Cardiovascular Research

Cardiovascular Research 2005 65(3):674-682; doi:10.1016/j.cardiores.2004.10.031

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Peppel, K. Articles by Freedman, N. J. Search for Related Content PubMed PubMed Citation Articles by Peppel, K. Articles by Freedman, N. J. Social Bookmarking          What's this?

Copyright © 2004, European Society of Cardiology

Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms

Karsten Peppel^a,*, Lisheng Zhang^a, Eric S. Orman^a, Per-Otto Hagen^b, Andrea Amalfitano^c, Leigh Brian^a and Neil J. Freedman^a,*

^aDepartment of Medicine (Cardiology), Duke University Medical Center, Box 3187, Durham, NC 27710, United States
^bDepartment of Surgery, Duke University Medical Center, United States
^cDepartment of Pediatrics and Molecular Genetics and Microbiology, Duke University Medical Center, United States

^* Corresponding authors. Tel.: +1 919 684 6876; fax: +1 919 684 6870. Email address: karsten.peppel{at}duke.edu neil.freedman{at}duke.edu

Received 7 July 2004; revised 8 October 2004; accepted 22 October 2004

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objective: Because tumor necrosis factor- (TNF) has been implicated in the pathogenesis of vein graft neointimal hyperplasia, we sought to determine mechanisms by which TNF could induce proliferative and migratory responses in smooth muscle cells (SMCs).

Methods and results: In rabbit jugulocarotid interposition vein grafts, SMCs expressed TNF as early as four days postoperatively. In rabbit aortic SMCs, TNF and platelet-derived growth factor (PDGF) elicited comparable migration (1.7-fold/basal), and their effects were partially additive. In contrast, while TNF failed to promote SMC [³H]thymidine incorporation alone, it doubled the [³H]thymidine incorporation observed with PDGF alone. To gain mechanistic insight into these phenomena, we found that TNF and PDGF each activated p38^mapk equivalently in SMCs, but that PDGF was two to three times more efficacious than TNF in activating SMC extracellular signal-regulated kinases (ERK) 1 and 2 and phosphoinositide 3-kinase. However, only TNF activated NFB. SMC [³H]thymidine incorporation that depended on TNF, but not PDGF, was abolished by overexpression of a dominant-negative IB mutant. Inhibition of ERK activation by U0126 reduced SMC migration stimulated only by PDGF (by 35%, P<0.05), but not by TNF. Inhibition of phosphoinositide 3-kinase by LY294002, however, significantly reduced both TNF- and PDGF-stimulated chemotaxis (by 38–54%, P<0.05). In contrast, both U0126 and LY294002 abolished SMC [³H]thymidine incorporation induced by either TNF, PDGF, or both agonists.

Conclusions: In primary rabbit SMCs, TNF promotes migration and mitogenesis through signaling mechanisms that are both distinct from and overlapping with those employed by PDGF. TNF-induced SMC mitogenesis requires complementary co-stimulation with other growth factors.

KEYWORDS Cytokines; Growth factors; Receptors; Signal transduction; Veins

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Vein graft neointimal hyperplasia is believed to form a nidus for accelerated atherosclerosis that leads to vein graft failure in >40% of cases within 10 years after operation [1,2]. Arterial grafting hyper-expands and injures veins; subsequently, platelets and leukocytes adhere to the vein graft luminal surface and secrete numerous growth factors and cytokines, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and tumor necrosis factor- (TNF), among others [1,3]. Consequent activation of SMCs in the vein graft and recruitment of SMC progenitors from outside the vein graft [4] engender neointimal hyperplasia [5]. Cytokines are believed to contribute to neointimal hyperplasia, but the mechanisms involved remain largely obscure.

The time course of vein graft neointimal hyperplasia and vein graft cytokine/growth factor expression suggests that if the pro-inflammatory cytokine TNF promotes vein graft neointimal hyperplasia [6,7], it does so only in concert with other growth factors [8]. TNF expression in rat vein grafts increases during the most rapid phase of SMC proliferation (postoperative weeks 1 and 2), but TNF expression also persists for at least 4 weeks postoperatively, when neointimal hyperplasia has ceased to progress [8]. In contrast, PDGF expression peaks one week postoperatively, and falls to near-baseline levels by 4 weeks postoperatively. Thus, while vein graft PDGF expression correlates with the time course of vein graft neointimal hyperplasia, TNF expression does not [8]. PDGF is among the most potent stimuli for SMC migration and proliferation in vitro [9], and inhibition of PDGF receptor-β signaling diminishes neointimal hyperplasia in balloon-injured and atherosclerotic arteries [10,11] as well as in autologous vein grafts [12].

Could TNF in the vein graft promote neointimal hyperplasia by potentiating pro-mitogenic and pro-migratory signaling elicited by the receptor protein tyrosine kinases for PDGF? If so, we would expect TNF and PDGF to activate SMCs in complementary fashion. Although TNF reduces mitogenic signaling by the receptor protein tyrosine kinase for insulin [13], its effects on PDGF-stimulated signaling are unclear. To address this issue with in vitro models of neointimal hyperplasia, we studied how TNF and PDGF together influence the migration and proliferation of primary SMCs, and examined SMC signal transduction mechanisms elicited by TNF and PDGF.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Rabbit vein graft surgery
Right common carotid interposition grafting with the ipsilateral jugular vein was performed on male New Zealand White (NZW) rabbits, and grafts were harvested as described [12]. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Primary SMC isolation, culture and infection
Primary rabbit aortic and inferior vena cava SMCs were isolated by explant outgrowth, and grown as described [14]. For serum starvation, SMCs were incubated 72 h in serum-free medium (SFM: DMEM containing 0.1% fatty acid-free bovine serum albumin, and 50 µg/ml gentamicin). SMCs were used before passage 7, and were infected with adenoviruses as described [14].

