Absence of Akt1 Reduces Vascular Smooth Muscle Cell Migration and Survival and Induces Features of Plaque Vulnerability and Cardiac Dysfunction During Atherosclerosis
Objective— Deletion of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Vascular smooth muscle cells (VSMCs) are an important component of atherosclerotic plaques, responsible for promoting plaque stability in advanced lesions. Fibrous caps of unstable plaques contain less collagen and ECM components and fewer VSMCs than caps from stable lesions. Here, we investigated the role of Akt1 in VSMC proliferation, migration, and oxidative stress–induced apoptosis. In addition, we also characterized the atherosclerotic plaque morphology and cardiac function in an atherosclerosis-prone mouse model deficient in Akt1.
Methods and Results— Absence of Akt1 reduces VSMC proliferation and migration. Mechanistically, the proliferation and migratory phenotype found in Akt1-null VSMCs was linked to reduced Rac-1 activity and MMP-2 secretion. Serum starvation and stress-induced apoptosis was enhanced in Akt1 null VSMCs as determined by flow cytometry using Annexin V/PI staining. Immunohistochemical analysis of atherosclerotic plaques from Akt1−/−ApoE−/− mice showed a dramatic increase in plaque vulnerability characteristics such as enlarged necrotic core and reduced fibrous cap and collagen content. Finally, we show evidence of myocardial infarcts and cardiac dysfunction in Akt1−/−ApoE−/− mice analyzed by immunohistochemistry and echocardiography, respectively.
Conclusion— Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress–induced apoptosis. Absence of Akt1 induces features of plaque vulnerability and cardiac dysfunction in a mouse model of atherosclerosis.
Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries.1–3 This complex disease involves interactions of modified lipoproteins, monocyte-derived macrophages or foam cells, T-lymphocytes, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and fibroblast.4,5 VSMCs are an important component of atherosclerotic plaques, responsible for promoting plaque stability in advanced lesions.4 The structural components of atherosclerotic plaque caps consist primarily of VSMC-derived collagen, elastin, proteoglycans, and extracellular matrix (ECM).4,5 Fibrous caps of unstable plaques contain less collagen and ECM components and fewer VSMCs than caps from stable lesions. Apoptosis of VSMCs may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death.6,7
The serine/threonine kinase Akt (also known as protein kinase B [PKB]) plays a central role in the regulation of cellular growth, survival, and metabolism.8,9 In mammalian cells, there are 3 distinct Akt isoforms (Akt1,-2, and -3) which are products of different genes.10 Gene knockout studies have shown that despite significant sequence homology, the 3 Akt isoforms have some nonredundant functions. In the cardiovascular system, Akt1 plays an important role in the regulation of cardiac hypertrophy, angiogenesis, and apoptosis.11–13 More importantly, we have recently reported that absence of Akt1 leads to severe atherosclerosis and occlusive artery disease.14 The increased atherosclerosis observed in mice lacking Akt1 is mechanistically linked to endothelial cell dysfunction. However, the role of Akt1 in VSMC biology and its implication in the progression of atherosclerosis remain unknown. Here, we report that Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress–induced apoptosis. Moreover, we show that absence of Akt1 in atherosclerosis-prone apolipoprotein E (ApoE) knockout mice induces features of plaque vulnerability such as increased necrotic core formation and reduced fibrous cap. Finally, we show evidences of spontaneous infarcts in mice lacking Akt1, a rare phenotype observed in murine models of atherosclerosis. The presence of infarcts in Akt1−/−ApoE−/− mice provides a new model for studying atherosclerosis-induce coronary syndromes in mice.
Materials and Methods
Akt1−/− mice were generated as previously described.15 Akt1−/− mice that have been backcrossed 6 generations onto a C57BL/6 background were crossed with ApoE-deficient mice, also on the C57BL/6 background, to generate mice heterozygous at both loci. These ApoE+/− Akt1−/− mice were crossed a second time with ApoE−/− mice. The ApoE−/− Akt1−/+ progeny from this round of breeding were then intercrossed to produce ApoE−/− Akt1−/− and ApoE−/− Akt1+/+ littermates that were used as controls for all studies. Accelerated atherosclerosis was induced by feeding the mice for 14 weeks with a high-fat Western-type diet containing 1.25% cholesterol (ResearchDiets, D12108). All animal protocols were approved by the Institutional Animal Care Use Committee of Yale University.
