Cardiovascular Research

Cardiovascular Research 2000 48(1):148-157; doi:10.1016/S0008-6363(00)00152-8
© 2000 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Jung, F. Articles by Dimmeler, S. Search for Related Content PubMed PubMed Citation Articles by Jung, F. Articles by Dimmeler, S. Social Bookmarking          What's this?

Copyright © 2000, European Society of Cardiology

Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death

Frank Jung, Judith Haendeler, Christine Goebel, Andreas M. Zeiher and Stefanie Dimmeler^*

Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany

^* Corresponding author. Tel.: +49-69-6301-7440; fax: +49-69-6301-7113 Dimmeler{at}em.uni-frankfurt.de

Received 20 April 1999; accepted 15 June 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The signaling pathways mediating proliferation and apoptosis in vascular smooth muscle cells (VSMC) are not well established. It has previously been shown that activation of the phosphoinositide 3-OH kinase (PI3K)/Akt pathway or the ERK 1/2 pathway can mediate anti-apoptotic function in different cell types. This study determined the specific contribution of the PI3K/Akt and ERK pathway in the regulation of apoptosis and proliferation of VSMC. Methods and results: Incubation of rat VSMC with FCS, insulin or IGF-1 time-dependently stimulated the phosphorylation of Akt, however FCS but not insulin or IGF-1 activated the MAP-kinase ERK 1/2. Moreover, insulin inhibited H₂O₂-induced apoptosis via the Akt pathway as demonstrated by pharmacological inhibition of the PI3K or overexpression of a dominant negative Akt mutant. In contrast, FCS inhibited H₂O₂-induced apoptosis via the Akt and also the ERK pathway. FCS, but not insulin or IGF-1 induced VSMC proliferation, suggesting that Akt activation is necessary but not sufficient for VSMC proliferation. FCS-induced proliferation of VSMC was only mediated via the Akt pathway and not the ERK pathway. Conclusions: These results define a link between cell proliferation and programmed cell death in VSMC via the same signal transduction pathway, namely activation of the serine/threonine kinase Akt, which may have significant implication for the development of vascular diseases or remodeling.

KEYWORDS Apoptosis; Growth factors; Protein kinases; Signal transduction; Smooth muscle

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The differentiated state of vascular smooth muscle cells (VSMC) within atherosclerotic and restenotic lesions in man as well as lesions generated following vascular injury in animals is altered with regard to normal medial VSMC [1–3]. This is characterized not only by extracellular matrix production and cell migration, but also by contrasting phenomena such as cell death and cell proliferation [4–6], which has been the classical paradigm for the development of vascular diseases [7,8]. However, recently it has become more evident that the balance between changes in regulation of cell growth and cell death is an important determinant of vascular integrity and lesion formation.

It is now well known that in response to a variety of different stimuli, cells can initiate highly conserved signaling events, which lead to either cell proliferation or programmed cell death (apoptosis). Growth factors such as PDGF [9], transforming growth factor beta-1 [10], basic fibroblast growth factor [11] or angiotensin II [12], have been shown to activate the mitogen-activated protein kinase (MAPK) cascade via ERK 1/2, which is critical to the mitogenic response and cellular differentiation [13,14]. Recent studies provide evidence that another kinase, the serine/threonine kinase Akt, plays a key role in matrix adhesion and integrin-mediated signal transduction, as well as suppression of apoptotic cell death induced by growth factor deprivation [15–17]. The activation of Akt seems to be mediated by the phosphoinositide 3-OH kinase (PI3K), which stimulates phosphorylation of Akt by activating protein kinase B/Akt kinases (PDK-1 and PDK-2) [18–20]. The down-stream targets of Akt include the glycogen synthase kinase-3 and possibly the p70 ribosomal S6 kinase [21,22], although neither of these substrates accounts for the involvement of Akt in cell attachment. Furthermore, Akt has recently been shown to phosphorylate the pro-apoptotic protein Bad, thereby inhibiting its pro-apoptotic function, which may account for the anti-apoptotic effect of Akt [23–25].

Oxidative stress, inflammation, endothelial denudation, have all been implicated in the promotion of proliferation on one hand and programmed cell death on the other hand in VSMC [26,27]. However, the mechanisms and autocrine–paracrine factors leading to proliferation or apoptotic cell death in VSMC are not well established. Recent studies have demonstrated that insulin or insulin-like growth factor IGF-1 and other signaling molecules, such as the cytoplasmic insulin receptor substrate-1 (IRS-1) exert anti-apoptotic effect via the PI3K/Akt kinase pathway and less frequently the ERK pathway in different cell types [28–30].

Therefore, one aim of the present study was to investigate the effect of insulin on H₂O₂-induced apoptosis in VSMC and to determine, if the PI3K/Akt kinase pathway acts as the downstream signal transduction pathway. We demonstrate that insulin as well as IGF-1 stimulate phosphorylation of Akt in a time-dependent manner, which was mediated by PI3K. Additionally, insulin-mediated Akt activation resulted in suppression of H₂O₂-induced apoptosis in VSMC. Furthermore, we show that growth factor-induced smooth muscle cell proliferation is mediated via the PI3K/Akt signal transduction pathway.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Cell culture
Rat vascular aortic smooth muscle cells were plated 24 h before transfection at a seeding density of 1.2x10⁵ cells per cm² in DMEM F-12 medium (GIBCO, Grand Island, NY, USA), supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY, USA), 100 U/ml penicillin and 100 µg/ml streptomycin.

