ABSTRACT years. Fasting serum lipid profile and homocysteine

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Last updated: September 18, 2019

ABSTRACTBackground: Cytochrome P450 (CYP)epoxygenase metabolise arachidonic acid (AA) into four epoxyeicosatrienoicacids (EETs) 5,6- EETs, 8,9 EETs, 11,12-EETs and 14,15-EETs.  Since, EETs are unstable eicosanoids theyrapidly get converted into dihydroxyeicosanoid trienoicacids (DHETs) by solublehydrolase.

These eicosanoids promote defence mechanism against inflammatoryatherosclerosis process. However, 11,12-EETs are more potent eicosanoids inmaintaining anti-atherosclerotic  activity.Endothelial dysfunction is the key step in the pathogenesis of atherosclerosis.Polymorphism in CYP epoxygenase can alter individual’s risk for events incoronary artery disease (CAD) patients. Therefore, we examined the impact ofCYP epoxygenase polymorphism indirectly through CYP metabolites on endothelialdysfunction to predict the risk of events in CAD patients.Methods:Itis a prospective case-control study consisting of 84 acute coronary syndrome (ACS)patients and 84 healthy controls of either gender aged above 18 years.

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Fastingserum lipid profile and homocysteine levels were measured in all subjects. Wemeasured plasma 11,12-dihydroxyeicosatrienoic acid (11,12-DHET) as indicativeof 11,12-EETs. Genotyping of CYP putative exons of CYP2C9, CYP2C19 and CYP2J2epoxygenase were carried out by Polymerase Chain Reaction–Single StrandConformation Polymorphism (PCR-SSCP) method.

Sanger’s sequencing chaintermination method was carried out for the SSCP subtle samples. All the dataobtained were analysed by using Ms-Excel, 2007 and SPSS, version 24.Software,IBM, USA.Results:Weobserved significantly higher levels of  homocysteinein CAD group (35.1 ± 13.

8 µmol/L) indicating higher inflammatory condition inpatients compared to control group (8.1 ± 2.9 µmol/L, p < 0.001). We also foundhigher 11,12-DHET levels  in CAD group  (628.6 ± 324.

3 pg/mL) compared to healthycontrols (332.1 pg/mL ± 203.2 pg/mL, p = 0.0001). In this connection, weobserved positive correlation between homocysteine levels and 11,12- DHETs inCAD group (p = 0.01).

Genotyping of CYP exons revealed 11 patients (13%)reporting 12 single nucleotide polymorphisms (SNPs). Further, we observednegative correlation between homocysteine levels and 11,12-DHETs in CADpatients reporting CYP polymorphisms indicating decline of DHET mediatedanti-atherosclerotic activityConclusions:Reduced 11,12-DHET levels due to CYP2C9, CYP2C19 and CYP2J2 gene polymorphismseems to have considerable effect on endothelial dysfunction and risk of events.Therefore, screening of these cardiac epoxygenase polymorphisms can berecommended to be used as prognostic marker for risk stratification in CADpatients.Keywords:Polymorphism, Cytochrome P450 epoxygenase, Endothelial function and Acutecoronary syndrome patients. Introduction:Despite the advancements in medical therapies forthe past one decade, coronary artery disease (CAD) is still the leading causeof cardiovascular morbidity and mortality worldwide.1 CAD is a heterogenic, multi-factorial diseaseand varies with different ethnic populations. There is an exponentialincrease in the incidence of CAD in all age groups which may be attributed togenetic predisposition besides common risk factors.

2 It is noteworthy thatCAD patients are prone for cardiac events depending upon patient’s individualrisk. Most of the risk factors such as diabetes mellitus, hypertension, smokingand genetic defects for CAD are reported to act through atherosclerosisprocess.3 In human cardiovascular system, nitric oxide(NO) is a powerful vasodilator that prevents the vascular damage. However, whencoronary endothelium is exposed to risk factors, NO production gets impairedand lack of its bioavailability.4,5 Endothelial dysfunction is thekey process that precedes the development of CAD through atherosclerosisprocess. It can be characterised as an imbalance between the humoral andcellular factors that distract the structure and function of coronary wall.