Atherosclerotic Lesion Analysis
After 12 weeks of being fed a Western-type diet, male mice (7 ApoE−/− Akt1+/+ and 6 ApoE−/− Akt1−/−) were anesthetized and euthanized. Mouse hearts were perfused using 10 mL of PBS (Invitrogen) followed by 10 mL of 4% paraformaldehyde (PFA). After overnight incubation in 4% PFA, washed tissues were embedded in OCT blocks. The proximal aorta was cut in 10-μm-thick cryosections and stained with hematoxylin/eosin and Masson trichrome. Images were captured with an Eclipse 80i microscope (Nikon), DMX 1200C digital camera (Nikon) using NIS-elements D 3.0 and analyzed using Image J software (NIH). Fibrous caps were defined as the VSMC- and proteoglycan-rich area overlying the cholesterol-rich, matrix-poor, acellular regions of the necrotic cores.
Primary Aortic Smooth Muscle Cells Isolation and Culture
Mouse primary VSMCs were isolated from normal aorta of male mice by combined collagenase and elastase digestion method. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and used until 10 passages. Human aortic VSMCs were isolated by explant outgrowth and serially cultured in M199 medium supplemented with 20% FBS, L-glutamine, and antibiotics. Human VSMCs were used at passages 3 and 4.
VSMC Proliferation Assay
VSMCs were grown in DMEM medium supplemented with 10% of FBS. At the indicated times, viable cells were determined by Trypan blue dye exclusion using a hemocytometer.
VSMC Migration Assay
Transwell inserts (Costar transwell inserts; Corning) were coated with 0.1% gelatin (Sigma). Platelet-derived growth factor (PDGF) at 1 and 10 ng/mL dissolved in DMEM medium containing 0.1% FBS was added in the bottom chamber. VSMCs (5×104 cells per well) suspended in 100 μL of DMEM containing 0.1% BSA was added to the upper chamber. After 5 hours of incubation, cells on both side of the membrane were fixed and stained with Diff-Quick staining kit (Baxter Healthcare Corp). Cells on the upper side of the membrane were removed with a cotton swab. The average number of cells from 5 randomly chosen high power (200×) fields on the lower side of the membrane was counted.
Cells were fixed with 2% formaldehyde and permeabilized with 0.1% Triton X-100 in phosphate buffered saline, pH 7.5. F-actin was examined using Alexa 568-conjugated phalloidin (Invitrogen).
Rac Activity Assay
Pull-down of GTP-bound Rac was performed by incubating cell lysates with GST-fusion protein containing the p21-binding domain of PAK-1 bound to glutathione agarose for 1 hour at 4°C. The amount of GTP-bound Rac was examined by immunoblotting using a Rac monoclonal antibody (BD Transduction Laboratories).
Flow Cytometry Analysis of Apoptotic Cells
Apoptotic cells were determined by their hypochromic subdiploid staining profiles (SubG1 population). VSMCs cultured in absence of serum or in presence of different doses of hydrogen peroxide (H2O2) were stained with propidium iodide (PI) and analyzed by flow cytometry (FACSort, Becton Dickinson). For staining, 1 million cells were harvested, washed in PBS, and then fixed with ethanol 70% ethanol. Fixed cells were treated with DNase-free (Roche) for 30 minutes at 37°C, washed in PBS, centrifuged, and incubated in PBS containing PI (25 μg/mL; Sigma). Forward light scatter characteristics were used to exclude cell debris from the analysis. For the estimation of early apoptotic cells, Alexa fluorescein isothiocyanate (FITC)-conjugated annexin V (Molecular Probes) was used together with PI dead cell counterstain according to the manufacturer’s recommendations.
Western Blot Analysis
VSMCs were resuspended in lysis buffer: 50 mmol/L Tris-HCl, pH 7.4, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1% NP-40, 0,1% sodium deoxycholate, 0,1% SDS, 100 mmol/L NaCl, 10 mmol/L NaF, 1 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L Pefabloc SC, and 2 mg/mL protease inhibitor cocktail (Roche Diagnostics Corp). Protein concentrations were determined using the DC Protein assay kit (Bio-Rad Laboratories). Lysates containing 30 μg of protein were analyzed by SDS-PAGE and immunoblotting. Primary antibodies used include the following: Akt, p-Akt473, p-Akt308, Akt1, p-FoxO1, FoxO1, p-GSK3, GSK3, p-MDM2, MDM2, ERK, p-ERK, p38 MAPK, p-p38 MAPK (Cell Signaling), PDGFR, Hsp90 (BD Transduction Laboratories; BD Biosciences-Pharmingen). Secondary antibodies were fluorescence-labeled antibodies (LI-COR Biotechnology). Bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology).
