Published online before print February 11, 2008, doi: 10.1161/CIRCULATIONAHA.107.720466
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(Circulation. 2008;117:1310-1317.)
© 2008 American Heart Association, Inc.
Vascular Medicine |
Genetic and Pharmacological Targeting of Phosphoinositide 3-Kinase-
Reduces Atherosclerosis and Favors Plaque Stability by Modulating Inflammatory Processes
From INSERM U563, Département Lipoprotéines et Médiateurs Lipidiques, Toulouse, France (A.F., S.G., J.-P.S., B.P., M.B.-D., M.L.); Université Paul Sabatier-Toulouse III, Toulouse, F-31000 France (A.F., S.G., P.G., A.S., J.-F.A., J.-P.S., B.P., M.B.-D., M.L.); CHU Toulouse, Hôpital des Enfants, Unité d’Endocrinologie, Toulouse, F31000 France (J.-P.S.); INSERM U858, Institut de Médecine Moléculaire de Rangueil, Toulouse, France (P.G., A.S., J.-F.A.); CHU Toulouse, Hôpital de Rangueil, Département Explorations fonctionnelles physiologiques, Toulouse, F-31000 France (J.-F.A.); CHU Toulouse, Hôpital de Rangueil, Service Diabétologie-Maladies Métaboliques-Nutrition, Toulouse, F-31000 France (P.G.); CHU Toulouse, Hôpital Purpan, Institut Fédératif de Biologie, Laboratoire de Biochimie, Toulouse, F-31000 France (B.P.); Merck Serono Geneva Research Center, Geneva, Switzerland (T.R., M.K.S., C.R.); University of Turin, Department of Genetics, Biology and Biochemistry, Turin, Italy (E.H.); and Institut of Biochemistry and Genetics, University of Basel, Department of Biomedicine, Basel, Switzerland (M.P.W.).
Correspondence to Muriel Laffargue, INSERM U563, Département Lipoprotéines et Médiateurs Lipidiques, Toulouse-Purpan, BP 3028, 31024 Toulouse Cedex 3, France. E-mail muriel.laffargue{at}toulouse.inserm.fr
Received June 8, 2007; accepted December 27, 2007.
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Abstract |
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Background— The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a notable quest for novel and innovative drugs for the treatment of atherosclerosis. The lipid kinase phosphoinositide 3-kinase-
(PI3K) is thought to be a key player in various inflammatory, autoimmune, and allergic processes. These properties and the expression of PI3K in the cardiovascular system suggest that PI3K plays a role in atherosclerosis.
Methods and Results— Here, we demonstrate that a specific PI3K inhibitor (AS605240) is effective in murine models of established atherosclerosis. Intraperitoneal administration of AS605240 (10 mg/kg daily) significantly decreased early atherosclerotic lesions in apolipoprotein E–deficient mice and attenuated advanced atherosclerosis in low-density lipoprotein receptor–deficient mice. Furthermore, PI3K levels were elevated in both human and murine atherosclerotic lesions. Comparison of low-density lipoprotein receptor–deficient mice transplanted with wild-type or PI3K-deficient bone marrow demonstrated that functional PI3K in the hematopoietic lineage is required for atherosclerotic progression. Alleviation of atherosclerosis by targeting of PI3K activity was accompanied by decreased macrophage and T-cell infiltration, as well as increased plaque stabilization.
Conclusions— These data identify PI3K as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment.
