atherosclerosisDeletion of the phosphoinositide 3-kinase p110{gamma} gene attenuates murine

J. Field, Caitlin Kennedy, Swetha Madhavarapu, Ji Luo, Dianqing Wu, and Lewis C. Cantley James D. Chang, Galina K. Sukhova, Peter Libby, Eugenia Schvartz, Alice H. Lichtenstein, Seth

doi:10.1073/pnas.0702663104 2007;104;8077-8082; originally published online May 2, 2007; PNAS

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Deletion of the phosphoinositide 3-kinase p110�gene attenuates murine atherosclerosisJames D. Chang*†‡, Galina K. Sukhova‡, Peter Libby‡, Eugenia Schvartz‡, Alice H. Lichtenstein§, Seth J. Field¶,Caitlin Kennedy*, Swetha Madhavarapu*, Ji Luo†, Dianqing Wu�, and Lewis C. Cantley*†‡**

*Beth Israel Deaconess Medical Center (Signal Transduction and Cardiovascular Divisions), Boston, MA 02115; †Department of Systems Biology, HarvardMedical School, Boston, MA 02115; ‡Donald W. Reynolds Cardiovascular Clinical Research Center, Brigham and Women’s Hospital (Cardiovascular Division),Boston, MA 02115; §U.S. Department of Agriculture Human Nutrition Research Center on Aging (Cardiovascular Nutrition Laboratory), Tufts University,Boston, MA 02111; ¶Massachusetts General Hospital (Endocrine Unit), Boston, MA 02114; and �Department of Genetics and Developmental Biology,University of Connecticut Health Center, Farmington, CT 06030

Contributed by Lewis C. Cantley, March 21, 2007 (sent for review March 1, 2007)

Inflammatory cell activation by chemokines requires intracellularsignaling through phosphoinositide 3-kinase (PI3-kinase) and thePI3-kinase-dependent protein serine/threonine kinase Akt. Athero-sclerosis is a chronic inflammatory process driven by oxidativelymodified (atherogenic) lipoproteins, chemokines, and other ago-nists that activate PI3-kinase. Here we show that macrophagePI3-kinase/Akt is activated by oxidized low-density lipoprotein,inflammatory chemokines, and angiotensin II. This activation ismarkedly reduced or absent in macrophages lacking p110�, thecatalytic subunit of class Ib PI3-kinase. We further demonstrateactivation of macrophage/foam cell PI3-kinase/Akt in atheroscle-rotic plaques from apolipoprotein E (apoE)-null mice, which man-ifest an aggressive form of atherosclerosis, whereas activation ofPI3-kinase/Akt was undetectable in lesions from apoE-null micelacking p110� despite the presence of class Ia PI3-kinase. Moreover,plaques were significantly smaller in apoE�/�p110��/� mice thanin apoE�/�p110��/� or apoE�/�p110��/�mice at all ages studied.In marked contrast to the embryonic lethality seen in mice lackingclass Ia PI3-kinase, germ-line deletion of p110� results in mice thatexhibit normal viability, longevity, and fertility, with relativelywell tolerated defects in innate immune and inflammatory re-sponses that may play a role in diseases such as atherosclerosis andmultiple sclerosis. Our results not only shed mechanistic light oninflammatory signaling during atherogenesis, but further identifyp110� as a possible target for pharmacological intervention in theprimary and secondary prevention of human atherosclerotic car-diovascular disease.

Akt � inflammation � macrophage � lipoproteins �G protein-coupled receptors

Akt regulates downstream signaling and effector molecules inthe phosphoinositide 3-kinase (PI3-kinase) pathway and is

