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Circulation Research. 2007;101:792-801
Published online before print August 17, 2007, doi: 10.1161/CIRCRESAHA.107.152736
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(Circulation Research. 2007;101:792.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Oxidized Phospholipids Induce Phenotypic Switching of Vascular Smooth Muscle Cells In Vivo and In Vitro

Nataliya A. Pidkovka, Olga A. Cherepanova, Tadashi Yoshida, Matthew R. Alexander, Rebecca A. Deaton, James A. Thomas, Norbert Leitinger, Gary K. Owens

From the Department of Molecular Physiology and Biophysics, Cardiovascular Research Center, University of Virginia, Charlottesville.

Correspondence to Dr Gary K. Owens, University of Virginia, Cardiovascular Research Center, 415 Lane Road, MR5 RM 1328, Charlottesville, VA 22908. E-mail gko{at}virginia.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Atherosclerosis is a vascular disease characterized by lipid deposition and inflammation within the arterial wall. Oxidized phospholipids (oxPLs), such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) and its constituents 1-palmytoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are concentrated within atherosclerotic lesions and are known to be potent proinflammatory mediators. Phenotypic switching of smooth muscle cells (SMCs) plays a critical role in the development, progression, and end-stage clinical consequences of atherosclerosis, yet little is known regarding the effects of specific oxPLs on SMC phenotype. The present studies were focused on determining whether oxPLs regulate expression of SMC differentiation marker genes and the molecular mechanisms involved. Results showed that POVPC and PGPC induced profound suppression of smooth muscle (SM) {alpha}-actin and SM myosin heavy chain expression while simultaneously increasing expression of MCP-1, MCP-3, and cytolysin. OxPLs also induced nuclear translocation of Krüppel-like transcription factor 4 (KLF4), a known repressor of SMC marker genes. siRNA targeting of KLF4 nearly blocked POVPC-induced suppression of SMC marker genes, and myocardin. POVPC-induced repression of SMC marker genes was also significantly attenuated in KLF4 knockout SMCs. Taken together, these results suggest a novel role for oxPLs in phenotypic modulation of SMCs and indicate that these effects are dependent on the transcription factor, KLF4. These results may have important novel implications for the mechanisms by which oxPLs contribute to the pathogenesis of atherosclerosis.


Key Words: atherosclerosis • smooth muscle cell phenotypic switching • oxidized phospholipids


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular smooth muscle cells (SMCs) are a predominant cell type in atherosclerotic plaques and are crucial in the development of vascular disease.1 Unlike either terminally-differentiated skeletal or cardiac muscle cells, SMCs within adult animals retain remarkable plasticity and can undergo profound and reversible changes in phenotype in response to changes in local environmental cues.1 During early atherogenesis, SMCs undergo phenotypic switching which is characterized by decreased expression of SMC differentiation marker proteins, a high rate of cellular proliferation, and increased synthesis of extracellular matrix proteins.1 In contrast, in late disease stage, SMCs within the fibrous cap of vulnerable atherosclerotic plaques exhibit reduced rates of proliferation and matrix production. These cells also undergo apoptosis and activation of matrix metalloproteinases, which may contribute to plaque rupture, thrombosis, and myocardial infarction.2 Despite the importance of SMC phenotypic switching in the pathogenesis of atherosclerosis, virtually nothing is known regarding specific factors and mechanisms that control this process.

Oxidized lipids, particularly oxidized phospholipids (oxPLs), accumulate in atherosclerotic lesions and there is evidence that changes in the plasma oxPLs/apoB ratio may reflect the extent of atherosclerotic disease burden.3 The presence of antibodies to oxPLs in patients with atherosclerosis, diabetes, hypertension, antiphospholipid syndrome, and other chronic diseases also underlines the potential importance of these molecules.4–6 OxPLs promote chronic inflammation at least in part by inducing expression of inflammatory cytokines in endothelial cells.7,8 The major bioactive lipids in minimally modified low-density lipoprotein (MM-LDL) are derived from the oxidation of arachidonate-containing phospholipids such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), namely: 1-palmytoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC).9–12 These oxidized phospholipids are found at high concentrations within fatty streak lesions of cholesterol-fed rabbits and antibodies to these factors are present in the sera of apoE-null mice.5,11,13 Unregulated uptake of oxidized LDL via scavenger LDL receptor pathways contributes to development of foam cells within atherosclerotic lesions. MM-LDL and its derivative phospholipids including POVPC and PGPC have also been shown to increase adhesion and trafficking of monocytes to endothelial cells and to be involved in development of experimental atherosclerosis.4–6,14 Finally, oxPLs have been implicated in control of end-stage disease consequences including plaque rupture.2,15