Aliquots of cell lysates and conditioned media were analyzed on a 0.1% gelatin-10% zymogram. Cells were lysed in SDS-sample buffer. Appropriately diluted media and cell lysate were loaded without reduction on gels at 10 μL and at an equivalent of 2.5 μg proteins per lane, respectively. After electrophoresis, gels were washed with enzyme renaturing buffer (2.5% TritonX100 in 50 mmol/L tris pH 7.4, 5 mmol/L CaCl2, 1 microM ZnCl2) and incubated in developing buffer (50 mmol/L Tris pH 7.4, 5 mmol/L CaCl, 1 microM ZnCl2) overnight at 37°C. The gel was then stained with Coomassie blue (25% methanol, 15% acetic acid, 0.1% Coomassie blue) for 1 hour at room temperature, and destained in 25% methanol/15% acetic acid for at least 2 hour with multiple changes of destaining solution. The different MMPs were identified by size.
Mice from different groups were anesthetized with isoflurane (0.2% in O2) and subjected to transthoracic echocardiography with a Vevo770 (VisualSonics) ecocardiograph (Perrino C et al 2006). In brief, left ventricular (LV) M-mode echocardiography was performed (VisualSonics). All measurements were made in 3 to 6 consecutive cardiac cycles, and the averaged values used for analysis. LV end-diastolic (LVDd) and end-systolic (LVDs) dimensions as well as the thickness of the interventricular septum (IVST) and posterior wall (PWT) were measured from the M-mode tracings, and fractional shortening (FS) was calculated as (LVDd−LVDs)/LVDd. Diastolic measurements were performed at the point of greatest cavity dimension, and systolic measurements were made at the point of minimal cavity dimension, using the leading-edge method of the American Society of Echocardiography.16
After sectioning, slides were immediately immersed in 2% of 2,3,5-Triphenyltetrazolium chloride (TTC, Sigma-Aldrich) in 0.9% NaCl at 37°C for 10 minutes, photographed, and transferred in 4% PFA.
Data are presented as mean±SEM (n is noted in the figure legends). Comparison of mean values between groups was evaluated by 2-tailed Student t test and Mann–Whitney U test. A probability value of less than 0.05 was considered significant.
Absence of Akt1 Decreased VSMC Proliferation and Migration
VSMC migration and proliferation are critical at different stages of atherosclerosis progression.1 To investigate whether Akt1 is important for regulating proliferation and migration, we analyzed VSMC proliferation at different time points and cell migration using a transwell assay. As shown in Figure 1A, VSMCs isolated from Akt1−/− mice did not grow as well as cells isolated from WT mice. Next, we examined the role of Akt1 in VSMC migration in response to either FBS or PDGF. As shown in Figure 1B the absence of Akt1 reduces FBS- and PDGF-induced migration of VSMCs. Furthermore, the reintroduction of Akt1, but not GFP (by retrovirus) into Akt1−/− VSMCs rescued the defective migratory phenotype in response to serum and PDGF (Figure 1C). Similar results were obtained using human aortic VSMCs after silencing Akt1 with siRNA (Figure 1D).
Akt1 Is Necessary for PDGF-Mediated Dorsal Ruffle Formation, Rac Activation, and MMP-2 Activity in VSMCs
Formations of lamellipodia and membrane ruffles at the leading edge are early events regulating directional cell migration. Because Akt1 has been shown to be important for cell spreading and directional migration in ECs and fibroblasts,17 we determined whether Akt1 is necessary for VSMC dorsal ruffle formation in response to PDGF. As shown in Figure 2A, and quantified in the right panel, VSMCs lacking Akt1 exhibit a significant reduction in dorsal ruffle formation after PDGF treatment. Previous studies in fibroblast and ECs have also demonstrated that Akt1 activity is necessary for Rac1-Pac signaling.17 As seen in Figure 2B, absence of Akt1 significantly reduces Rac1 activity (assayed as Rac1-GTP) after PDGF stimulation. Matrix metalloproteinase-2 (MMP-2) is known to play several important roles during changes is vascular structure associated with both normal and disease processes. The stimulation of VSMCs with PDGF leads to increased expression of MMP-2,18 and this upregulation was linked to increased cell migration.18,19 PDGF-induced MMP-2 has been shown to be dependent on the action of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt.20 To test whether Akt1 is necessary for MMP-2 expression, we stimulated VSMCs with PDGF and analyzed the metalloproteinase activity by zymography. As seen in Figure 2C, and quantified in the right panel, PDGF weakly induced MMP-2, but cells lacking Akt1 had a marked lower level of MMP-2 activity, indicating that Akt1 is important for the expression of MMP-2 in VSMCs.