activated by phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3)-dependent phosphorylation and localization. The catalytic sub-units (p110�, p110�, and p110�) of class Ia PI3-kinase producePIP3 in response to growth factors, insulin, and other receptortyrosine kinase agonists. p110�, the catalytic subunit of class IbPI3-kinase, produces PIP3 in response to chemokines and otherG protein-coupled receptor agonists (1). Germ-line deletion ofp110� or p110� results in embryonic lethality, suggesting afundamental role in the development of class Ia PI3-kinase(2–5). In contrast, germ-line deletion of p110� results in micethat display normal viability, longevity, and fertility (6–8). p110�and p110� are ubiquitous, whereas p110� is expressed primarilyin hematopoietic cells, muscle, and the pancreas. p110�-deficientmice display defects in responses of immune and inflammatorycells to agonists of heterotrimeric G protein-coupled receptors(6–8). Mice lacking p110� display impaired neutrophil chemo-taxis and respiratory burst in response to fMLP and C5a, as wellas reduced thymocyte survival and activation of mature Tlymphocytes (6–8). PIP3 production and activation of Akt aremarkedly reduced in p110��/� neutrophils compared with wild-

type neutrophils (8). p110��/� peritoneal macrophages displayreduced migration toward many chemotactic factors, as well asdiminished accumulation in a septic peritonitis model (8). Thesestudies in p110��/� mice demonstrate the critical role of p110�in the activation of cells that participate in a wide range ofinflammatory processes.

Numerous studies have established the importance of PI3-kinase in responses of macrophages, vascular smooth musclecells, and T lymphocytes to atherogenic mediators. Oxidativelymodified low-density lipoprotein (LDL), one of the most potentof such mediators, exerts multiple effects on these cell types,including induction of protein tyrosine phosphorylation andPI3-kinase activation (9). Oxidized LDL is a survival factor forbone marrow-derived macrophages (BMDMs) in culture, inhib-iting apoptosis after withdrawal of macrophage colony-stimulating factor (M-CSF), whereas native and acetylated LDLhave no effect on apoptosis (10). Two specific, structurallyunrelated inhibitors of PI3-kinase, LY294002 and wortmannin,block the survival-promoting effect of oxidized LDL as wellas phosphorylation of Akt, a major PI3-kinase-dependentantiapoptotic mediator. In addition to having a direct effect onmacrophage survival, oxidized LDL plays a priming role inmacrophage proliferation in atherosclerotic lesions by triggeringthe release of granulocyte/M-CSF (GM-CSF) via activation ofPI3-kinase and protein kinase C (PKC) (11–13). GM-CSFrelease from macrophages in response to oxidized LDL inducesproliferation in an autocrine or a paracrine fashion, suggestingthat both the antiapoptotic and proliferative effects are PI3-kinase-dependent. Because inflammatory cell migration, prolif-eration, and activation require signaling through PI3-kinase, wehypothesized that, to the extent that it is driven by theseprocesses, atherogenesis should be attenuated by blockingp110�, a major PI3-kinase isoform in inflammatory cells.

ResultsThe ability of oxidized LDL and atherogenic cytokines/chemokines to trigger PI3-kinase signaling and Akt activation ininflammatory cells is well established (11, 12, 14). To determinewhether p110� is the specific isoform of PI3-kinase involved inthis pathway, we exposed BMDMs to these G protein-coupled

Author contributions: J.D.C. and L.C.C. designed research; J.D.C., G.K.S., E.S., A.H.L., C.K.,and S.M. performed research; D.W. and L.C.C. contributed new reagents/analytic tools;J.D.C., G.K.S., P.L., A.H.L., S.J.F., J.L., and L.C.C. analyzed data; and J.D.C. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: PI3-kinase, phosphoinositide 3-kinase; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte/M-CSF; BMDM, bone marrow-derived macro-phage; GSK3, glycogen synthase kinase 3.

**To whom correspondence should be addressed. E-mail: lewis�cantley@hms.harvard.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0702663104/DC1.

