Mol Cell Biochem (2008) 308:209217 DOI 10.

1007/s11010-007-9630-3

Atorvastatin accelerates extracellular nucleotide degradation in human endothelial cells

Lana Osman Mohamed Amrani Charles Ilsley Magdi H. Yacoub Ryszard T. Smolenski

Received: 10 April 2006 / Accepted: 18 October 2007 / Published online: 25 December 2007 Springer Science+Business Media, LLC. 2007

Abstract HMG-CoA reductase inhibitors (statins) exert pleiotropic effects in the cardiovascular system beyond its cholesterol-lowering action. We aimed to investigate how atorvastatin affects extracellular nucleotide degradation in human endothelial cells, as increased activity of this pathway would facilitate conversion of pro-inammatory nucleotides into anti-inammatory adenosine. Primary cultures of human endothelial cells were treated with 1 lM, 10 lM and 100 lM atorvastatin for 24 h. Enzyme assays were performed as well as intact cell studies, to evaluate capacity of cells to degrade ATP to adenosine. Atorvastatin signicantly increased ATP breakdown and adenosine formation in the medium of intact cells in a dose-dependent manner. The activities of ATPase, ADPase and ecto-50 -nucleotidase (eN) in cell homogenates following Atorvastatin treatment were also increased while no change was observed in the lactate dehydrogenase activity. We suggest a new mechanism of protective effect of atorvastatin by activation of endothelial enzymes involved in extracellular nucleotide degradation in human endothelial cells.

Keywords Atorvastatin Adenosine Endothelium Ecto-50 -nucleotidase Nucleotide degradation enzymes

Introduction Atorvastatin inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme responsible for the synthesis of cholesterol [14]. It exerts well established benecial effects in cardiovascular disease [5, 6]. However, atorvastatin seems to be a pleiotropic drug exerting other effects independent of lowering the cholesterol concentration. These effects plays an important role in the early step of endothelial dysfunction, which proceeds to the development of atherosclerosis [7, 8]. The mechanisms underlying the pleiotropic effects of statins are not completely understood, but several studies have suggested that statins may induce apoptosis, inhibit cell cycle progression, reduce the proliferation of vascular smooth muscle cells (VSMCs) [911], decrease inammatory processes [1214], increase the production of NO in endothelial cells by the upregulation of endothelial cell NO synthase (ecNOS) expression [15], reduce the production of endothelin-1 [16] and benecially inuence the coagulation processes [17]. Above processes, in particular, regulations of inammation or thrombosis are predominantly controlled by endothelium. Major focus of research on cellular effects of statins was on vascular smooth muscle cells and only few studies addressed the effect of these drugs in endothelium despite obvious relevance to cardiovascular pathology. An important mechanism in maintaining the endothelial function is the degradation of extracellular purine nucleotides. Adenosine is an important intermediate of purine

L. Osman M. H. Yacoub R. T. Smolenski (&) Heart Science Centre, Imperial College at Hareeld Hospital, Hareeld, Middlesex UB9 6JH, UK e-mail: r.smolenski@imperial.ac.uk M. Amrani Department of Cardiothoracic Surgery, Royal Brompton and Hareeld NHS Trust, Hareeld, Middlesex UB9 6JH, UK C. Ilsley Department of Cardiology, Royal Brompton and Hareeld NHS Trust, Hareeld, Middlesex UB9 6JH, UK

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nucleotide metabolism with effects that are often opposite to ATP or ADP. It is known that increased activity of the enzymes involved in formation of adenosine has protective effects on ischemia-induced endothelial injury, maintenance of endothelial barrier function, support vasodilation and suppresses leukocyte adhesion to the vascular endothelium [1821]. These anti-inammatory effects are realised through Gs-protein coupled adenosine receptors [22]. Ecto-ATPDase/CD39 is the key enzyme responsible for the pro-inammatory nucleotide breakdown of ATP and ADP into AMP [2325], and ecto-50 -nucleotidase (eN) is another key enzyme responsible for the formation of anti-inammatory and immunosuppressive adenosine from extracellular nucleotides [26, 27]. Previous studies reported a 6-fold increase in CD39 mRNA after exposure of HUVEC to atorvastatin [28], however no work was carried out to assess the functional effect of atorvastatin on the enzyme activities involved in extracellular nucleotides degradation. In this study, we performed a comprehensive biochemical analysis of the effect of atorvastatin on extracellular ATP dephosphorylation to adenosine in human endothelial cells. We were able to conrm that atorvastatin enhances this pathway, which identies new benecial protective effects of atorvastatin in the human endothelium independent of lowering the cholesterol.