Although there is extensive evidence that oxidized lipids promote vascular inflammation and contribute to development of atherosclerosis,7,8,16,17 virtually nothing is known regarding how these atherogenic compounds impact SMC differentiation or phenotypic switching. The aims of the present study were to: (1) determine whether oxPLs regulate SMC differentiation marker genes in cultured SMCs; (2) determine whether POVPC has an effect on SMC differentiation marker genes in vivo; and (3) test the hypothesis that the suppressive effects of oxPLs on SM {alpha}-actin and SM myosin heavy chain (SM MHC) expression are mediated at least in part by the potent repressor of SMC differentiation marker genes Krüppel-like transcription factor 4 (KLF4).


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
For detailed Materials and Methods, please see the supplemental materials (available online at http://circres.ahajournals.org).

Cell Culture and Treatment of SMCs With Phospholipids
Rat aortic SMCs were isolated and cultured as previously described.18 Cells were grown to 100% confluence and then switched to serum-free media. After culture in serum-free media for 3 days, passages 8 to 15 of postconfluent rat aortic SMCs were treated with vehicle (DMSO), nonoxidized phospholipids 1-palmitoyl 2-arachidonoyl phosphatidylcholine (PAPC), 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) (Avanti Polar Lipids), oxPAPC, PGPC, or POVPC (Cayman Chemical).

siRNA Oligonucleotides and Transfection
siRNA oligonucleotides specific for KLF4 (siKLF #13; GUACAAUGGUUUAUUCCA-3', siKLF4 #8; 5'-CGAUCUACAUUUAUGACCU-3') and EGFP (5'-GAACGGCAUCAAGGUGAAC-3') were purchased from MWG-Biotech, and transient transfection of siRNA oligonucleotide was performed using Oligofectamine (Invitrogen) according to the manufacturer’s protocol.

Application of POVPC to Rat Carotid Arteries Using F-127 Pluronic Gel System
The animal protocol was approved by the Animal Care and Use Committee at the University of Virginia. 150 µL of ice-cold F-127 pluronic gel (Molecular Probes) containing POVPC 30 µg (n=6 rats) or vehicle (n=6 rats) was applied for 24 hours to the adventitial surface of rat carotid arteries as described previously.10,26 The Fast Prep FP120 (Q-Biogene) was used to homogenize tissues, and total RNA was prepared from both treated and untreated carotid arteries, aorta, and liver. SM {alpha}-actin, SM MHC, myocardin, KLF4, and ß-actin expression in the each sample were normalized to 18S rRNA levels and to the contralateral untreated control vessel.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
oxPAPC Decreased SMC Marker Gene Promoter Activity and mRNA Level in Cultured SMCs
To determine whether oxPLs alter SMC phenotype, cultured SMCs were transfected with various SMC promoter-reporter constructs and treated for 24 hours with oxPAPC at concentrations present within atherosclerotic lesions.11,13,19 OxPAPC markedly suppressed SM {alpha}-actin promoter activity and SM MHC promoter activity by 70% and 30%, respectively, at a dose ≥10 µg/mL (Figure 1A). To determine whether oxPAPC also altered endogenous gene expression, quantitative real time RT-PCR analyses were performed on cultured SMCs treated with oxPAPC for 24 hours (Figure 1B). Results showed that oxPAPC caused dose-dependent suppression of SM {alpha}-actin and SM MHC mRNA expression.


Figure 1
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  		Figure 1. oxPAPC suppressed expression of SMC marker genes in cultured SMCs. A, SM {alpha}-actin-luc and SM MHC-luc promoter-luciferase constructs were transiently transfected into SMCs and cells were treated for 24 hours with the indicated range of oxPAPC concentrations. Luciferase activity was measured, and normalized for protein contents and background luminescence, and then expressed as fold-increase over vehicle. Values represent the mean±SD. *P<0.05; **P≤0.001 vs vehicle. B, SMCs were treated for 24 hours with the indicated range of oxPAPC concentrations. Expression of SM {alpha}-actin and SM MHC mRNA was measured by real-time RT-PCR and normalized to 18S rRNA, and expressed as fold-increase over vehicle. Values represent the mean±SD. *P<0.005; **P<0.005 vs vehicle. Each experiment is representative of 3 independent studies.

