it is characterized by acute inflammatory response which causes coronary plaque rupture with subsequent thrombosis .
accumulating evidence showed that inflammation and immune cell activation play a key role in collagen loss in the fibrous cap which is a prelude to fibrous cap rupture [ 3 , 4 ] . the extent of cardiac damage due to this inflammatory response is reflected by peripheral levels of inflammatory biomarkers involved in the process of plaque instability .
moreover , markers of plaque destabilization and plaque rupture such as high sensitivity c - reactive protein ( hscrp ) may be used to predict future cardiovascular events not only in apparently healthy subjects , but also in patients with acs .  
granzyme b ( gzb ) is a serine protease released from cytotoxic t lymphocytes ( ctls ) and natural killer ( nk ) cells , playing an important role in cellular apoptosis by activating intracellular caspases .
furthermore , gzb is also identified as an extracellular protease degrading specific extracellular substrates such as fibronectin , vitronectin , and laminin , which implies the role of gzb in extracellular matrix ( ecm ) remodeling .
therefore , the mechanisms by which gzb may contribute to plaque instability may include ecm degradation and/or induction of macrophage or smooth muscle cells apoptosis in the fibrous cap .
interleukin 18 ( il-18 ) , previously known as interferon - gamma ( ifn- ) inducing factor , is a proinflammatory member of il-1 superfamily which plays a role in the initiation and progression of atherosclerosis .
both clinical and experimental studies have supported its role in atherosclerotic plaque progression and destabilization [ 11 , 12 ] .
nevertheless , it was described as an independent predictor of future adverse events in patients with acs .
another contributing factor to the formation of atherosclerotic plaques and may participate in their destabilization is chemokines . in this regard , fractalkine ( fkn ) or cx3cl1 is a unique dual function chemokine that exists in two forms ; a soluble form which acts as a chemoattractant and a membrane bound form acting as an adhesion molecule .
fkn was recently identified as an independent key factor in the pathogenesis of plaque vulnerability and subsequent plaque rupture .   
hence , the current study was designed to determine the circulating levels of gzb in patients with acs being compared with healthy control subjects .
furthermore , the associations between gzb with markers of inflammation and plaque destabilization ( hscrp , il-18 , and fkn ) as well as other metabolic , anthropometric , and risk factors are being evaluated .
  a total of 48 subjects ( 36 men and 12 postmenopausal women ) were enrolled in the study , of which 36 nondiabetic acs patients , being compared to 12 age- and sex - matched apparently healthy subjects as the control group .
twenty - two patients ( 61% ) were diagnosed with st segment elevation myocardial infarction ( stemi ) and 14 ( 39% ) with non st segment elevation myocardial infarction ( nstemi ) or unstable angina ( ua ) .
stemi , nstemi , and ua were diagnosed according to criteria stated by the consensus document of the joint european society of cardiology / american college of cardiology committee for the redefinition of myocardial infarction .
patients were recruited from the intensive care unit , cardiology department , el - demerdash hospital , ain shams university educational hospitals , cairo , egypt .
subjects with fasting plasma glucose 7.0  mmol / l or with a previous history of diabetes mellitus , inflammatory diseases , autoimmune disorders , malignancy , hematological diseases , hepatic or renal diseases , acute or chronic infections , or administration of immunosuppressive drugs were excluded from this study .  
the study was carried out in accordance with the regulations and recommendations of the declaration of helsinki , being approved by the committee on medical ethics of el - demerdash hospital as well as faculty of pharmacy , ain shams university ethical committee and informed consent was obtained from all participants .   a detailed family and medical history and drug treatment(s )
systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , heart rate ( hr ) , left ventricular ejection fraction ( lvef ) , and the incidence of prior acs were obtained from medical records .
routine serum samples for creatine kinase ( ck ) and ck - mb isoenzyme were obtained at admission and at 6- to 8-hour intervals .
hypertension ( htn ) was defined as sbp > 140  mm  hg and/or dbp > 90  mm  hg and/or initiation of antihypertensive medication .
demographic data and biochemical parameters of stemi and nstemi / ua groups are demonstrated in table 2 .
moreover , beta blockers , calcium channel blockers , angiotensin converting enzyme inhibitors , diuretics , nitrates , and statins were described according to the current guidelines .   peripheral blood samples ( 5  ml )
were taken from acs patients on day 1 ( day of onset ) and day 3 after onset .
samples were divided into 2 aliquots ; the first part was collected on plain vacutainer tubes for serum preparation used for lipids profile , hscrp , and il-18 assay .
both plasma and serum were divided into several aliquots , being stored at 80c for subsequent assay .
lipids profile was measured by enzymatic method according to kits provided by hannover , germany .
serum hscrp and il-18 were quantified by enzyme linked immunosorbent assay ( elisa ) technique using commercial available kit ( hscrp : accubind , momobind inc .