Key Words: phosphoinositide 3-kinase- • atherosclerosis • inflammation • leukocytes • fibrous cap
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Introduction |
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Atherosclerosis is a chronic disease of large arteries and is the primary cause of myocardial infarction and stroke. Atherosclerosis is the underlying cause of
50% of all deaths in most occidental countries and is increasing dramatically in developing countries. The disease starts with the formation of fatty streaks and culminates with plaque disruption, thrombosis, and cardiac infarction.1 The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a growing quest for novel and innovative drugs for the treatment of atherosclerosis.2 Vascular cells release chemokines, which recruit immune cells by their action on the G-protein–coupled receptor family.1 Via phosphoinositide 3-kinase (PI3K), G-protein–coupled receptors regulate several inflammatory and immune functions, eg, recruitment of macrophages, neutrophils, and lymphocytes,3–5 mast cell reactivity,6 platelet aggregation,7 and T-cell function,5 as demonstrated in mice lacking functional PI3K.8 The -isoform belongs to the class I PI3K family, which includes 2 subclasses: class IA (
, β, and 
), activated via the tyrosine kinase receptor, and class IB (), mainly activated by G-protein–coupled receptors.8 Initial studies proposed that PI3K-selective inhibitors possess immunomodulatory potential, pinpointing PI3K as a good candidate for drug development in chronic inflammation and autoimmune diseases.9 In murine models of rheumatoid arthritis, oral administration of selective PI3K inhibitors successfully prevented progression of joint inflammation and neutrophil accumulation.10 Moreover, it has been demonstrated that intraperitoneal administration of PI3K inhibitor in a murine model of systemic lupus erythematosus led to a reduction in pathogenic CD4+ memory cells, which resulted in a blockade of glomerulonephritis and an extended lifespan.11 PI3K expression is high in hematopoietic cells, but recent studies have demonstrated low levels of PI3K in endothelial cells,12 cardiomyocytes,13 and smooth muscle cells.14 PI3K expression in arterial cells is of particular interest because mice that lack PI3K showed modified E-selectin–dependent adhesion of neutrophils to endothelial cells12 and were protected against the hypertensive damage of chronic angiotensin II exposure.14 Here, we demonstrate that inhibition of PI3K alleviates atherosclerotic plaque development in 2 murine models of atherosclerosis. Furthermore, we provide evidence that loss of PI3K function in the hematopoietic lineage is sufficient to explain the observed antiatherosclerotic effect.
Clinical Perspective p 1317
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Methods |
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Biological Materials
Human carotid artery sections were obtained from surgical pieces of endarterectomy procedures performed in patients with carotid stenosis in the Department of Vascular Surgery, Toulouse University Hospital. PI3K
-deficient mice generated previously on a 129sv inbred genetic background3 were backcrossed for 10 generations onto the C57Bl/6 background. The resulting heterozygotes for PI3K were intercrossed to obtain PI3K+/+ and PI3K–/– littermates. As models of in vivo atherosclerosis, C57Bl/6 ApoE–/– mice and C57Bl/6 low-density lipoprotein receptor–deficient (LDLR–/–) mice were obtained from Charles River Laboratories, (Wilmington, Mass). To inhibit PI3K, we used AS605240 from Merck Serono (Geneva, Switzerland).10 AS605240 was used as described previously.11 Experiments were performed in accordance with institutional guidelines and national legislation.
Bone Marrow Transplantation
Eight-week-old C57Bl/6 LDLR–/– mice were subjected to medullar aplasia by 9-Gy lethal total-body irradiation. We repopulated mice with intravenous injection of bone marrow cells isolated from femurs and tibias of donor PI3K+/+ and PI3K–/– littermates. After 6 weeks of recovery, mice were fed a proatherogenic diet (15% fat, 1.25% cholesterol, 0% cholate) for an additional 10 weeks. Mice were euthanized at 24 weeks of age. Blood and tissues were collected as described below.
Tissue Preparation and Characterization of Atherosclerotic Lesions
Hearts were removed and aortic root sections prepared as described previously.15 Surface lesion area was measured after oil red O staining by computer-assisted image quantification with Leica QWin software (Leica Microsystems, Wetzlar, Germany). Images were captured with a Sony (Tokyo, Japan) 3CCD video camera. Collagen fibers were stained with Sirius red. At least 5 sections per mouse were examined for each staining.
Immunohistochemistry
Frozen sections from the aortic root were fixed in acetone/methanol, air-dried, and incubated with 10% of relevant serum for 30 minutes. For mouse primary antibodies on murine tissues, a mouse Ig-blocking reagent was used (Vector mouse-on-mouse [M.O.M.] immunodetection kit, Vector Laboratories, Burlingame, Calif). The following specific primary antibodies were used: clone MOMA-2 (Serotec, Oxford, United Kingdom) for macrophage staining; anti-CD3 (clone M-20, Santa Cruz Biotechnology, Santa Cruz, Calif); anti-PI3K,14 anti-P-PKB Ser473 (immunohistochemistry specific), and anti-PKB (pan; Cell Signaling, Danvers, Mass); and anti-SM -actin (clone 1A4, Sigma, St Louis, Mo). Then, sections were incubated with the corresponding secondary biotinylated antibodies (Vector Laboratories) and visualized with an avidin-biotin–horseradish peroxidase complex (Vectastain ABC kit, Vector Laboratories) and DAB peroxidase substrate kit (DakoCytomation, Glostrup, Denmark). Countercoloration was performed with Mayer’s hematoxylin. Irrelevant IgGs were used as negative controls.
