Published online before print April 24, 2008, doi: 10.1161/CIRCRESAHA.108.171728
© 2008 American Heart Association, Inc.
Integrative Physiology |
Heparin-Binding Epidermal Growth Factor–Like Growth Factor Signaling in Flow-Induced Arterial Remodeling
From the Departments of Cell & Molecular Physiology (H.Z., K.K.M., J.E.F.) and Biochemistry & Biophysics (S.W.S.), University of North Carolina, Chapel Hill; Ludwig Institute for Cancer Research (T.G.J.), Heidelberg, Australia; and University of Georgia (D.C.L.), Athens.
Correspondence to James E. Faber, PhD, Department of Cell and Molecular Physiology, 6309 MBRB, University of North Carolina, Chapel Hill, NC 27599-7545. E-mail jefaber{at}med.unc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is activated by reduced endothelial shear stress and stimulates smooth muscle cell proliferation in vitro. Moreover, HB-EGF is augmented at sites of intimal hyperplasia and atherosclerosis, conditions favored by low/disturbed shear stress. We thus tested whether HB-EGF contributes to low flow-induced negative hypertrophic remodeling (FINR) of a mouse carotid artery. Blood flow was surgically decreased in the left and increased in the right common carotid arteries. After 21 days, the left carotid artery exhibited lumen narrowing, thickening of intima–media and adventitia, and increased circumference that were inhibited by
50% in HB-EGF+/– and 90% in HB-EGF–/– mice. FINR was also inhibited by the EGF receptor inhibitor AG1478. In contrast, eutrophic outward remodeling of the right carotid artery was unaffected in HB-EGF+/– and HB-EGF–/– mice, nor by AG1478. FINR-induced proliferation and leukocyte accumulation were reduced in HB-EGF–/–. FINR was associated with increased reactive oxygen species, increased expression of pro-HB-EGF and tumor necrosis factor 
–converting enzyme (pro-HB-EGF sheddase), increased phosphorylation of EGF receptor and extracellular signal-regulated kinase 1/2, and increased nuclear factor 
B activity. Apocynin and deletion of p47phox inhibited FINR, whereas deletion of HB-EGF abolished nuclear factor B activation in smooth muscle cells. These findings suggest that HB-EGF signaling is required for low flow-induced hypertrophic remodeling and may participate in vascular wall disease and remodeling.
Key Words: artery • flow-mediated remodeling • HB-EGF • reactive oxygen species • NF-B
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vascular remodeling is directed by signals from vascular wall and blood-borne cells in response to changes in pressure, flow (shear stress), cyclic strain, and pathological processes.1 High flow-induced positive remodeling (eutrophic vessel enlargement) and low flow-induced negative remodeling (FINR) (hypertrophic vessel narrowing) are important during normal embryonic and postnatal growth and use-dependent hypertrophy and atrophy of tissues.2 Flow-induced proliferation and remodeling are also important in atherosclerosis, intimal hyperplasia, restenosis, and bypass graft failure.3,4 For example, branch points that are normally exposed to low and disturbed (oscillatory) shear stress are susceptible to lipid accumulation, smooth muscle cell (SMC) proliferation, and atherogenesis.3–5
Altered shear stress on endothelial cells (ECs) initiates flow-remodeling mediated by mechanosensitive elements and downstream signals including, proinflammatory pathways (eg, nuclear factor [NF]-B).4,6,7 In fact, chronic low shear is known to promote endothelial dysfunction in arteries, characterized by increased reactive oxygen species (ROS) generation, reduced bioavailability of NO, and increased prothrombotic, proinflammatory, and promigratory factors.5,8 Areas of disturbed shear stress have reduced barrier function, increased lipoprotein uptake, and increased adhesiveness for leukocytes5 that contribute, along with ECs and SMCs, to increased local ROS generation.8
Study of vessel restructuring is largely confined to in vivo models; thus, many of the underlying mechanisms are not well understood. In mouse,9,10 FINR of the carotid artery can be produced by ligating all of its distal branches except the thyroid or occipital arteries, which strongly reduces blood flow and shear stress.9,10 The low and hyperoscillatory shear stress induces intima–media hyperplasia, wall hypertrophy, and lumen loss. Korshunov et al reported that Axl-mediated inhibition of apoptosis contributes to FINR,7 whereas Sullivan and Hoying found that FGF2 deletion had no effect.9 Yu et al showed that endothelial NO synthase and NO protect against FINR, whereas caveolin-1 promotes FINR, possibly as a mechanoelement.11 In mesenteric artery, Bakker et al linked FINR and tissue transglutaminase.12
