12/15 Lipoxygenase Mediates Monocyte Adhesion to Aortic Endothelium in Apolipoprotein E-Deficient Mice Through Activation of RhoA and NF- B

【摘要】  Objectives- 12/15 lipoxygenase (12/15LO) has been implicated as a mediator of inflammation and atherosclerosis. In the current study, we identified mechanisms through which 12/15LO mediates monocyte:endothelial interactions in vivo in apolipoprotein E-deficient mice (apoEKO), a well-characterized mouse model of atherosclerosis.

Methods and Results- In apoEKO mice that are also deficient in 12/15LO (doubleKO), monocyte adhesion to aorta in vivo was reduced by 95% in doubleKO mice compared with apoEKO mice. Inhibition of 12/15LO in apoEKO mice in vivo using CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate) prevented monocyte adhesion to aortic endothelium in apoEKO mice. Aortic endothelium of apoEKO mice had significant activation of rhoA compared with doubleKO aortic endothelium. Further, apoEKO aorta displayed significant activation of NF- B. DoubleKO aorta displayed little nuclear localization of NF- B. Finally, we found significant upregulation of intercellular adhesion molecule-1 (ICAM-1) on apoEKO aortic endothelium compared with doubleKO endothelium. Inhibition of rhoA and PKC significantly reduced NF- B activation, ICAM-1 induction, and monocyte adhesion to aorta.

Conclusions- We conclude that 12/15LO products activate endothelial rhoA and PKC. Activation of rhoA and PKC cause activation and translocation of NF- B to the nucleus, which, in turn, results in induction of ICAM-1. Induction of ICAM-1 on aortic endothelium stimulates monocyte:endothelial adhesion in vivo in apoEKO mice.

L12/15 lipoxygenase (12/15LO) products activate endothelial rhoA and PKC. Activation of rhoA and PKC causes activation and translocation of NF- B to the nucleus, which, in turn, results in induction of ICAM-1. Induction of ICAM-1 on aortic endothelium stimulates monocyte:endothelial adhesion in vivo in apoEKO mice.

【关键词】  lipoxygenase NF B ICAM endothelium

Introduction

Akey early event in atherosclerosis development is the interaction of monocytes and endothelial cells in the vessel wall. 1 Activated monocytes interact with selectins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell surface to roll along and firmly adhere to the endothelium. 2,3 Monocytes are the primary inflammatory cells localized to human atherosclerotic plaques and play a major role in atherosclerotic plaque progression. 4 The apolipoprotein E-deficient mouse (apoEKO) is a well-characterized model of atherosclerosis that develops extensive aortic and coronary atherosclerosis and severe hypercholesterolemia when fed a Western-type diet. 5,6 Studies have reported increased P-selectin-mediated and VCAM-1-mediated monocyte rolling along apoE-deficient mouse endothelium, 7,8 and have illustrated that VCAM-1 and ICAM-1 are critical for atherosclerosis development in apoEKO mice. 9,10 See page 1204

12/15 lipoxygenase (12/15LO) incorporates molecular oxygen in a stereo-specific manner into arachidonic and linoleic acids to generate 12-S- and 15-S-hydroxyeicosatetraenoic acids (12SHETE/15SHETE) and 13-S-hydroxyoctadecadienoic acid (13SHODE), respectively. 11,12 Sources of 12/15LO eicosanoids in vascular cells are endothelial cells, smooth muscle cells, monocytes, and platelets. 13 Several groups have shown that the human 12/15LO enzyme oxidizes low-density lipoprotein (LDL) in vitro, and that 12/15LO inhibitors decrease the ability of macrophages to oxidize LDL. 14-17 12/15LO protein has been localized to aortic atherosclerotic lesions in rabbits and in humans. 18,19 We have shown that activated human endothelial cells (ECs) have increased expression of 12/15LO protein that mediates monocyte adhesion. 20 We have also shown that exogenous addition of 12SHETE and 15SHETE to EC significantly increased binding of monocytes. 20 Importantly, using a catalytic ribozyme to inactivate 12/15LO mRNA in ECs, we reduced 12SHETE production and blocked monocyte adhesion. 21 Striking evidence for a role for 12/15LO in atherogenesis came from the studies of Funk et al, who showed that disruption of the 12/15LO gene in apoE-deficient and LDL receptor-deficient mice significantly reduced atherosclerosis development in vivo. 22,23 The mechanism of action of 12/15LO in atherosclerosis remains unclear, and probably relates to signaling by several 12/15LO products in the endothelium of the vessel wall and in monocyte/macrophages.

