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(Circulation Research. 2002;90:420.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells

Implication of Rho-Family GTPases

S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, G. Friedlander

From INSERM U426 (S.L., D.L., M.E., I.R., G.F.), Faculté de Médecine Xavier Bichat, Université Paris 7; INSERM U538 (G.T.), Faculté de Médecine Saint-Antoine; and INSERM U460 (J.B.M.), Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France.

Correspondence to Séverine Ledoux, INSERM U426, Faculté Xavier Bichat, BP416, 16 rue Henri Huchard, 75870 Paris, Cedex 18, France. E-mail ledoux{at}bichat.inserm.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Extracellular adenosine production by the GPI-anchored Ecto-5'-Nucleotidase (Ecto-5'-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5'Nu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5'-Nu stimulation by HRIs, Ecto-5'-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5'-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5'-Nu and decreased endocytosis of Ecto-5'-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5'-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5'-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5'-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5'-Nu endocytosis through a decrease of Rho-GTPases isoprenylation.

Key Words: HMG-CoA reductase inhibitors • Ecto-5'-Nucleotidase • isoprenoids • Rho-GTPases • endocytosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many studies have demonstrated the major beneficial effects of HMG-CoA reductase inhibitors (HRIs) in cardiovascular diseases.^1–3 This benefit is already apparent at the early step of endothelial dysfunction, which precedes the development of atherosclerosis.^4,5 Some of the effects of these cholesterol-lowering drugs, particularly those observed in short-term exposure, could be supported by mechanisms independent of lowering of plasma LDL concentration.^1,6

Induction of adenosine production in the extracellular space by the GPI-anchored Ecto-5'-Nu could be one of these mechanisms. Indeed, the production of adenosine by Ecto-5'-Nu is implicated in the regulation of endothelial functions^7–9 and in defense against hypoxia.^9–12 Ecto-5'-Nu activity is increased by tissular hypoxia, notably in cardiomyocytes and endothelial cells.^13,14 The enzyme has been implicated in myocardial preconditioning,^13,15 and it was recently shown that HRIs potentiate the stimulation of Ecto-5'-Nu activity induced by ischemia in cardiomyocytes.¹⁶

However, it is not known whether HRIs also enhance Ecto-5'-Nu activity in endothelium, a main site of adenosine production,¹⁷ and the mechanisms whereby HRIs enhance Ecto-5'-Nu have not been studied. Some hypotheses can be put forward. First, it has been shown that HRIs can modulate the activity of enzymes, such as endothelial nitric oxide synthase, at the transcriptional level.⁶ On the other hand, HRIs can induce posttranslational events, which influence cell surface expression of proteins. Several studies^18,19 have pointed out the critical role of decreased cholesterol level in plasma membrane sorting of GPI-anchored proteins and in raft-mediated endocytosis.^20,21 By inhibition of isoprenoids synthesis, HRIs could also decrease the activity of Rho-GTPases, which are implicated in actin organization and clathrin-independent endocytosis.^22,23

In order to elucidate the mechanisms of enhancement of Ecto-5'-Nu activity by HRIs, we have studied the effect of lovastatin on Ecto-5'-Nu activity and expression in aortic endothelial cells. The results show that lovastatin enhances plasma membrane expression of Ecto-5'-Nu through posttranslational events and suggest that this effect could be mediated by an inhibition of Rho-GTPases isoprenylation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture products were from Gibco BRL (Cergy Pontoise, France); [¹⁴C]5'-AMP, ¹²⁵I-labeled goat anti-rabbit antibody was from Amersham (Amersham, UK). Lovastatin was from Merck Sharp and Dohme-Chibret and was converted to its active form as previously described.²⁴ Other products were from Sigma.

Endothelial Cells
SV 40 transfected aortic rat endothelial cells (SVARECs; J.B. Michel, INSERM U460, Paris, France) have been previously characterized.^24,25 Aortic rat primary endothelial cells were obtained as previously described.²⁶ All experiments were performed on confluent cells.

