Bcr kinase activation by Angiotensin II inhibits PPARγ transcriptional activity in vascular smooth muscle cells

doi:10.1161/CIRCRESAHA.108.188409

Circ Res. Author manuscript; available in PMC 2009 July 2.

Published in final edited form as:

Circ Res. 2009 January 2; 104(1): 69–78.

Published online 2008 November 20. doi: 10.1161/CIRCRESAHA.108.188409

PMCID: PMC2642522

NIHMSID: NIHMS80222

Bcr kinase activation by Angiotensin II inhibits PPARγ transcriptional activity in vascular smooth muscle cells

Jeffrey D. Alexis,^* Nadan Wang, Wenyi Che,^∀ Nicole Lerner-Marmarosh, Abha Sahni, Vyacheslav A. Korshunov, Yiping Zou,^# Bo Ding, Chen Yan, Bradford C. Berk, and Jun-ichi Abe

Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

^* Correspondence to: Jeffrey Alexis, MD, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 211 Bailey Road, West Henrietta, NY 14586, Telephone (585) 276-9795, Fax (585) 276-9830, E-mail address: jeffrey_alexis/at/urmc.rochester.edu

^#Department of Surgery, Beijing PLA 309 Hospital, Beijing, China

^∀Carestream Health Inc. Rochester, NY, USA

Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

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Abstract

Bcr is a serine/threonine kinase activated by PDGF that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin II (AngII) and PDGF mediated inflammatory responses in vascular smooth muscle cells (VSMC). Among transcription factors that might regulate AngII-mediated inflammatory responses we found that ligand-mediated PPARγ transcriptional activity was significantly decreased by AngII. AngII increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARγ activity. In contrast, knockdown of Bcr using Bcr siRNA and a dominant negative form of Bcr (DN-Bcr) reversed AngII-mediated inhibition of PPARγ activity significantly, suggesting the critical role of Bcr in AngII-mediated inhibition of PPARγ activity. Point-mutation and in vitro kinase analysis showed that PPARγ was phosphorylated by Bcr at serine 82. Overexpression of WT-Bcr kinase did not inhibit ligand-mediated PPARγ 1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARγ activity via S82 phosphorylation. DN-Bcr and Bcr siRNA inhibited AngII-mediated NF-κB activation in VSMC. DN-PPARγ reversed DN-Bcr mediated inhibition of NF-κB activation, suggesting that PPARγ is downstream from Bcr. Intimal proliferation in low flow carotid arteries was decreased in Bcr knockout mice compared with wild type mice suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPARγ/NF-κB transcriptional activity.

Keywords: signal transduction, smooth muscle cell, inflammation

Introduction

It is well known that the renin-angiotensin system plays an important role in regulating pathophysiological processes of cardiovascular disease. Many clinical studies have shown that inhibition of the renin-angiotensin system reduces inflammation and oxidative stress. For example, treatment with the angiotensin II type 1 (AT1) receptor blocker, valsartan, reduced lipopolysaccharide-stimulated IL-1β production by peripheral blood monocytes, and candesartan another AT1 receptor blocker reduced inflammation and insulin resistance in hypertensive patients.¹^,² In the Valsartan Heart Failure Trial (Val-HeFT), valsartan treatment lowered plasma CRP concentrations.³ These clinical studies suggest that angiotensin II (AngII) acts as an inflammatory mediator. In animal studies, it has been reported that AngII-induced hypertension specifically increased the development of atherosclerosis in apolipoprotein E (apoE) knockout mice.⁴ Interestingly, infusion of AngII in apoE knockout mice results in abdominal aortic aneurysm (AAA) formation, and the AAAs exhibit inflammatory infiltration, MMP activation, thrombus formation and oxidative stress, suggesting the profound impact of AngII on aneurysm formation and inflammation.⁵^,⁶ AngII activates NF-κB, a key component of inflammation, in vascular smooth muscle cells (VSMC). However, the exact mechanism of AngII-mediated inflammation and NF-κB activation in VSMC remains unclear.