2.3. Immunofluorescence
Rabbit jugular veins and vein grafts were frozen in OCT and sliced at 10 µm. Sections were incubated with goat anti-TNF IgG (Santa Cruz Biotechnology), and then a mixture of anti-goat/Alexa 488 (Molecular Probes) and Cy3-conjugated 1A4 mouse IgG (against -SMC actin), as described [12]. To stain for cell surface TNF, SMCs grown on culture slides were fixed with formalin and stained for TNF, as above, except that the nuclear dye Hoechst 33258 (10µg/ml) was included in the secondary IgG incubation. Fluorophore-specific images were obtained as described previously [14].

2.4. Measurement of SMC TNF production
To induce TNF secretion by murine RAW 264.7 macrophages and SMCs, we added 20 µg/ml lipopolysaccharide (Sigma) to the medium (DMEM/10% heat-inactivated FBS). After 22 h (during which there was no evidence of cell death), conditioned medium was assayed for TNF bioactivity as described [15]: murine L929 cells were treated concurrently with 100 µg/ml cycloheximide and conditioned medium for 16 h (37 °C); L929 cell death was quantitated by crystal violet staining and subsequent solubilization. To verify that TNF was the cytotoxic agent, we used a soluble p55 TNF receptor [15] to neutralize the activity of the conditioned medium.

2.5. SMC migration
The migration of serum-starved SMCs was assessed using 24-well plate cell culture inserts with 8 µm pores (Falcon) as described [12]. Where indicated, the MEK1 inhibitor U0126 (10 µmol/l, Calbiochem) or the phosphoinositide-3 kinase inhibitor LY294002 (20 µmol/l, Calbiochem) were added to the inner and outer chambers 30 min prior to agonist treatment.

2.6. Immunoblotting
Quiescent SMCs were stimulated with murine TNF (Biosource) or PDGF (Upstate Biotechnology) at 37 °C for the indicated time, washed with ice-cold PBS, and lysed in 200 µl of 1 x SDS-PAGE loading buffer. Extracts were processed for immunoblotting as described [12]. Antibodies against the phosphorylated (activated) forms of ERK1/2, p38, p70 S6 kinase, Akt1, Stat3, and IB were from Cell Signaling. Anti-phospho-JNK polyclonal antibody was from Promega (Madison, WI), and anti-phospho-JAK2 antibody was from Transduction Labs. To quantitate immunoblots by densitometry, we normalized each band intensity to the cognate actin band intensity obtained after stripping and re-probing blots [12].

2.7. SMC [³H]thymidine incorporation
Arterial or venous SMCs plated in 96-well plates (10⁴ cells/well) were rendered quiescent, challenged with agonist(s) in SFM for a total of 24 h, and assayed for [³H]thymidine incorporation as described [14]. Inhibitors were added to SMCs 30 min before agonists: 10 µmol/l U0126 (for MEK1); 10 µmol/l SB202190 (for p38^mapk, Calbiochem); 20 µmol/l LY294002 (for phosphoinositide 3-kinase). Specificity of the inhibitors at these concentrations was demonstrated by their lack of effect on PDGF-induced Akt activation (U0126, SB202190) or PDGF-induced ERK activation (LY294002) (data not shown).

2.8. NFB nuclear translocation
SMCs were seeded in eight-well glass slides (Nunc Lab Tek II; 1.25 x 10⁴ cells/well), rendered quiescent, and then challenged with 0.25 ml SFM ± TNF or PDGF for the indicated times. SMCs were processed for indirect immunofluorescence as described [14], with IgG against the p65 subunit of NFB (Upstate Biotechnology). Control incubations that omitted the primary antibody yielded no signal (data not shown).

2.9. Recombinant adenovirus
The cDNA encoding dominant-negative (DN, S32A/S36A) IB was excised from pUSE/amp/DN-IB (Upstate Biotechnology) with EcoRI and inserted into pSKAC [16]. A PmeI–XbaI cassette from this construct [14] was excised and ligated to the 3' terminal 90.7 map units of the adenovirus "empty vector," as described [16]. This second-generation adenoviral vector lacks the E1, E3, and E2b genes [17]. With Lipofectamine® (Life Technologies), the ligation product was transfected into 293 C7 cells, which allow replication and packaging of this virus [17]. Resulting adenoviruses were clonally isolated by two rounds of plaque purification. We infected 293 C7 cells with a single clone, and isolated virus as described [16]. We generated a control virus expressing no transgene by using the PmeI–XbaI fragment of pSKAC (without any cDNA insert).

2.10. Data analysis
Data from SMCs treated with separate agonists, inhibitors, or adenoviruses were analyzed by repeated-measures one-way ANOVA with Tukey's post hoc test for multiple comparisons (Prism^TM, GraphPad). Values are presented as mean ± S.E. (figures), or ± S.D. (text).