In the current study, we identify how 12/15LO signals in apoE-deficient mice to upregulate monocyte adhesion to aortic endothelium. We found that 12/15LO activates rhoA and NF- B in vivo in apoEKO aorta to cause increased monocyte:EC interactions. Inhibition of 12/15LO activity in vivo blocks monocyte adhesion to apoE-deficient mouse aorta.

Methods

Detailed methods can be found in an online supplement at http://atvb.ahajournals.org.

Results

Decreased ICAM-1 Expression in ApoE-Deficient x 12/15LO-Deficient Mice

We have previously reported increased ICAM-1 expression in 12/15LO transgenic mice. 24 We first tested whether ICAM-1 was decreased in apoEKO x 12/15LO double knockout (doubleKO) mice compared with apoEKO mice. Total RNA was isolated from aortic endothelial cells (MAEC) from apoEKO and doubleKO aorta and quantitative real-time polymerase chain reaction was performed to measure ICAM-1 mRNA expression. MAEC from doubleKO had significantly lower ICAM-1 mRNA expression than apoEKO ( P <0.005) ( Figure 1 ). Levels of VCAM-1 and E-selectin mRNA were similar between both groups (data not shown).

Figure 1. ICAM-1 mRNA expression in apoE -/- mice is mediated by 12/15 LO products. Total cellular RNA from apoE -/- (apoEKO) and apoE -/- x 12/15 LO -/- (doubleKO) aortic EC were analyzed for murine ICAM-1 using quantitative real-time polymerase chain reaction. *Significantly lower ICAM-1 expression than apoE -/- mRNA, P <0.005 by Student t test.

Inhibition of 12/15LO In Vivo Blocks Monocyte Adhesion to ApoE Mouse Aorta

We next directly tested whether monocyte adhesion to aortic endothelium was altered in apoEKO versus doubleKO mice. Aortas from apoEKO and doubleKO mice were used in an ex vivo monocyte adhesion assay. Aortas from apoEKO bound 5-fold more monocytes than did doubleKO aortas ( P <0.001) ( Figure 2 ). To confirm that the increased monocyte adhesion to aorta observed in the apoEKO mice is caused by 12/15LO activity, we used the 12/15LO inhibitor CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate). The apoEKO mice were treated in vivo with a single intraperitoneal injection of 8 mg/kg CDC. Ma et al have shown that this dose of CDC reduces urinary 12SHETE concentrations by 60% in 24 hours in rodents. 25 After 24 hours, aortas were isolated and used in the monocyte adhesion assay ex vivo. Treatment of apoEKO mice with CDC in vivo caused a dramatic 75% reduction in monocyte adhesion to aorta ( Figure 2 ). Sesame oil-injected mice were used as vehicle controls and showed no reduction in monocyte adhesion (92±10 monocytes bound/field). The CDC-treated apoEKO mice had approximately the same numbers of monocytes adherent to aorta as did the doubleKO mice. To confirm that 12/15LO products were indeed mediating monocyte adhesion, we performed a series of "addback" experiments in which we incubated doubleKO mouse aortas for 4 hours with 12/15LO product eicosanoids before performing a monocyte adhesion assay. We found that addition of 100 nmol 12SHETE to doubleKO aortas significantly increased monocyte adhesion by 3-fold (12±2 monocytes bound/field for doubleKO aorta and 13±2 monocytes bound for ethanol vehicle control versus 39±6 monocytes bound/field for 12SHETE addition to doubleKO aorta ( P <0.001)). Addition of 13SHODE to doubleKO aorta resulted in a 2-fold increase in monocyte adhesion with 28±5 monocytes bound/field ( P <0.006). Addition of 15SHETE showed only a slight increase (19±2 monocytes/bound); however, this increase did not reach statistical significance. Addition of 12RHETE, which is not a product of the 12/15LO pathway, had no effect on adhesion (12±2 monocytes bound/field). Addition of eicosanoids to apoEKO aorta did not further increase monocyte adhesion, suggesting that the 12/15LO pathway is the primary pathway mediating the increased adhesion observed in apoEKO aorta. These findings are shown in supplemental Figure I (please see http://atvb.ahajournals.org). These data indicate that the majority of monocyte:endothelial interactions in apoEKO mouse aorta in vivo are mediated by 12/15LO.