The PPAR family consists of three different genes, PPARα, PPARβ/δ and PPARγ. These receptors exert anti-inflammatory activities in vascular and immune cells including endothelial cells, VSMC and monocytes. There are two isoforms of PPARγ-PPARγ1 and PPARγ2. PPARγ agonists include naturally occurring ligands such as 15-deoxy-Δ 12, 14-prostaglandin J2 (15d-PGJ2) and synthetic ligands such as the thiazolidinedione class of insulin sensitizing drugs.⁷^–⁹ PPARγ agonists inhibit the production of monocyte inflammatory cytokines (TNF-α, IL-6 and IL-1β) ¹⁰ and inhibit IFNγ, TNF-α and IL-2 production by human CD4+ T cells.¹¹ PPARγ agonists have also been shown to inhibit VSMC growth, migration and DNA synthesis and to inhibit neointimal proliferation following arterial injury.¹²^,¹³ PPARγ contains a MAP kinase consensus recognition site at Serine 82. Phosphorylation of PPARγ1 by MAP kinase has been shown to reduce growth factor mediated PPARγ transcriptional activity.¹⁴^,¹⁵

Bcr is a serine/threonine kinase originally defined as the breakpoint of the Philadelphia chromosome translocation associated with chronic myelogenous leukemia (CML). Bcr is expressed in many cell types and its cDNA sequence predicts several functional domains ¹⁶ including serine/threonine kinase activity,¹⁷ a region that binds Src-homology 2 (SH2) domains ¹⁸ and a GTPase-activating function for the small GTP-binding protein Rac.¹⁹ We previously reported that Bcr mediates platelet derived growth factor (PDGF) activation of Elk-1 in VSMC.²⁰ We also demonstrated that Bcr expression is increased in proliferating VSMC of the neointima.²⁰ Because inflammation is an important component of intimal formation ²¹ we studied the contribution of Bcr to vascular inflammation and intimal proliferation. In the current study, we found that increased Bcr expression and activation mediated by AngII induces inflammatory responses and enhances VSMC proliferation in part via a Bcr-mediated inhibitory effect against PPARγ transcriptional activity.

Methods

Cell culture

Rat and mouse VSMC were isolated as previously described²²^,²³ or were purchased from Cell Applications, Inc. VSMC were maintained in DMEM. Cells were treated with ciglitazone (Biomol), pioglitazone (Takeda Pharmaceuticals, North America, Inc. Lincolnshire, IL), PDGF (R&D Systems) and AngII (MP Biomedicals) as described in individual experiments.

Plasmids and transfection

Bcr wild-type and dominant negative Bcr (Y328F) plasmids were prepared as previously described.²⁴ The single or double mutations of PPARγ were created with the QuikChange site-directed mutagenesis kit (Stratagene) as previously described.²⁵ For transient expression experiments, cells were transfected with the lipofectamine plus method (Invitrogen) as previously described.²⁶ For siRNA experiments, VSMC were transfected with Bcr siRNA oligonucleotides (Invitrogen) using RNAiFect reagent (Qiagen).

Immunoprecipitation and Western blot

After treatment with reagents, the cells were washed with PBS and harvested in 0.5 mL of lysis buffer as previously described.²⁷ For immunoprecipitation, cell lysates were incubated with mouse anti-Bcr antibody (10 μl) as previously described.²⁷ For Western analysis the blots were incubated for 2 hours at room temperature with Bcr antibody (Santa Cruz) or α-tubulin antibody (Sigma) followed by incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Life Science).²⁷

Bcr in vitro kinase assay

Immunoprecipitation was performed using Bcr antibody, and in vitro kinase activity was measured at 30 °C for 30 minutes in a reaction mixture including 0.1 mg/ml of indicated substrates.

Immunofluorescence

VSMC were fixed with 4% paraformaldehyde, permeabilized with 0.1% PBS-Triton, and stained with relevant primary antibodies followed by secondary antibodies as indicated. Nuclei were stained with DAPI (Sigma). Cells were visualized with an Olympus (BX-51) fluorescent microscope.

[³H] Thymidine Incorporation assay

Measurement of [³H] thymidine incorporation into DNA was performed as described.²⁸

Carotid ligation and immunohistochemistry

Mice were used in accordance with the guidelines of the National Institutes of Health and the American Heart Association for the care and use of laboratory animals. All procedures were approved by the University of Rochester Animal Care Committee. Mice were anesthetized with an intraperitoneal injection of ketamine (130 mg/kg) and xylazine (8.8 mg/kg) in saline (10 mL/kg). The left external and internal carotid branches were ligated so that left carotid blood flow was reduced to flow via the occipital artery.²⁹ Carotid arteries were harvested two weeks after ligation. Cross sections were stained with hematoxylin and eosin and were analyzed using MCID image software (MCID Elite 6.0, Imaging Research). Representative samples were evaluated with Ki-67 antibody ( DAKO, 1:500 dilution).

