|Home | About | Journals | Submit | Contact Us | Français|
We have previously demonstrated that TGF-β in the presence of elevated levels of its primary signaling protein, Smad3, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. Moreover, we have shown that the mechanism in part, is through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluate the role of the ERK mitogen-activated protein kinase (ERK MAPK) in TGF-β-induced VSMC proliferation.
Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with ansiRNA. For in vivo experiments, Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK and Proliferating Cell Nuclear Antigen (PCNA).
In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time and concentration-dependent manner. Overexpression of the signaling protein, Smad3 enhanced TGF-β-induced activation of ERK MAPK whereas inhibition of Smad3 with ansiRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by PCNA.
Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that through a Smad3/ERK MAPK signaling pathway, TGF-β enhances VSMC proliferation. These findings explain at least in part, the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.
According to the Centers for Disease Control and Prevention, atherosclerotic-associated diseases are the leading cause of death in the United States. However, interventional treatments of atherosclerosis are often complicated by restenosis or the development of intimal hyperplasia. Rates of restenosis range from 20 to 80% following coronary or peripheral angioplasty.1 Restenosis is the sequela of a complex process dominated by the development of plaque in the sub-intimal space. Intimal plaque is highly cellular and is in part the consequence of migration and proliferation of vascular smooth muscle cells (VSMC) from the arterial media, the adventitia as well as potentially from the bone marrow.2–4
The role of TGF-β in the pathogenesis of restenosis has been well established.5–8 The prevailing theory has been that TGF-β, which is known for its fibrotic effects, enhances intimal hyperplasia through the production of extracellular matrix proteins including the various collagens.6,8 Alternatively, our own laboratory has recently demonstrated that TGF-β, in the presence of its primary signaling protein, Smad3, enhances VSMC proliferation, and thus in this manner may contribute to the cellularity of intimal plaque.8 This was a surprising finding in that we and others have shown in vitro that TGF-β inhibits proliferation of VSMCs as well as other cell types.23 From our studies, it appears that persistently high levels of Smad3, which are coincidentally present at the time of arterial injury, are required for TGF-β to produce its proliferative effect.8
TGF-β signals primarily through the Smad pathway although a number of non-Smad signaling pathways have also been described.9,10 TGF-β transduces its signal by binding with type I and type II receptors leading to Smad2/3 phosphorylation. The phosphorylated Smad2/3 proteins interact with Smad4, and the entire complex then translocates and accumulates in the nucleus where it is regulates transcription of various target genes.11,12 In addition to the Smad pathway, TGF-β signals through various Smad-independent signaling pathways such as protein kinase C (PKC), protein kinase A (PKA) and PI3K/Akt.13 We have previously demonstrated that TGF-β’s growth inhibitory effects are mediated through a non-Smad pathway involving the regulation of Cyclin A.
The mitogen-activated protein kinases (MAPKs) are a family of intracellular signaling proteins that regulate a variety of cellular activities. Extracellular signal-related kinases 1 and 2 (ERK 1/2) are a subfamily of MAPKs that have been shown to regulate cellular processes such as transcription and proliferation.14 Following binding and activation of a variety of growth factors, including PDGF, a cascade of events leads to dual phosphorylation of the two isoforms of ERK MAPK. Once phosphorylated, ERK MAPK becomes an active kinase which translocates to the nucleus where it has been shown to regulate transcription, specifically of genes such as cyclin D1 leading to cell-cycle progression through cytoplasmic sequestration of p27.15
In VSMCs, ERK MAPK has been demonstrated to increase proliferation, and has been implicated in the development of restenosis.16 Therefore we chose to explore whether TGF-β/Smad3 might produce its proliferative effect through the induction of the ERK MAPK pathway. We report that TGF-β through Smad3 is a potent activator of ERK MAPK both in vitro and in vivo. We found that activation of ERK MAPK by TGF-β is a process that is greatly enhanced by overexpression of Smad3 and inhibited by an siRNA to Smad3. Moreover, blockade of ERK MAPK decreases TGF-β/Smad3-induced VSMC proliferation. Finally, we demonstrate that overexpression of Smad3 in vivo enhances expression of activated ERK MAPK which is associated with VSMC proliferation. These data suggest a novel mechanism by which TGF-β through Smad3 and ERK MAPK regulates VSMC proliferation and the formation of intimal hyperplasia.