Figure 2. Inhibition of 12/15LO in vivo reduces monocyte adhesion to aorta in apoE -/- mice. Aortas were isolated from apoE -/- (apoEKO), apoE -/- x12/15 LO -/- (doubleKO), and apoE -/- mice injected for 24 hours with 8 mg/kg CDC (ApoEKO+CDC). Fluorescently labeled monocytes were added to the aortas for an adhesion assay and counted by blinded observers using a fluorescent microscope. *Significantly lower than apoE -/-, P <0.0002; # significantly lower than apoE -/-, P <0.0001 by ANOVA. Data represent the mean±SE of 3 counted grids/aorta from 3 mice per group.

NF- B Is Activated in ApoEKO Aortic Endothelium

Activation of NF- B increases endothelial ICAM-1 expression as well as monocyte adhesion to endothelium. 26-28 On NF- B activation, the I B kinase complex phosphorylates I B, resulting in its degradation. This unmasks a nuclear localization signal on the p65 subunit resulting in its translocation to the nucleus. In the first set of experiments, we obtained nuclear extracts from apoEKO and doubleKO mouse aortic endothelium. We performed Western immunoblotting to look for expression of the p65 subunit of NF- B in the nucleus. We found that apoEKO endothelium had increased p65 expression in the nucleus compared with doubleKO endothelium, ( Figure 3 a), indicating translocation of NF- B from the cytosol to the nucleus to mediate inflammatory gene transcription. Interestingly, doubleKO aortic endothelium had almost no p65 expression in the nucleus ( Figure 3 a), suggesting that NF- B was not activated in doubleKO mouse aortas.

Figure 3. Lack of 12/15LO reduces NF- B nuclear translocation. A, Nuclear lysates. Nuclear extracts were collected from apoE -/- (apoEKO) and apoE -/- x 12/15 LO -/- (doubleKO) mouse aortic endothelium. Aortas were treated for 4 hours with either a PKC inhibitor GO6976 (+GO6976) or a rho kinase inhibitor Y27632 (+Y27632). Expression of the p65 subunit of NF- B in the nucleus was determined by immunoblotting. B, Fluorescence microscopy of nuclear p65 staining. ApoE -/- (apoEKO) and apoE -/- x 12/15 LO -/- (doubleKO) aortic EC were treated with 10 µmol/L GO6976 (+GO6976) or 10 µmol/L Y27632 (+Y27632) for 4 hours. NF- B translocation was visualized by fluorescent microscopy using an antibody specific for the p65 subunit of NF- B together with an Alexa 594-conjugated secondary antibody.

The Rho family of GTPases can activate NF- B-dependent gene expression in EC. 26 RhoA stimulates NF- B through its downstream effector Rho kinase (ROCK). 29 Interestingly, we found that both a rho kinase inhibitor (Y27632 ) and a PKC inhibitor (GO6976) dramatically reduced NF- B activation in apoEKO mouse aortas.

We also confirmed the lack of p65 translocation to the nucleus of doubleKO aortic endothelium using fluorescence microscopy. Using an anti-p65 antibody together with an Alexa 594-conjugated secondary antibody, we found that NF- B is located primarily in the nucleus of apoEKO mouse endothelium, again confirming NF- B activation and translocation ( Figure 3 b). In contrast, doubleKO endothelium had little expression of nuclear p65, indicating the NF- B was not activated in the doubleKO mice ( Figure 3 b). Pretreatment of apoEKO MAEC with the rho kinase inhibitor, the PKC inhibitor, or both compounds together dramatically reduced p65 nuclear localization. The percentage of nuclei positive for NF- B p65 was 77±4% for apoEKO, 22±2% for apoEKO+Y27632, 20±1% for apoEKO+Go6976, 18±1% for apoEKO+both inhibitors together; 12±1% for doubleKO, 8±1% for doubleKO+ Y27632, 10±2% for doubleKO+Go6976, and 8±1% for doubleKO+both inhibitors together. Taken together, these data indicate that 12/15LO enzyme activity causes activation of NF- B in apoEKO mouse aorta.

