Stem Cell Factor Deficiency Is Vasculoprotective
http://www.100md.com
Chao-Hung Wang, Nicole Anderson, Shu-Hon
参见附件。
Cardiology and Internal Medicine (C.-H.W., W.-J.C.), Chang Gung Memorial Hospital, Keelung, Chang Gung University College of Medicine, Taiwan
the Institute of Medical Science (N.A., W.L.S.), Department of Internal Medicine (P.E.S.), Institute of Biomaterials and Biomedical Engineering (W.L.S.)
Department of Chemical Engineering and Applied Chemistry (W.L.S.), University of Toronto, Canada
Cardiac Surgery (C.-H.W., S.-H.L., P.W.M.F., S.F., R.-K.L., T.M.Y., R.D.W.), Toronto General Hospital, Canada
Division of Cardiac Surgery (S.V.), St. Michael’s Hospital, Toronto, Canada.
Abstract
Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth muscle cells (SMCs) that contribute to intimal hyperplasia after vascular injury; however, the molecular pathways involved and the timeline of these events remain poorly characterized. We hypothesized that the stem cell factor (SCF)/c-Kit tyrosine kinase signaling pathway is critical to neointimal formation by BM-derived progenitors. Wire-induced femoral artery injury in mice reconstituted with wild-type BM cells expressing yellow fluorescent protein was performed, which revealed that 66±12% of the SMCs (-smooth muscle actin-positive [SMA+] cells) in the neointima were from BM. To characterize the role of the SCF/c-Kit pathway, we used c-Kit deficient W/Wv and SCF-deficient Steel-Dickie mice. Strikingly, vascular injury in these mice resulted in almost a complete inhibition of neointimal formation, whereas wild-type BM reconstitution of c-Kit mutant mice led to neointimal formation in a similar fashion as wild-type animals, as did chronic administration of SCF in matrix metalloproteinase-9–deficient mice, a model of soluble SCF deficiency. Pharmacological antagonism of the SCF/c-Kit pathway with imatinib mesylate (Gleevec) or ACK2 (c-Kit antibody) also resulted in a marked reduction in intimal hyperplasia. Vascular injury resulted in the local upregulation of SCF expression. c-Kit+ progenitor cells (PCs) homed to the injured vascular wall and differentiated into SMA+ cells. Vascular injury also caused an increase in circulating SCF levels which promoted CD34+ PC mobilization, a response that was blunted in mutant and imatinib mesylate-treated mice. In vitro, SCF promoted adhesion of BM PCs to fibronectin. Additionally, anti-SCF antibodies inhibited adhesion of BM PCs to activated SMCs and diminished SMC differentiation. These data indicate that SCF/c-Kit signaling plays a pivotal role in the development of neointima by BM-derived PCs and that the inhibition of this pathway may serve as a novel therapeutic target to limit aberrant vascular remodeling.
Key Words: vascular remodeling genetic mice models vascular biology vascular smooth muscle
Introduction
The majority of hematopoietic stem cells (HSCs) and various progenitors, including endothelial progenitor cells (EPCs), reside within the osteoblastic zone of the bone marrow (BM), the so-called stem cell niche. Tissue injury releases chemical cues that induce mobilization of cells within the niche into the peripheral blood. Along with inflammatory cells, stem cells and progenitors released from the niche follow chemotactic signals to the injured tissue. Myocardial infarction (MI) or vascular injury elicit BM cell mobilization and localized inflammatory responses, required for cardiovascular repair and remodeling.1–3 In addition to innate immune cell participation in repair, mobilized vascular endothelial and smooth muscle progenitors participate in angiogenesis and vascular repair.1,4 Other regenerative roles of BM-derived cells in the heart remain controversial and have not been clearly defined, but include cardiogenic differentiation,5–8 cardiac cell rescue by myelomonocytic cell fusion,7 or the release of anti-apoptotic paracrine factors.9 Regardless of the mechanism, induced mobilization or intracardiac injection of BM cells improves cardiac function following MI in animal models,6,10 which has stimulated numerous clinical trials testing the ability of BM cells either directly infused into hearts or mobilized into the periphery to improve cardiac function following MI.11,12
However, not all BM responses to cardiovascular injury are regenerative in nature, with recent studies demonstrating that mobilized BM cells can be a source of pathogenic vascular remodeling. Neointimal formation, with resultant vascular remodeling, is a unifying pathological event, complicating chronic atherosclerosis, restenosis, and transplant arteriopathy, and remains the major limiting factor for the long-term efficacy of vascular interventions, such as angioplasty13 and coronary artery bypass graft surgery.14 Although the process of neointimal formation following vascular injury was formerly ascribed solely to a local smooth muscle proliferative response, it has been demonstrated to be regulated significantly by transplantable BM-derived cells.1,8,15–17 Yet, the fundamental mechanism of neointimal hyperplasia by mobilized BM cells is unknown. Thus, the identification of the molecular and cellular mechanisms underlying regenerative and pathogenic cardiovascular remodeling by mobilized BM cells is critical so that new therapeutic approaches can be used to augment the regenerative potential of BM cells while preventing restenosis.
Stem cell factor (SCF) (also known as Steel factor) is expressed as a soluble or membrane-bound form and promotes survival,18 proliferation,19 mobilization,20 and adhesion21 of hematopoietic stem cells and progenitors through dimerization of its cognate receptor, the tyrosine kinase c-Kit.22 Granulocyte colony stimulating factor (G-CSF), stromal cell-derived factor 1 (SDF-1), or vascular endothelial growth factor (VEGF) mobilizes c-Kit+ progenitors and stem cells by inducing matrix metalloproteinase-9 (MMP-9) expression, which then cleaves membrane bound SCF (mSCF), activating c-Kit-induced proliferation and mobilization.23,24 Thus, the release of mSCF by MMP-9 following vascular injury, and the resulting c-Kit tyrosine kinase signaling, may play a mechanistic role in orchestrating the contribution of stem cell-derived vascular progenitors to vascular disease. We used both mutant and BM-reconstituted mice to provide definitive evidence that the SCF/c-Kit signaling pathway is critical to pathological neointimal formation following vascular injury.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animals Studies
Wild-type 129S6 mice, MMP-9–/–, W/Wv (WBB6F1 hybrid strain), colony control WBB6F1 (+/+), Sl/Sld (Steel-Dickie; WCB6F1hybrid strain), and colony control WCB6F1 (+/+) mice were used for the vascular injury studies. W/Wv mice have a relative deficiency of c-Kit kinase activity although the affected cells express normal to elevated levels of c-Kit receptor and Steel-Dickie mice have a complete deficiency of mSCF and reduced serum levels of soluble SCF (sSCF).
Results
BM Cells Contribute to Neointimal Formation
Following vascular injury, vascular smooth muscle cells (VSMCs) underwent extensive apoptosis (Figure I in the online data supplement), and vessels were completely denuded of endothelial cells. A significant increase in the number of VSMCs, or neointimal formation, occurred over a 4-week period (Figure 1A and supplemental Figure II and II1). In mice reconstituted with wild-type BM cells expressing yellow fluorescent protein (eYFP) (BMTYfpWild mice), 66±12% of the VSMCs (-smooth muscle actin-positive [SMA+] cells) in the neointima were eYFP+SMA+ (Figure 1B), confirming earlier reports that a significant portion of the neointima is formed by BM-derived cells.1 In addition to SMA expression, the BM-derived SMA+ cells express calponin, and caldesmon, but not for myelomonocytic markers.25 Local vascular injury has a positive impact on progenitor cell (PC) mobilization from the BM. CD45, a pan leukocyte marker, was used to estimate the contribution of inflammatory cells to the neointima. Many CD45+ BM-derived cells were present in the early phase after vascular injury (Figure 1C). However, as cells differentiated into SMA+ cells, few CD45+ cells could be identified (Figure 1D) suggesting that SMA+ progenitors were CD45–/low or that SMA+ progenitors were CD45+ but lost CD45 expression during differentiation.
