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Vol. 44, No. 4, 2007
Issue release date: June 2007

Free access is sponsored by an educational grant of the European Society for Microcirculation
J Vasc Res 2007;44:303–312
(DOI:10.1159/000101776)

Neuregulin-1 Attenuates Neointimal Formation following Vascular Injury and Inhibits the Proliferation of Vascular Smooth Muscle Cells

Clement C.M.a · Thomas L.K.b · Mou Y.b · Croslan D.R.a · Gibbons G.H.b · Ford B.D.a
aDepartment of Anatomy and Neurobiology, Neuroscience Institute, and bDepartment of Medicine, Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Ga., USA
email Corresponding Author

Abstract

Neuregulin-1 (NRG-1) is expressed in vascular endothelial cells, and its receptors are localized to the underlying smooth muscle cells. However, the role of NRG-1 in vascular function and injury is largely unknown. First, the expression of NRG-1 and its receptors (erbB receptors) was analyzed after balloon injury to the rat carotid artery. NRG-1 and erbB expression levels were low in uninjured vessels; however, NRG-1 and erbB4 were upregulated following injury. We then examined the effect of NRG-1 on neointimal formation following balloon injury. NRG-1 was administered by tail-vein injection prior to injury and every 2 days following injury. Two weeks after injury, NRG-1-treated animals demonstrated a 50% reduction in lesion size compared with controls receiving the vehicle. To examine possible mechanisms for NRG-1 action, we examined its effects on vascular smooth muscle cell (VSMC) function. Rat VSMC cultures were pretreated with NRG-1 for 24 h and then stimulated with platelet-derived growth factor. NRG-1 significantly decreased platelet-derived growth factor-stimulated VSMC proliferation and migration. These findings suggest that NRG-1 may be a novel therapeutic candidate for the treatment of restenosis and atherosclerosis.


 Outline


 goto top of outline Key Words

  • Angioplasty
  • erbB receptor
  • Vascular injury
  • Vascular smooth muscle cells
  • Restenosis
  • Atherosclerosis

 goto top of outline Abstract

Neuregulin-1 (NRG-1) is expressed in vascular endothelial cells, and its receptors are localized to the underlying smooth muscle cells. However, the role of NRG-1 in vascular function and injury is largely unknown. First, the expression of NRG-1 and its receptors (erbB receptors) was analyzed after balloon injury to the rat carotid artery. NRG-1 and erbB expression levels were low in uninjured vessels; however, NRG-1 and erbB4 were upregulated following injury. We then examined the effect of NRG-1 on neointimal formation following balloon injury. NRG-1 was administered by tail-vein injection prior to injury and every 2 days following injury. Two weeks after injury, NRG-1-treated animals demonstrated a 50% reduction in lesion size compared with controls receiving the vehicle. To examine possible mechanisms for NRG-1 action, we examined its effects on vascular smooth muscle cell (VSMC) function. Rat VSMC cultures were pretreated with NRG-1 for 24 h and then stimulated with platelet-derived growth factor. NRG-1 significantly decreased platelet-derived growth factor-stimulated VSMC proliferation and migration. These findings suggest that NRG-1 may be a novel therapeutic candidate for the treatment of restenosis and atherosclerosis.

Copyright © 2007 S. Karger AG, Basel


goto top of outline Introduction

Atherosclerosis is a major cause of death in Western civilizations, leading to both heart attack and strokes. Atherosclerosis is a complex, chronic inflammatory disease of the arterial vessel wall which involves multiple processes including endothelial dysfunction, inflammation, vascular smooth muscle cell (VSMC) proliferation and matrix alteration [1]. Damage to the endothelial lining of the arterial wall due to angioplasty, in-stent techniques and atherosclerosis induces the release of proinflammatory cytokines and growth factors that stimulate normally quiescent VSMCs to migrate and proliferate. VSMCs proliferate and migrate from the medial layer of the vessel into the intima resulting in neointimal hyperplasia, which is also a major cause of restenosis after angioplasty [1,2,3,4]. Mitogens, such as platelet-derived growth factor (PDGF), are potent stimulators of VSMC proliferation and migration following vascular injury [1, 5]. PDGF is produced by platelets, endothelial cells, smooth muscle cells and macrophages that infiltrate the artery in response to injury, and the release of PDGF after injury contributes significantly to the formation of the neointima. Reagents that block the activity of PDGF have been found to prevent restenosis and atherosclerotic lesion formation [6, 7].

Neuregulins are a family of multipotent epidermal growth factor (EGF)-like proteins that promote cellular growth, differentiation and survival during development [8,9,10,11]. Neuregulin-1 (NRG-1) is expressed in a variety of cells including vascular endothelial cells and endocardial cells [8,12,13,14]. They activate members of the EGF family of tyrosine kinase receptors, which include erbB2, erbB3 and erbB4, that are expressed in smooth muscle cells [15, 16]. However, the function of the NRG-1/erbB signaling pathway in vascular injury and VSMC function is not well understood. NRG-1 has been shown to stimulate angiogenesis, and erbB receptors were found to be essential for the angiopoietin-regulated recruitment of VSMCs by endothelial cells during angiogenesis [16,17,18,19,20]. Previous studies have shown that various EGF receptor ligands activated erbB2 and stimulated DNA synthesis in human airway smooth muscle cells; however, NRG-1 and its receptors were not directly involved in stimulating smooth muscle cell proliferation [15]. Also, it has been shown that NRG-1 is expressed in atherosclerotic lesions; however, its exact role and function in the development of coronary artery disease has yet to be determined [13].