Activation of RhoA in ApoEKO Aortic EC

We have reported that 12/15LO transgenic mice have increased rhoA activation caused by increased 12SHETE production. 28 Based on these previous data as well as the data in Figure 3, we directly examined activation of rhoA in both apoEKO and doubleKO aortic endothelium. RhoA activity was measured by affinity precipitation of active RhoA with the -binding domain of rhotekin as previously described. 30 We found increased rhoA activation in apoEKO endothelium compared with doubleKO endothelium, which showed little or no rhoA activation ( Figure 4 ). These data suggest that the activation of rhoA by the 12/15LO pathway in apoEKO mice significantly contributes to increased monocyte:EC interactions in these mice. Taken together, these data in Figures 3 and 4 illustrate that endothelial NF- B activation is most likely mediated through rhoA and PKC activation.

Figure 4. RhoA activation is modulated by 12/15LO. RhoA activation was measured in aortic EC isolated from apoE -/- (apoEKO) and apoE -/- x 12/15 LO -/- (doubleKO). RhoA activation in apoE -/- x 12/15 LO -/- (doubleKO) vs apoE -/- (apoEKO) is shown.

Mechanisms Contributing to Accelerated Monocyte Adhesion by 12/15LO

We next examined whether the rho kinase inhibitor or the PKC inhibitor modulated ICAM-1 expression in apoEKO cells. Expression of ICAM-1 in MAEC was measured by quantitative real-time polymerase chain reaction. MAEC from apoEKO had significantly higher ICAM-1 expression than doubleKO (supplemental Figure IIA). Pretreament of apoEKO MAEC with the rho kinase inhibitor, the PKC inhibitor, or both compounds together significantly reduced ICAM-1 mRNA ( P <0.0001). We also analyzed ICAM-1 surface expression in apoEKO and doubleKO MAEC by flow cytometry. ICAM-1 is constitutively expressed on endothelium; the percentage of ICAM-1 expression on EC ranged from 73% in doubleKO to 78% in apoEKO mouse EC. The mean fluorescence intensity (MFI) values indicated dramatic changes in ICAM-1 expression on EC. The MFI of doubleKO EC was 76, apoEKO was 151, apoEKO+Y27632 was 114, apoEKO+Go6976 was 123, and apoEKO+both inhibitors was 101. These data are illustrated graphically in supplemental Figure IIB. MAEC from doubleKO mice (shown in red) had significantly lower expression of ICAM-1 compared with apoEKO (shown in blue). Pretreatment of apoEKO MAEC with the PKC inhibitor (shown in orange), the rho kinase inhibitor (shown in turquoise), or both compounds together (shown in green) significantly reduced surface ICAM-1 ( P <0.0001; supplemental Figure IIB).

RhoA and PKC Inhibition Decrease Monocyte Adhesion to Aorta of ApoEKO Mice

Finally, we directly tested whether inhibition of rhoA or PKC activation altered monocyte adhesion to aortic endothelium in apoEKO mice. Aortas were isolated from apoEKO and doubleKO mice and were used in an ex vivo monocyte adhesion assay. As shown in Figure 5 a, there was a dramatic reduction ( 85%) in the number of monocytes bound to doubleKO endothelium compared with apoEKO endothelium. Treatment of apoEKO aortas for 4 hours with either the Rho kinase inhibitor or with the PKC inhibitor reduced monocyte adhesion by 70% ( Figure 5 a). Treatment of apoE aortas with both compounds together significantly reduced monocyte adhesion by 75% ( Figure 5a and 5 b). The use of both compounds blocked monocyte adhesion to a level similar as to that observed in the double KO mice ( Figure 5a and 5 b). Interestingly, neither the PKC inhibitor nor the rhoA inhibitor had any effect on monocyte adhesion in doubleKO aortas ( Figure 5 b), again supporting the concept that 12/15LO products signal through a rhoA/PKC pathway to mediate monocyte adhesion. These data indicate that rhoA and PKC signaling are involved in mediating monocyte:EC adhesion to apoE aorta.

Figure 5. PKC and rho kinase inhibitors reduce monocyte:EC adhesion to apoE -/- aorta. A, Fluorescence microscopy of aorta monocyte adhesion assay. Aortas were isolated from apoE -/- (apoEKO) and apoE -/- x 12/15 LO -/- (doubleKO) mice and incubated for 4 hours in the absence and presence of 10 µmol/L GO6976 (+GO6976), 10 µmol/L Y27632 (+Y27632), or both (+BOTH) inhibitors. Fluorescently labeled monocytes were added to the aortas for an adhesion assay. B, Quantification of monocyte adhesion assay. Using a fluorescent microscope, the number of monocytes firmly adhered to the endothelium were counted by blinded observers in 3 different fields. *Significantly higher adhesion than apoE -/- x 12/15 LO -/-, P <0.0001; **significantly lower than apoE -/-, P <0.0001 by ANOVA. Data represent the mean±SE of 3 counted grids/aorta from 3 mice per group.