© 2007 by The National Academy of Sciences of the USA

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receptor agonists and immunoblotted lysates with a phosphospe-cific antibody that recognizes activated Akt. Akt phosphoryla-tion in response to oxidized LDL, fMLP, IL-8, MCP-1, andlysophosphatidic acid was strongly induced in wild-type macro-phages, but absent or barely detectable in p110��/� macro-phages (Fig. 1a). C-reactive protein, an acute-phase reactant thatrises in inflammatory conditions, and C-peptide, a cleavageproduct of proinsulin that is elevated in type 2 diabetes mellitus,have been implicated recently as proatherogenic agents (15–18)that can activate PI3-kinase (15, 18–20). Both C-reactive protein(10 �g/ml) and C-peptide (1 nM) induced PI3-kinase/Akt acti-vation in wild-type but not in p110��/� macrophages (Fig. 1a).Angiotensin II, which elicits numerous responses in the cardio-vascular system, some of which are deleterious when the renin-angiotensin system is chronically hyperactivated, induced phos-phorylation of Akt in a dose-dependent manner in p110��/� butnot in p110��/� BMDMs (Fig. 1b). In contrast, both p110��/�

and wild-type BMDMs showed similar Akt activation in re-sponse to IGF-1 (Fig. 1a), demonstrating that class Ia PI3-kinaseis present and capable of activating Akt in p110�-null BMDMs.Wortmannin, an inhibitor of all class I PI3-kinases, blocked Aktphosphorylation in response to all agonists tested in bothwild-type and p110��/� BMDMs (Fig. 1b and data not shown).The presence or absence of apoE had no effect on PI3-kinaseactivation because Akt phosphorylation in response to all ago-nists used was similar in apoE�/� and apoE�/� BMDMs (datanot shown). In addition, Akt activation in p110��/� andp110��/� macrophages in response to all agonists tested wasidentical (data not shown).

Deletion of the apoE gene in mice causes marked elevation ofproatherogenic lipoproteins and marked reduction of anti-atherogenic high-density lipoprotein (HDL), resulting in anaggressive form of atherosclerosis (21). We performed an anal-ysis of atherosclerotic lesions in animals fed a standard labora-tory chow diet rather than in those fed a ‘‘Western’’-type diet(rich in cholesterol and saturated fat) because feeding apoE�/�

mice a Western diet is known to accelerate and exacerbate anatherosclerotic process that is already highly exuberant inapoE�/� animals fed a standard chow diet. Hence, we felt thatbiologically important differences in atherogenesis betweenapoE�/�p110��/� and apoE�/�p110��/� mice would tend to beobscured if the animals were fed a Western diet.

To determine the contribution of p110� to atheroscleroticplaque progression, we cross-bred p110��/� and apoE�/� mice.At 36 and 55 weeks, plasma total cholesterol, HDL cholesterol,non-HDL cholesterol, and triglycerides were similar amongapoE�/�p110��/�, apoE�/�p110��/�, and apoE�/�p110��/�

mice, although after 60 weeks there was a modest but statisticallysignificant reduction of plasma total and non-HDL cholesterolin apoE�/�p110��/� mice [supporting information (SI) Table 1].Most notably, atherosclerotic lesions were smaller inapoE�/�p110��/� mice than in age-matched apoE�/�p110��/�

or apoE�/�p110��/� littermates at all time points. InapoE�/�p110��/� mice, aortic root lesion size was reduced by52% (P � 0.0001), 32% (P � 0.0400), and 36% (P � 0.0385) at35, 53, and 60 weeks of age, respectively (Fig. 2). Reduction oflesion size, although statistically significant in older mice (53 and60 weeks), was less than in younger mice (35 weeks) due toprogression of atherosclerosis in the double knockout mice. Ourhypothesis was that reduced reactivity of inflammatory cellsdeficient in p110� in response to atherogenic agonists andchemokines should result in attenuation of atherosclerosis inapoE knockout mice. p110��/� and p110��/� macrophagesdisplayed similar Akt activation in response to these agonists/chemokines when tested in vitro. Furthermore, immunohisto-chemical analysis of PI3-kinase/Akt activation in aortic rootlesions from apoE�/�p110��/� and apoE�/�p110��/� miceyielded similar results (Figs. 3 and 4). Therefore, in quantify-ing aortic root atherosclerosis, we pooled results fromapoE�/�p110��/� and apoE�/�p110��/� mice. The mice in-cluded in the quantitative analysis of aortic root atherosclerosis(Fig. 2) were all females. Qualitatively similar results wereobtained when male animals were analyzed (data not shown).

