Bcr Kinase Activation by Angiotensin II Inhibits Peroxisome Proliferator-Activated Receptor γ Transcriptional Activity in Vascular Smooth Muscle Cells
Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor–mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II–mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)γ transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARγ activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II–mediated inhibition of PPARγ activity significantly, suggesting the critical role of Bcr in Ang II–mediated inhibition of PPARγ activity. Point-mutation and in vitro kinase analyses showed that PPARγ was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPARγ1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARγ activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II–mediated nuclear factor κB activation in VSMCs. DN-PPARγ reversed DN-Bcr–mediated inhibition of nuclear factor 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/nuclear factor κB transcriptional activity.
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 (Ang) II type 1 receptor blocker valsartan reduced lipopolysaccharide (LPS)-stimulated interleukin (IL)-1β production by peripheral blood monocytes, and candesartan, another Ang II type 1 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 Ang II acts as an inflammatory mediator. In animal studies, it has been reported that Ang II–induced hypertension specifically increased the development of atherosclerosis in apolipoprotein (apo)E knockout mice.4 Interestingly, infusion of Ang II in apoE knockout mice results in abdominal aortic aneurysm formation, and the abdominal aortic aneurysms exhibit inflammatory infiltration, matrix metalloproteinase activation, thrombus formation, and oxidative stress, suggesting the profound impact of Ang II on aneurysm formation and inflammation.5,6 Ang II activates nuclear factor (NF)-κB, a key component of inflammation, in vascular smooth muscle cells (VSMCs). However, the exact mechanism of Ang II–mediated inflammation and NF-κB activation in VSMCs remains unclear.
The PPAR family consists of 3 different genes, PPARα, PPARβ/δ, and PPARγ. These receptors exert antiinflammatory activities in vascular and immune cells including endothelial cells, VSMCs, and monocytes. There are 2 isoforms of PPARγ- PPARγ1 and PPARγ2. PPARγ agonists include naturally occurring ligands such as 15-deoxy-Δ12,14-prostaglandin (15d-PG)J2 and synthetic ligands such as the thiazolidinedione class of insulin-sensitizing drugs.7–9 PPARγ agonists inhibit the production of monocyte inflammatory cytokines (tumor necrosis factor [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 mitogen-activated protein kinase consensus recognition site at serine 82. Phosphorylation of PPARγ1 by mitogen-activated protein 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. Bcr is expressed in many cell types and its cDNA sequence predicts several functional domains16 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 VSMCs.20 We also demonstrated that Bcr expression is increased in proliferating VSMCs of the neointima.20 Because inflammation is an important component of intimal formation21 we studied the contribution of Bcr to vascular inflammation and intimal proliferation. In the present study, we found that increased Bcr expression and activation mediated by Ang II induces inflammatory responses and enhances VSMC proliferation in part via a Bcr-mediated inhibitory effect against PPARγ transcriptional activity.
Materials and Methods
Rat and mouse VSMCs were isolated as described previously22,23 or were purchased from Cell Applications Inc. VSMCs were maintained in DMEM. Cells were treated with ciglitazone (Biomol), pioglitazone (Takeda Pharmaceuticals, North America Inc, Lincolnshire, Ill), PDGF (R&D Systems), and Ang II (MP Biomedicals) as described in individual experiments.
Plasmids and Transfection
Bcr wild-type (WT) and dominant-negative Bcr (DN-Bcr) (Y328F) plasmids were prepared as described previously.24 The single or double mutations of PPARγ were created with the QuikChange site-directed mutagenesis kit (Stratagene) as described previously.25 For transient expression experiments, cells were transfected with the Lipofectamine Plus method (Invitrogen) as described previously.26 For small interfering (si)RNA experiments, VSMCs 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 described previously.27 For immunoprecipitation, cell lysates were incubated with mouse anti-Bcr antibody (10 μL) as described previously.27 For Western analysis, the blots were incubated for 2 hours at room temperature with Bcr antibody (Santa Cruz Biotechnology) 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 indicated substrates.
VSMCs 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 NIH 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 2 weeks after ligation. Cross-sections were stained with hematoxylin/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).
Numeric data are expressed as means±SEM or SD as indicated in the figure legends. Statistical analysis was performed with the StatView 5.0 package (ABACUS Concepts, Berkeley, Calif). Differences were analyzed with a 1-way or a 2-way repeated-measures ANOVA as appropriate, followed by Scheffé’s correction for multiple comparisons. A probability value of <0.05 was considered significant.
An expanded Materials and Methods section appears in the online data supplement at http://circres.ahajournals.org.