6Therefore in order to maintain vascular homeostasis, coronary endotheliumexpresses Cytochrome P450 (CYP) that metabolises arachidonic acid (AA) toproduce potent epoxyeicosatrienoic acids (EETs).These eicosanoids exertsanti-inflammatory activity on vascular system that promote artery dilation,angiogenesis and protects ischemic myocardium.7 Most of the riskfactors for cardiovascular disease such as hypertension, dyslipedemia,hyperglycemia, smoking and obesity can be controlled and treated through ameliorationof endothelial function. A detailed review of Raja B S et al reveals theimportance of novel risk factors and genetic predisposition that has furtherbroadened our understanding of the pathogenesis of atherosclerosis.

8 Inthis context, genetic factors have also been increasingly recognised as importantcontributors for risk stratification and patient prognostication. Thus, it isnecessary to study molecular mechanism involved in genetic predisposition thatmay pave way for selecting optimal therapies and prevention of complications inCAD patients.9 Therefore, we sought to evaluate the effect of CYP polymorphismand its association with endothelial dysfunction in patients with coronaryartery disease.2.

Methods:2.1 Study populationIt is a singlecenter, prospective case-control study. Eighty four patients diagnosed withacute coronary syndromes, ST – elevated myocardial infarction (STEMI), Non – STelevated myocardial infarction (NSTEMI) and unstable angina (UA) were recruitedin to study from department of cardiology, Sri Venkateswara Institute ofMedical Sciences, Tirupati, Andhra Pradesh, India and consequently 84 healthyvolunteers without any reported cardiovascular risk factors were also includedin the study.2.2 EthicalClearanceThis studyfollowed the ethical guidelines and it was approved by the Institutionalethical committee EC Regn. No. ECR/488/Inst/AP/2013 with IEC approval no.

407. Oral and written informed consent was obtained fromall the study participants. 2.

3 SamplingAll the studyparticipants were recruited between November 2015 and June 2016. Sampling wasdone on 2nd day of myocardial infarction (MI) from all the studypatients. Similarly, sampling was carried out from the healthy volunteers at 12hours fasting state.

Six millilitres (mL) of peripheral venous blood wascollected from all the subjects and aliquated into separate sterile labelled vialsfor biochemical and genetic analysis. All the separated serum, plasma and wholeblood samples were stored at -40°C until analysis.2.4 BiochemicalanalysisFasting totalcholesterol, high density lipid-cholesterol, triglycerides and homocysteinewere evaluated in all the study participants using appropriate commercially availablekits on DXC600 Beckmann auto analyser.2.5 Measurementof plasma 11,12-DHET levelsWe measuredplasma 11,12-DHET forms as a representative of 11,12-EETs (unstable).

Anti-11,12-DHET competitive ELISA kit from Detroit R,Inc.,USA10was used to quantify 11,12-DHETs of both the groups. Plasma samples wereprocessed as per the manufacturer’s instruction manual.

One mL of plasma and1mL ethyl acetate were mixed thoroughly and centrifuged at 2000 rpm for 10 min.The resultant upper organic phase were collected and allowed to evaporate anddry up at room temperature. Dried sample was dissolved in 2 mL of 20% potassiumhydroxide (KOH) and incubated for 1 hour at 50° C. The pH was adjusted toapproximately 5.5 with the help of formic acid. An equal volume of ethylacetate was added to the mixture and centrifugation was repeated.

