GTMB 7 - Gene Therapy & Molecular Biology

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Chavakis et al: Leukocyte adhesion and statinsTable 1: Influence of lovastatin on integrin and uPAR expression.Receptors Control LovastatinCD11a 100+8.2 92.6+3.1CD11b 100+6.4 97.3+5.3CD18 100+12.9 110.7+9.1CD29 100+8.4 106.7+4.5uPAR 100+8.9 97.9+1.7The expression of CD11a, CD11b, CD18, CD29 and uPAR on U937 cells that were preincubated for 18 h in the absence or presence oflovastatin (40 µM) as measured by FACS-analysis is shown. The expression of the various integrins or uPAR is presented as percent ofcontrol, which relates to the expression of the respective adhesion molecule in the absence of lovastatin. Data are mean ± SEM (n=3) ofa typical experiment; similar results were obtained in three separate experiments.IV. DiscussionLeukocyte activation and adhesion to theendothelium and the subsequent transendothelial migrationare pivotal steps in the recruitment of cells to theinflammatory /injured tissue. This highly coordinatedmultistep process requires tight regulation of adhesiveevents (Carlos and Harlan, 1994; Springer, 1994)including the induction of genes coding for participatingadhesion receptors including integrins, their change inavidity as well as the modification of ligand-bindingproperties (Porter and Hogg, 1998; Woods and Couchman,2000). Conversely, in pathological situations associatedwith organ transplantation, atherosclerosis andischemia/reperfusion injury, arthritis and psoriasis theantagonism of these adhesive leukocytic interactions maybecome a promising therapeutic appproach (Nahakura etal, 1996; Issekutz, 1998; Kruegeret al, 2000; Martin et al,2000; Poston et al, 2000). In this respect, recent evidencepoints to an immunomodulatory role of statins (Katznelsonand Kobashigawa, 1995; Maron et al, 2000; Kwak andMach, 2001) which are commonly used to reduce plasmacholesterol levels in order to decrease the risk ofcardiovascular disease (Corsini et al, 1995). In this studywe define the direct and indirect role of statins inleukocyte adhesion and the possible underlyingmechanisms. Two distinct pathways, a HMG-CoAreductase-dependent and an –independent weredistinguished and appear to be relevant for theantiadhesive effects of statins.In particular, coincubation of monocytes withlovastatin resulted in a dramatic reduction of LFA-1-dependent cell adhesion to ICAM-1, but not of Mac-1-dependent adhesion to FBG or uPAR-dependent adhesionto VN. This direct antiadhesive effect of lovastatin wasunrelated to HMG-CoA reductase inhibition, as it was notreversed by mevalonate or other isoprenoid metabolites.Rather, it was attributed to the direct inhibition of theLFA-1/ICAM-1 interaction by lovastatin as corroboratedin a purified system. Whereas Mac-1 binding to its ligandsICAM-1 and FBG as well as uPAR interaction with VNwere not directly affected by lovastatin, binding of anotherβ2-integrin, p150.95, to FBG and ICAM-1 was partiallyblocked directly by lovastatin. Our data are in accordancewith and extend a recent report showing that statins inhibitLFA-1 by binding to an allosteric L-site located within theI-domain of the α chain (Weitz-Schmidt et al, 2001).Thus, lovastatin binds to LFA-1 as well as with loweraffinity to p150.95, but not to Mac-1, thereby directlyaffecting leukocyte adhesion.When lovastatin was preincubated with monocytesfor up to 18 h, a different inhibition profile was observed:Lovastatin completely blocked all three adhesive events,namely LFA-1/Mac-1-dependent adhesion to ICAM-1,Mac-1-dependent adhesion to FBG and uPAR-dependentadhesion to VN. Inhibition of ICAM-1-related adhesioncould be partially attributed to the direct LFA-1 bindingproperty of lovastatin, as (i) a significant inhibition by50% occured already after 2 h, and was not reversed bymevalonate and (ii) complete inhibition was observed afterlonger preincubation times (12-18 h) and could bepartially reversed by mevalonate up to the adhesion