Skip Navigation

Cardiovascular Research 2003 57(1):253-264; doi:10.1016/S0008-6363(02)00618-1
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Skaletz-Rorowski, A.
Right arrow Articles by Walsh, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skaletz-Rorowski, A.
Right arrow Articles by Walsh, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

HMG-CoA reductase inhibitors promote cholesterol-dependent Akt/PKB translocation to membrane domains in endothelial cells

Adriane Skaletz-Rorowskia,c,1, Mohini Lutchmana,b,1, Yasuko Kureishia, David J. Leferd, Jerry R. Fauste and Kenneth Walsha,e,f,*

aDivision of Cardiovascular Research, St. Elizabeth's Medical Center of Boston, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA 02135, USA
bDepartment of Cell Biology, SGM 411, Harvard Medical, 240 Longwood Avenue, Boston, MA 02111, USA
cInstitute for Arteriosclerosis Research, University of Muenster, Domagkstrasse 3, 48149 Muenster, Germany
dDepartment of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
eDepartment of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
fMolecular Cardiology/CVI, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA

kwalsh{at}world.std.com

* Corresponding author. Molecular Cardiology/CVI, Boston University School of Medicine, 715 Albany Street, W611, Boston, MA 02118, USA. Tel.: +1-617-414-2392; fax: +1-617-414-2391.

Received 14 May 2002; accepted 5 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Recent results have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors referred to as statins rapidly activate the protein kinase Akt/PKB in endothelial cells (ECs) and endothelial precursor cells (EPCs). This pathway is critical for cellular responses that contribute to angiogenesis and EC function including nitric oxide production, cellular survival and migration. Methods: Here we tested whether statins control the translocation of recombinant and endogenous Akt to the plasma membrane of endothelial cells in a cholesterol-dependent manner. Results: Low doses of statins rapidly induce the translocation of Akt to discrete sites in endothelial cell plasma membrane that colocalize with F-actin-positive, focal adhesion kinase (FAK)-negative lamellipodia and filopodia. This translocation event requires the lipid-binding, pleckstrin homology domain of Akt. Treatment with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or the HMG-CoA reductase reaction product L-mevalonate blocks the translocation of Akt in response to statin stimulation. Furthermore, the ability of statins to promote Akt activation and translocation to the membrane is inhibited by cholesterol delivery to cells, but cholesterol loading had no effect on VEGF-induced Akt activation. Conclusions: These results suggest that statin activation of Akt signaling is mediated by the translocation of Akt to cholesterol-sensitive membrane structures within activated ECs.

KEYWORDS Cholesterol; Endothelial function; Lipid metabolism; Protein kinases; Protein phosphorylation; Statins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The 3-hydroxyl-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors referred to as statins are widely prescribed to lower cholesterol levels in patients at risk of coronary artery disease. Recent studies, however, suggest that statin therapy has an additional cardiovascular protective activity that may function independently of their ability to lower serum cholesterol levels [1]. Statin administration has been shown to rapidly improve vasomotor responses in patients [2–5] and animal models [6–8], suggesting that the target of this beneficial side effect may be vascular endothelial cells (ECs). This concept is reinforced by studies in normocholesterolemic animals that have shown that statin therapy can protect against stroke [9], preserve ischemic-reperfused myocardium [10] and inhibit vascular inflammatory responses in the microcirculation [11], through mechanisms that may be mediated by an increase in endothelium-derived NO.

Recently, it has been shown that low doses of statins can activate the Akt protein kinase, also referred to as protein kinase B (PKB), in ECs [12–14]. Statin treatment induces a rapid Akt-mediated phosphorylation of eNOS, that results in NO production with no accompanying change in the total level of eNOS protein. Low doses of statins promote Akt-dependent endothelial cell survival in serum-deprived media and induce vascular structure formation in vitro. Activation of angiogenesis by low doses of statins has been reported in a number of animal models [12,15–17], consistent with their Akt-activating function. It has also been reported that statins promote the survival, migration and differentiation of adult bone marrow-derived endothelial progenitor cells (EPCs) through an Akt-dependent mechanism in vitro and that statins will enhance EPC incorporation into sites of neovascularization and reendothelialization in animal models [13,17–19]. Moreover, statin administration will mobilize circulating CD34-positive EPCs in patients with stable coronary artery disease [20].