We recently reported that 1-adrenergic activity contributes strongly to FINR13 and, in other studies, that the trophic action of 1-adrenergic stimulation on vascular SMCs is mediated by HB-EGF transactivation of the EGF receptor (EGFR).14 Certain other G protein–coupled receptors (GPCRs) and receptor tyrosine kinases potentially involved in FINR may also signal through HB-EGF.15 Membrane-anchored pro-HB-EGF is cleaved by the metalloproteinase ADAM1716 (tumor necrosis factor [TNF]-–converting enzyme [TACE], the major sheddase for HB-EGF)17 and by matrix metalloproteinases (MMPs).18 Soluble HB-EGF binds ErbB1 (EGF receptor [EGFR]) and ErbB418 and stimulates SMC and fibroblast proliferation and migration with potencies comparable to platelet-derived growth factor-B.18 Pro-HB-EGF is induced by low shear stress in cultured ECs19 and at aneurysms in vivo.20 Balloon injury also activates HB-EGF in media and neointimal cells,21 and neutralizing antibody to EGFR reduces medial SMC proliferation and intimal hyperplasia.22 HB-EGF–dependent transactivation of EGFR by certain GPCRs, ie, the angiotensin II type 1 receptor,15,23 contributes to vascular remodeling, which may result from increased GPCR signaling produced by EC dysfunction.3,4
Atherogenesis has been associated with increased HB-EGF activity. Plasma HB-EGF is elevated in coronary artery disease.18 Proliferation of SMCs induced by remnant lipoproteins is accompanied by HB-EGF shedding and EGFR transactivation.24 HB-EGF induces expression of oxidized LDL receptor-1,25 is released by activated leukocytes, especially macrophages, and is further elevated by oxidized LDL.25 Expression of pro-HB-EGF, EGFR, and TACE is increased in shoulder regions of atheromas.17,26
Collectively, these findings suggest HB-EGF may be a key signaling nexus in vascular trophic responses to disturbed shear stress, GPCR signaling, injury, and atheroinflammation. Thus, the purpose of the present study was to test the hypothesis that HB-EGF signaling contributes to flow-mediated remodeling.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Wild-type littermates and mice targeted for EGFR ligands (C57Bl/6x129Sv background) are detailed in the expanded Materials and Methods section in the online data supplement at http://circres.ahajournals.org. The left external carotid, internal carotid, and occipital arteries were ligated, with the thyroid artery left intact. Carotid arteries were harvested 21 days later. Administration of AG1478-mesylate27 was via minipump, and administration of apocynin was via drinking water. Carotid arteries were maximally dilated and perfusion-fixed for histology. In situ ROS detection was by dihydroethidium or MitoTracker Red. Real-time RT-PCR used 18S for normalization. Leukocyte chemotaxis to 4-O-tetradecanoyphorbol-13-acetate (TPA) was by transwell assay using cells harvested from the peritoneum 4 days after thioglycollate.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Flow-Induced Negative Hypertrophic Remodeling Is Dependent on HB-EGF Signaling
Twenty-one days after ligation, the left carotid artery of wild-type mice exhibited a 28% decrease in lumen area, 325% and 142% increases in intima–media and adventitia thickness, respectively, and a 12% increase in circumference of the external elastic lamina (Figures 1A and 2
A). Flow-induced negative hypertrophic remodeling (FINR) was partially inhibited in HB-EGF–/+ mice (excepting lumen reduction), which express 50% less HB-EGF,28 and further inhibited in HB-EGF–/– mice (Figure 2A), suggesting that HB-EGF mediates FINR in a dose (allele)-dependent manner. Increased right carotid flow caused eutrophic outward remodeling (Figure 2B, control group). Outward remodeling was unaltered in HB-EGF+/– or HB-EGF–/– mice (Figure 2B), which provides an internal control for specificity.
|
|
We also evaluated amphiregulin and betacellulin, other EGF family ligands that bind EGFR. Pro-amphiregulin, like pro-HB-EGF, is proteolytically cleaved by TACE to yield mature growth factor, whereas betacellulin is shed by ADAM1018. Neither FINR nor right carotid outward remodeling was altered in amphiregulin–/– or betacellulin–/– mice (Figure 2). These results underscore the specificity of HB-EGF in FINR.