Vascular Injury Activates SCF Expression
Vascular injury resulted in the local upregulation of SCF in the adventitia and remaining VSMCs (Figure 1E and supplemental Figure III). Within the adventitia, most of the SCF+ cells were found to be vimentin+ (likely fibroblasts) and some of them were CD45+ (likely inflammatory cells; supplemental Figure IV), but none were CD31+ (an endothelial cell marker). In vitro, preincubation with SCF enhanced platelet-derived growth factor (PDGF) secretion by platelets on activation in a dose-dependent manner (Figure 1F). PDGF is a known stimulator of VSMC migration and promoter of neointimal proliferation.26–28 In turn, PDGF upregulated SCF expression by VSMCs (Figure 1G), suggesting the creation of a positive feedback signaling loop between SCF and PDGF. Thus, the local platelet response shortly after vascular injury may serve to secrete PDGF followed by upregulation of SCF expression, which may trigger the ensuing systemic response to injury, setting the stage for vascular remodeling. Consistent with this hypothesis, c-Kit+ PCs homed to the injured vascular wall and differentiated into SMA+ cells (Figure 1H).
SCF and c-Kit Mutations in Mice Attenuate Neointimal Hyperplasia
To determine whether SCF/c-Kit signaling was an essential for pathological vascular remodeling in vivo, we made use of naturally occurring mutant strains of c-Kit and SCF, the W/Wv and Sl/Sld strains, respectively. W/Wv mice demonstrate 15% of wild-type c-Kit activity a severe reduction in hematopoietic stem cells. Sl/Sld mice express low levels of SCF and are deficient in membrane bound SCF (mSCF). Both W/Wv and Sl/Sld mice are anemic; however, the platelet and white blood cell counts are not significantly reduced. W/Wv and Sl/Sld and littermate control mice underwent femoral artery wire injury. Strikingly, in W/Wv and Sl/Sld mice, neointimal formation was significantly less than that occurring in littermate control mice (Figure 2A and 2B). To further test a causal role of SCF/c-Kit signaling in neointimal formation, we performed vascular injury following wild-type BM transplantation into W/Wv mice. These transplanted mice, having BM PCs with functioning c-Kit receptor tyrosine kinase, demonstrated marked intimal hyperplasia 28 days after wire injury, similar to wild-type mice. Thus, c-Kit appears to be absolutely required for postinjury vascular remodeling. Attempting to restore normal levels of SCF to Sl/Sld mice, we administered SCF (100 ng/kg per day) subcutaneously to these mice. Despite inducing extreme increases in plasma sSCF levels (from 0.52 ng/mL at baseline up to 10749, 4432 and 3329 ng/mL, respectively, at 6, 12, and 24 hours after injection), sSCF supplementation did not result in a significant increase in neointimal thickness (Figure 2A; Sl/Sld+SCF). Thus, SCF, namely mSCF, appears to also be required for pathological vascular remodeling to occur. sSCF cannot replace the role of mSCF in promoting neointimal formation.
Vascular Injury Increases Blood SCF Levels
Vascular injury not only upregulated local SCF expression but also caused an increase in circulating SCF levels. In wild-type mice, sSCF levels peaked on day 3 after injury and gradually returned to baseline levels (Figure 3A). In W/WV mice, sSCF tended to increase after injury, but baseline sSCF concentrations were more than 3.5-fold higher than in wild-type mice because of feedback mechanisms compensating for markedly reduced c-Kit activity. However, W/WV mice rescued by wild-type BM transplantation had much lower levels of sSCF than the W/WV mice (146±8 versus 315±10 ng/mL, P<0.001; wild-type values are 91±9 ng/mL). This phenomenon could explain why rescued W/Wv mice had only similar rather than more intimal hyperplasia compared with wild-type mice (Figure 2C). Plasma sSCF levels in Sl/Sld mice were substantially lower than in wild-type mice and only increased slightly on day 7 after injury. MMP-9–/– mice exhibited sSCF baseline concentrations that were similar to wild-type mice; however, the levels of sSCF did not change in response to vascular injury, as expected, because MMP-9 facilitates the shedding of mSCF.23,24 Plasma G-CSF levels transiently increased 1 day after vascular injury in all mice (Figure 3B), but the rate of decline in the vascular injured wild-type mice was slower than in the sham-operated mice.
MMP-9 Facilitates SCF Shedding
SCF levels increased following vascular injury because of upregulated MMP-9 activity within the injured vasculature. At 1 and 7 days after injury, SCF expression was detected at areas that also contained higher levels of MMP-9 (Figure 4A). In addition, our data suggested that MMP-9 was activated to cleave mSCF, which was expressed throughout the vessel wall (data not shown). Zymography demonstrated a marked upregulation of pro-MMP-9 and active MMP-9 1 day after injury, with the majority of MMP-9 being in the active form by day 7 (Figure 4B). Vascular injury in MMP-9–/– mice resulted in modest neointimal formation; however, when the mice were supplemented with sSCF for 2 weeks after vascular injury, a 2.5-fold increase in neointimal formation was observed (Figure 4C). Thus, MMP-9–induced release of SCF appears to be a prerequisite for aberrant vascular remodeling following injury. In the BM, femoral artery wire injury also induced a significant increase in MMP-9 activity. Pharmacological inhibition of MMP activity led to significantly increased mSCF+ cell numbers in response to the injury. However, without MMP inhibition, mSCF+ cell numbers increased transiently and then significantly decreased at later time points. These findings suggested that MMP-9 was activated inside the BM and cleaved mSCF, contributing in part to the increase in circulating sSCF levels (supplemental Figure V).
Vascular Injury Promotes Progenitor Cell Mobilization
As of yet, the markers of smooth muscle PCs have not been clearly defined. However, Simper et al reported that circulating smooth muscle PCs appear to originate from CD34+ cells.29 Thus, CD34 was used as a surrogate for PC mobilization in this study. The increase in sSCF levels following vascular injury in wild-type mice coincided with a significant increase in the number of circulating BM-derived CD34+ PCs (Figure 3C). The level of circulating CD34+ cells appeared to increase in 2 waves, rising immediately after vascular injury and again 9 days later. The effect of sSCF on CD34+ cell mobilization was confirmed (Figure 3D). However, injection of sSCF (100 ng/kg per day) could only increase the level of circulating CD34+ cells in MMP-9–/– mice and not in Sl/Sld mice, suggesting a requirement for mSCF in mobilization rather than only a change in serum SCF. Treatment with imatinib mesylate (Gleevec), a c-Kit-kinase inhibitor, inhibited both early and late CD34+ cell mobilization (Figure 3C). Similarly, Sl/Sld mice displayed impaired cell mobilization.
SCF Contributes to the Differentiation of Progenitor Cells After Vascular Injury
To ascertain whether SCF contributed to the homing and differentiation of PCs after vascular injury, we directly tested the influence of SCF on BM PC adhesion (because cell adhesion is required in homing) and smooth muscle differentiation. Negatively selected Lin– cells, which were enriched with c-Kit+ cells, were used in the adhesion experiments. Lin– cells rather than directly isolated c-Kit+ cells were used to avoid blocking the c-Kit receptor by the primary anti-c-Kit antibody used for the positive selection processes. SCF (200 ng/mL) induced a 2.3-fold increase in the adhesion of Lin– BM cells to fibronectin (Figure 5A). This effect was completely blocked by an anti-51 integrin antibody. In a second adhesion assay, rat VSMCs were activated by PDGF (10 ng/mL) to upregulate the expression of SCF. Activated VSMCs greatly increased the adhesion of Lin– BM cells, an interaction that was blocked by administration of anti-SCF antibody (Figure 5B and 5C). Furthermore, the coculture of eYFP+ Lin– BM cells with activated VSMCs for 11 days showed clusters of cells that expressed both eYFP and SMA (Figure 5D, top). Adding anti-SCF antibody to the coculture decreased the number of double-positive cells (Figure 5D, bottom), suggesting that VSMC activation, marked by SCF expression, is not only required for PC homing but also PC differentiation toward the smooth muscle cell (SMC) lineage.
c-Kit+ Cells Give Rise to SMA+ Cells
c-Kit+ and c-Kit– BM cells were purified and tested for their potential to differentiate into SMA+ cells in low-serum endothelial growth medium-2 (EGM-2). Compared with c-Kit– cells a week after plating, c-Kit+ cells exhibited increased survival and gave rise to more SMA+ cell colonies (approximately 5-fold increase) (Figure 6A). Once c-Kit+ cells expressed SMA+, they lost both the c-Kit and CD45 phenotypes. This also appeared to be the case in vivo. Although a significant amount of c-Kit+ cells were noted to adhere to the injured vascular wall in the littermate control mice (Figure 6B and 6C), once these c-Kit+ cells differentiated into SMA+ cell, c-Kit expression became very weak or disappeared (Figure 6D). These cells underwent a remarkable extent of proliferation to cause intimal hyperplasia (Figure 6E). However, in the Sl/Sld mice, essentially no c-Kit+ cells could be detected in the neointima at the early time point after injury (Figure 6C).
