CD44 Regulates Vascular Gene Expression in a Proatherogenic Environment
Objective— To identify early changes in vascular gene expression mediated by CD44 that promote atherosclerotic disease in apolipoprotein E (apoE)–deficient (apoE−/−) mice.
Methods and Results— We demonstrate that CD44 is upregulated and functionally activated in aortic arch in the atherogenic environment of apoE−/− mice relative to wild-type (C57BL/6) controls. Moreover, CD44 activation even in apoE−/− mice is selective to lesion-prone regions because neither the thoracic aorta from apoE−/− mice nor the aortic arch of C57BL/6 mice exhibited upregulation of CD44 compared with thoracic aorta of CD57BL/6 mice. Consistent with these observations, gene expression profiling using cDNA microarrays and quantitative polymerase chain reaction revealed that ≈155 of 19 200 genes analyzed were differentially regulated in the aortic arch, but not in the thoracic aorta, in apoE−/− CD44−/− mice compared with apoE−/− CD44+/+ mice. However, these genes were not regulated by CD44 in the context of a C57BL/6 background, illustrating the selective impact of CD44 on gene expression in a proatherogenic environment. The patterns of differential gene expression implicate CD44 in focal adhesion formation, extracellular matrix deposition, and angiogenesis, processes critical to atherosclerosis.
Conclusions— CD44 is an early mediator of atherogenesis by virtue of its ability to regulate vascular gene expression in response to a proatherogenic environment.
Atherosclerosis is a vascular disease initiated by mechanical, biochemical, or immunologic injury to the arterial wall. Such insults induce changes in the phenotype1 and transcription profile2 of vascular cells, triggering a cascade of events that includes endothelial cell (EC) dysfunction and inflammation as well as vascular smooth muscle cell (VSMC) dedifferentiation, activation, and proliferation. Many of these processes are regulated by the actions of cellular adhesion molecules.3
CD44 is a widely expressed cellular adhesion molecule that serves as a principal receptor for extracellular matrix (ECM) components such as hyaluronan (HA), which accumulates in atherosclerotic lesions.4,5 CD44 was demonstrated to play a role in cellular growth, activation, and migration, mediates signal transduction, and regulates gene expression.6 CD44 promotes adhesion of leukocytes to ECs,7,8 induces the release of inflammatory mediators from macrophages, and regulates the proliferation and migration of VSMCs.9,10
We demonstrated that CD44-deficient apolipoprotein E (apoE)–null mice developed markedly reduced atherosclerosis compared with CD44 wild-type apoE-null littermates.11 Data presented in that study, together with previous reports, indicated that CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and the activation of leukocytes and VSMCs.8,12
To identify vascular genes regulated by CD44 that may predispose the vessel wall to development of atherosclerosis, we compared the global gene expression profile between CD44 wild-type and CD44-null apoE-deficient (apoE−/−) mice before the development of lesion in regions of aorta that are susceptible to the development of atherosclerosis to varying degrees (arch >abdominal >thoracic). We identified genes that were selectively regulated by CD44 in aortic arch but not in thoracic aorta of apoE−/− mice. Genes identified by this global approach indicated that CD44 may promote atherogenesis by regulating focal adhesion formation, ECM deposition, and angiogenesis. In contrast, the majority of these genes were not regulated by CD44 in the aortic arch of C57BL/6 mice. We conclude that CD44 contributes to atherogenesis in part by modulating vascular gene expression in response to a proatherogenic environment.
Materials and Methods
The expression and functionality of vascular CD44 were compared between apoE−/− and C57BL/6 mice (10th generation) at 10 to 12 weeks of age by ELISA and immunoblotting. Analyses of gene expression profiles by microarray were performed in aortic arch and thoracic aorta between age- and sex-matched apoE−/− and apoE/CD44 double knockout (DKO) mice at 10 to 12 weeks of age. The regulation of vascular gene expressions by CD44 was validated by quantitative polymerase chain reaction (qPCR), immunoblots, and immunohistochemistry (http://atvb.ahajournals.org).
