Published online before print April 17, 2008, doi: 10.1161/ATVBAHA.108.165753
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
CD44 Expressed on Both Bone Marrow–Derived and Non–Bone Marrow–Derived Cells Promotes Atherogenesis in ApoE-Deficient Mice
From the The Wistar Institute (L.Z., E.L., A.M.Z., M.K.M., M.K., P.S.A., J.A.H., E.P.), Philadelphia, Pa; the Department of Medicine (P.S.A., D.J.R., E.P.) and the Immunology Graduate Group (M.K.M., M.K., E.P.), University of Pennsylvania School of Medicine, Philadelphia; and the Ludwig Institute for Cancer Research (E.P.), Philadelphia, Pa.
Correspondence to Ellen Puré, PhD, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail pure{at}wistar.org
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Abstract |
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Objective— The purpose of this study was to distinguish the contributions of CD44 expressed on bone marrow–derived and non–bone marrow–derived cells to atherosclerosis.
Methods and Results— Using bone marrow chimeras, we compared the contributions of CD44 expressed on bone marrow–derived cells versus non–bone marrow–derived cells to the vascular inflammation underlying atherosclerosis. We show that CD44 in both bone marrow–derived and non–bone marrow–derived compartments promotes atherosclerosis in apoE–/– mice and mediates macrophage and T cell recruitment to lesions in vivo. We also demonstrate that CD44 on endothelial cells (ECs) as well as on macrophages and T cells enhances leukocyte-endothelial cell adhesion and transendothelial migration in vitro. Furthermore, CD44 on vascular smooth muscle cells (VSMCs) regulates their hyaluronan (HA)-dependent migration. Interestingly, in mice lacking CD44 in both compartments, where we observed the least inflammation, we also observed enhanced fibrous cap formation.
Conclusions— CD44 expressed on bone marrow–derived and non–bone marrow–derived cells both promote atherosclerosis in apoE-deficient mice. Furthermore, CD44 plays a pivotal role in determining the balance between inflammation and fibrosis in atherosclerotic lesions which can impact clinical outcome in humans.
Key Words: CD44 • atherosclerosis • apoE • bone marrow chimera • fibrous cap
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Introduction |
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Both vascular and inflammatory cells are critical to atherogenesis, but the distinct mechanisms by which these lineages contribute to disease are incompletely understood. Atherosclerosis is initiated by proinflammatory changes in the endothelium caused by insults such as the accumulation of oxidized or other modified lipoproteins and flow-induced mechano-signal transduction.1,2 The resulting upregulation of endothelial cell (EC) adhesion molecules and production of proinflammatory cytokines and chemokines promotes recruitment of inflammatory cells including monocytes and T cells.3,4 Vascular injury also promotes vascular remodeling by inducing proliferation and migration of vascular smooth muscle cells (VSMCs). Interestingly, evidence suggests that the balance between the fibrotic response, which promotes the formation of fibrous caps, and the inflammatory response, which inhibits formation of fibrous caps, governs the structure of the atheroma. The overall structure of atheroma in turn determines the susceptibility of plaques to rupture.5
CD44 is known to be upregulated in human and mouse atherosclerotic lesions and is expressed on all cell types known to play a role in atherogenesis.6,7 Moreover, ligands for CD44, such as the extra-/pericellular matrix glycosaminoglycan hyaluronan (HA) and osteopontin, accumulate in atherosclerotic lesions.8,9 We previously demonstrated that CD44-null apoE–/– mice develop less extensive atherosclerosis attributable to impaired recruitment of macrophages to atherosclerotic lesions and regulation of vascular smooth muscle cell (VSMC) proliferation.6,10 However, these studies did not distinguish the impact of CD44 expressed in various cellular compartments on atherogenesis. For example, we and others demonstrated that CD44 directly regulates VSMC proliferation in vitro6 and in an injury model in vivo,11 and that CD44 regulates macrophage inflammatory gene expression and vascular gene expression in a proatherogenic environment.12,13 Furthermore, vascular endothelial cell CD44 has been shown to promote leukocyte recruitment to sites of inflammation.14
To distinguish the contributions of CD44 expressed on bone marrow–derived versus non–bone marrow–derived cells to atherogenesis, we generated bone marrow chimeras between apoE–/–.CD44+/+ and apoE–/–.CD44–/– mice. We demonstrate that CD44 expressed in either compartment promotes atherogenesis. Furthermore, CD44 expression in either compartment promotes macrophage and T cell recruitment to lesions, at least in part by mediating macrophage and T cell adhesion and transendothelial migration. We also show that CD44 regulates VSMC migration in response to exogenous low molecular weight HA (LMW-HA). Interestingly, the impact of CD44 on inflammation correlated inversely with its impact on the extent of fibrosis in lesions. Taken together, these data indicate that CD44 expressed on bone marrow–derived and non–bone marrow–derived cells promotes atherogenesis in apoE-deficient mice and that CD44-deficiency in both compartments promotes fibrosis in atherosclerotic lesions.