Determination of Ecto-5'-Nu Activity
The activity of Ecto-5'-Nu was determined on intact cells with [¹⁴C]5'-AMP as a substrate, as previously described.²⁷ ß-Methylene-ADP (ßMADP, 2x10^-4 mol/L) was added in 1 well for each experimental condition for determination of nonspecific activity. Cells were lysed by 0.1 N NaOH to determine protein content in each well by the Bradford technique modified for microtitration.²⁸

Inhibition of Platelet Aggregation
Inhibition of platelet aggregation was performed on platelet rich plasma (PRP) obtained from healthy volunteers as previously described.⁷ After a 18 hours preincubation without or with 25 µmol/L lovastatin, SVARECs were rinsed and then incubated for 10 minutes with 50 µmol/L AMP, without or with 2x10^-4 mol/L ßMADP, in the same conditions as for measurement of Ecto-5'-Nu activity.²⁷ Cell supernatants (200 µL) or 200 µL of buffer alone or containing 50 µmol/L adenosine or 50 µmol/L AMP (without preincubation with SVARECs) were added to 200 µL of PRP. Platelet activation was started by the addition of 3 µmol/L ADP and monitored by the increase of light transmission using an aggregometer (Chrono-log Corps).

Isolation of Detergent-Resistant Membranes
Isolation of detergent-resistant membranes (DRM) was performed by flotation on a sucrose gradient.²⁹ After ultracentrifugation of cell lysates in a SW41 rotor (Beckman instruments, Inc) at 120 000g for 18 hours, the DRM were visible at the interface between 5% and 35% sucrose layers. Twelve fractions of 1 mL were collected from top to bottom of the sucrose gradient. In some experiments, fractions 4 to 7 were pooled to quantify Ecto-5'-Nu expression in whole DRM.

Western Blot
Western blot analysis was performed as previously described.²⁷ Ecto-5'-Nu and Caveolin contents were determined with rabbit polyclonal anti-5'-Nu antibody (1:5000, kind gift of B. Kaissling, Zurich, Switzerland) and monoclonal anti-caveolin 1 antibody (1:1000, Transduction laboratories, Lexington, Kentucky), respectively. Actin expression was used as an internal standard.

Immunoquantification of Ecto-5'-Nu
Quantification of Ecto-5'-Nu membrane expression was performed on nonpermeabilized fixed cells (4% formaldehyde for 15 minutes at room temperature) incubated overnight at 4°C with polyclonal anti-5'-Nu antibody (1:1000). For studies of Ecto-5'-Nu endocytosis, nonfixed cells were incubated for 1 hour at 4°C with the same antibody, rinsed 3 times, and then fixed (for T₀) or preincubated without or with 25 µmol/L lovastatin for 18 hours at 37°C before being fixed. The binding of the primary antibody was quantified with ¹²⁵I-labeled goat anti-rabbit antibody (4x10⁵ cpm/well for 2 hours at room temperature).³⁰ Nonspecific binding was less than 10% of specific binding.

Fluorescent Staining of Ecto-5'-Nu and Actin
For Ecto-5'-Nu staining, cells grown on glass slides were fixed with formaldehyde and incubated overnight with polyclonal anti-5'-Nu antibody with the same procedure for immunoquantification and then incubated for 30 minutes at 37°C with TRITC-labeled goat anti-rabbit antibody (1:100). For actin staining, fixed cells were permeabilized with 0.05% saponin for 10 minutes and then incubated for 30 minutes with 0.1 µmol/L of phalloidin-TRITC. The cells were then mounted in Dako Faramount aqueous medium and examined by confocal fluorescent microscopy.

Determination of F-Actin Content
Total F-actin content was measured, as previously described,³¹ on cells fixed with 4% formaldehyde and permeabilized with 0.05% saponin. The fixed cells were incubated for 1 hour with 0.5 µmol/L phalloidin-TRITC and then washed with PBS. Bound phalloidin was extracted with methanol. Extracts were quantified in a spectrofluorometer with excitation and emission wavelengths of 485 and 535 nm, respectively.