In this study, we analyzed the effect of NRG-1 on neointimal formation following balloon-injured carotid artery of the rat. In addition, we examined the effects of NRG-1 on PDGF-stimulated VSMC function in vitro. We report that NRG-1 significantly attenuated lesion formation following vascular injury. Our results also revealed that NRG-1 prevented mitogen-stimulated VSMC proliferation and migration. These data suggest that NRG-1 may serve to limit neointimal formation following vascular injury. Therefore, this property of NRG-1 may represent a potential therapy for treatment of restenosis and atherosclerosis.

 

goto top of outline Methods

goto top of outline Experimental Injury, Harvest and Tissue Preparation of Rat Carotid Arteries

Male Sprague-Dawley rats (350–400 g) were balloon-injured using methods as previously described in accordance with a protocol approved by the Standing Committee on Animals, Morehouse School of Medicine [21]. Rats were anesthetized with an intraperitoneal injection of xylazine (5 mg/kg body weight) and ketamine hydrochloride (90 mg/kg body weight). The left common carotid artery was exposed by a 6-cm midline cervical incision. Proximal and distal blood flow was occluded by clamping. Polyethylene 10 tubing was inserted retrogradely into the internal carotid artery and advanced into the left common carotid artery. After gentle flushing of the artery with normal saline, the tubing was removed and a 2-french Fogarty embolectomy balloon catheter was inserted. Balloon inflation to 1.5–1.8 times the external diameter of the artery was achieved by caliper measurement under stereomicroscopy. After holding the inflation for 30 s, the catheter was removed. The uninjured right carotid artery was used as the control. Rats were treated with NRG-1β or NRG-1α (EGF-like domain, dissolved in 1% bovine serum albumin/phosphate-buffered saline, PBS; R&D Systems, Minneapolis, Minn., USA) by tail-vein administration at a dose of 2.5 ng/kg body weight, starting at day 0 before injury and continuing for every 2 days for the next 14 days. Control rats were treated with vehicle (1% bovine serum albumin/PBS). The animals were weighed before the procedure and at sacrifice to evaluate the possible adverse effects of NRG-1. Vessels were harvested at time points 0 and 14 days for mRNA analysis or histology. Injured vessels were compared with their contralateral controls.

goto top of outline Immunohistology

Both uninjured and injured vessels were washed in ice-cold PBS, fixed in 2% formalin. Fixed arteries were transversely bisected, segments from each half were embedded in paraffin, and sections were cut from both the distal and proximal segments encompassing the center of the injured area. We assessed immunohistochemical localization of NRG-1 and its receptors using Santa Cruz (Santa Cruz, Calif., USA) polyclonal antibodies raised against erbB2 (sc-03), erbB3 (sc-285), erbB4 (sc-283), NRG-1α (sc-348) and NRG-1β (sc-1792) in samples obtained from rat carotid arteries. Paraffin-embedded carotid artery sections were prepared and subsequently cleared with xylene and hydrated with ethanol. After hydration, sections were washed 3 times with PBS and treated for 30 min with 0.9% H2O2 in methanol to block endogenous peroxidase activity. Sections were incubated with normal goat serum (15%, v/v) for 1 h at 37°C to block nonspecific binding. Sections were then incubated overnight at 4°C with primary antibodies at 1:400 concentration and then incubated for 1 h with a biotinylated goat anti-rabbit antibody (1:1,000) using a Santa Cruz ABC staining kit. Sections were counterstained with hematoxylin, mounted with Vectashield (Vector Labs, Burlingame, Calif., USA) and analyzed with an Olympus light microscope.

goto top of outline Tissue Processing and Quantitative Histomorphometric Analysis

Animals were euthanized with CO2 14 days after injury. Carotid arteries were washed with saline to clear blood, embedded in Tissue-Tek OCT medium and frozen using liquid nitrogen. Carotid sections were cut with a cryostat into cross-sections of 12 μm taken from the center and distal portion of the vessels and stained with hematoxylin and eosin. The medial thickness was determined by the area of the internal elastic lamina subtracted from the external elastic lamina. Morphometry was performed using at least 6 individual sections of each arterial segment and used to determine the lesion size expressed as intima/media ratio. The intimal and medial layer thicknesses were measured using a computer-based image analyzing program (Image J, NIH).

goto top of outline Rat VSMC Cultures

A7r5 rat aortic VSMCs (ATCC CRL-1444) were obtained from American Tissue Type Culture (Manassas, Va., USA) and grown in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, Calif., USA) supplemented with glutamine, 10% fetal calf serum and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2. Cells were passaged weekly. All experiments were performed on cells from passages 9–12.