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Materials and Methods |
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Bone marrow chimeras were generated between age- and sex-matched apoE–/–.CD44+/+ and apoE–/–.CD44–/– mice. Reconstitution was confirmed based on CD44 expression in splenocytes and aortic tissues isolated from recipients as determined by flow cytometry analysis and immunohistochemical staining, respectively. The atherosclerotic lesion in aortic root and on the luminal surface of aorta was quantified independently, both by computer assisted analyses.32 CD44 mediated leukocyte recruitment to atherosclerotic lesions was evaluated by in vivo macrophage and T cell homing.33 The leukocyte adhesion to endothelial cells and transendothelial migration were assayed in vitro by CytoSelect Leukocyte Transmigration Assay (Cell Biolabs, Inc) according to manufacturer’s recommendations. The scratch wound assays were performed as described previously.34 Migration of VSMCs was determined using CytoSelect 96-well 8-µm Cell Migration Assay (Cell Biolabs, Inc) and detected by CyQuant GR Dye. The fibrous cap formation in advanced lesions was determined by IHC staining for
-SMA and Trichrome staining for collagen. Data were normalized by Kolmogorov-Smirnov and Shapiro-Wilk normality tests. The parametric data were analyzed by 1-way ANOVA followed by Bonferroni multiple comparison, whereas nonparametric data were analyzed by Kruskal-Wallis ANOVA followed by Dunns multiple comparison. The mean velocity was compared by t test, and the instantaneous velocity (Vinst) was analyzed by the Mann-Whitney test. Probability values less than 0.05 was considered statistically significant in all analyses. Data are presented as mean±SD, and the error bar in the figure represents standard deviation. All methods are described in greater details in the supplemental materials (available online at http://atvb.ahajournals.org).
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Results |
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CD44 Expressed on Both Bone Marrow–Derived and Non–Bone Marrow–Derived Cells Promotes Atherosclerosis in ApoE-Deificient Mice
To distinguish the contributions of CD44 expressed on bone marrow–derived and non–bone marrow–derived cells, we generated bone marrow chimeras between CD44-wild-type and CD44-null apoE–/– mice. We determined that full reconstitution of the peripheral hematopoietic compartment was established by 7 weeks post–bone marrow transfer and resulted in >90% chimerism based on CD44 phenotype of splenocytes, as monitored by fluorescence activated flow cytometry. Furthermore, within the limits of sensitivity of immunohistochemistry, we found that the vast majority of hematopoietic cells in the atherosclerotic lesions of chimeric mice were donor-derived, whereas the vast majority of the vascular cells retained the phenotype of the recipients (supplemental Figure I).
To compare the effects of CD44 expressed by bone marrow–derived versus non–bone marrow–derived cells on atherogenesis, the extent of atherosclerosis was independently quantified by en face analysis of the aorta and cross-sectional analysis of the aortic roots of chimeric mice 14 weeks posttransplantation. We found that among the 4 types of chimeras generated, the apoE–/– 
apoE–/– control group exhibited the greatest amount of lesion, whereas the extent of atherosclerosis in the DKO DKO chimeric mice was most markedly reduced. Interestingly, the extent of lesion in 2 experimental groups, apoE–/– DKO and DKO apoE–/–, was intermediate compared to the DKO DKO and apoE–/– apoE–/– controls (Figure 1A and 1B). Total plasma cholesterol levels were comparable in all groups (Figure 1C), indicating that CD44 on bone marrow–derived and non–bone marrow–derived cells promotes atherogenesis in apoE–/– mice independent of effects on total plasma cholesterol levels.