microvascular endothelial cells (HMEC-1) were cultured in MCDB131 media containing the same amount of supplements for HUVEC, but containing only 10% FCS. HMEC1 were allowed to grow and be plated on to a 24-well plate for the experiment [29].

Treatment of human endothelial cells with atorvastatin, mevalonate and geranylgeranyl pyrophosphate Atorvastatin was dissolved in 100% DMSO and was further diluted with fresh appropriate media to prepare 1 lM, 10 lM and 100 lM concentrations. The volume of DMSO never exceeded 0.1% (v/v) in all prepared concentrations. Conuent cultures of HUVEC and HMEC-1 were then treated with atorvastatin at the concentrations indicated and were incubated for 24 h at 37C. Control cells were also cultured for 24 h in the appropriate media containing 0.1% DMSO, but in the absence of atorvastatin. Mevalonate and GGP were dissolved in water and 100 lM concentration was used to treat the cells.

Intact cell studies of nucleotide dephosphorylation Immediately after treatments, culture medium in the 24-well plate was removed and the cultured cells were washed twice with warm HANKs balanced salt solution (HBSS) (2 ml/well), followed by an addition of 1 ml of HBSS containing 5 lM deoxycorfomycin (dCf), an inhibitor of adenosine deaminase. Cells were then incubated for 30 min at 37C. Substrate concentrations of 10 lM of ATP or AMP containing 10 lM of AA, an inhibitor of adenosine kinase, were added to the cell medium and 50 ll aliquots of the incubation medium were collected at 0, 5, 15 and 30 min of incubation. The remaining medium was then completely removed and cells were prepared for enzyme assays. Incubation medium was analysed using the HPLC method as prescribed in detail previously [30].

Materials and methods Materials Atorvastatin (calcium salt) was kindly provided by Pzer (UK). Cell culture media M199, antibiotics, gelatine, foetal calf serum, endothelial cell growth supplements, heparin, HANKs Balanced Salt Solution (HBSS), DMSO, ATP, AMP, 50 -amino-50 -deoxyadenosine (AA), deoxycoformycin (dCf), ADP, adenosine, inosine, mevalonate (as mevalonolactone) and geranylgeranyl pyrophosphate ammonium salt (GGP) were all purchased from Sigma, UK. Cell culture media MCDB131 was purchased from GIBCO, UK.

Enzyme assays Cell culture Human umbilical vein endothelial cells (HUVEC) were grown in gelatine-coated tissue culture asks at 37C in a humidied environment containing 5% CO2. Complete M199 media was supplemented with 4 mM L-glutamine, 100 U/ml penicillin, 100 lM streptomycin, 15 ng/ml EC growth supplements, 5 ng/ml heparin and 20% FCS. HUVEC at passage 4 were used to grow in this experiment and were plated on to a gelatine-coated 24-well plate. Human To determine adenosine metabolizing enzyme activities, cultures of HUVEC and HMEC-1 were lysed in homogenisation buffer containing 0.1% Triton. To maximise cell recovery, adherent cells were scraped off using the end of a pipette tip and further aliquots of homogenisation buffer were collected. Enzyme activities of ATPase, ADPase, eN and PNP were determined by diluting the prepared cell homogenates with the appropriate incubation buffer. The enzyme reactions were initiated by the addition of 20 ll of cell homogenate and incubating it with 20 ll of the

