β-Arrestins Regulate Atherosclerosis and Neointimal Hyperplasia by Controlling Smooth Muscle Cell Proliferation and Migration
Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins β-arrestin1 and -2 might regulate this pathological process. Deficiency of β-arrestin2 in ldlr−/− mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that β-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, β-arrestin1−/−, and β-arrestin2−/− mice. Neointimal hyperplasia was enhanced in β-arrestin1−/− mice, and diminished in β-arrestin2−/− mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in β-arrestin2−/− mice was not altered by transplantation with either wild-type or β-arrestin2−/− bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in β-arrestin1−/− and decreased in β-arrestin2−/− mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in β-arrestin1−/− SMCs and less in β-arrestin2−/− SMCs. Proliferation was less than wild type in β-arrestin2−/− SMCs but not in β-arrestin2−/− endothelial cells. We conclude that β-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by β-arrestin2 and β-arrestin1. These findings identify inhibition of β-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.
In the context of atherosclerosis,1 or in response to arterial endothelial denudation,2 the normally quiescent smooth muscle cells (SMCs) of the tunica media of the artery proliferate and migrate into the subendothelial intima. This SMC proliferation and migration into the intima constitute a critical component of atherosclerosis: SMC-specific genetic changes that enhance these processes also enhance atherosclerosis considerably in low-density lipoprotein receptor (LDLR)-deficient mice.3 Collectively referred to as neointimal hyperplasia, SMC proliferation and migration into the intima continues to complicate angioplasty and stenting used to treat human atherosclerosis.4 The myriad stimuli that contribute to neointimal hyperplasia include agonists secreted from activated platelets, macrophages, and SMCs themselves; consequently, candidate molecular strategies to limit neointimal hyperplasia must target multiple receptor signaling systems and thereby affect SMC proliferation and migration, among other processes.
One such molecular strategy could involve proteins known as β-arrestin1 and β-arrestin2, ubiquitously expressed multifunctional scaffolding proteins that were originally discovered because of their ability to quench 7-transmembrane receptor signaling through heterotrimeric G proteins.5 In addition to this role in attenuating G protein signaling, however, β-arrestins also play multiple roles in diminishing, as well as activating, signaling downstream of not only 7-transmembrane receptors but also receptor protein tyrosine kinases, cytokine receptors, and ion channel receptors.5 Signal transduction pathways triggered or positively modulated by β-arrestins include the activation of Src family tyrosine kinases, phosphatidylinositol-3-kinase/Akt, IRS-1, RhoA, and the mitogen-activated protein kinase families, comprising extracellular signal-regulated kinase (ERK), JNK, and p38 isoforms.5 Findings obtained in physiological systems like striatal neurons and lymphocytes have corroborated antiapoptotic and chemotactic roles discovered for β-arrestin2 in immortalized cell lines.5
β-Arrestin1 and β-arrestin2 share 78% amino acid identity.5 It is, therefore, not surprising that many proteins interact with both β-arrestins (albeit with differing affinities), whereas several proteins interact preferentially, or even exclusively, with a single β-arrestin isoform.5,6 At the level of signal transduction, this β-arrestin–binding specificity manifests in 2 distinctive patterns. First, both β-arrestin isoforms may be required for full activation of signaling, as exemplified by G protein–independent activation of ERK1/2 signaling evoked by the β2-adrenergic receptor.7 Second, signaling may be promoted by 1 β-arrestin isoform and inhibited by the other, as exemplified by the angiotensin II type 1 receptor8 and the protease-activated receptor-1.5
Because β-arrestins affect the survival, antiapoptotic signaling, and migration of immune and other cells in response to diverse stimuli,5 we asked whether β-arrestins affect the neointimal hyperplasia that is associated with atherosclerosis or provoked by endothelial denudation, and whether β-arrestins affect the in vitro correlates of neointimal hyperplasia: migration, promitogenic signaling, and proliferation in primary SMCs. The relevance of these questions to human disease is supported by the finding that β-arrestin2 mRNA is upregulated in atherosclerotic, as compared with normal human coronary arteries.9 To address these questions, we used congenic LDLR-deficient, β-arrestin-deficient, and wild-type (WT) mice.
