lipoproteini velike gustine ( hdl ) imaju ateroprotektivne bioloke osobine : antioksidativne , antiapoptotike , anti - inflamatorne kao i kapacitet da izvlae holesterol iz elija . analiza plazmatske mrna moe da se koristi za ispitivanje plejotropnih efekata statina in vivo kao novo analitiko sredstvo za neinvazivnu procenu ekspresije gena u zidu krvnog suda . cilj ove studije je bio da se procene plejotropni efekti atorvastatina kod pacijenata sa stabilnom anginom sa visokorizinim vrednostima ( grupa a ) u odnosu na pacijente sa graninim i poeljnim vrednostima hdl holesterola ( hdl - c ) ( grupa b ) . etrdeset tri pacijenta sa stabilnom anginom su primala terapiju atorvastatinom ( 20 mg / dan ) 10 nedelja . mi smo ispitivali tri gena znaajna za plejotropno delovanje statina : intracelularni adhezioni molekul-1 , hemokin ( c - c motiv ) ligand 2 i katepsin s i procenjivali smo efekte atorvastatina na veliinu i raspodelu hdl subfrakcija pomou elektroforeze na poliakrilamidnom gradijent gelu . u grupi a , posle terapije , hdl - c koncentracija se znaajno poveala , ali ne i u grupi b. atorvastatin je snizio plazmatski nivo hemokin ( c - c motiv ) liganda 2 i intracelularnog adhezionog molekula-1 mrna u obe grupe , ali nije promenio plazmatski nivo gena za katepsin s. samo u grupi a , ukupni bilirubin je pokazao negativnu korelaciju sa genom za katepsin s ( r=0,506 ; p=0,023 ) pre zapoinjanja terapije i znaajni porast nakon terapije atorvastatinom . cell - free mrna ) u plazmi moe postati korisno sredstvo za procenu plejotropnog delovanja statina . high - density lipoproteins ( hdl ) have atheroprotective biological properties : antioxidative , antiapoptotic , antiinflammatory , and they have the efflux capacity of cellular cholesterol ( 1 ) . these plasma hdl particles have heterogeneous physicochemical properties , metabolism , and biological activity ( 1 ) . hdl is the next target in reducing cardiovascular risk in statin - treated high - risk patients , as evidenced from epidemiological data , the data based on animal studies and clinical trials ( 2 ) . prospective studies have shown that coronary heart disease ( chd ) risk was increased by 3% in women and by 2% in men per decrement of 1 therapeutic normalisation of attenuated antiatherogenic hdl function is the target of modern pharmacological approaches to hdl raising ( 1 ) . common cardiovascular prevention strategies emphasise therapeutic reductions in low - density lipoprotein cholesterol ( ldl - c ) ( 4 ) , however , attention is being focused on hdl cholesterol as a secondary prevention target to address the risk of residual cardiovascular disease ( cvd ) ( 5 ) . statins , the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase , are drugs that effectively reduce ldl - c levels . the studies indicate that the overall benefits observed with statins can be mediated through cholesterol - independent effects , the so - called pleiotropic effects ( 6 ) . plasma mrna analysis , as a new research tool for the nonin - vasive follow - up of gene expression in vascular walls , can be used to investigate statin pleiotropy in vivo ( 7 ) . monocyte chemoattractant protein-1 ( mcp-1 ) is the major chemokine responsible for attracting monocytes to sites of active inflammation , including the formation of atheromatous plaque ( 8) . the icam-1 is considered a major factor when circulating monocytes adhere to the endothelium and then transmigrate into the intima ( 9 ) . macrophages , smooth muscle cells , and endothelial cells synthesise cathepsin s ( ctss ) ( 10 ) . when released extracellularly , ctss exerts collagenolytic and elastolytic activities which have as effects : elastic lamina degradation , plaque rupture , and necrotic core formation ( 11 , 12 ) . in this study , we assessed the effects of atorvastatin on hdl size and subclasses . in addition , the aim of this study was to assess the pleiotropic effects of atorvastatin ( 20 mg / day , for 10 weeks ) on markers of inflammation , oxidative stress and destabilisation of atherosclerotic plaque in stable angina patients with high - risk values , as compared with patients who had borderline and desirable hdl - c values . we investigated three statin pleiotropy - targeted genes : intercellular adhesion molecule-1 ( icam1 ) , chemokine ( c - c motif ) ligand 2 ( ccl2 ) and cathepsin s ( ctss ) . the atorvastatin therapy ( 20 mg / day ) was given to forty - three patients with stable angina for 10 weeks . stable angina was diagnosed by clinical assessment , electrocardiogram evaluation and laboratory findings ( 13 ) . criteria for the inclusion of patients were a stable angina diagnosis and statin free treatment for at least one year . criteria for the exclusion of patients were ( by detailed history and clinical examination ) : inflammatory , endocrine , thyroid , liver , neoplastic and renal diseases ( serum creatinine concentration values  120 mol / l ) . the patients with stable angina from the health centre laktasi ( bosnia and herzegovina ) were consecutively enrolled in our study . the procedure for collecting cell - free plasma , a detailed description , has been published elsewhere ( 7 ) . rna was isolated from 3 ml of plasma using qiavac 24 plus ( qiagen ) and qiaamp circulating nucleic acid kit ( qiagen , hilden , germany ) according to the manufacturer s instructions , with certain modifications . the time of proteolysis was 5 min , and rna was eluted from the column with 47 l of rnase  free water . rna eluates were incubated in a 65 c water bath for 5 min and then immediately placed on ice for another 5 min . the eluted mrna was transcribed to cdna with a super script vilo cdna synthesis kit ( invitrogen , carlsbad , ca , usa ) . for more efficient mrna transcription to cdna the primers used for cdna transcript quantification ( taqman gene expression assay , applied biosystems , foster city , ca , usa ) were : hs00164932_m1 for icam1 ( exon boundary 23 , amplicon length 87 bp ) , hs00234140_m1 for ccl2 ( exon boundary 12 , amplicon length 101 bp ) , hs00175403_ml for ctss ( exon boundary 34 , amplicon lengh 82 bp ) and hs99999905_ml for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) as the reference gene . four pairs of samples were chosen for selection of the most appropriate reference gene among the 32 candidates available on the taqman human endogenous control plate ( applied biosystems ) . the cdna transcripts were amplified by pcr according to the manufacturer s protocol with faststart universal probe master ( rox ) ( roche applied science , mannheim , germany ) , using 11.25 l diluted ( 1:3 ) of cdna ( ccl2 , icam1 ) ; 11.25 l diluted ( 1:8 ) of cdna ( ctss ) and 10.00 l diluted ( 1:12 ) of cdna ( gapdh ) and the kit components in a total reaction volume of 25 l run in an abi prism sds pcr analyser ( applied biosystems ) . the possibility of dna interference was excluded by starting pcr reaction with samples of the subject but omitting the reverse transcription for every commercially available taqman gene expression assay . negative control ( blank ) was analysed to exclude the possibility of external mrna contamination . the coefficient of variation ( cv ) of ct was less than 2% between three averaged ct values ( 14 ) . the amount of each investigated transcript was nor - malised to the amount of the reference gene ( 15 ) . particle size and distribution of hdl subclasses were assessed by a method of polyacrylamide gradient gel electrophoresis , introduced by rainwater et al . briefly , in house made polyacrylamide gradient ( 331% ) gels were processed in a hoefer se 600 ruby electrophoresis unit ( amersham pharmacia biotech , vienna , austria ) for 20 h , at 8 c using tris ( 90 mmol / l)boric acid ( 80 mmol / l)na2edta ( 2.7 mmol / l ) buffer , ph 8.35 . for calibration purposes , we used pharmacia high molecular weight protein standards , carboxylated polystyrene microsphere beads and standard samples of human plasma , standardized in dr . david rainwater s laboratory ( southwest foundation for biomedical research , san antonio , tx , usa ) . following electrophoretic separation , the gels were stained for proteins with coomassie brilliant blue g-250 and for lipids with sudan black . image scanner ( amersham pharmacia biotech , vienna , austria ) with image quant software ( version 5.2 ; 1999 ; molecular dynamics ) was used as a device for densitometric analysis of the gels . by using the calibration curve created of migration distances of standards with known particle diameters , we calculated particle diameters corresponding to each absorbance peak in the hdl regions