Ang II Inhibits PPARγ Transcriptional Activity in VSMCs
It has been reported that PPARγ agonists can inhibit the development of hypertension in Ang II–infused rats, but it remains unclear whether Ang II can inhibit PPARγ transcriptional activity. Therefore, we examined the effect of Ang II on PPARγ activity and PPARγ expression. Using 2 different PPARγ agonists ciglitazone and pioglitazone, we demonstrated that Ang II inhibits PPARγ transcriptional activity in VSMCs (Figure 1A and 1B). Ang II 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 Ang II–mediated inflammation is via inhibiting PPARγ transcriptional activity.
Ang II Increased Bcr Expression and Bcr Kinase Activity in VSMCs
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 Ang II–mediated inhibition of PPARγ transcriptional activity, we investigated whether Ang II could regulate Bcr expression and Bcr kinase activity in VSMCs. Western blotting demonstrated that 200 nmol/L Ang II increased Bcr expression within 3 hours (Figure 2A). Ang II also rapidly stimulated Bcr kinase activity, detected by Bcr autophosphorylation as reported previously,20 with peak (2.0±0.12-fold increase versus no treatment) at 2 minutes (Figure 2B), suggesting the possible involvement of Bcr kinase in Ang II–mediated signaling.
Critical Role of Bcr Kinase on Ang II–Mediated Inhibition of PPARγ Transcriptional Activity
To investigate whether Bcr kinase is involved in Ang II–mediated inhibition of PPARγ transcriptional activity, VSMCs were cotransfected with a PPARγ reporter plasmid and either Bcr WT or a dominant-negative form of Bcr (Y328F). After stimulation with the PPARγ ligand ciglitazone (5 μmol/L or 10 μmol/L) 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 VSMCs (Figure 3A). This effect of WT-Bcr on PPARγ activity did not appear to be just an effect limited to exogenous PPARγ agonists because a similar result was obtained when we overexpressed PPARγ in the absence of exogenous PPARγ ligands (Figure 3B). Previously, we reported that PDGF-induced Bcr kinase activation is involved in extracellular signal-regulated kinase (ERK)1/2 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 c-Jun N-terminal kinase (JNK) activity (Figure I in the online data supplement). Therefore, a mechanism other than ERK1/2 or JNK activation is most likely involved in this PPARγ regulation.
To further confirm the role of Bcr kinase in Ang II–mediated inhibition of PPARγ transcriptional activity, we examined whether knockdown of Bcr would inhibit Ang II–mediated inhibition of PPARγ activation. Following cotransfection of VSMCs with DN-Bcr or Bcr siRNA and reporter plasmids, Ang II inhibition of PPARγ activity was significantly reversed by both DN-Bcr and deletion of Bcr expression with Bcr siRNA, suggesting that the effect of Ang II on PPARγ is mediated largely by Bcr (Figure 3C through 3E).
Bcr Phosphorylates PPARγ via Serine 82 and Inhibits PPARγ Transcriptional
Because phosphorylation of PPARγ (S82) inhibits PPARγ transcriptional activity, we hypothesized that Bcr kinase directly phosphorylates PPARγ. To examine this hypothesis, we used VSMCs to perform an in vitro kinase assay with glutathione S-transferase (GST)-PPARγ WT as substrate. VSMCs were treated with Ang II, and Bcr in vitro kinase assay was performed with GST-PPARγ WT. After 2 minutes of Ang II 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 nonspecifically 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 Ang II. We believe that this band represents another Bcr kinase substrate that coimmunoprecipitated with Bcr in VSMCs. Several candidate proteins have been identified using mass spectrometry analysis, but characterizing these proteins is beyond the scope of the present study.
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 VSMCs 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 1 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 overexpressed WT Bcr kinase with PPARγ1 WT or S82A mutant and PPRE-luc reporter gene. The cells were incubated with the PPARγ agonist ciglitazone (5 μmol/L) 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 ≈3-fold. Bcr WT significantly inhibited PPARγ WT transcriptional activity, whereas 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 VSMCs
Because PPARγ is a nuclear receptor, we next examined whether we could detect Bcr in the nucleus. As shown in supplemental Figure II, by using an anti-Bcr antibody, we observed significant immunostaining for Bcr in the nucleus in VSMCs. 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 to 80%) to select the Bcr downregulated cells from nontransfected cells, as performed previously.31 The beauty of this method is that we can compare transfected and nontransfected 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 nonspecific (supplemental Figure II). We did not observe significant changes in Bcr localization in cells stimulated by Ang II and PDGF-B (data not shown).