Analysis of Plasma Lipids and Lipoproteins
Cholesterol, triglyceride, and HDL levels were measured with commercial kits (CHOD-PAP for cholesterol and GPO-PAP for triglycerides; Randox Laboratories, Crumlin, County Antrim, Ireland). LDL/VLDL level was obtained by determining the difference between total cholesterol and HDL cholesterol.
Statistical Analysis
Comparisons between the different groups were performed with a Student t test for unpaired data. Statistical differences were considered significant for probability values <0.05. Data are shown as mean±SEM.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
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Results |
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Inhibitor of PI3K
Reduces Early Atherosclerotic Lesions in ApoE–/– MiceTo investigate the potential role of PI3K
activity in processes that lead to atherosclerosis, we treated mice with a selective inhibitor of PI3K, AS605240, which was injected intraperitoneally at 10 mg/kg daily. Apolipoprotein E–deficient (ApoE–/–) mice develop spontaneous atherosclerotic lesions that are well characterized as a model for the early stage of atherosclerosis. Monocyte adhesion and migration occur between weeks 7 and 10, whereas foam cell lesions form during week 9.16 Eight-week-old ApoE–/– mice were injected with PI3K inhibitor or vehicle for 5 weeks. To control AS605240 specificity, we investigated PKB phosphorylation, the main downstream target of PI3K, in monocyte chemoattractant protein-1 (MCP-1)– and granulocyte and macrophage-colony stimulating factor (GM-CSF)–stimulated leukocytes from AS605240- or vehicle-treated mice. We observed that PKB phosphorylation by MCP-1, known to directly recruit PI3K, was dramatically reduced in leukocytes from AS605240-treated mice (93.2±8.7%; Figure 1A). In contrast, stimulation by GM-CSF, an activator of class IA PI3K, did not result in any significant variation of PKB phosphorylation in leukocytes from AS605240-treated mice compared with vehicle-treated mice (Figure 1A). Analysis of lipid deposition in aortic root sections of control mice revealed formation of early lesions characterized by their small size (Figure 1B) and low collagen content (Figure 1C). Treatment with AS605240 dramatically reduced lesion formation without significantly affecting collagen levels. At the same time, plasma lipid levels were not changed, which suggests that the decreased lesion size observed was not accounted for by changes in plasma cholesterol levels or in the distribution of lipoprotein particles (Figure 1D).
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Advanced Atherosclerotic Lesions in LDLR–/– Mice Respond to PI3K Inhibition
LDLR–/– mice were fed an atherogenic diet high in fat and cholesterol for 8 weeks to trigger the development of an aggressive form of atherosclerosis that resembles progressive familial hypercholesterolemia. LDLR–/– mice treated with PI3K inhibitor during the last 5 weeks did not show any variation in plasma lipid levels compared with vehicle-treated controls (Figure 2A); however, AS605240 induced a significant decrease in advanced lesion size (84 900±6500 versus 138 000±17 900 µm2; Figure 2B). In these conditions, LDLR–/– mice developed more advanced plaques than in ApoE–/– experiments, as demonstrated by their collagen content (27% versus 5% of total lesion area; Figure 2C). Moreover, a significant increase in collagen was observed in AS605240-treated LDLR–/– mice compared with vehicle-treated mice (Figure 2C).