2.2 Transient transfection
The dominant negative Akt-mutant (Aktmt) [31] was digested with HincII/EcoRI and subcloned into the respective sites (EcoRV/EcoRI) of pcDNA3.1 (In vitrogen, NV Leek, The Netherlands). Cells were cotransfected with either the native pcDNA3.1, lacking an insert, and pcDNA3.1-lacZ as a control or pcDNA3.1-Aktmt and pcDNA3.1-lacZ (1 µg pcDNA3.1-lacZ and 2 µg pcDNA3.1-Aktmt or pcDNA3.1) and 30 µl Superfect (Qiagen GMBH, Hilden, Germany). After transfection, cells were incubated for 12 h and then starved in serum-free medium (SFM) for an additional 12 h prior to stimulation as described above. 12 h after stimulation, cells were fixed in 2% formalin/0.2% glutaraldehyde and viable or dead transfected cells were identified, using β-galactosidase staining, after incubation with 40 µg/ml X-gal for 6 h at 37°C. Cells were counted by two blind investigators and results were expressed as dead/viable cellsx100. Transfection efficiency was 8–10%.

2.3 Determination of Akt activation
VSMC were lysed (20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, 1 mM PMSF) for 5 min on ice. Cells were scraped off the plates and sonified (with a Branson Sonifier). Protein concentration was determined using Biorad reagent (München, Germany). Proteins (50 µg per lane) were loaded onto 9% SDS–polyacrylamide gels and blotted onto PVDF membranes. Incubation with phospho-Akt (Biolabs, Schwalbach, Germany) 1:500 or phospho-ERK-antibodies (Biolabs, Schwalbach, Germany) 1:1000 was performed at 4°C overnight in TBS (50 mM Tris/HCl, pH=8; 150 mM NaCl, 2.5 mM KCl), 0.1% Tween-20, 3% bovine serum albumin (BSA). After incubation with the second antibody (anti-rabbit: 1:4000) for 1 h, enhanced chemiluminescence was performed according to the instructions of the manufacturer (Amersham, Germany). Blots were re-probed with actin (Boehringer Mannheim, Germany) 1:2000 to determine equal protein loading. Blots were then scanned and semi-quantitatively analyzed.

2.4 Detection of apoptosis
For morphological staining of nuclei, cells were centrifuged for 10 min at 700xg, then fixed in 4% formaldehyde and stained with DAPI (0.2 µg/ml in 10 mM Tris–HCl, pH=7, 10 mM EDTA, 100 mM NaCl) for 20 min. Three visual fields were counted by two independent blind investigators and the percentage of apoptotic cells per total number of cells was determined.

2.5 FACS analysis
Cells were grown as described above and starved in serum-free medium for 48 h prior to stimulation with FCS. Cells were then resuspended in 900 µl PBS and precipitated in 2.1 ml of ice-cold 100% ethanol for 2 h at –20°C. Cells were then pelleted for 10 min at 200 rpm and resuspended in 640 µl PBS, 160 µl propidium iodide (20 µg/ml) and 8 µl RNase (100 µg/ml) for 30 min prior to FACS analysis.

2.6 Cell proliferation assay
Cells were grown in 96-well plates for 24 h and starved in serum-free medium for 48 h prior to stimulation with FCS, insulin or IGF-1. Cells were then grown for 24 h. Cells were then incubated with thiozolylblue for 3–4 h at 37 C°. Subsequently the medium was removed and the reaction was stopped with 0.04 N HCL. Extinction was measured in an ELISA-reader at 405 nm.

2.7 Statistical analysis
Data are expressed as means±S.E.M from at least three independent experiments. Statistical analysis was performed with t-test or ANOVA followed by modified LSD test (SPSS-software).

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effect of serum, IGF-1 and insulin on Akt phosphorylation in VSMC
As shown in Fig. 1A and B, FCS stimulated phosphorylation of Akt as early as 10 min lasting up to 1 h with a decrease starting at 2 h. The detection of phosphorylated Akt was shown to correlate with an increase of the enzymatic Akt activity (data not shown) [31]. Exposure of cells to IGF-1 and insulin promotes phosphorylation of Akt lasting up to 6 h (Fig. 2A and B). Next, we tested the involvement of FCS (Fig. 1A, upper panel), insulin (Fig. 4, middle panel) and IGF-1 (data not shown) on phosphorylation of the MAP kinase ERK in VSMC. FCS, but not insulin or IGF-1 resulted in phosphorylation of ERK 1/2 in VSMC.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of FCS on Akt phosphorylation. VSMC were incubated for 48 h in the absence of FCS and then exposed to 10% FCS containing medium for the times indicated. Proteins were obtained as described in the Methods section and western blot analysis was performed using an antibody directed against phosphorylated Akt (Akt-P). Phosphorylated Akt was used as positive and negative controls, respectively (data not shown). FCS-induced Akt phosphorylation is shown in (A) middle panel, at 10 min and 60 min. Blots were also re-probed with phospho-specific ERK-1 (p42) and ERK-2 (p44) antibodies (upper panel). (B) Demonstrates a time-dependent increase in Akt phosphorylation after stimulation with FCS (C=control). Results are shown as representative for three different experiments. Data are expressed as mean±S.D. after densitometric quantification (lower panel).

 

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of IGF and insulin on Akt phosphorylation in VSMC. VSMC were incubated for 48 h in the absence of FCS prior to exposure to IGF (10 ng/ml) (A) or insulin (100 nM) (B). Akt phosphorylation (Akt-P) was determined in a time-dependent manner by western blotting using a phospho-specific Akt-antibody. Results are shown as representative for three different experiments. Data are expressed as mean±S.D. after densitometric quantification (lower panel).

 

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of inhibition of PI3K on insulin-mediated Akt phosphorylation in VSMC. Cells were starved for 48 h in serum-free medium. Cells were then pre-incubated for 30 min in the presence or absence of Ly294002 (Ly) (10 µM), wortmannin (WM) (20 nM) or the MEK inhibitor PD98059 (PD) (10 µM) prior to stimulation with insulin (100 nM). Blots were probed using specific antibodies against phophorylated Akt (Akt-P) (upper panel). To test the involvement of insulin on phosphorylation of the MAP kinase, ERK-1 (p42) and ERK-2 (p44), western blots were re-probed using specific antibodies against phosphorylated ERK (middle panel). Actin re-probe served as a loading control (lower panel). Results are shown as representative for three different experiments. Data are expressed as mean±S.D. after densitometric quantification (lower panel).

