ABSTRACT years. Fasting serum lipid profile and homocysteine

ABSTRACT

Background: Cytochrome P450 (CYP)
epoxygenase metabolise arachidonic acid (AA) into four epoxyeicosatrienoic
acids (EETs) 5,6- EETs, 8,9 EETs, 11,12-EETs and 14,15-EETs.  Since, EETs are unstable eicosanoids they
rapidly get converted into dihydroxyeicosanoid trienoicacids (DHETs) by soluble
hydrolase. These eicosanoids promote defence mechanism against inflammatory
atherosclerosis process. However, 11,12-EETs are more potent eicosanoids in
maintaining 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 in
coronary artery disease (CAD) patients. Therefore, we examined the impact of
CYP epoxygenase polymorphism indirectly through CYP metabolites on endothelial
dysfunction to predict the risk of events in CAD patients.

Methods:
It
is a prospective case-control study consisting of 84 acute coronary syndrome (ACS)
patients and 84 healthy controls of either gender aged above 18 years. Fasting
serum lipid profile and homocysteine levels were measured in all subjects. We
measured plasma 11,12-dihydroxyeicosatrienoic acid (11,12-DHET) as indicative
of 11,12-EETs. Genotyping of CYP putative exons of CYP2C9, CYP2C19 and CYP2J2
epoxygenase were carried out by Polymerase Chain Reaction–Single Strand
Conformation Polymorphism (PCR-SSCP) method. Sanger’s sequencing chain
termination method was carried out for the SSCP subtle samples. All the data
obtained were analysed by using Ms-Excel, 2007 and SPSS, version 24.Software,
IBM, USA.