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appropriate substrate at 37C. Substrate concentrations for ATPase and ADPase were 50 lM ATP and 50 lM ADP respectively, in the presence of the adenosine deaminase inhibitor, EHNA. For the measurement of eN 0.2 mM AMP was used in the presence of 5 lM EHNA in the substrate solution and for PNP 1 mM inosine was used as substrate. All enzyme reactions were terminated at 5 min by the addition of 20 lM of 1.3 M HCLO4. Samples were then neutralised with *9 ll of 3 M K3PO4, centrifuged and were nally separated by reverse-phase high performance liquid chromatography (HPLC). The HPLC method used to determine ATP, ADP, AMP, adenosine, inosine and hypoxanthine in the cell homogenates was described in detail previously [31]. Lactate dehydrogenase activity was also measured in the cell homogenates. This was done by measuring the rate of production of NAD+ at 340 nm, at 30C temperature [31]. Total protein content of cell homogenates was assayed using the Bradford reagent kit from Sigma [32].

HUVEC pre-treated with the highest atorvastatin concentration (100 lM) was signicantly (two-fold) increased to 269.8 34.8 nmol/min/mg protein in comparison to the non-treated cells 93.2 41.1 nmol/min/mg protein. Adenosine formation was markedly increased (about 30%) in HUVEC in the presence of 100 lM atorvastatin in comparison to control from 51.4 0.5 to 74.8 6.1 nmol/min/mg protein. For all samples a two way ANOVA for repeated measures performed revealed statistically signicant (P \ 0.05 vs. control) group 9 time interaction.

Effect of atorvastatin exposure on the extracellular AMP breakdown To further study the mechanism of the effect of atorvastatin on adenosine formation, HUVEC were exposed to different concentrations of atorvastatin ranging from 1 to 100 lM for 24 h, followed by incubation with 10 lM AMP. The rate of conversion of AMP into adenosine was analysed by HPLC at 0, 5, 15 and 30 min time points. Figure 2 shows AMP breakdown and adenosine formation in HUVEC pretreated with atorvastatin demonstrating a concentrationdependent effect. AMP breakdown increased two-fold following 100 lM atorvastatin pre-treatment in comparison to control, accompanied by an acceleration in adenosine formation at 1 lM, 10 lM and 100 lM by the factor of 2, 4 and 8, respectively. All results following atorvastatin pre-treatment showed a signicant difference (P \ 0.05) in comparison to control, as shown by two-way ANOVA for repeated measures.

Determination of cell viability To determine possible cytotoxic effects of atorvastatin, mevalonate and GGP, lactate dehydrogenase (LDH) release to the culture medium was used as an index of cell injury. This has been performed as described for the cell homogenates and results were expressed as percent of total cell activity of LDH.

Statistical analysis Results are presented as mean SEM. Statistical analyses were performed using one way or two way ANOVA for repeated measures when appropriate. All statistical analyses were performed using GraphPad Prism TM software (version 3.0).

Effect of atorvastatin on the ATP breakdown in HUVEC lysates Following 24 h atorvastatin exposure of HUVEC cultures, ATPase, ADPase, eN and PNP activities were measured in HUVEC homogenates. A signicant increase in enzyme activity was observed following 24 h pre-treatment with atorvastatin. Figure 3 illustrates the increase in ATP metabolizing enzyme activity in a concentration-dependent manner in HUVEC cultures pre-treated with atorvastatin. In the absence of atorvastatin ATPase activity in HUVEC homogenates was low (0.21 0.02 nmol/min/mg protein, n = 8) in comparison to 100 lM pre-treated cells (0.60 0.05 nmol/min/mg protein, n = 8). However, an increase in ATPase activity was also observed in the 1 lM and 10 lM atorvastatin pre-treated endothelial cells in comparison to control (Fig. 3a).