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Thus, all pharmacological experiments performed in both mice models suggested that downmodulation of PI3K activity was able to reduce the size of both early and advanced atherosclerotic lesions in mice. In addition, the increase in collagen observed in AS605240-treated LDLR–/– mice suggests that PI3K from immune cells could participate in atherosclerosis development, because inhibition of the inflammatory process is responsible in part for an increased collagen content.17
PI3K Is Highly Expressed and Active in Human and Murine Atherosclerotic Lesions
PI3K was originally identified in hematopoietic cells, where it is highly expressed8; however, recent studies have demonstrated low levels of PI3K expression in other cells, especially in the cardiovascular system. We therefore performed immunohistochemical analyses of PI3K in atherosclerotic lesions. Expression of PI3K within healthy segments of human carotids was low, whereas high levels of PI3K were detected within atherosclerotic lesions (Figure 3A, left). High expression of PI3K was also observed in aortic sinus lesions from both ApoE–/– and LDLR–/– mice (Figure 3A, right). To precisely localize PI3K within the plaque, we then performed immunochemical staining of macrophages (MOMA-2) and T lymphocytes (CD3) in sections adjacent to those stained with anti-PI3K antibody (Figure 3B). Staining of PI3K, MOMA-2, and CD3 was perfectly superimposed, which suggests that most PI3K in the lesions was in immune cells. To assess PI3K activity in atherosclerotic lesions, we measured PKB phosphorylation in LDLR–/– aortic root sections treated or not treated with AS605240. Results showed an important phosphorylation of PKB (26.8±4.1% of the lesion area) that was largely inhibited by AS605240 treatment (3.22±2.5%) without a change in PKB expression (Figure 3C). These results further suggest that immune PI3K could be the main target of a pharmacological atheroprotective intervention.
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Loss of PI3K in Immune Cells Is Sufficient to Reduce Atherosclerosis
To evaluate the importance of hematopoietic PI3K in the development of atherosclerosis, we generated a mouse model of atherosclerosis that lacked PI3K exclusively in immune cells by bone marrow transplants from PI3K–/– mice to irradiated 
LDLR–/– recipient mice (PI3K–/–LDLR–/–). Analysis of atherosclerotic plaques revealed an important decrease in the size of lesions in PI3K–/–LDLR–/– compared with PI3K+/+ littermate transplanted mice (PI3K+/+LDLR–/–; 55 689±5185 µm2 vs 106 429± 10 055 µm2, respectively; Figure 4A). We did not observe any difference in plasma lipid levels either in PI3K–/– donor mice compared with wild-type donor mice (Figure 4B) or in PI3K–/–LDLR–/– compared with PI3K+/+LDLR–/– mice (Figure 4C) after hypercholesterolemic diet. These results illustrate that the expression of PI3K in immune cells is sufficient to drive formation of atherosclerotic lesions in mice.
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Loss of PI3K in Macrophages and T Cells Impairs Infiltration Within Atherosclerotic Lesions
Cellular composition of atherosclerotic lesions is of crucial importance in both atherogenesis and plaque rupture. We next performed immunohistochemical studies of atherosclerotic constituents to further delineate the effects of immune PI3K on lesion formation. Analyses were performed on transplanted mice fed an atherogenic diet for 10 weeks and were normalized to lesion area in the 2 groups. As shown with the anti-MOMA-2 antibody, there was a marked decrease in macrophage infiltration in atherosclerotic plaque in the PI3K–/–LDLR–/– mice compared with PI3K+/+ LDLR–/– control mice (30% reduction, P=0.019; Figure 5A). In addition to the observed effect on macrophage accumulation, anti-CD3 staining revealed an important reduction in T-cell infiltration in the absence of PI3K in the hematopoietic system (39% reduction, P=0.019; Figure 5B).
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Loss of Immune PI3K Favors Plaque Stabilization
In addition to reduction of lesion size, the constitution of a thick fibrous cap is an important feature of plaque stabilization that can prevent physical plaque disruption and subsequent thrombus formation. Indeed, in fatal myocardial infarction, the plaque has a large lipid core, an abundance of inflammatory cells, a relative paucity of smooth muscle cells, and a thin collagen fibrous cap.17 This type of plaque is called "vulnerable," and its stabilization has become a new therapeutic goal.18,19 To evaluate the potential benefit of the absence of immune PI3K on the biology of the plaque, we analyzed plaque collagen content and smooth muscle cell content in PI3K–/– bone marrow–transplanted LDLR–/– mice. A dramatic increase in collagen content (Figure 6A) accompanied by an increase in smooth muscle cell content (Figure 6B) in atherosclerotic lesions from PI3K–/– LDLR–/– mice emphasizes the fact that inactivation of PI3K in immune cells could have an additional beneficial effect on the stability of atherosclerotic plaque.
