atherogenesis is an inflammatory process in the vessel wall involving inflammatory cells like monocytes , macrophages , and cd4-positive lymphocytes [ 1 , 2 ] .   in early atherogenesis , cd4-positive lymphocytes are attracted by chemotactic proteins such as rantes and sdf-1 and enter the vessel wall as nave th0 cells . in the subendothelium , these cells then encounter antigens like oxidized ldl and differentiate into th1 cells , subsequently releasing proinflammatory mediators like tnf- and interferon- ( ifn ) . these cytokines then govern the inflammatory response in the vessel wall by activating other cells such as endothelial cells , macrophages , and vascular smooth muscle cells , thus promoting the inflammatory process in atherogenesis . moreover , experimental studies have shown that a reduction in cd4-positive lymphocyte recruitment hampers lesion development and plaque formation [ 3 , 4 ] . still , most of these studies targeted the effect of t - cell - specific chemokines , but hitherto little is known about modulatory effects on cd4-positive lymphocyte migration . epidemiological studies have shown that elevated heart rate represents a risk factor for cardiovascular morbidity both in primary prevention and in patients with hypertension , coronary artery disease , and myocardial infarction [ 58 ] . increased heart rate and reduced heart rate variability have been shown to be associated with coronary plaque rupture and subclinical inflammation in healthy middle - aged and elderly subjects [ 9 , 10 ] .   selective heart rate ( hr ) reduction by i(f)-channel inhibition is a recently developed pharmacological principle in cardiovascular therapy . among these newly identified hr - lowering drugs , i(f)-channel inhibition mainly reduces hr , thereby improving myocardial oxygen supply , energy balance , and cardiac function . ivabradine was well tolerated and revealed a good safety profile in the investigated study populations . recent data have shown that treatment with ivabradine reduces oxidative stress , improves endothelial function , and prevents atherosclerosis in apolipoprotein e - deficient mice . in this study , ivabradine treatment leads to a reduction in oxidative stress and to a potent downregulation of mcp-1 expression in atherosclerotic plaques , causing less inflammation and decreased lesion size . nothing is known about the direct effect of ivabradine on chemokine - induced migration . therefore , the current study examined the effect of ivabradine on cd4-positive lymphocyte migration and on intracellular signaling molecules involved . human cd4-positive lymphocytes were isolated from freshly drawn blood of healthy volunteers by ficoll - histopaque ( sigma - aldrich , germany ) gradient centrifugation to obtain mononuclear cells ( pbmcs ) and subsequent negative selection of cd4-positive t cells by magnetic bead separation ( miltenyi biotec , germany ) as described by the manufacturer 's protocol . the investigation conforms with the principles outlined in the declaration of helsinki and was granted by the university ethics review board . after isolation , cd4-positive cells were cultured in serum - free media for 16  h. t - cell chemotaxis was assayed under serum - free conditions in a 48-well microchemotaxis chamber ( neuroprobe , usa ) . wells in the upper and lower chamber were separated by a polyvinylpyrrolidone - free polycarbonate membrane ( pore size 5  m ; costar , cambridge , ma ) . cd4-positive cells at a density of 5  10/ml were pretreated for 15  min with ivabradine before 3 hours of incubation with sdf-1 or rantes ( sigma - aldrich , germany ) . migrated cells on the bottom face of the filter were stained and counted under the light microscope . cells were counted in 5 random high - power fields per well ( in control about 200300 cells and in sdf-1 or rantes - induced conditions about 600700 cells per 5 random power fields per well ) . cd4-positive lymphocytes were left untreated or incubated at 37c with 100  ng / ml rantes ( sigma ) or with 100  ng / ml sdf-1   ( upstate , lake placid , ny , usa ) for times indicated . cells were lysed in lysis buffer ( 50  mmol / l hepes ph 7.4 , 150  mmol / l nacl , 1% ( w / v ) np40 , 1% ( w / v ) glycerol , 1  mmol / l mgcl2 , 1  mmol / l mncl2 , 10  mmol / l naf , 1  mm na3vo4 , 10  g/l aprotinin , 10  g/l leupeptin , 0.1  mmol / l pmsf ) . aliquots of cell lysates were boiled in laemmli buffer before running on sds - page . immunoblotting was