High-Density Lipoprotein Promotes Endothelial Cell Migration and Reendothelialization via Scavenger Receptor-B Type I
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循环研究杂志 2006年第1期
the Departments of Pediatrics (D.S., C.M., A.K.G., L.L.G., K.L.C., L.D.H., M.L.C., P.W.S.) and Internal Medicine (W.V., R.L.K.) and the Donald W. Reynolds Cardiovascular Clinical Research Center (W.V.), University of Texas Southwestern Medical Center, Dallas, Tex
Lipoprotein and Atherosclerosis Research Group (Y.L.M.), University of Ottawa Heart Institute, Ottawa, Canada
the Department of Medicine (D.J.R.), University of Pennsylvania School of Medicine, Philadelphia, Pa.
Abstract
Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-IeC/eC mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BIeC/eC mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.
Key Words: high-density lipoprotein endothelium migration
Introduction
The risk of atherosclerosis is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol.1,2 HDL classically functions in reverse cholesterol transport (RCT), removing cholesterol from peripheral tissues and delivering it to the liver and steroidogenic organs by binding of the HDL apolipoprotein apolipoprotein A-I (apoA-I) to the HDL receptor scavenger receptor B type I (SR-BI).3,4 In mouse models of atherosclerosis, both apoA-I and SR-BI provide atheroprotection.5,6 However, as animal studies show that RCT is not dictated by circulating levels of HDL or apoA-I,7eC9 the changes in atherosclerosis risk related to varying HDL and apoA-I levels may not be explained by differences in RCT. In addition, clinical studies suggest that the risk for restenosis after vascular intervention may be inversely related to HDL levels,10,11 and the provision of apoA-I or HDL attenuates neointima formation after artery injury in hypercholesterolemic animal models.12,13 There is evidence that HDL also inhibits low-density lipoprotein oxidation and adhesion molecule expression in cultured endothelial cells, but the magnitude and impact of these processes in vivo is uncertain.14,15 As such, there is considerable evidence that HDL affords protection from vascular disease, but the mechanisms underlying the protection remain poorly understood.
Whereas it has been appreciated for some time that SR-BI is expressed in hepatocytes and steroidogenic tissues, we and others16eC20 have more recently demonstrated that SR-BI is also expressed in endothelium, where it mediates promodulatory effects of HDL on endothelial nitric oxide synthase (eNOS) to increase the abundance of NO. In addition to generating NO, an intact endothelial cell monolayer modulates local hemostasis and thrombolysis and provides a nonpermeable barrier protecting smooth muscle cells from circulating growth-promoting factors. Disruptions of endothelial cell monolayer integrity, either by gross denudation related to a vascular intervention or gap formation between cells due to disturbed shear stress, place the arterial wall at greater risk for vascular disease.21eC23 Furthermore, whereas repeated endothelial removal worsens vascular lesion severity,24 enhanced reendothelialization blunts lesion formation.25,26 To better understand the basis of HDL-related vascular protection, we designed experiments to determine whether HDL functioning through SR-BI promotes endothelial cell migration and the maintenance of endothelial monolayer integrity. HDL-induced migration, lamellipodia formation, and Rac GTPase activation were interrogated in cultured endothelium. Because HDL activation of eNOS is mediated by Src kinases, phosphatidylinositol 3-kinase (PI3K)/Akt kinase and p44/42 mitogen-activated protein kinase (MAPK),17 the roles of these signaling cascades in HDL modulation of Rac GTPase and migration were determined. Because NO is critically involved in endothelial migration and angiogenesis,27 dependence on NO was also evaluated. Furthermore, the impact of loss of SR-BI was investigated. Moreover, to determine whether these processes are operative in vivo, carotid artery reendothelialization was assessed after perivascular electric injury in mice in which apoA-I/HDL/SR-BI status was genetically manipulated.
Materials and Methods
Additional details are provided in the online-only data supplement, available at http://circres.ahajournals.org.
Cell Culture
Bovine aortic endothelial cells (BAECs) were harvested using procedures reported previously with minor modifications,28 cultured in endothelial growth medium-2 (Cambrex Bioscience) with 5% fetal bovine serum (Sigma), and studied at passages 5 to 9.
Endothelial Cell Migration Assay
BAECs were grown to near confluence, and a defined region of cells was removed with a razor blade. Cells were treated for 24 hours and then fixed, and the number of cells that had migrated past the wound edge was quantified.
Cytoskeletal Changes in Endothelial Cells
BAECs were plated onto glass coverslips, treated for 0 to 30 minutes, fixed and stained with Alexa 568-phalloidin (Molecular Probes, Inc), and viewed under a fluorescent microscope. The percent of cells with lamellipodia was quantified.
Rac Activity Assay
After treatment, Rac activity in BAECs was measured as previously described with minor modifications.29
Small Interfering RNA for Rac and SR-BI
Double-stranded RNA (dsRNA) sequences directed at bovine Rac or SR-BI were transfected into cells, and expression of the proteins and functional and signaling readouts were determined 24 to 48 hours later.
Carotid Artery Reendothelialization
Carotid artery reendothelialization was studied after perivascular electric injury in mice by assessing Evan’s blue dye uptake.30,31 Endothelial denudation and recovery after injury was confirmed by immunohistochemistry for von Willebrand factor (vWF). Study groups included wild-type mice, apoA-IeC/eC or SR-BIeC/eC mice, and apoA-IeC/eC mice with adenoviral reconstitution of apoA-I expression.
