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(Circulation. 2001;103:2102.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Oxidized LDL Inhibits Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration by an Inhibitory Effect on the Akt/Endothelial Nitric Oxide Synthase Pathway

Emmanouil Chavakis, MD; Elisabeth Dernbach, BSc; Corinna Hermann, PhD; Ulrich F. Mondorf, MD; Andreas M. Zeiher, MD; Stefanie Dimmeler, PhD

From the Division of Molecular Cardiology and the Division of Nephrology (U.F.M.), Department of Internal Medicine IV, University of Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Dept of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de

	Abstract

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Background—Oxidized LDL (oxLDL) inhibits endothelial cell (EC) migration. Stimulating ECs with vascular endothelial growth factor (VEGF) leads to the activation of Akt/protein kinase B, which in turn activates endothelial nitric oxide synthase (eNOS) by phosphorylation on serine 1177. VEGF-induced cell migration is dependent on the generation of nitric oxide (NO). Therefore, we investigated whether oxLDL affects EC migration by an inhibitory effect on the Akt/eNOS pathway.

Methods and Results—During an in vitro "scratched wound assay," oxLDL dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells. Western blot analysis revealed that oxLDL dose- and time-dependently led to dephosphorylation and thus deactivation of Akt. Moreover, oxLDL inhibited the VEGF-induced generation of NO, as detected and quantified using a fluorescent NO indicator, 4,5-diaminofluorescein diacetate. Overexpression of a constitutively active Akt construct (Akt T308D/S473D) or a phosphomimetic eNOS construct (eNOS S1177D) almost completely reversed the inhibitory effect of oxLDL on VEGF-induced EC migration and NO generation.

Conclusions—Our data indicate that oxLDL-induced dephosphorylation of Akt, followed by impaired eNOS activation, reduces the intracellular level of NO and thereby inhibits VEGF-induced EC migration.

Key Words: endothelium • lipoproteins • growth substances

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Oxidized LDL (oxLDL) plays a major role during atherogenesis.¹ OxLDL affects the endothelium in several ways by inducing the expression of adhesion molecules on endothelial cells (ECs),^{2 3} stimulating EC apoptosis,^{4 5} and impairing endothelial vasodilator function.^{6 7} In addition, oxLDL and its components, such as lysophosphatidylcholine, inhibit EC migration in a nontoxic manner.^{8 9} However, the precise mechanism of the migration-inhibitory effect remains unclear.

Stimulating ECs with specific growth factors like vascular endothelial growth factor (VEGF),^{10 11} angiopoietin-1,^{12 13 14} or fluid shear stress¹⁵ leads to the activation of the serine/threonine kinase Akt/protein kinase B in a phosphatidyl-inositol-3-kinase–dependent manner. The activation of Akt involves its phosphorylation on threonine 308 and on serine 473 by 3-phosphoinositide–dependent kinase-1 and -2, respectively.¹⁶ Besides mediating cell survival¹⁶ in ECs, Akt activates endothelial nitric oxide synthase (eNOS) by phosphorylation on serine 1177.^{17 18} Phosphorylation of eNOS on serine 1177 via the Akt/protein kinase B kinase is used by VEGF and shear stress to enhance NO generation in a calcium-independent manner.^{17 18}

Importantly, recent studies suggest that NO is an essential mediator of EC migration and VEGF-induced angiogenesis. Inhibiting NOS suppresses the mitogenic and migratory effects of VEGF on ECs in vitro^{19 20} and the VEGF-induced angiogenic response in vivo.²⁰ Moreover, NO was absolutely required for ischemia-induced angiogenesis in a model of hindlimb ischemia.²¹ Finally, we and others have recently demonstrated that Akt mediates the VEGF-induced migration^{22 23 24} of ECs via activation of eNOS.²⁴ Therefore, we hypothesized that oxLDL inhibits EC migration by affecting the Akt/eNOS pathway.

	Methods

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Materials
Human LDL was isolated by ultracentrifugation. LDL was oxidized by incubation for 6 hours with CuSO₄ (10 mmol/L). The oxidation was detected by the FOXII assay. Native LDL showed a concentration of 16 µmol of hydrogen peroxide per mg protein, whereas oxLDL generated a concentration of 578 µmol of hydrogen peroxide per mg protein. A monoclonal antibody against phospho-Akt (Ser 473) was purchased from New England Biolabs. Recombinant VEGF-165 was purchased from Biomol, N^G-mono-methyl-L-arginine (LNMA) was obtained from Alexis, and 4,5-diaminofluorescein diacetate (DAF-DA) was obtained from Calbiochem.

Cell Culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Cell Systems/Clonetics and cultured in endothelial basal medium supplemented with hydrocortisone (1 µg/mL), bovine brain extract (3 µg/mL), gentamicin (30 µg/mL), amphotericin B (50 µg/mL), epidermal growth factor (10 µg/mL), and 10% FCS until the third passage. After detachment with trypsin, cells were grown on 6-cm dishes for at least 18 hours.