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CD44 Expressed by Leukocytes and Non–Bone Marrow–Derived Cells Mediates Macrophage and T Cell Recruitment to Atherosclerotic Lesions In Vivo
Recruitment of monocytes into artery wall is a critical early event in atherosclerosis.15 We previously demonstrated that macrophage CD44 mediates trafficking of these cells to atherosclerotic lesions in apoE–/– mice.6 Furthermore, in the proatherogenic environment of apoE–/– mice, vascular CD44 is functionally activated with regard to HA binding activity, even before lesion development.12 We investigated the possibility that vascular CD44 may also play a role in recruiting macrophages to the vessel wall by quantifying the capacity of CD44+/+ and CD44–/– macrophages to home to atherosclerotic lesions not only in apoE–/–.CD44+/+ mice but also in apoE–/–.CD44–/– mice at 48 hours posttransfer. Because it would require a large number of mice to isolate sufficient numbers of peripheral blood monocytes, thioglycollate-elicited macrophages, which although differerent from circulating monocytes in their state of differentiation, are often used as a surrogate for homing assays. We observed homing of a significant number of CD44–/– macrophages to lesions when injected into apoE–/–.CD44+/+ mice. Indeed, the homing of macrophages in this case was as efficient as that of CD44+/+ macrophage homing to lesions in apoE–/–.CD44–/– mice and significantly more than homing of CD44–/– macrophages to lesions in apoE–/–.CD44–/– mice (Figure 2A). Thus, both macrophage and vascular CD44 is sufficient to promote macrophage recruitment to atherosclerotic lesions in apoE-deficient mice.
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T cells have been established as an integral component of atherogenesis,3 but the role of CD44 in T cell recruitment to atherosclerotic lesions has not been previously investigated. Therefore we investigated the in vivo homing of CD44+/+ and CD44–/– T cells to atherosclerotic lesions in both apoE–/–.CD44+/+ and apoE–/–.CD44–/– mice. Anti-CD3 antibody activated T cells were fluorescence-labeled, injected i.v. into apoE–/–.CD44+/+ and apoE–/–.CD44–/– mice, and homing was quantified 48 hours later. The most recruitment of T cells to atherosclerotic lesions was observed in apoE–/–.CD44+/+ mice that received CD44+/+ T cells, whereas the least recruitment was observed in apoE–/–.CD44–/– mice that received CD44–/– T cells. When CD44 was expressed only on either host non–bone marrow–derived cells or only on the transferred T cells, the efficiency of homing was intermediate and comparable (Figure 2B). Immunohistochemical staining of the fluorescently labeled cells indicated that both CD4+ and CD8+ T cells homed to atherosclerotic lesions in a CD44-dependent manner (data not shown). These data demonstrate that expression of CD44 in either non–bone marrow–derived cells or inflammatory leukocytes is sufficient to mediate macrophage and T cell recruitment to lesion.
To investigate the mechanism by which CD44 mediates leukocyte recruitment to atherosclerotic lesions, in vitro adhesion and transendothelial migration assays were performed between thioglycollate-activated macrophages or anti-CD3 antibody activated T cells and vascular ECs isolated from apoE–/–.CD44+/+ and apoE–/–.CD44–/– mice. The greatest adhesion and transendothelial migration was observed between CD44+/+ ECs with CD44+/+ T cells or macrophages, whereas the adhesion and transmigration was markedly reduced between CD44–/– ECs and CD44–/– T cells or macrophages (supplemental Figure II). These data suggest that the impact of CD44 on in vivo homing of macrophages and T cells to atherosclerotic lesions is at least in part attributable to the capacity of CD44 on either the vascular endothelium or on the leukocyte to mediate leukocyte adhesion and transendothelial migration.
CD44 Impacts VSMC Migration Through Interaction With Hyaluronan
VSMC modulation to a synthetic state, a hallmark of atherosclerosis, is characterized by increased proliferation, migration, and alterations in gene expression. There is downregulation of genes that define the contractile state, such as -smooth muscle actin (-SMA), and induction of genes that regulate cell adhesion and inflammation, such as vascular cell adhesion molecule (VCAM)-1 and matrix components.6,16,17 We reported decreased expression of VCAM-1 on VSMCs in atherosclerotic lesions of apoE–/–.CD44–/– mice6 and increased expression of genes associated with a contractile phenotype in aorta of apoE–/–.CD44–/– mice before lesion formation.12 In this study, we used live cell microscopy to analyze the migration of VSMCs isolated from CD44+/+ and CD44–/– mice in response to injury in a scratch wound assay. We found that CD44–/– VSMCs exhibited less directional migration in wound assays compared to CD44+/+ VSMCs (Figure 3A). A graphic depiction of the positional Y-coordinates of 10 representative KO and WT VSMCs (in a single experiment representative of >3 experiments performed), were tracked by live cell imaging and analyzed. These data indicated that CD44–/– VSMCs migrated in a less ordered and less directional manner (Figure 3B). However, CD44–/– VSMCs exhibited a significant increase in average mean velocity (Figure 3C) and in instantaneous velocity (Figure 3D) when compared to CD44+/+ VSMCs. These data establish that CD44–/– VSMCs have increased motility but are less directional, thus increasing the time necessary for wound closure, whereas CD44+/+ VSMCs are less motile but more directional providing for more "efficient" wound closure.