goto top of outline Determination of VSMC Proliferation

VSMCs were seeded at a density of 1 × 103 cells in triplicate wells of a 96-well plate. After 24 h, cells were serum starved in DMEM/F-12 (Gibco) containing 0.1% fetal calf serum (low serum medium, LSM) to induce quiescence. After 24 h of serum deprivation, cells were pretreated with 0–200 nM of NRG-1α or NRG-1β for 24 h. Cells were then treated with 10 ng/ml of PDGF-BB for 48 h to stimulate VSMC proliferation. For direct measure of cell numbers, cells were counted using a Coulter counter. VSMC proliferation and viability were also measured using the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, Wisc., USA) according to the manufacturer’s protocol. After incubation at 37°C in humidified 5% CO2 for 1 h, the absorbance was recorded at 490 nm using a plate reader. Measurement of DNA synthesis was performed using the BrDU Cell Proliferation Assay (Calbiochem, San Diego, Calif., USA) according to the manufacturer’s protocol.

goto top of outline Cell Migration Assay

Neuro Probe 48-well microchemotaxis chambers (Costar, Corning Inc., Corning, N.Y., USA) with a polyvinylpyrrolidone-free polycarbonate filter (8.0 μm pore size) were used to measure VSMC migration. Quiescent cells were trypsinized and resuspended in LSM with or without NRG-1 and incubated for 24 h at 37°C. Cells were then treated with PDGF which was added to the bottom well of the Boyden chamber and incubated for 48 h at 37°C. Cells that migrated to the lower side of the filters were fixed and stained with the Diff Quick staining kit (VWR Laboratory, West Chester, Pa., USA). The filters were mounted on glass slides and counted by light microscopy using ×100 magnification.

goto top of outline Protein Purification and Western Analysis

Reactions were terminated by placing the cells on ice, aspirating the medium and adding ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, pH 8.0) for 30 min at 4°C. Harvested lysates were denatured with loading buffer, resolved in SDS/5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, Mass., USA). Membranes were blocked with 3% nonfat dry milk in PBS 0.5% Tween-20 (PBST). Western blots were performed using primary antibodies for phosphorylated and unphosphorylated forms of ERK1/2 (Cell Signaling, Danvers, Mass., USA) diluted 1:250 in blocking buffer overnight at 4°C. For ERK1/2 phosphorylation, VSMCs were pretreated with NRG-1β for 24 h and stimulated for 15 min with PDGF. After incubation, membranes were washed with PBST. After the washing, membranes were exposed to an alkaline phosphatase-conjugated anti-rabbit secondary antibody for 1 h. Membranes were subsequently washed with PBST, incubated with chemiluminescence reagents and exposed to X-ray film.

goto top of outline Cell Viability Assay

Quantitative viability assessment was performed using 1% calcein-AM (Molecular Probes, Eugene, Oreg., USA), a fluorescent membrane integrity dye, diluted in Hanks’ balanced salt solution according to the manufacturer’s protocol. Qualitative assessment of cell viability in treated cells was performed using the trypan blue exclusion assay. Nonviable cells were quantified visually using a light microscopy.

goto top of outline Statistical Analysis

Each experiment was repeated a minimum of 3 times. Data are expressed as the mean ± standard deviation (SD) or standard error (SEM). An unpaired Student’s t test and ANOVA were performed to make comparisons between groups. A value of p < 0.05 was considered significant.

 

goto top of outline Results

goto top of outline Increased Expression of NRG-1 and erbB Receptors in Rat Carotid Arteries after Carotid Balloon Injury

As an initial step to define the role of NRG-1 in vascular injury, we examined the NRG-1 expression levels in injured vessels using the rat carotid balloon injury model. Our results demonstrated that neointimal formation in this model is associated with a significant increase in NRG-1 and erbB receptor expression as determined by immunohistochemistry (fig. 1). NRG-1α- and NRG-1β-specific antibodies were used for immunohistochemistry in sections of uninjured and injured rat carotid arteries to determine the pattern of NRG-1 expression in the developing lesion. In the uninjured artery, the intima consists of the single layer of endothelial cells (fig. 1a, c, e). However, following balloon injury, there was a dramatic thickening of the intima to form the postinjury neointima which primarily contained smooth muscle cells that have proliferated and migrated from the media (fig. 1b, d, f). Relatively low levels of NRG-1α (fig. 1a) and NRG-1β (fig. 1c) were seen in the uninjured control vessels. However, after vascular injury, the expression of both NRG-1α and NRG-1β was highly increased, particularly in the neointima (fig. 1b, d).

FIG01
Fig. 1. NRG-1 and erbB receptor expression in the carotid artery following balloon injury. Immunohistochemistry revealed that NRG-1α protein was expressed at low levels in uninjured arteries (a). However, 14 days after vascular injury, NRG-1α was increased (b). NRG-1β protein was expressed at low levels in uninjured arteries (c), and similarly, NRG-1β was increased after injury (d). Uninjured or control arteries showed little erbB4 (e); however, the erbB4 receptor was dramatically increased after injury (f). No staining is seen in IgG-negative controls (g). The arrows indicate the neointima.