Results Effect of atorvastatin on the extracellular ATP breakdown When HUVEC were pre-treated with atorvastatin for 24 h at concentrations between 1 and 100 lM, a marked increase was observed in the rate of ATP conversion to its metabolites ADP, AMP and adenosine (Fig. 1). Pre-incubation of HUVEC with atorvastatin caused a progressive ATP breakdown and formation of reaction products ADP to AMP and AMP to adenosine in a dose-dependent manner. The rate of extracellular ATP breakdown in

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212 Fig. 1 Effect of atorvastatin pre-treatment on the pattern of ATP breakdown in HUVEC medium. HUVEC in culture were incubated with 10 mM ATP following pre-treatment without (lled circle) or in the presence of 1 lM atorvastatin (open circle), 10 lM atorvastatin (lled triangle), or 100 lM atorvastatin (open triangle) for 24 h. Aliquots of medium were collected at 0, 5, 15 and 30 min of incubation and subsequently analysed by HPLC for ATP breakdown (a) and formation of ADP (b), AMP (c) and adenosine (d). Values are expressed as the means SEM, n = 4. *P \ 0.001, #P \ 0.01, P \ 0.05 vs. Control (closed circle)
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Mol Cell Biochem (2008) 308:209217

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Fig. 2 Effect of atorvastatin pre-treatment on 10 mM extracellular AMP breakdown by HUVECs. Cultured cells were pre-treated for 24 h in the absence (lled circle) or presence of 1 lM atorvastatin (open circle), 10 lM atorvastatin (lled triangle), or 100 lM atorvastatin (open triangle). Aliquots of medium were collected

following addition of AMP and were analysed by HPLC for AMP breakdown (a) and adenosine formation (b). Values are expressed as the mean SEM, n = 4. *P \ 0.001, #P \ 0.01, P \ 0.05 vs. Control (closed circle)

ADPase activity in HUVEC homogenates increased two-fold in the 100 lM atorvastatin pre-treated cells, whereas a low activity was seen in the lower atorvastatin concentrations used (Fig. 3b). Treatment of HUVEC with 1-100 lM atorvastatin also induced a marked increase in eN activity in cell homogenates. A signicant increase was observed when HUVEC were pre-treated with 10 and 100 lM atorvastatin by 2- and 4-folds respectively in comparison to the non-treated cells (Fig. 3c). Control eN activity measured in HUVEC homogenates was 2.5 0.23 nmol/min/mg protein in comparison to 8.47 0.41 nmol/min/mg protein after

24 h incubation with 100 lM atorvastatin (P \ 0.001 vs. control). Surprisingly, purine nucleoside phosphorylase (PNP) activity in HUVEC was also affected by the presence of atorvastatin. Under control conditions, the PNP activity was much lower (0.64 0.4 nmol/min/mg protein), while it was signicantly increased to 6.78 0.3, 8.09 0.85 and 9.93 0.52 nmol/min/mg protein after treatment with 1, 10 and 100 lM of atorvastatin, respectively (P \ 0.001 vs. control). PNP activity progressively increased up to ve-fold following exposure to the highest concentration of atorvastatin (Fig. 3d).

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Mol Cell Biochem (2008) 308:209217 Fig. 3 Effect of pre-treatment of HUVECs with atorvastatin for 24 h on the activities of ATPase (a), ADPase (b), E50 N (c) and PNP (d) in cell homogenates. Separate incubations were carried out for 5 min with the appropriate substrate and enzyme activities were immediately assayed and analysed by HPLC. Lactate dehydrogenase (LDH) activity (e) was also determined in all samples. Values are expressed as mean SEM (n = 8). *P \ 0.001 vs. Control
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Effect of atorvastatin exposure on the ATP breakdown in HMEC-1 homogenates To further conrm the effect of atorvastatin on the enhancement of ATP metabolizing enzyme activities in endothelial cells, we performed similar experiment with HMEC-1 cell line. Cultures of HMEC-1 were exposed to the same concentrations of atorvastatin used previously and were incubated for 24 h. Figure 4 demonstrate an upregulation of the activity of enzymes involved in ATP catabolism following pre-treatment with atorvastatin occurring in a dose-dependent manner. ATPase activity was two-fold increased in cells pre-treated with 100 lM atorvastatin as compared to control (Fig. 4a). This was accompanied by a signicant increase in ADPase activity from 0.34 0.03 to 0.67 0.03 nmol/min/mg protein after 24 h pre-treatment with the highest dose of atorvastatin (P \ 0.05 in comparison to control) (Fig. 4b). Figure 4c demonstrates a signicant increase in eN activity