Cholesterol Content Analysis
Cell lipids were extracted according to Bligh and Dyer³² and separated by thin-layer chromatography on silica gel plates. The cellular non-esterified cholesterol was assayed in the form of trimethylsilyl ether by gas chromatography.³³

RNase Protection Assay
RNase protection assay was performed with riboprobes for Ecto-5'-Nu and GAPDH as previously described.³⁴ The ratio of radioactivity for 5'-Nu to the radioactivity for GAPDH was calculated for each sample.

Data Presentation
Results are presented as mean±SEM. Statistical analyses were performed by using unpaired Student’s t test or by analysis of variance (ANOVA) when appropriate.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Lovastatin on Ecto-5'-Nu Activity
Ecto-5'-Nu activity measured in control conditions in SVARECs was 1.55±0.20 nmol/mg protein/min after a 5 minutes incubation with 50 µmol/L 5'-AMP. Lovastatin increased Ecto-5'-Nu activity in SVARECs. This effect was dependent on the concentration of lovastatin over the range of concentrations tested, from 3 to 25 µmol/L (Figure 1A). Simvastatin also increased Ecto-5'-Nu activity from 1.50±0.20 to 4.15±1.38 nmol/mg protein/min, after 18 hours incubation at 25 µmol/L (P<0.05). Stimulation of Ecto-5'-Nu activity became significant after 4 hours of incubation with lovastatin and was maximal at 8 hours (Figure 1B). Likewise, the reversion of lovastatin effect became significant after 4 hours, and Ecto-5'-Nu activity returned to control value at 8 hours (Figure 1B). For subsequent experiments we chose an 18-hour incubation period with 25 µmol/L lovastatin. As shown in Figure 2A, lovastatin increased V_max value without affecting apparent affinity (K_m) of Ecto-5'-Nu.

View larger version (29K): [in this window] [in a new window]   Figure 1. Effect of lovastatin on Ecto-5'-Nu activity. Ecto-5'-Nu activity was measured in SVARECs by incubating the cells with 50 µmol/L 5'-AMP for 5 minutes, after an 18-hour preincubation with different concentrations of lovastatin (lova) from 3 to 25 µmol/L (A). B, Ecto-5'-Nu activity was measured after an incubation period of 2 to 18 hours with 25 µmol/L lovastatin (hatched bars); for reversion studies, lovastatin was removed after an 18-hour incubation at 25 µmol/L, and fresh culture medium was added for an additional incubation of 2 to 8 hours (open bars). Results are expressed as mean±SEM of 3 different experiments. *P<0.05, **P<0.01 vs control; P<0.05, P<0.01 vs lovastatin for 18 hours.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of lovastatin on enzymatic parameters and synthesis of Ecto-5'-Nu. A, Cells were preincubated for 18 hours without (CT) or with 25 µmol/L lovastatin (lova) and then incubated for 5 minutes with different concentrations of 5'-AMP between 7.5 and 1000 µmol/L. Vmax and Km values were calculated after Eadie-Hoftee transformation (n=3). B, After incubation of SVARECs without (CT) or with 25 µmol/L lovastatin (lova) for 18 hours, Ecto-5'-Nu and GAPDH mRNA were quantified in SVARECs by RNAse protection assay. Results are expressed as arbitrary units (AU) of Ecto-5'-Nu/GAPDH mRNA ratio and are mean±SEM of 3 different experiments. C, Western blot analysis of Ecto-5'-Nu was performed in total cell membrane samples labeled with polyclonal anti-5'-Nu antibody (5'Nu: 70 kD) and monoclonal anti-actin antibody (actin: 35 kD); representative picture of 3 experiments.

In primary cultures of rat aortic endothelial cells, lovastatin also enhanced Ecto-5'-Nu activity (measured after 5 minutes incubation with 50 µmol/L 5'-AMP) from 0.68±0.07 pmol/mg protein/min in control conditions to 1.07±0.09 pmol/mg protein/min after 18 hours incubation with 25 µmol/L lovastatin (n=3, P<0.05).