Blocking c-Kit Signaling Attenuates Neointimal Hyperplasia
Imatinib mesylate is a protein-tyrosine kinase inhibitor, which selectively blocks Abl tyrosine kinase and the c-Kit and PDGF receptor (PDGF-R) tyrosine kinases.30,31 Early intervention with imatinib mesylate significantly attenuated neointimal formation, an average of 300% reduction, whereas imatinib mesylate treatment that started at the hyperplasia phase was ineffective (Figure 7A and 7B). Because imatinib mesylate also inhibits PDGF-R signaling, which is well known to mediate SMC proliferation, we analyzed the kinetics of PDGF-R expression following injury. Figure 7C demonstrates that PDGF-R was primarily expressed within the adventitia and to a lesser degree by VSMCs and that the expression of PDGF-R by medial SMCs was upregulated beginning 9 days after vascular injury. In contrast, c-Kit+ cells appeared within the vessel medial wall by 1 day after vascular injury (supplemental Figure VI). Imatinib mesylate did not affect the sSCF and G-CSF levels after vascular injury (data not shown). Finally, we hypothesized that if the imatinib mesylate-mediated inhibition of neointimal hyperplasia was acting through the c-Kit pathway, then blocking c-Kit activation using the blocking anti-c-Kit receptor monoclonal antibody ACK2 would generate similar results. In fact, we found a significant attenuating effect by ACK2 on intimal hyperplasia (Figure 7D).
Discussion
We have defined a new mechanistic pathway of neointimal formation and adverse vascular remodeling, critically dependent on the interaction between SCF/c-Kit receptor signaling and the subsequent mobilization of BM-derived PCs. Our conclusions are based on in vivo studies performed in mutant models of SCF deficiency and c-Kit receptor deficiency, with additional supportive data from BM transplantation and pharmacological antagonism studies with imatinib mesylate and blocking c-Kit antibodies. PC mobilization from the BM and subsequent recruitment to the area of injury occurs shortly after the vascular insult; however, intimal hyperplasia accelerates between 14 to 28 days postinjury, possibly secondary to proliferation of the newly recruited PCs. The mobilization and recruitment of PCs from the BM requires the release of SCF via MMP-9 activation. SCF, PDGF, and G-CSF have all been shown to activate MMP-9.32–34 Our data suggest 2 waves of PC mobilization following vascular injury: an early, immediate response and a delayed response (Figure 3C). Although the early episode may represent a nonspecific response to injury, only mice that underwent vascular injury maintained elevated sSCF levels and displayed the second phase, which became apparent approximately 9 days after vascular injury, just as the neointima was beginning to develop.
Both MMP-9–/– and Sl/Sld mice did not sustain changes to sSCF levels in response to vascular injury (Figure 3A and 3B). The observation that supplementation with sSCF only exerted an effect on MMP-9–/– mice, which have normal mSCF but are unable to release it, suggests that mSCF plays a critical role not only in PC mobilization but also in homing and differentiation. mSCF serves as a source of sSCF after wire injury, which may subsequently promote adhesion of PCs to the exposed matrix on the injured vessel and stimulate PDGF release on platelet activation. mSCF plays a critical role in the homing of PCs to the injured vessel wall and mediates the differentiation of c-Kit+ PCs to VSMCs (Figure 5). Either a lack of mSCF or the blockade of the c-Kit/mSCF interaction markedly attenuated PC mobilization, homing, and differentiation. Cell adhesion is only 1 component of PC homing, and we have not investigated directed migration of PCs, which may also play a role in modulating the effects of SCF on PC homing. Although wild-type BM repopulation of W/Wv mice restored neointimal hyperplasia following injury, demonstrating the requirement of the SCF/c-Kit pathway and BM cells in our model, the reciprocal experiment could not be performed. Classic BM transplantation experiments performed in the 1960s demonstrated that W/Wv hematopoietic stem cells are incompetent to repopulate wild-type mice. Furthermore, wild-type BM cannot repopulate Sl/Sld mice because of a lack of SCF expressed by the stromal cells within the hematopoietic stem cell niche.35
Although we have clearly shown that restenosis in our mouse model is dependent on the c-Kit signaling pathway, how do these findings translate to human pathology and clinical practice For example, the mouse femoral artery injury model represents a severe vascular injury model, which requires the participation of BM-derived progenitors in the repair process, and it has been shown that less severe injury models do not necessarily recruit BM-mobilized cells.36 Although it is important to not overextrapolate mouse data to human clinical data, there are several lines of evidence suggesting that c-Kit/SCF-induced mobilization of BM progenitors is clinically relevant and that imatinib mesylate therapy should be considered. First, although the extent of vascular injury is considerably less severe in human angioplasty, in this era of extensive vascular stent intervention, a similar stress on the vessel wall, as observed in the mouse model, may exist, attracting BM cells through the SCF/c-Kit pathway as atherosclerotic plaques are being pushed outward. Supporting this hypothesis are findings that BM-derived cells including monocytes are mobilized following coronary stent implantation and the number of circulating cells directly correlated with in-stent neointimal volume.37 More directly, c-Kit+ vascular progenitors have been identified within postangioplasty restenosis lesions.38 Moreover, a recent clinical trial performed on MI patients designed to test the efficacy and safety of mobilization of BM progenitors to improve cardiac function was halted because of the high rate of restenosis. The MAGIC clinical trial performed G-CSF-mediated mobilization of BM progenitors on patients with MI who underwent coronary stenting. Although cardiac function improved in these patients, the study ended prematurely because patients receiving G-CSF experienced an unexpectedly high rate of postangioplasty restenosis.11 As discussed above, G-CSF-induced mobilization acts through MMP-9 to cleave mSCF and activate c-Kit+ to proliferation and mobilize into the periphery.
Imatinib mesylate prevented adverse vascular remodeling when administered early after vascular injury, at a stage when c-Kit signaling is essential for PC mobilization and recruitment. This finding was mirrored by intervention with ACK2, a neutralizing anti-c-Kit receptor monoclonal antibody, further strengthening our conclusion that the action of imatinib mesylate by blocking the SCF/c-Kit pathway. However, imatinib mesylate may also have a role in blocking postangioplasty restenosis mediated by local vascular SMCs by blocking PDGFR tyrosine kinase activity. PDGF is a well-known potent mitogenic and migratory factor for vascular SMCs and has been implicated in vascular pathologies.39 Imatinib mesylate, either alone or in combination with other treatments, has in some cases been demonstrated to reduce the extent of restenosis through its effect on PDGF receptors.40–42 Our data suggest that c-Kit signaling is upstream of the PDGF-mediated pathway recruitment of perivascular cells; however, much more work must be done to discover the biochemical and cellular interactions between these 2 tyrosine kinases pathways in neointima formation.
In summary, we demonstrated that the SCF/c-Kit pathway is a novel, important, and logical target for therapeutic intervention to reduce neointimal formation and adverse vascular remodeling by BM-derived PCs. Using mutant models of SCF and c-Kit deficiency in addition to BM rescue and pharmacological antagonism approaches (imatinib mesylate or ACK2), we implicate a fundamental role of SCF/c-Kit signaling as a critical determinant of BM cell contribution to neointimal formation following arterial injury. Although preclinical4 and clinical evidence suggests that it is efficacious to induce mobilization of c-Kit+ cells into the heart to effect cardiac repair following MI, we have shown in this report that it is critical to avoid mobilization of c-Kit+ smooth muscle progenitors to sites of vascular injury.43 These data provide a rationale for testing directed therapies aimed at limiting the BM contribution to vascular injury (by interrupting the SCF/c-Kit pathway) in patients undergoing vascular interventions such as bypass grafting and angioplasty.