Materials and Methods
C57BL/6 ldlr−/− mice were crossed with C57BL/6-congenic β-arrestin2−/− mice6; progeny were interbred to obtain littermates used in atherosclerosis studies. For carotid endothelial denudation, we used congenic age- and sex-matched C57BL/6 WT, β-arrestin1−/−, β-arrestin2−/−, and green fluorescent protein (GFP)-transgenic mice. Bone marrow transplantation, SMC migration,10 RNA interference,11 and cell proliferation assays11 are described in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
β-Arrestin2 Promotes Atherogenesis
Because β-arrestins affect proliferative signaling and cellular migration, and because β-arrestin2 mRNA levels are higher in atherosclerotic than in normal human arteries,9 we hypothesized that β-arrestin2 activity contributes to the neointimal hyperplasia of atherosclerosis. To test this hypothesis, we examined aortic atherosclerosis in congenic female ldlr−/− mice that were either +/+ or −/− at the β-arrestin2 locus. The β-arrestin2+/+ and β-arrestin2−/− mice had equivalent systolic blood pressures (120±10 and 115±6 mm Hg, respectively) and equivalent serum cholesterol values on a “Western” diet (28±3 and 27±2 mmol/L, respectively). After 12 weeks on a Western diet, however, ldlr−/−/β-arrestin2−/− mice developed aortic atherosclerosis that was only 59% of that observed in ldlr−/−/β-arrestin2+/+ mice, as judged by en face analysis (P<0.02; Figure 1A and 1C).
β-Arrestin2 expression increased not only the extent of atherosclerosis but also the prevalence of atheroma SMCs. Measured as a fraction of all atheroma cells, aortic root SMCs in ldlr−/−/β-arrestin2−/− mice were only 65±10% as abundant as they were in ldlr−/−/β-arrestin2+/+ mice (P<0.01; Figure 1B and 1C). Strikingly, SMCs in the fibrous cap of aortic root atheromata expressed considerable levels of β-arrestins relative to the macrophage-rich components of the neointima (Figure 1D). Moreover, neointimal β-arrestin staining was much more prominent in mice that were +/+ at the β-arrestin2 locus, even though we stained specimens with an antibody that binds β-arrestin1 more avidly than β-arrestin2 (Figure 1D). These data accord well with human transcriptional profiling data from human atherosclerotic coronary arteries, in which β-arrestin2 mRNA levels are ≈2-fold higher than levels in nonatherosclerotic control coronaries.9 Thus, β-arrestin2 contributes to atherosclerosis through mechanisms that affect SMC recruitment to the neointima, and β-arrestin2 appears to be the predominant β-arrestin isoform in the atherosclerotic lesion.
To determine mechanisms by which β-arrestin2 may enhance the prevalence of SMCs in atherosclerotic lesions, we focused on pathological arterial SMC proliferation and migration using a simpler, nonatherosclerotic model system: neointimal hyperplasia induced by endothelial denudation in the absence of hyperlipidemia.2,12
β-Arrestins Regulate Neointimal Hyperplasia Through Effects in Arterial SMCs
We used 0.36-mm angioplasty guidewires to denude the endothelium of the mouse carotid artery.2 Arteries harvested within 2 hours of wire-mediated injury demonstrated scattered small luminal thrombi, no endothelial cells, and intact internal elastic laminae (data not shown). By 4 weeks after endothelial denudation, considerable neointimal hyperplasia developed (Figure I in the online data supplement). Neointimal lesions comprised SMCs, as judged by the high prevalence (>90%) of neointimal cell SMC myosin heavy chain expression (supplemental Figure I). As in our atherosclerotic lesions, the level of β-arrestin expression in these neointimal SMCs was consistently higher than that of the medial SMCs in WT mice (supplemental Figure I). We next studied neointimal hyperplasia in congenic WT and β-arrestin2−/− mice. Because β-arrestin1 can serve to antagonize β-arrestin2 activity in some signaling systems,5 we also included congenic β-arrestin1−/− mice in these studies.