The resultantorganic phase was collected and dried up at room temperature. The dried pelletwas reconstituted with 20 µL of ethyl alcohol for competitive enzyme assay.2.6 Evaluation of CYP Polymorphism Total genomicDNA of both the study group participants was extracted from ethylenediaminetetraacetic acid (EDTA) treated whole blood sample by using a standard phenol-chloroformextraction method.11 Genotyping was performed by polymerase chainreaction – single strand confirmation polymorphism (PCR-SSCP) technique. PCRamplification of three CYP exons, exon 3 of CYP2C9, exon 5 of CYP2C19 and exon4 of CYP2J2 genes were carried out with the help of suitable primers designedby using primer3 online software tool (Table 1).

PCRreaction mixture contained a final volume of 50?l, and consisted of 100µmolconcentration of each primer, 100?mol of dNTPs mix, 10mM Tris-HCl (pH 8.8), 1.5mM MgCl2, 1U of Taq DNA Polymerase and 0.50µg of genomic DNA. Theparameters for amplification included an initial denaturation for approximately10 minutes at 94°C, denaturation at 94°C for 60 seconds, 30 seconds ofcorresponding annealing at 64.4°C, 45.5°C and 61.

4°C respectively and 50 secondsof amplification at 72°C and this was followed by a final extension step for 5minutes at 72°C in a master cycler gradient thermo cycler. Amplified PCR productswere subjected to SSCP analysis using 6% polyacrylamide vertical gelelectrophoresis. Further, Sanger’s di-deoxy sequencing method was carried outfor the samples that showed mobility difference compared with control in SSCPanalysis.2.

7 Statistical analysisAll the dataobtained were tabulated in Microsoft (Ms) Excel spreadsheets. Descriptivestatistics including mean ± standard deviation (SD) for continuous variables andpercentages for categorical variables were calculated. Unpairedstudent’s t-test and ANOVA followed by multiple comparison tests for continuousdata were performed for the data following normal distribution. Pearson’scorrelation was performed to assess the correlation between the variables. For all the analyses, values of p ? 0.05were considered to be statistically significant. All statistical analysis wasperformed using Ms-Excel 2007 and SPSS 24.0 (IBM Corporation, Chicago, IL,USA).

Results:Male gender wasdominant in both the groups and accounted for 77% and 69% respectively. Meanage of the CAD group was 51.2 ± 9.3 years and 42.1 ± 8.1 years in control group.

Hypertension co-morbidity was more prevalent in 38 (45%) followed by smoking 37(44%) and diabetes mellitus 37 (44%) in our CAD group. Majority of the patients70(83%) presented with STEMI and single vessel disease (SVD) being predominanttype of lesion in 49(58%) patients. Fasting lipid profile including total cholesterol(TC), very low density lipid-cholesterol (VLDL-C) and triglycerides levels werehigher in CAD group compared to control group (Table 2).

We observedsignificantly higher levels of homocysteine in CAD group 35.1 ± 13.8 µmol/Lcompared to control group 8.1 ± 2.9 µmol/L, (p < 0.

001) (Figure 1). In ourstudy group, 73 (87%) of 84 CAD patients and only 2 (3%) of 84 control individualswere found to report hyperhomocysteinemia. We found significantly higher levelsof CYP derived 11,12-DHETs in CAD group 628.6 ± 324.3 pg/mL compared to healthycontrols 331.2 ± 203.2 pg/mL, (p=0.0001) (Table 3).

We also observed positivecorrelation between inflammatory marker homocysteine levels and vasoactive 11,12-DHET levels in CAD group (R2 = 0.087, p=0.01), (Figure 2).  PCRamplification of exon 3 of CYP2C9, exon 5 of CYP2C19 and exon 4 of CYP2J2 genesresulted in amplicon sizes of 308 base pair (bp), 287 bp and 157 bprespectively in both the groups. SSCP analysis of patient amplicons showedmobility differences in 11 patients compared with that of control amplicons. Further,Sanger’s sequencing analysis revealed 12 single nucleotide polymorphisms (SNPs)in 11(13%) patients constituting base substitutions and base insertions. Onepatient, Case 58 was found to report both CYP2C9 and CYP2C19 gene polymorphismbeing novel and reported mutations respectively.