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. TNF expression in vein grafts and cultured SMCs
In rat epigastric vein grafts to the femoral artery, TNF expression rose within postoperative week 1, plateaued by week 2, and persisted to at least week 4 [8]. We confirmed these results in rabbit autologous jugular vein grafts to the common carotid artery, and visualized the vein media by staining for -SMC actin (Fig. 1A–C). Ungrafted jugular veins expressed no detectable TNF (Fig. 1D). However, vein grafts demonstrated prominent TNF expression as soon as 4 days postoperatively–not only throughout the inflammatory cells of the adventitia, but also in the SMCs of the tunica media (Fig. 1B,E). In the SMCs of the neointima and media, TNF expression remained high for at least 28 days (Fig. 1C,F). Thus, TNF expression in this rabbit vein graft model commenced before vein graft SMC proliferation peaked [5], and persisted beyond the cessation of net SMC proliferation [12]–just as occurred in rat vein grafts [8]. Since TNF does not appear to inhibit neointimal hyperplasia [6], these findings raise the possibility that TNF promotes neointimal hyperplasia–but only in concert with other growth factors like PDGF, whose local concentration wanes over 28 days in rat [8] as well as rabbit vein grafts [5].

View larger version (46K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Both SMCs and inflammatory cells express TNF in rabbit vein grafts. Ungrafted rabbit jugular veins (A, D) and autologous jugular vein grafts harvested at the indicated postoperative times were stained for both TNF and -SMC actin. From each specimen, a single microscopic field is shown, imaged with two different fluorescence filter sets to identify either TNF (green, D, E, F), or -SMC actin (red, A, B, C) (original magnification x 220). Sections stained with only one of the fluorescent reagents demonstrated no "bleed-through" fluorescence in either the green or red fluorescence windows (data not shown). The luminal surface is upward in the depicted specimens, representative of results from three independent grafts.

 
To ascertain more directly that rabbit vascular SMCs can produce and secrete TNF, we first immunostained cultured SMCs for TNF. As seen in Fig. 2A, cultured SMCs do indeed express TNF on the cell surface. To determine whether this cell surface TNF could be secreted by SMCs as biologically active TNF, we used a TNF bioassay [15] to test conditioned medium from the SMCs. Quiescent SMCs secreted no detectable TNF (not shown). However, SMCs treated with bacterial lipopolysaccharide did secrete biologically active TNF–at levels 1000-fold lower than that evoked from RAW macrophages (Fig. 2B). To identify TNF as the mediator of cell toxicity in this bioassay, we used a soluble TNF receptor-1 fusion protein and eliminated the cell toxicity of conditioned media (Fig. 2) [15]. Thus, both in vein grafts and in vitro, vascular SMCs express TNF that may signal to adjacent cells (with cell surface TNF) [6] or, more generally, to neighboring cells (with secreted TNF)–like endothelial cells, macrophages, and other SMCs. To understand the contribution of TNF to vein graft neointimal hyperplasia, we sought to determine the SMC signal transduction pathways and activities engaged by TNF, and how TNF-evoked SMC signaling both compared with and was influenced by that of other growth factors.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Cultured SMCs produce and secrete TNF. (A) SMCs fixed in formalin were stained with the indicated primary antibody (± IgG-blocking peptide) and Hoechst 33258 (to display nuclei). Results are representative of three independent experiments. (B) TNF bioassay (means ± S.E. of three experiments performed in triplicate). L929 cells in 96-well plates were incubated (16 h, 37 °C) with the indicated concentration of conditioned media from lipopolysaccharide-provoked SMCs (upper) or lipopolysaccharide-provoked RAW macrophages (lower), along with 50 µl of conditioned medium from SMCs infected with adenoviruses encoding either no protein (closed circles, "None") or a TNF-neutralizing, secreted p55 TNF receptor-1 (open circles, "TNF-I") [15].

 
3.2. TNF promotes vascular SMC migration that is independent of ERK1/2 activation
Neointimal hyperplasia fundamentally involves migration of medial SMCs into the intima, and inhibitors of SMC migration attenuate neointimal hyperplasia in experimental animal models [18]. Among chemotactic stimuli for SMCs, PDGF-BB is believed to be most potent [9]. In a modified Boyden Chamber assay, TNF and PDGF evoked comparable SMC migration responses (Fig. 3)–an observation that highlights the potential importance of TNF-promoted SMC migration in neointimal hyperplasia. Moreover, TNF- and PDGF-evoked responses were partially additive (Fig. 3B), suggesting that TNF and PDGF receptors initiate pro-migratory signaling pathways that are only partially overlapping. Inhibition of phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) [19] with LY294002 reduced SMC migration elicited by both TNF and PDGF, by 40–50% (Fig. 3C). The same LY294002 concentration eliminated PDGF- and TNF-promoted [³H]thymidine incorporation and Akt activation, and had no effect on PDGF-promoted ERK1/2 activation (Fig. 3D and data not shown). Thus, both TNF- and PDGF-promoted migration appeared to involve PI3K and/or mTOR pathways to a comparable degree. By contrast, SMC chemotaxis induced by PDGF, but not TNF, was attenuated by inhibiting the ERK-activating kinase MEK1 with U0126 (at a concentration that abolished ERK activation by either PDGF or TNF, but not PI3K or p38 activation (Fig. 3C,D and data not shown)). Thus PDGF, but not TNF, engendered SMC migration through an ERK-dependent signaling pathway.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 TNF stimulates migration of SMCs in an ERK-independent manner. SMC migration was assayed (18 h, 37 °C) toward SFM without ("–," control) or with 10 ng/ml human PDGF-BB, 100 ng/ml murine TNF, or both agonists. (A) Representative fluorescence microscopy of nuclei from SMCs that migrated to the bottom surface of TranswellTM membranes. (B) The number of SMCs migrated is plotted (mean ± S.E. from 4 experiments). Compared with control: *, P 0.02; compared with PDGF or TNF alone: #, P<0.05 (paired analysis). (C) SMC migration was assayed in the absence (control) or presence of the MEK 1 inhibitor U0126 (10 µM) or the PI3K inhibitor LY294002 (20 µM) (see "Materials and methods"), and migration was normalized to that seen with control cells, and re-worked to obtain "% Inhibition": 100 x (1–(migration with inhibitor)/(control migration)). Shown are means ± S.E. from three experiments. Neither inhibitor affected migration of unstimulated SMCs. Compared with control: *P<0.05. (D) SMCs exposed to agonists and inhibitors as in panel C were lysed after 10 min (37 °C) and subjected to sequential immunoblotting (IB) for phosphorylated ("p")-ERK1/2, -Akt, and actin (not shown), which demonstrated equivalent protein loading (n=3 experiments).