 
Taken together, these results indicate that the Akt pathway and the ERK kinase-cascade are activated by distinct stimuli. FCS promotes phosphorylation of Akt and ERK, whereas insulin and IGF-1 selectively stimulate Akt phosphorylation.

3.2 Involvement of PI3K on FCS-, insulin- and IGF-1-mediated Akt phosphorylation in VSMC
To elucidate the signal transduction pathways underlying the activation of Akt phosphorylation by FCS, insulin and IGF-1, we tested the influence of the PI3K, which has been described to mediate Akt-stimulation in other cell systems [19,21]. Therefore, the effect of Ly294002 and wortmannin, two unrelated, but specific inhibitors of the PI3K, was determined on FCS- or insulin- and IGF-1-induced Akt phosphorylation. As shown in Fig. 3A (middle panel), Ly294002 (Ly) (10 µM) and wortmannin (Wm) (20 nM) inhibited Akt phosphorylation stimulated by exposure to FCS. In contrast, the PI3K inhibitors did not affect the FCS-induced phosphorylation of ERK 1/2 (Fig. 3A, upper panel). Co-incubation of cells with Ly294002 or wortmannin also reduced or prevented phosphorylation of Akt in cells stimulated with IGF-1 (Fig. 3B) or insulin (Fig. 4, upper panel), thus clearly demonstrating PI3K dependency.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of inhibition of PI3K on FCS and IGF-mediated Akt phosphorylation in VSMC. The effect of Ly294002 and wortmannin, specific inhibitors of the PI3K, on FCS (A, middle panel) or IGF-induced (B) Akt phosphorylation (Akt-P) was determined. Cells were starved for 48 h in serum-free medium prior to stimulation with FCS or IGF-1, with or without co-incubation of the specific PI3K inhibitors Ly294002 (Ly) (10 µM) or wortmannin (Wm) (20 nM). To also test the involvement of FCS on phosphorylation of the MAP-kinase, ERK-1 (p42) and ERK-2 (p44), western blots were re-probed using specific antibodies against phosphorylated ERK in the presence or absence of the specific PI3K inhibitors Ly294002 (Ly) (10 µM) or wortmannin (Wm) (20 nM) (Fig. 1A, upper panel). Results are shown as representative for three different experiments. Data are expressed as mean±S.D. after densitometric quantification (lower panel).

 
Moreover, PD98059, which prevents ERK-phosphorylation by inhibiting the up-stream kinase MEK, did not affect insulin-induced Akt phosphorylation (Fig. 4, upper panel). Thus, taken together, PI3K-dependent phosphorylation of Akt and phosphorylation of ERK are mediated by distinct pathways.

3.3 Involvement of insulin on H₂O₂-induced apoptosis in VSMC via the Akt pathway
Since insulin has been known to have anti-apoptotic effects via the PI3K/Akt pathway in other cell types, we tested whether insulin had anti-apoptotic effects on H₂O₂-induced apoptosis in VSMC. Stimulation of cells with H₂O₂ (100 µM) caused a significant increase of apoptotic cell death compared to non-stimulated cells, as assessed by morphological analysis of fluorescence-stained nuclei (Fig. 5A). Co-incubation of cells with insulin significantly reduced the rate of H₂O₂-induced apoptosis. Ly294002 and wortmannin partially inhibited the protective effect of insulin on H₂O₂-induced apoptosis in VSMC. These results indicate that insulin exerts its anti-apoptotic effect via the PI3K/Akt pathway. We also tested if FCS mediates anti-apoptotic effects via the PI3K/Akt pathway. Stimulation of cells with FCS prevented H₂O₂-induced apoptosis in VSMC, which was partially reversed by co-incubation of cells with the specific PI3K inhibitor wortmannin (Fig. 5B). As FCS was also shown to induce phosphorylation of ERK, cells were co-incubated with the specific MEK inhibitor PD98059 (PD), which partially reversed the anti-apoptotic effects of FCS on H₂O₂-induced apoptosis in VSMC. Addition of both inhibitors, WM and PD98059, had an additive inhibitory effect on the anti-apoptotic function of FCS (Fig. 5B). This suggests that the Akt and the ERK pathway mediate the anti-apoptotic function of FCS.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Involvement of insulin and FCS on H2O2-induced apoptosis in VSMC via the Akt pathway. Cells were starved for 48 h. Apoptosis was induced by stimulation of cells with H2O2 (100 µM) for 12 h prior to stimulation with insulin (100 nM) (A) or FCS (B). Cells were also preincubated with the specific PI3K inhibitors Ly (10 µM) and wortmannin (WM) (20 nM) or the MEK inhibitor PD98059 (PD) (10 µM). Apoptotic nuclei were counted by two blinded investigators after fluorescent nuclear staining and data are expressed as mean±S.D., n = 3. (* and **P<0.05 versus H2O2 and insulin in panel A; #, ## and ###P<0.05 versus H2O2 and FCS in panel B).