Statin activation of Akt signaling in ECs and EPCs may partly explain the improvements in vasomotor responses and tissue perfusion that are seen in patients who receive these drugs. Accordingly, it is important to understand the nature of statin-induced Akt activation because it may provide novel insights about the regulatory control of endothelial cell function and lead to the identification of new pharmacological targets for the control of blood vessel growth. Here, we assessed the ability of statins to promote the translocation of Akt to the plasma membrane of ECs and determined whether the translocation is regulated by the same experimental conditions that control statin-mediated Akt activation in biochemical assays [12]. We show for the first time that statin treatment induces Akt translocation to discrete sites within F-actin-positive lamellipodia and filopodia that are involved in cell movement. Furthermore, it is shown that loading ECs with cholesterol blocked statin-induced Akt translocation and activation, but has no effect on VEGF-induced Akt activation. These data suggest that localization of Akt to cholesterol-sensitive signaling domains within activated ECs is a proximal event in statin-mediated Akt signaling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture and reagents
Bovine aortic ECs (BAECs) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin G, 100 U/ml of streptomycin sulfate (Gibco BRL, Gaithersburg, MD, USA), and 10% fetal bovine serum (FBS) and used before passage 10. Before each experiment, BAECs were seeded on four-well chamber slides (Nalge Nunc International Corp., Naperville, IL, USA) at 2x104 cells/well in DMEM supplemented with 10% FBS and incubated at 37 °C for 48 h. Human µmbilical vein ECs (HUVECs) were cultured in endothelial cell growth medium (EGM, Clontech, Palo Alto, CA, USA). Before each experiment, HUVECs (passages 4 to 6) were placed in serum-depleted medium (endothelial cell basal medium, EBM, Clontech), supplemented with 3% FBS for 24 h. Subconfluent cultures (60 to 80%) of BAECs and HUVECs were analyzed. Rat vascular smooth muscle cells (VSMCs) were prepared and cultured as described previously [21]. Rat VSMCs were seeded at 2.5x104 cells/well in DMEM supplemented with 10% FBS and cultured for 2 days before transfection. BAECs or rat VSMCs were transiently transfected with 1 µg/ml of GFP-Akt, GFP-AktR25C or GFP-AHR25C expression plasmid by lipofection according to manufacturer's protocol (Gibco BRL). The full length and mutant forms of the GFP-Akt constructs contain the mouse Akt cDNA and were cloned into the pEGFP-C1 plasmid (Clontech). Control cells were transfected with the GFP expression plasmid pEGFP-C1. Following transfection and incubation in OPTI-MEM (Gibco BRL), a reduced serum medium, for 24 h, the cells were stimulated with 0.5 µM simvastatin, 1 µM pravastatin or 100 ng/ml recombinant human VEGF165 (R&D Systems, Minneapolis, MN, USA) typically for 30 min, although many experimental observations ranged from 5 to 60 min post-treatment. Every independent experiment also included an untreated (no statin or VEGF) control group. L-Mevalonate (200 µM) was activated by alkaline hydrolysis. In some experiments cells were repleted with cholesterol (Steraloids Inc., Newport, RI, USA) by incubating them in the presence of a cholesterol/2-hydroxypropyl-β-cyclodextrin (CD, Sigma, St. Louis, MO, USA) mixture as described by Furuchi and Anderson [22]. A stock solution of 0.4 mg/ml cholesterol and 10% cyclodextrin was prepared by vortexing at 40 °C in 10 ml of 10% cyclodextrin with 200 µl of cholesterol (20 mg/ml in ethanol solution). Each solution was filtered through a 0.2-µm filter before use. The cells were incubated in the presence of 16 µg/ml cholesterol/0.4% cyclodextrin complex and simvastatin (0.5 µM) for 1 h. Alternatively cells were treated with 80 µg/ml complex for 1 h prior to treatment with simvastatin (1 µM) for 1 h. In some experiments, BAECs were pretreated with LY294002 (Sigma) or wortmannin (Sigma) at the indicated doses for 1 h before stimulation with simvastatin. Simvastatin was provided by Merck (West Point, PA, USA) and activated by alkaline hydrolysis. Pravastatin was of pharmaceutical grade and it was solubilized in saline.

2.2 Immunocytochemistry
Cells were plated in chamber slides (2x104) or 60 mm dishes (1x105) and allowed to achieve about 60 to 70% confluency. At the end of each experiment, cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.2% Triton X-100 in PBS three times within 10 min at room temperature (RT). Then, cells were blocked with buffer A (0.1% Triton X-100, 10% FCS in PBS) for 30 min at RT. Using immunofluorescence microscopy, cells were analyzed for GFP localization or incubated with primary antibody [1:100 dilution for goat polyclonal anti-Akt1 antibody, 1:100 for mouse monoclonal anti-FAK (H-1) antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA)] overnight at 4 °C. After washing (0.1% Triton X-100 in PBS), cells were incubated with rhodamine-conjugated rabbit anti-goat IgG (1:100 dilution, Pierce, Rockford, IL, USA) or rhodamine-conjugated anti-mouse IgG (1:100 dilution). After washing, slides were mounted in a drop of mounting medium (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) to reduce photobleaching. Control experiments were performed in parallel with the omission of the secondary antibodies.

2.3 Microscopy
For immunofluorescence microscopy cells were washed three times in PBS, fixed in 4% PFA solution for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min at RT. F-actin was visualized using rhodamine phalloidin (Molecular Probes, Eugene, OR, USA). The captured images were then screened for co-localization. Overlayed images of GFP and rhodamine distinguished regions of co-localization (yellow), reflecting the additive effect of green and red pixels. Fluorescent images were recorded on a Nikon Diaphot microscope using a 60x oil immersion plan apo lens and a video graphic system (DEI-750 CE Digital Output Camera, Optronics, Goleta, CA, USA). Alternatively, transfected cells were examined using a Zeiss LSM-20 laser scanning confocal microscope equipped with a barrier filter for fluorescein (DTAF filter and Argon 488 nm as light source) and Cy3 epi-fluorescence (helium neon 543 nm as light source). A plan-neofluar 63x oil immersion objective (1.3 NA) was used for imaging of fluorescently labeled samples. Separate optical images of green fluorescence and rhodamine were captured from the same optical section. The captured images were then screened for colocalization. Image analysis was performed using the standard operating system software provided with the Zeiss LSM microscope (Version 2.08). For other experiments, images were acquired on a Nikon TE 300 microscope with a 60x plan apo objective (1.4 NA). Images were captured on a Hamamatsu ORCA-ER digital camera at 0.2 µm steps through the thickness of the cell (approximately 12 µm). Deconvolved images were captured using Openlab software program (Improvision, Inc., Coventry, UK). Typically, individual experiments were performed in duplicate on four-well chamber slides containing approximately 20,000 cells per well. Multiple microscopic fields were then analyzed in each well and photomicrographs of 20 to 30 representative cells were produced. Individual experiments were usually replicated under similar conditions on three or more occasions.