Discussion

The protein sequence of murine 12/15LO is most closely related to 15-LO in humans. 31-33 Although these enzymes share similarity, and the mouse enzyme is referred to as "12/15LO," there are subtle differences in the enzymes, especially in their generation of eicosanoid products. Human 15-LO generates 15SHETE and 12SHETE at a ratio of 9:1, yet murine 12/15LO generates 15SHETE and 12SHETE at a ratio of 1:4. 31,34 Most studies have reported 12SHETE to be pro-inflammatory, 13,20,24,28,35-40 whereas the inflammatory role of 15SHETE is unclear. 15SHETE can serve as a substrate for 5-LO to generate anti-inflammatory lipoxins, 41-43 and can also serve as a ligand for the nuclear hormone receptor PPAR. 44,45 Two studies in transgenic rabbits that overexpressed human 15-LO indicated that 15-LO expression reduced atherosclerosis development, 46,47 whereas 2 other studies indicated that treatment of rabbits with 15-LO inhibitors reduced atherosclerosis progression. 48,49 Although murine 12/15LO has been shown to mediate atherosclerosis development in mouse models of atherosclerosis, the exact mechanisms through which this occurs are unclear. Endothelial activation and subsequent monocyte recruitment and adhesion to aortic endothelium are key early events in atherosclerotic plaque initiation. 50,51 In the current study, we show that monocyte:endothelial adhesion to apoEKO mouse aorta is dependent on 12/15LO activity in vivo. Inhibition of 12/15LO activity dramatically reduces monocyte adhesion to apoEKO aorta. Further, we show that a primary mechanism by which 12/15LO mediates monocyte adhesion to aortic endothelium is through upregulation of ICAM-1 through activation of a rhoA/NF- B signaling pathway.

We have previously reported that rhoA and PKC work synergistically in endothelium to activate NF- B in response to 12/15LO products. 28 There are reports that RhoA and PKC work together in EC to regulate each other?s activity. 52,53 Furthermore, inhibition of RhoA can block PKC translocation and activation in EC. 52 Our previous data indicated that RhoA is upstream of PKC. 28 Thus, our data indicate that 12/15LO products activate endothelial rhoA and PKC, which, in turn, activate NF- B to upregulate endothelial ICAM-1 expression. Upregulation of ICAM-1 by this signaling pathway stimulates monocyte:EC interactions. Our data further illustrate that this RhoA/NF- B signaling pathway is a primary regulator of aortic endothelial activation in apoEKO mice. Inhibition of 12/15LO activity in vivo in the apoEKO mouse, either through pharmacological inhibition by CDC ( Figure 2 ) or through molecular gene targeting strategies by the use of knockout mice, prevents rhoA and NF- B activation, which subsequently prevents monocyte:EC adhesion in the apoEKO aorta.

The activation of endothelial rhoA by 12/15LO suggests that 12/15LO eicosanoid products bind to a specific G protein-coupled receptor on endothelium. We hypothesize that the 12/15LO eicosanoids are secreted by both activated endothelium and monocytes and bind to a specific G protein-coupled receptor on vascular cells, thus amplifying and prolonging the inflammatory response. A specific 12SHETE receptor has not yet been identified; however, Szekeres et al have reported the presence of putative low-affinity and high-affinity 12SHETE receptors in carcinoma cells. 54 We have previously reported that G12/G13 signaling is involved in the activation of rhoA by 12SHETE in endothelium, 28 further suggesting involvement of a G protein-coupled receptor. Studies to identify this receptor are currently ongoing in the laboratory.