Assessment of Akt Phosphorylation
HUVECs were stimulated with several concentrations of oxLDL for 1 hour in the presence or absence of 10 ng/mL VEGF or with 5 µg/mL oxLDL for 1 to 3 hours. Cellular proteins were prepared and separated on SDS-PAGE gels, as described previously.^{15 17} Immunoblotting was performed with a murine monoclonal phospho-Akt (Ser473) antibody (clone 4E2; 1:1000). Immunodetection was accomplished with an anti-mouse secondary antibody (1:4000) and the enhanced chemiluminescence kit (Amersham). The blots were reprobed with an anti-actin antibody (1:2000) and quantified by densitometry.

Cell Migration Assay
To assess cell migration, the "scratch" wound assay was used.^{24 25} Briefly, HUVECs were grown on 6-cm dishes previously labeled with a traced line. The cell monolayer was scraped with a disposable rubber policeman to create a cell-free zone. Thereafter, cells were washed with medium and stimulated with VEGF and/or oxLDL. EC migration was quantified by measuring the width of the cell-free zone (distance between the edges of the injured monolayer) before and 48 hours after stimulation using a computer-assisted microscope (Zeiss) at 5 distinct positions (every 5 mm).

Detection of Apoptosis and Necrosis
Cell apoptosis was detected morphologically by using DAPI staining of cell nuclei. Briefly, cells were centrifuged at 800g for 10 minutes, fixed in 4% paraformaldehyde, and incubated with the DAPI reagent (0.2 µg/mL in 10 mmol/L Tris-HCl [pH 7], 10 mmol/L EDTA, and 100 mmol/L NaCl) for 20 minutes. Five hundred cells were counted, and the percentage of apoptotic cells was determined per total number of cells. To detect necrosis, lactate dehydrogenase (LDH) activity in the supernatant of the cells was measured using a kit from Boehringer, according to the manufacturer’s instructions.

Detection of NO Generation
To assess NO release from ECs, a membrane-permeable fluorescent NO indicator, DAF-DA, was used as described previously.^{26 27} For that purpose, DAF-DA (10 µmol/L) was added to the medium of the cells, and fluorescence images were taken after 5 minutes of incubation using a computer-assisted microscope (Zeiss). The extent of the green fluorescence was achieved by densitometric quantification using image analysis software (KS 300 3.0, Zeiss) and expressed as the mean cell fluorescence density per mean cell area in arbitrary units.

Plasmids and Transfections
The plasmid encoding eNOS was a kind gift from Dr Masaki Nakane (Abbott Laboratories, Abbott Park, Ill), and the bovine Akt was a gift from Dr Julian Downward (Imperial Cancer Research Fund, London, UK). The plasmids were subcloned in pcDNA3.1-myc-his vector and mutated as previously described.¹⁷ HUVECs were transfected with the respective plasmids and Superfect reagent (Qiagen), as previously described.^{17 24} The migration experiments were started 2 hours after transfection. Directly after stimulation and after 48 hours, cell motility was determined as described above. Control experiments demonstrated the expression of the plasmids from 12 to 48 hours after transfection. To assess NO generation, transfected ECs were used 24 hours after transfection. The transfection rate was 50% (transfected cellsx100/total cell number) in all experiments, as detected 24 hours after transfection of HUVECs with the pcDNA3.1-GFP vector.

Statistical Analysis
Data are expressed as mean±SEM from at least 3 independent experiments. Statistical analysis was performed by 1-way ANOVA (LSD test) for multiple testing. Probability values were considered significant at P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
OxLDL Inhibits VEGF-Induced Cell Migration
Stimulating ECs with 10 ng/mL VEGF significantly increased EC migration in the scratched wound assay (Figure 1A). OxLDL dose-dependently reduced the EC migration induced by VEGF (Figure 1A). A dose of 5 µg/mL oxLDL completely abolished the stimulatory effect of VEGF on cell migration (Figure 1A). Increasing the VEGF concentration to 100 ng/mL slightly but significantly reduced the inhibitory effect of oxLDL (Figure 1B).

View larger version (29K): [in this window] [in a new window]   Figure 1. A, Dose-dependent inhibition of VEGF-stimulated EC migration by oxLDL. HUVECs were scratched and stimulated with 10 ng/mL VEGF and indicated concentrations of oxLDL. After 48 hours, cell migration was detected as described in Methods. Data are mean±SEM, n=5, with *P<0.01 vs control and #P<0.05 vs VEGF. B, Effect of oxLDL (5 µg/mL) on EC migration induced by increasing VEGF concentrations. Data are mean±SEM, n=4, with *P<0.01 vs respective VEGF concentration and #P<0.05 vs 10 ng/mL VEGF+oxLDL. C, Effect of oxLDL on apoptosis rate of ECs. HUVECs were stimulated with oxLDL for 48 hours. Percentage of apoptotic cells (fragmentation of nucleus) was determined per total number of cells by using DAPI staining. Data are mean±SEM, n=4, with *P<0.01 vs control. D, Effect of oxLDL on LDH activity of supernatant of HUVECs. LDH activity was expressed in percent (extracellular LDH/total LDH x100). Data are mean±SEM, n=4.