POVPC, a Specific Oxidative Product of PAPC, Decreased SMC Marker Gene Expression While Simultaneously Activating Expression of a Number of Proinflammatory Genes
OxPAPC has been shown to contain a variety of specific oxPLs including POVPC, which is one of the most abundant and bioactive oxPLs found in atherosclerotic lesions.11,12 POVPC induced concentration-dependent suppression of SM {alpha}-actin and SM MHC promoter activity, respectively (Figure 2A). Similarly, PGPC suppressed SM {alpha}-actin and SM MHC promoter activity (supplemental Figure I). Quantitative real-time RT-PCR analyses performed in cultured SMCs showed that POVPC induced dose-dependent repression of endogenous SM {alpha}-actin and SM MHC (Figure 2B) but had no effect on ß-actin expression (supplemental Figure IIA). POVPC dose-dependently decreased protein expression of SM {alpha}-actin and SM MHC after 72 hours of treatment (Figure 2C). In contrast, nonoxidized phospholipids such as PAPC and DMPC did not significantly affect SM {alpha}-actin and SM MHC promoter activity (supplemental Figure III), indicating that oxidative modification of phospholipids is necessary for downregulation of SM {alpha}-actin and SM MHC. Western blot analysis for caspase 3 activation and Bax expression, known markers of apoptosis (supplemental Figure IV), showed that POVPC concentrations of 50 µg/mL and greater caused apoptosis in SMCs, whereas no evidence of apoptosis was obtained at lower doses. Taken together, these results suggest that oxPAPC, POVPC, and PGPC induce profound concentration-dependent suppression of SMC marker gene expression.


Figure 2
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  		Figure 2. POVPC decreased expression of SMC marker genes in cultured SMCs. A, SM {alpha}-actin-luc and SM MHC-luc promoter-luciferase constructs were transiently transfected into SMCs, and SMCs were treated for 24 hours with the indicated range of POVPC concentration. Luciferase activity was measured and normalized to protein content and a promoterless construct, and expressed as fold-increase over vehicle. Values represent the mean±SEM. *P<0.005; **P<0.001 vs vehicle. B, SMCs were treated for 24 hours with the indicated range of POVPC concentration. Expression of SM {alpha}-actin and SM MHC mRNA was measured by real-time RT-PCR and normalized to 18S rRNA, and expressed as fold-increase over vehicle. Values represent the mean±SEM. *P<0.01; **P<0.005 vs vehicle. C, SMCs were treated for 24 and 72 hours with the indicated range of POVPC concentration. Total protein lysates of SMCs (0.3 µg and 10 µg) were subjected to Western blot analysis for SM {alpha}-actin and SM MHC protein expression, respectively.

To more fully characterize the nature of POVPC-induced changes in SMCs, we performed gene microarray and RayBio cytokine array analyses of POVPC- and vehicle-treated cultured rat aortic SMCs. Of major interest, POVPC induced expression of a number of proinflammatory genes including cytolysin (6.9-fold), chemokine ligand 2 (6.2-fold), TNF (5.8-fold), and MCP3 (3.1-fold; supplemental Figure V).

POVPC Decreased SM {alpha}-Actin and SM MHC mRNA Level in Rat Carotid Arteries In Vivo
To determine whether oxPLs also suppress SMC marker gene expression in vivo, POVPC or vehicle were applied to the adventitial surface of rat carotid arteries in vivo using an F-127 pluronic gel system.8,20 Results of RT-PCR analyses showed that POVPC induced marked suppression of SM {alpha}-actin (Figure 3A, upper panel) and SM MHC (Figure 3B, upper panel) mRNA levels after 24 hours of treatment relative to vehicle-treated vessels. In contrast, SMC marker gene expression in the aorta and liver was not changed, indicating a lack of systemic effects of POVPC (Figure 3A and B, lower 2 panels). Expression of ß-actin was not changed in POVPC-treated vessels (supplemental Figure IIC). These results show that POVPC decreases the expression of SMC differentiation marker genes within vessels in vivo.