The chemical inhibitor for ERK 1/2 MAPK (PD98059) was obtained from Calbiochem (San Diego, CA). Recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Dulbecco’s modified Eagle’s medium (DMEM) and cell culture reagents were from Invitrogen (Carlsbad, CA). Other reagents, if not specified, were purchased from Sigma (St. Louis, MO).
Adenoviral vectors expressing Smad3 (AdSmad3) and Green Fluorescent Protein (AdGFP) were constructed as previously described.17 Adenoviral vector expressing GFP was used as a control (AdGFP).
Rat aortic vascular smooth muscle cells (VSMC) were isolated from the thoracoabdominal aorta of male Sprague-Dawley rats based on a protocol described by Clowes et al. and maintained in DMEM containing 10% FBS at 37 °C with 5% CO2.18 Rat VSMCs were infected with adenovirus (3 × 104 particles/cell) in DMEM containing 2% FBS for 4 h at 37 °C followed by starvation in DMEM containing 0.5% FBS for 24 h. Efficiency of viral infection was evaluated using green fluorescent protein (GFP) on both control virus (AdGFP) and adenovirus expressing Smad3 (AdSmad3). In previous experiments, more than 80% of cells were infected and became GFP-positive (data not shown). The cells were then treated with recombinant TGF-β1 (5 ng/ml) or solvent (4mM HCl with 2% bovine serum albumin) for 1 h.
Rat VSMCs were plated at 50–60% confluence in DMEM containing 10% FBS in 6-well plates and incubated for 24 h. Cells were then transfected in Opti-MEM I medium with 100 pmol of siRNA for Smad3 or control siRNA using RNAiMax transfection reagent as described by the manufacturer’s protocol (Invitrogen, Carlsbad, CA). After 6 h, the Opti-MEM I medium was replaced with DMEM containing 10% FBS for 12 h.
Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 10 µg/ml aprotinin). Protein expression was confirmed by immunoblotting with the following antibodies: rabbit anti-phosphorylated ERK1/2 (p-ERK) or total ERK (t-ERK)(Cell Signaling, Boston, MA), rabbit anti-Smad3 (Invitrogen, Carlsbad, CA), and mouse anti-β-actin (Sigma, St. Louis, MO). After incubation with the appropriate primary and horseradish peroxidase-conjugated secondary antibodies, the membranes were developed with enhanced chemiluminescence reagent.
Immunoprecipitation was carried out as previously described.19 Briefly, cells were lysed in Nonidet P-40 buffer and incubated with 5 µg of anti-Smad3 or isotype matched IgG as control and incubated at 4°C with constant rotation overnight. Protein A-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added and incubated for an additional 4h at 4°C. Precipitated proteins were then separated by SDS-PAGE, then transferred to nitrocellulose membranes and immunoblotted with rabbit anti-phosphorylated-Smad3 or phosphorylated-ERK.
Cell viability was determined by modified MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.20 Cells were plated at 50–60% confluence on 24-well plates and treated as described above. Cells were then washed with PBS, and 25 µl MTT solution (0.5 mg/ml) in phenol red free culture medium (0.5% FCS) was added to each well and incubated for 2 h at 37 °C. The MTT was aspirated, and 500µl acidic isopropranol (0.04 M HCl) was added for 10 min to solubilize the intracellular formazan product. Absorbance was measured at 570 nm using absorbance at 690 nm to correct for background. MTT was measured as a fold-change from control cells.