Ang II Induced NF-κB Activation via Bcr Kinase Activation
Because it is well known that PPARγ ligands have comprehensive antiinflammatory effects, we next examined the effect of Bcr and PPARγ on Ang II mediated NF-κB activation. The proinflammatory effect of Ang II is mediated in part by NF-κB.32 To test the effect of PPARγ on Ang II–mediated NF-κB activation, VSMCs were transfected for 28 hours with a NF-κB reporter plasmid. VSMCs were treated with ciglitazone for 30 minutes and then stimulated with Ang II for 16 hours. Ciglitazone inhibited Ang II mediated NF-κB activation in a dose-dependent manner (Figure 5A). We then examined whether knockdown of Bcr would inhibit Ang II–mediated NF-κB activation. Following cotransfection of VSMCs 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 Ang II–mediated NF-κB activation (Figure 5B and 5C), suggesting the critical role of Bcr on Ang II–mediated NF-κB activation.
Bcr Kinase Increases NF-κB Activation via Inhibiting PPARγ Activity
To determine the involvement of Bcr-mediated inhibition of PPARγ activity in Ang II–induced NF-κB activation, we used a dominant-negative form of mouse PPARγ1 (DN-PPARγ1, L466A/E469A).33 Following cotransfection of VSMCs with DN-Bcr and/or DN-PPARγ1 and reporter plasmids, DN-PPARγ1 significantly reversed DN-Bcr mediated inhibition of Ang II–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 VSMCs (Figure 6A). Overexpression of PPARγ S82A mutant, but not PPARγ WT, blocked Bcr WT–mediated NF-κB activation (Figure 6B and 6C). 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.
Bcr siRNA Inhibits Ang II/PDGF-Induced DNA Synthesis
Ang II is an important regulator of VSMC growth and induces both protein synthesis and DNA synthesis in VSMCs 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 Ang II/PDGF-induced DNA synthesis assessed by [3H]thymidine incorporation. We used the combination of Ang II and PDGF to stimulate the cells, because we found that the combination of Ang II (200 nmol/L) 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 WT and Bcr knockout animals (Figure 7a and 7d). In ligated arteries, vascular remodeling was seen in WT animals (Figure 7a versus 7b) but less so in Bcr knockout animals (Figure 7d versus 7e). This difference in vascular remodeling was secondary to greater neointimal proliferation in WT animals compared with knockout animals (Figure 7c and 7f). These histological findings were confirmed by morphometry (Figure 7g through 7i). In addition, with Ki-67 staining, we showed a reduction in cell proliferation in Bcr knockout animals compared with WT animals (Figure 8A through 8C).
The major findings of this study are that Bcr kinase activation by Ang II inhibits PPARγ activation and that Ang II–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. Ang II 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 proinflammatory effects of NF-κB.36 Specifically, NF-κB has been shown to block PPARγ ligand-induced transactivation in adipocytes. Our data using VSMCs and previous reports30,37 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 VSMCs 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 Ang II inhibition of PPARγ (Figure 3) demonstrate that Bcr is a positive regulator of Ang II–mediated inflammation. We also found that both Bcr siRNA and DN-Bcr block Ang II–mediated NF-κB activation (Figure 5), demonstrating that Bcr regulation of NF-κB is a key component of regulation of inflammation by Bcr. Furthermore, both our in vitro and in vivo studies showing that knock down or absence of Bcr reduces Ang II/PDGF-induced [3H]thymidine incorporation and reduces cell growth and intimal thickening (Figures 7 and 8⇑ and supplemental Figure III) demonstrate that Bcr regulates proliferation.
Our data suggest that the proinflammatory and proliferative effects of Bcr are mediated, at least in part, by inhibition of PPARγ and suggest that Ang II–mediated Bcr kinase activation inhibits PPARγ by phosphorylation of S82. Our demonstration of nuclear localization of Bcr in VSMCs 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 to 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 antiproliferative effect of PPARγ has several possible mechanisms. One is a direct result of its antiinflammatory effect because cytokines and chemokines may promote lesion progression in a paracrine fashion.11 In addition, the PPARγ agonist troglitazone has been shown to inhibit basic fibroblast growth factor–induced DNA synthesis in VSMCs 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 kDa that correlated well with Bcr kinase activation induced by Ang II. 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 VSMCs 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.
Sources of Funding
This study was supported by NIH grants HL80938 (to J.D.A.), HL77789 (to B.C.B.), HL77789 (to C.Y.), and HL77789 (to J.-i.A.). J.-i.A. and C.Y. are recipients of Established Investigator Awards from the American Heart Association (0740013N and 0740021N).
Original received December 16, 2007; resubmission received September 29, 2008; revised resubmission received October 30, 2008; accepted November 6, 2008.
Anand IS, Latini R, Florea VG, Kuskowski MA, Rector T, Masson S, Signorini S, Mocarelli P, Hester A, Glazer R, Cohn JN. C-reactive protein in heart failure: prognostic value and the effect of valsartan. Circulation. 2005; 112: 1428–1434.
Weiss D, Kools JJ, Taylor WR. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in apoE-deficient mice. Circulation. 2001; 103: 448–454.