Before endothelial denudation, arteries from our 3 congenic mouse strains appeared histologically identical (data not shown). However, arteries harvested 2 to 4 weeks after endothelial denudation demonstrated clear β-arrestin isoform–specific differences (Figure 2). Two weeks after endothelial denudation, the average external carotid diameter was equivalent among all vessels (data not shown), and the extent of carotid reendothelialization was equivalent (≈35%) in all 3 genotypes (supplemental Figure II). However, compared with WT carotids, medial and neointimal area were 50% greater in β-arrestin1−/− carotids and 45% to 60% less in β-arrestin2−/− carotids (P<0.01; Figure 2). By 4 weeks after endothelial denudation, neointimal area was 37% larger than WT in β-arrestin1−/− carotids, and 47% smaller than WT in β-arrestin2−/− carotids. Moreover, compared with WT, the β-arrestin2−/− carotid media was 33% smaller (P<0.01; Figure 2) and the lumen area 44% larger (P<0.05), even though external carotid diameter remained equivalent across genotypes (data not shown). Thus, it appeared that whereas β-arrestin2 expression exacerbated neointimal hyperplasia, β-arrestin1 expression attenuated it.
This reciprocal regulation of neointimal hyperplasia by β-arrestin1 and β-arrestin2 could result from effects in two relatively distinct cellular compartments: (1) arterial SMCs, which proliferate and migrate into the neointima12; and (2) bone marrow–derived platelets, neutrophils, and monocyte/macrophages, which adhere to the basement membrane of endothelium-denuded arteries and release chemokines/cytokines that induce arterial SMC proliferation and migration.13 Certain models of vascular injury have implicated bone marrow–derived vascular precursor cells as a source of SMC-like cells in nonatherosclerotic neointimal hyperplasia,13 and β-arrestin2 is known to affect migration of hematopoietic lineage cells.5 Could β-arrestins affect neointimal hyperplasia by regulating possible recruitment and differentiation of bone marrow–derived cells into SMC-like cells of the arterial neointima? Alternatively (or in addition), could β-arrestins alter neointimal hyperplasia through mechanisms that affect platelet and leukocyte function?
To address these questions, we used WT and congenic transgenic mice that express GFP ubiquitously (WTGFP) as donors and recipients for bone marrow transplantation, and we performed carotid endothelial denudation 4 weeks later. In these experiments, GFP+ cells appeared in the neointima only when the artery wall (and thus the bone marrow recipient mouse) was WTGFP; GFP+ cells did not appear in the neointima when the bone marrow compartment alone was WTGFP (Figure 3A). Thus, neointimal cells in our model of endothelial denudation did not derive from bone marrow precursors. Further support for this inference emerges from bone marrow transplantation with either WT or β-arrestin2−/− cells, also depicted in Figure 3. Neointimal size correlated with the genotype of the bone marrow recipient only; it did not correlate with the genotype of the bone marrow cells themselves. Consequently, neointimal hyperplasia was reduced by ≈75% in β-arrestin2−/− mice as compared with WT mice, whether these mice were transplanted with WT or β-arrestin2−/− bone marrow cells (Figure 3B). Because β-arrestin2 expression in bone marrow–derived cells did not alter neointimal hyperplasia, we can also infer that β-arrestin2 augments neointimal hyperplasia through mechanisms fundamentally involving SMCs and not bone marrow–derived cells.
β-Arrestins Regulate SMC Proliferation and Apoptosis in Injured Arteries
Because neointimal hyperplasia in our model system involved principally arterial cells, we reasoned that arterial SMC proliferation should be diminished by β-arrestin1 expression and enhanced by β-arrestin2 expression. Moreover, the effect of β-arrestins on arterial SMC apoptosis, if any, should be the inverse of the β-arrestin effect on proliferation. To test these hypotheses, we examined the prevalence of proliferating-cell nuclear antigen (PCNA) and cleaved caspase-3 (for apoptosis) in SMCs of the carotid artery media 2 weeks after endothelial denudation (while neointimal hyperplasia was still developing). Deficiency of β-arrestin2 reduced medial SMC proliferation by ≈2.4-fold and augmented SMC apoptosis ≈2-fold (P<0.01; Figure 4). By contrast, deficiency of β-arrestin1 augmented medial SMC proliferation by ≈1.8-fold and reduced SMC apoptosis ≈2.5-fold (P<0.01; Figure 4). Thus, β-arrestin isoforms appeared to affect arterial SMC proliferation, and apoptosis, in a reciprocal manner.