Male Sprague-Dawley rats (300–350 g) underwent balloon injury of the left common carotid artery as described elsewhere in accordance with institutional guidelines and approval.19 Briefly, after induction of anesthesia with isoflurane, a 2-French balloon catheter was inserted through the left external carotid artery into the common carotid artery and insufflated with 2 atm of pressure three times. After injury the animals received intraluminal administration of adenovirus vectors (2.5 × 109 plaque-forming units in 200 µl of PBS over 20 min). The external carotid artery was then ligated, and flow was re-established through the common carotid and internal carotid arteries. Rats were sacrificed 3 days after injury and fixed in 4% paraformaldehyde overnight for paraffin-embedding.
Paraffin-embedded arteries were cut into 6 µm sections for analysis. Immunostaining for Smad3, phospho-ERK and Proliferating Cell Nuclear Antigen (PCNA) were performed as described previously.17 Antibody controls included detection of species-matched IgG.
Five immunostained sections from each animal were then chosen. Each section was then imaged from six different fields at 200×. Two independent investigators then manually counted the number of positive cells. These numbers were compounded to generate the mean and standard deviation for that animal. The means were then averaged and the standard error of the mean was calculated for each group.
Data are expressed as mean +/− SE. An unpaired Student’s t-test was used to evaluate the statistical differences between control and treated groups. Values of P <.05 were considered significant. All experiments were repeated at least three times.
Our previous studies have demonstrated that TGF-β/Smad3 enhances VSMC proliferation both in vitro and in vivo.8 ERK MAPK is known to stimulate proliferation in multiple cell types including VSMCs.14 Thus, we evaluated whether incubation of VSMCs with TGF-β would enhance phosphorylation of ERK MAPK (p-ERK) as measured by Western blotting. We first evaluated whether TGF-β-induced activation of ERK MAPK is in a concentration-dependent manner (Figure 1A). Stimulation for 1 hour with TGF-β at concentrations as low as 1 ng/ml enhanced activation of ERK MAPK with continued activation at concentrations as high as 10 ng/ml. We then evaluated the time course of ERK MAPK activation by TGF-β (Figure 1B). Utilizing a concentration of 5 ng/ml, TGF-β enhanced ERK MAPK phosphorylation as early as 15 minutes, peaking at 60 minutes and decreasing at 2 hours. Thus our findings demonstrate that despite TGF-β’s known anti-proliferative effect, this cytokine has a strong and reproducible ability to activate the pro-proliferative signaling protein ERK MAPK.
In the next series of experiments, we evaluated whether the observed activation of ERK MAPK by TGF-β might be mediated through the signaling protein Smad3. Our previous studies demonstrating that TGF-β induces proliferation in the presence of elevated levels of Smad3 suggest a possible role for Smad3 in TGF-β’s activation of ERK MAPK. We began by overexpressing Smad3 in VSMCs. VSMCs were infected with adenovirus expressing Smad3 or GFP (control) followed by stimulation with TGF-β (5ng/ml) for 1 hour (Figure 2A). As shown in Figure 2A, overexpression of Smad3 alone had little effect on ERK MAPK phosphorylation. However, stimulation of cells overexpressing Smad3 with TGF-β dramatically enhanced TGF-β-induced activation of ERK MAPK.
To further demonstrate the importance of Smad3 as a signaling intermediate in TGF-β-induced activation of ERK MAPK, we employed an siRNA to Smad3. VSMCs were pretreated for 24 hours with scrambled or Smad3 siRNA followed by incubation for 1 hour with TGF-β (5 ng/ml). Phosphorylated-ERK MAPK (p-ERK) was measured by Western blotting (Figure 2B). Inhibition of Smad3 resulted in a marked decrease in the activation of ERK MAPK. The foregoing data suggest that TGF-β-induced activation of ERK MAPK is dependent on Smad3.