of gels . dominant hdl particle diameter was assessed by estimation of the diameter of the major absorbance peak . the relative content of each hdl subclass was evaluated through determination of the areas under the peaks of densitometric scans . the relative proportion of small - sized hdl particles was assessed as the area of the densitometric scan at or below 8.8 nm ( 19 ) . increased small sized hdl particles were considered if more than 50% of total hdl absorbance was detected in the area 8.8 nm . oxidized low - density lipoprotein ( oxldl ) was assessed by means of a solid phase dissociation enhanced lanthanide time - resolved fluorescence immunoassay ( delfia ) based on the sandwich technique ( 20 ) , using the monoclonal antibody ob/04 ( 21 ) , and 24 hours oxidized ldl as a standard . the plates were precoated with ob04 , directed against an epitope generated on oxidized apolipoprotein b ( apo b ) upon oxidation . the plates were incubated with diluted plasma samples , applied in duplicate at room temperature on a plate shaker . after 2 hours the plates were washed 4 times using a plate washer ( anthos fluido laptec , salzburg , austria ) , to remove nonreactive plasma components . then , the plates were incubated for 1 hour at 4 c with a rabbit anti - apo b antibody ( behring , mannheim , germany ) , which recognized the oxldl bound to the solid phase . the second washing step ( 4 times ) removed unbound anti - apo b antibody . as the last step the wells were incubated with an eu - labeled antirabbit igg for 1 h at room temperature . the antibody ( sigma , st . louis , mo , usa ) was labeled with an eu - labeling kit ( perkin elmer , waltham , ma , usa ) . the fluorescence was developed using the enhancement solution , after washing the plates for 6 times . eu was dissociated from the labeled antibody by a low ph , whereafter the free lanthanide formed a fluorescent chelate with the components of the enhancement solution . hdl - c , ldl - c , tc , triglycerides and total bilirubin were measured by reagents from roche diagnostics ( roche diagnostics , mannheim , germany ) . commercial elisa kit ( invitrogen , frederick , md , usa ) was used for measurement of serum amyloid a ( saa ) . c - reactive protein ( crp ) was measured by beckman coulter crp latex reagent ( beckman coulter , inc . numerical data are shown as median and interquartile range for non - normally distributed variables . comparisons of the data before and after 10 weeks of atorvastatin therapy were performed by the wilcoxon s signed - rank test for non - normally distributed variables . the mann - whitney u - test was used to compare data between subgroups with hdl - c  1.2 mmol / l and hdl - c > 1.2 mmol / l . continuous variables are presented as meanstandard deviation and were compared by the student s t - test . categorical variables are presented as relative frequencies and were compared by the chi - square test . the spearman rank - order correlation test was used for determining relations between the variables . the atorvastatin therapy ( 20 mg / day ) was given to forty - three patients with stable angina for 10 weeks . stable angina was diagnosed by clinical assessment , electrocardiogram evaluation and laboratory findings ( 13 ) . criteria for the inclusion of patients were a stable angina diagnosis and statin free treatment for at least one year . criteria for the exclusion of patients were ( by detailed history and clinical examination ) : inflammatory , endocrine , thyroid , liver , neoplastic and renal diseases ( serum creatinine concentration values  120 mol / l ) . the patients with stable angina from the health centre laktasi ( bosnia and herzegovina ) were consecutively enrolled in our study . the procedure for collecting cell - free plasma , a detailed description , has been published elsewhere ( 7 ) . rna was isolated from 3 ml of plasma using qiavac 24 plus ( qiagen ) and qiaamp circulating nucleic acid kit ( qiagen , hilden , germany ) according to the manufacturer s instructions , with certain modifications . the time of proteolysis was 5 min , and rna was eluted from the column with 47 l of rnase  free water . rna eluates were incubated in a 65 c water bath for 5 min and then immediately placed on ice for another 5 min . the eluted mrna was transcribed