Figure 3
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  		Figure 3. Pluronic gel-based delivery of POVPC decreased expression of the endogenous SM {alpha}-actin and SM MHC genes in vivo. Pluronic gel with POVPC (30 µg/mL) or vehicle (DMSO) was applied for 24 hours to the adventitial surface of left common carotid arteries. Total RNA was isolated from the treated left carotid artery and the untreated right carotid artery as well as the aorta and liver. SMC marker gene expression in the each sample was normalized to the 18S rRNA level. Values for the left carotid artery were normalized to those for the contralateral control right carotid artery. Values represent the mean±SEM. *P<0.01; **P<0.01 vs vehicle. POVPC had no effect on ß-actin mRNA levels (see supplemental Figure II).

POVPC Increased Expression and Nuclear Translocation of the Potent SMC Differentiation Repressor Gene, KLF4
In contrast to the marked suppressive effects of POVPC on SMC differentiation marker genes, we found that POVPC increased expression of KLF4x35-fold (Figure 4A). Increases in KLF4 mRNA expression occurred within 2 hours of stimulation (Figure 4B) and were maximal at a concentration of POVPC 10 µg/mL. In addition, POVPC treatment resulted in rapid nuclear localization of KLF4 as determined by cell fractionation and Western blot analysis (Figure 4C), as well as by examining KLF4 subcellular localization by immunofluorescent microscopic analyses of SMCs transiently transfected with a pcDNA3-KLF4-FLAG expression plasmid and treated with POVPC (Figure 5A and 5B). Results showed that KLF4 was localized predominantly in the cytoplasm in cells treated with vehicle, whereas it translocated into the nucleus with POVPC treatment (Figure 5A and 5B). KLF4-specific antibodies were used to analyze endogenous KLF4 expression and localization by immunofluorescence. SMCs treated with vehicle exhibited diffuse staining (Figure 5C, upper panel). In contrast, POVPC caused nuclear localization of KLF4 (Figure 5C, low panel). These results provide evidence that POVPC induces not only increased expression of KLF4 but also its nuclear localization.


Figure 4
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  		Figure 4. POVPC induced KLF4 expression in cultured SMCs. A, Total RNA samples were prepared from SMCs treated with the various POVPC concentrations indicated for 2 hours. KLF4 mRNA expression was analyzed by real-time RT-PCR and normalized to 18S rRNA level, and expressed as fold-increase over vehicle. Values represent the mean±SD. *P<0.001 vs vehicle. B, Cultured SMCs were treated with 10 µg/mL POVPC or vehicle for the indicated time. Total KLF4 mRNA level was determined by real-time RT-PCR. KLF4 expression in the each sample was normalized to 18S rRNA level, and expressed as fold-increase over vehicle. Values represent the mean±SD. *P<0.01; **P<0.005 vs vehicle. Experiment is representative of 3 independent studies. C, Cultured SMCs were treated with 10 µg/mL POVPC or vehicle for 24 hours. Nuclear and cytoplasmic protein extracts were isolated as described in Methods and 10 µg of each sample were analyzed by Western blotting with anti-KLF4 and anti-Lamin A antibodies. Total protein extract of SMCs transfected with pcDNA3/KLF4 was used as a positive control.


Figure 5
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  		Figure 5. POVPC-induced KLF4 nuclear translocation in SMCs. A, POVPC induced nuclear translocation of FLAG-tagged KLF4. SMCs were transfected with pcDNA3-KLF4-FLAG expression plasmid and treated with vehicle and 20 µg/mL of POVPC for 24 hours. Immunofluorescence assays were performed using anti-FLAG antibody and Cy3 secondary antibody (Red). DAPI was used for nuclear counterstaining (Blue). B, Quantification of change in KLF4-FLAG localization on POVPC treatment. C, Endogenous KLF4 expression and localization after treatment of SMCs for 24 hours with vehicle and 20 µg/mL POVPC. Immunofluorescence assays were performed with KLF4 antibody and Cy3 secondary antibody (Red). DAPI was used for nuclear counterstaining (Blue). The experiment was repeated 3 times, and the representative data are shown.