Like NRG-1, we observed that the expression levels of all erbB receptors were low in uninjured carotid arteries. However, erbB4 expression was highly increased in the neointima after injury (fig. 1f). erbB2 and erbB3 expression was barely detectable in either uninjured arteries or after injury (data not shown). No staining was observed in the IgG-negative controls (fig. 1g).

goto top of outline NRG-1 Attenuates Neointima Formation after Rat Carotid Balloon Injury

As demonstrated above, neointimal hyperplasia was histologically evident in the carotid arteries 14 days after balloon injury (fig. 2c, d) compared with uninjured contralateral controls (fig. 2a, b). The neointima of the rats receiving intravenous administration of NRG-1 was significantly reduced compared with balloon-injured animals (fig. 2e, f). Morphometric analysis showed that NRG-1 reduced the size of the lesion by 70% compared with vehicle-treated control animals (control = 1.89 × 105 μm2, NRG-1 = 0.55 × 105 μm2; p < 0.001) (fig. 3). NRG-1 slightly reduced the diameter of the media resulting in an intima/media ratio of 1.36 ± 0.19 for vehicle-treated animals and 0.67 ± 0.09 for NRG-treated animals (a 50% reduction in the intima/media ratio; p < 0.001). Treatment of animals with NRG-1 showed no overt negative side effects and there was no significant difference in body weight observed among the control and NRG-1-treated rats.

FIG02
Fig. 2. Representative photomicrographs of perfusion-fixed balloon-injured left carotid arteries 14 days after injury. Cross-section of H&E-stained carotid arteries from an uninjured artery (a, b) and an injured artery (c, d) from a vehicle-treated rat are shown. Treatment with NRG-1β significantly reduced lesion size following balloon injury (e, f). The double arrows indicate the neointima.

FIG03
Fig. 3. Quantitative analysis of neointimal formation following NRG-1 treatment. Effect of NRG-1β on neointimal formation 14 days after balloon injury. Morphometry was performed using at least 6 individual sections of each arterial segment and used to determine the lesion size expressed as intima/media ratio. The intimal and medial layer thicknesses were measured using a computer-based image analyzing program (Image J, NIH). * p < 0.05 versus vehicle-treated control group (n = 6–8 rats/experimental group).

goto top of outline NRG-1 Inhibits Proliferation in VSMCs

One possible mechanism for the inhibitory effect of NRG-1 on neointimal formation is the regulation of pathological VSMC functions. To examine the effects of NRG-1 on VSMC proliferation, serum-starved A7r5 VSMCs were pretreated with NRG-1 for 24 h, then stimulated with PDGF for an additional 48 h. Stimulation of cells with PDGF increased proliferation of VSMCs by 2-fold (fig. 4). Pretreatment with either NRG-1β (fig. 4a) or NRG-1α (fig. 4b) resulted in a dose-dependent decrease in baseline and PDGF-stimulated proliferation as measured by MTS activity. Direct cell counting using Coulter counter demonstrated that NRG-1 reduced PDGF-stimulated VSMC proliferation, but not baseline cell numbers (fig. 4c). Analysis of bromodeoxyuridine (BrDU) incorporation revealed a similar pattern to the Coulter counter demonstrating that NRG-1 significantly inhibited PDGF-induced proliferation, but did not alter baseline DNA synthesis (fig. 4d). To determine whether the growth inhibitory effects of NRG-1 were due to toxicity or damage to the cells rather than proliferation, calcein-AM and trypan blue viability assays were carried out in cells pretreated with NRG-1 with or without PDGF. Both assays demonstrated that treatment of VSMCs with NRG-1 did not alter cell viability (data not shown).

FIG04
Fig. 4. NRG-1 inhibits PDGF-induced VSMC proliferation. VSMCs were pretreated with control medium, NRG-1α or NRG-1β for 24 h and then with 10 ng/ml PDGF-BB or medium for 48 h. Proliferation was measured using the Cell titer 96 MTS-based proliferation assay (a, b). To measure the dose responsiveness of VSMCs to NRG-1, cells were pretreated with the indicated concentrations of NRG-1β (a) or NRG-1α (b) before treatment with PDGF or control medium. Direct cell counts were measured using a Coulter counter after treatment with PDGF and/or NRG-1β (100 nM) (c). The effect of NRG-1β (100 nM) on DNA synthesis was analyzed by BrDU incorporation (d). Each bar represents the mean ± SD in 3 independent experiments performed in triplicate. * p < 0.01 versus control; ** p < 0.05 versus PDGF; *** p < 0.01 versus PDGF.

goto top of outline NRG-1 Decreases VSMC Migration

The migration of VSMC was measured using a transwell migration assay. VSMCs were pretreated with 100 nM NRG-1α or NRG-1β and then stimulated with 10 ng/ml of PDGF-BB for 48 h. Our results show that NRG-1 alone does not alter baseline VSMC migration (fig. 5). VSMC treated with PDGF displayed a 2- to 3-fold increase in migration. Both NRG-1α and NRG-1β decreased PDGF-stimulated VSMC migration by 80 and 90%, respectively.