To determine mechanisms by which β-arrestin isoforms could effect this reciprocal regulation of net SMC proliferation, we examined the activation of ERK1/2 and Akt in SMCs of the arterial media. Signaling by ERK and Akt isoforms is critical for SMC survival and proliferation in response to diverse stimuli.14 Moreover, β-arrestin isoforms serve as scaffolds for the ERK cascade members c-raf, MEK, and ERK1/2, in addition to Akt, and β-arrestins affect ERK and Akt activation downstream of 7-transmembrane receptors as well as receptor tyrosine kinases.5 Two weeks after activation by endothelial denudation, carotid SMCs of the tunica media indeed demonstrated a β-arrestin isoform–dependent pattern of ERK and Akt activation: phosphorylated (activated) Akt and ERK1/2 were ≈1.5- to 2-fold more abundant in β-arrestin1−/− and 33% to 67% less abundant in β-arrestin2−/− than in WT arteries (P<0.05; Figure 5). Thus, β-arrestin isoform-dependent ERK and Akt activation in vivo correlated with β-arrestin isoform-dependent SMC proliferation and neointimal hyperplasia: SMC proliferative signaling and proliferation were enhanced by β-arrestin2 activity and diminished by β-arrestin1 activity.
β-Arrestin2 Affects SMC Migration and Proliferation In Vitro
To investigate further the role of β-arrestin isoforms in the SMC proliferation and migration that are critical to neointimal hyperplasia, we used aortic SMCs derived from our congenic WT, β-arrestin1−/−, and β-arrestin2−/− mice (2 to 3 distinct lines of each genotype). These congenic SMC lines demonstrated the expected, genotype-specific expression patterns of β-arrestins, assessed by immunoblotting (supplemental Figure IVA). However, these congenic SMC lines demonstrated equivalent expression of G protein–coupled receptor kinase-2 (GRK2) and GRK5, kinases whose activities can determine the extent and functional consequences of β-arrestin isoform binding to 7-transmembrane receptors (immunoblot data not shown).15 In addition, as assessed by semiquantitative PCR, the SMC lines expressed equivalent levels of the 7-transmembrane receptors for lysophosphatidic acid (LPA), LPA1, and LPA2 (supplemental Figure IVA), protease-activated (thrombin) receptors (PAR1 to -4), and sphingosine-1-phosphate receptors (S1P1–5) (data not shown). These receptors are believed to be important for SMC responsiveness to platelet-derived products in the context of vascular injury.16,17 In response to LPA, calcium signaling was comparable in β-arrestin–expressing and –deficient SMC lines (supplemental Figure IVB). Thus, to the extent that β-arrestins regulate LPA-induced calcium signaling, it appeared that expression of either β-arrestin1 or β-arrestin2 suffices and that β-arrestin1 may compensate for deficiency of β-arrestin2 and vice versa.
Could expression of either β-arrestin1 or β-arrestin2 also suffice for regulating SMC migration, which involves signaling not only through heterotrimeric G proteins but also small G proteins,18 phosphoinositide 3-kinase, and ERKs?14 To address this question, we induced SMC migration through 7-transmembrane receptors and receptor tyrosine kinases, respectively, for LPA and PDGF, both abundantly derived from the activated platelets involved in neointimal hyperplasia.18 PDGF-promoted SMC migration appeared to be independent of β-arrestin1 and β-arrestin2 (Figure 6A). However, LPA-promoted migration was 30% less in β-arrestin2−/− than in WT SMCs, even though it was equivalent in β-arrestin1−/− and WT SMCs (Figure 6A). Similarly, thrombin-promoted migration (2-fold/basal) was 35% less in β-arrestin2−/− than in WT SMCs (P<0.05; data not shown). Thus, the ability of β-arrestin2 to promote SMC migration in response to some platelet-derived products accords well with the ability of β-arrestin2 to promote neointimal hyperplasia (Figure 2) and atherosclerosis (Figure 1).