2.4 Akt and PI 3-kinase phosphorylation assays
Treated cells and control were lysed with modified RIPA buffer and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10%), subjected to Western immunoblot analysis by using rabbit polyclonal anti-phosphorylated serine 473 residue of Akt1 antibody (Cell Signaling, Beverly, MA, USA). To verify the amount of loaded proteins, each blot was reprobed with goat polyclonal anti-Akt1 antibody (Santa Cruz Biochemicals, Santa Cruz, CA, USA). PI 3-kinase phosphorylation assays were performed as previously described [23]. In brief, treated cells (plated on 100 mm) were washed with buffer A (PBS containing 1 mM Ca2+, 1 mM MgCl2, and 100 µM NaVO4) once, then lysed with 500 µl of buffer A plus 1% nonidet P-40 and protease inhibitor cocktail (Roche). The level of tyrosine-phosphorylated p85 subunit of PI 3-kinase in each protein lysate (constant protein) was detected by immunoprecipitation using anti-phosphotyrosine antibody (PY20: Transduction Laboratories) followed by Western blot of immunoprecipitated material using anti-p85/PI 3-kinase antibody (Upstate Biotechnology).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 HMG-CoA reductase inhibition promotes Akt translocation to the cell membrane
To investigate the role of statins on Akt translocation to the membrane, BAECs were transiently transfected with a plasmid encoding full-length Akt fused to GFP at its N-terminus (GFP-Akt) [18] and incubated in low serum media for 24 h. GFP-Akt translocation induced by statin stimulation was then observed by immunofluorescence microscopy. Prior to statin stimulation, GFP-Akt was localized to the nucleus and the cytoplasm, which provided an outline of the cell's shape (Fig. 1A). Treatment with 0.5 µM simvastatin for 30 min (standardized conditions) led to the formation of membrane protrusions and ruffles (Fig. 1E), and GFP-Akt signal accumulated at these sites (Fig. 1B, F). Movement to these structures was rapid and typically occurred between 10 and 30 min of stimulation. GFP-Akt localization at these sites was maintained for at least 1 h following stimulation (data not shown). These sites stained positive with phalloidin indicating regions of the actin meshwork that is characteristic of lamellipodia and filopodia (Fig. 1C, D, G, H). GFP-Akt also co-localized with phalloidin-positive stress fibers within cells (Fig. 1F–H); however, the GFP-Akt signal was much less intense when compared with the sites at membrane ruffles and protrusions, which typically occurred in portions of the cell with diminished stress fiber structures. In contrast, the GFP-Akt-positive domains in the plasma membrane were negative for focal adhesion kinase (FAK), suggesting that statins induce the translocation of Akt to mobile, rather than static, membrane structures (Fig. 2). Quantitative analyses revealed that 72% of BAECs displayed GFP-Akt localization at the membrane in cultures stimulated with 0.5 µM simvastatin for 30 min (Table 1). This represents a 19-fold increase relative to unstimulated, GFP-Akt-transfected cells.


Figure 1
View larger version (55K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 {alpha}-Actin re-organization in simvastatin treated GFP-Akt transfected BAECs. (a) GFP-Akt transfected BAECs, unstimulated (control). (b), (f) GFP-Akt translocation induced by simvastatin for 30 min (0.5 µM). (c), (g) Localization of F-actin (red) in the same cells (b, f), F-actin was stained with rhodamine-conjugated phalloidin. (d), (h) Merged image of GFP-Akt (green) and F-actin (red) in the same cells as in b and f. (e) Phase contrast of the same cell as in f, g, h. Lamellipodia are indicated by white arrows. Simvastatin induced GFP-Akt translocation was typically analyzed in duplicate in chamber slides containing approximately 20,000 cells per well. Multiple microscopic fields in each well were analyzed for GFP-Akt-transfected cells. Between 40 and 60% of cells were positive for transgene expression following the transfection protocol. Photomicrographs of 20 to 30 cells were typically produced for each experimental condition. Induction of GFP-Akt translocation by 0.5 µM simvastatin treatment for 30 min (standard conditions) served as a positive control for all following studies, and therefore was replicated in more than 25 separate experiments. Arrows indicate regions of GFP-Akt accumulation at discrete membrane domains.

 

Figure 2
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Subcellular localization of FAK in GFP-Akt transfected BAECs. (a) GFP-Akt transfected BAECs were stimulated with simvastatin (0.5 µM) for 30 min. (b) Localization of FAK (red) in the same cells. (c) Merge image of GFP-Akt (green) and FAK (red) in the same cells as in a and b. FAK localization experiments were performed in duplicate and were analyzed twice.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Quantitation of cells characterized by Akt translocation to membrane domains

 
Confocal microscopic analysis revealed cytoplasmic and nuclear localization of GFP-Akt in non-stimulated BAECs, which provided an outline of the cell's shape (Fig. 3A). A 30 min activation with simvastatin (0.5 µM) induced the accumulation of GFP-Akt at discrete sites within membrane protrusions (Fig. 3B). Stimulation with VEGF also promoted GFP-Akt translocation to the membrane; however, the fluorescence intensity was more evenly distributed throughout the plasma membrane (Fig. 3C). The rapid simvastatin-induced translocation of endogenous Akt could also be detected by the immunofluorescence analysis with fixed cells. For these experiments, HUVECs were mock-treated or stimulated with 0.5 µM simvastatin for 30 min, prior to fixation and immunodetection of Akt with anti-Akt1 antibody and rhodamine-conjugated secondary antibody (Fig. 4). Similar to observations of GFP-Akt localization in transiently-transfected BAEC, endogenous Akt rapidly translocated to discrete sites at membrane ruffles in the simvastatin-treated cells. Treatment with simvastatin led to a 7.4-fold increase in the number of HUVECs displaying membrane-associated domains of Akt localization (Table 1). Quantitatively, this effect is similar to that seen in BAECs transduced with GFP-Akt. The sites of membrane-associated Akt accumulation appeared more focal when endogenous protein was examined relative to GFP-Akt, perhaps due to the difference in the expression levels of the endogenous and exogenous proteins (Fig. 4B). As with GFP-Akt, only a portion of the total endogenous Akt was translocated to the membrane following statin stimulation, and the cytoplasmic Akt signal provided an outline of the cell's shape. Deconvolved images captured at 200 nM steps revealed high local concentrations of Akt in discrete domains associated with the ruffled membranes of statin-treated cells (Fig. 4C).