FIG05
Fig. 5. NRG-1 prevents VSMC migration. VSMCs were grown onto Neuro Probe 48-well microchemotaxis chambers with polyvinylpyrrolidone-free polycarbonate filters (8.0-μm pore size) in LSM with or without NRG-1 and incubated for 24 h. After 24 h, cells were treated with PDGF and incubated for 48 h. Cells that migrated to the lower side of the filters were fixed and stained with the Diff Quick staining kit (VWR Laboratory). Data are shown as means of the results of triplicate experiments ± SD. * p < 0.05 versus PDGF; ** p < 0.01 versus control.

goto top of outline NRG-1 Inhibits PDGF-Induced Phosphorylation of ERK1/2

Several studies have shown that PDGF-induced VSMC proliferation involves the ERK signaling pathway [21,22,23]. Regulation of the phosphorylation of these kinases was used to determine whether NRG-1 could inhibit PDGF activity in VSMC by interfering with ERK activity. PDGF stimulation of VSMC resulted in an induction of ERK1/2 phosphorylation (fig. 6). Treatment with NRG-1 alone did not alter ERK1/2 phosphorylation compared with control untreated VSMCs. However, NRG-1 prevented PDGF-induced phosphorylation of ERK1/2. Densitometric analysis revealed that NRG-1 reduced PDGF-stimulated ERK1/2 phosphorylation in VSMCs by 70% (fig. 6b).

FIG06
Fig. 6. NRG-1 inhibits ERK1/2 phosphorylation in PDGF-stimulated VSMCs. VSMCs were pretreated with NRG-1β for 24 h and stimulated for 15 min with PDGF. A representative Western blot shows a decrease in the PDGF-stimulated phosphorylation of ERK1/2 protein by NRG-1 (a). Densitometric analysis of ERK1/2 phosphorylation is shown as the mean of the relative intensity values ± SD derived from 3 independent experiments. Phospho-ERK1/2 (pERK1/2) levels are normalized using tubulin expression (b). * p < 0.01 versus control; ** p < 0.05 versus PDGF.

 

goto top of outline Discussion

The NRG-1/erbB system has been shown to modulate various biological activities including cell survival, proliferation and migration [8, 9, 11, 24], which are critical for normal development and pathology in a variety of tissues. However, the role of NRG-1 in vascular function and injury has not been clearly elucidated. Here, we investigated the potential role of NRG-1 in lesion formation following vascular injury. In this study, we demonstrated that NRG-1 attenuates neointimal formation and vascular injury in the carotid artery balloon injury rat model. NRG-1 reduced the size of the lesion by approximately 50% compared with vehicle-treated control animals. This novel finding suggests that NRG-1 may be useful in the prevention of vascular diseases such as restenosis and atherosclerosis. NRG-1 also slightly reduced the area of the media, and studies are underway to determine whether NRG-1 affects VSMC proliferation in the media or regulation of vascular contraction.

A recent study showed that NRG-1 expression was increased in human atherosclerotic lesions and may play an important role in the atherosclerotic plaque [13]. Here, we also observed that NRG-1 and erbB receptors are upregulated following balloon injury to the carotid artery in the rat. Since exogenous NRG-1 prevented neointimal formation, we propose that the upregulation of NRG-1 may be an immediate protective response mechanism to inhibit local VSMC proliferation and inflammatory response after vascular injury. This would suggest that there exists a balance between pro- and antiatherogenic forces after vascular injury. Mitogenic molecules include additional ligands for erbB receptors, such as heparin-binding EGF, betacellulin and amphiregulin that are upregulated during vascular injury and may promote lesion formation [1, 25]. Therefore, administration of exogenous NRG-1 may shift the balance toward an antiatherogenic state and prevent neointimal formation.

To address the cellular mechanisms involved in the effect of NRG-1 on neointimal formation, we examined whether NRG-1 could influence VSMC proliferation. The accumulation of VSMC in the neointima is one of the central features of atherosclerosis and restenosis. The proliferation of VSMC contributes to the pathobiology of atherosclerosis and restenosis and is a major component of arterial remodeling in vascular disease. Injury to the vascular wall by atherosclerosis and postangioplasty restenosis stimulate the release of mitogenic growth factors, such as PDGF, that induce VSMCs to proliferate and migrate into the intima of the vessel, and thus, form the vascular lesion or neointima. Using the MTS-based proliferation assay, we showed that NRG-1 blocks PDGF- induced proliferation of VSMCs in a dose-dependent manner. The inhibitory effects of NRG-1 on VSMC proliferation were confirmed by direct cell counting and measuring DNA synthesis by BrDU incorporation. An intriguing observation was the difference in the effect of NRG-1 on baseline VSMC proliferation using the MTS-based assay compared with the other methods. In the cell-counting and BrDU approaches, PDGF-increased VSMC proliferation was blocked by NRG-1; however, baseline VSMC numbers were not altered. Using the MTS-based assay, a 50% decrease in baseline MTS activity was seen after NRG-1 administration. Since the MTS assay measures metabolic activity, it is possible that NRG-1 may reduce baseline MTS levels by decreasing VSMC metabolic activity or promoting VSMC differentiation. Induction of apoptosis by NRG-1 is unlikely to be a reason for attenuated VSMC baseline or PDGF-stimulated proliferation by NRG-1 since there was no evidence of increased dead or nonviable cells after NRG-1 treatment. Consistent with our findings, previous studies in visceral smooth muscle cells and VSMCs demonstrated that NRG-1 was not mitogenic to VSMCs; however, these studies did not investigate the antiproliferative effects of NRG-1 [26].