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We previously reported that HA, which accumulates in atherosclerotic lesions, regulates VSMC growth in a CD44-dependent manner.8 HA has also been implicated in regulating the migration of mesenchymal cells.18 We determined the effect of HA on migration of VSMCs isolated from CD44 wild-type and CD44-null mice in a dual chamber migration assay. Whereas PDGF induced comparable migration of CD44+/+ and CD44–/– VSMCs, HMW-HA had no effect on the migration. However, LMW-HA induced migration of CD44+/+ VSMCs but not CD44–/– VSMCs (Figure 4). Furthermore, the migration of wild-type cells induced by LMW-HA was ablated in the presence of anti-CD44 antibody KM81, which blocks HA binding to the receptor. These results suggest that CD44-LMW-HA interactions provide 1 mechanism underlying VSMC migration in atherosclerotic lesions and that the impact on atherosclerosis may reflect a balance of an array of direct effects of CD44 on the growth and migration of VSMCs.
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Lesions in Chimeras Lacking CD44 in both Non–Bone Marrow–Derived and Bone Marrow–Derived Compartments Exhibit Enhanced Fibrotic Cap Formation
To determine the impact of CD44 expressed in bone marrow–derived and non–bone marrow–derived cells on the composition and morphology of atherosclerotic lesions, we performed immunohistochemical analysis of lesions in aortic root from each of the groups of chimeric mice. Lesions were characterized based on morphology according to previously published criteria.19 We did not observe any differences in the types of early lesions between the various groups. However, based on -SMA expression as detected by immunohistochemical staining and total collagen content as determined by trichrome staining, we found that among more advanced lesions, mice deficient in CD44 in both compartments (DKO DKO) had significantly more fibroatheromas compared to the chimeras that expressed CD44 in both compartments (apoE–/– apoE–/–; Figure 5). However, the fibrotic areas in the mixed chimeras were not statistically different than that observed in the DKO DKO or apoE–/– apoE–/– control groups. Thus, in addition to impact on the extent of atherosclerosis, CD44 expression also appears to impact the composition and architecture of atheromas by reducing fibrous cap formation.
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Discussion |
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In a previous study, apoE–/– mice deficient in CD44 were shown to be protected against atherosclerosis.6 However, because CD44 can regulate the function of both inflammatory cells and parencyhmal cells,20 it was not possible from that study to determine the relative contributions of CD44 in the 2 compartments to atherogenesis. The current study provides insight into these questions by independently defining the impact of CD44 expressed on bone marrow–derived cells and non–bone marrow–derived cells. We demonstrate that CD44 on either bone marrow–derived cells or non–bone marrow–derived cells is sufficient to promote vascular inflammation by mediating T cell and macrophage recruitment to lesions and thereby the development of atherosclerosis. Moreover, CD44-deficiency in both compartments promotes fibrous cap formation which may reflect the impact of CD44 on plaque inflammation and the impact on VSMC functions.
Recruitment of circulating monocytes and lymphocytes is critical to early events in the development of atherosclerosis.15,21 The current study demonstrates that CD44 expressed on both bone marrow–derived and non–bone marrow–derived cells promotes atherogenesis in apoE-deficient mice at least in part through recruiting T cells and macrophages to sites of arterial inflammation. A role for inflammatory cell CD44 in leukocyte recruitment to sites of inflammation is well established.6,22,23 However, the role of CD44 expressed by non–bone marrow derived cells in atherosclerosis is not well understood. We recently demonstrated that CD44, in lesion-prone aorta of apoE-deficient mice, is upregulated before lesion formation and is functionally activated resulting in expression of the receptor in a state of high affinity for its ligand, HA.12 HA docking to ECs, possibly through CD44, has been shown to contribute to leukocyte recruitment to the vessel wall.24 Thus, HA accumulated in atherosclerotic lesions may serve as a bridge between CD44 on activated endothelium and CD44 on circulating leukocytes.8,25 The data presented herein support this model. Interestingly, whereas this model emphasizes HA expressed by ECs as the source of the "ligand bridge," we also detected moderate levels of cell surface-associated HA on macrophages and T cells that may serve a similar function (data not shown). Thus, taken together with our previous observation that vascular CD44 is upregulated before lesion formation, the data we present herein indicate that non–bone marrow–derived CD44 plays an equally important role as leukocyte CD44, in CD44-mediated inflammatory cell recruitment to atherosclerotic lesions.