following 24 h atorvastatin pre-treatment in comparison to control from 6.38 0.23 to 10.24 1.12 nmol/min/mg protein (n = 8, P \ 0.05 vs. control). As with HUVEC, PNP activity was also signicantly increased following pre-treatment with atorvastatin from 3.51 0.39 to 10.3 0.63 nmol/min/mg protein (n = 8, P \ 0.05 vs. control), (Fig. 4d).

Effect of mevalonate, geranylgeranyl pyrophosphate and atorvastatin on ecto-50 -nucleotidase activity Treatment of HMEC-1 with mevalonate or GGP reversed the effect of atorvastatin on eN activity (Fig. 5a). The eN activity in HMEC-1 homogenates increased after treatment with atorvastatin from 48.2 12.4 to 109.4 10.5 nmol/ min/mg protein (n = 4, P \ 0.05 vs. control). This increase was signicantly reduced by mevalonate treatment to 60.9 3.7 nmol/min/mg protein. Cells treated with

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214 Fig. 4 Effect of pre-treatment of HMEC-1 cells with atorvastatin for 24 h on the enzymes of purine nucleotide catabolism in cell homogenates. Activities of ATPase (a), ADPase (b), E50 N (c) and PNP (d) were determined after 5 min incubation of cell homogenates with the appropriate substrate. LDH activity (e) was also analysed in all samples. Values are means SEM (n = 8). *P \ 0.001 vs. Control
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GGP revealed a lower eN activity (79.8 4.6 nmol/min/ mg protein) in comparison the atorvastatin treatment, but did not signicantly reverse the effect of atorvastatin.

Effect of atorvastatin on lactate dehydrogenase activity in the cells and its release into the medium As can be seen from (Figs. 3e, 4e, and 5b), lactate dehydrogenase activity in both HUVEC and HMEC-1 did not change under any conditions studied. Release of lactate dehydrogenase from cells into the medium was not observed under any conditions studied.

Discussion This study demonstrates that increased extracellular nucleotide degradation could be yet another unknown pharmacological effect of atorvastatin. Analysis of two

different endothelial cell types exposed to atorvastatin for 24 h revealed an increased catalytic capacity to degrade extracellular ATP and generate adenosine. This nding was conrmed by analysis of both dephosphorylation of extracellular ATP by intact cells and enzyme activities in cell homogenates. The increase in eN activity by atorvastatin was reversed with mevalonate and partially by GGP treatment, indicating that this effect is directly related to inhibition of HMG-CoA reductase pathway. Nucleotides are known to be predominantly compartmentized inside the cell, but several specic and unspecic mechanisms that have been described lead to release of nucleotides into extracellular space. A cascade of extracellular enzymes predominantly expressed on the surface of endothelial cells is responsible for its sequential dephosphorylation: ATP ? ADP ? AMP ? adenosine [3335]. The rst two steps are catalyzed by single enzyme: ecto-ATPDase also known as CD39. The last step of AMP hydrolysis to adenosine can be attributed to the GPI-anchored ecto-50 -nucleotidase (eN/CD73) [36].