Atherosclerotic plaques in the aortic root of 35-, 53-, and60-week-old apoE�/�p110��/� and apoE�/�p110��/� mice thatconsumed a standard chow diet were complex and extensive,occupying most of the sinuses of Valsalva (Fig. 3). In age-matched apoE�/�p110��/� mice, the lesions were significantlysmaller. Numerically, macrophage/foam cells were the predom-inant cell type within lesions of both apoE�/�p110��/� and

Fig. 1. Western blot analysis of macrophage lysates from p110��/� andp110��/� mice. (a) Lysates of BMDMs exposed (15 min at 37°C) to variousagonists after 24 h of serum deprivation were separated by SDS/PAGE andimmunoblotted with phosphospecific Akt (Ser473) antibody (Upper). Separatebut identical blots were probed with nonphosphospecific Akt antibody todemonstrate equal loading of lanes (Lower). Agonist concentrations wereoxidized LDL, 1 and 10 �g/ml; fMLP, 10 �M; IL-8, 10 ng/ml; MCP-1, 20 ng/ml;lysophosphatidic acid, 10 �M; C-reactive protein, 10 �g/ml; C-peptide, 1 nM;IGF-1, 20 nM. (b) Cultured BMDMs were exposed to angiotensin II at theconcentrations indicated, for 5 min at 37°C before cell lysis, in the presence (20nM) or absence of wortmannin. wt, wild type; ko, p110��/�.

Fig. 2. Aortic root atherosclerosis was quantified on anti-Mac-3-stainedcryosections by digital histomorphometry using ImagePro Plus software. Ath-erosclerosis in apoE�/�p110��/� or apoE�/�p110��/� mice was compared withthat in apoE�/�p110��/� mice. At 35 weeks (n � 8 for apoE�/�p110��/� orapoE�/�p110��/� mice; n � 12 for apoE�/�p110��/� mice), double knockoutmice displayed 52% reduction of lesion area (P � 0.0001). At 53 weeks (n � 8for apoE�/�p110��/� mice; n � 7 for apoE�/�p110��/� mice), double knockoutmice displayed 32% reduction of lesion area (P � 0.04). At 60 weeks (n � 7 forapoE�/�p110��/� or apoE�/�p110��/� mice; n � 8 for apoE�/�p110��/� mice),double knockout mice displayed 36% reduction of lesion area (P � 0.0385).Error bars represent the SE of the mean.

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apoE�/�p110��/� mice, with a relative paucity of helper Tlymphocytes, as determined by staining with Mac-3 and CD4,respectively (Fig. 3 b, c, f, and g). The fully developed lesions inapoE�/�p110��/� and apoE�/�p110��/� mice ages 35 weeks andolder stained strongly with the phosphospecific Akt antibody(Fig. 3h). In striking contrast, the aortic root lesions of age-matched apoE�/�p110��/� mice showed no detectable Aktphosphorylation (Fig. 3d). These immunohistochemical findingscorrelate precisely with the results of immunoblotting by usingthe same phosphospecific Akt antibody on lysates of culturedmacrophages stimulated with oxidized LDL or atherogeniccytokines/chemokines (Fig. 1). In the aortic root atheroscleroticlesions, phosphoAkt staining localized primarily in macrophage-rich areas of the lesions (SI Fig. 5), and dual immunohistochem-ical staining (SI Fig. 6) with anti-phosphoAkt and anti-Mac-3demonstrated that the subpopulation of macrophage/foam cellsresiding in the areas of highest cell density display activatedPI3-kinase/Akt. Compared with the number of macrophagesfound in aortic root lesions, very few smooth muscle cells werepresent in lesions stained with antismooth muscle cell �-actinfrom either apoE�/�p110��/� or apoE�/�p110��/� mice (datanot shown).