In addition to its contributions to leukocyte recruitment, CD44 has the potential to regulate leukocyte and vascular gene expression, VSMC proliferation and migration, which may promote disease progression, and fibrous cap formation. Interestingly, we found that expression of CD44 relates directly to inflammation and inversely correlates with the development of fibroatheromas. We did not previously observe significant morphological difference in less advanced lesions of apoE–/–.CD44–/– mice compared to apoE–/–.CD44+/+ controls.6 The different impact on lesion composition observed in this study may be because the lesions in this study were more advanced and notably more complex, in part because of acceleration of atherogenesis and inflammation as a consequence of the irradiation protocol required to generate the chimeras.26 Indeed, the effects of irradiation may have made the protective effect of CD44-deficiency more apparent.
We observed that CD44 deficiency increased fibrous cap formation, but whether this is the result of direct effects of CD44 on VSMC function or dedifferentiation, or reflects an indirect effect of the reduction in inflammation, remains to be determined. These 2 mechanisms may not be mutually exclusive. In addition to its impact on VSMC proliferation6,11 and migration, we demonstrated that in a proatherogenic environment, CD44 also regulates gene expression in aorta that are associated with the differentiate state of VSMCs.12 Thus, direct effects of CD44 on VSMC function may impact fibrous cap formation. However, our data suggest that the impact of CD44 on inflammation may also be an important contributing factor in terms of lesion composition. Inflammation inhibits fibrous cap formation in 2 ways: by blocking the production of collagen fibers and by the destruction of existing collagen. In the arterial wall, transforming growth factor (TGF)-β stimulates collagen production by VSMCs.27 T cells in the plaque inhibit collagen production by VSMCs and the stimulatory effects of TGF-β via secretion of IFN-
.27,28 Notably, several factors produced by inflammatory cells such as CD40 ligand and interleukin (IL)-1 induce the production of collagen-degrading enzymes by macrophages such as members of matrix metalloproteinase (MMP) family.29 Therefore, reduced plaque inflammation resulting from CD44-deficiency may be an indirect mechanism underlying the difference in plaque composition we observed. CD44 also inhibits production of extracellular matrix components such as collagens and latent TGF-β binding proteins in aorta in a proatherogenic environment.12 CD44 can also regulate MMP-mediated activation of TGF-β on tumor cells30 and fibroblasts.31 In future studies, it will be interesting to determine whether CD44 regulates TGF-β activity in atherosclerotic lesions. Taken together, our data indicate that CD44 expression in both compartments inhibits fibrous cap formation at least by mediating plaque inflammation and regulating vascular gene expression, and thus impacting VSMC function.
In summary, the findings reported in this study demonstrate that the deletion of CD44 in either the bone marrow–derived or non–bone marrow–derived compartments reduces lesion burden in apoE–/– deficient mice. It will be important in future studies to determine the impact of CD44 in other animal models of atherosclerosis such as LDLR–/– mice and in man. The effects of CD44 in promoting inflammatory cell recruitment and in regulating VSMC migration suggest that CD44 may play a pivotal role in enhancing the initiation and progression of atherosclerosis, and in regulating the composition of lesions so as to impact their susceptibility to rupture. These findings support the notion that functional inhibition of CD44 is of potential therapeutic value and that a combination of bone marrow–derived and non–bone marrow–derived targets may enhance the treatment of atherosclerosis and the acute events that result from cardiovascular disease.
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Acknowledgments |
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We thank Dr Wen-Hwai Horng in the Wistar Institute for his consultation of the data analysis.
Sources of Funding
This study was supported by Public Health Service grants from The National Heath Institute (HL65507, HL70121, PO10-HL-06225006), and a grant from Pennsylvania Department of Heath.
Disclosures
None.
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Footnotes |
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Original received October 15, 2007; final version accepted April 7, 2008.
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