to cdna with a super script vilo cdna synthesis kit ( invitrogen , carlsbad , ca , usa ) . for more efficient mrna transcription to cdna the primers used for cdna transcript quantification ( taqman gene expression assay , applied biosystems , foster city , ca , usa ) were : hs00164932_m1 for icam1 ( exon boundary 23 , amplicon length 87 bp ) , hs00234140_m1 for ccl2 ( exon boundary 12 , amplicon length 101 bp ) , hs00175403_ml for ctss ( exon boundary 34 , amplicon lengh 82 bp ) and hs99999905_ml for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) as the reference gene . four pairs of samples were chosen for selection of the most appropriate reference gene among the 32 candidates available on the taqman human endogenous control plate ( applied biosystems ) . the cdna transcripts were amplified by pcr according to the manufacturer s protocol with faststart universal probe master ( rox ) ( roche applied science , mannheim , germany ) , using 11.25 l diluted ( 1:3 ) of cdna ( ccl2 , icam1 ) ; 11.25 l diluted ( 1:8 ) of cdna ( ctss ) and 10.00 l diluted ( 1:12 ) of cdna ( gapdh ) and the kit components in a total reaction volume of 25 l run in an abi prism sds pcr analyser ( applied biosystems ) . the possibility of dna interference was excluded by starting pcr reaction with samples of the subject but omitting the reverse transcription for every commercially available taqman gene expression assay . negative control ( blank ) was analysed to exclude the possibility of external mrna contamination . the coefficient of variation ( cv ) of ct was less than 2% between three averaged ct values ( 14 ) . the amount of each investigated transcript was nor - malised to the amount of the reference gene ( 15 ) . particle size and distribution of hdl subclasses were assessed by a method of polyacrylamide gradient gel electrophoresis , introduced by rainwater et al . ( 16 ) . briefly , in house made polyacrylamide gradient ( 331% ) gels were processed in a hoefer se 600 ruby electrophoresis unit ( amersham pharmacia biotech , vienna , austria ) for 20 h , at 8 c using tris ( 90 mmol / l)boric acid ( 80 mmol / l)na2edta ( 2.7 mmol / l ) buffer , ph 8.35 . for calibration purposes , we used pharmacia high molecular weight protein standards , carboxylated polystyrene microsphere beads and standard samples of human plasma , standardized in dr . david rainwater s laboratory ( southwest foundation for biomedical research , san antonio , tx , usa ) . following electrophoretic separation , the gels were stained for proteins with coomassie brilliant blue g-250 and for lipids with sudan black . image scanner ( amersham pharmacia biotech , vienna , austria ) with image quant software ( version 5.2 ; 1999 ; molecular dynamics ) was used as a device for densitometric analysis of the gels . by using the calibration curve created of migration distances of standards with known particle diameters , we calculated particle diameters corresponding to each absorbance peak in the hdl regions of gels . dominant hdl particle diameter was assessed by estimation of the diameter of the major absorbance peak . the relative content of each hdl subclass was evaluated through determination of the areas under the peaks of densitometric scans . the relative proportion of small - sized hdl particles was assessed as the area of the densitometric scan at or below 8.8 nm ( 19 ) . increased small sized hdl particles were considered if more than 50% of total hdl absorbance was detected in the area 8.8 nm . oxidized low - density lipoprotein ( oxldl ) was assessed by means of a solid phase dissociation enhanced lanthanide time - resolved fluorescence immunoassay ( delfia ) based on the sandwich technique ( 20 ) , using the monoclonal antibody ob/04 ( 21 ) , and 24 hours oxidized ldl as a standard . the plates were precoated with ob04 , directed against an epitope generated on oxidized apolipoprotein b ( apo b ) upon oxidation . the plates were incubated with diluted plasma samples , applied in duplicate at room temperature on a plate shaker . after 2 hours the plates were washed 4 times using a plate washer ( anthos fluido laptec , salzburg , austria ) , to remove nonreactive plasma components . then , the plates were incubated for 1 hour at 4 c with a rabbit anti - apo b antibody ( behring , mannheim , germany ) , which recognized the oxldl bound to the solid phase . the second washing step ( 4 times ) removed unbound anti - apo b antibody . as the last step the wells were incubated with an eu - labeled antirabbit igg for 1 h at room temperature . the antibody ( sigma , st . louis , mo , usa ) was labeled with an eu - labeling kit ( perkin elmer , waltham , ma , usa ) . the fluorescence was developed using the enhancement solution , after washing the plates for 6 times . eu was dissociated from the labeled antibody by a low ph , whereafter the free lanthanide formed a fluorescent chelate with the components of the enhancement solution . hdl - c , ldl - c , tc , triglycerides and total bilirubin were measured by reagents from roche diagnostics ( roche diagnostics , mannheim , germany ) . commercial elisa kit ( invitrogen , frederick , md , usa ) was used for measurement of serum amyloid a ( saa ) . c - reactive protein ( crp ) was measured by beckman coulter crp latex reagent ( beckman coulter , inc . numerical data are shown as median and interquartile range for non - normally distributed variables . comparisons of the data before and after 10 weeks of atorvastatin therapy were performed by the wilcoxon s signed - rank test for non - normally distributed variables . the mann - whitney u - test was used to compare data between subgroups with hdl - c  1.2 mmol / l and hdl - c > 1.2 mmol / l . continuous variables are presented as meanstandard deviation and were compared by the student s t - test . categorical variables are presented as relative frequencies and were compared by the chi - square test . the spearman rank - order correlation test was used for determining relations between the variables . mmol / l ( < 1.0 mmol / l for men , < 1.2 mmol / l for women , n = 20 ) , also known as high - risk values , were represented by group a , and patients with borderline and desirable values , i.e. > 1.2 mmol / l were represented by group b ( n=23 ) . patients were taking other therapy prior to inclusion into this study ( table i ) . baseline laboratory parameters and their changes after 10 weeks of atorvastatin treatment are shown in table ii , table iii and figure 1 . after therapy there was a significant difference between small - sized hdl particles and total bilirubin values between the two groups . likewise , hdl 2b was different in group a and group b , at baseline ( median 39.28 vs. 43.87 , p=0.054 ) and after the therapy ( median 40.92 vs. 47.13 , p=0.013 ) . as shown , atorvastatin treatment significantly increased hdl 2b in group b only ( p=0.004 ) . there was no correlation between the degree of changes in the plasma ldl - c after 10 weeks of statin treatment and the degree of changes in the following plasma biochemistry markers : ccl2 mrna , icam1 mrna , ctss mrna , crp and saa ( results not shown ) . in group a , hdl - c negatively correlated with small - sized hdl at baseline ( r=0.486 ; p=0.030 ) and positively correlated with dominant particle diameters hdl ( r=0.555 ; p=0.011 ) . as expected , hdl - c correlated with tg ( r=0.529 ; p=0.011 ) . in group b , hdl - c positively correlated with hdl dominant particle diameter at baseline ( r=0.433 ; p=0.039 ) and , as expected , hdl - c negatively correlated with tg ( r=0.514 ; p=0.012 ) . in group a only , baseline correlation was impressive : total bilirubin showed correlation with ctss mrna ( r=0.506 ; p=0.023 ) . atorvastatin ( 20 mg / day , 10 weeks ) was more effective in patients with high - risk values ( group a ) than in the group with borderline and desirable hdl - c values ( group b ) . in group a , after therapy , hdl - c was significantly increased but not in group b ( table ii ) . a drug induced increase of hdl - c levels the antiatherogenic function of hdl relates to their ability to promote the efflux of cholesterol from cells , so as to their antiinflammatory and antioxidant properties ( 1 ) . however , the atheroprotective function of hdl is related not only to the plasma concentration of hdl - c , but also to the distribution and functional characteristics of hdl particles . it is known that altered small - sized hdl particles have a diminished ability to protect ldl from oxidation , a reduced reverse cholesterol transport capacity and antiinflammatory properties ( 1 ) . small - sized hdl particles were more prevalent in group a than in group b , reaching statistically significant difference after therapy ( table ii ) . a relatively high baseline proportion of small - sized hdl particles in group a ( table ii ) could not be significantly reduced by atorvastatin , unlike in group b. in this study , atorvastatin s beneficent effect on the hdl 2b subclasses was present in both groups , but significantly more so in group b ( figure 1 ) . results of some studies indicated that hdl 2b , which is highly correlated with hdl - c , is strongly inversely related to chd risk ( 23 , 24 ) . in our study there was a difference in hdl 2b between group a and group b , at baseline and after therapy . hdl 2b subclasses were more prevalent in group b where the patients had a more favourable risk profile . in both groups , the treatment shifted hdl subclasses distribution toward larger particles which is in agreement with the study by asztalos et al . in addition , our finding of significantly increased dominant hdl diameter after atorvastatin therapy in group a ( table ii ) is in line with the studies ( 26 , 27 ) which showed beneficial effects of statins through the increase of large hdl particles that have considerably stronger antiatherogenic properties . although the literature is indicative of hdl - c antioxidative , antiapoptotic and antiinflammatory activities , in the study there were no correlations of hdl - c with bilirubin and hdl - c with genes that are significant for the aforementioned effects ( results not shown ) . the reason for this may be the limitations in our study , such as the small number of samples , the short treatment time of 10 weeks and the monitoring of pleiotropic effects only once , after 10 weeks of therapy ( a one - time assessment ) . this study indicates that atorvastatin in stable angina patients lowered the plasma mrna levels of statin pleiotropy - targeted genes icam1 and ccl2 in both groups . this reduction was obviously independent from the ldl - c reduction ( results not shown ) . this paper , and the previous paper ( 7 ) , shows that short - term atorvastatin treatment results in anti - inflammatory effects , presumably by different mechanisms , including the inhibition of nuclear factor kappa b ( nf-b ) ( 28 ) and the interference with the prenylation of small g proteins ( such as rho and ras ) ( 29 ) . current studies suggest that statins increase ctss in the plasma , by statin - induced apoptosis of vascular cells ( 30 ) . however , this suggestion has limitations because statin treatment did not change the activity of ctss in the wall of abdominal aortic aneurysma in one small case - control study ( 31 ) and ctss deficiency by itself reduces apoptosis of vascular macrophages ( 11 ) . in our study atorvastatin an alternative explanation could be that atorvastatin in our patients downregulated inflammation and oxidative stress , which further lowered ctss expression , or atorvastatin in our study improved the balance between ctss activity and cystatin c ( a potent endogenous ctss inhibitor ) , but again , this suggestion has limitations because we could not recognise any change in the plasma ctss mrna level . however , it is significant that in the group a subjects , the plasma total bilirubin concentration was associated with the baseline plasma ctss mrna . bilirubin is recognised as a potent antioxidant ( 32 ) and a lower total bilirubin could mean higher oxidative stress . in the development of vascular disease , the apoptotic cell death is caused by cellular depletion of bilirubin ( 34 ) and consequently increases ctss in the plasma . a negative correlation between bilirubin and ctss mrna in our clinical study ( in line with in vitro studies ) is significant since it leads to novel therapy directives aimed at preventing the clinical progression of atheroma . bilirubin correlation with ctss mrna raises the question as to whether lower values of bilirubin could be a therapeutic target in the field of atherosclerosis . some studies suggest that cathepsin s is involved in the complex pathways of cardiovascular disease and cancer . ( 35 ) revealed higher serum cathepsin s levels in elderly individuals in 2 independent groups were associated with an increased mortality risk . it is evident that