We acknowledge that the concentrations of NRG-1 required to prevent VSMC proliferation in vitro may be relatively high compared with the low dose used in the in vivo studies, although we currently have no assays available for measuring NRG-1 levels in vivo. There are a number of reasons that could account for relatively lower concentrations of NRG-1 required in the in vivo studies compared with the amount required to block proliferation in the VSMC cultures, including the upregulation of NRG-1 and erbB receptors in the lesion area after injury. This upregulation of both NRG-1 and its receptors could make cells more responsive to NRG-1 in the injured areas and require less exogenous NRG-1 to see an effect. It is plausible that NRG-1 may also have an effect on other cells in vivo in addition to VSMCs, such as macrophages, that may account for the differences in concentrations in vivo versus in vitro. In addition, the difference may be related to the relatively high concentrations of PDGF used in the in vitro assays.

It is well documented that neuroprotective factors and their receptors are upregulated in the peri-infarct regions following focal ischemia that serve to protect neurons from ischemic damage [27,28,29] including nerve growth factor, brain-derived neurotrophic factor and transforming growth factor-β1 [27, 28, 30]. The upregulation of NRG-1 and erbB receptors in the lesion area after injury may also account for the relatively lower concentrations of NRG-1 required in the in vivo studies compared with the amount required to block proliferation in the VSMC cultures. Similarly, the upregulation of NRG-1 and its erbB receptors has also been reported after ischemia and central nervous system injury [31,32,33,34,35,36]. As seen in the vascular injury model, we showed that administration of exogenous NRG-1 was neuroprotective and prevented inflammatory responses caused by ischemia [32, 37].

The signaling mechanisms by which NRG-1 prevents proliferation of VSMC are not clearly understood. At the moment, different mechanisms may be proposed for NRG-1 inhibitory effect of PDGF action in VSMCs. It is possible that NRG-1 may signal inhibitory pathways that block downstream growth factor receptor signals which upregulate proliferative pathways in VSMCs. To begin investigating the molecular signaling mechanisms underlying the antiproliferative effects of NRG-1, we examined the activity of a signaling molecule known to mediate mitogen-induced VSMC proliferation. PDGF has been shown to stimulated smooth muscle cell proliferation by activating the ERK1/2 pathway [21,22,23]. Our results confirmed that PDGF stimulates the phosphorylation of ERK1/2. PDGF-stimulated ERK1/2 phosphorylation was suppressed by NRG-1. Therefore, we propose that NRG-1 blocks signaling mechanisms from PDGF receptor-initiated proliferative pathways involving ERK1/2. Further studies are required to determine the exact location of NRG-1 action in other signaling pathways, including whether it acts as a PDGF antagonist by modulating receptor activity or expression levels. In addition to direct effects on smooth muscle cells, it is also possible that NRG-1 may downregulate proinflammatory responses that exacerbate the processes involved in neointimal formation. In fact, we have recently shown that NRG-1 prevents proinflammatory responses in monocyte/macrophage cultures [38]. In addition, NRG-1 could prevent extracellular matrix production required to facilitate the expansion of the neointimal lesion.

In conclusion, we have demonstrated that NRG-1 has an inhibitory effect on neointimal formation after vascular injury and prevents mitogen-induced VSMC proliferation and migration. Taken together, these results suggest that NRG-1 may be a novel therapeutic candidate for the prevention of neointimal formation in diseases, such as atherosclerosis and restenosis.

 

goto top of outline Acknowledgements

This work was supported by NIH grants NS34194, HL067702, HL003676 and the W.M. Keck Foundation. The investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant No. C06 RR-07571 from the National Center for Research Resources, NIH. We thank Dr. Yan Xiao and Mr. Tabari Baker for their technical assistance.