Blood Pressure by Radiotelemetry
Chronic blood pressure measurements were performed in 30- to 35-week-old SR-BI+/+ and SR-BIeC/eC mice by radiotelemetry.32
Statistical Analysis
All data are presented as mean±SEM. ANOVA with Neuman-Keuls post hoc testing was used to assess differences between 3 or more groups. Differences in reendothelialization were evaluated by Mann-Whitney tests. Significance was set at P<0.05.
Results
HDL and Endothelial Cell Migration
To determine the effect of HDL on endothelial cell migration, BAECs were wounded and treated with HDL. The lipoprotein caused a marked increase in migration (Figure 1A), with 2.8-, 3.6-, and 4-fold increases noted with 20, 50, and 100 e/mL HDL, respectively (Figure 1B), and the response to HDL was comparable to the response to vascular endothelial growth factor (VEGF) (Figure 1C). It has been previously reported that HDL activates endothelial cell migration33 and that this may be dependent on cargo molecules such as sphingosine-1-phosphate (S-1-P), which induces migration in a pertussis toxin (Ptx)-sensitive manner.34 To determine the contribution of S-1-P in HDL-induced migration, responses to HDL or S-1-P were assessed in the absence or presence of Ptx. Whereas S-1-P-induced migration was prevented by Ptx, HDL-mediated migration was not affected (Figure 1D). To determine whether the apolipoprotein, phospholipid, and cholesterol components of HDL are sufficient to stimulate migration, BAECs were treated with lipoprotein (Lp)2A-I particles reconstituted with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), cholesterol, and lipid-free recombinant apoA-I. Lp2A-I containing POPC, cholesterol, and apoA-I at molar ratios of 80:5:1 caused migration comparable to that promoted by native HDL (Figure 1E). These observations indicate that HDL stimulates endothelial cell migration and that the phospholipid, cholesterol, and apoA-I components are sufficient to mediate the process.
HDL, Lamellipodia Formation, and Rac GTPase
Because cell migration begins with key changes in the actin cytoskeleton, including the formation of lamellipodia,35,36 we tested the initial effects of HDL on the cytoskeleton. Under control conditions, BAEC displayed stress fibers and few lamellipodia (Figure 2A). Within 1 minute of HDL exposure, there was a decrease in stress fibers, an increase in lamellipodia, and membrane ruffling. This effect became more apparent at 5 minutes, the number of lamellipodia decreased at 15 minutes, and cells appeared similar to control at 30 minutes. As Rac GTPase mediates lamellipodia formation,37 we determined whether Rac is activated by HDL. Paralleling the findings for lamellipodia formation, there was an increase in Rac activity with HDL within 1 minute (Figure 2B). Activity was maximal at 5 minutes and decreased thereafter.
The role of Rac in HDL-induced endothelial cell migration and lamellipodia formation was then tested by depletion of Rac with small interfering RNA (siRNA) (Figure 2C). Whereas BAECs transfected with control siRNA migrated (Figure 2D) and had initial lamellipodia formation in response to HDL (Figure 2E), cells transfected with Rac siRNA did not. Thus, HDL causes lamellipodia formation in concert with Rac activation in endothelial cells, and both the lamellipodia formation and ensuing migration are mediated by Rac.
Role of Kinase Activation
To further delineate the mechanisms by which HDL stimulates endothelial cell migration, we determined whether the process entails the kinases that have been implicated in HDL-mediated eNOS activation.17 Whereas the Src kinase inhibitor PP2 did not affect basal migration, HDL-stimulated migration was blunted by 72% by PP2 (Figure 3A, left panel). PP3, the negative control compound for PP2, had no effect (data not shown). Treatment with the PI3K inhibitor LY294002 did not affect basal migration, but HDL-stimulated migration was fully inhibited (Figure 3A, middle panel). PD 98059, which inhibits the MAPK pathway, did not alter basal migration, but HDL-induced migration was attenuated by 84% (Figure 3A, right panel). Therefore, HDL-induced endothelial cell migration is dependent on Src family kinases, PI3K, and MAPK.
The roles of Src kinases, PI3K, and MAPK in HDL-induced lamellipodia formation were also evaluated. BAEC pretreatment with PP2 had no effect on the cytoskeleton at baseline. However, the 14-fold increase in lamellipodia formation with HDL (Figure 3B, left panel) was blunted by 87% by PP2. PP3 had no effect (data not shown). In contrast, both LY294002 and PD98059 did not modify lamellipodia formation with HDL (Figure 3B, middle and right panels, respectively). The efficacy of LY294002 and PD98059 was confirmed in studies of HDL-stimulated Akt and MAPK phosphorylation (data not shown).
The role of kinases in Rac activation by HDL was also investigated. Whereas treatment with PP2 did not alter basal activity, HDL-induced Rac activation was blunted by 67% (Figure 3C, left panel). Treatment with LY294002 did not affect basal Rac activity but it attenuated HDL-mediated Rac activation by 86% (Figure 3C, middle panel). Basal Rac activity was not affected by PD98059, but HDL-induced Rac activation was diminished by 82% (Figure 3C, right panel). Therefore, Src family kinases, PI3K, and MAPK are required for HDL-induced Rac activity. To verify the sequence of kinase and Rac activation, Rac was knocked-down with siRNA and HDL-induced kinase phosphorylation was tested. HDL caused comparable phosphorylation of Src, Akt, and MAPK in cells transfected with control and Rac siRNA (Figure 3D). Thus, Rac resides downstream of the kinases in the signaling cascade by which HDL activates endothelial cell migration.