Because oxLDL induces EC apoptosis,^{4 5} the apoptosis rate of ECs was determined to rule out the possibility that the inhibitory effect of oxLDL on cell migration was secondary to apoptosis induction. However, in this setting, oxLDL in concentrations <10 µg/mL did not increase the rate of EC apoptosis, although 10 µg/mL and 50 µg/mL oxLDL led to a significant increase in EC apoptosis (Figure 1C).

In addition, oxLDL concentrations up to 50 µg/mL did not induce an increase in extracellular LDH activity as an indicator of necrosis (Figure 1D). Thus, the effects of oxLDL on cell migration, which was significant at 5 µg/mL, cannot be attributed to the induction of cell death, suggesting a specific nontoxic effect of oxLDL on EC migration.

OxLDL Induces Dephosphorylation of Akt
To test our hypothesis that oxLDL influences Akt activity, HUVECs were incubated with oxLDL for 1 hour, and immunoblots were performed with a phosphospecific Akt antibody directed at the Ser 473 phosphorylation site, which correlates with the activity of Akt.^{15 28} OxLDL induced a dose-dependent dephosphorylation of Akt, with a 51±8% reduction at a dose of 5 µg/mL (Figure 2A). Moreover, dephosphorylation of Akt by 5 µg/mL oxLDL was time-dependent, with a maximal 63±9% reduction occurring at 3 hours after stimulation (Figure 2B). Because VEGF signals via the Akt pathway,¹⁰ we tested whether oxLDL could reduce Akt phosphorylation in the presence of VEGF. For this purpose, HUVECs were stimulated for 1 hour with 10 ng/mL VEGF and several doses of oxLDL. Indeed, as illustrated in Figure 2C, oxLDL led to a dose-dependent dephosphorylation of Akt under simultaneous stimulation with VEGF, with a 45±9% reduction at 5 µg/mL oxLDL.

View larger version (30K): [in this window] [in a new window]   Figure 2. Effect of oxidized LDL on Akt phosphorylation on Ser 473. A, Dose-dependent dephosphorylation of Akt by oxLDL. ECs were stimulated for 1 hour with indicated doses of oxLDL, and phosphorylation of Akt was determined with a phosphospecific Akt antibody. A representative blot from 4 independent experiments is shown. B, Time-dependent dephosphorylation of Akt by oxLDL. ECs were stimulated with 5 µg/mL oxLDL for indicated time periods, and phosphorylation of Akt was determined with a phosphospecific Akt antibody. A representative blot from 4 independent experiments is shown. C, Dose-dependent dephosphorylation of Akt by oxLDL in presence of VEGF. ECs were stimulated for 1 hour with indicated doses of oxLDL and 10 ng/mL VEGF, and phosphorylation of Akt was determined with a phosphospecific Akt antibody. A representative blot from 4 independent experiments is shown.

Effect of Phosphomimetic Akt and eNOS on oxLDL-Induced Inhibition of Cell Migration and NO Generation
The oxLDL-induced dephosphorylation of Akt associated with the inhibition of EC migration prompted us to investigate whether Akt dephosphorylation plays a causal role in the inhibitory effects of oxLDL on cell migration. Therefore, HUVECs were transfected with a phosphomimetic, constitutively active Akt mutant (T308D/S473D), which cannot become dephosphorylated and, therefore, inactivated. As illustrated in Figure 3A, overexpression of constitutively active Akt (T308D/S473D) was sufficient to stimulate EC migration, even in the absence of VEGF, to a level comparable to that seen on VEGF stimulation. In addition, VEGF stimulation of cells transfected with constitutively active Akt did not further increase cell migration (Figure 3A).

View larger version (26K): [in this window] [in a new window]   Figure 3. A, Effect of overexpression of a constitutively active Akt construct or a phosphomimetic eNOS construct on oxLDL-induced inhibition of VEGF-stimulated cell migration. HUVECs were transfected with a phosphomimetic eNOS construct (S1777D; P-eNOS), a constitutively active Akt mutant (T308D/S473; active Akt), or empty control vector (pcDNA3.1; vector). ECs were stimulated with oxLDL (5 µg/mL) and VEGF (10 ng/mL) where indicated and allowed to migrate for 48 hours. Migration was determined as described in Methods. Data are mean±SEM, n=4, with #P<0.01 vs vector+VEGF and *P<0.01 vs vector+oxLDL+VEGF. B, Restoration of oxLDL-induced impairment of NO release by overexpression of active Akt construct (T308D/S473D) or phosphomimetic eNOS construct (S1177D). HUVECs were also transfected with respective constructs of control vector (pcDNA3.1; vector). After 24 hours, ECs were preincubated with oxLDL (5 µg/mL) or LNMA (1 mmol/L) for 1 hour before stimulation with VEGF for 1 hour (10 ng/mL) as indicated. NO generation was determined as described in Methods. Data are mean±SEM, n=3, with *P<0.01 vs vector alone, #P<0.01 vs vector+VEGF, P<0.01 vs active Akt alone, **P<0.01 vs phosphomimetic eNOS, and +P<0.01 vs vector+oxLDL+VEGF.

Most important, although in vector-transfected cells VEGF-induced EC migration was significantly inhibited by oxLDL, overexpression of active Akt (T308D/S473D) almost completely blocked the oxLDL-induced inhibition of EC migration (Figure 3A).

