Mohammad Saifur Rohman

279 Val→Phe Polymorphism of lipoprotein-associated phospholipase A2 resulted in changes of folding kinetics and recognition to substrate

INTRODUCTION PLA2G7 encodes Lp-PLA2 having role in the formation of atherosclerotic plaques by catalyzing its substrate, phosphatydilcholine (PC), to be pro-inflammatory substances. The increased risk for coronary artery disease (CAD) in Asian population has been related with this enzyme. 279(Val→Phe) variant was reported to have a protective role against CAD due to, in part, secretion defect or loss of enzymatic function. Therefore, We study folding kinetics and enzyme-substrate interaction in 279(Val→Phe) by using clinical and computational biology approach. METHODS Polymorphisms were detected by genotyping among 103 acute myocardial infarction patients and 37 controls. Folding Lp-PLA2 was simulated using GROMACS software by assessing helicity, hydrogen bond formation and stability. The interactions of Lp-PLA2 and its substrate were simulated using Pyrx software followed by molecular dynamics simulation using YASARA software. RESULT Polymorphism of 279(Val→Phe) was represented by the change of nucleotide from G to T of 994th PLA2G7 gene. The folding simulation suggested a decreased percentage of α-helix, hydrogen bond formation, hydrogen bond stability and hydrophobicity in 279(Val→Phe). The PC did not interact with active site of 279(Val→Phe) as paradoxically observed in 279 valine. 279(Val→Phe) polymorphism is likely to cause unstable binding to the substrate and decrease the enzymatic activity as observed in molecular dynamics simulations. The results of our computational biology study supported a protected effect of 279(Val→Phe) Polymorphism showed by the odd ratio for MI of 0.22 (CI 95% 0.035-1.37) in this study. CONCLUSION 279(Val→Phe) Polymorphism of Lp-PLA2 may lead to decrease the enzymatic activity via changes of folding kinetics and recognition to its substrate.

Genetic Variants of C-5312T REN Increased Renin Levels and Diastolic Blood Pressure Response to Angiotensin Receptor Blockers

Renin catalyzes the cleavage of angiotensinogen into angiotensin I. Genetic variant C-5312T of renin enhancer has been reported to increase in vitro renin gene transcription. However, no obvious in vivo study was performed to see the renin level in C-5312T when treated with angiotensin receptor blockers (ARB). Therefore, this study aimed to investigate the serum renin level and blood pressure response in ARB treated hypertensive patients. Single nucleotide polymorphism (SNP) of C-5312T was identified in 55 hypertensive patients by using multiplex PCR and renin serum level was assayed by ELISA. The data showed that the increase of serum renin levels after 5 months of ARB treatment was significantly higher in patients with CT/TT genotype (10 pg/mL) than those with CC genotype (4.08 pg/mL) (P = 0.025). Hypertensive patients with CT/TT genotypes also showed less diastolic pressure reduction than CC genotypes in hypertensive patients with valsartan treatment (P = 0.04) or telmisartan treatment (P = 0.03). Finally, these findings suggested that SNP of C-5312T REN enhancer might contribute to higher increased renin serum levels and less diastolic blood pressure response to ARB treatment.

Genetic Variant of C-5312T Can Change Binding Pattern of Sp1 to Renin Enhancer that are Very Likely to Affect Renin Gene Expression.

Renin distal enhancer plays a pivotal role in renin gene expression, and the genetic variants C-5312T of renin enhancer can affect renin gene transcription level. However, the mechanism associated with the transcription level changes remains unknown. Therefore, it is of interest to investigate the possible role of distal enhancer in regulating the expression of renin gene. Single nucleotide polymorphism in renin distal enhancer was identified in 34 hypertensive patients by automatic sequencing. The data showed that the renin enhancer from the patients have genetic variants C-5312T or C-5312T SNP. Hence, the functionality of the renin enhancer and influence of the genetic variants C-5312T on binding to Sp1 is studied. These results from the binding study suggested that Sp1 binds to the DNA in GC rich region. Thus, the genetic variant C-5312T has changed the binding pattern of Sp1 to renin enhancer. This is likely to influence Sp1 activity to stimulate the expression of renin gene. The binding of Sp1 to the cis-element will enhance transcription of renin gene. Thus, polymorphism within C-5312T might contribute to the reduction of renin transcription.