CD44 Expression Is Upregulated and Functionally Activated in the Aortic Arch of ApoE−/− Mice
CD44 is expressed on the majority of cells. In many primary cells, activation leads to increased expression and transition of the receptor from a low-affinity state to a high-affinity state.13,14 We reported that deficiency in CD44 attenuates the extent of atherosclerosis in apoE−/− mice. To determine whether vascular CD44 plays an early role in atherogenesis, we compared the expression of CD44 and its HA binding capacity in aortae of apoE−/− mice before development of lesions compared with aortae from age- and sex-matched wild-type mice. Because various regions of aorta differ in their relative resistance/susceptibility to the development of atherosclerosis, tissue from each region was evaluated separately. Quantification of total levels of CD44 in aortic extracts by ELISA demonstrated that CD44 expression in aortic arch of apoE−/− mice was upregulated ≈2-fold when compared with the aortic arch from C57BL/6 mice and compared with thoracic and abdominal aorta from apoE−/− and C57BL/6 mice, which were comparable to each other (Figure 1A). These results were confirmed by immunoblotting with KM81 antibody, which recognizes all isoforms of CD44. Furthermore, immunoblotting indicated that the ≈85-kDa standard form of CD44 is the predominant isoform of CD44 expressed in all regions of aorta from apoE−/− and C57BL/6 mice at this age (Figure 1B, top).
To determine whether the receptor was functionally activated, CD44 from aortic tissue was analyzed using a functional HA binding assay as described.15 HA binding to a single ≈85-kDa species that comigrated with the standard form of CD44 was markedly upregulated in CD44 immune complexes isolated from aortic arch of apoE−/− mice compared with aortic arch of C57BL/6 and thoracic and abdominal aorta from apoE−/− and C57BL/6 mice (Figure 1B, bottom). HA binding was shown to be specific to CD44 by inhibition of binding in the presence of a blocking antibody (KM81) against the HA binding site of the receptor (data not shown). The extent of the increase in HA binding, ≈6-fold (Figure 1C), cannot be explained simply by the increase in expression of CD44 (≈2-fold), indicating that CD44 was functionally activated.
CD44-Dependent Regulation of Vascular Gene Expression in ApoE−/− Mice
To investigate the mechanisms by which vascular CD44 impacts atherogenesis, we compared the gene expression profiles in aortic arch and thoracic aorta isolated from CD44-null and CD44-wild type apoE−/− mice. To focus on the role of vascular CD44 in the absence of potentially confounding contributions from infiltrating inflammatory cells, gene expression analysis was performed on RNA isolated from aortic tissue of 10-week-old mice before lesion formation. The lack of lesion was confirmed by en face analysis of aorta from age- and sex-matched apoE−/− mice (data not shown).
Furthermore, we did not detect expression of RNA of hematopoietic lineage-specific markers in the aortic tissues used in the microarray analysis nor by qPCR for CD68 as a marker for macrophages or CD3 for T cells. On the other hand, qPCR analysis of VSMC- and EC-specific genes, α-smooth muscle cell actin, and CD31 (platelet endothelial cell adhesion molecule [PECAM]-1), respectively, indicated that the expression analysis reflected EC and VSMC transcripts (supplemental Figure I, available online at http://atvb.ahajournals.org). Thus, it can be concluded that the differential gene expression we describe largely reflects CD44-dependent regulation in vascular cells such as VSMCs or ECs.
In view of the gender bias in atherosclerosis, the data from male and female mice were analyzed separately. However, we focused our analysis on genes that were differentially regulated by CD44 in a gender-independent fashion. Based on these criteria, 207 genes were identified by microarray analysis as regulated by CD44 in aortic arch of apoE−/− CD44−/− mice relative to apoE−/− CD44+/+ mice (supplemental Table I) including ECM proteins, adhesion receptors, intracellular signaling molecules, and transcription factors.
qPCR Validation of Differentially Expressed Genes
qPCR was performed for a randomly selected subset of the genes identified in the microarray analysis as differentially regulated in the aortic arch of apoE−/− CD44−/− mice compared with apoE−/− CD44+/+ mice. Of a total of 65 genes analyzed by qPCR, 49 genes was confirmed (Table). This represents a validation rate of 75% of the genes tested. Based on this validation rate, without taking into account genes not represented on our arrays or the potential for false negatives in the array analysis, we predict that ≈155 genes are regulated by CD44 in the aortic arch of apoE−/− mice independent of gender. Interestingly, another HA receptor, HA-mediated motility receptor, which was not in the gene list of the array, also was upregulated in aortic arch of apoE−/− CD44−/− mice as validated by qPCR (Table), indicating a compensatory role of HA-mediated motility receptor for CD44-deficiency.
We also performed gene ontology analysis using IPA tool. Six networks and 9 functions were identified that are associated with the differentially expressed genes and that are also associated with processes involve in atherosclerosis, including cellular assembly and organization, cell-cell interaction and signaling, and cellular movement. The details of the analysis results were shown in online supplements.