Numerous signaling and effector proteins are regulated byPI3-kinase/Akt-dependent serine/threonine phosphorylation.These include glycogen synthase kinase 3 (GSK3), PKC, p70S6kinase,and FKHR (a member of the FOXO family of transcriptionfactors), which regulate cellular energy metabolism, protein trans-lation, and gene expression. Staining with phosphospecific antibod-ies against GSK3�/�, PKC�, p70S6kinase, FKHR, AFX-1 (anothermember of the FOXO family of transcription factors), and S6ribosomal protein was seen in aortic root atherosclerotic lesionsfrom apoE�/�p110��/�, and also in apoE�/�p110��/� mice (data

not shown), but not in lesions from apoE�/�p110��/� mice (Fig. 4),demonstrating that not only are formation of D3-phosphorylatedphosphoinositides and Akt activation absent or markedly reducedin plaque macrophages when p110� is absent, but also that PI3-kinase/Akt-dependent phosphorylation of downstream moleculesin this pathway is abolished.

The FOXO family transcription factor FKHR is regulated byPI3-kinase/Akt (22). FKHR and other members of the FOXOfamily are phosphorylated by Akt when PI3-kinase is activated,resulting in their exclusion from the nucleus (22). We used phos-phospecific and nonphosphospecific antibodies against FKHR todetermine simultaneously the localization (nuclear vs. cytoplasmic)and phosphorylation state of FKHR in atherosclerotic plaquemacrophage/foam cells. FKHR was phosphorylated and localizedin the cytosol in plaque macrophages from apoE�/�p110��/� mice(SI Fig. 7 a and b), whereas it was unphosphorylated and localizedin the nucleus in plaque macrophages from apoE�/�p110��/� mice(SI Fig. 7 c and d), corroborating the absence of PI3-kinase/Aktactivation in foam cell/macrophages of p110�-deficient mice.

DiscussionAtherosclerosis and its sequelae, including myocardial infarctionand stroke, are the leading causes of mortality and morbidity in thedeveloped world (23–25). Here we have demonstrated that p110�is required for activation of Akt in macrophages in response tooxidized LDL, atherogenic cytokines, and angiotensin II in vitro, aswell as in atherosclerotic lesions of hypercholesterolemic mice invivo. Moreover, we have shown that abrogation of Akt activation inmacrophage/foam cells by germ-line deletion of the p110� genecorrelates with the persistent reduction of plaque size. Double-mutant mice had smaller lesions with fewer Mac-3-positive cells(macrophages and macrophage-derived foam cells), but the overall

Fig. 3. Lipid content, cellular composition, and Akt activation were examined in atherosclerotic lesions. Aortic root cryosections from a 53-week-old femaleapoE�/�p110��/� mouse (a–d) and a female apoE�/�p110��/� littermate (e–h) stained with oil-red-O to visualize lipids (a and e), anti-Mac-3 to visualizemacrophages (b and f ), anti-CD4 to visualize T lymphocytes (c and g), and phosphoSer473 Akt antibody to detect Akt activation (d and h). Boxes in a and e indicateareas of higher magnification than in b–d and f–h, respectively. Arrows indicate position of endothelial and medial boundaries of lesions. Av, aortic valve leaflet;lum, lumen of aorta.

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density of macrophage/foam cells was similar in lesions fromapoE�/�p110��/� and apoE�/�p110��/� mice (data not shown).However, as shown in SI Figs. 5 and 6, the density of macrophage/foam cells varied considerably within lesions, and PI3-kinase/Aktactivation only occurred in areas of high cell density, suggesting thatautocrine or paracrine stimulation of macrophage/foam cell acti-vation and proliferation contributes to atherogenesis, a notion thatis well supported in the literature (see refs. 11 and 12). Thesefindings are consistent with a model of macrophage proliferationwithin atherosclerotic plaques proposed by Biwa et al. (11, 12),wherein the initial effect of oxidized LDL may be to trigger (PKC-and p110�-dependent) synthesis and release of GM-CSF by resi-dent macrophage/foam cells. Locally released GM-CSF then stim-ulates (class Ia PI3-kinase-dependent) proliferation of adjacentmacrophages in an autocrine/paracrine manner. We studied theeffect of p110� deficiency on macrophage expression of GM-CSFboth in macrophage culture and in situ in frozen sections of lesions.We chose to focus on GM-CSF because this cytokine has beenshown not only to be expressed by macrophages in response toatherogenic mediators such as oxidized LDL, but also to be at leastpartially dependent on PI3-kinase activity in macrophages. Fur-thermore, it has been hypothesized that macrophage-derived GM-CSF plays an important role in driving plaque progression throughan autocrine or paracrine mechanism (see refs. 11 and 12). How-

ever, in our hands, the levels of GM-CSF in culture medium (bothat baseline and after stimulation with oxidized LDL and otherproatherogenic cytokines and chemokines) and in frozen sectionsof actual lesions was below the level of detection by RIA and in situhybridization.