Serine 1177 of eNOS is the functionally relevant acceptor amino acid for Akt-induced eNOS phosphorylation and activation.^{17 18} On replacement of serine 1177 of eNOS by aspartate (S1177D), eNOS becomes resistant to dephosphorylation (inactivation) and is constitutively activated, resulting in continuous, calcium-independent NO generation.^{17 18} Therefore, we investigated whether a phosphomimetic (constitutively active) eNOS construct (S1177D) inhibits the oxLDL effects on EC migration. Overexpression of the phosphomimetic eNOS construct (S1177D) in the absence of oxLDL significantly increased EC migration to the extent observed after VEGF stimulation (Figure 3A). Moreover, overexpression of the phosphomimetic eNOS construct (S1177D) completely restored the impaired migratory capacity of ECs treated with oxLDL, indicating that the phosphorylation events of the Akt-dependent amino acid within eNOS is of fundamental importance for oxLDL-induced inhibition of cell migration (Figure 3A).

Because it is well established that VEGF-stimulated EC migration and angiogenesis is NO-dependent,^{19 20 21} we investigated the influence of transfection with the phosphomimetic Akt construct (T308D/S473D) or the phosphomimetic eNOS construct (S1177D) on the effects of oxLDL on NO generation. In vector-transfected cells, VEGF significantly increased NO generation, whereas oxLDL significantly inhibited both the basal and the VEGF-stimulated NO release (Figure 3B). Adding LNMA inhibited the basal and the VEGF-induced NO generation (Figure 3B). Overexpression of constitutively active Akt (T308D/S473D) was sufficient to stimulate NO generation in the absence of VEGF to the extent observed on VEGF stimulation (Figure 3B). Most important, transfection with the phosphomimetic Akt construct almost completely blocked the oxLDL-induced inhibition of NO generation (Figure 3B). Furthermore, overexpression of the phosphomimetic eNOS construct (S1177D) in the absence of oxLDL significantly increased the generation of NO in ECs to a level comparable to that observed on VEGF stimulation (Figure 3B). Moreover, overexpression of the phosphomimetic eNOS construct (S1177D) completely restored the capacity of ECs treated with oxLDL to generate NO.

Taken together, these results demonstrate that the oxLDL-induced inhibition of VEGF-stimulated cell migration and NO generation requires the dephosphorylation of Akt and eNOS on Ser1177, suggesting that the inhibitory effect of oxLDL on EC migration is mediated by the inactivation of Akt and of eNOS, leading to reduced generation of NO.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
EC migration is crucial for angiogenesis and reendothelialization after denuding injuries of arteries. VEGF signaling stimulates the generation of NO, which is essential for VEGF-induced angiogenesis and EC migration.^{19 20 21} We²⁴ and others^{22 23} demonstrated that VEGF-induced cell migration is mediated by the Akt pathway and is dependent on the phosphorylation of Akt on Thr308/Ser473.²⁴ Moreover, Akt-mediated EC migration is dependent on subsequent phosphorylation of eNOS on Ser1177 and on NO generation.²⁴ However, a study by Morales-Ruiz et al²³ in another model of cell migration suggested that constitutively active Akt may affect additional NO-independent pathways to regulate cell migration.

In the present study, we provide evidence that oxLDL inhibits VEGF-induced cell migration through an effect on the Akt/eNOS pathway. In detail, we demonstrate that oxLDL induced the dephosphorylation of the Akt kinase on Ser473. Dephosphorylation of Akt on Ser473 leads to inactivation of Akt¹⁶ and may affect eNOS activity, because eNOS is a downstream target of Akt.^{17 18} Functionally, oxLDL completely inhibited the stimulatory effect of VEGF on EC migration, without inducing apoptosis or necrosis. These results are consistent with the observation of Murugesan et al⁸ that oxLDL inhibits EC migration in the presence of basic fibroblast growth factor in a nontoxic manner. Lysophosphatidylcholine, a component of oxLDL, also blocks the motility of ECs,⁹ and it inhibits basic fibroblast growth factor–induced EC motility by inhibiting the Ras/extracellular signal–regulated kinase pathway.²⁹ Moreover, oxLDL inhibits angiogenesis.³⁰ Interestingly, simvastatin, a lipid-lowering drug, was recently shown to activate the Akt/eNOS pathway and to promote angiogenesis.³¹ The scratched wound assay predominantly assesses EC migration, not proliferation, because BrdU-staining reveals only a very low proliferation rate in the migration zone.²⁴ Consequently, the effect of oxLDL in this assay can be mainly classified as an effect on EC migration. Furthermore, our results demonstrate that overexpression of a constitutively active, non-dephosphorylatable Akt construct (Akt T308D/S473D) or a phosphomimetic, non-dephosphorylatable eNOS construct (eNOS S1177D) completely reversed the oxLDL-induced inhibition of reendothelialization to the level observed under VEGF stimulation, establishing causality between the oxLDL-induced dephosphorylation of Akt and the oxLDL-induced inhibition of EC migration. Both phosphomimetic constructs could also reverse the inhibitory effect of oxLDL on VEGF-induced NO generation. Taken together, these results, in association with the NO dependence of the signaling pathways mediating the migratory effects of VEGF^{19 20 21} and Akt,²⁴ indicate that the oxLDL-induced dephosphorylation/inactivation of Akt on Ser473 and of eNOS on Ser1177 reduce the intracellular level of NO, thereby inhibiting VEGF-induced EC migration.