Acknowledgments
We thank Shin-Yi Wang for analyzing histological images and John Dick, Mike Long, Fabio Rossi, and Alan Bernstein for critical reading of the manuscript.
Sources of Funding
Supported by the National Science Council of Taiwan (C.H.W. and W.J.C.), the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research (S.V. and W.L.S), and Physicians Services Inc Ontario (P.E.S.). W.L.S. is a Canadian Research Chair in Stem Cell Biology and Functional Genomics.
Disclosures
None.
Footnotes
Original received December 4, 2005; revision received August 8, 2006; accepted August 16, 2006.
References
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. [Order article via Infotrieve]
Wojakowski W, Tendera M, Michalowska A, Majka M, Kucia M, Maslankiewicz K, Wyderka R, Ochala A, Ratajczak MZ. Mobilization of CD34/CXCR4+, CD34/CD117+, c-met+ stem cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction. Circulation. 2004; 110: 3213–3220.
Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 2001; 103: 2776–2779.
Ayach BB, Yoshimitsu M, Dawood F, Sun M, Arab S, Chen M, Higuchi K, Siatskas C, Lee P, Lim H, Zhang J, Cukerman E, Stanford WL, Medin JA, Liu PP. Stem cell factor receptor induces progenitor and natural killer cell-mediated cardiac survival and repair after myocardial infarction. Proc Natl Acad Sci U S A. 2006; 103: 2304–2309.
Kajstura J, Rota M, Whang B, Cascapera S, Hosoda T, Bearzi C, Nurzynska D, Kasahara H, Zias E, Bonafe M, Nadal-Ginard B, Torella D, Nascimbene A, Quaini F, Urbanek K, Leri A, Anversa P. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion. Circ Res. 2005; 96: 127–137.
Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S. 2001; 98: 10344–10349.
Zhang S, Wang D, Estrov Z, Raj S, Willerson JT, Yeh ETH. Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo. Circulation. 2004; 110: 3803–3807.
Yeh ET, Zhang S, Wu HD, Korbling M, Willerson JT, Estrov Z. Transdifferentiation of human peripheral blood CD34+-enriched cell population into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. Circulation. 2003; 108: 2070–2073.
Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med. 2005; 11: 367–368. [Order article via Infotrieve]
Kim BO, Tian H, Prasongsukarn K, Wu J, Angoulvant D, Wnendt S, Muhs A, Spitkovsky D, Li RK. Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation. 2005; 112 (suppl I): I-96–I-104. [Order article via Infotrieve]
Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet. 2004; 363: 751–756. [Order article via Infotrieve]
Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, Vogl TJ, Martin H, Schachinger V, Dimmeler S, Zeiher AM. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003; 108: 2212–2218.
Babapulle MN, Eisenberg MJ. Coated stents for the prevention of restenosis. Part I. Circulation. 2002; 106: 2734–2740.
Savage MP, Douglas JS Jr, Fischman DL, Pepine CJ, King SB, III, Werner JA, Bailey SR, Overlie PA, Fenton SH, Brinker JA, Leon MB, Goldberg S. Stent placement compared with balloon angioplasty for obstructed coronary bypass grafts. Saphenous Vein De Novo Trial Investigators. N Engl J Med. 1997; 337: 740–747.
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.
Campbell JH, Han CL, Campbell GR. Neointimal formation by circulating bone marrow cells. Ann N Y Acad Sci. 2001; 947: 18–25.
Hoofnagle MH, Wamhoff BR, Owens GK. Lost in transdifferentiation. J Clin Invest. 2004; 113: 1249–1251.
Domen J, Weissman IL. Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the other. J Exp Med. 2000; 192: 1707–1718.
Leary AG, Zeng HQ, Clark SC, Ogawa M. Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors. Proc Natl Acad Sci U S A. 1992; 89: 4013–4017.
Fleming WH, Alpern EJ, Uchida N, Ikuta K, Weissman IL. Steel factor influences the distribution and activity of murine hematopoietic stem cells in vivo. Proc Natl Acad Sci U S A. 1993; 90: 3760–3764.
Levesque JP, Leavesley DI, Niutta S, Vadas M, Simmons PJ. Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med. 1995; 181: 1805–1815.
Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature. 1988; 335: 88–89. [Order article via Infotrieve]
Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002; 109: 625–637. [Order article via Infotrieve]
Rabbany SY, Heissig B, Hattori K, Rafii S. Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization. Trends Mol Med. 2003; 9: 109–117. [Order article via Infotrieve]
Wang CH, Ciliberti N, Li SH, Szmitko PE, Weisel RD, Fedak PW, Al-Omran M, Cherng WJ, Li RK, Stanford WL, Verma S. Rosiglitazone facilitates angiogenic progenitor cell differentiation toward endothelial lineage: a new paradigm in glitazone pleiotropy. Circulation. 2004; 109: 1392–1400.
Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991; 253: 1129–1132.
Ross R, Masuda J, Raines EW. Cellular interactions, growth factors, and smooth muscle proliferation in atherogenesis. Ann N Y Acad Sci. 1990; 598: 102–112. [Order article via Infotrieve]
Anderson HV, McNatt J, Clubb FJ, Herman M, Maffrand JP, DeClerck F, Ahn C, Buja LM, Willerson JT. Platelet inhibition reduces cyclic flow variations and neointimal proliferation in normal and hypercholesterolemic-atherosclerotic canine coronary arteries. Circulation. 2001; 104: 2331–2337.
Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation. 2002; 106: 1199–1204.
Bartolovic K, Balabanov S, Hartmann U, Komor M, Boehmler AM, Buhring HJ, Mohle R, Hoelzer D, Kanz L, Hofmann WK, Brummendorf TH. Inhibitory effect of imatinib on normal progenitor cells in vitro. Blood. 2004; 103: 523–529.
George D. Targeting PDGF receptors in cancer—rationales and proof of concept clinical trials. Adv Exp Med Biol. 2003; 532: 141–151. [Order article via Infotrieve]
Cho A, Graves J, Reidy MA. Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2000; 20: 2527–2532.
Janowska-Wieczorek A, Marquez LA, Nabholtz JM, Cabuhat ML, Montano J, Chang H, Rozmus J, Russell JA, Edwards DR, Turner AR. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane. Blood. 1999; 93: 3379–3390.
Carstanjen D, Ulbricht N, Iacone A, Regenfus M, Salama A. Matrix metalloproteinase-9 (gelatinase B) is elevated during mobilization of peripheral blood progenitor cells by G-CSF. Transfusion. 2002; 42: 588–596. [Order article via Infotrieve]
Russell ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genet. 1979; 20: 357–459. [Order article via Infotrieve]
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.
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.
Hibbert B, Chen YX, O’Brien ER. c-kit-Immunopositive vascular progenitor cells populate human coronary in-stent restenosis but not primary atherosclerotic lesions. Am J Physiol Heart Circ Physiol. 2004; 287: H518–H524.
Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev. 2004; 15: 237–254. [Order article via Infotrieve]
Myllarniemi M, Frosen J, Calderon Ramirez LG, Buchdunger E, Lemstrom K, Hayry P. Selective tyrosine kinase inhibitor for the platelet-derived growth factor receptor in vitro inhibits smooth muscle cell proliferation after reinjury of arterial intima in vivo. Cardiovasc Drugs Ther. 1999; 13: 159–168. [Order article via Infotrieve]
Savikko J, Von Willebrand E, Hayry P. Leflunomide analogue FK778 is vasculoprotective independent of its immunosuppressive effect: potential applications for restenosis and chronic rejection. Transplantation. 2003; 76: 455–458. [Order article via Infotrieve]
Leppanen O, Rutanen J, Hiltunen MO, Rissanen TT, Turunen MP, Sjoblom T, Bruggen J, Backstrom G, Carlsson M, Buchdunger E, Bergqvist D, Alitalo K, Heldin CH, Ostman A, Yla-Herttuala S. Oral imatinib mesylate (STI571/Gleevec) improves the efficacy of local intravascular vascular endothelial growth factor-C gene transfer in reducing neointimal growth in hypercholesterolemic rabbits. Circulation. 2004; 109: 1140–1146.
Wang CH. Stem cell factor attenuates vascular smooth muscle apoptosis and increases intimal hyperplasia after vascular injury. Circulation. 2005; 112 (suppl II): II-46 Abstract.