Val279Phe variant of Lp-PLA2 is a risk factor for a subpopulation of Indonesia patients with acute myocardial infarction

Lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of the phospholipase A2 superfamily, is an enzyme that hydrolyses phospholipids, is found in blood circulation as a sign of inflammation, and takes a role in atherogenesis. There is an epidemiologic relation between increased Lp-PLA2 levels and coronary heart disease. Lp-PLA2 is an enzyme that is produced by macrophages and takes a role as an independent predictor of a coronary event. A genetic variant of Val279Phe on the Lp-PLA2 gene has been reported with various results in Japan, China, Korea, and Caucasian populations. This study aims to analyse the influence of the Val279Phe genetic variant on acute myocardial infarction (AMI) at Saiful Anwar Hospital, Indonesia. This study was conducted on 151 patients (111 AMI patients and 40 non-AMI patients). The genetic variant of Val279Phe was identified through a genotyping method. There were no significant differences in age, total cholesterol level, LDL-C (low-density lipoprotein cholesterol) level, and family history data between AMI and non-AMI patients. However, AMI patients had low HDL-C (high-density lipoprotein cholesterol), triglyceride levels, dyslipidaemia, and hypertension risk factors compared to non-AMI patients. The frequency of the GG genotype (279Val) was dominant in both AMI and non-AMI groups. Further analysis suggested that the GG genotype has a 2.9 times greater risk of AMI compared to the GT/TT genotype (279Phe). This study concluded that the Val279Phe genetic variant undoubtedly influenced AMI risk, which is a warrant for further development of early detection and improving strategy to prevent AMI in patients.

Genetic Variant of C-5434T REN Enhancer on Serum Renin Levels and Binding Pattern of Signal Transducers and Activators Transcription 3.

The human renin gene has been widely known to be involved in essential hypertension (EH) pathogenesis. Genetic variant C-5434T of REN enhancer contributed to renin gene transcription and serum renin regulation. However, the mechanism associated with the transcription level changes remains unknown, and only a few reports exist that discussed serum renin levels on C-5434T of REN. Thus, this study aims to investigate the relationship between genetic variant C-5434T of REN enhancer and serum renin levels in Indonesian hypertensive patients. SNP of C-5434T was genotyped in 56 hypertensive patients by using RFLP. The data showed that serum renin is slightly higher in hypertensive patients with the TT genotype (39 ± 10.3) than patients with the CC genotype (33 ± 10.6) but the difference was not statistically significant (p = 0.35). Here, we also present a docking approach for predicting interaction between genetic variant -5434C/T and STAT3 (Signal Transducers and Activators Transcription 3), the predicted transcription factor that regulates renin gene enhancer. The results showed that STAT3-DNA allele T more favorably binds to DNA than STAT3-DNA allele C. These data suggest that the presence of genetic variant C-5434T has changed the binding pattern of STAT3 to REN enhancer. This is likely to influence STAT3 activity to stimulate the expression of renin gene in producing renin.

21 ANTIHYPERTENSIVE MEDICATION ADHERENCE REDUCE REHOSPITALIZATION AMONG HYPERTENSIVE HEART FAILURE PATIENT AT SAIFUL ANWAR HOSPITAL

Background: Previous studies suggest that medication adherence strongly related to cardiovascular outcome, including rehospitalization event. Objective: The study aims to determine the correlation between antihypertensive medication adherence and rehospitalization rate. Method: We conducted a cross sectional research of hypertensive heart failure patient, who were hospitalized in Saiful Anwar Hospital from January to December 2017. Data were collected based on patient demographic, medical history, laboratory findings, antihypertensive therapies, and readmission events. The association between medication adherence and rehospitalization events were examined using logistic regression. Result: Hypertension was noted among 57,8% heart failure patient. Among hypertensive patient, as much as 39,8% were controlled. Compliance between controlled patient only 21,1%. The risk of hospitalization was increased stepwise with decreasing adherence. The hazard risk for the risk of hospitalization were estimated as 5.578 (CI 95% 2.561 – 12.945) for the poor adherence group. We also examine the precipitating factors related to rehospitalization event. They were acute coronary syndrome (31,3%), poor compliance (21,1%), infection (12,7%), hypertension crisis (12,0%), arrhythmia (11,4%), and others (11,4%). Conclusion: Poor medication adherence was associated with a greater risk of hospitalization.