increased bilirubin will cause lower expression of the ctss mrna , the gene associated with a higher mortality risk . thus , it is not easy to form an exact image of the mechanism of negative correlation between bilirubin and ctss mrna in the group a patients , and we have to propose that more detailed studies are needed to elucidate this issue . our study shows that , in stable angina patients , atorvastatin increased the plasma total bilirubin concentrations in group a only . these results ( table iii ) are in line with the abovementioned increase of dominant hdl diameter and large hdl 2b subclasses ( table ii ) . ( 36 ) showed that elevated circulating bilirubin is associated with a reduced concentration of proatherogenic lipids and inflammation biomarkers . statins increase total bilirubin that protects ldl from oxidation ( 37 ) , which then reduces oxidative stress . on the other hand , a more favourable oxidative status after 10 weeks of therapy ( lower concentration of ox ldl ) could lead to savings of bilirubin , a potent antioxidant , which is reflected in a higher concentration of bilirubin in the plasma . altogether , our results indicated favourable effects of statins on two compounds of the antioxidative defence system : bilirubin and large hdl 2b subclasses . the fact that elevations of both the concentration of bilirubin and the proportion of hdl 2b were more prominent in group a , which is more vulnerable in terms of antiatherogenic protection , is especially important . these findings suggest that low hdl - c concentration may also be a target for beneficial effects of statins . results from table iii raise the question about the necessary length of therapy before evaluating the antioxidant activity of statins . likewise , our results suggest that a significant decrease of saa and crp in plasma is not due to the atorvastatin effect on ldl cholesterol , but is a consequence of the pleiotropic effects of atorvastatin . atorvastatin exerts a number of pleiotropic effects , which include anti - inflammatory and antioxidative activities . however , further , well planned studies are needed to evaluate our currently observed results . the results suggest that routine care in stable angina patients could be complemented with new therapeutic approaches to raise hdl - c . hdl - c may become a therapeutic target equal to ldl - c in the field of cvd and atherosclerosis . the beneficial effect of atorvastatin on bilirubin also suggests that bilirubin can be a new therapeutic target in cvd . analysis of cell - free mrna in plasma could be a new tool in non - invasive assessment of statin pleiotropy .
summarybackgroundhigh - density lipoproteins ( hdl ) have athero - protective biological properties : antioxidative , anti - apoptotic , anti - inflammatory , and they have the efflux capacity of cellular cholesterol . 
 plasma mrna analysis can be used to investigate statin pleiotropy in vivo as a new analytical tool for non - invasive assessment of gene expression in vascular beds . 
 the aim of this study was to assess the pleiotropic effects of atorvastatin in stable angina patients with high - risk values ( group a ) as compared with patients who had borderline and desirable hdl - cholesterol ( hdl - c ) values ( group b).methodsthe atorvastatin therapy ( 20 mg / day ) was given to forty - three patients with stable angina for 10 weeks . 
 we investigated three statin pleiotropy - targeted genes : inter - cellular adhesion molecule-1 , chemokine ( c - c motif ) ligand 2 and cathepsin s and assessed by gel electrophoresis gradient the effects of atorvastatin on hdl size and subclasses.resultsin group a , after therapy , hdl - c concentration was significantly increased but not in group b. atorvastatin lowered plasma chemokine ( c - c motif ) ligand 2 and intercellular adhesion molecule-1 mrna levels in both groups , but did not change the plasma cathepsin s mrna levels . in group a only , baseline total bilirubin showed negative correlations with the genes of cathepsin s ( r=0.506 ; p=0.023 ) and significantly increased after therapy.conclusionshdl-c and bilirubin can be promising therapeutic targets in the treatment of cardiovascular diseases . 
 analysis of cell - free mrna in plasma might become a useful tool for estimating statin pleiotropy .