Loss of Akt1 Leads to a Reduction in PAK1 Phosphorylation Without Changing p38 and ERK Signaling Pathways
To directly examine cellular substrate that may explain the impaired migratory response in VSMCs, we studied Akt substrate phosphorylation (PAK, FoxO1, GSK3β, and MDM2) after PDGF stimulation. As shown in Figure 3A and quantified in the right panels of 3B, all substrates were basally phosphorylated on their respective Akt phosphorylation sites. The loss of Akt1 markedly reduced the phosphorylation of PAK1 and total-Akt, but not FoxO1, GSK3, and MDM2. In addition, absence of Akt1 did not affect the phosphorylation levels of ERK and p38 and the total PDGFR expression (Figure 3).
Absence of Akt1 Reduces VSMC Survival, and the Loss of Akt1 In Vivo Induces Features of Plaque Vulnerability and Acute Myocardial Infarction
To investigate whether Akt1 is important for regulating VSMC viability, we analyzed VSMC survival after treatment with H2O2 (as an apoptogen) using double staining (propidium iodide/ annexin V) analysis by flow cytometry. As shown in Figure 4A and quantified in Figure 4B, the absence of Akt1 causes a dramatic increase of annexin V–positive cells, indicating that this Akt isoform is crucial for sustaining VSMC survival. Similar results were obtained when we determined viable and apoptotic cells by their hypochromic subdiploid staining profile (subG1 population) after incubation of WT and Akt1−/− VSMCs in absence of serum at different time points (Figure 4C).
VSMC apoptosis has been implicated in a number of deleterious consequences in atherosclerosis, including plaque rupture, vessel remodeling, coagulation, inflammation, and calcification.4,21 In advanced plaques, the fibrous cap is thinned from loss of VSMCs, and thin-cap fibroatheromatas are the most prevalent lesions that rupture to produce fatal heart attacks.6 We recently showed that mice deficient in Akt1 in an ApoE-null background exhibit enhanced atherosclerosis,14 however the role of Akt1 on plaque morphology remains unknown. To assess the potential role of Akt1 on plaque composition, we subjected double knockout ApoE−/−Akt1−/− and corresponding ApoE−/− mice to high-cholesterol diet for 14 weeks and analyzed the aortic root lesion areas by hematoxylin/eosin (cellular composition) and Masson trichrome (collagen composition) staining. In ApoE−/−, plaques contained a thick VSMC-rich fibrous cap with abundant collagen and matrix overlying small necrotic cores (Figure 5A). However, fibrous caps were markedly thinner in ApoE−/−Akt1−/− mice, with smaller cap areas per plaque area and displayed reduced collagen composition (Figure 5A). Moreover, in mice lacking Akt1 necrotic core areas (NC) were markedly larger (Figure 5A).
Impaired Cardiac Function and Myocardial Infarcts in Mice Lacking Akt1
Previously we found that ApoE−/−Akt1−/− mice have coronary occlusions and approximately 20% of the mice die spontaneously.14 Similar findings were reporting coronary lesions were found in ApoE-deficient mice maintained on chow diet for 10 months22,23,24. To determine whether coronary artery disease may have contributed to cardiac performance, we performed echocardiographic studies in both groups of mice. As seen in Figure 5B, 42% percent of the ApoE−/−Akt1−/− double knockout mice analyzed had increased left ventricular systolic diameter (LVSd) and reduced fractional shortening consistent with a reduction in contractile function. The reduction in contractibility was independent of changes in left ventricular diastolic diameter (LVDd), posterior wall thickness (PWT), and interventricular wall septal thickness (IVWS; data not shown). Further analysis showed that all the mice with contractile dysfunction showed a dramatic interstitial fibrosis and distal coronary atherosclerosis as revealed by Masson trichrome staining of heart sections (supplemental Figure I, available online at http://atvb.ahajournals.org) and obvious spontaneous infarcts as showed after TTC staining (Figure 5C, representative image observed in 2 additional mice with cardiac dysfunction and not observed in all mice with normal cardiac function).