, ny , usa elisa kit and plasma fkn elisa kit was supplied by r&d systems , inc , minneapolis , mn , usa . all elisa procedures were done by hyprep automated elisa system ( hyperion inc , miami , fl ) according to the manufacturer 's instructions .
the ibm statistical package for social sciences ( spss ) statistics ( v.19.0 , ibm corp .
continuous variables were presented as mean  sem and categorical ones as actual numbers and percentages .
comparisons between two independent normally distributed mean groups were done using independent - samples t - test .
changes in the serum or plasma levels of the inflammatory markers were evaluated with paired t - test or wilcoxon matched pairs test . correlations between markers were ascertained with ranked spearman test . multiple stepwise regression analysis was done to identify risk factors that contribute to plasma gzb levels .
serum levels of hscrp , gzb , il-18 , and fkn showed significant elevation in acs group when compared to the healthy control group at p < 0.001 ( table 1 ) .
stepwise in the nstemi / ua and stemi groups , hscrp and gzb were significantly increased in the later group in comparison to the former ( table 2 ) .  
comparisons between hscrp , gzb , il-18 , and fkn levels on day 1 and day 3 being compared to the control group levels are given in table 3 .
both stemi and nstemi / ua groups showed the same dynamic changes in the measured biomarkers but with different degrees . in comparison to day 1
, hscrp levels significantly decreased on day 3 in either nstemi / ua or stemi groups to 74% and 65% , respectively .
moreover , plasma gzb levels significantly increased to 118% in nstemi / ua and to 126% in the stemi group in day 3 compared to day 1 in both groups , respectively .
however , plasma fkn decreased significantly on day 3 by 8% in nstemi / ua and by 11% in stemi group in comparison to its level in day 1 .
as shown in figure 1 , a significant positive correlation is observed between gzb and fkn ( r = 0.58 , p < 0.001 ) .
furthermore , il-18 levels displayed a significant positive correlation with fkn ( r = 0.345 , p < 0.05 ) and gzb ( r = 0.416 , p < 0.01 ) . for the clinical parameters of acs patients ,
as depicted in table 4 , correlation analyses also revealed significant correlations between gzb and sbp , dbp , peak ck , and peak ck - mb .
however , serum il-18 did not show significant correlation with any of anthropometric or clinical parameters in the acs patient group .
on the other hand , fkn levels showed significant positive correlations with bmi and peak ck - mb only .  
next , we conducted multivariate regression analysis to examine the cardiovascular risk factors contributing to plasma granzyme b levels .
we set plasma granzyme b levels as a dependent variable and age , gender , bmi , htn , hypercholesterolemia , current smoker , and patient history of previous acs as independent variables .
as depicted in table 5 , htn and patient history were independent risk factors that significantly affect plasma granzyme b levels in patients with acs .
increased systemic inflammatory mediators in patients with stable atherosclerotic plaques are a major driving force for plaque disruption and hence the incidence of acs syndrome . however , other inflammatory conditions such as diabetes mellitus may affect circulating levels of those biomarkers [ 19 , 20 ] .
therefore , the need for assessment of the inflammatory state in patients with acs in absence of such interfering condition has been rising .
since extracellular gzb was discovered , several in vitro studies have been done to verify its mechanisms of action but only a limited number of clinical studies evaluated its role as a circulating biomarker for cardiovascular diseases [ 2123 ] .
these studies attributed the rise in gzb levels to its nature as proapoptotic protein not as a novel proinflammatory marker .  
accordingly , our study evaluated the association between the blood levels of known inflammatory cytokines like hscrp , il-18 , and fkn with gzb in nondiabetic patients with acute cardiac events .
we also focused on the temporal changes in levels of those biomarkers in patients with stemi versus nstemi / ua to highlight the differential inflammatory response in different types of acs and moreover to explain the impact of sample timing ( day 1 and 3 ) on the recorded results .
the present study demonstrated a significant increase in blood levels of hscrp , gzb , il-18 , and fkn in acs patients compared to the control group .
elevated levels of gzb were detected in the plasma of patients with atherosclerosis , with the highest levels detected in patients with unstable plaques , lending support to the hypothesis that granzyme b influences plaque instability .
in addition , the consequences of elevated granzyme b levels may extend beyond plaque rupture .
had described the novel role of elevated levels of gzb at day 14 after acute myocardial infarction ( ami ) in postinfarct ventricular remodeling .
moreover , the authors found that gzb protein expression was increased at the infarcted myocardium from day 3 to 17 after ami . within the atherosclerotic plaques ,
macrophages , foam cells , and smooth muscle cells ( smcs ) also express gzb as well as it also can be detected extracellularly [ 9 , 25 ] .
interestingly , gzb expression in the atherosclerotic plaque was found to be increased with disease severity .
recently , hendel et al . also identified that the reduced expression of the gzb endogenous inhibitor , proteinase inhibitor 9 , in vascular smcs is associated with the disease progression and that may increase smcs susceptibility to gzb - induced apoptosis within the plaque .
therefore , we found that gzb levels were increased significantly in stemi group in comparison to nstemi / ua group .
although kondo and coworkers have proved that plasma gzb is elevated in patients with ami , however , tsuru et al . could not evaluate changes in plasma gzb concentration in ua patients where only few patients showed detectable results .