All the identified mutationsof exon 3 of CYP2C9, exon 5 of CYP2C19 and exon 5 of CYP2J2 genes werecommunicated to GenBank and their corresponding accession numbers were depictedin Table 4. CYP2C9 gene base substitutions were found in 3 patients of whichone patient was found to report CYP2C9*2 allele, c.430C>T (Case 47, Figure 3-A) and novel basesubstitutions in other two patients (Case 58 and Case 73).

CYP2C19 gene SNP basesubstitutions were found in 5 patients of which 3 patients were found to reportCYP2C19*2, c.681G>A(Figure 3-B) and two patients reported novel base substitutions (Case 5 andCase 10). CYP2J2 genepolymorphism constituting novel base substitutions (Figure 3-C) were found in 4patients and with Cytosine base insertions in Case 31.

In this connection, wealso noticed comparatively reduced levels of 11,12-DHETs in the patientsreporting CYP gene polymorphisms (Table 4).Further,data analysis revealed significant differences in 11,12-DHET levels between patientsreporting CYP polymorphism  and  patients without CYP polymorphism comparedwith the control individuals (p < 0.001),(Figure 4). We also observednegative correlation between homocysteine and 11,12-DHETs levels (R2= 0.222, p = 0.143) with the patients reporting CYP polymorphism (Figure 5).

However,we did not find any association between diabetic and hypertensive CAD patientsand 11,12-DHETs levels.                               Discussion:There is an increasing appreciation on the importanceof EET/DHET mediated vasodilation.12,13 CYP2C9, CYP2C19 and CYP2J2 are the main epoxygenases in humancardiovascular system particularly found in cardiomyocytes, endothelium andsmooth muscle cells (SMCs).14 CYP epoxygenase derived EETs/DHETs promotedefence activity against the inflammatory stimulus.

Inter-individualdifferential expression of regulatory enzymes can influence the function andrisk of developing disease.15 Thus, differential gene expression of CYP epoxygenase canlead to functional alterations of epoxygenase activity that may increase theindividual’s risk. In our study group, we observed hypertension, smoking and diabetesmellitus co-morbidities are more prevalent in CAD group that can increase therisk of developing events. Hyperhomocysteinemia is also well establishedindependent novel risk factor associated with early onset of CAD and risk ofvenous thrombosis.16,17  JatinD P et al reported age wise hyperhomocysteinemia in 78-82% of CAD patients andonly 5% in controls.18 Consistently, we also found higher levels of homocysteinein 87% of CAD patients and only 3% in control individuals.

Higher levels ofhomocysteine can cause endothelial damage and increase the risk of developingevents in CAD patients. Akasaka T et al in 2016 reported that higher levels of EETs/DHETsin CAD patients represent the protective defence mechanism against theinflammatory endothelial damage.19In this connection, we also observedsignificantly higher levels of 11,12-DHETs in response to the higherinflammatory stimuli in CAD group compared to control group. Interestingly, wenoticed significant positive correlation between homocysteine levels and 11,12-DHETsin CAD patients depicting the protective anti-inflammatory activity of DHET againsthigher homocysteine levels in CAD group. Similar observations were found with thestudy of Yang T et al in 2013 who reported higher levels of DHETs correspondingto higher levels of high sensitive – C reactive protein (hs-CRP) in CAD patients.10However, we did not find any correlation between 11,12-DHET levels and otherrisk factors like hypertension, diabetes and blood lipoproteins. It isnote worthy that about 20% of the CAD patients report with low or no prevalenceof traditional risk factors.

Thus, it isnecessary to concentrate on novel risk factors including genetic polymorphismthat may pave way to develop new strategy. A study by Arun kumar A S et al.,(2015) suggests that the individuals with any confounding risk factors for CVDalong with CYP epoxygenase polymorphism may be predisposed to risk of CAD.20 Genetic polymorphism in these CYP epoxygenasescan alter the epoxygenase activity and decline of DHET mediated vasodilation. CYP2C9 is an important epoxygenasein endothelial cells that contributes higher 11,12-EET/DHET mediated vascularhomeostasis.