Our study provides in vitro and in vivo data supporting a critical role for Akt1 in regulating proliferation, survival, and migration of VSMC and its implication in atherosclerotic plaque formation. Lack of Akt1 in VSMCs causes a reduction in proliferation, PDGF-induced migration, and increased apoptosis after serum starvation and oxidative stress. Mechanistically, the proliferative and migratory phenotype found in Akt1 null VSMCs was linked to a reduced Rac-1 activation and MMP-2 expression and secretion. The importance of Akt1 for proliferation and migration has previously been reported in different cell types.17 The absence of Akt1 causes impaired integrin activation, migration, and formation of actin-based structures such as membrane ruffles in ECs and fibroblast. Similarly, we show that loss of Akt1 in VSMCs results in impaired cell migration, Rac activation, and PDGF-induce dorsal ruffle formation. These results identify Akt1 as the key Akt isoform in regulating VSMC proliferation, migration, and cell survival.
Akt is a multifunctional kinase implicated in a broad range of cellular functions including survival.10 We previously demonstrated that Akt1 is critical in sustaining ECs and monocyte/macrophage survival.14 Because VSMC apoptosis has important implications in atherosclerosis and is responsible for promoting plaque stability in advanced lesions,4 we studied the potential role of this Akt isoform in cell death. VSMCs lacking Akt1 were more susceptible to apoptosis after treatment with H2O2 (as an apoptogen) or serum starvation. The potential role of Akt signaling pathway on VSMC survival has also been explored in cells derived from human atherosclerotic lesions.25 Plaque-derived VSMCs showed increased sensitivity to proapoptotic stimuli and reduced insulin-like growth factor 1 receptor (IGF1R)/Akt signaling. Moreover, Allard et al demonstrated that Akt-dependent phosphorylation and subsequent inactivation of FoxoO3a and GSK3 were important for VSMC survival.25 Similarly, we described here reduced GSK3 phosphorylation in response to PDGF in VSMC lacking Akt1.
VSMC apoptosis alone in mouse models is sufficient to induce multiple features of atherosclerotic plaque vulnerability including reduced VSMC content, thin fibrous caps, as well as increased plaque lipid content and necrotic cores compared to stable lesions.4 Here, we found that the absence of Akt1 increases necrotic cores and reduces collagen content and fibrous cap thickness in atherosclerotic plaques from ApoE/Akt1 deficient mice. The increased necrotic core observed in this animal model could also be attributable to an increase in macrophage apoptosis as we previously described.14 In agreement with this idea is that insulin-resistant macrophages have reduced Akt activity and are more susceptible to endoplasmic reticulum stress–associated apoptosis contributing to macrophage death and necrotic core formation in atherosclerotic plaques.26,27 Collectively, these data suggest that the absence of Akt1 in VSMCs and macrophages increases the apoptotic susceptibility of these cells in vivo and may lead to a dramatic increase in necrotic core formation in atherosclerotic plaques. We also showed that absence of Akt1 in vivo causes a significant reduction in collagen content and fibrous cap thickness, which correlates with the reduced growth and increased apoptosis found in VSMC lacking Akt1.
In humans, plaques with advanced necrotic cores are responsible for acute atherothrombotic events.28 We previously reported that global absence of Akt1 leads to severe atherosclerosis, coronary artery disease, and significant mortality (20%) in the doubly mutant mice.14 In the present study using prospective monitoring of cardiac function, approximately 50% of the doubly mutant mice had compromised cardiac function before the end of the high-fat diet feeding period. This suggests that the global loss of Akt1 does not influence cardiac function, per se; however, all mice with impaired cardiac function had intramyocardial lesions and infarcts on sacrifice. Importantly, it is not known why a subgroup of ApoE−/−Akt1−/− progress to infarction, whereas others do not, despite all ApoE−/−Akt1−/− mice having distal coronary lesions (on sacrifice). Additional studies monitoring electric changes in the heart during paradigms of cardiac stress (dobutamine challenge or exercise) would be interesting to test the relationship between coronary lesions and cardiac function.
The authors acknowledge the generosity of Dr Howard Rockman for assistance with echocardiographic training and measurements.
Sources of Funding
This work was supported by grants R01 HL64793, R01 HL61371, R01 HL57665, P01 Hl70295, and Contract No. N01-HV-28186 (NHLBI-Yale Proteomics Contract) from the National Institutes of Health to W.C. Sessa, and by a Scientist Development Grant from American Heart Association to C. Fernández-Hernando. L. József is supported by a postdoctoral fellowship from the Canadian Institutes of Health Research.
C.F.-H. AND L.J. contributed equally to this study.
Received July 17, 2009; revision accepted August 27, 2009.
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