Effect of Lovastatin on Ecto-5'-Nu Protein Synthesis
To evaluate whether the enhancement of Ecto-5'-Nu activity by lovastatin resulted from increased synthesis of the enzyme, mRNA and protein levels of Ecto-5'-Nu were assessed. Ecto-5'-Nu mRNA level, quantified by RNase protection assay, was not increased by lovastatin (Figure 2B). Instead, a small decrease of mRNA Ecto-5'-Nu/GAPDH ratio was observed after incubation with lovastatin but did not reach statistical significance. Analysis of Ecto-5'-Nu protein expression by Western blot in total membrane fractions (Figure 2C) did not show any difference between lovastatin and control conditions (Ecto-5'-Nu/actin ratio: 0.87±0.20 versus 1.01±0.43 for lovastatin-treated cells and controls, respectively, n=3, NS).

Effect of Lovastatin on Ecto-5'-Nu Expression to the Plasma Membrane
Cell surface expression of Ecto-5'-Nu was studied by immunoquantification. As shown in Figure 3A, fluorescent staining of Ecto-5'-Nu with specific polyclonal antibody was located exclusively at the plasma membrane and was enhanced by lovastatin. Quantification of plasma membrane Ecto-5'-Nu, using a ¹²⁵I-labeled secondary antibody, showed that lovastatin induced a 2-fold increase of Ecto-5'-Nu cell surface expression (Figure 3B). This effect was reproduced by 25 µmol/L simvastatin, which enhanced Ecto-5'-Nu cell surface expression to 276±53% of control (P<0.001).

View larger version (31K): [in this window] [in a new window]   Figure 3. Plasma membrane staining of Ecto-5'-Nu. After 18 hours of preincubation without (CT) or with 25 µmol/L lovastatin (lova), fixed cells were incubated overnight with polyclonal anti-5'-Nu antibody, and then stained with a FITC-conjugated secondary antibody for immunofluorescence study (A) or with a 123I-labeled anti-rabbit antibody for immunoquantification of Ecto-5'-Nu (B). Nonfixed cells were incubated with anti-5'-Nu antibody for 1 hour and then fixed (T0) or incubated for 18 hours with or without lovastatin before fixation. Endocytosis (hatched part of the columns) was estimated as Ecto-5'-Nu cell surface staining at T0 minus the staining that persists at plasma membrane after the 18-hour incubation (open part of the columns) (C). Results are mean±SEM of 3 different experiments. *P<0.05 vs control.

We performed subsequent experiments to determine whether this plasma membrane overexpression was supported by an enhancement of plasma membrane sorting or by a decrease of Ecto-5'-Nu endocytosis. Nonfixed cells were labeled with anti-5'-Nu antibody, before incubation with lovastatin, to allow internalization. The amount of labeled Ecto-5'-Nu that remained at the plasma membrane after 18 hours of incubation was about 2-fold higher in lovastatin-treated cells than in control condition (Figure 3C). This suggests that lovastatin induced a decrease of endocytosis of cell surface-exposed (labeled) Ecto-5'-Nu, rather than an increase of sorting of an intracellular (unlabeled) pool.

Because GPI-anchor proteins are mainly located in DRM,²⁹ we assessed the effect of lovastatin on Ecto-5'-Nu content in DRM. As shown in Figure 4, Ecto-5'-Nu expression in whole DRM and partition of the enzyme in sucrose gradient fractions were not significantly modified by lovastatin. Ecto-5'-Nu/actin ratio in DRM was 6.81±1.37 for lovastatin-treated cells and 6.01±1.49 for control cells (n=3, NS).

View larger version (32K): [in this window] [in a new window]   Figure 4. Ecto-5'-Nu expression in sucrose gradient fractions. After incubation of SVARECs without (CT) or with 25 µmol/L lovastatin (lova) for 18 hours, Western blot analyses of pooled DRM fractions (A) or of each sucrose gradient fraction (B) were performed with polyclonal anti-5'-Nu antibody (5'Nu: 70 kD), polyclonal anti-caveolin antibody (cav: 23 kD), and monoclonal anti-actin antibody (actin: 35 kD); representative picture of 3 experiments.