 
3.3. PDGF and TNF signal via both common and divergent mechanisms in SMCs
The ERK dependence of SMC chemotaxis induced by PDGF, but not TNF, suggested that these two SMC agonists might signal differently through ERK1/2. While TNF and PDGF each activated ERK1/2 in SMCs (Fig. 4A), TNF-induced activation was only 50 ± 20% of that induced by PDGF (P<0.05). Thus, the relative weakness of TNF-promoted ERK activation may explain why inhibition of ERK activation failed to reduce TNF-induced SMC migration. Alternatively, the relative ERK-independence of TNF-promoted SMC migration could arise from the way in which ERK signaling interacts with other signal transduction pathways elicited by TNF, as contrasted with PDGF.

View larger version (53K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 PDGF and TNF engage similar and distinct signaling pathways in SMCs, in a non-additive manner. Serum-starved SMCs were treated with SFM lacking ("none") or containing either 100 ng/ml murine TNF, 10 ng/ml human PDGF-BB, or both ("T+P") for 3 min (A, left), 10 min (A, right), or the indicated times (B), and then lysed. Cell extracts were immunoblotted for phosphorylated ("p") forms of the indicated proteins. To ascertain equivalent protein loading in each lane, membranes were stripped and re-probed for actin. Immunoblots are from individual experiments, representative of three performed at all time points.

 
To examine this possibility, we tested activation of pathways engaged by TNF receptors in other cell types [20]. PDGF activated both p38^mapk (4-fold/basal) and c-jun N terminal kinase (JNK) (3-fold/basal) to a degree comparable with TNF (3- and 2-fold/basal, respectively). However, despite the ability of TNF to activate the JAK/Stat pathway in other cell types [21], only PDGF activated JAK-2 and Stat-3 in SMCs (Fig. 4A). While PDGF activated Akt/PKB and p70 S6 kinase in SMCs, TNF did so to levels that were only 35 ± 16% and 38 ± 24%, respectively, of that achieved by PDGF stimulation (Fig. 4B, P<0.05)–suggesting that the rabbit SMC PDGF β receptor [14] activated PI3K more efficaciously than the rabbit SMC TNF receptors (Fig. 4B). Nonetheless, inhibition of this TNF-induced PI3K activation diminished SMC migration evoked by TNF (Fig. 3). Lastly, TNF- and PDGF-evoked signaling through these pathways was not additive (Fig. 4A and data not shown). Taken together, these data demonstrate considerable diversity in the signaling mechanisms emanating from receptors for PDGF and TNF in vascular SMCs.

3.4. TNF promotes SMC [³H]thymidine incorporation synergistically with PDGF, via NFB
DNA synthesis is an integral component of neointimal hyperplasia, and we have shown it can be estimated by [³H]thymidine incorporation in our rabbit SMCs [14]. Whether TNF promotes SMC proliferation or not remains controversial [22,23]. Moreover, the relationship between TNF-elicited [³H]thymidine incorporation and that elicited by other growth factors implicated in neointimal hyperplasia remains obscure [22]. Accordingly, we assessed rabbit vascular SMC [³H]thymidine incorporation stimulated by TNF, PDGF, and the combination of these SMC growth factors. While TNF alone induced essentially no SMC [³H]thymidine incorporation, PDGF induced robust [³H]thymidine incorporation (13-fold/basal, Fig. 5), as expected [14]. The addition of TNF to PDGF dramatically increased [³H]thymidine incorporation, to 29-fold/basal (Fig. 5). Thus, TNF-induced SMC mitogenesis was revealed only by co-stimulation with PDGF. In this way, TNF mimics the behavior of G_q-coupled heptahelical receptor agonists implicated in neointimal hyperplasia [12,14].