Protein Level Validation of Differentially Expressed Genes
Of the differentially expressed genes validated by qPCR, several were also analyzed at the protein level by immunoblotting (Figure 2A and 2B) and immunohistochemistry (Figure 2C). Differential protein expression was observed for the majority of cases tested, including Hsp90, Actb, and Zyxin, for example. We were particularly interested in the impact of CD44 on the expression of the antiangiogenic factor endostatin because its precursor protein, collagen XVIII, was one of the genes most affected at the RNA level and was implicated recently in atherogenesis.16,17 We found that endostatin was indeed elevated in aortic arch of apoE−/− CD44−/− mice compared with apoE−/− CD44+/+ mice.
Immunohistochemistry showed comparably high levels of Actb in ECs, whereas VSMCs in the tunica media had enriched levels of Actb in apoE−/− CD44−/− mice. Hsp90 and Zyxin appeared to be upregulated in the aortic arch of apoE−/− CD44−/− mice in ECs and VSMCs. We also noted a marked increase in the intensity of nuclear staining of Zyxin and Hsp90 in the aortae of apoE−/− CD44−/− mice, suggesting that CD44 may regulate their subcellular localization and thereby their functions.
CD44-Dependent Regulation of Vascular Gene Expression Is Associated With the Focal Susceptibility of ApoE−/− Mice to Develop Atherosclerosis
As described above, vascular CD44 is functionally activated in a proatherogenic environment. An important question is whether the CD44-dependent regulation of vascular gene expression we observed in the aortic arch of apoE−/− mice is constitutive or induced by a proatherogenic environment. It was also critical to define whether CD44-mediated gene regulation is selective to vascular regions susceptible to the development of lesions.
To address the first of these issues, we performed qPCR analysis for a subset of the genes in a nonatherogenic environment by comparing vascular gene expression in CD44-deficient and CD44 wild-type C57BL/6 mice. Of 16 genes tested (from the set of 49 genes validated by qPCR in the context of an apoE-null background), we found that the majority were not differentially expressed in the aortic arch of CD44-deficient mice compared with wild-type C57BL/6 mice (Figure 3A). Together with the data in Figure 1, these results indicate that the differential gene expression in aortic arch between apoE−/− CD44−/− mice and apoE−/− CD44+/+ mice reflects a role of CD44 in the response to early proatherogenic processes in apoE−/− mice, such as hypercholesterolemia or early inflammatory signals.
To determine whether regulation of gene expression by CD44 in apoE−/− mice is selective to regions susceptible to atherosclerotic lesion formation, we compared gene expression in thoracic aorta between age- and sex-matched apoE−/− CD44−/− mice and apoE−/− CD44+/+ mice. Of the 207 differentially expressed genes regulated by CD44 in aortic arch, only 7 of these genes were also differentially expressed in the thoracic aorta of apoE−/− CD44−/− compared with apoE−/− CD44+/+ mice (Figure 3B; supplemental Table I). These results indicate that CD44-dependent effects on vascular gene expression in apoE−/− mice are largely selective to atherosclerosis-prone regions of the aorta, providing additional support for the notion that CD44-dependent regulation of gene expression is induced in response to local proatherogenic signals.
To understand the molecular basis by which vascular CD44 promotes atherogenesis, we investigated the functional state of CD44 in prelesion aorta and compared the gene expression profiles of aortic arch isolated from apoE−/− CD44−/− and apoE−/− CD44+/+ mice before lesion development. We demonstrate that a proatherogenic environment results in early upregulation and functional activation of vascular CD44. Furthermore, in the context of such an environment, CD44 regulates vascular gene expression. Many of these genes have been reported to be involved in atherosclerosis. Thus, the early impact of CD44 on vascular gene expression is likely to be in part responsible for the difference in the extent of atherosclerosis we observed in apoE−/− CD44−/− and apoE−/− CD44+/+ mice.11
The apoE−/− mouse model shares many early events associated with atherogenesis in man, including hypercholesterolemia, lipoprotein accumulation, localization to regions of disturbed blood flow,18 and inflammation.19,20 Our results indicate that the expression and function of CD44 and CD44-dependent regulation of vascular gene expression is invoked by one or more of these proatherogenic stimuli. However, comparable levels of total plasma cholesterol between apoE−/− CD44−/− and apoE−/− CD44+/+ mice exclude hypercholesterolemia as a direct cause sufficient to induce the differential gene regulation observed (data not shown). That the CD44-dependent effects on vascular gene expression are largely selective to atherosclerosis-prone regions in apoE−/− but not C57BL/6 mice, further supports our conclusion that CD44 regulates vascular gene expression in response to a local proatherogenic environment. This is in keeping with our previous observations that CD44 plays important roles that are evident in disease states rather than under normal homeostatic conditions.11
The finding that CD44 is an early regulator of atherosclerotic disease begs the question as to what causes its upregulation in prelesion vasculature. The focal regulation of CD44 we observed suggests that mechanosignal transduction attributable to decreased shear stress at sites of disturbed flow may induce and activate CD44 on vascular cells, which is consistent with a recent report that CD44 is upregulated in vascular wall in response to hypercholesterolemia21 and disturbed flow.2 Furthermore, genes regulated by mechanosignal transduction independent of CD44 may regulate CD44 expression and activity in atherosclerosis-prone areas before lesion formation. Regardless of the mechanism by which CD44 is regulated, our study indicates that the accumulation of HA22 and the upregulation of vascular CD44 promote atherogenesis by regulating vascular gene expression.