levelobtained after 2 h preincubation with lovastatin. Incontrast, both Mac-1- and uPAR-dependent cell adhesionwere partially inhibited after 6 h preincubation withlovastatin and were completely blocked after 12-18 h. Thiseffect of lovastatin was dependent on HMG-CoAreductase inhibition, as it was completely reversible in thepresence of mevalonate. Interestingly, the IC50 of theHMG-CoA reductase-dependent effect of lovastatin wasapproximately 1 µM, which is about 20 times (LFA-1) or70 times (p150.95) lower than the IC50 of the HMG-CoAreductase-independent direct abrogation of both integrinmediatedadhesion reactions. Thus, the antiinflammatoryaction of statins implied in clinical studies are very likelyattributable to the HMG-CoA reductase-dependentpathway, as the higher concentrations of statins requiredfor the direct inhibition of the LFA-1/ICAM-1-, thep150.95/FBG- and the p150.95/ICAM-1-interactions maynot be reached with the standard doses (nanomolar range)of approved statin drugs (Frenette, 2001). Indeed, a recentreport demonstrated that mevalonate-derived isoprenoidmetabolites mediate the antiinflammatory activity ofstatins in the in vivo air-pouch model of localinflammation (Diomede et al, 2001). Finally, the antiinflammatorycapacity of statins may vary dependent ontheir individual structure (Weitz-Schmidt et al, 2001).While direct binding to LFA-1 and p150.95sufficiently explains the HMG-CoA reductaseindependentantiadhesive effect of lovastatin, differentmechanisms might be involved in the HMG-CoAreductase-dependent anti-adhesive property of lovastatin:(i) Lowering the plasma membrane cholesterol content canaffect cell adhesion by disrupting lipid raft integrity(Krauss and Altevogt, 1999; Simons and Toomre, 2000).Recently, the assembly of adhesion complexes containing108
Gene Therapy and Molecular Biology Vol 7, page 109adhesion receptors as well as signaling molecules such asfocal adhesion kinase or src kinases has been proposed tobe confined to glycosphingolipid- and cholesterol-rich,detergent insoluble microdomains of the cell membrane.The antiadhesive effect of lovastatin preincubationpresented here could very well be due to raft disruption bycholesterol depletion, as other approaches to disrupt thesemembrane microdomains result in a very similardownregulation of β2-integrin and uPAR mediatedleukocyte adhesion (Chavakis et al., unpublishedobservations). Moreover, as lipid rafts have beenimplicated in T-cell receptor-, EGF-receptor-, insulinreceptor-, H-Ras-, eNOS- and integrin-dependentsignalling phenomena (Simons and Toomre, 2000), thepotential modulatory role of HMG-CoA-reductaseinhibitors on raft integrity and associated vital cellularfunctions renders these drugs very attractive for severaltherapeutic interventions in vascular medicine. (ii)Although conflicting results have been reported as to theinfluence of statins on the cell type specific integrin anduPAR expression (Weber et al, 1997; Liu et al, 1999;Wojeiak-Stothard, 1999; Yoschida et al, 2001), our dataare in accordance with these reports showing no change inintegrin expression in e.g. myelo-monocytic U937 cells bylovastatin (Weber et al, 1995; Liu et al, 1999). (iii) It hasbeen demonstrated that protein geranyl-geranylation isrequired for β1-integrin-dependent adhesion of leukocytes.It is thus conceivable that statin treatment may affectintegrin-dependent leukocyte adhesion via inhibition ofthe geranyl-geranylation of RhoA, which is thought to beone of the most important effectors involved in regulationof the cytoskeleton network, including the clustering ofadhesion molecules during monocyte adherence (Liu et al,1999; Wojciak-Stothard et al, 1999; Kwak and Mach,2001; Yoshida et al, 2001). The possibility that statintreatment could thereby directly inhibit RhoA activationand disrupt actin polymerization leading to failure ofintegrin clustering is a likely interpretation of thepresented data, since isoprenoid metabolites could reversethe antiadhesive