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  14. Corfas G, Rosen KM, Aratake H, Krauss R, Fischbach GD: Differential expression of ARIA isoforms in the rat brain. Neuron 1995;14:103–115.
  15. Krymskaya VP, Hoffman R, Eszterhas A, Kane S, Ciocca V, Panettieri RA Jr: EGF activates ErbB-2 and stimulates phosphatidylinositol 3-kinase in human airway smooth muscle cells. Am J Physiol 1999;276:L246–L255.
  16. Iivanainen E, Nelimarkka L, Elenius V, Heikkinen SM, Junttila TT, Sihombing L, Sundvall M, Maatta JA, Laine VJ, Yla-Herttuala S, Higashiyama S, Alitalo K, Elenius K: Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor. FASEB J 2003;17:1609–1621.
  17. Bagheri-Yarmand R, Vadlamudi RK, Wang RA, Mendelsohn J, Kumar R: Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-β1-mediated angiogenesis. J Biol Chem 2000;275:39451–39457.
  18. Kumar R, Yarmand-Bagheri R: The role of HER2 in angiogenesis. Semin Oncol 2001;28:27–32.
  19. Russell KS, Stern DF, Polverini PJ, Bender JR: Neuregulin activation of ErbB receptors in vascular endothelium leads to angiogenesis. Am J Physiol 1999;277:H2205–H2211.
  20. Yen L, You XL, Al Moustafa AE, Batist G, Hynes NE, Mader S, Meloche S, Alaoui-Jamali MA: Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene 2000;19:3460–3469.
  21. Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H: Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 2003;23:795–801.
  22. Force T, Pombo CM, Avruch JA, Bonventre JV, Kyriakis JM: Stress-activated protein kinases in cardiovascular disease. Circ Res 1996;78:947–953.
  23. Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, Law RE: Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells. Hypertension 1997;29:334–339.
  24. Falls DL: Neuregulins: functions, forms, and signaling strategies. Exp Cell Res 2003;284:14–30.
  25. Shin HS, Lee HJ, Nishida M, Lee MS, Tamura R, Yamashita S, Matsuzawa Y, Lee IK, Koh GY: Betacellulin and amphiregulin induce upregulation of cyclin D1 and DNA synthesis activity through differential signaling pathways in vascular smooth muscle cells. Circ Res 2003;93:302–310.
  26. Yamanaka Y, Hayashi K, Komurasaki T, Morimoto S, Ogihara T, Sobue K: EGF family ligand-dependent phenotypic modulation of smooth muscle cells through EGF receptor. Biochem Biophys Res Commun 2001;281:373–377.
  27. Ferrer I, Planas AM: Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol 2003;62:329–339.
  28. Guegan C, Sola B: Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice. Brain Res 2000;856:93–100.
  29. Matsushima K, Schmidt-Kastner R, Hogan MJ, Hakim AM: Cortical spreading depression activates trophic factor expression in neurons and astrocytes and protects against subsequent focal brain ischemia. Brain Res 1998;807:47–60.
  30. Guegan C, Ceballos-Picot I, Nicole A, Kato H, Onteniente B, Sola B: Recruitment of several neuroprotective pathways after permanent focal ischemia in mice. Exp Neurol 1998;154:371–380.
  31. Xu Z, Ford BD: Upregulation of erbB receptors in rat brain after middle cerebral arterial occlusion. Neurosci Lett 2005;375:181–186.
  32. Xu Z, Jiang J, Ford G, Ford BD: Neuregulin-1 is neuroprotective and attenuates inflammatory responses induced by ischemic stroke. Biochem Biophys Res Commun 2004;322:440–446.
  33. Parker MW, Chen Y, Hallenbeck JM, Ford BD: Neuregulin expression after focal stroke in the rat. Neurosci Lett 2002;334:169–172.
  34. Erlich S, Shohami E, Pinkas-Kramarski R: Closed head injury induces up-regulation of ErbB-4 receptor at the site of injury. Mol Cell Neurosci 2000;16:597–608.
  35. Tokita Y, Keino H, Matsui F, Aono S, Ishiguro H, Higashiyama S, Oohira A: Regulation of neuregulin expression in the injured rat brain and cultured astrocytes. J Neurosci 2001;21:1257–1264.
  36. Lindholm T, Cullheim S, Deckner M, Carlstedt T, Risling M: Expression of neuregulin and ErbB3 and ErbB4 after a traumatic lesion in the ventral funiculus of the spinal cord and in the intact primary olfactory system. Exp Brain Res 2002;142:81–90.
  37. Shyu WC, Lin SZ, Chiang MF, Yang HI, Thajeb P, Li H: Neuregulin-1 reduces ischemia-induced brain damage in rats. Neurobiol Aging 2004;25:935–944.
  38. Xu Z, Ford GD, Croslan DR, Jiang J, Gates A, Allen R, Ford BD: Neuroprotection by neuregulin-1 following focal stroke is associated with the attenuation of ischemia-induced pro-inflammatory and stress gene expression. Neurobiol Dis 2005;19:461–470.

 goto top of outline Author Contacts

Dr. Byron D. Ford
Department of Anatomy and Neurobiology, Neuroscience Institute
Morehouse School of Medicine, 720 Westview Drive, SW, MRC 222
Atlanta, GA 30310 (USA)
Tel. +1 404 756 5222, Fax +1 404 752 1041, E-Mail bford@msm.edu


 goto top of outline Article Information

Received: October 18, 2006
Accepted after revision: January 16, 2007
Published online: April 16, 2007
Number of Print Pages : 10
Number of Figures : 6, Number of Tables : 0, Number of References : 38


 goto top of outline Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 44, No. 4, Year 2007 (Cover Date: June 2007)

Journal Editor: Pohl, U. (Munich)
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Neuregulin-1 (NRG-1) is expressed in vascular endothelial cells, and its receptors are localized to the underlying smooth muscle cells. However, the role of NRG-1 in vascular function and injury is largely unknown. First, the expression of NRG-1 and its receptors (erbB receptors) was analyzed after balloon injury to the rat carotid artery. NRG-1 and erbB expression levels were low in uninjured vessels; however, NRG-1 and erbB4 were upregulated following injury. We then examined the effect of NRG-1 on neointimal formation following balloon injury. NRG-1 was administered by tail-vein injection prior to injury and every 2 days following injury. Two weeks after injury, NRG-1-treated animals demonstrated a 50% reduction in lesion size compared with controls receiving the vehicle. To examine possible mechanisms for NRG-1 action, we examined its effects on vascular smooth muscle cell (VSMC) function. Rat VSMC cultures were pretreated with NRG-1 for 24 h and then stimulated with platelet-derived growth factor. NRG-1 significantly decreased platelet-derived growth factor-stimulated VSMC proliferation and migration. These findings suggest that NRG-1 may be a novel therapeutic candidate for the treatment of restenosis and atherosclerosis.