Control for Cardiac Hypertrophic Phenotype in HB-EGF–/– Mice
HB-EGF–/– mice are born with cardiac valve deformities, modest reduction in lung alveolar number and increase in pulmonary interstitial tissue.29 The valvulopathy leads, with varying penetrance, to stenosis, cardiac hypertrophy, and progression to heart failure with aging.29 No effects have been associated with the pulmonary changes.29 Heart failure could affect FINR through hemodynamic and/or humoral disturbances. However, we did not find any evidence of heart failure. (1) In a separate experiment, electromagnetic flowmetry (Transonic) in isoflurane-anesthetized 4.5-month-old mice showed that carotid flows were comparable in wild type and HB-EGF–/– before and after ligation (Figure 1B), in agreement with their similar arterial pressures (see below). (2) We also correlated heart and lung weight with FINR 21 days after ligation in several strains (Figure IA in the online data supplement). Only HB-EGF–/– mice had cardiac hypertrophy. However, this was without pulmonary edema, ie, lung dry/wet weight in wild type was 0.086±0.0024 and in HB-EGF–/– was 0.079±0.006 (P=0.30; n=5 each), which were comparable to the other groups (supplemental Figure IA). The small increase in dry weight in HB-EGF+/– and HB-EGF–/– likely reflects the phenotypic increase in pulmonary interstitium.29 (3) We previously reported30 similar heart/lung data in HB-EGF–/– mice that were older (5 months), in which cardiac hypertrophy was greater (74% versus the 56% in supplemental Figure IB), and arterial pressure, heart rate, and renin–angiotensin activity in conscious unrestrained and anesthetized states were not different from sham-treated wild-type littermates, arguing against heart failure. These and the present results indicate that the HB-EGF–/– mice studied herein had compensatory hypertrophy that had not progressed to congestive heart failure. (4) This conclusion is supported by comparable percentage decreases in body weight before versus 3 weeks after surgery in wild type (–1.5±2.6%) and HB-EGF–/– (–1.9±1.3) (P=0.99). (5) Moreover, no correlations were present between any parameter of FINR and the amount of cardiac hypertrophy in HB-EGF–/– (supplemental Figure IB). (6) In addition, outward remodeling in the right carotid artery was not affected in any group (Figure 2B). (7) Lastly and importantly, FINR was partially inhibited in HB-EGF+/– and AG1478-treated mice (discussed below) despite absence of hypertrophy. Thus, inhibition of FINR by genetic or pharmacological reduction of HB-EGF signaling (Figures 1 and 2
A) cannot be ascribed to a secondary effect caused by cardiac hypertrophy.
HB-EGF Expression, EGFR, and Extracellular Signal-Regulated Kinase 1/2 Are Augmented During FINR
The above data support the following pathway: low flow
HB- EGFEGFRextracellular signal-regulated kinase (ERK)1/2hypertrophic remodeling. To examine major elements in this pathway, we first quantified pro-HB-EGF mRNA, as done by others.17,19–21,26 Pro-HB-EGF increased 2.2 and 4.7 fold at 36 and 72 hours after ligation (Figure 3A). Immunohistochemistry detected increased activity in ECs and diffusely in the media (Figure 3B; see supplemental Figure III for positive and negative controls). Because HB-EGF binding to EGFR induces phosphorylation,18 we measured EGFR phosphorylation. Adult male Sprague-Dawley rats were used rather than mice because of availability of antibodies against rat EGFR and protein quantity required for immunoprecipitation/-blot for results shown in Figures 3B through 3D, 5, and 8A. FINR in rats and mice are similar,9 however the mechanisms may not be identical. Phospho-EGFR increased transiently at 36 hours (Figure 3C and 3D). In normal carotid arteries (sham surgery), immunoreactivity was detected in ECs and medial SMCs close to the lumen, but not adventitial cells which are mostly fibroblasts (Figure 3C). However, phospho-EGFR-positive cells were evident in all layers after ligation, including mono/macrophage-like cells in the adventitia and lumen (Figure 3C). ERK1/2, which is activated by EGFR and important in vascular wall growth and remodeling,14 exhibited sustained activation (Figure 3D).
|
Inhibition of EGFR Reduces FINR
HB-EGF binds EGFR (ErbB1) and ErbB4, and ECs and monocytes/macrophages express EGFR, whereas medial SMCs express all four receptors (ErbB1-B4).18 To examine EGFR involvement, AG1478 mesylate, a highly selective inhibitor of EGFR tyrosine kinase,15 was administered via subcutaneous mini-pump. Mesylated AG1478 has increased solubility27 that was further increased by Captisol.27 AG1478 inhibited intima–media thickening by 54%, circumference increase by 91%, and adventitial thickening by 58%, but lumen decrease was unaffected (Figure 3E). Partial inhibition of FINR may reflect incomplete blockade of EGFR (see Discussion). We did not test a higher dose because body weight declines27; herein it was unaffected. Consistent with results obtained in HB-EGF+/– and HB-EGF–/– mice (Figure 2), right carotid outward remodeling was unaffected by AG1478 (data not shown).
HB-EGF–/– Mice Display Reduced Proliferation and Leukocyte Infiltration
Proliferation and leukocyte accumulation occur early in FINR.9,13 Likewise, wild-type mice had increased proliferation in intima, media and adventitia, and leukocyte accumulation in lumen, media and adventitia at day 5, whereas, overall, cell density did not increase (Figure 4) because proliferation and apoptosis increase similarly.13 In HB-EGF–/– mice, proliferation in media and leukocyte accumulation in media and adventitia were reduced (Figure 4).
|
HB-EGF and EGFR are expressed by ECs, SMCs, circulating leukocytes and bone marrow hematopoietic cells.18 It is not known if HB-EGF expression in the latter cells influences circulating leukocytes. Complete blood counts were done to determine whether reduced leukocyte accumulation during FINR in HB-EGF–/– mice might arise from diminished plasma levels. No significant differences were found between wild-type and HB-EGF–/– in total white blood cells (4.6±1.0 versus 5.1±0.6x103/µL), granulocytes (0.9±0.2 versus 0.9±0.1), lymphocytes (3.0±0.8 versus 3.7±0.5) or monocytes (0.7±0.1 versus 0.6±0.1) (n=5 to 6). To determine whether leukocytes migration is reduced in HB-EGF–/– mice to explain reduced accumulation, we examined TPA-induced chemotaxis of leukocytes isolated from the peritoneum. No difference was found in transwell migration between wild-type and HB-EGF–/– mice (Figure 4D). These data are consistent with evidence that increased local tissue HB-EGF promotes macrophage accumulation31 and suggest that reduced leukocytes in carotid arteries of HB-EGF–/– mice undergoing FINR (Figure 4B) extends from lack of HB-EGF production by vascular wall cells.