this may also support the significant difference in our plasma gzb levels between stemi and nstemi / ua which probably may be due to proapoptotic nature of gzb associated with the significantly higher degree of cardiac necrosis , reflected by significant peak ck and peak ck - mb , and inflammation ( hscrp ) observed in stemi compared to nstemi / ua group .  
there is no expression of fkn in the normal coronary artery ; however , fkn and its receptor cx3cr1 are highly expressed in atherosclerotic lesions . as was the case in our study , li et al .
found higher fkn levels in patients with acs when compared to control subjects or even when compared to patients with stable angina .  
ziakas et al . identified higher peak values of acute phase reactants in patients with stemi versus nstemi / ua . for hscrp , previous studies demonstrated significantly higher admission and peak values in subjects with stemi compared to nstemi and in ami compared to ua [ 31 , 32 ] .
this comes in line with our finding where hscrp levels were significantly higher in stemi patients compared to nstemi / ua patients . for il-18 ,
our finding was confirmed by brunetti et al . who reported that serum levels of il-18 are not affected by acs diagnosis .
furthermore , a recent study revealed that the circulating levels of fkn were not statistically different between ami and ua groups .
an in vitro study identified that the production of gzb from cultured peripheral blood mononuclear cells is time dependent , where gzb levels in culture media gradually increased up to 48 hours after incubation . moreover , gzb showed peak plasma levels on day 7 after ami .
this comes in agreement with our results , where plasma gzb levels significantly increased on day 3 compared to day 1 in both stemi and nstemi / ua groups . in the study conducted by ghanavatian et al .
our study hscrp decreased after 3 days in comparison to day 1 in both groups . to the best of our knowledge ,
this is the first study to compare plasma fkn levels in timed samples after acs . on the other hand
, a study of inflammatory cytokines imbalance in patients with acs revealed that il-18 levels were significantly high on admission and remained unchanged after 12 and 24 hours .
this was the case in our study where serum il-18 did not change significantly from day 1 to day 3 in both groups .
this association could be explained in context of the results of kyaw and coworkers who concluded that cd8 + t lymphocytes , the most abundant inflammatory cells in advanced atherosclerotic lesions , promote plaque vulnerability via gzb - mediated apoptosis of macrophages , smcs , and endothelial cells .
previously , it was shown that the interaction between fkn and its receptor is the main player in trafficking such gzb producing cd8 + t lymphocytes into the vascular lesions   that suggests a strong relationship between fkn and gzb levels as was demonstrated in our results .
the extracellular role of gzb indicates that gzb may have an indirect mechanism for eliciting cytokine release .
recently , it was demonstrated that granzyme b processes il-1 into a significantly more potent proinflammatory fragment .
additionally , a direct relationship between il-18 and gzb was identified by omoto et al . who found that gzb as a serine protease cleaves inactive pro - il-18 at the same residue cleaved by its documented activator caspase-1 ;
hence gzb could be considered as a novel activator of the proinflammatory il-18 . as a result ,
accumulation of the extracellular gzb may result in elevation of serum il-18 levels which is consistent with our results . to the best of our knowledge
however , previous studies showed that ifn- stimulates fkn expression in vascular endothelial cells [ 39 , 40 ] .
moreover , other members of il-1 superfamily such as il-1 were also involved in fkn induction .
these relationships may illustrate the positive significant correlation between fkn and il-18 in our study .
there might be some sort of interplay between gzb , il-18 , and fkn in the pathogenesis of acs .
the current study also confirmed the different inflammatory response in stemi patients with respect to nstemi / ua patients .
indeed , further large scale prospective studies are required to demonstrate the possible use of plasma gzb in risk stratification after myocardial ischemia .
 
 objective . the proapoptotic protein , granzyme b ( gzb ) , was identified as a contributor to the atherosclerotic plaque instability and recently as inflammatory activator . 
 we studied the release kinetics of gzb and other markers of inflammation such as high sensitivity c reactive protein ( hscrp ) , interleukin 18 ( il-18 ) , and fractalkine ( fkn ) in the early phase after acute cardiac events in different acs subgroups . 
 methods . thirty - six nondiabetic patients with acs 
 were compared to 12 control subjects . according to acs diagnosis , 
 the patients were classified into 22 patients with st elevation myocardial infarction ( stemi ) and 14 patients with non - st elevation myocardial infarction or unstable angina ( nstemi / ua ) . 
 blood samples were taken on day 1 ( day of onset ) and day 3 to measure hscrp , il-18 , fkn , and gzb by elisa . 
 results . 
 patients with acs showed significantly higher gzb , il-18 , and fkn levels than the controls . 
 stemi group showed significantly higher gzb levels than nstemi / ua group . on day 3 , fkn levels displayed a significant decrease , while gzb levels were significantly increased . 
 il-18 levels were more or less constant . 
 gzb levels were positively correlated with il-18 ( r = 0.416 , p < 0.01 ) and fkn ( r = 0.58 , p < 0.001 ) . conclusions . 
 unlike il-18 and fkn , plasma gzb may be a marker of acs disease severity . 
1. Introduction
2. Subjects and Methods
3. Results
4. Discussion
5. Conclusions