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 TNF promotes [3H]thymidine incorporation synergistically with PDGF in both aortic and venous SMCs. (A) Aortic SMCs were exposed to SFM without (basal) or with the indicated agonists (as in Fig. 4) for 24 h. [3H]Thymidine ([3H]TdR) incorporation is plotted as fold over basal (stimulated/unstimulated), mean ± S.E. of three experiments performed in quadruplicate. The basal DPM value (mean ± S.E.) was 295 ± 44/well. Compared with unstimulated SMCs: #,*P<0.05. Compared with PDGF-stimulated SMCs: *P<0.05 (pairwise analysis). (B) Venous SMCs were stimulated with the indicated concentration of TNF plus either 10 ng/ml PDGF-BB or 1 ng/ml EGF. [3H]Thymidine incorporation is plotted (mean ± S.E. of two experiments performed in quadruplicate) as in (A). The basal DPM value was 165 ± 8/well.

 
Because venous and arterial SMCs may demonstrate different responses to growth factor stimulation [24], and because we wished to model vein graft neointimal hyperplasia, we also tested TNF-promoted [³H]thymidine incorporation in SMCs derived from the rabbit inferior vena cava. These SMCs were indistinguishable from aortic SMCs with regard to both TNF responsiveness and the synergy between TNF and PDGF in stimulating [³H]thymidine incorporation (Fig. 5B).

Is the [³H]thymidine incorporation synergy between TNF receptors and receptor tyrosine kinases unique to PDGF receptors? To address this question, we stimulated receptors for TNF and EGF, which can engage signaling pathways distinct from PDGF [25]. Similar to PDGF, though, EGF demonstrated considerable synergy with TNF in evoking SMC [³H]thymidine incorporation (Fig. 5B). In aggregate, these results suggest that SMC TNF receptors can substantially amplify the SMC activation achieved by PDGF, EGF, and perhaps other growth factor receptors–and thereby augment SMC migration and DNA synthesis that contribute to neointimal hyperplasia.

To understand the synergy between TNF- and PDGF-evoked [³H]thymidine incorporation in SMCs, we sought to identify signaling pathways that would differentiate TNF- from PDGF-promoted SMC [³H]thymidine incorporation. We found that all SMC [³H]thymidine incorporation-elicited by either PDGF or TNF plus PDGF-was abolished when we prevented the activation of ERK1/2 (with U0126), inhibited p38^mapk (with SB202190), or inhibited PI3K and/or mTOR [19] (with LY294002, data not shown). These results implicate ERKs, p38^mapk, and PI3K (and/or mTOR) in PDGF-promoted [³H]thymidine incorporation, but do not allow clear inferences about the role of these kinases in TNF-induced [³H]thymidine incorporation.

To dissect out TNF- from PDGF-promoted pro-mitogenic signaling, we exploited the essential role of NFB in the responses of cells to TNF [26]. In comparing TNF- with PDGF-induced NFB activation in SMCs, we found that PDGF stimulated nuclear translocation of NFB only weakly, and did not stimulate phosphorylation of IB- detectably (Fig. 6A). In contrast, TNF rapidly and robustly engendered nuclear translocation of NFB that persisted for over 1 h, and provoked easily detectable IB- phosphorylation (Fig. 6A). Thus, NFB activation differentiated TNF- from PDGF-promoted SMC signaling. To investigate whether NFB activation by TNF could mediate TNF's [³H]thymidine incorporation synergy with PDGF, we inhibited SMC NFB activation by expressing a dominant negative mutant of IB (DN-IB). While expression of DN-IB had no significant effect on SMC [³H]thymidine incorporation induced by either PDGF or EGF, it abolished TNF-promoted synergy with these growth factors (Fig. 6B). Although TNF can promote apoptosis when NFB activation is inhibited [20], we saw no evidence of apoptosis in our DN-IB-expressing SMCs. After 24 h of TNF exposure, crystal violet staining of DN-IB-expressing SMCs was indistinguishable from control virus-infected cells (data not shown). Moreover, basal and TNF-induced [³H]thymidine incorporation were equivalent in DN-IB-expressing and control SMCs. Specificity of the DN-IB effect in our SMCs was also manifest by the failure of DN-IB to affect the synergy between EGF and PDGF in promoting SMC [³H]thymidine incorporation (Fig. 6B). Thus, activation of NFB appears necessary for TNF-promoted SMC [³H]thymidine incorporation, just as it appears necessary for TNF-induced SMC migration [27].

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 TNF-promoted SMC [3H]thymidine incorporation requires NFB activity. (A) Agonist-induced NFB nuclear translocation is depicted in SMCs stimulated (37 °C) with either TNF or PDGF-BB as in Fig. 5, for the times indicated. SMCs were then either lysed and immunoblotted sequentially for phosphorylated IB- and actin (top panel), or fixed, permeabilized, and immunostained for the NFB p65 subunit. Immunoblots and fluorescence photomicrographs (original magnification x 400) are from a single experiment, representative of three performed. (B) SMCs infected with adenoviruses encoding either a dominant negative IB (DN IB) or no protein (Control, "ctl") were stimulated with TNF, PDGF-BB, and/or EGF as in Fig. 5, and assayed for [3H]thymidine incorporation. Parallel aliquots of SMCs were immunoblotted (10 µg protein) for IB (inset). Displayed are the absolute SMC [3H]thymidine incorporation values (mean ± S.E.) from a single experiment, representative of two performed in quadruplicate.

 

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
This study provides the first evidence that TNF and PDGF evoke both common and complementary signaling mechanisms that together can contribute to SMC migration and mitogenesis, and thereby to neointimal hyperplasia. Furthermore, this study also illuminates potential reasons for which TNF expressed by SMCs in mature vein grafts fails to promote further neointimal hyperplasia: the ability of TNF to promote SMC DNA synthesis requires co-stimulation by other growth factors, like PDGF, whose levels in the vein graft fall dramatically when vein graft neointimal hyperplasia reaches steady state [8,12].