 
To further demonstrate that the PI3K-stimulated Akt phosphorylation accounts for the anti-apoptotic effect of insulin in H₂O₂-stimulated cells, endogenous Akt was specifically inhibited by expression of a dominant negative mutant in VSMC. Thus, VSMC were cotransfected with β-galactosidase and a dominant negative Akt-mutant. Subsequently, apoptosis was induced by stimulation with H₂O₂. Transfected cells were identified by β-galactosidase staining and viable versus dead cells were counted (Fig. 6). Stimulation with H₂O₂-triggered cell death of mock transfected cells to a similar extent as compared to non-transfected cells. Again, co-incubation of cells with insulin partially reduced H₂O₂-induced apoptosis in those transfected cells. Most importantly, however, inhibition of Akt by expression of the dominant negative mutant significantly reduced the suppressive effect of insulin on apoptosis, thus demonstrating that the anti-apoptotic effect of insulin on H₂O₂-induced programmed cell death in VSMC is at least in part mediated by the PI3K/Akt pathway.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Transfection of VSMC with a dominant negative Akt-mutant reduced the anti-apoptotic effects of insulin on H2O2-induced apoptosis. VSMC were transiently cotransfected with pcDNA3.1 (vector, 2 µg) or pcDNA3.1-Aktmt (Aktmt, 2 µg) and pcDNA3.1-β-galactosidase (1 µg) and incubated for 12 h to allow protein expression. Cells were then starved in serum-free medium for 12 h and subsequently apoptosis was induced by stimulation with H2O2 (100 µM) in the presence or absence of insulin (100 nM). Transfected cells were identified by β-galactosidase staining and viable versus dead cells were counted. Dead cells were additionally analyzed under higher magnification to confirm the morphological alterations typical for apoptotic cell death. Data were expressed as mean±S.D., n = 3, *P = 0.025 versus vector/H2O2/insulin.

 
3.4 Involvement of the Akt kinase pathway on growth factor-induced VSMC proliferation
It has been previously shown that different growth factors can induce proliferation of VSMC. We therefore tested the involvement of FCS, insulin and IGF-1 on proliferation of VSMC. Using a cell proliferation assay, FCS was shown to induce proliferation of VSMC, whereas no significant proliferation was observed, when cells were stimulated with insulin or IGF-1 (Fig. 7A). To test the involvement of the PI3K/Akt pathway on FCS-induced proliferation of VSMC, FACS analysis was used. Stimulation of cells with FCS resulted in an almost ten-fold increase of cells entering the S-phase of the cell cycle (Fig. 7B). Co-incubation of cells with the specific PI3K inhibitor Ly294002 partially reversed the proliferative effect of FCS on cell cycle progression in VSMC (Fig. 7B and C), thus demonstrating that the effect of FCS on proliferation of VSMC is at least in part mediated via the PI3K/Akt pathway and that Akt is necessary but not sufficient to mediate proliferation in VSMC.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Growth factor-induced VSMC proliferation and the involvement of the PI3K/Akt-pathway: Cells were starved in serum-free medium for 48 h prior to stimulation of VSMC with FCS, insulin or IGF for 24 h. (A) Proliferation of VSMC was determined using a MTT cell proliferation assay. Proliferation was expressed in percent relative to non-stimulated cells. (B) To also determine the effect of the PI3K on FCS-induced proliferation of VSMC, cells were co-incubated with the specific PI3K inhibitor Ly294002 (Ly) (10 µM) and subsequently subjected to FACS analysis as described in the Methods section. Cells in S-phase were quantified and data are expressed as mean±S.D., n = 4, with *P<0.05 versus +FCS (B). A representative FACS analysis is shown in panel C.

 
To test if the ERK 1/2 pathway has additional effects on FCS-mediated cell proliferation, VSMC were co-incubated with FCS and the specific MEK inhibitor PD98059, which had no significant effect on FCS-induced cell proliferation. Moreover, co-incubation of cells with PD98059 and the specific PI3K inhibitor WM did not result in a significantly greater reduction of the FCS-induced cell proliferation, than with WM alone. These results suggest that FCS induces cell proliferation mainly via the PI3K/Akt pathway (Fig. 8).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Involvement of the ERK pathway on FCS-induced VSMC proliferation. Cells were starved in serum-free medium for 48 h prior to stimulation of VSMC with FCS. Proliferation of VSMC was determined using a MTT cell proliferation assay. Proliferation was expressed in percent relative to non-stimulated cells. To also determine the effect of the Akt and the ERK pathway on FCS-induced proliferation of VSMC, cells were co-incubated with the specific PI3K inhibitor wortmannin (WM) (20 nM) and the MEK inhibitor PD98059 (10 µM). Data are expressed as mean±S.D., n = 3. Statistics are shown between cells stimulated with FCS and cells stimulated with FCS and inhibitors.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Proliferation and apoptotic cell death of smooth muscle cells play a critical role in the response to vascular injury [2,4,32]. Therefore, identifying factors that regulate cell proliferation on one hand or cell death on the other hand may have important implications for the treatment of vascular diseases. To date, several growth factors have been identified in promoting cell proliferation [33,34] and cell survival of VSMC [35]. The signaling pathways mediating these effects, however, are not entirely characterized. Previous studies have shown that the ERK pathway and potentially the PI3K/Akt pathway may be involved in mediating these effects [36–41]. The major finding of this study is that stimulation of VSMC with different growth factors, such as insulin, IGF-1 or FCS, all mediate phosphorylation of the Akt-kinase via activation of PI3K, although with different cellular morphological alterations in VSMC. Stimulation of cells with FCS causes cell proliferation and promotes cell survival, whereas stimulation with insulin and IGF-1 only promotes cell survival via protection from programmed cell death.