Results:
We
observed significantly higher levels of  homocysteine
in CAD group (35.1 ± 13.8 µmol/L) indicating higher inflammatory condition in
patients compared to control group (8.1 ± 2.9 µmol/L, p < 0.001). We also found higher 11,12-DHET levels  in CAD group  (628.6 ± 324.3 pg/mL) compared to healthy controls (332.1 pg/mL ± 203.2 pg/mL, p = 0.0001). In this connection, we observed positive correlation between homocysteine levels and 11,12- DHETs in CAD group (p = 0.01). Genotyping of CYP exons revealed 11 patients (13%) reporting 12 single nucleotide polymorphisms (SNPs). Further, we observed negative correlation between homocysteine levels and 11,12-DHETs in CAD patients reporting CYP polymorphisms indicating decline of DHET mediated anti-atherosclerotic activity Conclusions: Reduced 11,12-DHET levels due to CYP2C9, CYP2C19 and CYP2J2 gene polymorphism seems to have considerable effect on endothelial dysfunction and risk of events. Therefore, screening of these cardiac epoxygenase polymorphisms can be recommended to be used as prognostic marker for risk stratification in CAD patients. Keywords: Polymorphism, Cytochrome P450 epoxygenase, Endothelial function and Acute coronary syndrome patients.  Introduction: Despite the advancements in medical therapies for the past one decade, coronary artery disease (CAD) is still the leading cause of cardiovascular morbidity and mortality worldwide.1 CAD is a heterogenic, multi-factorial disease and varies with different ethnic populations. There is an exponential increase in the incidence of CAD in all age groups which may be attributed to genetic predisposition besides common risk factors.2 It is noteworthy that CAD patients are prone for cardiac events depending upon patient's individual risk. Most of the risk factors such as diabetes mellitus, hypertension, smoking and genetic defects for CAD are reported to act through atherosclerosis process.3 In human cardiovascular system, nitric oxide (NO) is a powerful vasodilator that prevents the vascular damage. However, when coronary endothelium is exposed to risk factors, NO production gets impaired and lack of its bioavailability.4,5 Endothelial dysfunction is the key process that precedes the development of CAD through atherosclerosis process. It can be characterised as an imbalance between the humoral and cellular factors that distract the structure and function of coronary wall.6 Therefore in order to maintain vascular homeostasis, coronary endothelium expresses Cytochrome P450 (CYP) that metabolises arachidonic acid (AA) to produce potent epoxyeicosatrienoic acids (EETs).These eicosanoids exerts anti-inflammatory activity on vascular system that promote artery dilation, angiogenesis and protects ischemic myocardium.7 Most of the risk factors for cardiovascular disease such as hypertension, dyslipedemia, hyperglycemia, smoking and obesity can be controlled and treated through amelioration of endothelial function. A detailed review of Raja B S et al reveals the importance of novel risk factors and genetic predisposition that has further broadened our understanding of the pathogenesis of atherosclerosis.8 In this context, genetic factors have also been increasingly recognised as important contributors for risk stratification and patient prognostication. Thus, it is necessary to study molecular mechanism involved in genetic predisposition that may pave way for selecting optimal therapies and prevention of complications in CAD patients.9 Therefore, we sought to evaluate the effect of CYP polymorphism and its association with endothelial dysfunction in patients with coronary artery disease. 2. Methods: 2.1 Study population It is a single center, prospective case-control study. Eighty four patients diagnosed with acute coronary syndromes, ST - elevated myocardial infarction (STEMI), Non - ST elevated myocardial infarction (NSTEMI) and unstable angina (UA) were recruited in to study from department of cardiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India and consequently 84 healthy volunteers without any reported cardiovascular risk factors were also included in the study. 2.2 Ethical Clearance This study followed the ethical guidelines and it was approved by the Institutional ethical committee EC Regn. No. ECR/488/Inst/AP/2013 with IEC approval no. 407. Oral and written informed consent was obtained from all the study participants. 2.3 Sampling All the study participants were recruited between November 2015 and June 2016. Sampling was done on 2nd day of myocardial infarction (MI) from all the study patients. Similarly, sampling was carried out from the healthy volunteers at 12 hours fasting state. Six millilitres (mL) of peripheral venous blood was collected from all the subjects and aliquated into separate sterile labelled vials for biochemical and genetic analysis. All the separated serum, plasma and whole blood samples were stored at -40°C until analysis. 2.4 Biochemical analysis Fasting total cholesterol, high density lipid-cholesterol, triglycerides and homocysteine were evaluated in all the study participants using appropriate commercially available kits on DXC600 Beckmann auto analyser. 2.5 Measurement of plasma 11,12-DHET levels We measured plasma 11,12-DHET forms as a representative of 11,12-EETs (unstable). Anti-11,12-DHET competitive ELISA kit from Detroit R,Inc.,USA10 was used to quantify 11,12-DHETs of both the groups. Plasma samples were processed as per the manufacturer's instruction manual. One mL of plasma and 1mL ethyl acetate were mixed thoroughly and centrifuged at 2000 rpm for 10 min. The resultant upper organic phase were collected and allowed to evaporate and dry up at room temperature. Dried sample was dissolved in 2 mL of 20% potassium hydroxide (KOH) and incubated for 1 hour at 50° C. The pH was adjusted to approximately 5.5 with the help of formic acid. An equal volume of ethyl acetate was added to the mixture and centrifugation was repeated. The resultant organic phase was collected and dried up at room temperature. The dried pellet was reconstituted with 20 µL of ethyl alcohol for competitive enzyme assay. 2.6 Evaluation of CYP Polymorphism Total genomic DNA of both the study group participants was extracted from ethylenediamine tetraacetic acid (EDTA) treated whole blood sample by using a standard phenol-chloroform extraction method.11 Genotyping was performed by polymerase chain reaction - single strand confirmation polymorphism (PCR-SSCP) technique. PCR amplification of three CYP exons, exon 3 of CYP2C9, exon 5 of CYP2C19 and exon 4 of CYP2J2 genes were carried out with the help of suitable primers designed by using primer3 online software tool (Table 1). PCR reaction mixture contained a final volume of 50?l, and consisted of 100µmol concentration of each primer, 100?mol of dNTPs mix, 10mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 1U of Taq DNA Polymerase and 0.50µg of genomic DNA. The parameters for amplification included an initial denaturation for approximately 10 minutes at 94°C, denaturation at 94°C for 60 seconds, 30 seconds of corresponding annealing at 64.4°C, 45.5°C and 61.4°C respectively and 50 seconds of amplification at 72°C and this was followed by a final extension step for 5 minutes at 72°C in a master cycler gradient thermo cycler. Amplified PCR products were subjected to SSCP analysis using 6% polyacrylamide vertical gel electrophoresis. Further, Sanger's di-deoxy sequencing method was carried out for the samples that showed mobility difference compared with control in SSCP analysis. 2.7 Statistical analysis All the data obtained were tabulated in Microsoft (Ms) Excel spreadsheets. Descriptive statistics including mean ± standard deviation (SD) for continuous variables and percentages for categorical variables were calculated. Unpaired student's t-test and ANOVA followed by multiple comparison tests for continuous data were performed for the data following normal distribution. Pearson's correlation was performed to assess the correlation between the variables. For all the analyses, values of p ? 0.05 were considered to be statistically significant. All statistical analysis was performed using Ms-Excel 2007 and SPSS 24.0 (IBM Corporation, Chicago, IL, USA). Results: Male gender was dominant in both the groups and accounted for 77% and 69% respectively. Mean age 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 patients 70(83%) presented with STEMI and single vessel disease (SVD) being predominant type of lesion in 49(58%) patients. Fasting lipid profile including total cholesterol (TC), very low density lipid-cholesterol (VLDL-C) and triglycerides levels were higher in CAD group compared to control group (Table 2). We observed significantly higher levels of homocysteine in CAD group 35.1 ± 13.8 µmol/L compared to control group 8.1 ± 2.9 µmol/L, (p < 0.001) (Figure 1). In our study group, 73 (87%) of 84 CAD patients and only 2 (3%) of 84 control individuals were found to report hyperhomocysteinemia. We found significantly higher levels of CYP derived 11,12-DHETs in CAD group 628.6 ± 324.3 pg/mL compared to healthy controls 331.2 ± 203.2 pg/mL, (p=0.0001) (Table 3).We also observed positive correlation between inflammatory marker homocysteine levels and vasoactive 11,12- DHET levels in CAD group (R2 = 0.087, p=0.01), (Figure 2).   PCR amplification of exon 3 of CYP2C9, exon 5 of CYP2C19 and exon 4 of CYP2J2 genes resulted in amplicon sizes of 308 base pair (bp), 287 bp and 157 bp respectively in both the groups. SSCP analysis of patient amplicons showed mobility 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. One patient, Case 58 was found to report both CYP2C9 and CYP2C19 gene polymorphism being novel and reported mutations respectively. All the identified mutations of exon 3 of CYP2C9, exon 5 of CYP2C19 and exon 5 of CYP2J2 genes were communicated to GenBank and their corresponding accession numbers were depicted in Table 4. CYP2C9 gene base substitutions were found in 3 patients of which one patient was found to report CYP2C9*2 allele, c.430C>T (Case 47, Figure 3-A) and novel base
substitutions in other two patients (Case 58 and Case 73). CYP2C19 gene SNP base
substitutions were found in 5 patients of which 3 patients were found to report
CYP2C19*2, c.681G>A
(Figure 3-B) and two patients reported novel base substitutions (Case 5 and
Case 10). CYP2J2 gene
polymorphism constituting novel base substitutions (Figure 3-C) were found in 4
patients and with Cytosine base insertions in Case 31. In this connection, we
also noticed comparatively reduced levels of 11,12-DHETs in the patients
reporting CYP gene polymorphisms (Table 4).