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Discussion |
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PI3K
has been shown to possess immunomodulatory potential and is expressed in the cardiovascular system, which provides a rationale for pharmacological targeting of PI3K in atherogenesis in vivo. In agreement with the results of a study by Chang et al20 reporting reduced plaque size in PI3K–/–/ApoE–/– double-knockout mice compared with PI3K+/+/ApoE–/– mice, we found that PI3K was essential to the development of atherosclerosis. Here, we provide new insights into the role of PI3K in inflammatory cells in the development of atherosclerotic lesions. To test a PI3K-based therapy, mice susceptible to atherosclerosis were treated with AS605240, a specific PI3K inhibitor. PI3K dependence was observed in early atherosclerotic lesions in ApoE–/– mice and in advanced lesions created in LDLR–/– mice on an atherogenic diet without any toxic effect at a dose of 10 mg/kg daily. Pharmacological studies with an AS605240 inhibitor have been described in mouse models of rheumatoid arthritis and systemic lupus erythematosus. In both models, PI3K-inhibitor administration blocked the progression of the pathology by modulating immune responses without inducing adverse effects.10,11 AS605240 went through standard regulatory toxicity studies with increasing oral dosing up to 300 mg/kg. Toxicological examination showed that AS605240 had no adverse secondary effects after the 5-day treatment period, with a cumulative dose of 500 mg/kg (data not shown). Whereas in rheumatoid arthritis, a PI3K inhibitor prevented joint inflammation and tissue damage via defective neutrophil migration,10 in lupus erythematosus, PI3K inhibition reduced levels of pathogenic CD4+ memory cells, thus blocking glomerulonephritis.11 Interestingly, successful treatment of these autoimmune diseases was obtained at elevated doses of PI3K inhibitor (30 mg · kg–1 · d–1). Here, we demonstrate that an important reduction of atherosclerotic lesions was already observed at a lower dose (10 mg · kg–1 · d–1). In addition to its function in the immune system, PI3K has also been found at lower levels in endothelial and smooth muscle cells. Endothelial PI3K activity has been demonstrated as essential for interaction of neutrophils with the inflamed vessel walls that result from a perturbation in selectin-mediated adhesion of neutrophils to vessel wall.12 In smooth muscle cells, PI3K was involved in angiotensin II–mediated contraction, and mice deficient in PI3K were protected against the hypertensive damage of chronic angiotensin II exposure.14 Consistent with these data, we found weak PI3K expression in healthy segments of human coronary arteries. Nevertheless, a high signal was observed in macrophage and T-lymphocyte–rich regions of atherosclerotic lesions in humans and mice, which suggests that immune PI3K could be responsible for the pharmacological effects observed. According to the present transplantation experiments, inhibition of lesion formation in the absence of PI3K from immune cells attenuates atherosclerosis as effectively as a systemic inhibition of PI3K by pharmacological means with AS605240; however, we cannot completely exclude that a small portion of the observed effects can also be attributed to endothelial PI3K activity.
The present study focused on aortic root atherosclerotic lesions; however, it was demonstrated that immune cells could differentially regulate atherosclerosis development, depending on the lesion site considered.21 In the present model, en face aorta analysis showed only a few lesions apart from the aortic roots (data not shown). Nevertheless, aortic arch lesions were only observed in aortas from PI3K+/+LDLR–/– mice (2% of aortic arch area) and not in PI3K–/–LDLR–/– mice.