Cardiology and Internal Medicine (C.-H.W., W.-J.C.), Chang Gung Memorial Hospital, Keelung, Chang Gung University College of Medicine, Taiwan
the Institute of Medical Science (N.A., W.L.S.), Department of Internal Medicine (P.E.S.), Institute of Biomaterials and Biomedical Engineering (W.L.S.)
Department of Chemical Engineering and Applied Chemistry (W.L.S.), University of Toronto, Canada
Cardiac Surgery (C.-H.W., S.-H.L., P.W.M.F., S.F., R.-K.L., T.M.Y., R.D.W.), Toronto General Hospital, Canada
Division of Cardiac Surgery (S.V.), St. Michael’s Hospital, Toronto, Canada.
Abstract
Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth muscle cells (SMCs) that contribute to intimal hyperplasia after vascular injury; however, the molecular pathways involved and the timeline of these events remain poorly characterized. We hypothesized that the stem cell factor (SCF)/c-Kit tyrosine kinase signaling pathway is critical to neointimal formation by BM-derived progenitors. Wire-induced femoral artery injury in mice reconstituted with wild-type BM cells expressing yellow fluorescent protein was performed, which revealed that 66±12% of the SMCs (-smooth muscle actin-positive [SMA+] cells) in the neointima were from BM. To characterize the role of the SCF/c-Kit pathway, we used c-Kit deficient W/Wv and SCF-deficient Steel-Dickie mice. Strikingly, vascular injury in these mice resulted in almost a complete inhibition of neointimal formation, whereas wild-type BM reconstitution of c-Kit mutant mice led to neointimal formation in a similar fashion as wild-type animals, as did chronic administration of SCF in matrix metalloproteinase-9–deficient mice, a model of soluble SCF deficiency. Pharmacological antagonism of the SCF/c-Kit pathway with imatinib mesylate (Gleevec) or ACK2 (c-Kit antibody) also resulted in a marked reduction in intimal hyperplasia. Vascular injury resulted in the local upregulation of SCF expression. c-Kit+ progenitor cells (PCs) homed to the injured vascular wall and differentiated into SMA+ cells. Vascular injury also caused an increase in circulating SCF levels which promoted CD34+ PC mobilization, a response that was blunted in mutant and imatinib mesylate-treated mice. In vitro, SCF promoted adhesion of BM PCs to fibronectin. Additionally, anti-SCF antibodies inhibited adhesion of BM PCs to activated SMCs and diminished SMC differentiation. These data indicate that SCF/c-Kit signaling plays a pivotal role in the development of neointima by BM-derived PCs and that the inhibition of this pathway may serve as a novel therapeutic target to limit aberrant vascular remodeling.
Key Words: vascular remodeling genetic mice models vascular biology vascular smooth muscle
Introduction
The majority of hematopoietic stem cells (HSCs) and various progenitors, including endothelial progenitor cells (EPCs), reside within the osteoblastic zone of the bone marrow (BM), the so-called stem cell niche. Tissue injury releases chemical cues that induce mobilization of cells within the niche into the peripheral blood. Along with inflammatory cells, stem cells and progenitors released from the niche follow chemotactic signals to the injured tissue. Myocardial infarction (MI) or vascular injury elicit BM cell mobilization and localized inflammatory responses, required for cardiovascular repair and remodeling.1–3 In addition to innate immune cell participation in repair, mobilized vascular endothelial and smooth muscle progenitors participate in angiogenesis and vascular repair.1,4 Other regenerative roles of BM-derived cells in the heart remain controversial and have not been clearly defined, but include cardiogenic differentiation,5–8 cardiac cell rescue by myelomonocytic cell fusion,7 or the release of anti-apoptotic paracrine factors.9 Regardless of the mechanism, induced mobilization or intracardiac injection of BM cells improves cardiac function following MI in animal models,6,10 which has stimulated numerous clinical trials testing the ability of BM cells either directly infused into hearts or mobilized into the periphery to improve cardiac function following MI.11,12
However, not all BM responses to cardiovascular injury are regenerative in nature, with recent studies demonstrating that mobilized BM cells can be a source of pathogenic vascular remodeling. Neointimal formation, with resultant vascular remodeling, is a unifying pathological event, complicating chronic atherosclerosis, restenosis, and transplant arteriopathy, and remains the major limiting factor for the long-term efficacy of vascular interventions, such as angioplasty13 and coronary artery bypass graft surgery.14 Although the process of neointimal formation following vascular injury was formerly ascribed solely to a local smooth muscle proliferative response, it has been demonstrated to be regulated significantly by transplantable BM-derived cells.1,8,15–17 Yet, the fundamental mechanism of neointimal hyperplasia by mobilized BM cells is unknown. Thus, the identification of the molecular and cellular mechanisms underlying regenerative and pathogenic cardiovascular remodeling by mobilized BM cells is critical so that new therapeutic approaches can be used to augment the regenerative potential of BM cells while preventing restenosis.
Stem cell factor (SCF) (also known as Steel factor) is expressed as a soluble or membrane-bound form and promotes survival,18 proliferation,19 mobilization,20 and adhesion21 of hematopoietic stem cells and progenitors through dimerization of its cognate receptor, the tyrosine kinase c-Kit.22 Granulocyte colony stimulating factor (G-CSF), stromal cell-derived factor 1 (SDF-1), or vascular endothelial growth factor (VEGF) mobilizes c-Kit+ progenitors and stem cells by inducing matrix metalloproteinase-9 (MMP-9) expression, which then cleaves membrane bound SCF (mSCF), activating c-Kit-induced proliferation and mobilization.23,24 Thus, the release of mSCF by MMP-9 following vascular injury, and the resulting c-Kit tyrosine kinase signaling, may play a mechanistic role in orchestrating the contribution of stem cell-derived vascular progenitors to vascular disease. We used both mutant and BM-reconstituted mice to provide definitive evidence that the SCF/c-Kit signaling pathway is critical to pathological neointimal formation following vascular injury.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animals Studies
Wild-type 129S6 mice, MMP-9–/–, W/Wv (WBB6F1 hybrid strain), colony control WBB6F1 (+/+), Sl/Sld (Steel-Dickie; WCB6F1hybrid strain), and colony control WCB6F1 (+/+) mice were used for the vascular injury studies. W/Wv mice have a relative deficiency of c-Kit kinase activity although the affected cells express normal to elevated levels of c-Kit receptor and Steel-Dickie mice have a complete deficiency of mSCF and reduced serum levels of soluble SCF (sSCF).
Results
BM Cells Contribute to Neointimal Formation
Following vascular injury, vascular smooth muscle cells (VSMCs) underwent extensive apoptosis (Figure I in the online data supplement), and vessels were completely denuded of endothelial cells. A significant increase in the number of VSMCs, or neointimal formation, occurred over a 4-week period (Figure 1A and supplemental Figure II and II1). In mice reconstituted with wild-type BM cells expressing yellow fluorescent protein (eYFP) (BMTYfpWild mice), 66±12% of the VSMCs (-smooth muscle actin-positive [SMA+] cells) in the neointima were eYFP+SMA+ (Figure 1B), confirming earlier reports that a significant portion of the neointima is formed by BM-derived cells.1 In addition to SMA expression, the BM-derived SMA+ cells express calponin, and caldesmon, but not for myelomonocytic markers.25 Local vascular injury has a positive impact on progenitor cell (PC) mobilization from the BM. CD45, a pan leukocyte marker, was used to estimate the contribution of inflammatory cells to the neointima. Many CD45+ BM-derived cells were present in the early phase after vascular injury (Figure 1C). However, as cells differentiated into SMA+ cells, few CD45+ cells could be identified (Figure 1D) suggesting that SMA+ progenitors were CD45–/low or that SMA+ progenitors were CD45+ but lost CD45 expression during differentiation.