Role of Cholesterol and Mevalonate Pathway Products
Cholesterol membrane content was reported to be critical for plasma membrane expression of GPI-anchored proteins.^18,19 Methyl-ß-cyclodextrin, a chelator of cholesterol,^20,21 increased Ecto-5'-Nu activity (168.93±18.26% of control, P<0.05, n=5) and Ecto-5'-Nu membrane expression (216±28.29% of control, P<0.01, n=3) in SVARECs, after 18 hours incubation at 10 mmol/L. However, methyl-ß-cyclodextrin significantly reduced cholesterol content to 47.64±3.41% of control (P<0.001), whereas the decrease of cholesterol content after 18 hours incubation with 25 µmol/L lovastatin did not reach statistical significance (73.91±13.67% of control). Furthermore, addition of 0.5 mg/mL soluble cholesterol, which raised cholesterol content to 229.39±37.14% of control (P<0.05), did not reverse the stimulation of Ecto-5'-Nu activity induced by lovastatin (Figure 5).

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of mevalonate pathway products on Ecto-5'-Nu activity. SVARECs were incubated for 18 hours without (open bars) or with 25 µmol/L lovastatin (hatched bars) in the presence of 0.5 mg/mL soluble cholesterol (chol), or 500 µmol/L mevalonate (meva), or 15 µmol/L geranyl-geranyl pyrophosphate (GGP), or 15 µmol/L farnesyl pyrophosphate (FPP), prior to determination of Ecto-5'-Nu activity (50 µmol/L 5'-AMP for 5 minutes).

However, lovastatin stimulation of Ecto-5'-Nu was reversed by mevalonate (Figure 5), the product of HMG-CoA reductase.³⁵ We then tested the hypothesis that inhibition of isoprenoids synthesis is implicated in the regulation of Ecto-5'-Nu by lovastatin. Farnesyl-pyrophosphate (FPP) did not affect Ecto-5'-Nu stimulation, whereas geranyl-geranyl pyrophosphate (GGP) completely reversed lovastatin stimulation of Ecto-5'-Nu activity (Figure 5). Furthermore, GGP also reversed the enhancement of Ecto-5'-Nu cell surface expression induced by lovastatin (Figure 6).

View larger version (29K): [in this window] [in a new window]   Figure 6. Effect of modulation of Rho-GTPases on Ecto-5'-Nu activity and membrane expression. Cells were coincubated for 18 hours without (open bars) or with 25 µmol/L lovastatin (hatched bars), and with 1 µg/mL C3 exoenzyme (C3), 15 µmol/L geranyl-geranyl pyrophosphate (GGP), and 0.1 nmol/L cytotoxic necrotizing factor (CNF). Ecto-5'-Nu activity (A) was measured with 50 µmol/L 5'-AMP for 5 minutes. Immunoquantification of Ecto-5'Nu (B) was performed with polyclonal anti-5'-Nu antibody and a secondary 123I-labeled anti-rabbit antibody. Results are expressed as percent of control, and are mean±SEM of 3 different experiments. *P<0.05 vs control; P<0.05, P<0.01 vs lovastatin alone; and P<0.05, P<0.01 vs lovastatin plus GGP.

Implication of Rho-GTPases
Because one of the main actions of GGP is to activate Rho-GTPases, we tested whether modulation of Rho-GTPases activity affected Ecto-5'-Nu. As shown in Figure 6, C3 exoenzyme (kind gift of M.R. Popoff, Institut Pasteur, Paris, France), an inhibitor of Rho-GTPases,³⁶ partially reproduced the enhancement of Ecto-5'-Nu activity and membrane expression observed with lovastatin and abolished the reversion of lovastatin effect induced by GGP. Furthermore, CNF (kind gift of P. Boquet, INSERM, Nice, France), an activator of Rho-GTPases,³⁷ reversed the stimulation of Ecto-5'-Nu induced by lovastatin.