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Fig. 5 Effect of pre-treatment of HMEC-1 homogenates with atorvastatin, mevalonate and GGP on the eN activity after 24 h of treatment (a). Enzyme activity was determined after 5 min incubation of cell homogenates with the appropriate substrate. LDH activity (b) was also analysed in all samples. Values are means SEM (n = 4). *P \ 0.05 vs. Control, #P \ .0.05 vs. atorvastatin treatment

A number of studies demonstrated that extracellular adenosine is a potent immunosuppressive, anti-inammatory and anti-aggregatory agent [20]. These effects are mediated predominantly by A2A receptors and increased cAMP concentration within target cells such as polymorphonuclear leucocytes, lymphocytes, NK cells [37, 38]. The A2A adenosine receptor and hypoxia-inducible factor have been shown to play important roles in the attenuation of pro-inammatory process, which could potentially have clinical implications [39]. Interestingly, nucleotides are potent pro-inammatory triggers. ADP is also most potent inducer of platelet aggregation. These effects are mediated by array of nucleotide receptors classied as P2. The increase process of extracellular ATP conversions to adenosine was demonstrated by intact assays, where the rate of ATP breakdown and adenosine formation in the medium was dramatically increased HUVEC pre-treated with atorvastatin for 24 h. It should be noted that relatively low concentrations of ATP metabolites were observed in the absence of atorvastatin in HUVEC and that there was no signicant change in the enzyme activities after 3 h

pre-treatment with atorvastatin. Furthermore, an increased activity of purine nucleoside phosphorylase (PNP) in the endothelium was also observed in the presence of atorvastatin. PNP is the enzyme of nucleotide degradation that converts inosine into hypoxanthine. The signicance of the increase in PNP activity merits further investigation since the role of PNP in ischemic heart has not yet been claried, although it may be involved in regulation of the intracellular phosphate concentration that is one of the substrates of the enzyme. In all conditions, LDH activity was not detectable in the medium and remained unchanged in the cell homogenates. Light microscopic examinations revealed no sign of cell damage or change in cell morphology in the cultures. Although direct benecial effects of atorvastatin on the endothelium have been previously reported, the mechanisms involved remain poorly characterized. It is therefore important to investigate further, the mechanism of atorvastatin effect on extracellular nucleotide degradation. Others studies demonstrated that statins increase nitric oxide (NO) production by stimulating and upregulating endothelial NO synthase (eNOS) [15]. It does so by inhibiting HMG-CoA reductase and blocking the synthesis of isoprenoids and cholesterol. The isoprenoid intermediate; geranylgeranyl pyrophosphate (GGP), is an important lipid attachment for the small GTP-binding protein Rho. Studies have shown that by using inhibitors of Rho geranylgeranylation such as geranylgeranyl transferase inhibitor (GGTi), clostridium botulinum C3 transferase and Rho kinase inhibitors, the eNOS expression in the endothelium would increase [40]. Since GGP is an intermediate in the cholesterol biosynthesis pathway, it is thought that the pleiotropic effects of statins are mediated by its ability to block the production of GGP and cause an upregulation in eNOS. The ability of statins to prolong eNOS half-life is very important under conditions that down-regulate eNOS expression such as hypoxia, effects of oxidized LDL and cytokines including tumor-necrosis factor-a (TNF-a) [41]. Moreover, it has been previously shown that lovastatin enhances eN and membrane expression in rat endothelial cells, by an inhibition of ecto-50 -nucleotide endocytosis through a decrease of Rho-GTPases isoprenylation [42]. Other reports also proposed that simvastatin inhibits the prenylation of the Rho family GTPases [43]. These small GTPases act as molecular switches controlling various cellular events, including actomyosin rearrangement. This revealed further benecial effects of simvastatin by decreasing the actin stress bre formation, which subsequently leads to passive relaxation of the cytoskeleton [44]. These studies reveal the importance of the Rho in regulating intracellular enzymatic targets including endothelial NOS and protecting the cytoskeleton arrangement. Therefore, with regards to our data the increase in the endothelial