Signaling pathways activated in our primary SMCs by either PDGF or TNF included those involving ERK, p38, and PI3K. However, TNF and PDGF were not equally efficacious in activating these signaling pathways. Indeed, PDGF activated ERK and PI3K pathways to levels two to three times higher than those achieved by TNF alone. Perhaps as a result, ERK activation proved necessary for full SMC migration promoted by PDGF, but not by TNF (Fig. 3). Similarly, since SMC NFB was activated by TNF to an extent far greater than that achieved with PDGF, NFB activation proved necessary (but not sufficient) for SMC DNA synthesis induced by TNF, but not by PDGF (Fig. 6). The complementary effects we observed in primary SMCs stimulated with PDGF and TNF may also be explained by relatively higher levels of ERK, PI3K, and JAK/Stat activation by PDGF, and relatively higher levels of NFB activation by TNF. Specifically, TNF and PDGF promoted SMC migration additively (Fig. 3 and Ref. [27]), and SMC [³H]thymidine incorporation synergistically (Fig. 6). We have summarized these observations and inferences in the Table 1.

View this table: [in this window] [in a new window]   Table 1 Summary of SMC signaling triggered by TNF and PDGF

 
Complementarity of TNF and PDGF has also been demonstrated with regard to inducing SMC production of matrix metalloproteinase-9, a process that also involved agonist-specific signaling through members of the mitogen-activated protein kinase (MAPK) family [28]. Like SMC migration and proliferation, SMC matrix metalloproteinase production is also considered integral to neointimal hyperplasia. Thus, complementarity of PDGF and TNF in promoting all three of these SMC functions suggests a generalizable process with substantial implications for the pathogenesis of neointimal hyperplasia. Small discrepancies in the relative apparent activation of MAPKs by TNF and PDGF among these studies may result from differences in SMC species [13,28,29], passage number [28], and absence [29], presence, or duration [28] of SMC serum starvation.

While TNF by itself appears to stimulate SMC migration, a co-stimulatory role for PDGF in this process cannot be entirely excluded. In our experiments, trypsinization of quiescent SMCs was quenched with medium containing 10% fetal bovine serum (FBS) before medium was changed to SFM and SMCs were plated onto polycarbonate membranes [12]. Substitution of soybean trypsin inhibitor for (PDGF-containing) FBS in this protocol essentially eliminated TNF-induced SMC migration (data not shown). Other groups studying TNF-induced vascular SMC migration have employed methods that almost certainly introduce PDGF into the assay, either by physically damaging SMCs [30] or by adding FBS to the medium [27,31]. (Indeed, the inclusion of (PDGF-containing) FBS during TNF stimulation may explain why Goetze et al. found that SMC migration attributed to TNF was sensitive to inhibition of MEK1.) The only study to conclude that TNF fails to induce SMC migration was performed with baboon aortic explants [22]. In this model, endogenous (explant) PDGF activity [22] may be below the "permissive threshold" necessary for TNF to induce SMC migration.

Although NFB activation has long been known to protect against TNF-induced apoptosis in a variety of cell lines [26], our study is the first to demonstrate that NFB specifically mediates TNF-induced augmentation of SMC [³H]thymidine incorporation. Previously, liposome-delivered IB was shown to abrogate TNF-promoted SMC tetrazolium reduction (an index for proliferation) [23]. In these studies, however, the signaling pathway specificity of IB remained uncertain, because no proliferative signal was shown to resist the effect of IB. By contrast, DN-IB did not inhibit SMC [³H]thymidine incorporation promoted by either PDGF or EGF in our experiments, and thereby demonstrated signaling pathway specificity of its action.

While our work has demonstrated PDGF-BB to be a critical co-stimulant in SMCs for TNF, additional important co-stimulatory factors undoubtedly exist (like EGF in Figs. 5 and 6). Despite its apparent dependence on these co-stimulatory factors to promote SMC mitogenesis (and perhaps even migration), TNF itself contributes significantly to neointimal hyperplasia in at least two arterial models employing TNF-transgenic and -knockout mice [6,7]. Whether these observations will prove generalizable to vein grafting remains to be determined.

	Acknowledgements

 
This work was supported by NIH grants HL 63288 (NJF) and HL 64744 (KP), and American Heart Association Grants-in-Aid (KP, NJF). ESO was supported by a Eugene Stead Medical Student Scholarship.