The mechanism by which oxLDL induces Akt dephosphorylation is unclear. Osmotic stress³² and ceramide³³ have been found to lead to Akt-dephosphorylation and inactivation by stimulating protein phosphatase 2A (PP2A)-like activity. Moreover, other stimuli such as tumor necrosis factor- and angiotensin II are also capable of leading to Akt-dephosphorylation in ECs.³⁴ It is possible that oxLDL leads to the activation of a serine/threonine phosphatase, which subsequently dephosphorylates Akt. Because it is well established that oxLDL leads to increased intracellular ceramide generation,⁵ it is conceivable that oxLDL could activate, via ceramide, a protein phosphatase with PP2A-like activity, which dephosphorylates Akt. Calyculin A (a PP1/PP2A inhibitor) and okadaic acid (a PP2A inhibitor) both induced maximal phosphorylation of Akt on serine 473 in HUVECs, suggesting that PP2A-like activity is involved in the dephosphorylation and inactivation of Akt in ECs (data not shown). Furthermore, it is possible that oxLDL activates a lipid phosphatase such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or SH2-domain–containing inositol 5-phosphatase-2 (SHIP-2), which inactivate the phospholipid products of phosphatidyl-inositol-3-kinase and block the activation of Akt upstream.³⁵ Further studies are required to elucidate the oxLDL-transduction pathway leading to Akt dephosphorylation in ECs. Finally, we cannot exclude the possibility that oxLDL may also, in addition to its effects on Akt phosphorylation, directly affect the phosphorylation of eNOS on Ser1177 via a specific phosphatase.

In summary, in this report we provide evidence that oxLDL blocks EC migration by inhibiting the Akt/eNOS pathway. This may be of clinical relevance for patients with hypercholesterolemia who undergo revascularization procedures. Gene therapy with VEGF constructs in such patients may not lead to a maximal angiogenetic response because the downstream signaling of VEGF is impaired. The finding that increasing VEGF concentrations at least in part ameliorates the impairment of EC migration by oxLDL suggests that the balance between VEGF and oxLDL may determine the migratory capacity of ECs. However, even high VEGF concentrations did not completely compensate for the reduction of EC migration by oxLDL. Therefore, it is conceivable that gene therapy with downstream targets of VEGF, such as a phosphomimetic eNOS construct, may be more effective in inducing neovascularization, because this construct potently induces EC migration to the extent observed under VEGF stimulation and is able to completely overcome the inhibition of the VEGF transduction pathway by oxLDL.

	Acknowledgments

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 335, B6). The authors thank Susanne Ficus, Christiane Mildner-Rihm, and Jutta Siegers for expert technical assistance.

Received September 13, 2000; revision received November 14, 2000; accepted November 16, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;340:115–126.[Free Full Text]

2. Khan BV, Parthasarathy SS, Alexander RW, et al. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995;95:1262–1270.

3. Liao L, Starzyk RM, Granger DN. Molecular determinants of oxidized low-density lipoprotein-induced leukocyte adhesion and microvascular dysfunction. Arterioscler Thromb Vasc Biol. 1997;17:437–444.[Abstract/Free Full Text]

4. Dimmeler S, Haendeler J, Galle J, et al. Oxidized low density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases: a mechanistic clue to the response to injury hypothesis. Circulation. 1997;95:1760–1763.[Abstract/Free Full Text]

5. Harada-Shiba M, Kinoshita M, Kamido H, et al. Oxidized low density lipoprotein induces apoptosis in cultured human umbilical venous endothelial cells by common and unique mechanisms. J Biol Chem. 1998;273:9681–9687.[Abstract/Free Full Text]

6. Kugiyama K, Kerns SA, Morrisett JD, et al. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990;344:160–162.[Medline] [Order article via Infotrieve]

7. Simon BC, Cunningham LD, Cohen RA. Oxidized low density lipoproteins cause contraction and inhibit endothelium-dependent relaxation in the pig coronary artery. J Clin Invest. 1990;86:75–79.

8. Murugesan G, Chisolm GM, Fox PL. Oxidized low density lipoprotein inhibits the migration of aortic endothelial cells in vitro. J Cell Biol. 1993;120:1011–1019.[Abstract/Free Full Text]

9. Murugesan G, Fox PL. Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein. J Clin Invest. 1996;97:2736–2744.[Medline] [Order article via Infotrieve]

10. Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway: requirement for Flk-1/KDR activation. J Biol Chem. 1998;273:30336–30343.[Abstract/Free Full Text]

11. 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]

12. Papapetropoulos A, Fulton D, Mahboubi K, et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem. 2000;275:9102–9105.[Abstract/Free Full Text]

13. Fujikawa K, de Aos Scherpenseel I, Jain SK, et al. Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells [published erratum appears in Exp Cell Res. 2000;255:133]. Exp Cell Res. 1999;253:663–672.