Figure 3
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Detection of subcellular localization of GFP-Akt in BAECs using confocal microscopy. (a) Representative photomicrograph of unstimulated GFP-Akt transfected BAECs. GFP-Akt transfected BAECs, stimulated with simvastatin (0.5 µM). GFP-Akt (green), F-actin (rhodamine, red). (c) GFP-Akt transfected BAECs, stimulated with VEGF165 (100 ng/ml) for 30 min. For confocal analysis multiple fields from two sets of slides were analyzed for each condition. 100 nM steps taken through each cell were analyzed for representative images that revealed membranous patterns of fluorescence. Arrows indicate GFP-Akt accumulation in discrete membrane domains in simvastatin-treated cells (b) and uniform GFP-Akt distribution in the membranes of VEGF-treated cells (c).

 

Figure 4
View larger version (62K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Simvastatin induces the translocation of the endogenous Akt to discrete sites at membrane ruffles in HUVECs. (a) Localization of endogenous Akt (rhodamine) in HUVECs, unstimulated (control). (b) Endogenous Akt translocation induced by simvastatin (0.5 µM). (c) Localization of endogenous Akt in simvastatin-stimulated cells, using the deconvolution system. HUVEC cultures were plated on chamber slides at a density of 2x104 cells/well or in 60 mm dishes at a density of 1x105 cells/dish. After an incubation in serum-depleted media for 24 h, cells were treated with simvastatin (0.5 µM) for 30 min. Statin activation of endogenous Akt was assayed in duplicate wells for each experiment, and this experiment was replicated four times under similar conditions. Arrows indicate regions of Akt accumulation in discrete membrane domains.

 
The effect of simvastatin on the GFP-Akt translocation in transiently-transfected BAECs was dose-dependent (Fig. 5). Membrane protrusions and GFP-Akt translocation occurred over a range of simvastatin concentrations from 0.1 to 10 µM, although toxicity by the highest dose (10 µM) could be observed at later time points [12].


Figure 5
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Dose-dependent effect of simvastatin on the subcellular localization of GFP-Akt. (a) Representative photomicrograph of unstimulated GFP-Akt transfected BAECs. (b)–(f) Dose-dependent GFP-Akt translocation by simvastatin. GFP-Akt transfected BAECs were treated with simvastatin (0.01–10 µM) for 30 min. The simvastatin dose–response curve was performed on duplicate slides and was replicated twice.

 
Cells transfected with a vector expressing the GFP marker alone did not display statin- or VEGF-induced translocation of the fluorescence signal to the membrane (Fig. 6A). Furthermore, GFP did not colocalize with stress fibers as was sometimes seen with the GFP-Akt fusion protein (Fig. 1F–H). To further assess the specificity of the fusion protein translocation, a GFP-AktR25C construct was assessed where the arginine at position 25 in the pleckstrin homology domain was mutated to a cysteine. The pleckstrin homology domain binds to the lipid products of the PI 3-kinase reaction and serves as a membrane-targeting module [24]. The GFP-AktR25C failed to translocate to the membrane in response to simvastatin treatment, whereas the wild-type GFP-Akt construct translocated to focal regions within the membrane (Fig. 6B, C). A 30 min stimulation with VEGF (100 ng/ml) also promoted GFP-Akt translocation to the membrane (Fig. 6E). VEGF had no affect on the localization of the GFP marker alone (Fig. 6D) nor on the localization of GFP-AktR25C (Fig. 6F). Under conditions of VEGF-stimulation, GFP-Akt appeared uniformly distributed along the whole extent of the plasma membrane (Fig. 6E).


Figure 6
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Subcellular localization of GFP, GFP-Akt and GFP-AktR25C after simvastatin or VEGF treatment. (a) GFP-vector-transduced cells treated with 0.5 µM simvastatin for 30 min (standard conditions). GFP-vector transduced cells served as a negative control in most experiments, and was replicated on more than 18 separate occasions. (b) GFP-Akt translocation induced by simvastatin under standard conditions. (c) GFP-AktR25C transfected cells, treated with simvastatin under standard conditions. (d) GFP-vector, treated with VEGF165 (100 ng/ml). (e) GFP-Akt transfected cells, stimulated with VEGF. (f) GFP-AktR25C transfected cells, stimulated with VEGF. All BAECs were treated with recombinant human VEGF165 (100 ng/ml) for 30 min. (g) VEGF activation of GFP-vector, GFP-Akt and GFP-AktR25C. VEGF activation of Akt translocation was analyzed in duplicate or quadruplicate at a single time point (30 min) or at five different time points (10, 15, 20, 30 and 60 min) in three separate experiments. Arrows indicate regions of GFP-Akt accumulation in discrete membrane domains of simvastatin-treated cells (d) and region of uniform GFP-Akt distribution in the membranes of VEGF-treated cells (e).

 
The rapid translocation of GFP-Akt to discrete membrane domains in BAEC was also detected upon stimulation with pravastatin and this localization pattern was similar to that obtained by treatment with simvastatin (Fig. 7A, B). Co-incubation with the product of the HMG-CoA reductase reaction L-mevalonate blocked GFP-Akt translocation induced by treatment with simvastatin (Fig. 7C) or pravastatin (data not shown).


Figure 7
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Specificity of statin effect on GFP-Akt translocation. Representative images of the GFP-Akt translocation are shown under various conditions as follows: (a) GFP-Akt translocation in BAEC induced by simvastatin (0.5 µM), 30 min treatment. (b) GFP-Akt translocation in BAEC induced by pravastatin (0.5 µM), 30 min treatment. Pravastatin-induced GFP-Akt translocation was analyzed in duplicate at 5, 10, 20 and 30 min. (c) Reversal of the simvastatin-induced GFP-Akt translocation in BAECs by L-mevalonate (200 µM). L-Mevalonate effects on simvastatin-induced GFP-Akt translocation was analyzed in duplicate at 100, 200 and 300 µM L-mevalonate in two separate experiments. (d), (e) Inhibition of the simvastatin induced translocation of GFP-Akt in wortmannin (d) or LY294002 (e) pretreated BAECs. BAECs were pretreated with wortmannin (250 nM) or LY294002 (7.5 µM) for 1 h, before 30 min stimulation with simvastatin (0.5 µM). Wortmannin and LY294002 effects on simvastatin-induced GFP-Akt translocation was analyzed at different doses in duplicate in three separate experiments. (f) Simvastatin has no effect on GFP-Akt localization in VSMCs. Rat aortic VSMCs were treated with simvastatin (0.5 µM) for 30 min. GFP-Akt localization in VSMCs was analyzed in duplicate at 0.5, 1.0, 2.5, 5.0, 7.5 and 10 µM simvastatin for 30 min, and at 0.5 µM for 15, 30, 60 and 360 min. F-Actin (rhodamine). Arrows indicate regions of GFP-Akt accumulation in discrete membrane domains of statin-treated cells.