The modest reduction of total and non-HDL cholesterol inapoE�/�p110��/� compared with apoE�/�p110��/� mice at thevery late time point of 60 weeks is intriguing but unlikely to haveplayed a major role in the attenuation of plaque size in p110��/�

mice because the greatest difference in plaque size occurred at35 weeks, when cholesterol levels were similar. That cholesterolbound to atherogenic (non-HDL) lipoproteins as well as totalcholesterol was reduced in apoE�/�p110��/� mice at 60 weeksof age implies a (late-appearing) reduction of cholesterol ab-sorption or biosynthesis as result of p110� deficiency rather thanincreased elimination. It is unlikely that increased reverse cho-lesterol transport was responsible for reduced atherogenesis inapoE�/�p110��/� mice because an increased level of HDLcholesterol in apoE�/�p110��/� mice would be expected if thiswere the case.

The complete absence of immunohistochemically detectableAkt activation and of PI3-kinase-dependent phosphorylation ofdownstream effector molecules in cells of atherosclerotic lesionsfrom apoE�/�p110��/� mice was unexpected because p110� is

Fig. 4. The activation state of downstream effector and signaling molecules in the PI3-kinase/Akt pathway was assayed by immunohistochemistry. Immuno-histochemistry of adjacent aortic root cryosections from 36-week-old apoE�/�p110��/� (a, c, e, g, i, k, m, o) and apoE�/�p110��/� (b, d, f, h, j, l, n, p) mice. Sectionswere stained with phosphospecific antibodies against Akt (Magnification: a and b, �4), Akt (Magnification: c and d, �20), GSK3�/� (e and f ), AFX (g and h), FKHR(i and j), S6 ribosomal protein (k and l), p70S6kinase (m and n), and PKC� (o and p). av, aortic valve leaflet.

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only one of several agonist-activated (class I) isoforms of PI3-kinase found in inflammatory cells. Deletion of the p110� genealone was sufficient to abrogate all detectable PI3-kinase/Aktactivation as well as PI3-kinase-dependent phosphorylation ofdownstream effector molecules FKHR, AFX, GSK3, p70S6kinase,S6 ribosomal protein, and PKC� within the context of athero-sclerotic lesions in apoE�/� mice. This remarkable phenomenonwas associated with a significant reduction of atheroscleroticlesion size. In the present study, we demonstrated an absoluterequirement for p110� in Akt activation in cultured BMDMs inresponse to many of the agonists, chemokines, and inflammatorymediators commonly implicated in atherogenesis. This findinglends strong support for the critical role of p110� in atheroscle-rosis progression and may in part explain the absence of Aktactivation in lesions from apoE�/�p110��/� mice.

Oxidative stress in general, and oxidatively modified lipopro-teins in particular, play a major role in driving atheroscleroticplaque progression. Here we have demonstrated that oxidizedLDL is a potent macrophage agonist, inducing PI3-kinase/Aktactivation, and that this activation requires p110�. Becauseactivation of inflammatory cells results in stimulation of respi-ratory burst and generation of oxygen-derived free radicals, theinteraction of oxidatively modified lipoproteins with plaquemacrophages can be expected to result in cellular activation,generation of oxygen-derived free radicals, increased formationof oxidatively modified lipoproteins, and so on in a self-reinforcing process of feed-forward stimulation. Because p110�-deficient inflammatory cells are severely impaired in their abilityto generate superoxide in response to chemokines and otheragonists (6–8), it is likely that deficiency (or inhibition) of p110�in vivo would abrogate this process, reducing oxidative stresswithin the atherosclerotic plaque, reducing lesion size, andpossibly increasing plaque stability.