 goto top of outline Author Contacts

Dr. Byron D. Ford
Department of Anatomy and Neurobiology, Neuroscience Institute
Morehouse School of Medicine, 720 Westview Drive, SW, MRC 222
Atlanta, GA 30310 (USA)
Tel. +1 404 756 5222, Fax +1 404 752 1041, E-Mail bford@msm.edu


 goto top of outline Article Information

Received: October 18, 2006
Accepted after revision: January 16, 2007
Published online: April 16, 2007
Number of Print Pages : 10
Number of Figures : 6, Number of Tables : 0, Number of References : 38


 goto top of outline Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 44, No. 4, Year 2007 (Cover Date: June 2007)

Journal Editor: Pohl, U. (Munich)
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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  16. Iivanainen E, Nelimarkka L, Elenius V, Heikkinen SM, Junttila TT, Sihombing L, Sundvall M, Maatta JA, Laine VJ, Yla-Herttuala S, Higashiyama S, Alitalo K, Elenius K: Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor. FASEB J 2003;17:1609–1621.
  17. Bagheri-Yarmand R, Vadlamudi RK, Wang RA, Mendelsohn J, Kumar R: Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-β1-mediated angiogenesis. J Biol Chem 2000;275:39451–39457.
  18. Kumar R, Yarmand-Bagheri R: The role of HER2 in angiogenesis. Semin Oncol 2001;28:27–32.
  19. Russell KS, Stern DF, Polverini PJ, Bender JR: Neuregulin activation of ErbB receptors in vascular endothelium leads to angiogenesis. Am J Physiol 1999;277:H2205–H2211.
  20. Yen L, You XL, Al Moustafa AE, Batist G, Hynes NE, Mader S, Meloche S, Alaoui-Jamali MA: Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene 2000;19:3460–3469.
  21. Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H: Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 2003;23:795–801.
  22. Force T, Pombo CM, Avruch JA, Bonventre JV, Kyriakis JM: Stress-activated protein kinases in cardiovascular disease. Circ Res 1996;78:947–953.
  23. Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, Law RE: Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells. Hypertension 1997;29:334–339.
  24. Falls DL: Neuregulins: functions, forms, and signaling strategies. Exp Cell Res 2003;284:14–30.
  25. Shin HS, Lee HJ, Nishida M, Lee MS, Tamura R, Yamashita S, Matsuzawa Y, Lee IK, Koh GY: Betacellulin and amphiregulin induce upregulation of cyclin D1 and DNA synthesis activity through differential signaling pathways in vascular smooth muscle cells. Circ Res 2003;93:302–310.
  26. Yamanaka Y, Hayashi K, Komurasaki T, Morimoto S, Ogihara T, Sobue K: EGF family ligand-dependent phenotypic modulation of smooth muscle cells through EGF receptor. Biochem Biophys Res Commun 2001;281:373–377.
  27. Ferrer I, Planas AM: Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol 2003;62:329–339.
  28. Guegan C, Sola B: Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice. Brain Res 2000;856:93–100.
  29. Matsushima K, Schmidt-Kastner R, Hogan MJ, Hakim AM: Cortical spreading depression activates trophic factor expression in neurons and astrocytes and protects against subsequent focal brain ischemia. Brain Res 1998;807:47–60.
  30. Guegan C, Ceballos-Picot I, Nicole A, Kato H, Onteniente B, Sola B: Recruitment of several neuroprotective pathways after permanent focal ischemia in mice. Exp Neurol 1998;154:371–380.
  31. Xu Z, Ford BD: Upregulation of erbB receptors in rat brain after middle cerebral arterial occlusion. Neurosci Lett 2005;375:181–186.
  32. Xu Z, Jiang J, Ford G, Ford BD: Neuregulin-1 is neuroprotective and attenuates inflammatory responses induced by ischemic stroke. Biochem Biophys Res Commun 2004;322:440–446.
  33. Parker MW, Chen Y, Hallenbeck JM, Ford BD: Neuregulin expression after focal stroke in the rat. Neurosci Lett 2002;334:169–172.
  34. Erlich S, Shohami E, Pinkas-Kramarski R: Closed head injury induces up-regulation of ErbB-4 receptor at the site of injury. Mol Cell Neurosci 2000;16:597–608.
  35. Tokita Y, Keino H, Matsui F, Aono S, Ishiguro H, Higashiyama S, Oohira A: Regulation of neuregulin expression in the injured rat brain and cultured astrocytes. J Neurosci 2001;21:1257–1264.
  36. Lindholm T, Cullheim S, Deckner M, Carlstedt T, Risling M: Expression of neuregulin and ErbB3 and ErbB4 after a traumatic lesion in the ventral funiculus of the spinal cord and in the intact primary olfactory system. Exp Brain Res 2002;142:81–90.
  37. Shyu WC, Lin SZ, Chiang MF, Yang HI, Thajeb P, Li H: Neuregulin-1 reduces ischemia-induced brain damage in rats. Neurobiol Aging 2004;25:935–944.
  38. Xu Z, Ford GD, Croslan DR, Jiang J, Gates A, Allen R, Ford BD: Neuroprotection by neuregulin-1 following focal stroke is associated with the attenuation of ischemia-induced pro-inflammatory and stress gene expression. Neurobiol Dis 2005;19:461–470.