siRNAs Specific to KLF4 Inhibited POVPC-Induced Suppression of SMC Marker Genes
To determine whether KLF4 is required for POVPC-induced suppression of SMC marker genes, SMCs were treated with POVPC in the presence of 2 different siRNA oligonucleotides specific for KLF4. Results showed that anti-KLF4 siRNA, but not a control anti-EGFP siRNA, inhibited POVPC-induced suppression of SM {alpha}-actin and SM MHC (Figure 6A). KLF4 mRNA expression was induced by 16.5-fold after POVPC treatment of SMCs, and was nearly completely suppressed by either of 2 KLF4 siRNAs (Figure 6B). The efficacy of the KLF4 siRNA oligonucleotide used in these studies was documented at both the mRNA (Figure 6B) and protein (Figure 6C) levels. In the latter case, total protein extracts of SMCs transfected with either EGFP siRNA or KLF4 siRNA and treated with PDGF-BB were used as positive controls because our laboratory has previously shown KLF4 induction by PDGF-BB.21,22 Endogenous KLF4 protein level was markedly increased in control cells transfected with nonspecific siRNA (siEGFP) and treated with POVPC (Figure 6C). This increase was virtually eliminated by the KLF4 siRNA. Expression of ß-actin mRNA was not changed after siEGFP or siKLF4 transfection (supplemental Figure IIB). We also tested the contribution of KLF4 to POVPC-induced suppression of SMC marker genes in primary cultures of mouse aortic SMCs. Aortic SMCs were isolated from the aorta of KLF4 floxed mice,23 and cells infected with adenovirus expressing Cre-recombinase (KLF4 KO SMCs) or control adenovirus (Control SMCs). Treatment with 10 µg/mL POVPC dramatically decreased expression of SM {alpha}-actin and SM-MHC in control mouse aortic SMCs (Figure 6D). In contrast, effects of POVPC on SMC marker gene expression were markedly attenuated in KLF4 KO SMCs. Taken together, the preceding results provide evidence that KLF4 is a key down-stream effector of POVPC-induced SMC phenotypic switching.


Figure 6
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  		Figure 6. POVPC-induced suppression of SMC marker genes was mediated by KLF4. A, Cultured SMCs were transfected with KLF4 siRNA oligonucleotides (siKLF4 #13 and siKLF4 #8) or nonspecific control oligonucleotides (siEGFP) with POVPC or vehicle treatment as described in Methods. Total RNA samples were isolated, and SM {alpha}-actin and SM MHC mRNA expression analyzed by real-time RT-PCR. Values were normalized for 18S rRNA level. The experiment was repeated 3 times and the representative data are shown. Values represent the mean±SEM. *P<0.001. B, To validate the knockdown efficacy of KLF4 siRNA, the endogenous KLF4 mRNA expression was analyzed by real-time RT-PCR and normalized for 18S rRNA level, and expressed as fold-increase over vehicle. The data are representative of 4 experiments. Values represent the mean±SD. *P<0.005 vs vehicle. C, Twenty micrograms of total protein from lysates of SMCs transfected with KLF4 siRNA or EGFP siRNA were subjected to Western blotting using anti-KLF4 and anti-GAPDH antibody. The band intensity was quantified by densitometry. Relative band intensity was normalized to the band intensity of GAPDH and presented as fold-increase over vehicle. D, SMCs were isolated from the aorta of KLF4 floxed mice and infected with adenovirus expressing Cre recombinase (KLF4 KO SMCs) or control adenovirus (Control SMCs). KLF4 KO SMCs and Control SMCs were treated with 10 µg/mL POVPC for 24 hours and expression of SM {alpha}-actin and SM MHC was determined by real-time RT-PCR. Values were normalized to 18S rRNA level and expressed as fold-increase over vehicle. Values represent the mean±SEM. *P<0.01 vs vehicle.