Statistics

Numerical data are expressed as mean ± SEM or SD as indicated in figure legends. Statistical analysis was performed with the StatView 5.0 package (ABACUS Concepts, Berkeley, CA). Differences were analyzed with a one-way or a two way repeated–measure analysis of variance as appropriate, followed by Scheffé’s correction for multiple comparisons. A probability value < 0.05 was considered significant.

RESULTS

AngII inhibits PPARγ transcriptional activity in VSMC

It has been reported that PPARγ agonists can inhibit the development of hypertension in AngII-infused rats, but it remains unclear whether AngII can inhibit PPARγ transcriptional activity. Therefore, we examined the effect of AngII on PPARγ activity and PPARγ expression. Using two different PPARγ agonists ciglitazone and pioglitazone we demonstrated that AngII inhibits PPARγ transcriptional activity in VSMC (Figure 1A and 1B). AngII did not alter PPARγ expression (data not shown). Because PPARγ activation has a critical role in regulating inflammatory responses,¹⁰^,³⁰ these data suggest a mechanism of AngII-mediated inflammation is via inhibiting PPARγ transcriptional activity.

Figure 1

AngII inhibited PPARγ transcriptional activity in VSMC

AngII increased Bcr expression and Bcr kinase activity in VSMC

Previously, we reported that Bcr kinase activation can regulate Elk-1, which may have a significant impact on inflammation.²⁰ To determine the role of Bcr kinase on AngII-mediated inhibition of PPARγ transcriptional activity, we investigated whether AngII could regulate Bcr expression and Bcr kinase activity in VSMC. Western blotting demonstrated that 200nM AngII increased Bcr expression within 3 hours (Figure 2A). AngII also rapidly stimulated Bcr kinase activity, detected by Bcr autophosphorylation as previously reported,²⁰ with peak (2.0 ± 0.12, fold-increase vs. no treatment) at 2 minutes (Figure 2B), suggesting the possible involvement of Bcr kinase in AngII-mediated signaling.

Figure 2

AngII increased Bcr expression and increased Bcr activity in VSMC

Critical role of Bcr kinase on AngII-mediated inhibition of PPARγ transcriptional activity

To investigate whether Bcr kinase is involved in AngII-mediated inhibition of PPARγ transcriptional activity, VSMC were co-transfected with a PPARγ reporter plasmid and either Bcr wild type (WT) or a dominant negative (DN) form of Bcr (Y328F). After stimulation with PPARγ ligand ciglitazone (5μM or 10 μM) or vehicle for 16 hours the cells were harvested and dual-luciferase reporter assay performed. Overexpression of Bcr WT inhibited PPARγ transcriptional activity (Figure 3A). In contrast, DN-Bcr did not result in a change of PPARγ transcriptional activity in VSMC (Figure 3A). This effect of WT-Bcr on PPARγ activity did not appear to be just an effect limited to exogenous PPARγ agonists as a similar result was obtained when we over expressed PPARγ in the absence of exogenous PPARγ ligands (Figure 3B). Previously we reported that PDGF-induced Bcr kinase activation is involved in ERK1/2 activation. It may be possible that overexpression of Bcr inhibits PPARγ activity via ERK1/2 activation, but we did not find significant ERK1/2 activation by Bcr overexpression alone.²⁰ In addition, overexpression of WT Bcr does not increase c-Jun transcriptional activity, which represents JNK activity (Supplemental Figure I). Therefore, a mechanism other than ERK1/2 or JNK activation is most likely involved in this PPARγ regulation.

Figure 3

Critical role of Bcr on AngII-mediated inhibition of PPARγ transcriptional activity

To further confirm the role of Bcr kinase in AngII-mediated inhibition of PPARγ transcriptional activity we examined whether knockdown of Bcr would inhibit AngII-mediated inhibition of PPARγ activation. Following co-transfection of VSMC with DN-Bcr or Bcr siRNA and reporter plasmids, AngII inhibition of PPARγ activity was significantly reversed by both DN-Bcr and deletion of Bcr expression with Bcr siRNA suggesting that the effect of AngII on PPARγ is mediated largely by Bcr (Figure 3C–E).