Because β-arrestins significantly influenced SMC proliferation in vivo, we sought to determine which SMC signaling pathways could confer β-arrestin isoform–specific proliferative responses. To that end, we tested SMC thymidine incorporation elicited by receptors for platelet-derived serum constituents implicated in neointimal hyperplasia: the 7-transmembrane receptors for LPA, thrombin, and sphingosine-1-phosphate, as well as by the receptor protein tyrosine kinases for PDGF and epidermal growth factor (EGF).16,19–21 As with SMC migration, β-arrestin1 and β-arrestin2 activity appeared to have no effect on SMC thymidine incorporation stimulated by PDGF (Figure 6B) or EGF (data not shown). In contrast, LPA evoked 33% more thymidine incorporation in β-arrestin1−/− than in WT SMCs, whereas LPA, S1P, and thrombin evoked 20% to 40% less thymidine incorporation in β-arrestin2−/− than in WT SMCs (Figure 6B; P<0.05). Concordantly, serum evoked ≈40% less proliferation from β-arrestin2−/− than from WT SMCs (Figure 6C), and serum deprivation engendered 50±10% more cell death in β-arrestin2−/− than in WT SMCs (n=7, P<0.01, data not shown). Thus, it appears that β-arrestin1 activity attenuates, whereas β-arrestin2 activity potentiates mitogenic signaling evoked through 7-transmembrane and perhaps other growth factor receptors in SMCs. Consequently, the activities of the β-arrestin isoforms in vitro appear concordant with those observed with neointimal hyperplasia in vivo, and we can infer that β-arrestin isoform–mediated signaling downstream of key 7-transmembrane receptors may be responsible for β-arrestin isoform–specific effects on neointimal hyperplasia.
Unlike SMCs, β-arrestin2−/− and WT endothelial cells demonstrated equivalent proliferation and apoptosis in response to serum and serum deprivation, respectively (supplemental Figure V and data not shown), just as β-arrestin2−/− and WT mice demonstrated equivalent carotid reendothelialization (supplemental Figure II). This failure of β-arrestin2 deficiency to affect endothelial cell proliferation likely results from the undetectably low β-arestin2 expression levels in mouse (supplemental Figure VA) and human umbilical vein endothelial cells (data not shown).
β-Arrestins Affect SMC Signaling Through ERK
Proliferative and chemotactic signaling downstream of β-arrestins include both Raf/MEK/ERK and phosphoinositide 3-kinase/Akt pathways.5 In light of the β-arrestin isoform–specific effects on migration and proliferation observed in Figure 6, we asked whether ERK, Akt, or both pathways confer β-arrestin isoform–specific proliferation or migration in SMCs. To address this question in the context of a mixture of agonists that promote neointimal hyperplasia, we challenged our congenic SMCs with FBS, which contains all of the agonists used in Figure 6.16,18 Serum evoked equivalent Akt activation in WT and β-arrestin–deficient SMCs, but it evoked 32% less ERK activation in β-arrestin2−/− than in WT SMCs (P<0.01; Figure 7). Serum similarly evoked 38±10% less ERK activation in WT SMCs when β-arrestin2 expression was reduced (79±8%) by acute treatment with small interfering RNA (P<0.05; Figure 7C). In contrast, serum activated Akt and ERK equivalently in β-arrestin2−/− and WT endothelial cells (supplemental Figure VC). Thus, SMCs and endothelial cells demonstrate cell type–specific roles of β-arrestin2, and serum-stimulated SMCs recapitulate only a subset of the β-arrestin isoform–dependent ERK and Akt findings we obtained in neointimal hyperplasia (Figure 5). Differences between in vivo and in vitro findings likely relate to greater inflammatory cytokine levels present in injured arteries as compared with purified serum, as well as the time course of the assays (weeks versus minutes). Moreover, whereas SMC Akt activity promotes the “contractile” SMC phenotype when ERK is not concomitantly activated, SMC ERK activity engenders the “activated” SMC phenotype associated with migration, proliferation, and neointimal hyperplasia.14,18 We therefore sought to characterize further the effects of β-arrestins on SMC ERK activation induced by the components of serum used in Figure 6.