Although plaque rupture occurs only sporadically in thecommonly studied mouse models of atherosclerosis, including inthe apoE-deficient mice used in the present study, the possibilitythat inhibition of PI3-kinase in atherosclerotic lesions mightaffect plaque stability (in addition to retarding plaque progres-sion) is clinically relevant. Inherent in the unstable plaque is ahigher propensity to rupture, thereby causing acute thromboticocclusion of arteries that leads to clinical syndromes such asmyocardial infarction and stroke (26). Oxidative stress withinatherosclerotic lesions, a potential contributor to plaque desta-bilization (27–29), should decline in the absence of p110�, giventhe markedly reduced respiratory burst seen in p110�-nullinflammatory cells (6–8). Here we have demonstrated thatangiotensin II signaling, which contributes to oxidative stress,plaque progression, and plaque destabilization (30, 31), is mark-edly attenuated in p110�-null macrophages.

Until recently, exploitation of PI3-kinase as a target forpreventative or therapeutic intervention in the numerous diseaseprocesses in which it has been implicated has been hampered bythe lack of isoform-specificity of the available inhibitors such aswortmannin and LY294002. In marked contrast to these non-specific but widely used inhibitors, AS605240, a recently de-scribed thiazolidinedione derivative that is an ATP-competitiveinhibitor of p110� with a ki of 0.0078 �M, displays 30-fold higherpotency against p110� in vitro compared with the other PI3-kinases. AS605240 has been shown to block glomerulonephritisin a mouse model of systemic lupus erythematosus as well as jointinflammation in a mouse model of rheumatoid arthritis (32, 33).It would be of interest to test this, or related compounds, forpotential antiatherogenic activity in LDL receptor- or apoEknockout mice.

In summary, the present study shows that p110� is required forAkt activation in macrophages in response to oxidized LDL,atherogenic chemokines, and angiotensin II in vitro and inatherosclerotic lesions of hypercholesterolemic mice in vivo, and

that loss of p110� results in reduced lesion size. Moreover, itprovides evidence to support the possibility that inhibition ofp110� could also stabilize atherosclerotic plaques. Mice lackingp110� or expressing in its place a catalytically inactive mutantdisplay normal viability, fertility, and longevity despite thepresence of specific defects in inflammatory and immune celldevelopment and activation (6–8, 34). In light of the welltolerated nature of these defects, p110� is a potential target fora drug that could be distributed systemically yet maintain a hightherapeutic index. Pharmacologic suppression of inflammatoryand immune responses is a cornerstone for the treatment of awide variety of conditions such as rheumatoid arthritis, multiplesclerosis, Crohn’s disease, and organ transplant rejection. Ourfindings provide a compelling rationale for extending such anapproach to the prevention and treatment of atheroscleroticcardiovascular disease.

MethodsMice. Mice were handled in accordance with guidelines of theBeth Israel Deaconess Medical Center Institutional AnimalCare and Use Committee. ApoE�/� mice (C57BL/6J) wereobtained from The Jackson Laboratory (Bar Harbor, ME).p110��/� mice (C57BL/6J) were generated as described previ-ously (7). Both lines of mice had previously been backcrossedonto a C57BL/6 background for �12 generations. Mice con-suming standard laboratory chow (Harlan Teklad F6 RodentDiet) containing 6% crude fat were killed at various time pointsfrom 22 to 60 weeks of age. Aortas were removed en bloc, andcryosections of the aortic root were obtained at the level of theaortic valve and sinuses of Valsalva. The cryosections werestained for lipid content with oil-red-O and for immunoperox-idase with antibodies against macrophages (Mac-3), smoothmuscle cells (�-actin), and T lymphocytes (CD4), as well as forAkt phosphoSer473.

Measurement of Cholesterol and Triglycerides. Serum was separatedfrom erythrocytes by centrifugation at 1,100 � g at 4°C andassayed for total cholesterol, HDL cholesterol, and triglycerideson a Hitachi 911 automated analyzer (Roche Diagnostics, Indi-anapolis, IN) by using enzymatic methods (35). The assays arestandardized through the Lipid Standardization Program of theCenters for Disease Control (Atlanta, GA). Non-HDL choles-terol was computed as the difference between total and HDLcholesterol.