14. Kim I, Kim HG, Moon SO, et al. Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion. Circ Res. 2000;86:952–959.[Abstract/Free Full Text]

15. Dimmeler S, Assmus B, Hermann C, et al. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res. 1998;83:334–342.[Abstract/Free Full Text]

16. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999;13:2905–2927.[Free Full Text]

17. Dimmeler S, Fisslthaler B, Fleming I, et al. Activation of nitric oxide synthase in endothelial cells via Akt-dependent phosphorylation. Nature. 1999;399:601–605.[Medline] [Order article via Infotrieve]

18. Fulton D, Gratton JP, McCabe TJ, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601.[Medline] [Order article via Infotrieve]

19. Papapetropoulos A, Garcia-Cardena G, Madri JA, et al. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997;100:3131–3139.[Medline] [Order article via Infotrieve]

20. Ziche M, Morbidelli L, Choudhuri R, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99:2625–2634.[Medline] [Order article via Infotrieve]

21. Murohara T, Asahara T, Silver M, et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 1998;101:2567–2578.[Medline] [Order article via Infotrieve]

22. Radisavljevic Z, Avraham H, Avraham S. Vascular endothelial growth factor up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/nitric oxide pathway and modulates migration of brain microvascular endothelial cells. J Biol Chem. 2000;275:20770–20774.[Abstract/Free Full Text]

23. Morales-Ruiz M, Fulton D, 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]

24. Dimmeler S, Dernbach E, Zeiher AM. Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett. 2000;477:258–262.[Medline] [Order article via Infotrieve]

25. Tamura M, Gu J, Matsumoto K, et al. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science. 1998;280:1614–1617.[Abstract/Free Full Text]

26. Nakatsubo N, Kojima H, Kikuchi K, et al. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett. 1998;427:263–266.[Medline] [Order article via Infotrieve]

27. Igarashi J, Thatte HS, Prabhakar P, et al. Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci U S A. 1999;96:12583–12588.[Abstract/Free Full Text]

28. Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541–6551.[Medline] [Order article via Infotrieve]

29. Rikitake Y, Kawashima S, Yamashita T, et al. Lysophosphatidylcholine inhibits endothelial cell migration and proliferation via inhibition of the extracellular signal-regulated kinase pathway. Arterioscler Thromb Vasc Biol. 2000;20:1006–1012.[Abstract/Free Full Text]

30. Chen CH, Cartwright J Jr, Li Z, et al. Inhibitory effects of hypercholesterolemia and ox-LDL on angiogenesis-like endothelial growth in rabbit aortic explants: essential role of basic fibroblast growth factor. Arterioscler Thromb Vasc Biol. 1997;17:1303–1312.[Abstract/Free Full Text]

31. 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.[Medline] [Order article via Infotrieve]

32. Meier R, Thelen M, Hemmings BA. Inactivation and dephosphorylation of protein kinase B (PKB) promoted by hyperosmotic stress. Embo J. 1998;17:7294–7303.[Medline] [Order article via Infotrieve]

33. Schubert KM, Scheid MP, Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem. 2000;275:13330–13335.[Abstract/Free Full Text]

34. Hermann C, Assmus B, Urbich C, et al. Insulin-mediated stimulation of protein kinase Akt: a potent survival signaling cascade for endothelial cells. Arterioscler Thromb Vasc Biol. 2000;20:402–409.[Abstract/Free Full Text]

35. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A. 1999;96:4240–4245. [Abstract/Free Full Text]

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				T. Kinnaird, E. Stabile, S. Zbinden, M.-S. Burnett, and S. E. Epstein Cardiovascular risk factors impair native collateral development and may impair efficacy of therapeutic interventions Cardiovasc Res, May 1, 2008; 78(2): 257 - 264. [Abstract] [Full Text] [PDF]

				E. Chavakis, G. Carmona, C. Urbich, S. Gottig, R. Henschler, J. M. Penninger, A. M. Zeiher, T. Chavakis, and S. Dimmeler Phosphatidylinositol-3-Kinase-{gamma} Is Integral to Homing Functions of Progenitor Cells Circ. Res., April 25, 2008; 102(8): 942 - 949. [Abstract] [Full Text] [PDF]

				G. Carmona, E. Chavakis, U. Koehl, A. M. Zeiher, and S. Dimmeler Activation of Epac stimulates integrin-dependent homing of progenitor cells Blood, March 1, 2008; 111(5): 2640 - 2646. [Abstract] [Full Text] [PDF]

				J. Lu, W. Jiang, J.-H. Yang, P.-Y. Chang, J. P. Walterscheid, H.-H. Chen, M. Marcelli, D. Tang, Y.-T. Lee, W. S.L. Liao, et al. Electronegative LDL Impairs Vascular Endothelial Cell Integrity in Diabetes by Disrupting Fibroblast Growth Factor 2 (FGF2) Autoregulation Diabetes, January 1, 2008; 57(1): 158 - 166. [Abstract] [Full Text] [PDF]

				S.-E. Chow, Y.-C. Hshu, J.-S. Wang, and J.-K. Chen Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages J Appl Physiol, April 1, 2007; 102(4): 1520 - 1527. [Abstract] [Full Text] [PDF]

