039213, 16046204, and 16659194 to Dr Sata).
References
Jacobs AK. Coronary stents: Have they fulfilled their promise; N Engl J Med. 1999; 341: 2005–2006.
Marks AR. Sirolimus for the prevention of in-stent restenosis in a coronary artery. N Engl J Med. 2003; 349: 1307–1309.
Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med. 2003; 349: 1315–1323.
Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002; 8: 403–409.
Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation. 2002; 106: 1199–1204.
Caplice NM, Bunch TJ, Stalboerger PG, Wang S, Simper D, Miller DV, Russell SJ, Litzow MR, Edwards WD. Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc Natl Acad Sci U S A. 2003; 100: 4754–4759.
Fukuda D, Shimada K, Tanaka A, Kawarabayashi T, Yoshiyama M, Yoshikawa J. Circulating monocytes and in-stent neointima after coronary stent implantation. J Am Coll Cardiol. 2004; 43: 18–23.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
Boehm M, Olive M, True AL, Crook MF, San H, Qu X, Nabel EG. Bone marrow–derived immune cells regulate vascular disease through a p27(Kip1)-dependent mechanism. J Clin Invest. 2004; 114: 419–426.
Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A. 1997; 94: 3789–3794.
Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res. 2003; 93: 783–790.
Sata M, Maejima Y, Adachi F, Fukino K, Saiura A, Sugiura S, Aoyagi T, Imai Y, Kurihara H, Kimura K, Omata M, Makuuchi M, Hirata Y, Nagai R. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J Mol Cell Cardiol. 2000; 32: 2097–2104.
Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. Circ Res. 1995; 76: 412–417.
Zohlnhofer D, Klein CA, Richter T, Brandl R, Murr A, Nuhrenberg T, Schomig A, Baeuerle PA, Neumann FJ. Gene expression profiling of human stent-induced neointima by cDNA array analysis of microscopic specimens retrieved by helix cutter atherectomy: detection of FK506-binding protein 12 upregulation. Circulation. 2001; 103: 1396–1402.
Arakawa E, Hasegawa K, Irie J, Ide S, Ushiki J, Yamaguchi K, Oda S, Matsuda Y. L-ascorbic acid stimulates expression of smooth muscle–specific markers in smooth muscle cells both in vitro and in vivo. J Cardiovasc Pharmacol. 2003; 42: 745–751.
Zohlnhofer D, Klein CA, Richter T, Brandl R, Murr A, Nuhrenberg T, Schomig A, Baeuerle PA, Neumann FJ. Gene expression profiling of human stent-induced neointima by cDNA array analysis of microscopic specimens retrieved by helix cutter atherectomy: detection of FK506-binding protein 12 upregulation. Circulation. 2001; 103: 1396–1402.
Marx SO, Marks AR. Bench to bedside: the development of rapamycin and its application to stent restenosis. Circulation. 2001; 104: 852–855.
Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003; 348: 593–600.
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1–E7.
Farb A, Burke AP, Kolodgie FD, Virmani R. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation. 2003; 108: 1701–1706.
Farb A, John M, Acampado E, Kolodgie FD, Prescott MF, Virmani R. Oral everolimus inhibits in-stent neointimal growth. Circulation. 2002; 106: 2379–2384.
Virmani R, Guagliumi G, Farb A, Musumeci G, Grieco N, Motta T, Mihalcsik L, Tespili M, Valsecchi O, Kolodgie FD. Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious; Circulation. 2004; 109: 701–705.
Guagliumi G, Virmani R, Musumeci G, Motta T, Valsecchi O, Bonaldi G, Saino A, Tespili M, Greco N, Farb A. Drug-eluting versus bare metal coronary stents: long-term human pathology: findings from different coronary arteries in the same patient. Ital Heart J. 2003; 4: 713–720.
Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R, Helisch A, Schaper W. Bone marrow–derived cells do not incorporate into the adult growing vasculature. Circ Res. 2004; 94: 230–238.
Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002; 297: 2256–2259.
Walsh K, Perlman H. Molecular strategies to inhibit restenosis: modulation of the vascular myocyte phenotype. Semin Interv Cardiol. 1996; 1: 173–179.
Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 2004; 114: 438–446.(Daiju Fukuda, MD; Masatak)