Role of eNOS
Because HDL stimulates eNOS19 and NO promotes endothelial cell migration and angiogenesis,27 we determined whether eNOS is required for HDL-mediated migration. The treatment of BAECs with the NOS antagonist N-Nitro-L-arginine methyl ester (L-NAME) did not affect basal migration, and HDL-induced endothelial cell migration was also not attenuated (Figure 4A). In contrast, VEGF-stimulated migration was blocked by L-NAME (Figure 4B). L-NAME did not alter the basal number of cells displaying lamellipodia, and lamellipodia formation in response to HDL was not changed (Figure 4C). The efficacy of L-NAME was verified by confirming inhibition of eNOS enzymatic activity (data not shown). These findings indicate that, in contrast to the mechanisms of action of multiple known stimuli including VEGF,38 the promotion of endothelial cell migration by HDL is NO-independent.
Role of SR-BI
To determine whether SR-BI plays a role in HDL-mediated endothelial cell migration, SR-BI was knocked down by siRNA (Figure 5A). Whereas cells transfected with control siRNA displayed an 8.5-fold increase in migration with HDL (Figure 5B), cells transfected with SR-BI siRNA did not. In parallel, cells transfected with control siRNA had lamellipodia formation induced 5.3-fold by HDL (Figure 5C), but after knockdown of SR-BI, lamellipodia formation did not occur. Similarly, cells transfected with control siRNA displayed Rac activation by HDL (Figure 5D), and Rac activation was not demonstrable after SR-BI knockdown. As such, SR-BI is required for HDL-induced Rac activation and the resulting changes in the actin cytoskeleton that promote migration.
HDL and Reendothelialization In Vivo
To determine whether the mechanisms revealed in vitro are operative in vivo, carotid artery reendothelialization studies were performed in mice. The area of remaining denudation was determined after perivascular electric injury by the injection of Evan’s blue dye, which is incorporated in the region of denudation (supplemental Figure IA and IB).30,31 In control C57BL/6 mice, reendothelialization was complete by 7 days (supplemental Figure IC). Denudation of the endothelium after injury (1 day) and reendothelialization after injury (8 days) was confirmed by immunostaining for vWF (Data supplemental Figure ID). To determine the role of HDL in reendothelialization in vivo, reendothelialization was compared 5 days after injury in apoA-I+/+ and apoA-IeC/eC mice, which have HDL cholesterol levels that are decreased by 83% compared with wild-type.39 Greater reendothelialization occurred in apoA-I+/+ (Figure 6A) versus apoA-IeC/eC (Figure 6B) mice, as indicated by the larger area of remaining denudation in apoA-IeC/eC mice. Cumulative studies revealed that the area of remaining denudation was 52% larger in apoA-IeC/eC versus apoA-I+/+ mice (Figure 6C). Differences in reendothelialization 5 days after injury were confirmed by assessments of endothelial cell density in the region of prior injury by vWF immunostaining (data not shown).
To test wheter normal reendothelialization can be rescued by reconstitution of apoA-I expression in apoA-IeC/eC mice, liver-directed gene transfer of human apoA-I was performed at the time of artery injury. Five days after injection, apoA-IeC/eC mice given control adenovirus had HDL levels of 19.6±7.7 mg/dL, whereas mice receiving apoA-I-containing adenovirus had apoA-I levels of 148.4±49.4 mg/dL (P<0.05 versus control) and HDL levels of 69.7±25.2 mg/dL (P<0.05 versus control). Evan’s blue dye incorporation 5 days after injury demonstrated that in comparison with apoA-IeC/eC mice given control adenovirus (Figure 6D), mice injected with apoA-I-containing adenovirus displayed greater reendothelialization (Figure 6E). Quantitation in multiple mice indicated that there was 40% less denudation after the reconstitution of apoA-I expression in apoA-IeC/eC mice (Figure 6F). These findings indicate that apoA-I and HDL promote endothelial monolayer integrity in vivo.
SR-BI and Reendothelialization In Vivo
Having demonstrated that there is a major contribution of apoA-I/HDL to reendothelialization in vivo, we next determined the role of SR-BI in studies of SR-BI+/+ and SR-BIeC/eC mice 5 days after thermal injury. Greater reendothelialization occurred in SR-BI+/+ (Figure 7A) versus SR-BIeC/eC mice (Figure 7B) as indicated by the larger area of Evan’s blue dye incorporation in the latter. Cumulative studies indicated that the area of remaining denudation was 44% larger in SR-BIeC/eC versus SR-BI+/+ (Figure 7C). Comparable findings were obtained in older mice studied at 30 to 35 weeks of age (data not shown). Differences in reendothelialization 5 days after injury were confirmed by assessments of endothelial cell density in the region of prior injury by vWF immunostaining (data not shown). Thus, the phenotype of attenuated reendothelialization observed with lowered apoA-I and HDL was recapitulated by loss of SR-BI, thereby providing mechanistic linkage of apoA-I, HDL, and SR-BI in the promotion of endothelial monolayer integrity in vivo.
Because diminished eNOS function causes hypertension40 and hypertension is associated with endothelial injury,41 we determined whether the blunted reendothelialization in SR-BIeC/eC mice is related to hypertension. Systolic, diastolic, and mean blood pressure and heart rate measured by radiotelemetry were similar in SR-BI+/+ and SR-BIeC/eC mice (supplemental Table I). Therefore, the attenuation of reendothelialization in SR-BIeC/eC mice is not due to hypertension.