Vascular Injury Activates SCF Expression
Vascular injury resulted in the local upregulation of SCF in the adventitia and remaining VSMCs (Figure 1E and supplemental Figure III). Within the adventitia, most of the SCF+ cells were found to be vimentin+ (likely fibroblasts) and some of them were CD45+ (likely inflammatory cells; supplemental Figure IV), but none were CD31+ (an endothelial cell marker). In vitro, preincubation with SCF enhanced platelet-derived growth factor (PDGF) secretion by platelets on activation in a dose-dependent manner (Figure 1F). PDGF is a known stimulator of VSMC migration and promoter of neointimal proliferation.26–28 In turn, PDGF upregulated SCF expression by VSMCs (Figure 1G), suggesting the creation of a positive feedback signaling loop between SCF and PDGF. Thus, the local platelet response shortly after vascular injury may serve to secrete PDGF followed by upregulation of SCF expression, which may trigger the ensuing systemic response to injury, setting the stage for vascular remodeling. Consistent with this hypothesis, c-Kit+ PCs homed to the injured vascular wall and differentiated into SMA+ cells (Figure 1H).
SCF and c-Kit Mutations in Mice Attenuate Neointimal Hyperplasia
To determine whether SCF/c-Kit signaling was an essential for pathological vascular remodeling in vivo, we made use of naturally occurring mutant strains of c-Kit and SCF, the W/Wv and Sl/Sld strains, respectively. W/Wv mice demonstrate 15% of wild-type c-Kit activity a severe reduction in hematopoietic stem cells. Sl/Sld mice express low levels of SCF and are deficient in membrane bound SCF (mSCF). Both W/Wv and Sl/Sld mice are anemic; however, the platelet and white blood cell counts are not significantly reduced. W/Wv and Sl/Sld and littermate control mice underwent femoral artery wire injury. Strikingly, in W/Wv and Sl/Sld mice, neointimal formation was significantly less than that occurring in littermate control mice (Figure 2A and 2B). To further test a causal role of SCF/c-Kit signaling in neointimal formation, we performed vascular injury following wild-type BM transplantation into W/Wv mice. These transplanted mice, having BM PCs with functioning c-Kit receptor tyrosine kinase, demonstrated marked intimal hyperplasia 28 days after wire injury, similar to wild-type mice. Thus, c-Kit appears to be absolutely required for postinjury vascular remodeling. Attempting to restore normal levels of SCF to Sl/Sld mice, we administered SCF (100 ng/kg per day) subcutaneously to these mice. Despite inducing extreme increases in plasma sSCF levels (from 0.52 ng/mL at baseline up to 10749, 4432 and 3329 ng/mL, respectively, at 6, 12, and 24 hours after injection), sSCF supplementation did not result in a significant increase in neointimal thickness (Figure 2A; Sl/Sld+SCF). Thus, SCF, namely mSCF, appears to also be required for pathological vascular remodeling to occur. sSCF cannot replace the role of mSCF in promoting neointimal formation.
Vascular Injury Increases Blood SCF Levels
Vascular injury not only upregulated local SCF expression but also caused an increase in circulating SCF levels. In wild-type mice, sSCF levels peaked on day 3 after injury and gradually returned to baseline levels (Figure 3A). In W/WV mice, sSCF tended to increase after injury, but baseline sSCF concentrations were more than 3.5-fold higher than in wild-type mice because of feedback mechanisms compensating for markedly reduced c-Kit activity. However, W/WV mice rescued by wild-type BM transplantation had much lower levels of sSCF than the W/WV mice (146±8 versus 315±10 ng/mL, P<0.001; wild-type values are 91±9 ng/mL). This phenomenon could explain why rescued W/Wv mice had only similar rather than more intimal hyperplasia compared with wild-type mice (Figure 2C). Plasma sSCF levels in Sl/Sld mice were substantially lower than in wild-type mice and only increased slightly on day 7 after injury. MMP-9–/– mice exhibited sSCF baseline concentrations that were similar to wild-type mice; however, the levels of sSCF did not change in response to vascular injury, as expected, because MMP-9 facilitates the shedding of mSCF.23,24 Plasma G-CSF levels transiently increased 1 day after vascular injury in all mice (Figure 3B), but the rate of decline in the vascular injured wild-type mice was slower than in the sham-operated mice.
MMP-9 Facilitates SCF Shedding
SCF levels increased following vascular injury because of upregulated MMP-9 activity within the injured vasculature. At 1 and 7 days after injury, SCF expression was detected at areas that also contained higher levels of MMP-9 (Figure 4A). In addition, our data suggested that MMP-9 was activated to cleave mSCF, which was expressed throughout the vessel wall (data not shown). Zymography demonstrated a marked upregulation of pro-MMP-9 and active MMP-9 1 day after injury, with the majority of MMP-9 being in the active form by day 7 (Figure 4B). Vascular injury in MMP-9–/– mice resulted in modest neointimal formation; however, when the mice were supplemented with sSCF for 2 weeks after vascular injury, a 2.5-fold increase in neointimal formation was observed (Figure 4C). Thus, MMP-9–induced release of SCF appears to be a prerequisite for aberrant vascular remodeling following injury. In the BM, femoral artery wire injury also induced a significant increase in MMP-9 activity. Pharmacological inhibition of MMP activity led to significantly increased mSCF+ cell numbers in response to the injury. However, without MMP inhibition, mSCF+ cell numbers increased transiently and then significantly decreased at later time points. These findings suggested that MMP-9 was activated inside the BM and cleaved mSCF, contributing in part to the increase in circulating sSCF levels (supplemental Figure V).
Vascular Injury Promotes Progenitor Cell Mobilization
As of yet, the markers of smooth muscle PCs have not been clearly defined. However, Simper et al reported that circulating smooth muscle PCs appear to originate from CD34+ cells.29 Thus, CD34 was used as a surrogate for PC mobilization in this study. The increase in sSCF levels following vascular injury in wild-type mice coincided with a significant increase in the number of circulating BM-derived CD34+ PCs (Figure 3C). The level of circulating CD34+ cells appeared to increase in 2 waves, rising immediately after vascular injury and again 9 days later. The effect of sSCF on CD34+ cell mobilization was confirmed (Figure 3D). However, injection of sSCF (100 ng/kg per day) could only increase the level of circulating CD34+ cells in MMP-9–/– mice and not in Sl/Sld mice, suggesting a requirement for mSCF in mobilization rather than only a change in serum SCF. Treatment with imatinib mesylate (Gleevec), a c-Kit-kinase inhibitor, inhibited both early and late CD34+ cell mobilization (Figure 3C). Similarly, Sl/Sld mice displayed impaired cell mobilization.
SCF Contributes to the Differentiation of Progenitor Cells After Vascular Injury
To ascertain whether SCF contributed to the homing and differentiation of PCs after vascular injury, we directly tested the influence of SCF on BM PC adhesion (because cell adhesion is required in homing) and smooth muscle differentiation. Negatively selected Lin– cells, which were enriched with c-Kit+ cells, were used in the adhesion experiments. Lin– cells rather than directly isolated c-Kit+ cells were used to avoid blocking the c-Kit receptor by the primary anti-c-Kit antibody used for the positive selection processes. SCF (200 ng/mL) induced a 2.3-fold increase in the adhesion of Lin– BM cells to fibronectin (Figure 5A). This effect was completely blocked by an anti-51 integrin antibody. In a second adhesion assay, rat VSMCs were activated by PDGF (10 ng/mL) to upregulate the expression of SCF. Activated VSMCs greatly increased the adhesion of Lin– BM cells, an interaction that was blocked by administration of anti-SCF antibody (Figure 5B and 5C). Furthermore, the coculture of eYFP+ Lin– BM cells with activated VSMCs for 11 days showed clusters of cells that expressed both eYFP and SMA (Figure 5D, top). Adding anti-SCF antibody to the coculture decreased the number of double-positive cells (Figure 5D, bottom), suggesting that VSMC activation, marked by SCF expression, is not only required for PC homing but also PC differentiation toward the smooth muscle cell (SMC) lineage.
c-Kit+ Cells Give Rise to SMA+ Cells
c-Kit+ and c-Kit– BM cells were purified and tested for their potential to differentiate into SMA+ cells in low-serum endothelial growth medium-2 (EGM-2). Compared with c-Kit– cells a week after plating, c-Kit+ cells exhibited increased survival and gave rise to more SMA+ cell colonies (approximately 5-fold increase) (Figure 6A). Once c-Kit+ cells expressed SMA+, they lost both the c-Kit and CD45 phenotypes. This also appeared to be the case in vivo. Although a significant amount of c-Kit+ cells were noted to adhere to the injured vascular wall in the littermate control mice (Figure 6B and 6C), once these c-Kit+ cells differentiated into SMA+ cell, c-Kit expression became very weak or disappeared (Figure 6D). These cells underwent a remarkable extent of proliferation to cause intimal hyperplasia (Figure 6E). However, in the Sl/Sld mice, essentially no c-Kit+ cells could be detected in the neointima at the early time point after injury (Figure 6C).