effect of lovastatin pretreatment.Together, our findings help to decipher the mechanismsunderlying the postulated antiinflammatory effects ofstatins, which, besides atherothrombosis, may prove to bebeneficial in arthritis, organ transplantation or psoriasis.AcknowledgmentsThis work was supported in part by a grant from theNovartis Foundation for Therapeutical Research to TC andKTP (Nürnberg, Germany), by a grant from the DeutscheForschungsgemeinschaft to TC (CH279/1-1) and by agrant from Vascular Genomics-Kerckhoff Klinik GmbH toKTP (Bad Nauheim, Germany). We acknowledge thegenerous gift of reagents from Drs. D.B. Cines(Philadelphia, PA), G. Hoyer-Hansen and N. Behrendt(Copenhagen, Denmark), S. Bodary (San Francisco, CA)and J. Harlan (Seattle, WA) and Ms M. Economopouloufor help during manuscript preparation.ReferencesAikawa M, Rabkin E, Okada Y, Voglic SJ, Clinton SK,Brinckerhoff CE, Sukhova GK, Libby P (1998) Lipidlowering by diet reduces matrix metalloproteinase activityand increases collagen content of rabbit atheroma: a potentialmechanism of lesion stabilization. Circulation 97,2433–2444Blackford J, Reid HW, Pappin DJ, Bowers FS, Wilkinson JM(1996) A monoclonal antibody, 3/22 to rabbit CD11c whichinduces homotypic T cell aggregation: evidence that ICAM-1is a ligand for CD11c/CD18. Eur J Immunol 26, 525-531Carlos TM, Harlan JM (1994) Leukocyte-endothelial adhesionmolecules. Blood 84, 2068-2101Chavakis T, Kanse SM, Yutzy B, Lijnen HR, Preissner KT(1998) Vitronectin concentrates proteolytic activity on thecell surface and extracellular matrix by trapping solubleurokinase receptor-urokinase complexes. Blood 91, 2305-2312Chavakis T, May AE, Preissner KT, Kanse SM (1999) Molecularmechanisms of zinc-dependent leukocyte adhesion involvingthe urokinase receptor and β2-integrins. Blood 93, 2976-2983Chavakis T, Kanse SM, Lupu F, Hammes HP, Muller-Esterl W,Pixley RA, Colman RW, Preissner KT (2000) Differentmechanisms define the antiadhesive function of highmolecular weight kininogen in integrin- and urokinasereceptor-dependent interactions. Blood 96, 514-522Chavakis T, Kanse SM, Pixley RA, May AE, Isordia-Salas I,Colman RW, Preissner KT (2001) Regulation of leukocyterecruitment by polypeptides derived from high molecularweight kininogen. FASEB J 15 2365-2376Chavakis T, Hussain M, Kanse SM, Peters G, Bretzel RG, FlockJI, Herrmann M, Preissner KT (2002) Staphylococcus aureusextracellular adherence protein (Eap) serves as antiinflammatoryfactor by inhibiting the recruitment of hostleukocytes. Nature Medicine 8, 687-693Corsini A, Maggi FM, Catapano AL (1995) Pharmacology ofcompetitive inhibitors of HMG-CoA reductase. PharmacolRes 31, 9–27Diomede L, Albani D, Sottocorno M, Polentarutti N, Donati MB,Bianchi M, Fruscella P, Salmona M (2001) The in vivo antiinflammatoryeffect of statins is mediated by nonsterolmevalonate products. Arterioscler Thromb Vasc Biol 21,1327–1332Essig M, Nguyen G, Prie D, Escoubet B, Sraer JD, Friedlander G(1998) 3-Hydroxy-3-methylglutaryl coenzyme A reductaseinhibitors increase fibrinolytic activity in rat aorticendothelial cells: role of geranylgeranylation and Rhoproteins. Circ Res 83, 683–690Frenette PS (2001) Locking a leukocyte integrin with statins. NEngl J Med 345, 1419-1421Gahmberg CG (1997) Leukocyte adhesion: CD11/CD18integrins and intercellular adhesion molecules. Curr OpinCell Biol 9, 643-650Ganne F, Vasse M, Beaudeux JL, Peynet J, Francois A, MishalZ, Chartier A, Tobelem G, Vannier JP, Soria J, Soria C(2000) Cerivastatin, an inhibitor of HMG-CoA reductase,inhibits urokinase/urokinase-receptor expression and MMP-9secretion by peripheral blood monocytes-a possibleprotective mechanism against atherothrombosis. ThrombHaemost 84, 680-688Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A,Plaza JJ, Diaz C, Hernandez G, Edigo J (1998) 3-Hydroxy-3-109
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GTMB 7 - Gene Therapy & Molecular Biology

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