Activation of Potential Upstream Effectors of HB-EGF During FINR
The above results suggest that low shear stress activates HB-EGF signaling. To further explore this pathway, we examined TACE, the primary metalloproteinase implicated in HB-EGF shedding.16 TACE immunoreactivity increased transiently, with a time course that preceded EGFR activation and, like phospho-EGFR (Figure 3D), returned to control by day 5 (Figure 5). TACE immunoreactivity (Figure 5) also followed a pattern similar to EGFR (Figure 3C), ie, staining in the intima and inner layers of the media in sham-ligated carotid artery, as well as in circulating monocytic cells, increased 36 hour after ligation. Like HB-EGF and EGFR, little staining was evident in adventitial fibroblasts. The data in Figures 4 and 6 suggest leukocytes, ECs, and SMCs are involved in TACEHB-EGFEGFR signaling in FINR.
|
|
No studies have examined whether reduced shear stress enhances ROS in vivo, although this has been observed in cultured endothelial cells.32 We thus examined intracellular ROS by in situ staining for dihydroethidium, which binds ROS and concentrates it in the nucleus. Ligation increased ROS in the low-flow left carotid artery and also, as expected (positive control),33 in the high-flow right carotid artery (Figure 6A through 6D). ROS activity in adherent monocytic cells was also evident in the left carotid artery, along with in ECs (Figure 6C). However, mitochondrial ROS was not increased (Figure 6E and 6F; see supplemental Figure III for quantification). Because NAD(P)H oxidase is the major source of inducible ROS in vascular wall cells, we tested FINR in mice receiving apocynin (an inhibitor of oxidase subunit assembly) in the drinking water. Apocynin inhibited intima–media and adventitia hypertrophy and increase in circumference but had no effect on lumen reduction (Figure 7A). Essentially identical results were obtained in p47phox–/– mice deficient, a regulatory subunit of the oxidase (Figure 7B). Similar to the HB-EGF–/–, HB-EGF+/– and AG1478 experiments, outward remodeling of the right carotid artery was unaffected by apocynin or in p47phox–/– mice (data not shown).
|
Evidence in ECs and SMCs suggests that NF-B is activated by low/disturbed shear stress,32 ROS,34 interleukin-8, and TNF-18 and that these factors increase pro-HB-EGF expression, in part, through NF-B. We thus examined whether NF-B is activated (p65 subunit translocation) during reduced flow. In ECs of carotid arteries with normal flow, p65 immunoreactivity appeared abundant in the cytoplasm but absent in the nucleus (supplemental Figure IV). However, 6 hours after ligation, nuclear translocation appeared evident. Staining was weak in medial SMCs and adventitial fibroblasts in both normal and low-flow conditions, precluding detection of translocation (supplemental Figure IV). Because this assay is limited for quantification, NF-B activation in both ECs and SMCs was confirmed with semiquantitative immunohistochemistry for activated p65 (Figure 8A and 8B). To determine whether HB-EGF is upstream or downstream of NF-B, we also compared activated NF-B in wild-type and HB-EGF–/– mice 5 days after ligation (Figure 8B and 8C). NF-B activation in ECs of wild-type and HB-EGF–/– were similar; however, activation was reduced in SMCs of HB-EGF–/– mice. These data demonstrate that a more rapid and sustained activation of NF-B activation occurs in ECs compared to SMCs during low flow and suggest that HB-EGF may reside, relative to NF-B, downstream in ECs and upstream in SMCs.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Evidence suggests flow-induced arterial remodeling involves factors released from cells that are intrinsic to the vessel wall and recruited from the bloodstream. Understanding the molecular details has been hampered by the need to study the process in vivo. The present findings suggest that HB-EGF, which has primarily been studied in epithelial and tumor cells, plays a pivotal role in low FINR of the mouse carotid artery. Sustained low flow activated or increased the following elements within the HB-EGF signaling pathway: ROS, the ROS-sensitive HB-EGF sheddase TACE, expression of pro-HB-EGF, HB-EGF immunoreactivity, the HB-EGF receptor EGFR, ERK1/2, and the transcription factor NF-
B. These changes were associated with proliferation, increased leukocyte density, wall hypertrophy, and lumen narrowing. Heterozygous and homozygous deletion of HB-EGF alleles caused "dose-dependent-like" inhibition of FINR (although inhibition of lumen narrowing was in some situations spared (see below), where proliferation and leukocyte accumulation were reduced and FINR almost abolished in HB-EGF–/–. Inhibition was also obtained by genetic and pharmacological reduction in ROS generation and inhibition of EGFR activation. FINR was unaffected in mice deficient in the EGFR ligand family members betacellulin and amphiregulin. Interestingly, none of the above interventions affected high flow-induced eutrophic positive remodeling of the right carotid artery. This suggests that low and high flow-mediated remodeling are achieved by unique mechanisms. A second intriguing observation is that in FINR, significant inhibition of wall hypertrophy in HB-EGF+/– and p47phox–/– mice and, with AG1478 or apocynin treatment, did not inhibit lumen narrowing (an exception was in HB-EGF–/– mice, but their baseline diameters trended lower for unapparent reasons). This suggests that HB-EGF signaling is necessary for the hypertrophic response in FINR but not for the mechanisms that cause lumen narrowing. The latter mechanisms may be driven in a negative feedback manner to normalize shear stress. This is the first report identifying a role for the HB-EGF pathway in flow-induced remodeling.