Activation of the PI3K/Akt pathway has been shown to inhibit apoptosis in a variety of other cell types [15,17,23,30]. Similarly the present data demonstrate that Akt phosphorylation mediates the anti-apoptotic effects of insulin on H₂O₂-induced apoptosis in VSMC. This was confirmed by overexpression of a dominant negative Akt-mutant, clearly indicating that the down-stream target of the PI3K, Akt, mediates this effects. Interestingly, the PI3K/Akt pathway seems only partially responsible for the apoptosis-suppressive effect of insulin. The MAP-kinases ERK1/2 are known to exert potent anti-apoptotic effects and therefore could contribute to the protection achieved by insulin/IGF-1. However, in contrast to the stimulation of Akt, insulin and IGF-1 failed to activate the ERK 1/2 pathway in VSMC, which is in accordance with recent studies by Pukac et al. [42]. Therefore, the ERK 1/2 pathway is unlikely to participate in insulin-mediated apoptosis suppression. However, controversial effects of insulin on MAP-kinase signaling have been demonstrated by other groups [43]. Cospedal and coworkers described an increase in ERK 1/2 activity after stimulation of rabbit VSMC with IGF-1 [44]. Discrepancies between these studies may be due to differences in species and phenotype. This is supported by a recent publication demonstrating that the cross talk between Akt and ERK 1/2 depends on the differentiation stage and that Akt can, under certain condition, even inhibit the raf-ERK 1/2 pathway [45], which indicates that the signal transduction pathways are not only cell type-specific but also highly selective for differentiation phenotypes. However, the question remains, why the PI3K/Akt pathway only partially accounts for the inhibition of VSMC apoptosis by insulin/IGF-1. IGF-1 has been shown to translocate Raf-1 to the mitochondria via the interaction of the IGF-receptors with 14-3-3-proteins thereby providing an additional anti-apoptotic signal [43]. Furthermore, insulin/IGF-1 may transcriptionally up-regulate anti-apoptotic proteins such as Bcl-2 [46]. According to our data, activation of the PI3K/Akt pathway seems only partially to be responsible for the effect of insulin on suppression of apoptosis in VSMC, thus suggesting that additional pathways may still be involved.

Different downstream cellular targets of Akt have been described, which may indicate the apoptosis-suppressive effect of Akt, including phosphorylation of the pro-apoptotic protein Bad [23,24]. During the preparation of this manuscript, Bai et al. reported that stimulation of VSMC with IGF-1 resulted in phosphorylation and thus inhibition of the pro-apoptotic protein Bad [25]. However, other studies failed to demonstrate a causative role of Bad-phosphorylation in Akt-mediated cell survival in other cell types [47,48]. Alternatively, Akt has been shown to directly inhibit activation of apoptosis executing caspase cascade, e.g. via phosphorylation of the caspase-9 [49]. Thus, multiple pathways may be involved in Akt-mediated suppression of apoptosis [50].

The second part of our study evaluated the role of the PI3K/Akt pathway in growth factor-induced cell proliferation, which is less well established. Using FACS analysis and MTT cell proliferation assays, our data demonstrate, that serum-induced proliferation of VSMC was mainly mediated via the PI3K/Akt kinase pathway. These data complement other recent studies, which have shown increased growth factor-induced DNA synthesis or amino acid uptake mediated via the PI3K/Akt pathway in VSMC [51,52]. Likewise, the phenotype of VSMC appears to be determined by the balance of the PI3K/Akt and the MAPK (ERK/p38) pathways [53]. Furthermore, the downstream mechanisms underlying the Akt-mediated proliferative response are not entirely clear. It has been recently shown that expression of activated Akt rescues G₁ arrest and stimulates cell cycle progression in the absence of growth factors, in part by affecting the expression of c-myc and Bcl-2 [54]. Moreover, the PI3K/Akt pathway may increase the protein expression and stability of cyclin D [55–57], suggesting a crucial connection between the PI3K/Akt pathway and the cell machinery [58,59]. In contrast, there is evidence that increased phosphatase activity could suppress the ERK and PI3K/Akt pathway, resulting in exit from the cell cycle by downregulation of cyclins D, A, E and upregulation of p27(kip1) [60]. In our study, insulin and IGF-1 stimulated Akt to a similar extent as compared to FCS, but did not promote cell cycle progression, therefore indicating that other signals are required for successful proliferation of VSMC. Given that PD98059 did not inhibit FCS-induced proliferation, ERK 1/2 are unlikely to be involved.

In summary, the present study demonstrates that insulin/IGF-1, mediating anti-apoptotic effects and FCS, mediating cell proliferation and anti-apoptotic effects, all three stimulate the same signaling pathway in VSMC, the PI3K/Akt. Thus, activation of the PI3K/Akt pathway can mediate several functional and morphological alterations of VSMC, which may contribute to maintain VSMC integrity. Further characterization of the physiological consequences of Akt phosphorylation in VSMC will provide important information to clarify the role of the PI3K/Akt pathway in the development of vascular diseases.

Time for primary review 35 days.