	Notes

 
Time for primary review 20 days

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

1. Motwani J.G., Topol E.J. Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention. Circulation (1998) 97:916–931.[Abstract/Free Full Text]
2. Mann M.J., Gibbons G.H., Kernoff R.S., Diet F.P., Tsao P.S., Cooke J.P., et al. Genetic engineering of vein grafts resistant to atherosclerosis. Proc. Natl. Acad. Sci. U. S. A. (1995) 92:4502–4506.[Abstract/Free Full Text]
3. Epstein S., Speir E., Unger E., Guzman R., Finkel T. The basis of molecular strategies for treating coronary restenosis after angioplasty. J. Am. Coll. Cardiol. (1994) 23:1278–1288.[Abstract]
4. Zhang L., Freedman N.J., Brian L., Peppel K. Graft-extrinsic cells predominate in vein graft arterialization. Arterioscler. Thromb. Vasc. Biol. (2004) 24:470–476.[Abstract/Free Full Text]
5. Davies M.G., Hagen P.O. Pathobiology of intimal hyperplasia. Br. J. Surg. (1994) 81:1254–1269.[ISI][Medline]
6. Rectenwald J.E., Moldawer L.L., Huber T.S., Seeger J.M., Ozaki C.K. Direct evidence for cytokine involvement in neointimal hyperplasia. Circulation (2000) 102:1697–1702.[Abstract/Free Full Text]
7. Zimmerman M.A., Selzman C.H., Reznikov L.L., Miller S.A., Raeburn C.D., Emmick J., et al. Lack of TNF-alpha attenuates intimal hyperplasia after mouse carotid artery injury. Am. J. Physiol. Regul. Integr. Comp. Physiol. (2002) 283:R505–R512.[Abstract/Free Full Text]
8. Faries P.L., Marin M.L., Veith F.J., Ramirez J.A., Suggs W.D., Parsons R.E., et al. Immunolocalization and temporal distribution of cytokine expression during the development of vein graft intimal hyperplasia in an experimental model. J. Vasc. Surg. (1996) 24:463–471.[CrossRef][ISI][Medline]
9. Heldin C.H., Ostman A., Ronnstrand L. Signal transduction via platelet-derived growth factor receptors. Biochim. Biophys. Acta (1998) 1378:F79–F113.[Medline]
10. Hart C.E., Kraiss L.W., Vergel S., Gilbertson D., Kenagy R., Kirkman T., et al. PDGFbeta receptor blockade inhibits intimal hyperplasia in the baboon. Circulation (1999) 99:564–569.[Abstract/Free Full Text]
11. Sano H., Sudo T., Yokode M., Murayama T., Kataoka H., Takakura N., et al. Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein E-deficient mice. Circulation (2001) 103:2955–2960.[Abstract/Free Full Text]
12. Peppel K., Zhang L., Huynh T.T., Huang X., Jacobson A., Brian L., et al. Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia. J. Mol. Cell. Cardiol. (2002) 34:1399–1409.[CrossRef][ISI][Medline]
13. Goetze S., Kintscher U., Kawano H., Kawano Y., Wakino S., Fleck E., et al. Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells. J. Biol. Chem. (2000) 275:18279–18283.[Abstract/Free Full Text]
14. Peppel K., Jacobson A., Huang X., Murray J.P., Oppermann M., Freedman N.J. Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells attenuates mitogenic signaling via G protein-coupled and platelet-derived growth factor receptors. Circulation (2000) 102:793–799.[Abstract/Free Full Text]
15. Peppel K., Crawford D., Beutler B. A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity. J. Exp. Med. (1991) 174:1483–1489.[Abstract/Free Full Text]
16. Drazner M.H., Peppel K.C., Dyer S., Grant A.O., Koch W.J., Lefkowitz R.J. Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes. J. Clin. Invest. (1997) 99:288–296.[ISI][Medline]
17. Amalfitano A., Hauser M.A., Hu H., Serra D., Begy C.R., Chamberlain J.S. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted. J. Virol. (1998) 72:926–933.[Abstract/Free Full Text]
18. Axel D.I., Kunert W., Goggelmann C., Oberhoff M., Herdeg C., Kuttner A., et al. Paclitaxel inhibits arterial smooth muscle cell proliferation and migration in vitro and in vivo using local drug delivery. Circulation (1997) 96:636–645.[Abstract/Free Full Text]
19. Brunn G.J., Williams J., Sabers C., Wiederrecht G., Lawrence J.C. Jr., Abraham R.T. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. (1996) 15:5256–5267.[ISI][Medline]
20. Baker S.J., Reddy E.P. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene (1996) 12:1–9.[ISI][Medline]
21. Guo D., Dunbar J.D., Yang C.H., Pfeffer L.M., Donner D.B. Induction of Jak/STAT signaling by activation of the type 1 TNF receptor. J. Immunol. (1998) 160:2742–2750.[Abstract/Free Full Text]
22. Kenagy R.D., Clowes A.W. Blockade of smooth muscle cell migration and proliferation in baboon aortic explants by interleukin-1beta and tumor necrosis factor-alpha is nitric oxide-dependent and nitric oxide-independent. J. Vasc. Res. (2000) 37:381–389.[CrossRef][ISI][Medline]
23. Selzman C.H., Shames B.D., Reznikov L.L., Miller S.A., Meng X., Barton H.A., et al. Liposomal delivery of purified inhibitory-kappaBalpha inhibits tumor necrosis factor-alpha-induced human vascular smooth muscle proliferation. Circ. Res. (1999) 84:867–875.[Abstract/Free Full Text]
24. Yang Z., Oemar B.S., Carrel T., Kipfer B., Julmy F., Luscher T.F. Different proliferative properties of smooth muscle cells of human arterial and venous bypass vessels: role of PDGF receptors, mitogen-activated protein kinase, and cyclin-dependent kinase inhibitors. Circulation (1998) 97:181–187.[Abstract/Free Full Text]
25. Rani C.S., Wang F., Fuior E., Berger A., Wu J., Sturgill T.W., et al. Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling. J. Biol. Chem. (1997) 272:10777–10783.[Abstract/Free Full Text]
26. Natoli G., Costanzo A., Guido F., Moretti F., Levrero M. Apoptotic, non-apoptotic, and anti-apoptotic pathways of tumor necrosis factor signalling. Biochem. Pharmacol. (1998) 56:915–920.[CrossRef][ISI][Medline]
27. Wang Z., Castresana M.R., Newman W.H. NF-kappaB is required for TNF-alpha-directed smooth muscle cell migration. FEBS Lett. (2001) 508:360–364.[CrossRef][ISI][Medline]
28. Cho A., Graves J., Reidy M.A. Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. (2000) 20:2527–2532.[Abstract/Free Full Text]
29. Li M., Mossman B.T., Kolpa E., Timblin C.R., Shukla A., Taatjes D.J., et al. Age-related differences in MAP kinase activity in VSMC in response to glucose or TNF-alpha. J. Cell. Physiol. (2003) 197:418–425.[CrossRef][ISI][Medline]
30. Jovinge S., Hultgardh-Nilsson A., Regnstrom J., Nilsson J. Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler. Thromb. Vasc. Biol. (1997) 17:490–497.[Abstract/Free Full Text]
31. Goetze S., Xi X.P., Kawano Y., Kawano H., Fleck E., Hsueh W.A., et al. TNF-alpha-induced migration of vascular smooth muscle cells is MAPK dependent. Hypertension (1999) 33:183–189.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				J. Kim, L. Zhang, K. Peppel, J.-H. Wu, D. A. Zidar, L. Brian, S. M. DeWire, S. T. Exum, R. J. Lefkowitz, and N. J. Freedman {beta}-Arrestins Regulate Atherosclerosis and Neointimal Hyperplasia by Controlling Smooth Muscle Cell Proliferation and Migration Circ. Res., July 3, 2008; 103(1): 70 - 79. [Abstract] [Full Text] [PDF]