Although HB-EGF is well known to activate EGFR and downstream mechanisms including Akt and ERK1/2 in SMCs, ECs, and other cell types, few studies have investigated possible links among reduced shear stress, ROS, and HB-EGF activation. Reduced shear stress causes rapid increase in HB-EGF expression in ECs.19 Low or disturbed shear stress promotes inflammatory-like conditions associated with increased ROS,32 in particular, reduced NO,35 increased expression of attractant and adhesion molecules that promote leukocyte transmigration,3 and increased local angiotensin, endothelin, and norepinephrine activity that induce ROS- and HB-EGF–dependent GPCR transactivation of EGFR.14,15,23 Although angiotensin and endothelin have not been examined in FINR, norepinephrine-induced trophic activity contributes significantly to FINR.13 In agreement with the prominent role of SMC proliferation in FINR,7,10,15 proliferation was inhibited in the media but not intima in HB-EGF–/– mice (Figure 4A), a finding consistent with the mitogenic action of HB-EGF on SMCs but not ECs.18 Besides SMCs, leukocytes and ECs (and possibly adventitial fibroblasts) also release HB-EGF and express EGFR in vitro.18,19 Indeed, our histological results confirm ROS, TACE and EGFR activity in each vascular layer. Thus, autocrine/paracrine HB-EGF activity in FINR could involve all 4 cell types. Additional studies are required to determine their relative contributions. It should be noted that only associative evidence is provided for 2 of the elements (TACE, ERK1/2) in the signaling pathway proposed herein, because, in the case of TACE, specific antagonists and genetic models (TACE–/– mice are embryonic lethal) are currently unavailable.
Primarily in vitro studies have shown that pro-HB-EGF is cleaved by ADAM family proteases and MMPs.18 Evidence suggests that TACE (ADAM-17) is the primary mediator of HB-EGF shedding in many cells types, including SMCs, where proliferation and hypertrophy result.23 In the present study, TACE immunoreactivity was elevated at the earliest time point examined after flow reduction (12 hours). ROS can increase MMP expression through ERK1/2, JNK, AP-1, and Ets-1.36 Although this pathway has not been examined for TACE, TACE may be directly activated by a ROS-sensitive cysteine switch mechanism.37 In the present study, systemic administration of the NAD(P)H oxidase inhibitor apocynin, as well as in p47phox–/– mice, resulted in potent inhibition of FINR, whereas increased mitochondrial ROS activity was not evident. Although the role of other sources of ROS and the specific ROS molecule(s) and pro-HB-EGF protease(s) that direct FINR remain to be determined, our findings demonstrate a primary role for NAD(P)H oxidase.
The observation that pro-HB-EGF expression occurs rapidly (hours) in response to reduced shear stress in ECs,19 and to other stimuli in other cells, suggests that HB-EGF behaves as an immediate-early gene.21 The HB-EGF promoter contains a consensus site for NF-B.21 Binding of NF- B is redox-sensitive, and activation of NF-B by TNF- and interleukin-1β has been linked to induction of ROS molecules.34 Indeed, low shear stress in cultured ECs activates NF-B by a NAD(P)H oxidase–dependent mechanism.32 Our results support the presence of this signaling sequence in arteries exposed to low flow in vivo. Interestingly, in HB-EGF–/– mice, activation of NF-B was unaffected in ECs but abolished in SMCs (Figure 8D), suggesting that low shear-induced activation of NF-B is upstream of HB-EGF in ECs but downstream in SMCs. Consistent with this, in SMCs NF-B did not induce expression of EGFR mRNA or protein,38 whereas EGFR stimulation induced NF-B activation.39 Others have found that NF-B in SMCs resides downstream of EGFR in a pathway inducing ERK1/2- and Akt-mediated proliferation and apoptosis.40 Also, peak activation of NF-B occurred earlier in ECs than in SMCs (Figure 8B), possibly because ECs are more directly coupled mechanically to fluid shear stress than are SMCs.