performed by running samples on sds - page with subsequent electrotransfer onto nitrocellulose membranes ( amersham pharmacia biotech , amersham , england ) , blocking with 5% skim milk in tbs buffer with 0.1% tween 20 for 1  h , and incubating with primary antibody anti - rac1 was from upstate ( lake placid , ny , usa ) , anti - phospho - mlc2 , anti - phospho - akt , and anti - gapdh were from cell signaling ( beverly , ma , usa ) , anti--tubulin was from sigma and 1  :  2000 dilution of the secondary antibody ( antigoat , antirabbit , or antimouse horseradish peroxidase ( dako , glostrup , denmark ) ) . development was done by using enhanced chemiluminescence reagents ( pearce , rockford , il , usa ) according to the manufacturer 's specifications . after isolation , human t cells were incubated for 16 hours in rpmi medium without serum . cells pretreated for 15  min with or without ivabradine were stimulated with 100  ng / ml sdf-1 . standard pi 3-kinase activity assays were performed   using goat antihuman p85 ( santa cruz ) antibodies . for detection of gtp - bound rac1 , isolated cd4-positive lymphocytes were treated at 37c with 100  ng / ml sdf-1 in presence or absence of ivabradine . cells were lysed for rac1-gtp pulldown experiments in 500  l rac1-ripa buffer ( 50  mmol / l tris / hcl , ph 7.2 , 150  mmol / l nacl , 10  mmol / l mgcl2 , 1% ( v / v ) triton x-100 , 0.5% ( w / v ) sodium deoxyholate , 10  g/l aprotinin , 10  g/l leupeptin , and 0.1  mmol / l pmsf ) . cell lysates were cleared by centrifugation in a benchtop centrifuge at 15800    g for 15 minutes at + 4c . lysates were then incubated with 2040  g glutathione s - transferase- ( gst- ) fusion proteins immobilized to glutathione sepharose 4b beads for 45 minutes at + 4c . the active gtp - bound form of rac1 was determined by using a gst - pak fusion protein . beads were washed four times with washing buffer ( 50  mmol / l tris / hcl , ph 7.2 , 150  mmol / l nacl , 10  mmol / l mgcl2 , 1% ( v / v ) triton x-100 , 10  g/l aprotinin , 10  g/l leupeptin , and 0.1  mmol / l pmsf ) . precipitated proteins were separated on 12% sds - polyacrylamide gels , transferred to membrane , and detected by using appropriate antibodies . 10 cd-4 positive lymphocytes were incubated in special 4-well plates ( costar corp . , cambridge , ma ) in a final volume of 500  l complete medium on coverslips coated with collagen . before treatment with sdf-1 ( 100  ng / ml , 30  min ) cells were then fixed with 3.7% ( wt / vol ) paraformaldehyde in pbs , ph 7.4 , at room temperature and rinsed in tbs . cells were incubated with a specific antibody against icam3 ( mouse antihuman icam3 , caltag , uk ) and after washing with pbs , carboxymethylindocyanine 3-cy3-coupled ( dianova , hamburg , germany ) goat antimouse iggs were added as secondary antibodies ( dilution 1  :  1,000 ) for 45  min . images were recorded with a fluorescence microscope ( leica , wetzlar , germany ) , and cells in 5 randomly chosen fields were analyzed . icam-3 translocation was considered to be present when a clear clustering of icam3 at the uropod was visible . cd4-positive lymphocytes ( 2  10  cells / ml ) were treated with sdf-1 at 100  ng / ml for times indicated after 15  min pretreatment with ivabradine . after stimulation , cells were fixed in 3.7% ( wt / vol ) paraformaldehyde in pbs , ph 7.4 , and then washed at room temperature in tbs ( 50  mmol / l tris - hcl , ph 7.6 , 150  mmol / l nacl , 0.1% nan3 ) . for intracellular staining of f - actin , cells were permeabilized with 0.1% triton x-100 for 10  min before application of the first antibody . cell suspensions were incubated for 30  min in pbs with fitc - conjugated phalloidin ( sigma no . flow cytometry analysis was performed in an facscan cytofluorometer ( becton dickinson , heidelberg , germany ) , and induction of f - actin was measured . migration of human cd4-positive lymphocytes across human endothelial cell monolayers was performed as previously described . cd4-positive lymphocytes ( 0.5  10 cells per well ) , pretreated for 30 minutes with and without ivabradine ( 0.05 or 0.1 mol / l ) , were added to the upper chamber of transwells inserts containing 1500  l hbss . 