Bcr phosphorylates PPARγ via serine 82and inhibits PPARγ transcriptional activity

Because phosphorylation of PPARγ (S82) inhibits PPARγ transcriptional activity, we hypothesized that Bcr kinase directly phosphorylates PPARγ. To examine this hypothesis we used VSMC to perform an in vitro kinase assay with GST-PPARγ wild type (WT) as substrate. VSMC were treated with AngII, and Bcr in vitro kinase assay was performed with GST-PPARγ WT. After 2 min of AngII stimulation Bcr kinase significantly phosphorylated GST-PPARγ WT (Figure 4A) with a time course similar to the Bcr autophosphorylation assay in Figure 2B. IgG was also non-specifically phosphorylated, but it did not relate to Bcr kinase activity. We observed a phosphorylated protein around 60 kDa (asterisk), which correlated well with Bcr kinase activation induced by AngII. We believe that this band represents another Bcr kinase substrate that co-immunoprecipitated with Bcr in VSMC. Several candidate proteins have been identified using mass spectrometry analysis, but characterizing these proteins is beyond the scope of the current study.

Figure 4

Bcr kinase phosphorylates S82 site of PPARγ and inhibits PPARγ transcriptional activity

To determine the possible role of S82 phosphorylation by Bcr kinase, we generated a point mutation replacing serine with alanine and created a GST-tagged fusion protein with the PPARγ S82A mutant. Following immunoprecipitation of VSMC with Bcr antibody, Bcr in vitro kinase assay was performed with GST-PPARγ WT, S82A mutant, and GST control as substrate (Figure 4B). In vitro kinase assay revealed that Bcr phosphorylates PPARγ WT, but phosphorylation of GST-PPARγ S82A was significantly reduced compared with GST-PPARγ WT, suggesting that S82 is one of the phosphorylation sites of Bcr kinase. No phosphorylation of GST alone was observed.

To examine whether the inhibition of PPARγ activity by Bcr kinase is via phosphorylation of PPARγ1 S82, we determined the effect of Bcr kinase on PPARγ transcriptional activity with mutation of S82. We over expressed wild type Bcr kinase with PPARγ1 wild type or S82A mutant and PPRE-luc reporter gene. The cells were incubated with PPARγ agonist ciglitazone (5 μM) 24 hours after transfection. After 16 hours of ciglitazone stimulation, luciferase PPARγ transcriptional activity was assayed. As shown in Figure 4C, ciglitazone stimulated transcriptional activity of both PPARγ WT and PPARγ S82 by approximately 3 fold. Bcr WT significantly inhibited PPARγ WT transcriptional activity while PPARγ S82A transcriptional activity was not decreased by Bcr WT. Surprisingly, we found that Bcr WT could increase PPARγ S82A transcriptional activity, which may reflect a positive effect of Bcr WT on PPARγ transcriptional activity via a S82 phosphorylation-independent mechanism. Combined with our in vitro kinase assay result, these data suggest that Bcr inhibits PPARγ activation by PPARγ S82 phosphorylation.

Bcr localizes in the nucleus in VSMC

Because PPARγ is a nuclear receptor we next examined whether we could detect Bcr in the nucleus. As shown in supplemental Figure II, utilizing an anti-Bcr antibody we observed significant immunostaining for Bcr in the nucleus in VSMC. To confirm the specificity of Bcr antibody for endogenous Bcr, we used Bcr siRNA and determined whether Bcr siRNA, specifically designed to inhibit Bcr expression, could reduce immunostaining detected by Bcr antibody used in this study. Bcr siRNA, but not control siRNA, significantly decreased Bcr immunostaining in the nucleus as shown in Supplemental Figure II, supporting the specificity of anti-Bcr antibody. We intentionally transfected Bcr siRNA at moderate transfection efficiency (70–80 %) to select the Bcr down-regulated cells from non-transfected cells as performed previously.³¹ The beauty of this method is that we can compare transfected and non-transfected cells in the same optical field, meaning that both cells are under the same condition, and we can observe the immunofluorescence signals of the cells under the same conditions. Therefore, the residual immunostaining in the cytosol should be non-specific (Supplemental Figure II). We did not observe significant changes in Bcr localization in cells stimulated by AngII and PDGF-B (data not shown).

AngII induced NF-κB activation via Bcr kinase activation

Since it is well known that PPARγ ligands have comprehensive anti-inflammatory effects, we next examined the effect of Bcr and PPARγ on AngII mediated NF-κB activation. The pro-inflammatory effect of AngII is mediated in part by NF-κB.³² To test the effect of PPARγ on AngII-mediated NF-κB activation, VSMC were transfected for 28 hours with a NF-κB reporter plasmid. VSMC were treated with ciglitazone for 30 minutes and then stimulated with AngII for 16 hours. Ciglitazone inhibited AngII mediated NF-κB activation in a dose dependent manner (Figure 5A). We then examined whether knockdown of Bcr would inhibit AngII-mediated NF-κB activation. Following co-transfection of VSMC with DN-Bcr or Bcr siRNA and reporter plasmids, we found that DN-Bcr as well as deletion of Bcr expression with Bcr siRNA inhibited AngII mediated NF-κB activation (Figure 5B and 5C), suggesting the critical role of Bcr on AngII-mediated NF-κB activation.