It was previously shown in our laboratory²⁴ that lovastatin affects actin organization in SVARECs through an inhibition of Rho-GTPases. Fluorescent staining of actin (Figure 7A) showed a rarefaction of stress fibers and irregularity of subcortical actin after incubation with lovastatin.

View larger version (42K): [in this window] [in a new window]   Figure 7. Effect of lovastatin on actin organization and actin content. After 18 hours of preincubation without (CT) or with 25 µmol/L lovastatin (lova), fixed cells were incubated with phalloidin-TRITC. Actin stress fibers (top) and subcortical actin (bottom) were examined by confocal fluorescent microscopy. Representative pictures of 5 experiments (A). F-actin content was quantified in a spectrofluorometer, after extraction of bound phalloidin-TRITC with methanol (B). Results are expressed as pmol of bound phalloidin (BPh) and are mean±SEM of 3 different experiments. *P<0.05, **P<0.01 vs control.

Quantification of bound phalloidin (Figure 7B) showed a decrease of F-actin content after lovastatin treatment. Furthermore, cytochalasin D, which disorganizes actin fibers, reproduced the stimulation of Ecto-5'-Nu activity observed with lovastatin (3.35 ±0.7 pmol/mg protein/min in cells treated with 10^-6 mol/L cytochalasin D for 18 hours versus 1.39±0.22 pmol/mg protein/min in controls, respectively, n=4, P<0.001).

Effect of Ecto-5'-Nu Stimulation on Platelet Aggregation
Conversion of AMP into adenosine by endothelial Ecto-5'-Nu has been implicated in inhibition of platelet aggregation.⁷ We then asked whether stimulation of Ecto-5'-Nu by lovastatin in SVARECs could affect platelet aggregation. Addition of 50 µmol/L adenosine solution to PRP decreased platelet aggregation induced by 3 µmol/L ADP, with a inhibition of 47.37±4.02% as compared with buffer alone (P<0.001). The 50 µmol/L AMP solution, added to PRP before cell incubation induced a weaker but significant inhibition of aggregation in our experimental condition (24.56±4.64%, P<0.05), which could reflect a degradation of AMP by PRP. As shown in Figure 8, preincubation of the AMP solution with lovastatin-treated cells significantly enhanced the inhibition of platelet aggregation as compared with control cells. Addition of 2x10^-4 mol/L ßMADP totally reversed the effect of cell preincubation on AMP-induced inhibition of platelet aggregation.

View larger version (27K): [in this window] [in a new window]   Figure 8. Effect of Ecto-5'-Nu stimulation on platelet aggregation. After 18 hours of preincubation without (CT) or with 25 µmol/L lovastatin (lova), cells were incubated with 50 µmol/L 5'-AMP for 10 minutes, without or with 2x10-4 mol/L ßMADP (+ß). Cell supernatants (200 µL) were added to 200 µL PRP. Aggregation, induced by addition of 3 µmol/L ADP, was quantified in an aggregometer. A, Representative picture of 2 separate experiments with duplicates for each condition. B, Results were expressed as percent of inhibition of the aggregation measured in presence of 200 µL of 50 µmol/L AMP, without cell preincubation. *P<0.05 vs control; P<0.05 vs lovastatin.

Effect of Lovastatin on Hypoxic Stimulation of Ecto-5'-Nu
Hypoxia enhanced Ecto-5'-Nu activity in endothelial cells.^13,14 In order to evaluate the putative role of lovastatin in adaptation to hypoxia, we asked whether lovastatin potentiates the effect of hypoxia on Ecto-5'-Nu activity in SVARECs. Incubation for 18 hours with an hypoxic gas mixture (0% O₂, 5% CO₂, 95% N₂) increased Ecto-5'-Nu activity from 1.06±0.15 pmol/mg protein/min in normoxic cells to 3.42±0.23 pmol/mg protein/min in hypoxic cells (n=3, P<0.01). Lovastatin, added at 25 µmol/L during the period of hypoxia, further increased Ecto-5'-Nu activity to 4.77±0.46 pmol/mg protein/min (P<0.05 versus hypoxia).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of this study was to elucidate the mechanisms by which HRIs enhance Ecto-5'-Nu activity. We found that lovastatin stimulation of Ecto-5'-Nu activity in endothelial cells was supported by an increase of the enzyme cell surface expression. Whereas synthesis of the enzyme was unaffected, lovastatin decreased Ecto-5'-Nu endocytosis, and activators of Rho-GTPases reversed the stimulation of Ecto-5'-Nu induced by lovastatin.