Genetic data have provided evidence for the importance of chemokine receptors in the early steps of atherosclerosis, because they recruit monocytes22 and T cells,23–25 which have been identified recently as key regulators of atherosclerosis. Several reports show that T-helper type 1–driven responses promote plaque formation,26–28 whereas natural regulatory T cells are able to attenuate these responses and decrease the progression of atherosclerotic lesions.29 PI3K plays a major role in recruiting inflammatory cells, acting downstream of chemokine receptors, because attraction of macrophages to inflamed tissue was highly reduced in PI3K-null mice.3–5 The analyses of cell content in atherosclerotic lesion from PI3K–/–LDLR–/– mice compared with P3K+/+ LDLR–/– mice demonstrated a decrease in macrophage and T-cell infiltration, which provides further evidence that interference with PI3K signaling modifies the inflammatory processes responsible for the development of atherosclerosis. These results could be explained by the important role of PI3K downstream of chemokine receptors for macrophage recruitment at the lesion site,3–5 but its involvement in T-cell migration is controversial. Indeed, in vitro studies demonstrated that migration of PI3K-deficient CD4+ and CD8+ T cells to CCL19, CXCL12, and low doses of CCL21 was reduced by 30% to 65%.30,31 In contrast, recent observations with multiphoton intravital microscopy revealed that lack of PI3K had negligible effects on migration velocities but resulted in increased turning angles of T cells.32 Moreover, systemic lupus erythematosus induced by an enhanced activation of class IA PI3K in T cells was ameliorated by PI3K deficiency without effects on T-cell invasion, which suggests that PI3K was not involved in T-cell migration in vivo.33 Nevertheless, recent data indicating that PI3K is responsible for a modification of the CD4/CD8 differentiation ratio34 are particularly relevant in the pathology of atherosclerosis. It would be of great interest to discriminate the importance of PI3K activity in macrophages from its effects in T-cell differentiation during atherosclerosis development.
An important finding of the present study is that immune deletion of PI3K not only reduces plaque size formation but also promotes plaque stabilization. Although plaque rupture is rarely observed in mice, whereas it is fairly common in humans,16 collagen content and smooth muscle cells are good indicators of plaque stability. The observed increase in collagen and smooth muscle cells in plaques from PI3K-null mice may thus be clinically relevant, because stable plaque could keep atherosclerosis nonsymptomatic, prevent thrombus formation, and prevent subsequent myocardial infarction and stroke.
It has also been reported that PI3K plays an important role in platelet aggregation downstream of the ADP receptor P2Y12. The absence of PI3K did not affect bleeding time but protected mice from death caused by ADP-induced thromboembolic vascular occlusion.7 Currently, P2Y12 receptor inhibitors such as ticlopidine and clopidogrel are used as antithrombotic therapy.35 The present results led us to speculate that pharmacological inhibition of PI3K could also have beneficial effects on thrombi formation when plaque is disrupted without inducing critical adverse effects, especially the bleeding and thrombocytopenic purpura observed in patients treated with clopidrogrel.36
In summary, the results of the present study identify PI3K as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment. Current treatments for atherosclerosis are mainly based on drugs that lower plasma cholesterol concentration and blood pressure. Statins, which target 3-hydroxy-3-methylglutaryl coenzyme A reductase, have well-documented advantageous effects on atherosclerosis. In addition to their cholesterol-lowering effect, recent reports suggest that statins also possess antiinflammatory and immunomodulatory actions.37,38 Nevertheless, several adverse events have been reported, such as renal toxicity and rhabdomyolysis, in patients treated with statins.39,40 Although the precise reason for these effects is not fully understood, it appears that they are in part the result of mitochondrial toxicity and selenoprotein deficiency.41,42 The identification and development of promising new antiinflammatory therapy is therefore of great medical interest. In light of the immunomodulatory properties of PI3K inhibitors and the known function of PI3K in controlling vascular smooth muscle tone and cardiac cell contractility,43 our present finding is the last major step needed to promote the development of PI3K inhibitors for cardioprotective therapy.
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Acknowledgments |
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We are grateful to J.C. Thiers for providing surgical samples from human endarterectomy procedures and M.J. Fouque for technical assistance.
Sources of Funding
This work was supported by the Institut National de la Santé et de la Recherche Médicale and the Fondation de France (grant 2004004845). A. Fougerat was supported by the Nouvelle Société Française d’Athérosclérose. Dr Wymann was supported by the Swiss National Science Foundation and an FP7 project MAIN LSHG-CT-2003-502935.
Disclosures
None.
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*The first 2 authors contributed equally to this work.
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Circulation 2008 117: 1247.
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