Because SR-BIeC/eC mice have heterogeneous, enlarged HDL particles with increased cholesterol content compared with those of SR-BI+/+ mice,42 it is possible that differences in reendothelialization in SR-BI+/+ and SR-BIeC/eC are due to disparities in the HDL particle. To address this possibility, BAEC migration responses to human HDL (positive control) or to HDL from SR-BI+/+ versus SR-BIeC/eC mice were evaluated. Endothelial cell migration with HDL from SR-BIeC/eC mice was at least as robust as that with HDL from SR-BI+/+ mice (Figure 7D). In studies of lamellipodia formation, responses were identical for HDL from SR-BI+/+ and SR-BIeC/eC mice (Figure 7E). Thus, the features of HDL required to activate endothelial cell migration are not altered in SR-BIeC/eC mice. In addition, it is important to note that HDL levels in SR-BIeC/eC mice are higher than in wild-type mice,42 yet reendothelialization is blunted. Therefore, it is not differences in the quantity or nature of the "ligand" for SR-BI that underlie the attenuation in reendothelialization in SR-BIeC/eC mice, and the evidence for a major role for SR-BI in reendothelialization is further strengthened.
Discussion
Circulating levels of HDL and apoA-I are associated with lower risk for vascular disease.1,2,10,11 In addition, trials with agents such as benzafibrate or gemfibrozil that raise HDL levels indicate that modest elevations in HDL are associated with a significant reduction in overall cardiovascular events.43,44 Thus, HDL is not simply a marker of decreased vascular disease risk but an important mediator of vascular health. The classical actions of HDL to promote RCT may not fully explain the protective nature of HDL/apoA-I, as RCT is not dictated by circulating levels of HDL or apoA-I.7eC9 In addition, the impact of the antioxidant and antiinflammatory properties of HDL is yet to be clarified.14,15 In the present study, we show that HDL and SR-BI stimulate endothelial cell migration in vitro with potency equivalent to VEGF. We further demonstrate that the phospholipid, apoA-I, and cholesterol components of HDL are sufficient to initiate this cellular response. Importantly, we also show in the context of all other factors regulating endothelial cell phenotype in vivo that HDL/apoA-I and SR-BI promote endothelial monolayer integrity. This represents an entirely novel role for the HDL/SR-BI tandem, complementing the capacity of the lipoprotein and receptor to regulate cholesterol flux and endothelial NO production.3,4,16eC19
The processes underlying HDL and SR-BI stimulation of endothelial cell migration were also investigated (Figure 8). Prior studies of HDL activation of eNOS revealed that the lipoprotein activates Src family kinases that activate PI3 kinase, leading to Akt kinase and MAPK stimulation and greater eNOS enzymatic activity.17 In the present study, we demonstrate that the activation of these kinases by HDL-SR-BI also promotes endothelial cell migration, but that this response is independent of eNOS. In addition, we show that HDL causes rapid Rac activation in concert with lamellipodia formation in an SR-BI-dependent manner, and that Rac is required for both increased lamellipodia formation and ultimate cell migration. We further demonstrate that the kinases reside upstream of Rac in the series of events by which HDL and SR-BI regulate endothelial cell motility (Figure 8, solid arrows). Although antagonism of PI3 kinase or MAPK activity prevented HDL-induced Rac activation and cell migration, these interventions did not alter lamellipodia formation. As such, alternative SR-BI- and Src family kinase-dependent processes may also mediate initial lamellipodia formation (Figure 8, dashed arrows). Collectively, these studies have revealed that multiple, sequential signaling events occur in endothelium in response to the lipoprotein. When combined with our prior work on HDL activation of eNOS,17 they also indicate that after the activation of common upstream signaling events, NO production and cell migration are independently promoted by HDL, thereby enhancing both the integrity and the paracrine functions of the endothelium to optimize vascular health. Whether comparable signaling mediates HDL actions in other cell types is yet to be determined.
In a recent investigation of the most proximal events in HDL signaling to the kinases regulating eNOS, we found that the process requires cholesterol flux, the C-terminal transmembrane domain of SR-BI that directly binds cholesterol, and the C-terminal PDZ-interacting domain of SR-BI. In addition, comparable signaling was initiated by HDL and the cholesterol acceptor methyl-beta cyclodextrin, and both were dependent on SR-BI, further suggesting that SR-BI is a cholesterol sensor on the plasma membrane.45 However, it is yet to be determined how these C-terminal domains of SR-BI, which do not mediate cholesterol movement,46 initiate signaling. The current work provides in vitro and in vivo evidence that such signaling mediated by HDL and SR-BI is of physiological importance.
The present observations reveal a novel series of mechanisms by which apoA-I/HDL and SR-BI are positive modulators of endothelial cell motility. Our findings provide a new framework for understanding how HDL promotes vascular health. Further research in this realm will enhance both our understanding of HDL and SR-BI signaling and our efforts to harness the potent actions of HDL to develop new strategies to combat vascular disease.
Acknowledgments
This work was supported by National Institutes of Health grants HL58888 (P.W.S.), AI45896 (R.L.K.), and HL70128 and HL59407 (D.J.R.), and Canadian Institutes of Health Grants 44359 and 64519 (Y.L.M.). The project was also supported by the Crystal Charity Ball Center for Pediatric Critical Care Research and the Lowe Foundation (P.W.S.) and by the Donald W. Reynolds Cardiovascular Clinical Research Center (W.V.). We are indebted to Marilyn Dixon for preparing the manuscript.
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Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, Wilt TJ, Wittes J. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med. 1999; 341: 410eC418.