Figure 5

Critical role of Bcr/PPARγ on AngII-mediated NF-κ B activation

Bcr kinase increases NF-κB activation via inhibiting PPARγ activity

To determine the involvement of Bcr-mediated inhibition of PPARγ activity in AngII-induced NF-κB activation, we utilized a dominant negative form of mouse PPARγ1 ( DN-PPARγ1, L466A/E469A).³³ Following co-transfection of VSMC with DN-Bcr and/or DN-PPARγ1 and reporter plasmids, DN-PPARγ1 significantly reversed DN-Bcr mediated inhibition of AngII-mediated NF-κB activation, demonstrating that PPARγ is downstream of Bcr (Figure 5C). Next, we examined the effect of PPARγ S82A mutant on Bcr induced NF-κB activation. Bcr overexpression dose-dependently increased NF-κB activation in VSMC (Figure 6A). Overexpression of PPARγ S82A mutant, but not PPARγ WT, blocked Bcr WT-mediated NF-κB activation (Figure 6B and C). These data also suggest that phosphorylation of S82 by Bcr kinase inhibits PPARγ transcriptional activity, and mutation of S82 enables PPARγ to inhibit Bcr-induced NF-κB activation.

Figure 6

Bcr-induced NF-κB activation was inhibited by PPARγ S82 mutant but not by PPARγ WT in VSMC

Bcr siRNA inhibits AngII/PDGF induced DNA synthesis

AngII is an important regulator of VSMC growth and induces both protein synthesis and DNA synthesis in VSMC and enhances PDGF induced DNA synthesis.³⁴^,³⁵ As shown in Supplemental Figure III, we found that knockdown of Bcr by Bcr siRNA significantly blocked AngII/PDGF-induced DNA synthesis assessed by [³H] thymidine incorporation. We used the combination of AngII and PDGF to stimulate the cells, because we found that the combination of AngII (200 nM) and PDGF (10 ng/ml) maximized DNA synthesis.

Intimal proliferation after decreased blood flow in Bcr knockout mice

Our group has developed a reproducible mouse model of flow-dependent vascular remodeling that resembles human intima-media thickening.²⁹ In response to decreased blood flow intimal thickening occurs, which involves inflammation and VSMC proliferation. Based on the significant role of Bcr in VSMC inflammation and proliferation in vitro, we hypothesized that Bcr plays an important role in intimal thickening associated with decreased flow.

Immunohistochemical analysis demonstrated no difference between sham operated wild type and Bcr knockout animals (Figure 7a, d). In ligated arteries vascular remodeling was seen in wild type animals (Figure 7a vs. b) but less so in Bcr knockout animals (Figure 7d vs. e). This difference in vascular remodeling was secondary to greater neointimal proliferation in wild type animals compared with knockout animals (Figure 7c, f). These histological findings were confirmed by morphometry (Figure 7g–i). In addition, with Ki-67 staining we showed a reduction in cell proliferation in Bcr knockout animals compared with wild type animals (Figure 8A–C).

Figure 7

Knockdown or absence of Bcr inhibits cellular proliferation in vitro and in vivo

Figure 8

Bcr and cell growth

Discussion

The major findings of this study are that Bcr kinase activation by AngII inhibits PPARγ activation, and that AngII induced NF-κB activation occurs in part via Bcr kinase activation and subsequent inhibition of PPARγ activation. These data suggest that Bcr inhibits PPARγ activation via phosphorylation of S82. Furthermore, to our knowledge this is the first report to show that activation of Bcr kinase plays an important role in arterial proliferative disease in vivo. AngII is an inflammatory mediator that activates NF-κB, a key component of inflammation. Previously reported data show that crosstalk between NF-κB and PPARγ is important in the pro-inflammatory effects of NF-κB.³⁶ Specifically, NF-κB has been shown to block PPARγ ligand-induced transactivation in adipocytes. Our data using VSMC and previous reports ³⁰^,³⁷ show the converse, that PPARγ inhibits NF-κB activity. Therefore we propose that crosstalk between NF-κB and PPARγ, which are regulated by Bcr kinase, is important in regulating VSMC inflammatory gene expression. Given our novel findings that Bcr kinase inhibits PPARγ transcriptional activation and enhances NF-κB coupled with our finding that PPARγ inhibits NF-κB activation we believe that Bcr acts as a set point mechanism that regulates the sensitivity of VSMC to inflammatory stimuli.