Besides ECs and SMCs, FINR-induced leukocyte accumulation is also a potential source of HB-EGF.18,26 Activated macrophages exhibit increased HB-EGF expression and release.18,26 In the present study, we confirmed earlier reports13 for leukocyte accumulation in intima, media, and adventitia during FINR and detected reduction in HB-EGF–/– mice. Interestingly, HB-EGF promotes macrophage accumulation in ischemic heart.31 Thus, HB-EGF release from vascular wall cells may contribute to recruitment of inflammatory cells that further increase local HB-EGF activity. We did not find that reduced leukocyte density in HB-EGF–/– mice was accompanied by reduced circulating leukocyte counts or migratory capacity. However, pharmacological depletion of macrophages reduced FINR of small branches of the mesenteric artery.12 Whether the association of reduced leukocyte density and reduced FINR in that study12 and the present study extends from less HB-EGF in the wall coming from fewer infiltrated cells, or from the attendant lower levels of cytokines such as interleukin-1/6 and TNF- that induce HB-EGF in vascular wall cells, awaits studies using selective leukocyte depletion of HB-EGF.
Partial inhibition of FINR was obtained with chronic systemic administration of the EGFR antagonist AG1478-mesylate (Figure 3E). We did use a higher dose to test for incomplete blockade because of potential nonspecific effects.27 However, ErbB receptor-ligand interactions could also underlie the partial inhibition. ErbB1 (EGFR) and ErbB4 have different biological functions.18 Proliferation is mediated by ErbB1, whereas chemotaxis is mediated by ErbB4.18 Also, ErbB2 (for which no ligand has been identified) forms heterodimers with ErbB1 or ErbB4 which may induce crosstalk among downstream pathways promoting proliferation or migration.18 Neither ErbB2 nor ErbB4 is blocked by AG1478. In addition, Higashiyama and colleagues have recently identified an intracellular signaling pathway activated by pro-HB-EGF cleavage,41 wherein the intracellular carboxy-terminal remnant (HB-EGF-C) interacts with the transcriptional regulator promyelocytic leukemia zinc finger (PLZF), resulting in increased proliferation. This mechanism, which is independent of EGFR, thus not inhibited by AG1478, could contribute to the residual FINR in the presence of AG1478 that we observed.
In conclusion, the present study has identified a central role for ROSHB-EGFEGFR signaling in hypertrophic low flow-induced arterial remodeling. Besides physiological remodeling, this pathway may also contribute to pathological processes. For example, oxidized LDL and remnant lipoproteins induce HB-EGF,24 and HB-EGF increases expression of oxidized LDL receptor.25 TACE, pro-HB-EGF, and EGFR are increased in human atheromas and in those of animals with experimentally induced atherosclerosis.17,26 Furthermore, plasma HB-EGF is increased in patients with atherosclerosis.18 It is, therefore, intriguing to hypothesize that increased HB-EGF signaling may impair the important adaptive outward remodeling response that occurs at sites of expanding atheromas along arteries.3 If this were true, inhibition of pathway activity may enhance this process and promote preservation of lumen area. Our finding that HB-EGF signaling is not involved in flow-induced positive remodeling suggests that blocking adverse effects of excessive HB-EGF activity may leave this important physiological mechanism intact.
|
Acknowledgments |
|---|
We thank Carolyn Suitt for histological assistance, Wendy Salmon for assistance with confocal microscopy, and Drs Nageswara Madamanchi and Marshall Runge (University of North Carolina, Chapel Hill) for p47phox–/– mice.
Sources of Funding
This work was supported by NIH grants HL62584 (to J.E.F.) and CA43793 (to D.C.L. and S.W.S.).
Disclosures
None.
|
Footnotes |
|---|
Original received January 11, 2008; revision received April 1, 2008; accepted April 16, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Ward MR, Pasterkamp G, Yeung AC, Borst C. Arterial remodeling. Mechanisms and clinical implications. Circulation. 2000; 102: 1186–1191.
2. Prior BM, Yang HT, Terjung RL. What makes vessels grow with exercise training? J Appl Physiol. 2004; 97: 1119–1128.
3. Cunningham KS, Gotlieb AI. The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest. 2005; 85: 9–23.[Medline] [Order article via Infotrieve]
4. Chatzizisis Y, Giannoglou G. Pulsatile flow: a critical modulator of the natural history of atherosclerosis. Med Hypotheses. 2006; 67: 338–340.[CrossRef][Medline] [Order article via Infotrieve]
5. Stone P, Coskun A, Yeghiazarians Y, Kinlay S, Popma J, Kuntz R, Feldman C. Prediction of sites of coronary atherosclerosis progression: in vivo profiling of endothelial shear stress, lumen, and outer vessel wall characteristics to predict vascular behavior. Curr Opin Cardiol. 2003; 18: 458–470.[CrossRef][Medline] [Order article via Infotrieve]
6. Silver A, Vita J. Shear-stress-mediated arterial remodeling in atherosclerosis: too much of a good thing? Circulation. 2006; 113: 2787–2789.