Ecto-5'-Nu activity measured in SVARECs was close to that reported in other endothelial cells.¹⁷ Lovastatin enhanced Ecto-5'-Nu activity in SVARECs in a dose-dependent manner. The concentrations of lovastatin used in this study were in the same range as the concentrations of HRIs previously reported to inhibit HMG-CoA reductase in endothelial cell lines,^6,24,31 and the effect of lovastatin was reproduced by simvastatin, another HMG-CoA reductase inhibitor. Furthermore, a nonspecific toxic effect was ruled out by the reversion of lovastatin effect by mevalonate, the product of HMG-CoA reductase.³⁵ In the same line, this effect could not be related to SV40 transfection because it was reproduced in endothelial cells grown in primary culture.

Stimulation of Ecto-5'-Nu by lovastatin resulted from an increase of the enzyme V_max without modification of its affinity. HRIs have been shown to regulate protein synthesis, such as nitric oxide synthase or tissue plasminogen activator, at the transcriptional level in endothelial cells.^6,24 However, the short delay of appearance of lovastatin effect, in our study, does not argue for a transcriptional mechanism. Furthermore, the absence of increase of Ecto-5'-Nu mRNA and protein levels after lovastatin treatment is strong evidence against a stimulation of de novo synthesis of the enzyme by lovastatin.

In contrast, lovastatin enhanced cell surface expression of Ecto-5'-Nu. The 2-fold increase assessed by immunolabeling was consistent with the increase of the enzyme V_max. Ecto-5'-Nu permanently cycles between cell surface and intracellular compartments with a half-time of about 4 hours.³⁸ The increase of cell surface expression of the enzyme could then result from a decrease of endocytosis or from an increase of recycling from the endocytic compartment.^18,38 Because the disappearance of cell surface Ecto-5'-Nu-specific antibody was reduced after incubation with lovastatin, our results rather suggested that lovastatin inhibits Ecto-5'-Nu endocytosis.

GPI-anchored proteins are endocytosed through a clathrin-independent pathway, which is dependent, as reported by other investigators,^20,21 on association of GPI-anchored proteins with membrane microdomains enriched in cholesterol, termed rafts or DRM. HRIs could inhibit Ecto-5'-Nu endocytosis by 2 separate mechanisms. First, several reports have pointed out the critical role of cholesterol content on plasma membrane expression of GPI-anchored proteins,^18,19 and it was hypothesized^19–21 that cholesterol depletion could reduce endocytosis of GPI-anchored proteins through disorganization of DRM. The enhancement by methyl-ß-cyclodextrin of Ecto-5'-Nu activity and plasma membrane expression in SVARECs was consistent with this hypothesis. However, in our experimental conditions, lovastatin only slightly decreased cell cholesterol content, as compared with methyl-ß-cyclodextrin. Furthermore, lovastatin-induced stimulation of Ecto-5'-Nu activity was not reversed by addition of soluble cholesterol (or LDL-cholesterol, data not shown). Along the same line, we did not observe any significant modification of Ecto-5'-Nu expression in DRM after lovastatin treatment. These data argue against a major influence of cholesterol depletion on the effect of lovastatin.