 
3.2 Simvastatin-induced localization of GFP-Akt was disrupted by treatment with inhibitors of PI 3-kinase
Pretreatment with 250 nM wortmannin blocked the translocation induced by 0.5 µM simvastatin (Fig. 7D). Lower doses of wortmannin (5, 10, 50 and 100 nM) had little or no effect on statin-induced translocation, and a higher dose (400 nM) appeared to be toxic to BAECs based upon the appearance of morphological features including rounding and detachment and the appearance of cellular debris (data not shown). Similarly, pretreatment with the PI 3-kinase inhibitor LY294002 at 7.5 µM blocked GFP-Akt translocation induced by 0.5 simvastatin (Fig. 7E). Pretreatment with LY294002 for 1 h prior to a 30 min stimulation with simvastatin led to a reduction in GFP-Akt translocation by a factor of 7 (13 microscopic fields from three independent experiments, P<0.05 relative to simvastatin-stimulated BAECs in Table 1). Lower doses of LY294002 (0.05, 0.1, 1.0, 2.5 µM) had little or no effect on GFP-Akt translocation, whereas higher doses appeared to be toxic (data not shown).

In transiently-transfected rat smooth muscle cells, there was no change in the diffuse pattern of GFP-Akt distribution under standard conditions of statin stimulation (30 min incubation with 0.5 µM simvastatin) (Fig. 7F). Furthermore, no effect on GFP-Akt localization in smooth muscle cells could be observed at 0.5, 1.0, 2.5, 5.0, 7.5 and 10 µM simvastatin, nor at 15 min, 60 min or 6 h post-treatment with simvastatin.

Under the conditions of these assays, treatment with 0.5 µM simvastatin or pravastatin for 1 h significantly lowered 3H-acetate incorporation into cholesterol in BAECs or HUVECs between 50 and 95% (data not shown). Thus, to examine the role of cholesterol in statin-mediated Akt translocation, cells were incubated with a cholesterol/cyclodextrin complex at the time of simvastatin addition. This method of cholesterol repletion reversed the statin-induced translocation of GFP-Akt to the membrane of transiently-transfected BAECs when analyzed at 1 h following the addition of the cholesterol/cyclodextrin complex (Fig. 8A, B and Table 1). The effect of cholesterol repletion was time-dependent, and a 30 or 45 min incubation with the cholesterol/cyclodextrin complex partially reversed GFP-Akt translocation (data not shown). Analyses of endogenous Akt in HUVECs, also revealed that simvastatin-induced translocation was blocked by cholesterol repletion for 1 h (Fig. 8C, D and Table 1).


Figure 8
View larger version (117K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Reversal of the simvastatin induced Akt translocation by cholesterol repletion. (a) GFP-Akt transfected BAECs stimulated by simvastatin (0.5 µM) for 1 h. (b) Reversal of the simvastatin-induced GFP-Akt translocation by cholesterol/cyclodextrin complex. Effects of cholesterol/cyclodextrin complex on GFP-Akt localization were analyzed in duplicate in six experiments under two treatment regiments. Shown is a representative cell from an experiment where simvastatin and cholesterol/cyclodextrin complex were simultaneously added to cells and incubated for 1 h. In the second treatment regimen, cells were incubated for 30 min with simvastatin and then washed and treated with cholesterol/cyclodextrin for 30, 45 or 60 min. In these experiments, the 60 min incubation led to a complete reversal of Akt translocation to the membrane, whereas a partial effect was observed at 30 and 45 min. (c) Translocation of the endogenous Akt (rhodamine, red) in HUVEC induced by simvastatin (0.5 µM for 1 h). (d) Reversal of the simvastatin-induced translocation of the endogenous Akt by cholesterol/cyclodextrin complex. Effects of cholesterol/cyclodextrin complex on endogenous Akt translocation was analyzed in duplicate. For b and d cells were incubated in the presence of 16 µg/ml cholesterol/0.4% cyclodextrin complex for 1 h.

 
To further examine the consequences of cholesterol repletion of Akt activation, immunoblot analyses were performed on lysates from treated HUVEC cultures using an antibody that is specific for Akt phosphorylated at serine residue 473. Phosphorylation of this residue can result from an autophosphorylation event [25], and it reflects the status of Akt activation [26]. Consistent with previous data [12], stimulation of cultures with simvastatin or VEGF led to a rapid increase in Akt phosphorylation at this residue (Fig. 9A). Incubation with cholesterol/cyclodextrin complex blocked simvastatin-stimulated Akt phosphorylation, but this treatment had no detectable effect on VEGF-stimulated phosphorylation. None of the treatments altered total Akt expression. Both simvastatin and VEGF promoted association of tyrosine-phosphorylated protein with the p85 subunit of PI 3-kinase in coupled immunoprecipitation/immunoblot experiments (Fig. 9B). Cholesterol repletion diminished the statin-induced association, but this treatment had no effect on the VEGF-induced effect.