				L. Zhang, P. Sivashanmugam, J.-H. Wu, L. Brian, S. T. Exum, N. J. Freedman, and K. Peppel Tumor Necrosis Factor Receptor-2 Signaling Attenuates Vein Graft Neointima Formation by Promoting Endothelial Recovery Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 284 - 289. [Abstract] [Full Text] [PDF]

				K. Bostrom Osteopontin, a missing link in PDGF-induced smooth muscle cell migration Cardiovasc Res, September 1, 2007; 75(4): 634 - 635. [Full Text] [PDF]

				S. Jalvy, M.-A. Renault, L. L. S. Leen, I. Belloc, J. Bonnet, A.-P. Gadeau, and C. Desgranges Autocrine expression of osteopontin contributes to PDGF-mediated arterial smooth muscle cell migration Cardiovasc Res, September 1, 2007; 75(4): 738 - 747. [Abstract] [Full Text] [PDF]

				S. Koide, M. Okazaki, M. Tamura, K. Ozumi, H. Takatsu, F. Kamezaki, A. Tanimoto, H. Tasaki, Y. Sasaguri, Y. Nakashima, et al. PTEN reduces cuff-induced neointima formation and proinflammatory cytokines Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2824 - H2831. [Abstract] [Full Text] [PDF]

				L. Zhang, K. Peppel, P. Sivashanmugam, E. S. Orman, L. Brian, S. T. Exum, and N. J. Freedman Expression of Tumor Necrosis Factor Receptor-1 in Arterial Wall Cells Promotes Atherosclerosis Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 1087 - 1094. [Abstract] [Full Text] [PDF]

				J.-H. Wu, R. Goswami, X. Cai, S. T. Exum, X. Huang, L. Zhang, L. Brian, R. T. Premont, K. Peppel, and N. J. Freedman Regulation of the Platelet-derived Growth Factor Receptor-beta by G Protein-coupled Receptor Kinase-5 in Vascular Smooth Muscle Cells Involves the Phosphatase Shp2 J. Biol. Chem., December 8, 2006; 281(49): 37758 - 37772. [Abstract] [Full Text] [PDF]

				C. M. Mallawaarachchi, P. L. Weissberg, and R. C. M. Siow Antagonism of platelet-derived growth factor by perivascular gene transfer attenuates adventitial cell migration after vascular injury: new tricks for old dogs? FASEB J, August 1, 2006; 20(10): 1686 - 1688. [Abstract] [Full Text] [PDF]

				T. Khomenko, S. Szabo, X. Deng, M. R. Jadus, H. Ishikawa, K. Osapay, Z. Sandor, and L. Chen Suppression of early growth response factor-1 with egr-1 antisense oligodeoxynucleotide aggravates experimental duodenal ulcers Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1211 - G1218. [Abstract] [Full Text] [PDF]

				J.E. Ferguson III, R. W. Kelley, and C. Patterson Mechanisms of Endothelial Differentiation in Embryonic Vasculogenesis Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2246 - 2254. [Abstract] [Full Text] [PDF]

				S. Grundmann, I. Hoefer, S. Ulusans, N. van Royen, S. H. Schirmer, C. K. Ozaki, C. Bode, J. J. Piek, and I. Buschmann Anti-tumor necrosis factor-{alpha} therapies attenuate adaptive arteriogenesis in the rabbit Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1497 - H1505. [Abstract] [Full Text] [PDF]

				M. Post and J. Waltenberger Modulation of growth factor action in the cardiovascular system Cardiovasc Res, February 15, 2005; 65(3): 547 - 549. [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Peppel, K. Articles by Freedman, N. J. Search for Related Content PubMed