Assanasen C, Mineo C, Seetharam D, Yuhanna IS, Marcel YL, Connelly MA, Williams DL, Llera-Moya M, Shaul PW, Silver DL. Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling. J Clin Invest. 2005; 115: 969eC977.
Connelly MA, Llera-Moya M, Monzo P, Yancey PG, Drazul D, Stoudt G, Fournier N, Klein SM, Rothblat GH, Williams DL. Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain. Biochemistry. 2001; 40: 5249eC5259.(Divya Seetharam, Chieko M)
Lipoprotein and Atherosclerosis Research Group (Y.L.M.), University of Ottawa Heart Institute, Ottawa, Canada
the Department of Medicine (D.J.R.), University of Pennsylvania School of Medicine, Philadelphia, Pa.
Abstract
Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-IeC/eC mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BIeC/eC mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.
Key Words: high-density lipoprotein endothelium migration
Introduction
The risk of atherosclerosis is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol.1,2 HDL classically functions in reverse cholesterol transport (RCT), removing cholesterol from peripheral tissues and delivering it to the liver and steroidogenic organs by binding of the HDL apolipoprotein apolipoprotein A-I (apoA-I) to the HDL receptor scavenger receptor B type I (SR-BI).3,4 In mouse models of atherosclerosis, both apoA-I and SR-BI provide atheroprotection.5,6 However, as animal studies show that RCT is not dictated by circulating levels of HDL or apoA-I,7eC9 the changes in atherosclerosis risk related to varying HDL and apoA-I levels may not be explained by differences in RCT. In addition, clinical studies suggest that the risk for restenosis after vascular intervention may be inversely related to HDL levels,10,11 and the provision of apoA-I or HDL attenuates neointima formation after artery injury in hypercholesterolemic animal models.12,13 There is evidence that HDL also inhibits low-density lipoprotein oxidation and adhesion molecule expression in cultured endothelial cells, but the magnitude and impact of these processes in vivo is uncertain.14,15 As such, there is considerable evidence that HDL affords protection from vascular disease, but the mechanisms underlying the protection remain poorly understood.
Whereas it has been appreciated for some time that SR-BI is expressed in hepatocytes and steroidogenic tissues, we and others16eC20 have more recently demonstrated that SR-BI is also expressed in endothelium, where it mediates promodulatory effects of HDL on endothelial nitric oxide synthase (eNOS) to increase the abundance of NO. In addition to generating NO, an intact endothelial cell monolayer modulates local hemostasis and thrombolysis and provides a nonpermeable barrier protecting smooth muscle cells from circulating growth-promoting factors. Disruptions of endothelial cell monolayer integrity, either by gross denudation related to a vascular intervention or gap formation between cells due to disturbed shear stress, place the arterial wall at greater risk for vascular disease.21eC23 Furthermore, whereas repeated endothelial removal worsens vascular lesion severity,24 enhanced reendothelialization blunts lesion formation.25,26 To better understand the basis of HDL-related vascular protection, we designed experiments to determine whether HDL functioning through SR-BI promotes endothelial cell migration and the maintenance of endothelial monolayer integrity. HDL-induced migration, lamellipodia formation, and Rac GTPase activation were interrogated in cultured endothelium. Because HDL activation of eNOS is mediated by Src kinases, phosphatidylinositol 3-kinase (PI3K)/Akt kinase and p44/42 mitogen-activated protein kinase (MAPK),17 the roles of these signaling cascades in HDL modulation of Rac GTPase and migration were determined. Because NO is critically involved in endothelial migration and angiogenesis,27 dependence on NO was also evaluated. Furthermore, the impact of loss of SR-BI was investigated. Moreover, to determine whether these processes are operative in vivo, carotid artery reendothelialization was assessed after perivascular electric injury in mice in which apoA-I/HDL/SR-BI status was genetically manipulated.
Materials and Methods
Additional details are provided in the online-only data supplement, available at http://circres.ahajournals.org.
Cell Culture
Bovine aortic endothelial cells (BAECs) were harvested using procedures reported previously with minor modifications,28 cultured in endothelial growth medium-2 (Cambrex Bioscience) with 5% fetal bovine serum (Sigma), and studied at passages 5 to 9.
Endothelial Cell Migration Assay
BAECs were grown to near confluence, and a defined region of cells was removed with a razor blade. Cells were treated for 24 hours and then fixed, and the number of cells that had migrated past the wound edge was quantified.
Cytoskeletal Changes in Endothelial Cells
BAECs were plated onto glass coverslips, treated for 0 to 30 minutes, fixed and stained with Alexa 568-phalloidin (Molecular Probes, Inc), and viewed under a fluorescent microscope. The percent of cells with lamellipodia was quantified.
Rac Activity Assay
After treatment, Rac activity in BAECs was measured as previously described with minor modifications.29
Small Interfering RNA for Rac and SR-BI
Double-stranded RNA (dsRNA) sequences directed at bovine Rac or SR-BI were transfected into cells, and expression of the proteins and functional and signaling readouts were determined 24 to 48 hours later.
Carotid Artery Reendothelialization
Carotid artery reendothelialization was studied after perivascular electric injury in mice by assessing Evan’s blue dye uptake.30,31 Endothelial denudation and recovery after injury was confirmed by immunohistochemistry for von Willebrand factor (vWF). Study groups included wild-type mice, apoA-IeC/eC or SR-BIeC/eC mice, and apoA-IeC/eC mice with adenoviral reconstitution of apoA-I expression.