28 GREEN TEA EXTRACT ADMINISTRATION HAD A BENEFICIAL EFFECT ON PPAR ALPHA AND PPAR GAMMA GENE EXPRESSION IN METABOLIC SYNDROME RAT MODEL

Background: PPARs play important role in metabolic syndrome regulation thus, they become the molecular therapeutic target of some drugs. The benefit of green tea extract administration on these molecular targets needs further investigation. This study aimed to investigate the effect of green tea extract administration on PPAR&agr; and PPAR&ggr; gene expression in metabolic syndrome rat models. Methods: Thirty Sprague Dawley Metabolic Syndrome Rat Model weighed 300 – 400 grams were divided into EGCG 200 (n = 5), EGCG 300 (n = 5), and EGCG 400 (n = 5) groups. Moreover, as control groups fifteen rats were divided into normal control (NC) (n = 5), simvastatin and metformin treated (SM) (n = 5), and metabolic syndrome (MS) (n = 5) groups. Rats in the EGCG 200, 300, and 400 groups were treated once daily with green tea extract at a dose of 200, 300, and 400 mg/bw.t respectively. Moreover, Rats in the SM group were treated daily with 10 mg/bw.t of simvastatin and 200 mg/bw.t of metformin. The extract as well as the drugs were administrated for 9 weeks through oral gavage. Fasting blood glucose, triglyceride, and HDL level was measured every two weeks by colorimetric method. Blood pressure was determined by tail cuff blood pressure recorder. PPAR&agr; and PPAR&ggr; gene expression was analysed by RT-PCR methods. Result: After administration of green tea extract for 9 weeks, fasting blood glucose, triglyceride, and systolic blood pressure were significantly lower (p < 0.05), and HDL-Cholesterol was significantly higher in all EGCG groups compared to that of MS group. Moreover, PPAR&agr; gene expression significantly higher in all EGCG and SM group (p > 0.05) after 9 week compared to that of MS group. Moreover, PPAR&agr; gene expression in all EGCG group were similar with that of SM groups (p > 0.05). Furthermore, PPAR&ggr; gene expression were significantly higher in all EGCG group compared to that of MS groups (p < 0.05). On the other hand, PPAR&ggr; gene expression in SM group were significantly higher (p < 0.05) compared to those of all EGCG group. Conclusion: Green tea extract administration for 9 weeks have beneficial effect in increasing gene expression of PPAR&agr; and PPAR&ggr; in metabolic syndrome rat model

30 GREEN COFFEE EXTRACT INCREASED PPAR ALPHA AND PPAR GAMMA GENE EXPRESSION IN METABOLIC SYNDROME RAT MODEL

Background: PPARs act as pivotal regulators of metabolic syndrome. PPAR&agr; and PPAR&ggr; are the molecular targets of a number of marketed drugs for the treatment of metabolic syndrome. Green coffee administration is a new candidates of metabolic syndrome treatment which need further investigation of its mechanism. This study aimed to investigate the effect of green coffee extract administration on PPAR&agr; and PPAR&ggr; gene expression in metabolic syndrome rat models. Methods: Thirty Sprague Dawley Metabolic Syndrome Rat Model weighed 300 – 400 grams were divided into CGA 100 (n = 5), CGA 200 (n = 5), and CGA 250 (n = 5) groups. Moreover, as control groups fifteen rats were divided into normal control (NC) (n = 5), simvastatin and metformin treated (SM) (n = 5), and metabolic syndrome (MS) (n = 5) groups. Rats in the 100 groups were treated once daily with green coffee extract at a dose of 100, 200, and 250 mg/bw.t respectively. The extract was given for 9 weeks through oral gavage. Fasting blood glucose, triglyceride, and HDL level was measured every two weeks by colorimetric method. Blood pressure was determined by tail cuff blood pressure recorder. PPAR&agr; and PPAR&ggr; gene expression was analyzed by RT-PCR methods. Result: Fasting blood glucose, triglyceride, and systolic blood pressure significantly decreased (p < 0.05) and HDL-Cholesterol was significantly higher in all CGA groups after administration of green coffee extract for 9 weeks. Moreover, after administration of green coffee extract for 9 weeks, PPAR&agr; gene expression was significantly higher in all CGA and SM group (p > 0.05) compared to that of MS group. Furthermore, PPAR&agr; gene expression in CGA 100 and 200 group were not significantly different (p > 0.05) compared to that of SM group. Furthermore, PPAR&ggr; gene expression in all CGA group showed significantly higher expression compared to that of MS group(p < 0.05). On the other hand, PPAR&ggr; gene expression in SM group were significantly higher (p < 0.05) compared to that all CGA group after 9 week Conclusion: Administration of green coffe extract for 9 weeks have beneficial effect in improvement of PPAR&agr; and PPAR&ggr; gene expression in metabolic syndrome rat model