21 Any genevariations in this epoxygenase may result in poor AA metabolism. Presenceof CYP2C9*2 in exon 3 was reported to show 50% reduced activity compared to thewild type.22 Consistently, we observed reduced levels of 11,12 DHETsin the patients reporting CYP2C9 polymorphism depicted in table 4. A study by CrespiCL & Miller VP in 1997 also reported that presence of CYP2C9*2 altered theinteraction of epoxygenase with substrate and reduced metabolism.23 Inour study CYP2C9*2 allele (c.430C>T, R144C) was found only in one patientand accounted for 1.

2% which is less compared to the study by Jose R et al in2004 reporting 4%.24 clinicalImportance  of base substitution.CYP2C19 is also an importantepoxygenase and any variations in the gene encoding this enzyme may lead toreduced epoxygenase activity. CYP2C19*2 allele has clinical importance especiallyin acute coronary syndrome patients.12,25 Akasava T et al. in 2016 also reported that patients withCYP2C19*2 allele showed reduced levels of plasma 11,12- DHETs compared to thepatients without the mutant allele.19  Three of 84 CAD patients (case 8, Case 58& case 68) were found to report CYP2C19*2 allele (c.

681G>A, p.G228R) and accountedfor 3.6% and it was comparatively low with the studies reporting 12% and 10% byShuldiner AR et al 26 and Tantray JA et al 27respectively in south Indian population. Genetic polymorphismin CYP2C19 gene was reported to be independent risk factor for cardiovascularevents irrespective of clopidogrel resistance.28 Thus,reduced levels of DHETs in CAD patients result in decline of DHET-mediateddefence activity against vascular inflammation that can cause endothelial dysfunction.

CYP2J2 is cardiac specific epoxygenase expressedpredominately in vascular endothelial cells. Presence of CYP2J2*4 allele inexon 4 significantly decreases the CYP2J2 epoxygenase activity, especiallyresults in reduced expression of 11,12 DHETs compared to the wild type. However,Asians are rare to this allele accounting for 0-2%29 and it was consistent with our study none was foundto report CYP2J2*4 allele. Variations in gene sequences may lead to alteredenzyme activity and have effect on cardiovascular homeostasis and diseaseoutcomes. We observed CYP2J2 novel base substitutions and Cinsertions in the patients of case23 and case 40 reporting single base substitution c.646G>Aand in patients of case 31 & case 73 reporting with C insertions at c.664_665insC and c.673_674insCrespectively.

We noticed comparatively reduced levels of 11,12- DHETs in thepatients reporting C base insertions in case 31,  and case 73,  depicting reduced enzyme activity due to frameshift mutation. A study by Indrayan Ain 2013 reported that  gene variations inthese epoxygenases influence their activity that can act as important modifiersof cardiovascular risk in CAD patients.30            Interestingly,we observed negative correlation between 11,12-DHETs and  homocysteine levels in 11 patients reporting CY2C9,CYP2C19 and CYP2J2 polymorphisms depicting the reduced enzyme activity. Thus,our study findings showed that presence of CYP polymorphisms result in reducedlevels of 11,12-DHETs and decline of DHET mediated vasodilation that can causeendothelial dysfunction and risk of events. Conclusions:We observeddecreased levels of 11,12- DHETs in these patients  compared to the patients withoutpolymorphism.

Presence of lower levels of 11,12- DHETs is a reflection of poorreserve defence mechanism in CAD patients that might result in endothelialdysfunction and lead to cardiac events.Therefore,in acute coronary syndromes patients showing lower 11,12-DHET levels,genotyping of CYP2C9, CYP2C19 and CYP2J2 genes may serve as a prognostic markerfor  future events.

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