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Mol Cell Biochem (2008) 308:209217 10. Laufs U, Marra D, Node K, Liao JK (1999) 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced downregulation of p27(Kip1). J Biol Chem 274:2192621931 11. Negre-Aminou P, van Vliet AK, van Erck M, van Thiel GC, van Leeuwen RE, Cohen LH (1997) Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitors; comparison with other human cell types. Biochim Biophys Acta 1345:259268 12. Bustos C, Hernandez-Presa MA, Ortego M, Tunon J, Ortega L, Perez F, Diaz C, Hernandez G, Egido J (1998) HMG-CoA reductase inhibition by atorvastatin reduces neointimal inammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 32:20572064 13. Kothe H, Dalhoff K, Rupp J, Muller A, Kreuzer J, Maass M, Katus HA (2000) Hydroxymethylglutaryl coenzyme A reductase inhibitors modify the inammatory response of human macrophages and endothelial cells infected with Chlamydia pneumoniae. Circulation 101:17601763 14. Takeuchi S, Kawashima S, Rikitake Y, Ueyama T, Inoue N, Hirata K, Yokoyama M (2000) Cerivastatin suppresses lipopolysaccharide-induced ICAM-1 expression through inhibition of Rho GTPase in BAEC. Biochem Biophys Res Commun 269: 97102 15. Laufs U, La Fata V, Plutzky J, Liao JK (1998) Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97:11291135 16. Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J, SanchezPascuala R, Hernandez G, Diaz C, Lamas S (1998) Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J Clin Invest 101:27112719 17. Essig M, Nguyen G, Prie D, Escoubet B, Sraer JD, Friedlander G (1998) 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase brinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins. Circ Res 83:683690 18. Cronstein BN (1994) Adenosine, an endogenous anti-inammatory agent. J Appl Physiol 76:513 19. Ely SW, Berne RM (1992) Protective effects of adenosine in myocardial ischemia. Circulation 85:893904 20. Ohta A, Sitkovsky M (2001) Role of G-protein-coupled adenosine receptors in downregulation of inammation and protection from tissue damage. Nature 414:916920 21. Spychala J (2000) Tumor-promoting functions of adenosine. Pharmacol Ther 87:161173 22. Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413492 23. Plesner L (1995) Ecto-ATPases: identities and functions. Int Rev Cytol 158:141214 24. Zimmermann H (1996) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 49:589618 25. Zimmermann H (1999) Two novel families of ectonucleotidases: molecular structures, catalytic properties and a search for function. Trends Pharmacol Sci 20:231236 26. Resta R, Yamashita Y, Thompson LF (1998) Ecto-enzyme and signaling functions of lymphocyte CD73. Immunol Rev 161:95109 27. Zimmermann H (1992) 5-Nucleotidase: molecular structure and functional aspects. Biochem J 285(Pt 2):345365 28. Morikawa S, Takabe W, Mataki C, Kanke T, Itoh T, Wada Y, Izumi A, Saito Y, Hamakubo T, Kodama T (2002) The effect of statins on mRNA levels of genes related to inammation, coagulation, and vascular constriction in HUVEC. Human umbilical vein endothelial cells. J Atheroscler Thromb 9:178183

extracellular degradation by atorvastatin could occur through modulation of Rho-GTPases activity. The acceleration in extracellular nucleotide catabolism caused by atorvastatin could occur through its inhibitory effect on the GGP metabolism. In summary, our results provide evidence for the stimulatory effect of atorvastatin on extracellular nucleotide breakdown and production of protective adenosine. This was caused by an increase in the ecto-enzyme activities responsible for the breakdown of ATP (ATPase), ADP (ADPase) and AMP (eN) in the human endothelial cells. These ndings further support the ability of atorvastatin to protect endothelial function under pathological conditions. Although the major effect of atorvastatin is to lower cholesterol and other lipid concentrations, its effect on nucleotide metabolism may contribute to the overall benet of statin therapy.
Acknowledgements This work was supported by the Magdi Yacoub Institute (MYI) and the Royal Brompton & Hareeld NHS Trust. RTS is Senior Lecturer at Department of Biochemistry, Medical University of Gdansk, Poland.