29 DOSE-DEPENDENT EFFECTS OF GREEN TEA EXTRACT ON ADIPONECTIN LEVEL AND ADIPONECTIN RECEPTOR GENE EXPRESSION IN METABOLIC SYNDROME RAT MODELS

Background: Adiponectin and its receptor are considered as an important metabolic syndrome biomarker and therapeutic target. Green tea is widely consumed worldwide and its catechins is an attractive candidates in treating metabolic syndrome. This study aimed to investigate the effect of green tea extract administration on adiponectin and its receptor gene expression in metabolic syndrome rat models. Methods: Thirty Sprague Dawley Metabolic Syndrome Rat Model weighed 300 – 400 grams were divided into EGCG 200 (n = 5), EGCG 300 (n = 5), and EGCG 400 (n = 5) groups. Moreover, as control groups fifteen rats were divided into normal control (NC) (n = 5), simvastatin and metformin treated (SM) (n = 5), and metabolic syndrome (MS) (n = 5) groups. Rats in the EGCG groups were treated once daily with green tea extract at a dose of 200, 300, and 400 mg/bw.t respectively. Moreover, Rats in the SM group were treated daily with 10 mg/bw.t of simvastatin and 200 mg/bw.t of metformin. The extract as well as the drug were administrated for 9 weeks through oral gavage. Fasting blood glucose, triglyceride, and HDL level was measured every two weeks by colorimetric method. Blood pressure was determined by tail cuff blood pressure recorder. Adiponectin level was analyzed by ELISA method. Adiponectin receptor gene expression was analyzed by RT-PCR methods. Result: After administration of green tea extract for 9 weeks, fasting blood glucose, triglyceride, and systolic blood pressure were significantly lower (p < 0.05), and HDL-Cholesterol was significantly higher in all EGCG groups compared to that of MS group. Moreover, adiponectin level was similar between EGCG 200 grup and MS group (p > 0.05). In contrast, the adiponectin level in EGCG 300 and EGCG 400 group was significantly higher (p < 0.05) compared to that of MS group. On the other hand, the adiponectin level was similar among all EGCG group compared to that of SM group. Furthermore, adiponectin receptor gene expression in EGCG 300 and EGCG 400 group were significantly higher compared to that of MS group (p < 0.05). In contrast, adiponectin receptor gene expression in EGCG 200 group was not significantly higher compared to that MS group. Conclusion: Administration of green tea extract at a dose of 300 and 400 mg/bw.t for 9 weeks improved adiponectin level and adiponectin receptor gene expression in metabolic syndrome rat model

ABO Gene Polymorphism and Thrombomodulin −33G>A Polymorphism Were Not Risk Factors for Myocardial Infarction in Javanese Men

Genetic factors contribute to about a half of coronary artery diseases. During the last several decades, some studies suggested that non-O blood group and thrombomodulin polymorphism −33G>A are the risk factors of coronary artery disease especially in Asia. There was no prior study in Indonesia regarding this issue. Hence, this study was designed to investigate the correlation of ABO polymorphism and thrombomodulin polymorphism −33G>A with the incidence of acute myocardial infarction (AMI). A total of 192 subjects were enrolled in this case control study. AMI patients were diagnosed based on World Health Organization criteria. Healthy patients were subjects with AMI risk factor without any sign and symptoms of AMI. Patients with diabetes mellitus, cancer, and arrhythmia were excluded from this study. Genotyping for both polymorphisms was performed by PCR RFLP methods. The result of this study suggested that ABO polymorphism and thrombomodulin polymorphism −33G>A were not risk factors of AMI, p = 0.727 and p = 0.699, respectively. Furthermore, the analysis to identify the synergy of these polymorphisms failed to prove their correlation with AMI (p = 0.118). Conclusively, this study showed that ABO polymorphism and thrombomodulin polymorphism −33G>A were not risk factors of AMI.