				E. Chavakis, A. Hain, M. Vinci, G. Carmona, M. E. Bianchi, P. Vajkoczy, A. M. Zeiher, T. Chavakis, and S. Dimmeler High-Mobility Group Box 1 Activates Integrin-Dependent Homing of Endothelial Progenitor Cells Circ. Res., February 2, 2007; 100(2): 204 - 212. [Abstract] [Full Text] [PDF]

				C.-H. Chen and J. P. Walterscheid Plaque Angiogenesis Versus Compensatory Arteriogenesis in Atherosclerosis Circ. Res., October 13, 2006; 99(8): 787 - 789. [Full Text] [PDF]

				F. J. Byfield, S. Tikku, G. H. Rothblat, K. J. Gooch, and I. Levitan OxLDL increases endothelial stiffness, force generation, and network formation J. Lipid Res., April 1, 2006; 47(4): 715 - 723. [Abstract] [Full Text] [PDF]

				L. DeMaio, M. Rouhanizadeh, S. Reddy, A. Sevanian, J. Hwang, and T. K. Hsiai Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H674 - H683. [Abstract] [Full Text] [PDF]

				J. L. Mehta, J. Chen, P. L. Hermonat, F. Romeo, and G. Novelli Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): A critical player in the development of atherosclerosis and related disorders Cardiovasc Res, January 1, 2006; 69(1): 36 - 45. [Abstract] [Full Text] [PDF]

				J. S. Isenberg, L. A. Ridnour, E. M. Perruccio, M. G. Espey, D. A. Wink, and D. D. Roberts Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner PNAS, September 13, 2005; 102(37): 13141 - 13146. [Abstract] [Full Text] [PDF]

				E. Lopez-Rivera, T. R. Lizarbe, M. Martinez-Moreno, J. M. Lopez-Novoa, A. Rodriguez-Barbero, J. Rodrigo, A. P. Fernandez, A. Alvarez-Barrientos, S. Lamas, and C. Zaragoza Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration PNAS, March 8, 2005; 102(10): 3685 - 3690. [Abstract] [Full Text] [PDF]

				I. Fleming, A. Mohamed, J. Galle, L. Turchanowa, R. P. Brandes, B. Fisslthaler, and R. Busse Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKC{alpha} Cardiovasc Res, March 1, 2005; 65(4): 897 - 906. [Abstract] [Full Text] [PDF]

				J. Huang, X.-L. Niu, A. M. Pippen, B. H. Annex, and C. D. Kontos Adenovirus-Mediated Intraarterial Delivery of PTEN Inhibits Neointimal Hyperplasia Arterioscler Thromb Vasc Biol, February 1, 2005; 25(2): 354 - 358. [Abstract] [Full Text] [PDF]

				E. Chavakis, A. Aicher, C. Heeschen, K.-i. Sasaki, R. Kaiser, N. El Makhfi, C. Urbich, T. Peters, K. Scharffetter-Kochanek, A. M. Zeiher, et al. Role of {beta}2-integrins for homing and neovascularization capacity of endothelial progenitor cells J. Exp. Med., January 3, 2005; 201(1): 63 - 72. [Abstract] [Full Text] [PDF]

				J. Sun and J. K. Liao Induction of Angiogenesis by Heat Shock Protein 90 Mediated by Protein Kinase Akt and Endothelial Nitric Oxide Synthase Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2238 - 2244. [Abstract] [Full Text] [PDF]

				G. Marsche, R. Heller, G. Fauler, A. Kovacevic, A. Nuszkowski, W. Graier, W. Sattler, and E. Malle 2-Chlorohexadecanal Derived From Hypochlorite-Modified High-Density Lipoprotein-Associated Plasmalogen Is a Natural Inhibitor of Endothelial Nitric Oxide Biosynthesis Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2302 - 2306. [Abstract] [Full Text] [PDF]

				R.-H. Zhou, M. Yao, T.-S. Lee, Y. Zhu, M. Martins-Green, and J. Y.-J. Shyy Vascular Endothelial Growth Factor Activation of Sterol Regulatory Element Binding Protein: A Potential Role in Angiogenesis Circ. Res., September 3, 2004; 95(5): 471 - 478. [Abstract] [Full Text] [PDF]

				C. Skurk, H. Maatz, H.-S. Kim, J. Yang, M. R. Abid, W. C. Aird, and K. Walsh The Akt-regulated Forkhead Transcription Factor FOXO3a Controls Endothelial Cell Viability through Modulation of the Caspase-8 Inhibitor FLIP J. Biol. Chem., January 9, 2004; 279(2): 1513 - 1525. [Abstract] [Full Text] [PDF]

				Z. S. Katusic, N. M. Caplice, and K. A. Nath Nitric Oxide Synthase Gene Transfer as a Tool to Study Biology of Endothelial Cells Arterioscler Thromb Vasc Biol, November 1, 2003; 23(11): 1990 - 1994. [Abstract] [Full Text] [PDF]

				Y.-T. Tai, K. Podar, N. Mitsiades, B. Lin, C. Mitsiades, D. Gupta, M. Akiyama, L. Catley, T. Hideshima, N. C. Munshi, et al. CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling Blood, April 1, 2003; 101(7): 2762 - 2769. [Abstract] [Full Text] [PDF]