POVPC Markedly Suppressed Expression of the SMC Selective SRF Coactivator Myocardin in a KLF4 Dependent Manner
To further investigate mechanisms by which POVPC suppresses expression of SMC differentiation marker genes, we determined its effects on expression of the highly potent SRF coactivators, myocardin, and the myocardin-related factors, MKL1 and MKL2. Of major interest, POVPC treatment decreased myocardin mRNA expression by 40% and was blocked by the 2 KLF4 siRNAs (Figure 7A). Conversely, POVPC had no effect on MKL1 and MKL2 mRNA levels and the KLF4 siRNAs had no effect on expression of these genes (Figure 7B). We also tested the effect of POVPC on myocardin and KLF4 expression in rat carotid arteries in vivo by the application of pluronic gel with POVPC to rat carotid arteries for 6 hours. Although results did not achieve statistical significance, there was a trend toward POVPC increasing KLF4 expression while simultaneously decreasing myocardin expression (supplemental Figure VI). These results provide evidence that POVPC-induced suppression of SMC marker genes is attributable, at least in part, to KLF4-dependent suppression of myocardin but not MKL1/2 expression.


Figure 7
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  		Figure 7. POVPC decreased expression of myocardin, but not expression of MKL1 and MKL2 in cultured SMCs. Total RNA samples from Figure 6 were subjected to real-time RT-PCR for myocardin (A), MKL1, and MKL2 (B). Values were normalized for 18S rRNA level. Values represent the mean±SD. *P<0.01 vs vehicle.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Despite advances made in understanding mechanisms that promote proliferation and migration of SMCs associated with vascular disease, the mechanisms by which SMC marker gene expression is repressed in phenotypically modulated SMCs within lipid-rich atherosclerotic lesions are poorly understood. In this report, we provide novel evidence showing that a number of oxPLs including POVPC and oxPAPC, which are known to induce vascular inflammation,7,8 also regulate SMC phenotypic switching in vitro and in vivo as evidenced by downregulation of SMC differentiation marker genes. Results of the present studies are also the first, to our knowledge to identify a specific factor that is capable of inducing phenotypic switching of SMC in vivo (Figure 3). Observations in the present studies showing that various oxPLs that are present within atherosclerotic lesions induce marked suppression of SMC marker genes raise the possibility that these factors may play a key role in SMC phenotypic switching in the pathogenesis of atherosclerosis in addition to their well-documented role in controlling vascular inflammation.7,8,16,17 However, further studies will be needed to directly test the role of oxPL-induced SMC phenotypic switching in atherosclerotic disease progression in vivo, and in mediating late stage clinical events such as plaque rupture.

It is possible that the effects of oxPLs on SMC marker gene expression observed in our in vivo studies could be secondary to activation of other cell types as a part of an inflammatory response. A number of inflammation-related genes such as cytolisin, MCP-3, and hemeoxygenase-1 were induced in our cultured SMCs after POVPC treatment (supplemental Figure V). OxPAPC has also been shown to induce a subset of atherosclerotic genes in carotid arteries including MCP-1, IL-6, hemeoxygenase-1, and Egr-1, as well as enhanced adhesion of monocytes.24–26 The distribution of these chemokines was shown throughout the vessel wall as well as the induction of VCAM-1, MCP-1, and keratinocyte derived chemokine (KC), indicating that the oxPAPC effect was not restricted to the adventitial surface of the vessel but rather penetrated the entire vessel wall.7 As such, the effects of oxPLs on SMC we observed in vivo might be indirect through one or more of these secondary pathways. However, our observations that purified oxPLs could profoundly repress SMC marker gene expression in both rat and mouse vascular SMC in culture indicate that they are capable of exerting direct effects on SMC phenotype. We also observed that POVPC induced a modest increase in 3H-thymidine incorporation in cultured SMCs (supplemental Figure VII) and that POVPC induced enhanced repair in a scratch wound assay (supplemental Figure VIII), suggesting that oxPLs may also modulate functional properties of SMC important in lesion formation. There is also evidence that oxPLs stimulate angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids in endothelial cells.27 Results of our studies showed that POVPC increased VEGF and MCP-1 secretion by SMCs (supplemental Figure VB and VC). As such, it is possible that at least some of the effects of oxPLs on SMCs phenotypic switching may involve production of autocrine growth factors like VEGF.