Stimulated by individual agonist constituents of serum, SMCs demonstrated ERK activation patterns consistent with antagonistic roles of β-arrestin1 and β-arrestin2, as we observed with SMC thymidine incorporation in Figure 6. ERK activation triggered by the 7-transmembrane receptors for LPA, thrombin, and sphingosine-1-phosphate was diminished in β-arrestin2−/− SMCs, but it enhanced in β-arrestin1−/− SMCs (Figure 8). In contrast, deficiency of β-arrestin isoforms did not affect ERK activation elicited by SMC receptor tyrosine kinase(s) for PDGF (Figure 8B). These agonist-specific results for β-arrestin–dependent ERK activation and thymidine incorporation are highly congruent with β-arrestin isoform–specific effects on neointimal hyperplasia. Consequently, these agonist-specific results suggest that β-arrestin–dependent effects on neointimal hyperplasia are mediated substantially, if not exclusively, by effects on signaling through 7-transmembrane receptors. Together with ERK and Akt activation data from Figures 5 and 7⇑, these data in Figure 8 also support the inference that β-arrestins affect neointimal hyperplasia through ERK-mediated mechanisms.
Our studies demonstrate that the multifunctional scaffolding protein β-arrestin2 contributes to the pathogenesis of atherosclerosis. This novel finding in mice seems highly pertinent to human atherosclerosis, because atherosclerotic human coronary arteries show ≈2-fold higher β-arrestin2 mRNA levels than nonatherosclerotic coronary arteries.9 That β-arrestin2 activity augments atherosclerosis by augmenting SMC proliferation and migration derives credence from three concordant lines of evidence: atheroma SMC prevalence and β-arrestin2 expression, SMC-dependent neointimal hyperplasia triggered by endothelial denudation, and cultured primary SMCs. Moreover, we discovered that whereas β-arrestin2 promotes SMC proliferation, migration, and consequently neointimal hyperplasia, β-arrestin1 does not. Indeed, β-arrestin1 inhibits these processes in vivo. Thus, in response to the innumerable stimuli associated with vascular injury, the activities of β-arrestin1 and β-arrestin2 in SMCs appear to be antagonistic to each other, just as we found them to be with regard to SMC ERK signaling and proliferation induced in vitro by 7-transmembrane receptors for LPA, thrombin, and sphingosine-1-phosphate.
That atherosclerosis can be affected primarily, or exclusively, by changes in SMC physiology was first clearly demonstrated with the SMC-specific knockout of the multifunctional LDLR-related protein-1 (LRP1).3 As a result of SMC LRP1 deficiency, SMCs express more PDGF receptor (PDGFR)-β and demonstrate enhanced proliferation and migration. Consequently, SMC LRP1 deficiency substantially aggravates atherosclerosis in ldlr−/− mice, in a manner that can be largely eliminated by inhibiting PDGFRβ signaling.3 Although β-arrestin2 can exacerbate atherosclerosis by enhancing SMC proliferation and migration, our SMC findings certainly do not exclude possible atherogenic effects of β-arrestin2 on migration of CD4+ lymphocytes5 or monocytes or on macrophage proliferation.1
When 7-transmembrane receptors are activated in SMCs, β-arrestin1 and β-arrestin2 exert comparable effects on intracellular calcium signaling, but markedly different effects on ERK signaling and thymidine incorporation. These divergent, signal-specific effects of β-arrestins on 7-transmembrane receptor signaling characterize the LPA receptors studied in our SMCs, as well as the atherogenic22 angiotensin II type 1 receptor, and appear to result from divergent signaling mediated by heterotrimeric G proteins or other, distinct mechanisms. In mouse embryo fibroblasts and HEK cells, expression of either β-arrestin1 or β-arrestin2 suffices to achieve normal desensitization of angiotensin II–evoked phosphoinositide hydrolysis (just upstream of cytosolic calcium signaling).5,8 However, like our SMC ERK signaling evoked by LPA, thrombin, and sphingosine-1-phosphate, angiotensin II–induced ERK signaling in HEK cells is promoted by β-arrestin2 and reduced by β-arrestin1 activity.8 Although the association of either β-arrestin isoform with 7-transmembrane receptors terminates G protein–dependent signaling, this same association also triggers signaling through ERKs and other mechanisms, in a manner that depends not only on the specific β-arrestin isoform but also on the phosphorylation state of the 7-transmembrane receptor.5,15 In addition, the newly described role of β-arrestins in activating Wnt/Frizzled/β-catenin signaling may also contribute to β-arrestin2–promoted SMC proliferation.23