				X. Peng, S. Haldar, S. Deshpande, K. Irani, and D. A. Kass Wall Stiffness Suppresses Akt/eNOS and Cytoprotection in Pulse-Perfused Endothelium Hypertension, February 1, 2003; 41(2): 378 - 381. [Abstract] [Full Text] [PDF]

				I. Shiojima and K. Walsh Role of Akt Signaling in Vascular Homeostasis and Angiogenesis Circ. Res., June 28, 2002; 90(12): 1243 - 1250. [Abstract] [Full Text] [PDF]

				R. S. Scotland, M. Morales-Ruiz, Y. Chen, J. Yu, R. D. Rudic, D. Fulton, J.-P. Gratton, and W. C. Sessa Functional Reconstitution of Endothelial Nitric Oxide Synthase Reveals the Importance of Serine 1179 in Endothelium-Dependent Vasomotion Circ. Res., May 3, 2002; 90(8): 904 - 910. [Abstract] [Full Text] [PDF]

				C. Urbich, E. Dernbach, A. M. Zeiher, and S. Dimmeler Double-Edged Role of Statins in Angiogenesis Signaling Circ. Res., April 5, 2002; 90(6): 737 - 744. [Abstract] [Full Text] [PDF]

				K. Tanaga, H. Bujo, M. Inoue, K. Mikami, K. Kotani, K. Takahashi, T. Kanno, and Y. Saito Increased Circulating Malondialdehyde-Modified LDL Levels in Patients With Coronary Artery Diseases and Their Association With Peak Sizes of LDL Particles Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 662 - 666. [Abstract] [Full Text] [PDF]

				C. Urbich, E. Dernbach, A. Reissner, M. Vasa, A. M. Zeiher, and S. Dimmeler Shear Stress-Induced Endothelial Cell Migration Involves Integrin Signaling Via the Fibronectin Receptor Subunits {alpha}5 and {beta}1 Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 69 - 75. [Abstract] [Full Text] [PDF]

				S. Fichtlscherer, L. Rossig, S. Breuer, M. Vasa, S. Dimmeler, and A. M. Zeiher Tumor Necrosis Factor Antagonism With Etanercept Improves Systemic Endothelial Vasoreactivity in Patients With Advanced Heart Failure Circulation, December 18, 2001; 104(25): 3023 - 3025. [Abstract] [Full Text] [PDF]

				Y.-M. Go, A.-L. Levonen, D. Moellering, A. Ramachandran, R. P. Patel, H. Jo, and V. M. Darley-Usmar Endothelial NOS-dependent activation of c-Jun NH2- terminal kinase by oxidized low-density lipoprotein Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2705 - H2713. [Abstract] [Full Text] [PDF]

				S. Blum, K. Issbruker, A. Willuweit, S. Hehlgans, M. Lucerna, D. Mechtcheriakova, K. Walsh, D. von der Ahe, E. Hofer, and M. Clauss An Inhibitory Role of the Phosphatidylinositol 3-Kinase-signaling Pathway in Vascular Endothelial Growth Factor-induced Tissue Factor Expression J. Biol. Chem., August 31, 2001; 276(36): 33428 - 33434. [Abstract] [Full Text] [PDF]

				J. Igarashi and T. Michel Sphingosine 1-Phosphate and Isoform-specific Activation of Phosphoinositide 3-Kinase beta . EVIDENCE FOR DIVERGENCE AND CONVERGENCE OF RECEPTOR-REGULATED ENDOTHELIAL NITRIC-OXIDE SYNTHASE SIGNALING PATHWAYS J. Biol. Chem., September 21, 2001; 276(39): 36281 - 36288. [Abstract] [Full Text] [PDF]

				K. Tanaga, H. Bujo, M. Inoue, K. Mikami, K. Kotani, K. Takahashi, T. Kanno, and Y. Saito Increased Circulating Malondialdehyde-Modified LDL Levels in Patients With Coronary Artery Diseases and Their Association With Peak Sizes of LDL Particles Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 662 - 666. [Abstract] [Full Text] [PDF]

				C. Urbich, E. Dernbach, A. M. Zeiher, and S. Dimmeler Double-Edged Role of Statins in Angiogenesis Signaling Circ. Res., April 5, 2002; 90(6): 737 - 744. [Abstract] [Full Text] [PDF]

				R. S. Scotland, M. Morales-Ruiz, Y. Chen, J. Yu, R. D. Rudic, D. Fulton, J.-P. Gratton, and W. C. Sessa Functional Reconstitution of Endothelial Nitric Oxide Synthase Reveals the Importance of Serine 1179 in Endothelium-Dependent Vasomotion Circ. Res., May 3, 2002; 90(8): 904 - 910. [Abstract] [Full Text] [PDF]

				A. Burger-Kentischer, H. Goebel, R. Seiler, G. Fraedrich, H. E. Schaefer, S. Dimmeler, R. Kleemann, J. Bernhagen, and C. Ihling Expression of Macrophage Migration Inhibitory Factor in Different Stages of Human Atherosclerosis Circulation, April 2, 2002; 105(13): 1561 - 1566. [Abstract] [Full Text] [PDF]

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