100 after 1-hour migration , cells that had transmigrated to the lower chamber were harvested and counted by flow cytometry . results of the experimental studies are reported as mean  standard deviation ( sd ) . to examine the effect of ivabradine on cd4-positive lymphocyte migration , cells were stimulated with sdf-1 in the absence or presence of ivabradine , and lymphocyte migration was assessed in a modified boyden chamber . sdf-1 treatment significantly induced cell migration by 2.0  0.1-fold ( p < .01 ; n = 7 ) , and 15  min pretreatment of cells with ivabradine reduced this effect in a concentration - dependent manner to a maximal 1.2  0.1-fold induction at 0.1  mol / l ivabradine ( p < .01 compared with sdf-1-treated cells ; n = 7 ) ( figure 1(a ) ) . next , we examined the effect of ivabradine on rantes - induced lymphocyte migration . pretreatment with ivabradine for 15  min reduces rantes - induced migration in a concentration - dependent manner to a maximal 1.1  0.2-fold induction at 0.1  mol ivabradine ( * p < .01 compared with rantes - treated cells ; n = 7 ) ( figure 1(b ) ) . these results suggest that the effect of ivabradine on lymphocyte migration is independent of the stimulus employed . moreover , ivabradine did not affect cell viability and had no effect on the expression of the chemokine receptor cxcr4 as assessed by flow cytometry ( data not shown ) . activation of pi-3 kinase is a critical step in chemokine - induced t - cell migration downstream of the respective chemokine receptor . as demonstrated in figure 2(a ) , ivabradine limited sdf-1-induced pi-3 kinase activity , suggesting that ivabradine modulates a very upstream step in the chemokine - activated signaling cascade . downstream of pi-3 kinase phosphorylation of akt plays an important role in leucocyte migration [ 16 , 17 ] . sdf-1 treatment significantly induced phosphorylation of akt , and pretreatment with ivabradine reduced this effect in a concentration - dependent manner to a maximal 0.2  0.1-fold induction at 0.1  mol ivabradine ( * p < .01 compared with sdf-1-treated cells ; n = 3 ) ( figure 2(b ) ) . downstream of pi3k small rho gtpases are important signaling molecules involved in leukocyte migration [ 1820 ] . therefore , we assessed the effect of ivabradine on rac1 activity by performing affinity precipitation experiments with gst - pak to which only the active gtp - bound form of rac1 can bind . stimulation with ivabradine diminished sdf-1-induced rac1 activity in a concentration - dependent manner with a maximal effect at 0.1  mol / l ( * p < .01 compared with sdf-1-treated cells ; n = 5 ) ( figure 3(a ) ) .   at the uropod of the cell , phosphorylation of mlc is of critical importance for actin - myosin contractility . pretreatment of cd4-positive lymphocytes with ivabradine significantly reduced sdf-1-induced phosphorylation of mlc in a concentration - dependent manner to a maximal 0.5  0.1-fold induction at 0.1  mol / l ivabradine ( * p < .01 compared with sdf-1-treated cells ; n = 5 ) ( figure 3(b ) ) . downstream of rac1 , f - actin formation is a crucial mechanism in cell polarisation during migration [ 22 , 23 ] . ivabradine significantly reduced sdf-1-induced f - actin formation as demonstrated by flow cytometry ( figure 4 ) . next , we examined the effect of ivabradine on icam3 translocation at the uropod of migrating lymphocytes , a critical step in cell movement . as shown in figure 5 , ivabradine significantly diminished sdf-1-induced icam3 translocation ( figure 5 ) with a maximal reduction at 0.1  mol / l . to facilitate the inhibitory effect of ivabradine on sdf-1-induced migration of cd4-positive lymphocytes , we examined the effect of ivabradine on t cells in a transendothelial migration assay . mol / l or 0.1  mol / l reduces sdf-1-induced transendothelial migration of cd4-positive lymphocytes ( * p < .01 , compared with sdf-1-stimulated cells , n = 6 , figure 6 ) . the present study demonstrates that ivabradine inhibits chemokine - induced migration of cd4-positive lymphocytes by reducing pi-3 kinase activity , phosphorylation of akt , and activation of rac-1 with subsequent inhibition of mlc - phosphorylation , f - actin formation , and icam3 translocation . ivabradine exerts antianginal and anti - ischemic effects in patients with stable coronary artery disease . clinical trials revealed improved exercise tolerance , increased time to exercise - induced ischemia , and reduced frequency of ambient angina attacks after i(f)-channel inhibition [ 25 , 26 ] . ivabradine given orally to mice reduces their heart rates without influencing left ventricular contractile function and therefore is used as a tool to study the effects of heart rate reduction in the vasculature . ivabradine decreases markers of vascular oxidative stress , shown by reduced vascular nadph oxides activity and decreased markers of lipid peroxidation . furthermore , endothelial function was improved and atherosclerotic plaque formation was reduced in the ivabradine - treated group . the content of inflammatory cells was not analyzed , especially the effect on chemokine - induced cd4-positive cell migration . chemokine - induced t - cell migration is mediated by activation of g - protein - coupled receptors leading to an increase in pi-3 kinase activity   with subsequent polarization of cells and formation of a leading edge and the so - called uropod in the rear . the agonists employed in our study interfere with this signaling pathway by inhibiting pi-3 kinase activity and activation of rac . interestingly , 15  min pretreatment of cd4-positive cells with ivabradine already prevented the rapid effect of sdf-1 on pi 3-kinase activation , raising the question of a nontranscriptional effect of these agents in this context . this rapid effect described here was already seen for other nuclear receptors , like the ppar and ppar. previous studies from our own group showed similar results with ppar -activating substances . downstream of pi 3-kinase and rac1 , ivabradine leads to a reduction in f - actin formation and icam3 translocation at the uropod of the cell , thus counterbalancing critical steps in cell movement [ 2224 ] . in addition , the effect of ivabradine on cell migration does not depend on the chemotactic stimulus , since ivabradine diminished both sdf-1- as well as rantes - induced lymphocyte migration . further studies should show if this effect of ivabradine in vitro could be also observed in vivo and how this rapid response of ivabradine on chemokine - induced migration works . the importance of chemokine - induced migration and the chemokine receptor expression was shown in a mouse model with a genetic deletion of ccr5 . in these apoe/ mice , the deletion of ccr5 protects from diet - induced atherosclerosis , associated with a more stable plaque phenotype , reduced mononuclear cell infiltration , th1-type immune responses , and increased interleukin-10 expression . furthermore , blockade of mif ( macrophage migration inhibitory factor ) but not of canonical ligands of cxcr2 or cxcr4 in mice with advanced atherosclerosis led to plaque regression and reduced monocyte and t - cell content in plaques . cd4-positive cells , once recruited into the subendothelial space by chemokines like sdf-1 or rantes , differentiate to th1-cells , thus releasing proinflammatory cytokines which then promote inflammatory activation of other cells in the vessel wall [ 1 , 2 ] . therefore , modulating the migration of t - lymphocytes into the vasculature may target the inflammatory process in atherogenesis at a nodal point and as such modulate a critical step in atherosclerotic lesion development .
aims . 
 migration of cd4-positive lymphocytes into the vessel wall is a critical step in atherogenesis . 
 recent data suggest that ivabradine , a selective i(f)-channel blocker , reduces atherosclerotic plaque formation in apolipoprotein e - deficient mice , hitherto nothing is known about the mechanism by which ivabradine modulates plaque formation . therefore , the present study investigated whether ivabradine regulates chemokine - induced migration of lymphocytes . 
 methods and results . 
 stimulation of cd4-positive lymphocytes with sdf-1 leads to a 2.0  0.1 fold increase in cell migration ( p < .01 ; n = 7 ) . 
 pretreatment of cells with ivabradine reduces this effect to a maximal 1.2  0.1 fold induction at 0.1 mol / l ivabradine ( p < .01 compared to sdf-1-treated cells , n = 7 ) . 
 the effect of ivabradine on cd4-positive lymphocyte migration was mediated through an early inhibition of chemokine - induced pi-3 kinase activity as determined by pi-3 kinase activity assays . 
 downstream , ivabradine inhibits activation of the small gtpase rac and phosphorylation of the myosin light chain ( mlc ) . 
 moreover , ivabradine treatment reduces f - actin formation as well as icam3 translocation to the uropod of the cell , thus interfering with two important steps in t cell migration . conclusion . 
 ivabradine inhibits chemokine - induced migration of cd4-positive lymphocytes . 
 given the crucial importance of chemokine - induced t - cell migration in early atherogenesis , ivabradine may be a promising tool to modulate this effect .