Blood Pressure by Radiotelemetry
Chronic blood pressure measurements were performed in 30- to 35-week-old SR-BI+/+ and SR-BIeC/eC mice by radiotelemetry.32
Statistical Analysis
All data are presented as mean±SEM. ANOVA with Neuman-Keuls post hoc testing was used to assess differences between 3 or more groups. Differences in reendothelialization were evaluated by Mann-Whitney tests. Significance was set at P<0.05.
Results
HDL and Endothelial Cell Migration
To determine the effect of HDL on endothelial cell migration, BAECs were wounded and treated with HDL. The lipoprotein caused a marked increase in migration (Figure 1A), with 2.8-, 3.6-, and 4-fold increases noted with 20, 50, and 100 e/mL HDL, respectively (Figure 1B), and the response to HDL was comparable to the response to vascular endothelial growth factor (VEGF) (Figure 1C). It has been previously reported that HDL activates endothelial cell migration33 and that this may be dependent on cargo molecules such as sphingosine-1-phosphate (S-1-P), which induces migration in a pertussis toxin (Ptx)-sensitive manner.34 To determine the contribution of S-1-P in HDL-induced migration, responses to HDL or S-1-P were assessed in the absence or presence of Ptx. Whereas S-1-P-induced migration was prevented by Ptx, HDL-mediated migration was not affected (Figure 1D). To determine whether the apolipoprotein, phospholipid, and cholesterol components of HDL are sufficient to stimulate migration, BAECs were treated with lipoprotein (Lp)2A-I particles reconstituted with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), cholesterol, and lipid-free recombinant apoA-I. Lp2A-I containing POPC, cholesterol, and apoA-I at molar ratios of 80:5:1 caused migration comparable to that promoted by native HDL (Figure 1E). These observations indicate that HDL stimulates endothelial cell migration and that the phospholipid, cholesterol, and apoA-I components are sufficient to mediate the process.
HDL, Lamellipodia Formation, and Rac GTPase
Because cell migration begins with key changes in the actin cytoskeleton, including the formation of lamellipodia,35,36 we tested the initial effects of HDL on the cytoskeleton. Under control conditions, BAEC displayed stress fibers and few lamellipodia (Figure 2A). Within 1 minute of HDL exposure, there was a decrease in stress fibers, an increase in lamellipodia, and membrane ruffling. This effect became more apparent at 5 minutes, the number of lamellipodia decreased at 15 minutes, and cells appeared similar to control at 30 minutes. As Rac GTPase mediates lamellipodia formation,37 we determined whether Rac is activated by HDL. Paralleling the findings for lamellipodia formation, there was an increase in Rac activity with HDL within 1 minute (Figure 2B). Activity was maximal at 5 minutes and decreased thereafter.
The role of Rac in HDL-induced endothelial cell migration and lamellipodia formation was then tested by depletion of Rac with small interfering RNA (siRNA) (Figure 2C). Whereas BAECs transfected with control siRNA migrated (Figure 2D) and had initial lamellipodia formation in response to HDL (Figure 2E), cells transfected with Rac siRNA did not. Thus, HDL causes lamellipodia formation in concert with Rac activation in endothelial cells, and both the lamellipodia formation and ensuing migration are mediated by Rac.
Role of Kinase Activation
To further delineate the mechanisms by which HDL stimulates endothelial cell migration, we determined whether the process entails the kinases that have been implicated in HDL-mediated eNOS activation.17 Whereas the Src kinase inhibitor PP2 did not affect basal migration, HDL-stimulated migration was blunted by 72% by PP2 (Figure 3A, left panel). PP3, the negative control compound for PP2, had no effect (data not shown). Treatment with the PI3K inhibitor LY294002 did not affect basal migration, but HDL-stimulated migration was fully inhibited (Figure 3A, middle panel). PD 98059, which inhibits the MAPK pathway, did not alter basal migration, but HDL-induced migration was attenuated by 84% (Figure 3A, right panel). Therefore, HDL-induced endothelial cell migration is dependent on Src family kinases, PI3K, and MAPK.
The roles of Src kinases, PI3K, and MAPK in HDL-induced lamellipodia formation were also evaluated. BAEC pretreatment with PP2 had no effect on the cytoskeleton at baseline. However, the 14-fold increase in lamellipodia formation with HDL (Figure 3B, left panel) was blunted by 87% by PP2. PP3 had no effect (data not shown). In contrast, both LY294002 and PD98059 did not modify lamellipodia formation with HDL (Figure 3B, middle and right panels, respectively). The efficacy of LY294002 and PD98059 was confirmed in studies of HDL-stimulated Akt and MAPK phosphorylation (data not shown).
The role of kinases in Rac activation by HDL was also investigated. Whereas treatment with PP2 did not alter basal activity, HDL-induced Rac activation was blunted by 67% (Figure 3C, left panel). Treatment with LY294002 did not affect basal Rac activity but it attenuated HDL-mediated Rac activation by 86% (Figure 3C, middle panel). Basal Rac activity was not affected by PD98059, but HDL-induced Rac activation was diminished by 82% (Figure 3C, right panel). Therefore, Src family kinases, PI3K, and MAPK are required for HDL-induced Rac activity. To verify the sequence of kinase and Rac activation, Rac was knocked-down with siRNA and HDL-induced kinase phosphorylation was tested. HDL caused comparable phosphorylation of Src, Akt, and MAPK in cells transfected with control and Rac siRNA (Figure 3D). Thus, Rac resides downstream of the kinases in the signaling cascade by which HDL activates endothelial cell migration.