References
1. Bocan TM, Mueller SB, Brown EQ, Lee P, Bocan MJ, Rea T, Pape ME (1998) HMG-CoA reductase and ACAT inhibitors act synergistically to lower plasma cholesterol and limit atherosclerotic lesion development in the cholesterol-fed rabbit. Atherosclerosis 139:2130 2. Burnett JR, Wilcox LJ, Telford DE, Kleinstiver SJ, Barrett PH, Newton RS, Huff MW (1997) Inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apolipoprotein B production in miniature pigs. Arterioscler Thromb Vasc Biol 17:25892600 3. Howard BV, Howard WJ (1994) Dyslipidemia in non-insulindependent diabetes mellitus. Endocr Rev 15:263274 4. Yee HS, Fong NT (1998) Atorvastatin in the treatment of primary hypercholesterolemia and mixed dyslipidemias. Ann Pharmacother 32:10301043 5. Altieri DC (2001) Statins benets begin to sprout. J Clin Invest 108:365366 6. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, Macfarlane PW, McKillop JH, Packard CJ (1995) Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group. N Engl J Med 333:13011307 7. Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P (1995) The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med 332:488493 8. Treasure CB, Klein JL, Weintraub WS, Talley JD, Stillabower ME, Kosinski AS, Zhang J, Boccuzzi SJ, Cedarholm JC, Alexander RW (1995) Benecial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N Engl J Med 332:481487 9. Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernandez G, Egido J (1998) 3-Hydroxy-3methylglutaryl coenzyme a reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res 83:490500

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Mol Cell Biochem (2008) 308:209217 29. Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identication by morphologic and immunologic criteria. J Clin Invest 52:27452756 30. Smolenski RT, Lachno DR, Ledingham SJ, Yacoub MH (1990) Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation. J Chromatogr 527:414420 31. Wilson AC, Cahn RD, Kaplan NO (1963) Functions of the two forms of lactic dehydrogenase in the breast muscle of birds. Nature 197:331334 32. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254 33. Gordon EL, Pearson JD, Dickinson ES, Moreau D, Slakey LL (1989) The hydrolysis of extracellular adenine nucleotides by arterial smooth muscle cells. Regulation of adenosine production at the cell surface. J Biol Chem 264:1898618995 34. Meghji P, Pearson JD, Slakey LL (1995) Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart. Biochem J 308(Pt 3):725731 35. Pearson JD, Carleton JS, Gordon JL (1980) Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture. Biochem J 190:421429 36. Yegutkin G, Bodin P, Burnstock G (2000) Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5-nucleotidase along with endogenous ATP from vascular endothelial cells. Br J Pharmacol 129:921926 37. Caldwell CC, Kojima H, Lukashev D, Armstrong J, Farber M, Apasov SG, Sitkovsky MV (2001) Differential effects of

217 physiologically relevant hypoxic conditions on T lymphocyte development and effector functions. J Immunol 167:61406149 Lukashev D, Caldwell C, Ohta A, Chen P, Sitkovsky M (2001) Differential regulation of two alternatively spliced isoforms of hypoxia-inducible factor-1 alpha in activated T lymphocytes. J Biol Chem 276:4875448763 Lukashev D, Ohta A, Sitkovsky M (2004) Targeting hypoxiaA(2A) adenosine receptor-mediated mechanisms of tissue protection. Drug Discov Today 9:403409 Takemoto M, Liao JK (2001) Pleiotropic effects of 3-hydroxy-3methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol 21:17121719 Laufs U, La Fata V, Liao JK (1997) Inhibition of 3-hydroxy-3methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated down-regulation of endothelial nitric oxide synthase. J Biol Chem 272:3172531729 Ledoux S, Laouari D, Essig M, Runembert I, Trugnan G, Michel JB, Friedlander G (2002) Lovastatin enhances ecto-5-nucleotidase activity and cell surface expression in endothelial cells: implication of rho-family GTPases. Circ Res 90:420427 Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J, SanchezPascuala R, Hernandez G, Diaz C, Lamas S (1998) Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J Clin Invest 101:27112719 Jacobson JR, Dudek SM, Birukov KG, Ye SQ, Grigoryev DN, Girgis RE, Garcia JG (2004) Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. Am J Respir Cell Mol Biol 30:662670

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39.

40.

41.

42.

43.

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