7. Korshunov VA, Mohan AM, Georger MA, Berk BC. Axl, a receptor tyrosine kinase, mediates flow-induced vascular remodeling. Circ Res. 2006; 98: 1446–1452.
8. Fortuno A, Jose G, Moreno M, Diez J, Zalba G. Oxidative stress and vascular remodelling. Exp Physiol. 2005; 90: 457–462.
9. Sullivan C, Hoying J. Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice. Arterioscler Thromb Vasc Biol. 2002; 22: 1100–1105.
10. Korshunov VA, Berk BC. Strain-dependent vascular remodeling: the "Glagov phenomenon" is genetically determined. Circulation. 2004; 110: 220–226.
11. Yu J, Bergaya S, Murata T, Alp I, Bauer M, Lin M, Drab M, Kurzchalia T, Stan R, Sessa W. Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels. J Clin Invest. 2006; 116: 1284–1291.[CrossRef][Medline] [Order article via Infotrieve]
12. Bakker E, Pistea A, Spaan J, Rolf T, de Vries C, van Rooijen N, Candi E, VanBavel E. Flow-dependent remodeling of small arteries in mice deficient for tissue-type transglutaminase: possible compensation by macrophage-derived factor XIII. Circ Res. 2006; 99: 86–92.
13. Erami C, Zhang H, Tanoue A, Tsujimoto G, Thomas S, Faber J. Adrenergic catecholamine trophic activity contributes to flow-mediated arterial remodeling. Am J Physiol Heart Circ Physiol. 2005; 289: H744–H753.
14. Zhang H, Chalothorn D, Jackson L, Lee D, Faber J. Transactivation of epidermal growth factor receptor mediates catecholamine-induced growth of vascular smooth muscle. Circ Res. 2004; 95: 989–997.
15. Touyz R. Mitochondrial redox control of matrix metalloproteinase signaling in resistance arteries. Arterioscler Thromb Vasc Biol. 2006; 26: 685–688.
16. Sunnarborg S, Hinkle C, Stevenson M, Russell W, Raska C, Peschon J, Castner B, Gerhart M, Paxton R, Black R, Lee D. Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J Biol Chem. 2002; 277: 12838–12845.
17. Canault M, Peiretti F, Kopp F, Bonardo B, Bonzi M, Coudeyre J, Alessi M, Juhan-Vague I, Nalbone G. The TNF alpha converting enzyme (TACE/ADAM17) is expressed in the atherosclerotic lesions of apolipoprotein E-deficient mice: possible contribution to elevated plasma levels of soluble TNF alpha receptors. Atherosclerosis. 2006; 187: 82–91.[CrossRef][Medline] [Order article via Infotrieve]
18. Dreux A, Lamb D, Modjtahedi H, Ferns G. The epidermal growth factor receptors and their family of ligands: their putative role in atherogenesis. Atherosclerosis. 2006; 186: 38–53.[CrossRef][Medline] [Order article via Infotrieve]
19. Morita T, Yoshizumi M, Kurihara H, Maemura K, Nagai R, Yazaki Y. Shear stress increases heparin-binding epidermal growth factor-like growth factor mRNA levels in human vascular endothelial cells. Biochem Biophys Res Commun. 1993; 197: 256–262.[CrossRef][Medline] [Order article via Infotrieve]
20. Sho E, Sho M, Nanjo H, Kawamura K, Masuda H, Dalman R. Comparison of cell-type-specific vs transmural aortic gene expression in experimental aneurysms. J Vasc Surg. 2005; 41: 844–852.[CrossRef][Medline] [Order article via Infotrieve]
21. Igura T, Kawata S, Miyagawa J, Inui Y, Tamura S, Fukuda K, Isozaki K, Yamamori K, Taniguchi N, Higashiyama S, Matsuzawa Y. Expression of heparin-binding epidermal growth factor-like growth factor in neointimal cells induced by balloon injury in rat carotid arteries. Arterioscler Thromb Vasc Biol. 1996; 16: 1524–1531.
22. Chan A, Kalmes A, Hawkins S, Daum G, Clowes A. Blockade of the epidermal growth factor receptor decreases intimal hyperplasia in balloon-injured rat carotid artery. J Vasc Surg. 2003; 37: 644–649.[CrossRef][Medline] [Order article via Infotrieve]
23. Ohtsu H, Dempsey P, Frank G, Brailoiu E, Higuchi S, Suzuki H, Nakashima H, Eguchi K, Eguchi S. ADAM17 mediates epidermal growth factor receptor transactivation and vascular smooth muscle cell hypertrophy induced by angiotensin II. Arterioscler Thromb Vasc Biol. 2006; 26: e133–e137.