It has been previously shown that Smad3 produces its effect through gene transcription. Receptor activation by TGF-β leads to Smad2/3 phosphorylation, followed by translocation of these proteins along with Smad4 to the nucleus where this complex is directly involved in the transcriptional regulation of various target genes. Our finding that TGF-β through Smad3 produces ERK MAPK activation within 15 minutes would suggest that gene transcription is not involved. Thus, we postulated the requirement of a more direct protein-protein interaction between Smad3 and ERK MAPK. To test the presence of a physical association between these two proteins, we used the technique of immunoprecipitation. We overexpressed Smad3 in VSMCs and stimulated for 1 hour with TGF-β (5ng/ml). Cell lysate was immunoprecipitated with an antibody to Smad3 or isotype matched IgG control, and blotted for p-ERK and p-Smad3. As shown in Figure 3, Smad3 and p-ERK MAPK are associated with endogenous Smad3, and overexpression of Smad3 followed by stimulation with TGF-β further enhances the association. These data demonstrate that Smad3 and p-ERK MAPK co-associate suggesting that Smad3 activates ERK MAPK through a direct interaction.
We have previously demonstrated that TGF-β/Smad3 increases VSMC proliferation. Additionally, in the forgoing experiments we have shown that TGF-β/Smad3 activates ERK MAPK. The final link is to demonstrate that TGF-β/Smad3’s effect on VSMC proliferation is mediated by ERK MAPK. To accomplish this we employed a selective inhibitor of ERK MAPK, PD98059. VSMCs were infected with AdGFP or AdSmad3 and either pretreated or not with PD98059 (10 um/ml) for 30 minutes followed by stimulation with TGF-β (5ng/ml) for 96 hours. Proliferation was measured using an MTT assay. In Figure 4, we confirm that TGF-β/Smad3 increases VSMC proliferation compared to control. We then demonstrate that inhibition of ERK MAPK completely eliminates the enhancement in VSMC proliferation produced by TGF-β/Smad3. These and the preceding experiments provide conclusive proof that the stimulatory effect of TGF-β on VSMC proliferation is mediated through a pathway that involves both Smad3 as well as ERK MAPK.
Our in vitro findings suggest that TGF-β along with Smad3 enhances VSMC proliferation through activation of ERK MAPK. To verify these findings in vivo, we employed a rat carotid injury model of intimal hyperplasia. Rat carotid arteries were balloon injured as previously described and immediately following injury, infused with an adenovirus expressing Smad3 (AdSmad3) or GFP (AdGFP) for 30 minutes. Animals were then sacrificed at 3 days and immunohistochemistry performed using IgG as well as antibodies to Smad3, p-ERK MAPK and proliferating cell nuclear antigen (PCNA)(Figure 5). As anticipated, overexpression of Smad3 resulted in a dramatic increase in the number of Smad3 positive cells. Furthermore, in the animals overexpressing Smad3 there was an almost 50% increase in the number of cells expressing p-ERK MAPK (AdGFP 32.4±2.65 vs AdSmad3 63.5±6.5). Finally, overexpression of Smad3 with the resultant enhancement of p-ERK MAPK was associated with a substantial increase in VSMC proliferation as demonstrated by PCNA (AdGFP 43.1±2.4 vs AdSmad3 68.3±4.65). Our findings suggest that in vivo (although only through association), TGF-β/Smad3 enhances VSMC proliferation through a mechanism that involves ERK MAPK.
TGF-β plays an indisputable role in the development of intimal hyperplasia. Levels of TGF-β increase dramatically following arterial injury, and blocking TGF-β via a number of mechanisms very significantly inhibits the arterial hyperplastic response. Intimal hyperplasia is associated with enhanced VSMC proliferation and migration, posing a conundrum since TGF-β has been shown in vitro to be an inhibitor of both of these processes23. Potentially resolving this discrepancy, our recent studies suggest that under circumstances present at the time of arterial injury (specifically in the presence of elevated levels of Smad3), TGF-β significantly enhances proliferation of VSMCs. We, in addition to a number of other investigators, have demonstrated that Smad3 is upregulated following arterial injury in animals.8,41,42,43 Moreover, we have demonstrated that the majority of Smad3 expressing cells also express proliferating marker PCNA.8 These findings have been made by our laboratory in human restenotic lesions as well.44 Thus, the findings of our experiments where we have overexpressed Smad3 both in vitro and in vivo, likely have physiological relevance in that a similar state is found following arterial injury. We postulate that enhancement of VSMC proliferation by TGF-β may be the primary mechanism through which this cytokine mediates intimal hyperplasia.