Further,
data analysis revealed significant differences in 11,12-DHET levels between patients
reporting CYP polymorphism  and  patients without CYP polymorphism compared
with the control individuals (p < 0.001),(Figure 4). We also observed negative 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 patients and 11,12-DHETs levels.                                                       Discussion: There is an increasing appreciation on the importance of EET/DHET mediated vasodilation.12,13 CYP2C9, CYP2C19 and CYP2J2 are the main epoxygenases in human cardiovascular system particularly found in cardiomyocytes, endothelium and smooth muscle cells (SMCs).14 CYP epoxygenase derived EETs/DHETs promote defence activity against the inflammatory stimulus. Inter-individual differential expression of regulatory enzymes can influence the function and risk of developing disease.15 Thus, differential gene expression of CYP epoxygenase can lead to functional alterations of epoxygenase activity that may increase the individual's risk. In our study group, we observed hypertension, smoking and diabetes mellitus co-morbidities are more prevalent in CAD group that can increase the risk of developing events. Hyperhomocysteinemia is also well established independent novel risk factor associated with early onset of CAD and risk of venous thrombosis.16,17  Jatin D P et al reported age wise hyperhomocysteinemia in 78-82% of CAD patients and only 5% in controls.18 Consistently, we also found higher levels of homocysteine in 87% of CAD patients and only 3% in control individuals. Higher levels of homocysteine can cause endothelial damage and increase the risk of developing events in CAD patients. Akasaka T et al in 2016 reported that higher levels of EETs/DHETs in CAD patients represent the protective defence mechanism against the inflammatory endothelial damage.19 In this connection, we also observed significantly higher levels of 11,12-DHETs in response to the higher inflammatory stimuli in CAD group compared to control group. Interestingly, we noticed significant positive correlation between homocysteine levels and 11,12-DHETs in CAD patients depicting the protective anti-inflammatory activity of DHET against higher homocysteine levels in CAD group. Similar observations were found with the study of Yang T et al in 2013 who reported higher levels of DHETs corresponding to higher levels of high sensitive - C reactive protein (hs-CRP) in CAD patients.10 However, we did not find any correlation between 11,12-DHET levels and other risk factors like hypertension, diabetes and blood lipoproteins. It is note worthy that about 20% of the CAD patients report with low or no prevalence of traditional risk factors. Thus, it is necessary to concentrate on novel risk factors including genetic polymorphism that 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 CVD along with CYP epoxygenase polymorphism may be predisposed to risk of CAD.20 Genetic polymorphism in these CYP epoxygenases can alter the epoxygenase activity and decline of DHET mediated vasodilation. CYP2C9 is an important epoxygenase in endothelial cells that contributes higher 11,12-EET/DHET mediated vascular homeostasis.21 Any gene variations in this epoxygenase may result in poor AA metabolism. Presence of CYP2C9*2 in exon 3 was reported to show 50% reduced activity compared to the wild type.22 Consistently, we observed reduced levels of 11,12 DHETs in the patients reporting CYP2C9 polymorphism depicted in table 4. A study by Crespi CL & Miller VP in 1997 also reported that presence of CYP2C9*2 altered the interaction of epoxygenase with substrate and reduced metabolism.23 In our study CYP2C9*2 allele (c.430C>T, R144C) was found only in one patient
and accounted for 1.2% which is less compared to the study by Jose R et al in
2004 reporting 4%.24 clinical
Importance  of base substitution.

