Mifetika Lukitasari

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.

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.

YIA 03-05 BLOOD PRESSURE REDUCTION AND DIFFERENT C/EBPα-DNA BINDING PATTERN IN TELMISARTAN-TREATED ANGIOTENSINOGEN G-217A POLYMORPHISM HYPERTENSIVE PATIENTS.

Objective: To investigate whether the angiotensinogen (AGT) G-217A polymorphism affects blood pressure response of telmisartan and valsartan in hypertensive patients. Design and Method: A total of 46 primary hypertensive patients, 23 patients received telmisartan 80 mg and 23 patients received valsartan 160 mg, were observed and followed up for 4 months. The AGT G-217A polymorphism was determined by PCR-RFLP and direct sequencing method. Blood pressure response was measured daytime (6 am – 10 pm), night-time (10 pm – 6 am), and 24 hours using twenty-four hours ambulatory blood pressure monitoring (ABPM). Plasma AGT level were performed at baseline and after 4 months of ARB therapy. The docking procedure between C/EBP&agr; (1NWQ) and oligonucleotides containing -217G and -217A was analyzed by patchdock and firedock. The visualization of the structures were done by PyMol. Results: Significantly reduced blood pressure in both daytime and 24 hours mean systolic/diastolic blood pressure were observed in patients with -217AA/AG genotype who received telmisartan, but not valsartan, compared to those carrying GG genotype. In line with the change of blood pressure, the change of plasma AGT level in those carrying -217AA/AG were slightly lower compared to GG genotype in telmisartan-treated group, even it failed to reach stastistically significant (p = 0.17). We suggested the variability antihypertensive response in telmisartan-treated AGT G-217A polymorphism hypertensive patients might correlate with the change in AGT expression due to the PPAR&ggr;-activating property of telmisartan, that involved C/EBP&agr;. The adenine at position -217 formed a favoured hydrogen bond with Asn292 which is important for stabilizing the C/EBP&agr; – DNA interface. This likely influences C/EBP&agr; activity to repress AGT expression in response to PPAR&ggr;-activated telmisartan. Conclusions: AGT G-217A polymorphism may increases the antihypertensive response of telmisartan by forming a favoured hydrogen bond with Asn292 of C/EBP&agr; protein.OBJECTIVE To investigate whether the angiotensinogen (AGT) G-217A polymorphism affects blood pressure response of telmisartan and valsartan in hypertensive patients. DESIGN AND METHOD A total of 46 primary hypertensive patients, 23 patients received telmisartan 80 mg and 23 patients received valsartan 160 mg, were observed and followed up for 4 months. The AGT G-217A polymorphism was determined by PCR-RFLP and direct sequencing method. Blood pressure response was measured daytime (6 am - 10 pm), night-time (10 pm - 6 am), and 24 hours using twenty-four hours ambulatory blood pressure monitoring (ABPM). Plasma AGT level were performed at baseline and after 4 months of ARB therapy. The docking procedure between C/EBPα (1NWQ) and oligonucleotides containing -217G and -217A was analyzed by patchdock and firedock. The visualization of the structures were done by PyMol. RESULTS Significantly reduced blood pressure in both daytime and 24 hours mean systolic/diastolic blood pressure were observed in patients with -217AA/AG genotype who received telmisartan, but not valsartan, compared to those carrying GG genotype. In line with the change of blood pressure, the change of plasma AGT level in those carrying -217AA/AG were slightly lower compared to GG genotype in telmisartan-treated group, even it failed to reach stastistically significant (p = 0.17). We suggested the variability antihypertensive response in telmisartan-treated AGT G-217A polymorphism hypertensive patients might correlate with the change in AGT expression due to the PPARγ-activating property of telmisartan, that involved C/EBPα. The adenine at position -217 formed a favoured hydrogen bond with Asn which is important for stabilizing the C/EBPα - DNA interface. This likely influences C/EBPα activity to repress AGT expression in response to PPARγ-activated telmisartan. CONCLUSIONS AGT G-217A polymorphism may increases the antihypertensive response of telmisartan by forming a favoured hydrogen bond with Asn of C/EBPα protein.

BLOOD PRESSURE REDUCTION WITH ANGIOTENSIN RECEPTOR BLOCKER THERAPY IN REN C-5312T GENETIC VARIANT

Background: Renin plays a pivotal role in blood pressure regulation. Objective: This study designed to investigate whether blood pressure response to angiotensin receptor blocker (ARB)therapy is determined by REN C-5312T genetic variant in enhancer region. Methods: A total of fifty nine hypertensive patients was randomly selected from hypertensive patients in cardiology outpatient clinic of Saiful Anwar hospital Malang. Patients with known secondary hypertension, active bleeding, cirrhosis, pregnancy, on corticosteroid and/ or estrogen therapy were excluded. Twenty-four hours ambulatory blood pressure (ABP) monitoring was measured before and after angiotensin receptor blocker therapy. Baseline and after 3 months treatment plasma renin level was measured by ELISA method. Genomic DNA was analyzed by multiplex polymerase chain reaction to determine the presense of REN C-5312T genetic variant. Results: Patients with CT/TT genotype were found in 67.8% of total sample and the remaining samples were CC genotype. CT/TT genotype patients showed significant systolic and diastolic blood pressure reductionafter 3 months-ARB treatment (p = 0.003 and p = 0.018, respectively). Moreover, they showed significant renin level increase from the baseline (p = 0.003). On the other hand, CC genotype patients showed no significant reduction in either systolic or diastolic blood pressure. This group also showed significant renin level increase from the baseline (p = 0.005). Conclusion: REN C-5312T genetic variants predicts blood pressure response to ARB treatment.

SERUM RENIN LEVEL IN LEFT VENTRICULAR HYPERTROPHY AND DIPPING HYPERTENSIVE PATIENTS

Background: High prevalence of target organ damage in hypertensive patients might be preventable by early proper adequate treatment. Early recognation of blood pressure pattern using ambulatory blood pressure monitoring and serum renin level may correlate with development of target organ damage. Objective: The study aimed to examine ambulatory blood pressure pattern and left ventricular hypertrophy (LVH) as well as and serum renin level in hypertensive patients. Methods: A total of eighteen hypertensive patients were randomly selected from hypertensive patients in cardiology outpatient clinic of Saiful Anwar hospital Malang. Patients with known secondary hypertension, active bleeding, cirrhosis, pregnancy, on corticosteroid and/ or estrogen therapy were excluded. Twenty-four hours ambulatory blood pressure (ABP) monitoring was measured to determine blood pressure pattern. LVH measured by Trans Thoracic Echocardiography and serum renin level measured by ELISA. Results: Uncontrolled blood pressure observed in 55.6% of patients. Non-dipping pattern observed in 94.4%. Concentric hypertrophy/concentric remodelling (LVH) and normal was observed in 66.7%, and 33.3% respectevelly. No significant association observed between dipping patern and LVH (p = 0.70). There was no difference of serum renin level between dipping and non-dipping hypertensive patients (31.50 vs 36.99 ± 9.93, P = 0.598). No difference of serum renin level in hypertensive patient with and without LVH (39.25 ± 25 vs 35.40 ± 11.57, P = 0.314). Conclusion: No difference observed in serum renin level among hypertensive patients.