The apoE-deficient mice that are also deficient in 12/15LO (double KO) have decreased expression of ICAM-1 on aortic endothelium compared with apoEKO mice ( Figure 1 and supplemental Figure II). ICAM-1 has been strongly linked to atherosclerosis development. 8,55,56 In the current study, we did not examine other adhesion molecules that are regulated by NF- B and rhoA. We reported several years ago that 12SHETE stimulated expression of connecting segment-1 (CS-1) fibronectin on the apical surface of human endothelium, 20 and that CS-1 served as a counter-receptor for VLA-4 on monocytes. 57 Thus, it is conceivable that apoEKO mouse aorta has significant expression of CS-1, whereas expression of CS-1 is reduced in the doubleKO mice. Based on our earlier in vitro data with CS-1, such a situation would significantly impact monocyte:EC interactions. Downregulation of rhoA has been found to impact the interactions of CS-1 with integrins. 58 Reagents to accurately quantify CS-1 fibronectin expression in the mouse are not available, so we currently cannot rule out a contribution of CS-1.

We focused this present study on ICAM-1 expression in apoE-deficient mice in vivo, and the signaling mechanisms that contribute to increased ICAM-1 expression mediated by 12/15LO. The reasons that we focused exclusively on ICAM-1 are that we recently found that 12SHETE stimulates ICAM-1 expression in endothelium, 28 and that ICAM-1 expression is increased in 12/15LO transgenic mice in vivo. 24 However, VCAM-1 and E-selectin promote rolling and adhesion of monocytes along endothelium 8,56,59-61 and both are regulated by NF- B. 62-64 Therefore, it is quite conceivable that surface expression of these molecules is downregulated in 12/15LO-deficient mice. We have preliminary data to suggest that mRNA expression of VCAM-1 and E-selectin is not reduced in 12/15LO-deficient mice (data not shown), but we have not measured surface expression of these molecules by flow cytometry. Downregulation of VCAM-1 and E-selectin expression would result in decreased monocyte rolling and adhesion to endothelium. We anticipate that the reduction in monocyte adhesion to aortic endothelium in the absence of 12/15LO is not solely caused by ICAM-1, and that other adhesion molecules may contribute to this process.

We also cannot rule out changes in chemokine production in apoEKO versus doubleKO mice. Monocyte chemotactic protein-1 (MCP-1) is regulated by NF B activation and is secreted by activated endothelium. 65-68 We have preliminary evidence to suggest that MCP-1 is increased by 12/15LO activity (data not shown). Lee et al have shown induction of MCP-1 by oxidized phospholipid components of LDL. 69 The induction of MCP-1 by 12/15LO products and oxidized LDL cause increased monocyte recruitment to the activated aortic endothelium. Funk et al have shown that IL-12 signaling by macrophages is regulated by 12/15LO and is reduced in doubleKO mice. 70 Thus, reductions in cytokine production in 12/15LO-deficient mice could certainly contribute to the reduction in monocyte adhesion to endothelium.

We previously examined the contribution of macrophage versus endothelial-derived 12/15LO eicosanoids as critical mediators of atherogenesis in apoE-deficient mice in vivo. 40 We found that presence of 12/15LO in bone marrow-derived cells was critical for atherogenesis. 40 However, we could not rule out contribution of endothelial 12/15LO in this process. Thus, we conclude that 12/15LO products, either monocyte-derived or endothelial-derived, can activate an endothelial surface receptor to cause endothelial activation via a rhoA/NF- B signaling cascade. This activation of endothelium by 12/15LO dramatically increases monocyte:endothelial adhesion, a key initiating event in atherosclerotic plaque development.

In summary, we report that 12/15LO is a critical mediator of monocyte adhesion to aorta of apoEKO mice in vivo. 12/15LO activity activates a rhoA/NF- B signaling pathway that results in the upregulation of ICAM-1 on the endothelial surface, thereby facilitating monocyte adhesion. Inhibition of 12/15LO activity in vivo in apoE-deficient mice dramatically reduces monocyte adhesion to aorta, indicating that the 12/15LO enzyme is a critical regulator of these processes in the vessel wall.

Acknowledgments

This work was supported by NIH P01 HL55798 (Project 2; C.C.H.) and NIH R01 HL071141 (C.C.H). The authors thank Dr A. Wayne Orr (University of Virginia) for advice on rhoA signaling, Dr Rama Natarajan (Beckman Research Institute, City of Hope, Duarte, Calif) for advice on the CDC studies, and Dr Jerry L. Nadler (University of Virginia) and Dr Colin Funk (Queen?s University, Ontario, Canada) for the gift of the apoE -/- x 12/15LO -/- mice.

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作者单位：From Cardiovascular Research Center, University of Virginia, Charlottesville.