CYP2C19 is also an important
epoxygenase and any variations in the gene encoding this enzyme may lead to
reduced epoxygenase activity. CYP2C19*2 allele has clinical importance especially
in acute coronary syndrome patients.12,25 
Akasava T et al. in 2016 also reported that patients with
CYP2C19*2 allele showed reduced levels of plasma 11,12- DHETs compared to the
patients 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 accounted
for 3.6% and it was comparatively low with the studies reporting 12% and 10% by
Shuldiner AR et al 26 and Tantray JA et al 27
respectively in south Indian population. Genetic polymorphism
in CYP2C19 gene was reported to be independent risk factor for cardiovascular
events irrespective of clopidogrel resistance.28 Thus,
reduced levels of DHETs in CAD patients result in decline of DHET-mediated
defence activity against vascular inflammation that can cause endothelial dysfunction.

CYP2J2 is cardiac specific epoxygenase expressed
predominately in vascular endothelial cells. Presence of CYP2J2*4 allele in
exon 4 significantly decreases the CYP2J2 epoxygenase activity, especially
results 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 found
to report CYP2J2*4 allele. Variations in gene sequences may lead to altered
enzyme activity and have effect on cardiovascular homeostasis and disease
outcomes. We observed CYP2J2 novel base substitutions and C
insertions in the patients of case
23 and case 40 reporting single base substitution c.646G>A
and in patients of case 31 & case 73 reporting with C insertions at c.
664_665insC and c.673_674insC
respectively. We noticed comparatively reduced levels of 11,12- DHETs in the
patients reporting C base insertions in case 
31,  and case 73,  depicting reduced enzyme activity due to frame
shift mutation. A study by Indrayan A
in 2013 reported that  gene variations in
these epoxygenases influence their activity that can act as important modifiers
of 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 reduced
levels of 11,12-DHETs and decline of DHET mediated vasodilation that can cause
endothelial dysfunction and risk of events.

Conclusions:

We observed
decreased levels of 11,12- DHETs in these patients  compared to the patients without
polymorphism. Presence of lower levels of 11,12- DHETs is a reflection of poor
reserve defence mechanism in CAD patients that might result in endothelial
dysfunction 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 marker
for  future events.

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