Alternatively, lovastatin could decrease Ecto-5'-Nu endocytosis through inhibition of Rho-GTPases isoprenylation. These small G proteins are implicated in actin organization^39,40 and, by this way, can modulate clathrin-independent endocytosis.^22,23,41 Mevalonate is a common substrate for the synthesis of cholesterol and isoprenoids such as GGP and FPP,³⁵ and it has been well demonstrated that HRIs decrease the synthesis of isoprenoids, notably in endothelial cells, at concentrations close to those used in our study.^6,24 Whereas FPP did not influence lovastatin effect, GGP, which is implicated in isoprenylation of Rho-GTPases,⁶ completely reversed the activation of Ecto-5'-Nu in SVARECs. Furthermore, we observed that the reversion by GGP of lovastatin effect on Ecto-5'-Nu was abolished by C3 exoenzyme, an inhibitor of Rho-GTPase.^6,22,36 Finally, CNF, a bacterial toxin that activates Rho-GTPases,^22,37 blocked the enhancement of Ecto-5'-Nu activity and membrane expression induced by lovastatin. These results strongly suggest that modulation of Rho-GTPases plays a role in lovastatin effect.

As previously shown in our laboratory,²⁴ lovastatin inhibits actin stress fibers formation in SVARECs. Furthermore, cytochalasin D, a disruptor of actin fibers,^6,20,21 mimicked the stimulation of Ecto-5'-Nu activity by lovastatin. These results are concordant with the hypothesis that inhibition of Rho-GTPases isoprenylation by lovastatin reduced Ecto-5'-Nu endocytosis through actin disorganization. It has been previously shown that cytochalasin D inhibits raft-mediated endocytosis²¹ and, more specifically, endocytosis of GPI-anchored proteins.^42,43 We provide the first report of an implication of Rho-GTPases in the regulation of GPI-anchored protein endocytosis.

The preventive effect of HRIs on atherosclerosis lesions is well demonstrated,^1–3 and it has been shown that HRIs act before the development of atherosclerosis^4,5 by restoring endothelial functions. It is now clear that the beneficial effects of HRIs on cardiovascular events are not fully accounted for by their ability to lower plasma cholesterol and that other mechanisms are very likely.^1,6,24 Adenosine, the product of Ecto-5'-Nu, is implicated in the regulation of endothelial functions, such as adaptation of blood supply to metabolic demand, vascular permeability, and interaction of circulating cells with endothelium.⁹ Endothelial Ecto-5'-Nu has been notably implicated in promotion of endothelial barrier function during inflammation⁸ and inhibition of platelet aggregation by intravascular nucleotides.⁷ Here we show that stimulation of Ecto-5'-Nu by lovastatin enhanced inhibition of platelet aggregation by endothelial cells. Adenosine also seems to be crucial for adaptation to hypoxia,^9,11,12 notably in the cardiovascular system,^13–15 and hypoxic stimulation of Ecto-5'-Nu could be protective against hypoxic lesions.^10,13,15,44 Pravastatin, at a dose which did not normalize serum cholesterol level, increased Ecto-5'-Nu activity in rabbit myocardial ischemia in parallel with the decrease of infarct size.¹⁶ Here we confirm that hypoxia also enhances Ecto-5'-Nu activity in endothelial cells, and that lovastatin potentiates the hypoxic stimulation of Ecto-5'-Nu. It is therefore attractive to speculate that stimulation of Ecto-5'-Nu could be one of the mechanisms that mediate protective effects of HRIs on the cardiovascular system.

In conclusion, our results show that lovastatin enhances Ecto-5'-Nu activity in endothelial cells. Stimulation of Ecto-5'-Nu by lovastatin results from enhancement of cell surface expression of the enzyme, which could be related to a decrease of Ecto-5'-Nu endocytosis. In vivo, additional effects of HRIs on Ecto-5'-Nu, through lowering cholesterol, cannot not be ruled out. However, our results provide evidence that lovastatin stimulates Ecto-5'-Nu through an alternative pathway, which implicates modulation of Rho-GTPases activity. Upregulation of Ecto-5'-Nu by cholesterol-lowering drugs could contribute to their protective effect on cardiovascular events.

	Acknowledgments

 
Financial support for this study was provided by CRIT and CEGETEL. We thank K. Koumanov (Inserm U538, Paris, France) for cholesterol measurements and F. Adam (Inserm E9907, Paris, France) for his technical assistance for measurements of platelet aggregation.

Received September 20, 2001; revision received January 14, 2002; accepted January 14, 2002.

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