Figure 9
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 Cholesterol repletion abrogates simvastatin-induced Akt and PI 3-kinase phosphorylation. (a) Serum-deprived HUVECs were incubated in the absence or presence of cholesterol/cyclodextrin complex (Chol) for 1 h, prior to stimulation with 1 µM simvastatin (SIM) for 1 h, 50 ng/ml VEGF for 20 min or no stimulation. Lysates were analyzed by immunobloting using anti-phosphoAkt (serine473) (upper panel) and anti-Akt1 antibody (total Akt; lower panel). A representative immunoblot from one of three independent experiments is shown. (b) Simvastatin promotes tyrosine phosphorylation of the PI 3-kinase p85 subunit in a cholesterol-sensitive manner. Lysates from an Akt phosphorylation experiment described in a were subjected to immunoprecipitation using anti-phospho-tyrosine residue antibody (PY20) followed by immunoprecipitation with anti-p85/PI 3-kinase antibody (PI3K).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
A number of recent studies have documented that Akt/PKB signaling is an important regulator of endothelial cell NO production [27,28], migration [29] and survival [30] at a cellular level and that it controls vasomotor activity [31] and angiogenesis [12] in animal models. Recently, we and others have shown that statins rapidly activate Akt signaling in both ECs and EPCs isolated from adult peripheral blood [12–14,17,18]. Consistent with its Akt-activating function, low doses of statins promote neovascularization in a number of animal models [12,15–17]. In contrast, relatively higher doses of statins inhibit endothelial cell viability [12,13] and migration [32], and have a negative effect on angiogenesis in animal models [16] (and Kureishi and Walsh, unpublished observations). To develop better understanding of how statins activate this signaling pathway in ECs, we examined their effect on Akt translocation to the membrane, an essential feature of Akt activation that is associated with cellular migration [33]. In unstimulated ECs, a GFP-Akt fusion protein was localized to the nucleus and cytoplasm. Low doses of simvastatin or pravastatin rapidly (<30 min) promoted the formation of extended membrane processes and ruffled lamellipodia that develop from the reorganization of actin into bundles at the periphery of the ECs. Statin-stimulation also induced the accumulation of GFP-Akt to discrete membrane locations within these membrane protrusions. This translocation event was not observed in cells transfected with the vector expressing the GFP marker protein alone nor was it observed in cells expressing GFP-AktR25C that is mutated in the Akt pleckstrin homology domain which normally targets the protein to the membrane. Microscopic analyses of thin focal planes revealed that statin treatment promotes the accumulation of locally high concentrations of GFP-Akt at discrete sites within membrane protrusions rather than the accumulation of GFP-Akt in pockets of cytoplasm that might result from a collapse of adjacent regions of the cell. Furthermore, it is shown that simvastatin-induced Akt translocation occurred over a range of doses that promote the activating phosphorylation of Akt at serine 473 [12]. The lowest simvastatin dose to promote Akt translocation (0.1 µM) is comparable to the reported levels of simvastatin in plasma [34]. Akt translocation was inhibited by mevalonate, the product of the HMG-CoA reductase reaction, and by the PI 3-kinase inhibitors wortmannin and LY294002. Collectively, these data indicate that statin-mediated Akt translocation to the membrane is under the same or similar regulatory control as the statin-mediated activation of Akt protein kinase activity that was shown previously [12]. Since membrane translocation is critical in Akt activation, these data indicate that the statin-mediated translocation of Akt could represent a proximal step in the mechanism by which these drugs activate this signaling pathway.

The pattern of GFP-Akt localization at the membrane differed between VEGF- and statin-stimulated cells. VEGF promoted a relatively uniform pattern of GFP-Akt localization pattern along the plasma membrane, similar to the pattern described for Akt distribution in mitogen-stimulated 293 and NIH3T3 cells [24,35]. In contrast, statin-treatment resulted in a discrete aggregation of GFP-Akt within membrane protrusions. FAK did not colocalize with GFP-Akt at these membrane sites, suggesting that these bundled actin microfilaments occur at the leading edges of cell movement rather than at the trailing edge of migrating cells. Statins also induced the translocation of endogenous Akt to discrete membrane locations associated with ruffled lamellipodia. Statin-stimulation of non-transduced ECs gave rise to fewer filopodial extensions and the domains of endogenous Akt accumulation at the membrane edge appeared more focal, suggesting that the overexpression of Akt might lead to an increase in signaling and exaggerate the effect of statins on endothelial cell morphology. However, comparative analyses of GFP-Akt translocation in BAECs and endogenous Akt in HUVECs revealed quantitatively similar effects of statin-stimulation on translocation, with the majority of cells responding in either case. In contrast, smooth muscle cells did not respond to statin treatment by forming these membrane structures, nor did statins promote Akt translocation to the membrane in these cells.

The exact nature of the Akt-rich membrane domains observed in statin-activated cells is unknown, but it is tempting to speculate that they are related to "lipid rafts" [36], which can coalesce into "superrafts" that can occupy a large fraction of the total plasma membrane [37–39]. Lipid rafts are membrane domains that are rich in cholesterol and sphingolipids, and are more fluid than membranes that are comprised of unsaturated phospholipids. These domains serve as platforms for the assembly of signaling proteins at the leading edge of migrating cells [40] and they appear to be involved in the control of multiple cellular processes. Lipid raft-mediated regulation of insulin signaling has been reported [41,42], and they have been implicated in the regulation of MAPK and PI 3-kinase [22,43,44]. In some instances, it has been found that cholesterol repletion can inhibit intracellular signaling, whereas inhibition of cholesterol synthesis or cholesterol depletion can activate signaling.

An important finding in this study is the observation that loading ECs with cholesterol will block both statin-induced translocation of Akt to the membrane and the statin-induced activating phosphorylation of Akt. In contrast, cholesterol-loading had little or no effect on Akt activation in response to VEGF stimulation. With respect to statin-stimulation, these data indicate that endothelial Akt signaling is subject to regulation by a rapidly exchanging pool of cholesterol within these cells. We propose that this endothelial cholesterol pool in the intact organism is more sensitive to statin-mediated inhibition of endogenous cholesterol synthesis than it is to changes in exogenous cholesterol delivery from the serum by low-density lipoprotein. Furthermore, we suggest that the inhibition of ECs cholesterol biosynthesis by statins will alter cholesterol concentrations within membrane domains leading to Akt recruitment or retention at these sites. These effects may be partly mediated by PI 3-kinase since statin-activation of Akt kinase activity [12] and translocation to the membrane (this study) is blocked by the PI 3-kinase inhibitors wortmannin and LY294002. In support of this hypothesis, we also show that statin stimulation will promote the association of tyrosine phosphorylated protein with the p85 subunit of PI 3-kinase and that this effect is blocked by cholesterol repletion. In this regard, it may be relevant that PI 3-kinase activity in fibroblasts is negatively regulated by the recruitment of caveolin-1, an intracellular cholesterol transport protein, to PI 3-kinase-associated receptor complexes within lipid rafts [44]. It has also been shown that statin inhibition of cholesterol synthesis in ECs can ameliorate the inhibitory action of caveolin-1 on eNOS [45,46], whose activity is controlled by Akt-mediated phosphorylation [27,28].