Because PDGF plays such a prominent role in promoting neointimal hyperplasia provoked by arterial injury20 and atherosclerosis,1 it may seem surprising that β-arrestins affect neointimal hyperplasia significantly and yet have no effect on primary SMC mitogenic signaling or migration in response to PDGF (Figures 6 and 8⇑). However, this apparent paradox may be resolved if we consider that PDGF and 7-transmembrane receptors signal synergistically through many mechanisms, including NAD(P)H oxidases.22 Mitogenic signaling in primary SMCs more than doubles when agonists for Gq/11- and Gi-coupled 7-transmembrane receptors are added to PDGF, even at receptor-saturating PDGF concentrations.11,24 By altering SMC mitogenic signaling elicited by 7-transmembrane receptors, β-arrestin activity should augment (β-arrestin2) or reduce (β-arrestin1) mitogenic signaling elicited by the combination of PDGF and platelet-derived agonists for 7-transmembrane receptors. Furthermore, because PDGFRs can be transactivated by 7-transmembrane receptors,22 β-arrestin-mediated regulation of 7-transmembrane receptors may alter levels of SMC PDGFR activation. Although β-arrestins transactivate the EGFR in a manner that is 7-transmembrane receptor– and cell type–specific,25,26 the role of β-arrestins in transactivating the PDGFR remains obscure.
Our bone marrow transplantation data demonstrate (a) that β-arrestin2 expression in bone marrow–derived cells does not affect carotid injury-induced neointimal hyperplasia and (b) that neointimal cells do not derive from bone marrow precursors in our model of carotid deendothelialization. These findings may reflect the possibility that although β-arrestin2 is important for lymphocyte chemotaxis to CXCL12,5 it is not important for the migration of leukocytes in response to the diversity of stimuli encountered in carotid injury. To reconcile the absence and presence of bone marrow–derived cells in the neointima of the injured carotid and femoral13 arteries, respectively, we must consider arterial dimensions. Relative to the injury-inducing guide wire diameter (0.36 mm), the mouse femoral artery diameter is only 50%,27 whereas the carotid diameter is ≈90% (supplemental Figure I).10 Consequently, the abundant medial apoptosis and necrosis observed with femoral artery injury27 is greatly diminished (or absent) with carotid artery injury. It is therefore quite conceivable that guide wire injury of the carotid artery provides insufficient stimulus to recruit bone marrow–derived cells that differentiate into neointimal SMCs.
In light of the newly discovered role of β-arrestin2 in atherogenesis and in regulating injury-induced neointimal hyperplasia, novel therapeutic approaches to atherosclerosis and arterial restenosis after angioplasty could conceivably involve inhibition of SMC β-arrestin2 activity or enhancement of SMC β-arrestin1 activity. Our studies of SMCs and endothelial cells in vivo and in vitro suggest a salient potential advantage of anti–β-arrestin2 approaches: the possibility of inhibiting atherogenic SMC activity without inhibiting antiatherogenic endothelial cell activity. Whether these therapeutic possibilities will prove practicable, of course, remains to be ascertained.
Sources of Funding
This work was supported by NIH grants HL73005, HL77185, and AG25462 (to N.J.F.), HL16037 and HL70631 (to R.J.L.), and HL72842 (to K.P.), the Howard Hughes Medical Institute (R.J.L.); and a grant from the Pennsylvania Department of Health (to K.P.).
RJL is a founder of Trevena, Inc.
↵*Both authors contributed equally to this work.
The Department specifically disclaims responsibility for any analyses interpretations, or conclusions.
Original received January 17, 2008; revision received May 18, 2008; accepted May 20, 2008.
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