	Acknowledgements

 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft Ju 241/2-1.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Gabbiani G., Kocher O., Bloom W.S., Vandekerckhove J., Weber K. Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest (1984) 73:148–152.[Web of Science][Medline]
2. Hanke H., Strohschneider T., Oberhoff M., Betz E., Karsch K.R. Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty. Circ Res (1990) 67:651–659.[Abstract/Free Full Text]
3. Clowes A.W., Clowes M.M., Fingerle J., Reidy M.A. Kinetics of cellular proliferation after arterial injury. V. Role of acute distension in the induction of smooth muscle proliferation. Lab Invest (1989) 60:360–364.[Web of Science][Medline]
4. Clowes A.W., Clowes M.M., Fingerle J., Reidy M.A. Regulation of smooth muscle cell growth in injured artery. J Cardiovasc Pharmacol (1989) 14(Suppl_6):S12–S15.
5. Bochaton P.M.L., Gabbiani F., Redard M., Desmouliere A., Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol (1995) 146:1059–1064.[Abstract]
6. Isner J.M., Kearney M., Bortman S., Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation (1995) 91:2703–2711.[Abstract/Free Full Text]
7. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (1993) 362:801–809.[CrossRef][Medline]
8. Rao G.N., Berk B.C. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res (1992) 70:593–599.[Abstract/Free Full Text]
9. Jawien A., Bowen-Pope D.F., Lindner V., Schwartz S.M., Clowes A.W. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest (1992) 89:507–511.[Web of Science][Medline]
10. Nabel E.G., Shum L., Pompili V.J., et al. Direct gene transfer of transforming growth factor beta-1 into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci USA (1993) 90:10759–10763.[Abstract/Free Full Text]
11. Lindner V., Lappi D.A., Baird A., Majack R.A., Reidy M.A. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res (1991) 68:106–113.[Abstract/Free Full Text]
12. Duff J.L., Marrero M.B., Paxton W.G., et al. Angiotensin II signal transduction and the mitogen-activated protein kinase pathway. Cardiovasc Res (1995) 30:511–517.[Abstract/Free Full Text]
13. Sturgill T.W., Ray L.B., Erikson E., Maller J.L. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature (1988) 334:715–718.[CrossRef][Medline]
14. Denhardt D.T. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signaling. Biochem J (1996) 318:729–747.[Web of Science][Medline]
15. Khwaja A., Rodriguez-Viciana P., Wennstrom S., Warne P.H., Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J (1997) 16:2783–2793.[CrossRef][Web of Science][Medline]
16. Franke T.F., Kaplan D.R., Cantley L.C. PI3K: downstream AKTion blocks apoptosis. Cell (1997) 88:435–437.[CrossRef][Web of Science][Medline]
17. Kennedy S.G., Wagner A.J., Conzen S.D., et al. The PI3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev (1997) 11:701–713.[Abstract/Free Full Text]
18. Klippel A., Kavanaugh W.M., Pot D., Williams L.T. A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain. Mol Cell Biol (1997) 17:338–344.[Abstract]
19. Stephens L., Anderson K., Stokoe D., et al. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science (1998) 279:710–714.[Abstract/Free Full Text]
20. Downward J. Lipid-regulated kinases: some common themes at last. Science (1998) 279:673–674.[Free Full Text]
21. Burgering B.M., Coffer P.J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature (1995) 376:599–602.[CrossRef][Medline]
22. Cross D.A., Alessi D.R., Cohen P., Andjelkovich M., Hemmings B.A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature (1995) 378:785–789.[CrossRef][Medline]
23. Datta S.R., Dudek H., Tao X., et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell (1997) 91:231–241.[CrossRef][Web of Science][Medline]
24. del Peso L., Gonzalez-Garcia M., Page C., Herrera R., Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (1997) 278:687–689.[Abstract/Free Full Text]
25. Bai H., Pollman M.J., Inishi Y., Gibbons G.H. Regulation of vascular smooth muscle cell apoptosis; modulation of Bad by a phosphatidylinositol 3-kinase-dependent pathway. Circ Res (1999) 85:229–237.[Abstract/Free Full Text]
26. Ferns G.A., Stewart L.A., Anggard E.E. Arterial response to mechanical injury: balloon catheter de-endothelialization. Atherosclerosis (1992) 92:89–104.[CrossRef][Web of Science][Medline]
27. Hearse D.J., Maxwell L., Saldanha C., Gavin J.B. The myocardial vasculature during ischemia and reperfusion: a target for injury and protection. J Mol Cell Cardiol (1993) 25:759–800.[CrossRef][Web of Science][Medline]
28. Dudek H., Datta S.R., Franke T.F., et al. Regulation of neuronal survival by the serine–threonine protein kinase Akt. Science (1997) 275:661–665.[Abstract/Free Full Text]
29. Parrizas M., Saltiel A.R., LeRoith D. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem (1997) 272:154–161.[Abstract/Free Full Text]
30. Kulik G., Klippel A., Weber M.J. Anti-apoptotic signaling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol (1997) 17:1595–1606.[Abstract]
31. Dimmeler S., Assmus B., Hermann C., Haendeler J., Zeiher A.M. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res (1998) 83:334–341.[Abstract/Free Full Text]
32. Bennett M.R., Evan G.I., Schwartz S.M. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest (1995) 95:2266–2274.[Web of Science][Medline]
33. Walker L.N., Bowen-Pope D.F., Ross R., Reidy M.A. Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Proc Natl Acad Sci USA (1986) 83:7311–7315.[Abstract/Free Full Text]
34. Gibbons G.H., Pratt R.E., Dzau V.J. Vascular smooth muscle cell hypertrophy vs. hyperplasia: autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest (1992) 90:456–461.[Web of Science][Medline]
35. Rampalli A.M., Zelenka P.S. Insulin regulates expression of c-fos and c-jun and suppresses apoptosis of lens epithelial cells. Cell Growth Differ (1995) 6:945–953.[Abstract]
36. Force T., Bonventre J.V. Growth factors and mitogen-activated protein kinases. Hypertension (1998) 31(2):152–161.[Abstract/Free Full Text]
37. Wang Y., Rose P.M., Webb M.L., Dunn M.J. Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB. Am J Physiol (1994) 267:C1130–C1135.[Web of Science][Medline]
38. Liao D.F., Monia B., Dean N., Berk B.C. Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J Biol Chem. (1997) 272:6146–6150.[Abstract/Free Full Text]
39. Kim S., Izumi Y., Yano M., et al. Angiotensin blockade inhibits activation of mitogen-activated protein kinases in rat balloon-injured artery. Circulation (1998) 97:1731–1737.[Abstract/Free Full Text]
40. Andjelkovic M., Jakubowicz T., Cron P., et al. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci USA (1996) 93:5699–5704.[Abstract/Free Full Text]
41. Franke T.F., Yang S.I., Chan T.O., et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell (1995) 81:727–736.[CrossRef][Web of Science][Medline]
42. Pukac L., Huangpu J., Karnovsky M.J. Platelet-derived growth factor-BB, insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration. Exp Cell Res (1998) 242:548–560.[CrossRef][Web of Science][Medline]
43. Peruzzi F., Prisco M., Dews M., et al. Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis. Mol Cell Biol (1999) 19:7203–7215.[Abstract/Free Full Text]
44. Cospedal R., Abedi H., Zachary I. Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Cardiovasc Res (1999) 41:708–721.[Abstract/Free Full Text]
45. Zimmermann S., Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science (1999) 286:1741–1744.[Abstract/Free Full Text]
46. Pugazhenthi S., Miller E., Sable C., et al. Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein. J Biol Chem (1999) 274:27529–27535.[Abstract/Free Full Text]
47. Scheid M.P., Duronio V. Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation. Proc Natl Acad Sci USA (1998) 95:7439–7444.[Abstract/Free Full Text]
48. Hermann C., Zeiher A.M., Dimmeler S. Shear stress inhibits H₂O₂-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol (1997) 17:3588–3592.[Abstract/Free Full Text]
49. Cardone M.H., Roy N., Stennicke H.R., et al. Regulation of cell death protease caspase-9 by phosphorylation. Science (1998) 282:1318–1321.[Abstract/Free Full Text]
50. Neshat M.S., Raitano A.B., Wang H.G., Reed J.C., Sawyers C.L. The survival function of the bcr-Abl oncogene is mediated by bad-dependent and -independent pathways: roles for phosphatidylinositol 3-kinase and Raf. Mol Cell Biol (2000) 20:1179–1186.[Abstract/Free Full Text]
51. Higaki M., Shimokado K. Phosphatidylinositol 3-kinase is required for growth factor-induced amino acid uptake by vascular smooth muscle cells. Arterioscler Thromb Vasc Biol (1999) 19:2127–2132.[Abstract/Free Full Text]
52. Imai Y., Clemmons D.R. Roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in stimulation of vascular smooth muscle cell migration and deoxyribonucleic acid synthesis by insulin-like growth factor-I. Endocrinology (1999) 140:4228–4235.[Abstract/Free Full Text]
53. Hayashi K., Takahashi M., Kimura K., et al. Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells. J Cell Biol (1999) 145:727–740.[Abstract/Free Full Text]
54. Skorski T., Bellacosa A., Nieborowska-Skorska M., et al. Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway. EMBO J (1997) 16:6151–6161.[CrossRef][Web of Science][Medline]
55. Muise-Helmericks R.C., Grimes H.L., Bellacosa A., et al. Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-Kinase/Akt-dependent pathway. J Biol Chem (1998) 273:29864–29872.[Abstract/Free Full Text]
56. Diehl J.A., Cheng M., Roussel M.F., Sherr C.J. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev (1998) 12:3499–3511.[Abstract/Free Full Text]
57. Gille H., Downward J. Multiple ras effector pathways contribute to G[1] cell cycle progression. J Biol Chem (1999) 274:22033–22040.[Abstract/Free Full Text]
58. Weng L.P., Smith W.M., Dahia P.L., et al. PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death. Cancer Res (1999) 59:5808–5814.[Abstract/Free Full Text]
59. Paramio J.M., Navarro M., Segrelles C., Gomez-Casero E., Jorcano J.L. PTEN tumor suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene (1999) 18:7462–7468.[CrossRef][Web of Science][Medline]
60. Suzuki E., Nagata D., Yoshizumi M., et al. Re-entry into the cell cycle of contact-inhibited vascular endothelial cells by a phosphatase inhibitor. Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J Biol Chem (2000) 275:3637–3644.[Abstract/Free Full Text]