In this study we demonstrate that Bcr is a major regulator of SMC that sits at the cross roads of inflammation and proliferation (Figure 8D). Our findings that Bcr inhibits PPARγ transcriptional activation (Figure 3) and that knockdown of Bcr with Bcr siRNA or DN-Bcr reverses AngII inhibition of PPARγ (Figure 3) demonstrate that Bcr is a positive regulator of AngII mediated inflammation. We also found that both Bcr siRNA and DN-Bcr block AngII mediated NF-κB activation (Figure 5) demonstrating that Bcr regulation of NF-κB is a key component of Bcr’s regulation of inflammation. Furthermore both our in vitro and in vivo studies showing that knock down or absence of Bcr reduces AngII/PDGF induced [³H] thymidine incorporation and reduces cell growth and intimal thickening (Figure 7, Figure 8, and Supplemental Figure III) demonstrate that Bcr regulates proliferation.

Our data suggest that the pro-inflammatory and proliferative effects of Bcr are mediated, at least in part, by inhibition of PPARγ and suggest that AngII-mediated Bcr kinase activation inhibits PPARγ by phosphorylation of S82. Our demonstration of nuclear localization of Bcr in VSMC is consistent with this concept. It remains unclear whether there are distinct differences between nuclear and cytoplasmic Bcr. Interestingly, Bcr contains a putative nuclear localization signal at amino acid 802–819 (http://myhits.isb-sib.ch/cgi-bin/motif_scan), but the functional consequence of this domain needs further investigation.

Overexpression of Bcr can inhibit PPARγ activation without showing any ERK1/2 activation, suggesting that Bcr inhibited PPARγ activation in an ERK1/2-independent manner. PPARγ plays an important role in regulating inflammation. PPARγ is a negative regulator of macrophage activation ³⁰ and PPARγ agonists have been demonstrated to inhibit the production of monocyte inflammatory cytokines.¹⁰ The PPARγ agonist 15d-PGJ₂ has been shown to inhibit transcription factors including NF-κB.³⁰ The anti-proliferative effect of PPARγ has several possible mechanisms. One is a direct result of its anti-inflammatory effect as cytokines and chemokines may promote lesion progression in a paracrine fashion.¹¹ In addition, the PPARγ agonist troglitazone has been shown to inhibit bFGF induced DNA synthesis in VSMC and to inhibit intimal proliferation in a rat aortic balloon injury model.¹³ Troglitazone was shown to inhibit c-fos induction and to inhibit transactivation of the serum response element that regulates c-fos expression, but the exact inhibitory target of PPARγ agonists against inflammation and proliferation remains unclear.

Our results do not exclude the possibility of an effect of Bcr on inflammation and proliferation that is independent of PPARγ (Figure 8D). Indeed as noted, we did find a phosphorylated protein around 60 kD that correlated well with Bcr kinase activation induced by AngII. Future studies will focus on PPARγ independent effects of Bcr signaling.

In conclusion, our data suggest that Bcr is an important regulator of inflammation and proliferation in VSMC and that Bcr plays a key role in arterial proliferative disease. This effect of Bcr is mediated in part by inhibition of PPARγ transcriptional activation via phosphorylation of PPARγ by Bcr.

Supplementary Material

Supplement Mat

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Acknowledgments

Sources of Funding

This study was supported by grants from the National Institutes of Health to Dr. Alexis (HL80938), Dr. Berk (HL77789), Dr. Yan (HL77789) and Dr. Abe (HL77789). Drs. Abe and Yan are recipients of Established Investigator Awards of the American Heart Association (0740013N and 0740021N).

Footnotes

Disclosures

None

This is an un-copyedited author manuscript accepted for publication in Circulation Research, copyright The American Heart Association. This may not be duplicated or reproduced, other than for personal use or within the “Fair Use of Copyrighted Materials” (section 107, title 17, U.S. Code) without prior permission of the copyright owner, The American Heart Association. The final copyedited article, which is the version of record, can be found at http://circres.ahajournals.org/. The American Heart Association disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties.

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