The purpose of the current study is to better understand the signaling mechanism through which TGF-β produces its proliferative effect. Here within we have demonstrated a signaling mechanism through which TGF-β activates ERK MAPK through a pathway involving Smad3. We have demonstrated a protein-protein interaction between Smad3 and activated ERK MAPK. Moreover, we have shown that blocking ERK MAPK decreases TGF-β/Smad3-induced VSMC proliferation. Our in vivo data demonstrate that in injured arteries overexpressing Smad3, there is increased expression of activated ERK MAPK as well as enhanced VSMC proliferation. Together, these data suggest a pathway that involves TGF-β/Smad3/ERK MAPK may have a significant role in the development of intimal hyperplasia in response to TGF-β.
In our initial exploration of the signaling pathways through which TGF-β mediates SMC proliferation, we studied the cyclin-dependent kinase inhibitor p27.8 Nuclear p27 acts as a potent cell cycle inhibitor whereas phosphorylation of p27 results in translocation of this protein out of the nucleus allowing the cell cycle to progress. We found that TGF-β and Smad3 were able to significantly enhance VSMC proliferation by promoting nuclear exportation of p27. Phosphorylation of p27 is a very downstream event in the cellular pathways that lead to proliferation. Thus in evaluating ERK MAPK, we searched for a connection between Smad3 and p27. MAPK has been found to down-regulate p27 expression, increase the degradation of p27, and enhance cytoplasmic sequestration of p27. The effect of all of these events is to eliminate p27’s nuclear inhibition of cell cycle proteins resulting in enhancement of proliferation. Thus, the identification of a role for ERK MAPK in TGF-β induced VSMC proliferation reveals ERK MAPK to be the likely connection between TGF-β/Smad3 and p27.
The role of ERK MAPK in cellular proliferation, specifically VSMC proliferation, is well established. ERK MAPK activation occurs in response to a variety of cytokines and growth factors leading to both the passage of cell cycle checkpoints as well as the activation of transcription factors related to cell proliferation.21,22 We and others have shown that in cultured VSMCs, ERK MAPK plays a critical role in both proliferation and migration.23,24 In vivo studies have demonstrated the significance of ERK MAPK in the development of intimal hyperplasia. Hu et al. showed that in a rat carotid balloon injury model there was increased expression of activated ERK MAPK peaking at 5 minutes with elevated levels sustained for up to 7 days post injury.25 Furthermore, extraluminal application of the chemical inhibitor to ERK MAPK PD98059, which was used in our experiments, has been shown to decrease the development of intimal hyperplasia.26 Whether ERK MAPK is the sole pathway through which TGF-β enhances VSMC proliferation is not clear. Recent studies from our laboratory suggest that other members of the MAPK family, specifically p38, may act as intermediates between TGF-β/Smad3 and VSMC proliferation. These studies reveal a completely distinct pathway from ERK MAPK for proliferation involving p38 as well as Akt (unpublished data). Thus it may well be that TGF-β produces its proliferative effect through a number of parallel pathways.