Blocking c-Kit Signaling Attenuates Neointimal Hyperplasia
Imatinib mesylate is a protein-tyrosine kinase inhibitor, which selectively blocks Abl tyrosine kinase and the c-Kit and PDGF receptor (PDGF-R) tyrosine kinases.30,31 Early intervention with imatinib mesylate significantly attenuated neointimal formation, an average of 300% reduction, whereas imatinib mesylate treatment that started at the hyperplasia phase was ineffective (Figure 7A and 7B). Because imatinib mesylate also inhibits PDGF-R signaling, which is well known to mediate SMC proliferation, we analyzed the kinetics of PDGF-R expression following injury. Figure 7C demonstrates that PDGF-R was primarily expressed within the adventitia and to a lesser degree by VSMCs and that the expression of PDGF-R by medial SMCs was upregulated beginning 9 days after vascular injury. In contrast, c-Kit+ cells appeared within the vessel medial wall by 1 day after vascular injury (supplemental Figure VI). Imatinib mesylate did not affect the sSCF and G-CSF levels after vascular injury (data not shown). Finally, we hypothesized that if the imatinib mesylate-mediated inhibition of neointimal hyperplasia was acting through the c-Kit pathway, then blocking c-Kit activation using the blocking anti-c-Kit receptor monoclonal antibody ACK2 would generate similar results. In fact, we found a significant attenuating effect by ACK2 on intimal hyperplasia (Figure 7D).
Discussion
We have defined a new mechanistic pathway of neointimal formation and adverse vascular remodeling, critically dependent on the interaction between SCF/c-Kit receptor signaling and the subsequent mobilization of BM-derived PCs. Our conclusions are based on in vivo studies performed in mutant models of SCF deficiency and c-Kit receptor deficiency, with additional supportive data from BM transplantation and pharmacological antagonism studies with imatinib mesylate and blocking c-Kit antibodies. PC mobilization from the BM and subsequent recruitment to the area of injury occurs shortly after the vascular insult; however, intimal hyperplasia accelerates between 14 to 28 days postinjury, possibly secondary to proliferation of the newly recruited PCs. The mobilization and recruitment of PCs from the BM requires the release of SCF via MMP-9 activation. SCF, PDGF, and G-CSF have all been shown to activate MMP-9.32–34 Our data suggest 2 waves of PC mobilization following vascular injury: an early, immediate response and a delayed response (Figure 3C). Although the early episode may represent a nonspecific response to injury, only mice that underwent vascular injury maintained elevated sSCF levels and displayed the second phase, which became apparent approximately 9 days after vascular injury, just as the neointima was beginning to develop.
Both MMP-9–/– and Sl/Sld mice did not sustain changes to sSCF levels in response to vascular injury (Figure 3A and 3B). The observation that supplementation with sSCF only exerted an effect on MMP-9–/– mice, which have normal mSCF but are unable to release it, suggests that mSCF plays a critical role not only in PC mobilization but also in homing and differentiation. mSCF serves as a source of sSCF after wire injury, which may subsequently promote adhesion of PCs to the exposed matrix on the injured vessel and stimulate PDGF release on platelet activation. mSCF plays a critical role in the homing of PCs to the injured vessel wall and mediates the differentiation of c-Kit+ PCs to VSMCs (Figure 5). Either a lack of mSCF or the blockade of the c-Kit/mSCF interaction markedly attenuated PC mobilization, homing, and differentiation. Cell adhesion is only 1 component of PC homing, and we have not investigated directed migration of PCs, which may also play a role in modulating the effects of SCF on PC homing. Although wild-type BM repopulation of W/Wv mice restored neointimal hyperplasia following injury, demonstrating the requirement of the SCF/c-Kit pathway and BM cells in our model, the reciprocal experiment could not be performed. Classic BM transplantation experiments performed in the 1960s demonstrated that W/Wv hematopoietic stem cells are incompetent to repopulate wild-type mice. Furthermore, wild-type BM cannot repopulate Sl/Sld mice because of a lack of SCF expressed by the stromal cells within the hematopoietic stem cell niche.35
Although we have clearly shown that restenosis in our mouse model is dependent on the c-Kit signaling pathway, how do these findings translate to human pathology and clinical practice For example, the mouse femoral artery injury model represents a severe vascular injury model, which requires the participation of BM-derived progenitors in the repair process, and it has been shown that less severe injury models do not necessarily recruit BM-mobilized cells.36 Although it is important to not overextrapolate mouse data to human clinical data, there are several lines of evidence suggesting that c-Kit/SCF-induced mobilization of BM progenitors is clinically relevant and that imatinib mesylate therapy should be considered. First, although the extent of vascular injury is considerably less severe in human angioplasty, in this era of extensive vascular stent intervention, a similar stress on the vessel wall, as observed in the mouse model, may exist, attracting BM cells through the SCF/c-Kit pathway as atherosclerotic plaques are being pushed outward. Supporting this hypothesis are findings that BM-derived cells including monocytes are mobilized following coronary stent implantation and the number of circulating cells directly correlated with in-stent neointimal volume.37 More directly, c-Kit+ vascular progenitors have been identified within postangioplasty restenosis lesions.38 Moreover, a recent clinical trial performed on MI patients designed to test the efficacy and safety of mobilization of BM progenitors to improve cardiac function was halted because of the high rate of restenosis. The MAGIC clinical trial performed G-CSF-mediated mobilization of BM progenitors on patients with MI who underwent coronary stenting. Although cardiac function improved in these patients, the study ended prematurely because patients receiving G-CSF experienced an unexpectedly high rate of postangioplasty restenosis.11 As discussed above, G-CSF-induced mobilization acts through MMP-9 to cleave mSCF and activate c-Kit+ to proliferation and mobilize into the periphery.
Imatinib mesylate prevented adverse vascular remodeling when administered early after vascular injury, at a stage when c-Kit signaling is essential for PC mobilization and recruitment. This finding was mirrored by intervention with ACK2, a neutralizing anti-c-Kit receptor monoclonal antibody, further strengthening our conclusion that the action of imatinib mesylate by blocking the SCF/c-Kit pathway. However, imatinib mesylate may also have a role in blocking postangioplasty restenosis mediated by local vascular SMCs by blocking PDGFR tyrosine kinase activity. PDGF is a well-known potent mitogenic and migratory factor for vascular SMCs and has been implicated in vascular pathologies.39 Imatinib mesylate, either alone or in combination with other treatments, has in some cases been demonstrated to reduce the extent of restenosis through its effect on PDGF receptors.40–42 Our data suggest that c-Kit signaling is upstream of the PDGF-mediated pathway recruitment of perivascular cells; however, much more work must be done to discover the biochemical and cellular interactions between these 2 tyrosine kinases pathways in neointima formation.
In summary, we demonstrated that the SCF/c-Kit pathway is a novel, important, and logical target for therapeutic intervention to reduce neointimal formation and adverse vascular remodeling by BM-derived PCs. Using mutant models of SCF and c-Kit deficiency in addition to BM rescue and pharmacological antagonism approaches (imatinib mesylate or ACK2), we implicate a fundamental role of SCF/c-Kit signaling as a critical determinant of BM cell contribution to neointimal formation following arterial injury. Although preclinical4 and clinical evidence suggests that it is efficacious to induce mobilization of c-Kit+ cells into the heart to effect cardiac repair following MI, we have shown in this report that it is critical to avoid mobilization of c-Kit+ smooth muscle progenitors to sites of vascular injury.43 These data provide a rationale for testing directed therapies aimed at limiting the BM contribution to vascular injury (by interrupting the SCF/c-Kit pathway) in patients undergoing vascular interventions such as bypass grafting and angioplasty.
Acknowledgments
We thank Shin-Yi Wang for analyzing histological images and John Dick, Mike Long, Fabio Rossi, and Alan Bernstein for critical reading of the manuscript.
Sources of Funding
Supported by the National Science Council of Taiwan (C.H.W. and W.J.C.), the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research (S.V. and W.L.S), and Physicians Services Inc Ontario (P.E.S.). W.L.S. is a Canadian Research Chair in Stem Cell Biology and Functional Genomics.
Disclosures
None.
Footnotes
Original received December 4, 2005; revision received August 8, 2006; accepted August 16, 2006.
References
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. [Order article via Infotrieve]
Wojakowski W, Tendera M, Michalowska A, Majka M, Kucia M, Maslankiewicz K, Wyderka R, Ochala A, Ratajczak MZ. Mobilization of CD34/CXCR4+, CD34/CD117+, c-met+ stem cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction. Circulation. 2004; 110: 3213–3220.
Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 2001; 103: 2776–2779.
Ayach BB, Yoshimitsu M, Dawood F, Sun M, Arab S, Chen M, Higuchi K, Siatskas C, Lee P, Lim H, Zhang J, Cukerman E, Stanford WL, Medin JA, Liu PP. Stem cell factor receptor induces progenitor and natural killer cell-mediated cardiac survival and repair after myocardial infarction. Proc Natl Acad Sci U S A. 2006; 103: 2304–2309.
Kajstura J, Rota M, Whang B, Cascapera S, Hosoda T, Bearzi C, Nurzynska D, Kasahara H, Zias E, Bonafe M, Nadal-Ginard B, Torella D, Nascimbene A, Quaini F, Urbanek K, Leri A, Anversa P. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion. Circ Res. 2005; 96: 127–137.
Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S. 2001; 98: 10344–10349.
Zhang S, Wang D, Estrov Z, Raj S, Willerson JT, Yeh ETH. Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo. Circulation. 2004; 110: 3803–3807.
Yeh ET, Zhang S, Wu HD, Korbling M, Willerson JT, Estrov Z. Transdifferentiation of human peripheral blood CD34+-enriched cell population into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. Circulation. 2003; 108: 2070–2073.
Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med. 2005; 11: 367–368. [Order article via Infotrieve]
Kim BO, Tian H, Prasongsukarn K, Wu J, Angoulvant D, Wnendt S, Muhs A, Spitkovsky D, Li RK. Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation. 2005; 112 (suppl I): I-96–I-104. [Order article via Infotrieve]
Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo LD, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet. 2004; 363: 751–756. [Order article via Infotrieve]
Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, Vogl TJ, Martin H, Schachinger V, Dimmeler S, Zeiher AM. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003; 108: 2212–2218.
Babapulle MN, Eisenberg MJ. Coated stents for the prevention of restenosis. Part I. Circulation. 2002; 106: 2734–2740.
Savage MP, Douglas JS Jr, Fischman DL, Pepine CJ, King SB, III, Werner JA, Bailey SR, Overlie PA, Fenton SH, Brinker JA, Leon MB, Goldberg S. Stent placement compared with balloon angioplasty for obstructed coronary bypass grafts. Saphenous Vein De Novo Trial Investigators. N Engl J Med. 1997; 337: 740–747.
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.
Campbell JH, Han CL, Campbell GR. Neointimal formation by circulating bone marrow cells. Ann N Y Acad Sci. 2001; 947: 18–25.
Hoofnagle MH, Wamhoff BR, Owens GK. Lost in transdifferentiation. J Clin Invest. 2004; 113: 1249–1251.
Domen J, Weissman IL. Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the other. J Exp Med. 2000; 192: 1707–1718.
Leary AG, Zeng HQ, Clark SC, Ogawa M. Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors. Proc Natl Acad Sci U S A. 1992; 89: 4013–4017.
Fleming WH, Alpern EJ, Uchida N, Ikuta K, Weissman IL. Steel factor influences the distribution and activity of murine hematopoietic stem cells in vivo. Proc Natl Acad Sci U S A. 1993; 90: 3760–3764.
Levesque JP, Leavesley DI, Niutta S, Vadas M, Simmons PJ. Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med. 1995; 181: 1805–1815.
Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature. 1988; 335: 88–89. [Order article via Infotrieve]
Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002; 109: 625–637. [Order article via Infotrieve]
Rabbany SY, Heissig B, Hattori K, Rafii S. Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization. Trends Mol Med. 2003; 9: 109–117. [Order article via Infotrieve]
Wang CH, Ciliberti N, Li SH, Szmitko PE, Weisel RD, Fedak PW, Al-Omran M, Cherng WJ, Li RK, Stanford WL, Verma S. Rosiglitazone facilitates angiogenic progenitor cell differentiation toward endothelial lineage: a new paradigm in glitazone pleiotropy. Circulation. 2004; 109: 1392–1400.
Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991; 253: 1129–1132.
Ross R, Masuda J, Raines EW. Cellular interactions, growth factors, and smooth muscle proliferation in atherogenesis. Ann N Y Acad Sci. 1990; 598: 102–112. [Order article via Infotrieve]
Anderson HV, McNatt J, Clubb FJ, Herman M, Maffrand JP, DeClerck F, Ahn C, Buja LM, Willerson JT. Platelet inhibition reduces cyclic flow variations and neointimal proliferation in normal and hypercholesterolemic-atherosclerotic canine coronary arteries. Circulation. 2001; 104: 2331–2337.
Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation. 2002; 106: 1199–1204.
Bartolovic K, Balabanov S, Hartmann U, Komor M, Boehmler AM, Buhring HJ, Mohle R, Hoelzer D, Kanz L, Hofmann WK, Brummendorf TH. Inhibitory effect of imatinib on normal progenitor cells in vitro. Blood. 2004; 103: 523–529.
George D. Targeting PDGF receptors in cancer—rationales and proof of concept clinical trials. Adv Exp Med Biol. 2003; 532: 141–151. [Order article via Infotrieve]
Cho A, Graves J, Reidy MA. Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2000; 20: 2527–2532.
Janowska-Wieczorek A, Marquez LA, Nabholtz JM, Cabuhat ML, Montano J, Chang H, Rozmus J, Russell JA, Edwards DR, Turner AR. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane. Blood. 1999; 93: 3379–3390.
Carstanjen D, Ulbricht N, Iacone A, Regenfus M, Salama A. Matrix metalloproteinase-9 (gelatinase B) is elevated during mobilization of peripheral blood progenitor cells by G-CSF. Transfusion. 2002; 42: 588–596. [Order article via Infotrieve]
Russell ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genet. 1979; 20: 357–459. [Order article via Infotrieve]
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.
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.
Hibbert B, Chen YX, O’Brien ER. c-kit-Immunopositive vascular progenitor cells populate human coronary in-stent restenosis but not primary atherosclerotic lesions. Am J Physiol Heart Circ Physiol. 2004; 287: H518–H524.
Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev. 2004; 15: 237–254. [Order article via Infotrieve]
Myllarniemi M, Frosen J, Calderon Ramirez LG, Buchdunger E, Lemstrom K, Hayry P. Selective tyrosine kinase inhibitor for the platelet-derived growth factor receptor in vitro inhibits smooth muscle cell proliferation after reinjury of arterial intima in vivo. Cardiovasc Drugs Ther. 1999; 13: 159–168. [Order article via Infotrieve]
Savikko J, Von Willebrand E, Hayry P. Leflunomide analogue FK778 is vasculoprotective independent of its immunosuppressive effect: potential applications for restenosis and chronic rejection. Transplantation. 2003; 76: 455–458. [Order article via Infotrieve]
Leppanen O, Rutanen J, Hiltunen MO, Rissanen TT, Turunen MP, Sjoblom T, Bruggen J, Backstrom G, Carlsson M, Buchdunger E, Bergqvist D, Alitalo K, Heldin CH, Ostman A, Yla-Herttuala S. Oral imatinib mesylate (STI571/Gleevec) improves the efficacy of local intravascular vascular endothelial growth factor-C gene transfer in reducing neointimal growth in hypercholesterolemic rabbits. Circulation. 2004; 109: 1140–1146.
Wang CH. Stem cell factor attenuates vascular smooth muscle apoptosis and increases intimal hyperplasia after vascular injury. Circulation. 2005; 112 (suppl II): II-46 Abstract.
您现在查看是摘要介绍页，详见ORG附件。