Lipoprotein Purification and Oxidation. LDL, HDL, very LDL, andvery HDL were isolated by preparative ultracentrifugation fromhuman blood plasma according to the method of Hatch (36) andused within 2 weeks of purification. Oxidation of LDL wasperformed by incubation of LDL in PBS at a concentration of 5mg/ml (total protein) with 5 �M CuSO4 at 37°C for 24 h. Nativeand oxidized LDL were preserved with 1 mM EDTA and 0.01%sodium azide and stored at 0°C in tightly sealed tubes undernitrogen gas to minimize further oxidation.

Analysis of BMDMs. Femurs from 1-month-old mice were har-vested after pheonobarbital-induced killing. Marrow wasflushed out with DMEM and cultured in DMEM containing10% FBS and 20% L cell-conditioned medium, which supportsmonocyte/macrophage proliferation as a result of M-CSF se-creted by L cells. Cells were incubated overnight in bacterialPetri dishes, and nonadherent cells were collected and passagedfor further culture. Adherent cells were replated the next day ata density of 5 � 105 cells per well in 60-mm tissue culture plates.After overnight serum starvation, cells were exposed to agonistswith or without pretreatment (20 min) in 20 nM wortmannin.

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Western Blot Analysis of PI3-Kinase Activation. The primary anti-body, rabbit polyclonal anti-phosphoAkt (Ser473) (Cell SignalingTechnology, Danvers, MA), detects Akt only when Akt is phos-phorylated on serine in position 473. Phosphorylation of thisresidue is wortmannin-sensitive and PI3-kinase-dependent. TotalAkt was detected with a rabbit polyclonal Akt (Cell SignalingTechnology) that recognizes all phosphorylation states of Akt.

Analysis and Quantification of Aortic Atherosclerosis. Double knock-out and littermate control mice were killed at various time pointsto quantify early (nascent) and late (advanced) atheroscleroticlesions as described previously (37). Because our hypothesis wasthat reduced reactivity of inflammatory cells deficient in p110�to atherogenic agonists and chemokines should result in atten-uation of atherosclerosis in apoE knockout mice, and p110��/�

and p110��/� macrophages displayed similar Akt activation inresponse to these agonists/chemokines, we pooled results fromapoE�/�p110��/� and apoE�/�p110��/� mice. The mice in-cluded in a quantitative analysis of aortic root atherosclerosiswere all females. Qualitatively similar results were obtainedwhen male animals were analyzed. Similarly, immunohistochem-ical findings with phosphospecific antibody staining of aorticroot lesions from apoE�/�p110��/� and apoE�/�p110��/� micewere identical.

Immunohistochemistry. Histopathological and immunohistochem-ical analysis was performed on serial cryostat sections (6 �m)counterstained with hematoxylin as described previously (38).Antibodies against phosphoAkt Ser473 (IHC-specific), Akt,FKHR, phosphoFKHR, phosphoAFX, phosphoGSK3�/�,phosphoPKC�, phosphop70S6kinase, and phosphoS6 ribosomalprotein were obtained from Cell Signaling Technology and usedat a dilution of 1:100. Mac-3, CD4, and smooth muscle actinantibodies were obtained from BD Biosciences (San Jose, CA)and used at dilutions of 1:1,000, 1:200, and 1:1,000, respectively.

Statistical Analysis. Student’s two-tailed t test was used to deter-mine the statistical significance of differences in serum lipopro-tein/triglyceride levels and aortic root atherosclerosis betweenapoE�/�p110��/� or apoE�/�p110��/� mice and age-matchedapoE�/�p110��/� mice.

We thank Bradley Smith and Glen J. Bubley for discussion of immuno-histochemistry using phosphospecific antibodies, and Kenneth Swansonfor assistance with BMDM isolation and culture. This work was sup-ported by grants from the Donald W. Reynolds Foundation (to J.D.C.,P.L., and S.J.F.) and National Institutes of Health Grant GM41890 (toL.C.C.).

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