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

This article has been cited by other articles:

				E. Favaro, I. Miceli, B. Bussolati, M. Schimitt-Ney, P. Cavallo Perin, G. Camussi, and M. M. Zanone Hyperglycemia Induces Apoptosis of Human Pancreatic Islet Endothelial Cells: Effects of Pravastatin on the Akt Survival Pathway Am. J. Pathol., August 1, 2008; 173(2): 442 - 450. [Abstract] [Full Text] [PDF]

				D. Allard, N. Figg, M. R. Bennett, and T. D. Littlewood Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3 J. Biol. Chem., July 11, 2008; 283(28): 19739 - 19747. [Abstract] [Full Text] [PDF]

				S. Perrini, A. Natalicchio, L. Laviola, A. Cignarelli, M. Melchiorre, F. De Stefano, C. Caccioppoli, A. Leonardini, S. Martemucci, G. Belsanti, et al. Abnormalities of Insulin-Like Growth Factor-I Signaling and Impaired Cell Proliferation in Osteoblasts from Subjects with Osteoporosis Endocrinology, March 1, 2008; 149(3): 1302 - 1313. [Abstract] [Full Text] [PDF]

				W.-Z. Ying, H.-G. Zhang, and P. W. Sanders EGF Receptor Activity Modulates Apoptosis Induced by Inhibition of the Proteasome of Vascular Smooth Muscle Cells J. Am. Soc. Nephrol., January 1, 2007; 18(1): 131 - 142. [Abstract] [Full Text] [PDF]

				M. Li, J.-F. Chiu, B. T. Mossman, and N. K. Fukagawa Down-regulation of Manganese-Superoxide Dismutase through Phosphorylation of FOXO3a by Akt in Explanted Vascular Smooth Muscle Cells from Old Rats J. Biol. Chem., December 29, 2006; 281(52): 40429 - 40439. [Abstract] [Full Text] [PDF]

				R. Rexhepaj, F. Grahammer, H. Volkl, C. Remy, C. A. Wagner, D. Sandulache, F. Artunc, G. Henke, S. Nammi, G. Capasso, et al. Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice FASEB J, November 1, 2006; 20(13): 2214 - 2222. [Abstract] [Full Text] [PDF]

				D. J. Son, S. J. Ha, H. S. Song, Y. Lim, Y. P. Yun, J. W. Lee, D. C. Moon, Y. H. Park, B. S. Park, M. J. Song, et al. Melittin Inhibits Vascular Smooth Muscle Cell Proliferation through Induction of Apoptosis via Suppression of Nuclear Factor-{kappa}B and Akt Activation and Enhancement of Apoptotic Protein Expression J. Pharmacol. Exp. Ther., May 1, 2006; 317(2): 627 - 634. [Abstract] [Full Text] [PDF]

				M. Vantler, E. Caglayan, W. H. Zimmermann, A. T. Baumer, and S. Rosenkranz Systematic Evaluation of Anti-apoptotic Growth Factor Signaling in Vascular Smooth Muscle Cells: ONLY PHOSPHATIDYLINOSITOL 3'-KINASE IS IMPORTANT J. Biol. Chem., April 8, 2005; 280(14): 14168 - 14176. [Abstract] [Full Text] [PDF]