Miller FJ Jr, Sharp WJ, Fang X, Oberley LW, Oberley TD, Weintraub NL. Oxidative stress in human abdominal aortic aneurysms: a potential mediator of aneurysmal remodeling. Arterioscler Thromb Vasc Biol. 2002; 22: 560–565.
Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem. 1995; 270: 12953–12956.
Marx N, Kehrle B, Kohlhammer K, Grub M, Koenig W, Hombach V, Libby P, Plutzky J. PPAR activators as antiinflammatory mediators in human T lymphocytes: implications for atherosclerosis and transplantation-associated arteriosclerosis. Circ Res. 2002; 90: 703–710.
Marx N, Schonbeck U, Lazar MA, Libby P, Plutzky J. Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998; 83: 1097–1103.
Adams M, Reginato MJ, Shao D, Lazar MA, Chatterjee VK. Transcriptional activation by peroxisome proliferator-activated receptor gamma is inhibited by phosphorylation at a consensus mitogen-activated protein kinase site. J Biol Chem. 1997; 272: 5128–5132.
Camp HS, Tafuri SR. Regulation of peroxisome proliferator-activated receptor gamma activity by mitogen-activated protein kinase. J Biol Chem. 1997; 272: 10811–10816.
Che W, Abe J, Yoshizumi M, Huang Q, Glassman M, Ohta S, Melaragno MG, Poppa V, Yan C, Lerner-Marmarosh N, Zhang C, Wu Y, Arlinghaus R, Berk BC. p160 Bcr mediates platelet-derived growth factor activation of extracellular signal-regulated kinase in vascular smooth muscle cells. Circulation. 2001; 104: 1399–1406.
Korshunov VA, Nikonenko TA, Tkachuk VA, Brooks A, Berk BC. Interleukin-18 and macrophage migration inhibitory factor are associated with increased carotid intima-media thickening. Arterioscler Thromb Vasc Biol. 2006; 26: 295–300.
Duff JL, Marrero MB, Paxton WG, Charles CH, Lau LF, Bernstein KE, Berk BC. Angiotensin II induces 3CH134, a protein-tyrosine phosphatase, in vascular smooth muscle cells. J Biol Chem. 1993; 268: 26037–26040.
Akaike M, Che W, Marmarosh NL, Ohta S, Osawa M, Ding B, Berk BC, Yan C, Abe J. The hinge-helix 1 region of peroxisome proliferator-activated receptor gamma1 (PPARgamma1) mediates interaction with extracellular signal-regulated kinase 5 and PPARgamma1 transcriptional activation: involvement in flow-induced PPARgamma activation in endothelial cells. Mol Cell Biol. 2004; 24: 8691–8704.
Liao DF, Monia B, Dean N, Berk BC. Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J Biol Chem. 1997; 272: 6146–6150.
Abe J, Kusuhara M, Ulevitch RJ, Berk BC, Lee JD. Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase. J Biol Chem. 1996; 271: 16586–16590.
Abe J, Zhou W, Takuwa N, Taguchi J, Kurokawa K, Kumada M, Takuwa Y. A fumagillin derivative angiogenesis inhibitor, AGM-1470, inhibits activation of cyclin-dependent kinases and phosphorylation of retinoblastoma gene product but not protein tyrosyl phosphorylation or protooncogene expression in vascular endothelial cells. Cancer Res. 1994; 54: 3407–3412.
Korshunov VA, Berk BC. Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening. Arterioscler Thromb Vasc Biol. 2003; 23: 2185–2191.
Osawa M, Itoh S, Ohta S, Huang Q, Berk BC, Marmarosh NL, Che W, Ding B, Yan C, Abe J. ERK1/2 associates with the c-Met-binding domain of growth factor receptor-bound protein 2 (Grb2)-associated binder-1 (Gab1): role in ERK1/2 and early growth response factor-1 (Egr-1) nuclear accumulation. J Biol Chem. 2004; 279: 29691–29699.
Zhang L, Cheng J, Ma Y, Thomas W, Zhang J, Du J. Dual pathways for nuclear factor kappaB activation by angiotensin II in vascular smooth muscle: phosphorylation of p65 by IkappaB kinase and ribosomal kinase. Circ Res. 2005; 97: 975–982.
Gurnell M, Wentworth JM, Agostini M, Adams M, Collingwood TN, Provenzano C, Browne PO, Rajanayagam O, Burris TP, Schwabe JW, Lazar MA, Chatterjee VK. A dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutant is a constitutive repressor and inhibits PPARgamma-mediated adipogenesis. J Biol Chem. 2000; 275: 5754–5759.
Bunkenburg B, van Amelsvoort T, Rogg H, Wood JM. Receptor-mediated effects of angiotensin II on growth of vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension. 1992; 20: 746–754.