Results of the present studies demonstrated that POVPC-induced suppression of SMC marker genes was dependent on KLF4, a factor that we have previously shown potently suppresses expression of multiple SMC marker genes, and to be required for PDGF-BB induced phenotypic switching.21,28,29 Significantly, we previously showed that suppression of the SM22{alpha} gene in vivo in response to vascular injury30 and experimental atherosclerosis31 was dependent on a G/C repressor element that closely matches the consensus DNA binding motif for KLF4.32 G/C repressor elements also exist within other SMC marker genes including SM MHC,33,34 and the recently identified myocardin promoter35 contains multiple potential consensus KLF4 binding sites. As such, it is interesting to postulate that KLF4 binding to G/C repressor elements within SMC marker genes, or KLF4 binding sites within the myocardin promoter may contribute to coordinate repression of SMC marker genes under a variety of pathophysiological conditions. We previously demonstrated that KLF4 suppresses SMC marker gene expression through a variety of potential mechanisms including: (1) reducing SRF binding to CArG elements within intact chromatin through induction of histone hypoacetylation22; (2) decreasing expression of myocardin21; and (3) binding to SRF and reducing its binding to SMC promoter CArG elements.21 Results of the present studies have extended these findings by showing that POVPC not only activates expression of KLF4, but also induces its rapid nuclear localization.

Whereas KLF4 is required for POVPC-induced suppression of SMC marker genes, it may not be sufficient to fully account for its effects. Multiple repressor pathways have been shown to contribute to PDGF-BB-induced phenotypic switching of SMCs in addition to KLF4.36 Of interest, we found that ERK inhibitors partially blocked POVPC-induced repression of SMC marker genes (Pidkovka and Owens, unpublished observations, 2005), indicating that this pathway also contributes to effects of oxPLs. In contrast, effects of oxPLs were not blocked by overexpression of an I-{kappa}B super-repressor dominant negative construct, indicating that NF-{kappa}B is not involved in this response (data not shown). These latter observations are consistent with studies showing that oxPLs-induced activation of endothelial cells are also mediated by activation of MAP-kinase signaling, rather than NF-{kappa}B pathways.

We37–39 and others40,41 have shown that the SRF coactivators, myocardin, and the myocardin-related transcription factors, MKL1 and MKL2, markedly stimulate expression of CArG-dependent SMC marker genes. Moreover, siRNA-induced suppression of each of these genes showed that they act in a cooperative fashion to regulate expression of a large cohort of SMC differentiation marker genes including SM {alpha}-actin, SM MHC, and SM22{alpha}.39 Results of the present studies provided evidence that oxPL-induced suppression of SMC marker genes is mediated at least in part by reduced expression of myocardin. However, surprisingly, oxPLs had no effect on expression of MKL1 and MKL2 in our studies, at least at the mRNA level, indicating that suppression of these factors may not contribute to phenotypic switching in this model system, although it is possible that oxPLs may inhibit the function of MKL1 or MKL2 through posttranscriptional control mechanisms. Consistent with this possibility, our laboratory39 previously demonstrated that PDGF-BB–induced suppression of SMC marker genes was mediated through the combinatorial mechanisms including downregulation of myocardin expression, but also through inhibition of the association of myocardin/MKL factors to CArG-containing SMC marker gene promoters within intact chromatin. Moreover, we presented evidence that this occurred by competition between myocardin/MKL factors and phosphorylated Elk-1 for SRF binding.39 Further studies will be required to directly test whether oxPLs have similar effects.

In summary, results of the present studies provide novel evidence showing that oxPLs such as POVPC profoundly suppress expression of multiple SMC differentiation marker genes and that these effects, at least in cultured SMCs, are dependent on KLF4. In addition, we found that POVPC also induced expression of a number of proinflammatory genes, and increased repair within a SMC scratch wound assay by SMCs. These findings are of major interest, because they are the first to define a class of compounds known to be present at high concentrations within lipid-rich atherosclerotic lesions,5,16,42 that can induce a SMC inflammatory phenotype. Further studies are needed to directly test the role of these compounds in SMC phenotypic switching in experimental atherosclerosis and to extend our understanding of the functional consequences of this process during different stages of disease development, progression, and end-stage events including plaque rupture.


*    Acknowledgments
 
The authors acknowledge the expert technical assistance of Rupande Tripathi and Dominique Rose. We are grateful to Dr Abbes Belkhiri for the excellent ideas and contribution to immunofluorescence experiments.

Sources of Funding

This work was supported by NIH grants P01HL19242, R37HL57353, R01HL38854 (to G.K.O.), and RO1HL084422-01 (to N.L.).

Disclosures

None.


*    Footnotes
 
Original received March 24, 2007; revision received July 12, 2007; accepted August 8, 2007.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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