				T. Nakazawa, T. Chiba, E. Kaneko, K. Yui, M. Yoshida, and K. Shimokado Insulin Signaling in Arteries Prevents Smooth Muscle Apoptosis Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 760 - 765. [Abstract] [Full Text] [PDF]

				E. V. Gerasimovskaya, D. A. Tucker, and K. R. Stenmark Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation J Appl Physiol, February 1, 2005; 98(2): 722 - 731. [Abstract] [Full Text] [PDF]

				L. Ragolia, T. Palaia, T. B. Koutrouby, and J. K. Maesaka Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1273 - C1281. [Abstract] [Full Text] [PDF]

				A. H. Shih, C. Dai, X. Hu, M. K. Rosenblum, J. A. Koutcher, and E. C. Holland Dose-Dependent Effects of Platelet-Derived Growth Factor-B on Glial Tumorigenesis Cancer Res., July 15, 2004; 64(14): 4783 - 4789. [Abstract] [Full Text] [PDF]

				B Gonzalez-Timon, M Gonzalez-Munoz, C Zaragoza, S Lamas, and E.M Melian Native and oxidized low density lipoproteins oppositely modulate the effects of insulin-like growth factor I on VSMC Cardiovasc Res, February 1, 2004; 61(2): 247 - 255. [Abstract] [Full Text] [PDF]

				A. Ruiz-Torres, R. Lozano, J. Melon, and R. Carraro Age-Dependent Decline of In Vitro Migration (Basal and Stimulated by IGF-1 or Insulin) of Human Vascular Smooth Muscle Cells J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2003; 58(12): B1074 - 1077. [Abstract] [Full Text] [PDF]

				L. Bergandi, F. Silvagno, I. Russo, C. Riganti, G. Anfossi, E. Aldieri, D. Ghigo, M. Trovati, and A. Bosia Insulin Stimulates Glucose Transport Via Nitric Oxide/Cyclic GMP Pathway in Human Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2215 - 2221. [Abstract] [Full Text] [PDF]

				E. Stabile, Y. F. Zhou, M. Saji, M. Castagna, M. Shou, T. D. Kinnaird, R. Baffour, M. D. Ringel, S. E. Epstein, and S. Fuchs Akt Controls Vascular Smooth Muscle Cell Proliferation In Vitro and In Vivo by Delaying G1/S Exit Circ. Res., November 28, 2003; 93(11): 1059 - 1065. [Abstract] [Full Text] [PDF]

				W. Wolfsgruber, S. Feil, S. Brummer, O. Kuppinger, F. Hofmann, and R. Feil A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells PNAS, November 11, 2003; 100(23): 13519 - 13524. [Abstract] [Full Text] [PDF]

				N. Kaplan-Albuquerque, C. Garat, C. Desseva, P. L. Jones, and R. A. Nemenoff Platelet-derived Growth Factor-BB-mediated Activation of Akt Suppresses Smooth Muscle-specific Gene Expression through Inhibition of Mitogen-activated Protein Kinase and Redistribution of Serum Response Factor J. Biol. Chem., October 10, 2003; 278(41): 39830 - 39838. [Abstract] [Full Text] [PDF]

				T. Sasaoka, K. Kikuchi, T. Wada, A. Sato, H. Hori, S. Murakami, K. Fukui, H. Ishihara, R. Aota, I. Kimura, et al. Dual Role of Src Homology Domain 2-Containing Inositol Phosphatase 2 in the Regulation of Platelet-Derived Growth Factor and Insulin-Like Growth Factor I Signaling in Rat Vascular Smooth Muscle Cells Endocrinology, September 1, 2003; 144(9): 4204 - 4214. [Abstract] [Full Text] [PDF]

				L. Ragolia, T. Palaia, E. Paric, and J. K. Maesaka Prostaglandin D2 Synthase Inhibits the Exaggerated Growth Phenotype of Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells J. Biol. Chem., June 6, 2003; 278(24): 22175 - 22181. [Abstract] [Full Text] [PDF]

				J. L. Hall, G. H. Gibbons, and J. C. Chatham IGF-I promotes a shift in metabolic flux in vascular smooth muscle cells Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E465 - E471. [Abstract] [Full Text] [PDF]

				E. A. Goncharova, A. J. Ammit, C. Irani, R. G. Carroll, A. J. Eszterhas, R. A. Panettieri, and V. P. Krymskaya PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L354 - L363. [Abstract] [Full Text] [PDF]

				B. Stiles, V. Gilman, N. Khanzenzon, R. Lesche, A. Li, R. Qiao, X. Liu, and H. Wu Essential Role of AKT-1/Protein Kinase B{alpha} in PTEN-Controlled Tumorigenesis Mol. Cell. Biol., June 1, 2002; 22(11): 3842 - 3851. [Abstract] [Full Text] [PDF]

				N. A. Bourbon, L. Sandirasegarane, and M. Kester Ceramide-induced Inhibition of Akt Is Mediated through Protein Kinase Czeta . IMPLICATIONS FOR GROWTH ARREST J. Biol. Chem., January 25, 2002; 277(5): 3286 - 3292. [Abstract] [Full Text] [PDF]

				Y. Ido, D. Carling, and N. Ruderman Hyperglycemia-Induced Apoptosis in Human Umbilical Vein Endothelial Cells: Inhibition by the AMP-Activated Protein Kinase Activation Diabetes, January 1, 2002; 51(1): 159 - 167. [Abstract] [Full Text] [PDF]

				B. Gonzalez, S. Lamas, and E. M. Melian Cooperation between Low Density Lipoproteins and IGF-I in the Promotion of Mitogenesis in Vascular Smooth Muscle Cells Endocrinology, November 1, 2001; 142(11): 4852 - 4860. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Jung, F. Articles by Dimmeler, S. Search for Related Content PubMed PubMed Citation Articles by Jung, F. Articles by Dimmeler, S. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics