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Cellular Plasticity in the Cardiovascular System
Vascular Biology Laboratory, Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y4W7
Submitted 6 January 2004 ; accepted in final form 2 April 2004
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ABSTRACT |
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-actin, whereas the intima and media of PR lesions and normal arteries were devoid of c-kit-immunopositive cells. The abundance of peripheral blood mononuclear cell-derived CACs was assessed in 10 patients with ISR, 6 patients with angiographically verified patent stents, and 6 individuals with no clinical evidence of coronary artery disease. CACs were less abundant in ISR patients than in non-ISR controls (13.9 ± 3.1 vs. 22.3 ± 6.7 cells/high-power field, P < 0.05), and both of these groups had fewer CACs than non-coronary artery disease patients (37.6 ± 3.8, P < 0.05). These findings suggest a unique pathogenesis for ISR and RS lesions that involves c-kit-immunopositive smooth muscle cells. Moreover, the paucity of CACs in patients with ISR may contribute to the pathogenesis of ISR, perhaps because of attenuated reendothelialization.
stent; endothelial progenitor cells; cultured angiogenic cells
-actin-immunopositive cells surrounded by an abundant proteoglycan matrix, minimal cell proliferation, and a paucity or absence of endothelial cells (7, 8). Although our present understanding of intimal formation includes the proliferation and inward migration of medial smooth muscle cells (SMCs), more recent studies have prompted a reworking of these concepts to include the involvement of vascular precursor cells (11, 26). To some extent, neointimal SMCs appear to involve bone marrow-derived cells and circulating mononuclear cells (3, 10, 25, 27). Although various markers of vascular progenitors have been identified, few are practical for immunolabeling vascular tissue specimens (11). We chose to study c-kit as a marker of primitive, pluripotent cell types, inasmuch as previously it was documented that 
70% of CD34+ progenitor/stem cells in marrow express c-kit, even though only a very low percentage of cultured endothelial precursor cells (EPCs) express this antigen (2, 24). Immunodetection of c-kit has previously been used to demonstrate primitive cells in transplanted human hearts; however, c-kit protein and/or mRNA are also expressed by a select number of other cells and tissues (e.g., mast cells, melanocytes, testis, bone marrow, embryonic brain tissue, astrocytes, renal tubules, breast glandular epithelial cells, circulating CD34 stem/progenitor cells, and vascular endothelial cells) (14, 22, 33). Although the initial vascular biology interest in EPCs comes from the therapeutic angiogenesis literature, more recent studies show a strong correlation between EPC abundance and endothelial function and cardiovascular risk (1, 12, 29). When cultured in appropriate conditions, peripheral blood mononuclear cells (PBMCs) give rise to nonproliferative, secretory cells with morphological features and molecular expression profiles typical of endothelial cells (13, 24). Rehman and colleagues (24) suggest that the absence of specific endothelial or progenitor cell markers on cultured PBMCs gives reason to label these cells as cultured angiogenic cells (CACs), rather than EPCs. Recent studies show that intravenous transfusions of CACs mitigate neointima formation after vascular injury and prevent atherosclerosis in murine models (23, 32).
In this study, we attempted to determine whether 1) SMCs characteristically found in human ISR tissue express the primitive cell marker c-kit, thereby providing supportive evidence of a blood-borne origin for these cells, and 2) attenuated levels of CACs are found in patients with ISR. To ascertain the origin of SMCs, atherectomy tissue specimens from ISR, postangioplasty restenosis (RS), and primary (PR) atherosclerotic lesions and postmortem coronary artery cross sections from young individuals without atherosclerosis were studied. In the second part of this study, the abundance of CACs in patients with ISR was compared with that of individuals with angiographically verified patent stents. The data suggest a unique role for c-kit-immunopositive cells that coexpress smooth muscle -actin as well as attenuated CAC levels in the pathogenesis of ISR.
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MATERIALS AND METHODS |
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Immunolabeling.
Immunolabeling of tissue slides was performed using the following antibodies: rabbit anti-human CD-117 (c-kit, 1:100; Dako, Mississauga, ON, Canada), mouse anti-human -smooth muscle actin (1:100; Dako), mouse monoclonal antibody mast cell tryptase (1:200; Novocastra Laboratories, Benton Lane, UK), and biotinylated anti-mouse or anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA). Single-label immunohistochemistry was performed as described previously (4). Briefly, tissue sections were deparaffinized and incubated with 10% horse or goat serum (Vector Laboratories) for 20 min to minimize the nonspecific binding of the primary antibody before incubation overnight with one of the primary antibodies in a 4°C moisture chamber. Tissue sections were then incubated with the appropriate secondary antibody for 30 min at room temperature. To inhibit endogenous peroxidase activity, tissue sections were incubated with 3% H2O2 for 30 min before incubation with peroxidase-labeled streptavidin (Vector Laboratories) for 30 min. Visualization of a positive immunoreaction was made possible by the addition of the standard peroxidase enzyme substrate 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO), which resulted in a brown color reaction product. Hematoxylin was used as a nuclear counterstain. Phosphate-buffered saline washes were used between each of the above-described steps. For double immunostaining of c-kit and SMCs, an indirect immunofluorescence method was performed by combining a mouse anti-human smooth muscle actin antibody and an FITC-conjugated anti-mouse IgG antibody (1:100; Vector Laboratories) with a rabbit anti-human CD-117 (c-kit) antibody and a Texas red anti-rabbit IgG antibody (1:100; Vector Laboratories) before addition of the nuclear fluorochrome Hoechst 33258 (0.1 µg/ml; Sigma). Negative controls for all immunolabeling procedures were performed by incubation with nonimmune mouse or rabbit serum, instead of the primary antibody. To determine the percentage of c-kit-immunolabeled cells in ISR tissue, the total number of c-kit-immunopositive cells and the total number of cells were manually counted in the entire sample and expressed as a percentage. In addition, we used image analysis (Image-Pro software, Media Cybernetics, Silver Spring, MD) to quantify the total area of the specimens and divided the number of c-kit-immunopositive cells by the tissue area to arrive at the density of c-kit-immunopositive cells per square millimeter.
Isolation of CACs and culture assay. Blood samples were drawn from 10 ISR patients (70 ± 11 yr old, 9 men), 6 patients with angiographic evidence of a patent stent (69.7 ± 9.5 yr old), and 6 patients with no clinical evidence of CAD (43 ± 8.1 yr old). In the ISR population, four patients had diabetes mellitus and eight used statin drugs; in the non-ISR control population, one individual had diabetes mellitus and all patients were taking statin drugs. None of the individuals in the non-CAD group had diabetes mellitus or used statin drugs. CACs were isolated and cultured as previously described (13). Briefly, a 10-ml sample of blood was collected by venipuncture and anticoagulated with EDTA. PBMCs were isolated by density gradient centrifugation with Ficoll (Sigma). Cells were washed once with Hanks' balanced salt solution (HBSS). Fibronectin (Sigma)-coated six-well plates where then seeded with 2.5 x 106 PBMCs/well. Culture medium consisted of endothelial cell medium (EGM-2, Cambrex, East Rutherford, NJ). At 4 days after plating, nonadherent cells were removed and adherent cells were washed three times with HBSS and incubated with 1,1'-dioctadecyl-3,3,3',3'-tetramethyliodocarbocyanine-acetylated LDL (2.5 µg/ml; Molecular Probes, Eugene, OR) for 1 h at 37°C. Subsequently, cells were fixed with Cytofix buffer (Becton-Dickinson, Franklin Lakes, NJ) and then incubated with FITC-conjugated Ulex europaeus agglutinin I (5 µg/ml; Sigma) for 30 min at 37°C. After incubation, plates were washed three times with HBSS. CACs were defined by the uptake of acetylated LDL (acLDL+) and Ulex europeaeus agglutinin I binding (agglutinin I+); however, they have also been referred to as EPCs and culture-modified mononuclear cells. The abundance of CACs was assessed by manually counting the number of cells per x200 magnification optical field. Six optical fields were evaluated per patient, and the mean result is reported.
Statistics. Values are means ± SD. Comparison of two groups was performed using a t-test and Bonferroni's adjustment. Statistical significance was defined by P < 0.05.
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RESULTS |
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-actin revealed that the majority of the c-kit-immunopositive cells were also immunopositive for smooth muscle -actin and showed a stellate-shaped morphology of SMCs that is commonly thought to be characteristic of RS lesions (19) (Fig. 3). The percentage of c-kit-immunopositive cells in these specimens varied from 2.0 to 29.6% (Table 1). Moreover, the density of c-kit-immunopositive cells correlated inversely with the interval between stent placement and tissue resection with the atherectomy catheter (r = –0.733, P < 0.05; Fig. 4).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-actin in ISR and some RS tissue. This result, combined with the conspicuous absence of c-kit-immunopositive cells in primary atherosclerotic lesions or the intima and media of normal healthy arteries, advocates a unique role for these cells in the genesis of ISR and, perhaps, RS. Second, the abundance of CACs, as judged from the tissue culture adhesion assay using PBMCs, was lower in patients with ISR than in patients free of ISR or individuals without clinical evidence of CAD. Hence, our CAC data concur with other reports and support the concept that CACs may, indeed, be vasculoprotective (12, 29).
Previously, we showed that atherectomy specimens from RS and ISR lesions consisted primarily of SMCs in a proteoglycan-rich matrix, with occasional focal collections of CD68-immunopositive monocytes and organizing thrombus but a marked absence of proliferation (7, 20). Although stellate-shaped cells that coexpress smooth muscle -actin were routinely found in these lesions, little was known about their origin. In the restenosis literature, myxoid tissue with these stellate-shaped cells is commonly believed to be a characteristic finding of RS and ISR lesions and presumed evidence of the proliferative nature of this tissue (19). Moreover, Tjurmin and colleagues (28) demonstrated that stellate cells expressed CD1a and HLA-DR antigens and speculated that these cells may form part of an immune response during vascular lesion formation. Blood-borne inflammatory cells are certainly thought to play a key role in the genesis of ISR, and we recently demonstrated that antagonism of VLA-4 to prevent mononuclear cell attachment and transmigration into the vessel wall at least transiently inhibits stent intimal formation (16). However, gaps remain in our understanding of how these cells might transform into stellate-shaped smooth muscle-like intimal cells (31).
To the best of our knowledge, the data from our study demonstrate for the first time that circulating CAC levels are lower in ISR patients than in non-ISR patients with patent stents or individuals without clinical evidence of CAD. Although circulating CAC levels have been associated with endothelial function and cardiovascular risk, their precise involvement in vascular repair is yet to be causally established (12, 29). Recently, George and colleagues (6) used a similar culture assay and reported that patients with diffuse, as opposed to focal, ISR had lower levels of CACs; however, there was no difference in CAC abundance when ISR and non-ISR patients were compared. Perhaps the discordant results of their study and our study relate to the relatively small patient populations that were surveyed. Although statin drugs, which are known to augment CAC levels, were used in 6 of 6 of the non-ISR group compared with 8 of 10 of the ISR population, it is difficult to judge whether this difference in medication use is relevant to the study results (30). Moreover, it is unclear whether all statins are equal in their ability to boost CAC levels. Our finding that the non-CAD comparison group had much higher CACs than the stent recipients was also not surprising because of the age difference and absence of diabetes. Finally, the low levels of CACs in ISR patients are consistent with our previous observations on the histology of ISR tissue, in that we failed to observe vasa vasorum in ISR specimens (7). Taken together, these data may suggest that low CAC levels result not only in reduced (or delayed) reendothelialization of the lumen of the stented vessel segment but also impairment of formation of artery wall microvessels or vasa vasorum. As such, the oxygen tension in the ISR lesions may be reduced and, inasmuch as vessel wall hypoxia is known to result in altered gene regulation, could play a role in the genesis of ISR lesions.
Although the data presented here fail to provide mechanistic insights, the working hypothesis that we derive from the data is that the presence of c-kit-immunopositive cells and a relative dearth of CACs may serve as a template for the development of ISR lesions. Exactly how c-kit-immunopositive cells participate in this process is unknown; moreover, we cannot ignore the possibility that they may simply serve as an incidental marker of this disease process. Although we are unable to go beyond these postulates, there are circumstantial data in support of the notion that circulating progenitor cells may participate in vascular repair and lesion formation (3, 10, 23, 25, 32). However, as noted previously, the precise nature of endothelial and smooth muscle precursor cells and markers that might uniquely identify these cell types remains unclear (11). These studies are further complicated by the possibility that CACs may not represent a homogenous population of cells and could, in fact, originate from different sources. However, it is unlikely that these c-kit-immunopositive cells are of endothelial origin, inasmuch as we routinely noted a paucity of endothelial cells in these specimens, and cultured CACs do not express c-kit 4 days after isolation (7, 8, 24). Therefore, we cannot necessarily link the in vivo findings with the in vitro observations made in this study.
This study is not without limitations. First, although the study of human tissue is of paramount value, the number of available specimens is limited. Second, our ability to determine the exact lineage of the c-kit-immunopositive cells is limited by the facts that we are only obtaining "snapshots" of cells at one point in their life cycle and that the expression of smooth muscle or endothelial cell markers is a dynamic, inducible process, consistent with the plasticity of these cells. From our previous immunolabeling studies of this same tissue, we know that the frequency of inflammatory or endothelial cells (e.g., using anti-CD68 and anti-CD31 antibodies, respectively) is low, and the majority of the cells express SMC -actin. Moreover, unlike other studies in which the number of endothelial cells increased with the interval from stent implantation to tissue resection for ISR, in this study the density of the c-kit-immunopositive cells showed an inverse relation over time, thereby suggesting that these cells were not of endothelial lineage (9, 15). Finally, the CAC assay used in this study, although commonly described in the literature, may be of insufficient rigor to exclude 1) the possibility that PBMCs subjected to the artificial influences of tissue culture express these markers in vitro but not in vivo and 2) that these cells may, in fact, remain as a mononuclear cell population in vivo.
In summary, our findings suggest that c-kit-immunopositive cells that coexpress smooth muscle -actin are unique to ISR tissue and are not present in primary atherosclerotic lesions or normal arteries. Studies aimed at identifying the precursors that give rise to these neointimal cells and the signaling molecules involved in their mobilization and homing are ongoing. In addition, we found that patients with ISR had lower circulating levels of CACs, a feature that may attenuate reconstitution of a normal endothelium and, thereby, contribute to neointimal formation. With studies of CAC abundance continuing in our laboratory, we soon expect to clarify the predictive role of the CAC abundance in identifying patients at high risk of ISR.
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FOOTNOTES |
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* B. Hibbert and Y.-X. Chen contributed equally to this work. 
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4-integrin subunit attenuates the acute inflammatory response to stent implantation yet is insufficient to prevent late intimal formation. J Leukoc Biol 75: 1016–1021.
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