The low doses of statins employed in this study will predominantly affect cholesterol synthesis. In addition to cholesterol, mevalonic acid is an essential precursor for several cellular components including ubiquinone, isopentenylated tRNAs, and prenylated proteins. Interestingly, the utilization of mevalonate for cholesterol synthesis exhibits much lower affinity than mevalonate incorporation into its non-sterol products [47]. Consequently, low doses of statins will lead to preferential inhibition of cholesterol synthesis relative to the production of ubiquinone, isopentenylated tRNAs, and prenylated proteins.

It is becoming increasingly recognized that cholesterol has important functions as a modulator of cellular signaling in vertebrates and invertebrates [48–50]. In this study we have shown that inhibition of HMG-CoA reductase rapidly promotes the translocation of Akt to discrete membrane domains associated with the lamellipodia and filopodia of activated ECs. These data extend our earlier biochemical observations of statin-induced Akt activation, and they suggest that a rapidly-exchanging pool of cholesterol within ECs may be involved in the regulation of Akt signaling by controlling its association with specific membrane domains.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by National Institutes of Health grants AR40197, HL50692, AG15052 and AG17241 to K.W. A.S.-R. was supported by the Lise-Meitner fellowship of the Ministerium für Schule und Weiterbildung, Wissenschaft und Forschung des Landes Nordrhein-Westfalen of Germany. The authors thank K. Rosen for suggesting some of these experiments, B. Schneider (Improvision, Inc.) and B. Costello (Microvideo, Inc.) for aid with microscopy, R.C. Smith and M. Hixon for helpful discussions, and J. Downward for providing the GFP-Akt expression plasmids.