Interestingly, in most pathways, whereas the majority of the genes were negatively regulated by CD44, several genes were positively regulated in each connectivity; despite this finding, the net effect of CD44 regulation on signaling and vascular cell functions in each pathway is not necessarily negated. The pathway analysis indicates that CD44 is primarily involved in 3 critical pathological processes in the vessel wall: focal adhesion formation, deposition of ECM, and angiogenesis, all of which have been implicated in atherosclerosis.
Focal adhesion plaques are structures that form at the ends of actin fibers and serve as sites for force transmission, thereby playing a role in the initiation and progression of atherosclerosis in response to disturbed blood flow.23 A role for CD44 in focal adhesion is consistent with indirect evidence of cross-talk between CD44 and integrins. The gene expression profiling indicates that CD44 may regulate the assembly of adhesion complexes by regulating the expression of focal adhesion molecules such as zyxin, β-actin, syndecan 4, Thy1, and integrins.
The ECM participates in several key atherogenic events, such as cell migration and proliferation, lipoprotein retention, and thrombosis, as well as plaque stabilization.24,25 CD44 is critical to the formation of HA-rich pericellular matrix and turnover, thus influencing the adhesion and migration of cells.26 However, we did not detect a significant difference in HA content in the vessel wall between apoE−/− CD44−/− and apoE−/− CD44+/+ mice (data not shown). This may be attributable to the upregulation of hyaluronidase Mgea5 or a possible compensatory effect of upregulation of another HA receptor, HA-mediated motility receptor, in apoE−/− CD44−/− mice that can compensate for the loss of CD44 in HA turnover.27 The CD44-dependent differential expression of ECM components, such as ECM1, Col18α1, Ltbp, and Npnt, may also change the nature of the interactions with their receptors, thereby indirectly regulating related signaling pathways.
Increased neovascularization is also a feature of atherosclerosis.28 Several antiangiogenic and proangiogenic effects of CD44 have been described in the context of tumors.29 However, the role of CD44 in angiogenesis in atherosclerosis has not been investigated. We found several genes involved in angiogenesis are regulated by CD44, including Col18α1, ECM1, Itgb5, Hsp90, Sdc4, Thy1, and Wnt2. Some of these have reported proangiogenic roles such as Thy1, whereas others, such as endostatin, have antiangiogenic roles. Endostatin, the C-terminal fragment of col18α1, is a potent antiangiogenic peptide reported to reduce intimal neovascularization and plaque growth in apoE−/− mice16 and enhance lipoprotein retention in subendothelial matrix.17 Therefore, additional studies to determine the role of CD44 in angiogenesis in atherosclerosis will be of interest.
We used global analysis of gene expression in the aortae of apoE−/− mice to determine the functional significance of the early activation of CD44 in atherosclerosis-prone environment. These experiments led to our discovery of CD44-dependent regulation of multiple genes in various pathways that may play a critical role in the response to a local proatherogenic environment. The role of the CD44-regulated genes identified in this study in vascular function will be investigated using genetic approaches that target specific components of these genes. Alternatively, these genes can be targeted in vitro and in vivo using a pharmacological approach to determine their roles in vascular cell function. In addition, development of a conditional CD44 knockout mouse will help define the role of CD44 expressed in specific vascular cell types.
In summary, the putative links we established herein provide new insights into the potential mechanisms by which CD44 regulates atherogenesis and underscore its distinct roles in the homeostatic versus the inflammatory state. These data provide a framework for developing novel targets for the treatment of atherosclerotic disease.
Sources of Funding
This study was supported by public health service grants from the National Health Institute (HL65507, HL70121, HL62250, and PO1-HL-06225006) and a grant from the Pennsylvania Department of Health.
Original received July 14, 2006; final version accepted January 17, 2007.
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