Role of eNOS
Because HDL stimulates eNOS19 and NO promotes endothelial cell migration and angiogenesis,27 we determined whether eNOS is required for HDL-mediated migration. The treatment of BAECs with the NOS antagonist N-Nitro-L-arginine methyl ester (L-NAME) did not affect basal migration, and HDL-induced endothelial cell migration was also not attenuated (Figure 4A). In contrast, VEGF-stimulated migration was blocked by L-NAME (Figure 4B). L-NAME did not alter the basal number of cells displaying lamellipodia, and lamellipodia formation in response to HDL was not changed (Figure 4C). The efficacy of L-NAME was verified by confirming inhibition of eNOS enzymatic activity (data not shown). These findings indicate that, in contrast to the mechanisms of action of multiple known stimuli including VEGF,38 the promotion of endothelial cell migration by HDL is NO-independent.
Role of SR-BI
To determine whether SR-BI plays a role in HDL-mediated endothelial cell migration, SR-BI was knocked down by siRNA (Figure 5A). Whereas cells transfected with control siRNA displayed an 8.5-fold increase in migration with HDL (Figure 5B), cells transfected with SR-BI siRNA did not. In parallel, cells transfected with control siRNA had lamellipodia formation induced 5.3-fold by HDL (Figure 5C), but after knockdown of SR-BI, lamellipodia formation did not occur. Similarly, cells transfected with control siRNA displayed Rac activation by HDL (Figure 5D), and Rac activation was not demonstrable after SR-BI knockdown. As such, SR-BI is required for HDL-induced Rac activation and the resulting changes in the actin cytoskeleton that promote migration.
HDL and Reendothelialization In Vivo
To determine whether the mechanisms revealed in vitro are operative in vivo, carotid artery reendothelialization studies were performed in mice. The area of remaining denudation was determined after perivascular electric injury by the injection of Evan’s blue dye, which is incorporated in the region of denudation (supplemental Figure IA and IB).30,31 In control C57BL/6 mice, reendothelialization was complete by 7 days (supplemental Figure IC). Denudation of the endothelium after injury (1 day) and reendothelialization after injury (8 days) was confirmed by immunostaining for vWF (Data supplemental Figure ID). To determine the role of HDL in reendothelialization in vivo, reendothelialization was compared 5 days after injury in apoA-I+/+ and apoA-IeC/eC mice, which have HDL cholesterol levels that are decreased by 83% compared with wild-type.39 Greater reendothelialization occurred in apoA-I+/+ (Figure 6A) versus apoA-IeC/eC (Figure 6B) mice, as indicated by the larger area of remaining denudation in apoA-IeC/eC mice. Cumulative studies revealed that the area of remaining denudation was 52% larger in apoA-IeC/eC versus apoA-I+/+ mice (Figure 6C). Differences in reendothelialization 5 days after injury were confirmed by assessments of endothelial cell density in the region of prior injury by vWF immunostaining (data not shown).
To test wheter normal reendothelialization can be rescued by reconstitution of apoA-I expression in apoA-IeC/eC mice, liver-directed gene transfer of human apoA-I was performed at the time of artery injury. Five days after injection, apoA-IeC/eC mice given control adenovirus had HDL levels of 19.6±7.7 mg/dL, whereas mice receiving apoA-I-containing adenovirus had apoA-I levels of 148.4±49.4 mg/dL (P<0.05 versus control) and HDL levels of 69.7±25.2 mg/dL (P<0.05 versus control). Evan’s blue dye incorporation 5 days after injury demonstrated that in comparison with apoA-IeC/eC mice given control adenovirus (Figure 6D), mice injected with apoA-I-containing adenovirus displayed greater reendothelialization (Figure 6E). Quantitation in multiple mice indicated that there was 40% less denudation after the reconstitution of apoA-I expression in apoA-IeC/eC mice (Figure 6F). These findings indicate that apoA-I and HDL promote endothelial monolayer integrity in vivo.
SR-BI and Reendothelialization In Vivo
Having demonstrated that there is a major contribution of apoA-I/HDL to reendothelialization in vivo, we next determined the role of SR-BI in studies of SR-BI+/+ and SR-BIeC/eC mice 5 days after thermal injury. Greater reendothelialization occurred in SR-BI+/+ (Figure 7A) versus SR-BIeC/eC mice (Figure 7B) as indicated by the larger area of Evan’s blue dye incorporation in the latter. Cumulative studies indicated that the area of remaining denudation was 44% larger in SR-BIeC/eC versus SR-BI+/+ (Figure 7C). Comparable findings were obtained in older mice studied at 30 to 35 weeks of age (data not shown). Differences in reendothelialization 5 days after injury were confirmed by assessments of endothelial cell density in the region of prior injury by vWF immunostaining (data not shown). Thus, the phenotype of attenuated reendothelialization observed with lowered apoA-I and HDL was recapitulated by loss of SR-BI, thereby providing mechanistic linkage of apoA-I, HDL, and SR-BI in the promotion of endothelial monolayer integrity in vivo.
Because diminished eNOS function causes hypertension40 and hypertension is associated with endothelial injury,41 we determined whether the blunted reendothelialization in SR-BIeC/eC mice is related to hypertension. Systolic, diastolic, and mean blood pressure and heart rate measured by radiotelemetry were similar in SR-BI+/+ and SR-BIeC/eC mice (supplemental Table I). Therefore, the attenuation of reendothelialization in SR-BIeC/eC mice is not due to hypertension.