   Notes
 
1 Both authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. West of Scotland Coronary Prevention Study Group. Influence of pravastatin and plasma lipids of clinical events in the West of Scotland Coronary Prevention Study (WOSCOPS). Circulation (1998) 97:1440–1445.[Abstract/Free Full Text]
  2. Dupuis J., Tardif J.C., Cernacek P., Théroux P. Cholesterol reduction rapidly improves endothelial function after acute coronary syndromes: the RECIFE (Reduction of Cholesterol in Ischemia and Function of the Endothelium) trial. Circulation (1999) 99:3227–3233.[Abstract/Free Full Text]
  3. Muramatsu J., Kobayashi A., Hasegawa N., Yokouchi S. Hemodynamic changes associated with reduction in total cholesterol by treatment with the HMG-CoA reductase inhibitor pravastatin. Atherosclerosis (1997) 130:179–182.[CrossRef][ISI][Medline]
  4. Egashira K., Hirooka Y., Kai H., et al. Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia. Circulation (1994) 89:2519–2524.[Abstract/Free Full Text]
  5. Leung W.H., Lau C.P., Wong C.K. Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolemic patients. Lancet (1993) 341:1496–1500.[CrossRef][ISI][Medline]
  6. Kaesemeyer W.H., Caldwell R.B., Huang J., Caldwell R.W. Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions. J Am. Coll Cardiol (1999) 33:234–241.[Abstract/Free Full Text]
  7. Williams J.K., Sukhova G.K., Herrington D.M., Libby P. Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys. J Am Coll Cardiol (1998) 31:684–691.[Abstract/Free Full Text]
  8. Jorge P.A., Osaki M.R., de Almeida E. Rapid reversal of endothelial dysfunction in hypercholesterolaemic rabbits treated with simvastatin and pravastatin. Clin Exp Pharmacol Physiol (1997) 24:948–953.[ISI][Medline]
  9. Endres M., Laufs U., Huang Z., et al. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA (1998) 95:8880–8885.[Abstract/Free Full Text]
  10. Lefer A.M., Campbell B., Shin Y.-K., et al. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation (1999) 100:178–184.[Abstract/Free Full Text]
  11. Lefer A.M., Lefer D.J. The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion. Cardiovasc Res (1996) 32:743–751.[Abstract/Free Full Text]
  12. Kureishi Y., Luo Z., Shiojima I., et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med (2000) 6:1004–1010.[CrossRef][ISI][Medline]
  13. Urbich C., Dernbach E., Zeiher A.M., Dimmeler S. Double-edged role of statins in angiogenesis signaling. Circ Res (2002) 90:737–744.[Abstract/Free Full Text]
  14. Brouet A., Sonveaux P., Dessy C., et al. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res (2001) 89:866–873.[Abstract/Free Full Text]
  15. Sata M., Nishimatsu H., Suzuki E., et al. Endothelial nitric oxide synthase is essential for the HMG-CoA reductase inhibitor cerivastatin to promote collateral growth in response to ischemia. FASEB J (2001) 15:2530–2532.[Free Full Text]
  16. Weis M., Heeschen C., Glassford A.J., Cooke J.P. Statins have biphasic effects on angiogenesis. Circulation (2002) 105:739–745.[Abstract/Free Full Text]
  17. Llevadot J., Murasawa S., Kureishi Y., et al. HMG-CoA reductase inhibitor mobilizes bone-marrow derived endothelial progenitor cells. J Clin Invest (2001) 108:399–405.[CrossRef][ISI][Medline]
  18. Dimmeler S., Aicher A., Vasa M., et al. HMG-CoA-reductase inhibitors (statins) increase endothelial progenitor cells via the P.I. 3-kinase/Akt pathway. J Clin Invest (2001) 108:391–397.[CrossRef][ISI][Medline]
  19. Walter D.H., Rittig K., Bahlmann F.H., et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation (2002) 105:3017–3024.[Abstract/Free Full Text]
  20. Vasa M., Fichtlscherer S., Adler K., et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation (2001) 103:2885–2890.[Abstract/Free Full Text]
  21. Smith R.C., Branellec D., Gorski D.H., et al. p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene. Genes Dev (1997) 11:1674–1689.[Abstract/Free Full Text]
  22. Furuchi T., Anderson R.G. Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK). J Biol Chem (1998) 273:21099–21104.[Abstract/Free Full Text]
  23. Whitman M., Kaplan D., Roberts T., Cantley L. Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation. Biochem J (1987) 247:165–174.[ISI][Medline]
  24. Andjelkovic M., Alessi D.R., Meier R., et al. Role of translocation in the activation and function of protein kinase B. J Biol Chem (1997) 272:31515–31524.[Abstract/Free Full Text]
  25. Toker A., Newton A.C. Akt/Protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J Biol Chem (2000) 275:8271–8274.[Abstract/Free Full Text]
  26. Alessi D.R., Andjelkovic M., Caudwell B., et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J (1996) 15:6541–6551.[ISI][Medline]
  27. Fulton D., Gratton J.-P., McCabe T.J., et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature (1999) 399:597–601.[CrossRef][Medline]
  28. Dimmeler S., Fleming I., Fisslthaler B., et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature (1999) 399:601–605.[CrossRef][Medline]
  29. Morales-Ruiz M., Fulton G., Sowa G., et al. Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt. Circ Res (2000) 86:892–896.[Abstract/Free Full Text]
  30. Fujio Y., Walsh K. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner. J Biol Chem (1999) 274:16349–16354.[Abstract/Free Full Text]
  31. Luo Z., Fujio Y., Kureishi Y., et al. Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo. J Clin Invest (2000) 106:493–499.[ISI][Medline]
  32. Vincent L., Chen W., Hong L., et al. Inhibition of endothelial cell migration by cerivastatin, an HMG-CoA reductase inhibitor: contribution to its anti-angiogenic effect. FEBS Lett (2001) 495:159–166.[CrossRef][ISI][Medline]
  33. Servant G., Weiner O.D., Herzmark P., et al. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science (2000) 287:1037–1040.[Abstract/Free Full Text]
  34. Backman J.T., Kyrklund C., Kivisto K.T., Wang J.S., Neuvonen P.J. Plasma concentrations of active simvastatin acid are increased by gemfibrozil. Clin Pharmacol Ther (2000) 68:122–129.[CrossRef][ISI][Medline]
  35. Watton S.J., Downward J. Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell–matrix and cell–cell interaction. Curr Biol (1999) 9:433–436.[CrossRef][ISI][Medline]
  36. Brown D.A., London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem (2000) 275:17221–17224.[Free Full Text]
  37. Khan A.A., Bose C., Yam L.S., Soloski M.J., Rupp F. Physiological regulation of the immunological synapse by agrin. Science (2001) 292:1681–1686.[Abstract/Free Full Text]
  38. Varma R., Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature (1998) 394:798–801.[CrossRef][Medline]
  39. Nguyen D.H., Hildreth J.E. Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol (2000) 74:3264–3272.[Abstract/Free Full Text]
  40. Manes S., Mira E., Gomez-Mouton C., et al. Membrane raft microdomains mediate front–rear polarity in migrating cells. EMBO J (1999) 18:6211–6220.[CrossRef][ISI][Medline]
  41. Baumann C.A., Ribon V., Kanzaki M., et al. CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature (2000) 407:202–207.[CrossRef][Medline]
  42. Muller G., Jung C., Wied S., et al. Redistribution of glycolipid raft domain components induces insulin-mimetic signaling in rat adipocytes. Mol Cell Biol (2001) 21:4553–4567.[Abstract/Free Full Text]
  43. Rizzo M.A., Kraft C.A., Watkins S.C., Levitan E.S., Romero G. Agonist-dependent traffic of raft-associated ras and raf-1 is required for activation of the mitogen-activated protein kinase cascade. J Biol Chem (2001) 276:34928–34933.[Abstract/Free Full Text]
  44. Zundel W., Swiersz L.M., Giaccia A. Caveolin 1-mediated regulation of receptor tyrosine kinase-associated phosphatidylinositol 3-kinase activity by ceramide. Mol Cell Biol (2000) 20:1507–1514.[Abstract/Free Full Text]
  45. Feron O., Dessy C., Desager J.P., Balligand J.L. Hydroxy-methylglutaryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation (2001) 103:113–118.[Abstract/Free Full Text]
  46. Feron O., Saldana F., Michel J.B., Michel T. The endothelial nitric-oxide synthase-caveolin regulatory cycle. J Biol Chem (1998) 273:3125–3128.[Abstract/Free Full Text]
  47. Faust J.R., Brown M.S., Goldstein J.L. Synthesis of delta 2-isopentenyl tRNA from mevalonate in cultured human fibroblasts. J Biol Chem (1980) 255:6546–6548.[Abstract/Free Full Text]
  48. Burke R., Nellen D., Bellotto M., et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell (1999) 99:803–815.[CrossRef][ISI][Medline]
  49. Rietveld A., Neutz S., Simons K., Eaton S. Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains. J Biol Chem (1999) 274:12049–12054.[Abstract/Free Full Text]
  50. Incardona J.P., Eaton S. Cholesterol in signal transduction. Curr Opin Cell Biol (2000) 12:193–203.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Stem CellsHome page
H. Shao, Y. Tan, D. Eton, Z. Yang, M. G. Uberti, S. Li, A. Schulick, and H. Yu
Statin and Stromal Cell-Derived Factor-1 Additively Promote Angiogenesis by Enhancement of Progenitor Cells Incorporation into New Vessels
Stem Cells, May 1, 2008; 26(5): 1376 - 1384.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Skaletz-Rorowski, A.
Right arrow Articles by Walsh, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skaletz-Rorowski, A.
Right arrow Articles by Walsh, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us