24. Kawakami A, Yoshida M. Remnant lipoproteins and atherogenesis. J Atheroscler Thromb. 2005; 12: 73–76.[Medline] [Order article via Infotrieve]
25. Mukai E, Kume N, Hayashida K, Minami M, Yamada Y, Seino Y, Kita T. Heparin-binding EGF-like growth factor induces expression of lectin-like oxidized LDL receptor-1 in vascular smooth muscle cells. Atherosclerosis. 2004; 176: 289–296.[Medline] [Order article via Infotrieve]
26. Nakata A, Miyagawa J, Yamashita S, Nishida M, Tamura R, Yamamori K, Nakamura T, Nozaki S, Kameda-Takemura K, Kawata S, Taniguchi N, Higashiyama S, Matsuzawa Y. Localization of heparin-binding epidermal growth factor-like growth factor in human coronary arteries. Possible roles of HB-EGF in the formation of coronary atherosclerosis. Circulation. 1996; 94: 2778–2786.
27. Johns T, Luwor R, Murone C, Walker F, Weinstock J, Vitali A, Perera R, Jungbluth A, Stockert E, Old L, Nice E, Burgess A, Scott A. Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478. Proc Natl Acad Sci U S A. 2003; 100: 15871–15876.
28. Iwamoto R, Yamazaki S, Asakura M, Takashima S, Hasuwa H, Miyado K, Adachi S, Kitakaze M, Hashimoto K, Raab G, Nanba D, Higashiyama S, Hori M, Klagsbrun M, Mekada E. Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function. Proc Natl Acad Sci U S A. 2003; 100: 3221–3226.
29. Jackson L, Qiu T, Sunnarborg S, Chang A, Zhang C, Patterson C, Lee D. Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling. EMBO J. 2003; 22: 2704–2716.[CrossRef][Medline] [Order article via Infotrieve]
30. Chalothorn D, Moore S, Zhang H, Sunnarborg S, Lee D, Faber J. Heparin-binding epidermal growth factor-like growth factor, collateral vessel development, and angiogenesis in skeletal muscle ischemia. Arterioscler Thromb Vasc Biol. 2005; 25: 1884–1890.
31. Mahtouk K, Jourdan M, De Vos J, Hertogh C, Fiol G, Jourdan E, Rossi J, Klein B. Local overexpression of HB-EGF exacerbates remodeling following myocardial infarction by activating noncardiomyocytes. Lab Invest. 2005; 85: 862–873.[CrossRef][Medline] [Order article via Infotrieve]
32. Mohan S, Koyoma K, Thangasamy A, Nakano H, Glickman R, Mohan N. Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-kappaB in endothelial cells. Am J Physiol Cell Physiol. 2007; 292: C362–C371.
33. Castier Y, Brandes R, Leseche G, Tedgui A, Lehoux S. p47phox-dependent NADPH oxidase regulates flow-induced vascular remodeling. Circ Res. 2005; 97: 533–540.
34. Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol. 2006; 72: 1493–1505.[CrossRef][Medline] [Order article via Infotrieve]
35. Mohan S, Hamuro M, Sorescu G, Koyoma K, Sprague E, Jo H, Valente A, Prihoda T, Natarajan M. IkappaBalpha-dependent regulation of low-shear flow-induced NF-kappa B activity: role of nitric oxide. Am J Physiol Cell Physiol. 2003; 284: C1039–C1047.
36. Nelson K, Melendez J. Mitochondrial redox control of matrix metalloproteinases. Free Radic Biol Med. 2004; 37: 768–784.[CrossRef][Medline] [Order article via Infotrieve]
37. Zhang Z, Oliver P, Lancaster JJ, Schwarzenberger P, Joshi M, Cork J, Kolls J. Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate. FASEB J. 2001; 15: 303–305.
38. Nishi H, Neta G, Nishi KH, Akers LM, Rikiyama T, Proctor KN, Murphy BA, Johnson AC. Analysis of the epidermal growth factor receptor promoter: the effect of nuclear factor-kappaB. Int J Mol Med. 2003; 11: 49–55.[Medline] [Order article via Infotrieve]
39. Sethi G, Ahn KS, Chaturvedi MM, Aggarwal BB. Epidermal growth factor (EGF) activates nuclear factor-kappaB through IkappaBalpha kinase-independent but EGF receptor-kinase dependent tyrosine 42 phosphorylation of IkappaBalpha. Oncogene. 2007; 26: 7324–7332.[CrossRef][Medline] [Order article via Infotrieve]
40. Lemarie C, Tharaux P, Esposito B, Tedgui A, Lehoux S. Transforming growth factor-alpha mediates nuclear factor kappaB activation in strained arteries. Circ Res. 2006; 99: 434–441.
41. Nanba D, Inoue H, Shigemi Y, Shirakata Y, Hashimoto K, Higashiyama S. An intermediary role of proHB-EGF shedding in growth factor-induced c-Myc gene expression. J Cell Physiol. 2008; 214: 465–473.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  J. P. Edwards, X. Zhang, and D. M. Mosser The Expression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor by Regulatory Macrophages J. Immunol., February 15, 2009; 182(4): 1929 - 1939. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||