Our data reveal a pathway involving ERK MAPK activation that is downstream from Smad3. There are examples in other cell types where TGF-β has been found to modulate MAPK through a Smad-dependent pathway. Simeone et al. found in pancreatic acinar cells that TGF-β activates ERK MAPK through a pathway that involves Smad4. Moreover, Smad4 has been found to directly affect MAPK in other cell types.27,28
TGF-β is also known to signal through independent pathways. Smad-independent pathways utilized by TGF-β include JNK, PKA, PKC and PI3K/Akt. These pathways are thought to modulate the effects of TGF-β that require rapid activation. This is contrary to the Smad pathways that are typically involved in transcriptional regulation of genes.29 Accordingly, a relationship between TGF-β and ERK MAPK that is independent of Smads has been previously described in a number of cell types. In VSMCs TGF-β through a Smad-independent pathway can enhance production of collagen as well as biglycans.30,31 TGF-β, also in a Smad-independent manner can activate ERK MAPK in chondrocytes, epithelial cells, and hepatic stellate cells.32,33 Although the exact molecular mechanism by which TGF-β activates ERK MAPK in these cells has not been clearly defined, the Ras/Raf signaling pathway may act as an intermediate. Despite the existence of Smad independent signaling pathways however, our data fairly conclusively show that when vascular smooth muscle cell proliferation is the endpoint, TGF-β activated ERK MAPK through a Smad-dependent versus a Smad-independent pathway.
In another variation in the relationship between these two signaling proteins, ERK MAPK has been found to act upstream rather than downstream of the Smad proteins.34,35 Studies have shown that ERK MAPK, can enhance Smad activation and translocation into the nucleus.30,36 In VSMCs, for example, ERK MAPK has been shown to phosphorylate Smad2 at its linker region.30 A similar observation has been made in rat mesangial cells leading to an increase in collagen IV expression.37 There are examples of MAPK being upstream from other members of the TGF-β superfamily including bone morphogenetic proteins (BMP) in human umbilical vein endothelial cells (HUVECs).38,39 These data, along with our new observation that Smad3 can activate MAPK, suggests the potential for a positive feedback loop between these two proteins. This positive feedback loop, if it exists, might have the potential to greatly enhance TGF-β’s effect on VSMC proliferation.
Smads have been traditionally thought to influence cell function through gene regulation. In fact, Aoki et al. has demonstrated that in pancreatic stellate cells, TGF-β through Smad3 induces secretion of IL-1β which in turn signals through ERK MAPK to further enhance cell secretion.40 However, since stimulation of VSMCs with TGF-β results in phosphorylation of ERK MAPK as early as 15 minutes and since Smad3 is the intermediate, these findings raise the possibility that Smad3 may immediately and directly interact with ERK MAPK leading to its activation. Further supporting this hypothesis is the fact that our immunoprecipitation studies reveal a direct protein-protein interaction between Smad3 and ERK MAPK.
In conclusion, we demonstrate a mechanism in VSMCs by which TGF-β activates Smad3, which in turn activates ERK MAPK leading to VSMC proliferation. Moreover, our in vitro findings are supported by in vivo studies using a rat carotid injury model. Although the mechanism by which TGF-β enhances intimal hyperplasia has not been fully elucidated, TGF-β-induced VSMC proliferation through this signaling pathway is a likely contributor. Manipulation of TGF-β or the various components of its signaling pathway may prove useful in creating targets that allow inhibition of the devastating and ubiquitous process that leads to restenosis.
Figure S1. Smad3 expression is upregulated after infection with AdSmad3, and decreased after incubation with siRNA to Smad3. A. VSMCs were infected with control virus (AdGFP) or adenovirus expressing Smad3 (AdSmad3) followed by stimulation with TGF-β (5 ng/ml) for 1 hour. B. VSMCs were transfected with 100 pmol of scramble or Smad3 siRNA for 24 hours. Protein lysates were analyzedby Western blotting using antibodies forSmad3 or β-actin. Data shown are representative of three independent experiments.
Figure S2. TGF-β induces phosphorylation of Smad3 in VSMCs. A. VSMCs were treated with TGF-β for 1 hour at the indicated concentrations. B. VSMCs overexpressing Smad3 were treated with TGF-β (5 ng/ml) at the indicated time points. Protein lysates were analyzed by Western blotting using antibodies against phospho-Smad3 (p-Smad3) or β-actin. Data shown are representative of three independent experiments.
For Presentation: Midwestern Vascular Surgery Society Annual Meeting, September 16, 2011, Chicago, IL Plenary Session