Because SR-BIeC/eC mice have heterogeneous, enlarged HDL particles with increased cholesterol content compared with those of SR-BI+/+ mice,42 it is possible that differences in reendothelialization in SR-BI+/+ and SR-BIeC/eC are due to disparities in the HDL particle. To address this possibility, BAEC migration responses to human HDL (positive control) or to HDL from SR-BI+/+ versus SR-BIeC/eC mice were evaluated. Endothelial cell migration with HDL from SR-BIeC/eC mice was at least as robust as that with HDL from SR-BI+/+ mice (Figure 7D). In studies of lamellipodia formation, responses were identical for HDL from SR-BI+/+ and SR-BIeC/eC mice (Figure 7E). Thus, the features of HDL required to activate endothelial cell migration are not altered in SR-BIeC/eC mice. In addition, it is important to note that HDL levels in SR-BIeC/eC mice are higher than in wild-type mice,42 yet reendothelialization is blunted. Therefore, it is not differences in the quantity or nature of the "ligand" for SR-BI that underlie the attenuation in reendothelialization in SR-BIeC/eC mice, and the evidence for a major role for SR-BI in reendothelialization is further strengthened.
Discussion
Circulating levels of HDL and apoA-I are associated with lower risk for vascular disease.1,2,10,11 In addition, trials with agents such as benzafibrate or gemfibrozil that raise HDL levels indicate that modest elevations in HDL are associated with a significant reduction in overall cardiovascular events.43,44 Thus, HDL is not simply a marker of decreased vascular disease risk but an important mediator of vascular health. The classical actions of HDL to promote RCT may not fully explain the protective nature of HDL/apoA-I, as RCT is not dictated by circulating levels of HDL or apoA-I.7eC9 In addition, the impact of the antioxidant and antiinflammatory properties of HDL is yet to be clarified.14,15 In the present study, we show that HDL and SR-BI stimulate endothelial cell migration in vitro with potency equivalent to VEGF. We further demonstrate that the phospholipid, apoA-I, and cholesterol components of HDL are sufficient to initiate this cellular response. Importantly, we also show in the context of all other factors regulating endothelial cell phenotype in vivo that HDL/apoA-I and SR-BI promote endothelial monolayer integrity. This represents an entirely novel role for the HDL/SR-BI tandem, complementing the capacity of the lipoprotein and receptor to regulate cholesterol flux and endothelial NO production.3,4,16eC19
The processes underlying HDL and SR-BI stimulation of endothelial cell migration were also investigated (Figure 8). Prior studies of HDL activation of eNOS revealed that the lipoprotein activates Src family kinases that activate PI3 kinase, leading to Akt kinase and MAPK stimulation and greater eNOS enzymatic activity.17 In the present study, we demonstrate that the activation of these kinases by HDL-SR-BI also promotes endothelial cell migration, but that this response is independent of eNOS. In addition, we show that HDL causes rapid Rac activation in concert with lamellipodia formation in an SR-BI-dependent manner, and that Rac is required for both increased lamellipodia formation and ultimate cell migration. We further demonstrate that the kinases reside upstream of Rac in the series of events by which HDL and SR-BI regulate endothelial cell motility (Figure 8, solid arrows). Although antagonism of PI3 kinase or MAPK activity prevented HDL-induced Rac activation and cell migration, these interventions did not alter lamellipodia formation. As such, alternative SR-BI- and Src family kinase-dependent processes may also mediate initial lamellipodia formation (Figure 8, dashed arrows). Collectively, these studies have revealed that multiple, sequential signaling events occur in endothelium in response to the lipoprotein. When combined with our prior work on HDL activation of eNOS,17 they also indicate that after the activation of common upstream signaling events, NO production and cell migration are independently promoted by HDL, thereby enhancing both the integrity and the paracrine functions of the endothelium to optimize vascular health. Whether comparable signaling mediates HDL actions in other cell types is yet to be determined.
In a recent investigation of the most proximal events in HDL signaling to the kinases regulating eNOS, we found that the process requires cholesterol flux, the C-terminal transmembrane domain of SR-BI that directly binds cholesterol, and the C-terminal PDZ-interacting domain of SR-BI. In addition, comparable signaling was initiated by HDL and the cholesterol acceptor methyl-beta cyclodextrin, and both were dependent on SR-BI, further suggesting that SR-BI is a cholesterol sensor on the plasma membrane.45 However, it is yet to be determined how these C-terminal domains of SR-BI, which do not mediate cholesterol movement,46 initiate signaling. The current work provides in vitro and in vivo evidence that such signaling mediated by HDL and SR-BI is of physiological importance.
The present observations reveal a novel series of mechanisms by which apoA-I/HDL and SR-BI are positive modulators of endothelial cell motility. Our findings provide a new framework for understanding how HDL promotes vascular health. Further research in this realm will enhance both our understanding of HDL and SR-BI signaling and our efforts to harness the potent actions of HDL to develop new strategies to combat vascular disease.
Acknowledgments
This work was supported by National Institutes of Health grants HL58888 (P.W.S.), AI45896 (R.L.K.), and HL70128 and HL59407 (D.J.R.), and Canadian Institutes of Health Grants 44359 and 64519 (Y.L.M.). The project was also supported by the Crystal Charity Ball Center for Pediatric Critical Care Research and the Lowe Foundation (P.W.S.) and by the Donald W. Reynolds Cardiovascular Clinical Research Center (W.V.). We are indebted to Marilyn Dixon for preparing the manuscript.
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