Cardiovascular candidate genes within the oxidative stress pathway: rat and human studies

Combined congenic breeding strategy and microarray expression profiling studies from our group previously identified rat glutathione s-transferase mu type-1 (rGstm1) as a functional and positional candidate gene implicated in hypertension. In the previous studies, expression of rGstm1 was lower in the kidney from the hypertensive rat model, stroke-prone spontaneously hypertensive rat (SHRSP0, compared to the normotensive Wistar Kyoto (WKY) and a chromosome 2 congenic strain (SP. WKYG1a2c*). This project aims to characterise Gstm gene family in rat and human tissues. Results from this thesis demonstrate reduced mRNA expression of several members of rGstm gene family, in vascular and renal tissues. Most notably, expression of vascular and renal rGstm1 mRNA and protein, are lower in the SHRSP compared to WKY and SP.WKYG1a2c*. Vascular mRNA expression of rGstm2, rGstm5 and rGstm7 were reduced in the SHRSP males compared to the WKY males and SP.WKYG1a2c* males but not in the females. The expression profile of the human GSTM (hGSTM) gene family in vascular tissue from varicose vein ad saphenous vein have also been characterised in an attempt to identify the true human orthologue of rGstm1. There are five known members in the hGSTM gene family, all of which were expressed in vascular tissues. Results from characterisation of the vascular hGSTM gene family show that four hGSTM members are homologous to rGstm1. These four hGstm genes remain as potential orthologue for rGstm1. Functional roles of vascular and renal rGstm genes have been investigated using two pharmacological intervention studies. Olmesartan (AT1R antagonist) or hydralazine plus hydrochlorothiazide (direct vasodilator and diuretic) have been used to evaluate the oxidative stress status and gene expression of rGstm genes, in the reversal and prevention studies. Both drug treatments improved the BP of SHRSP rats in reversal study and prevented the rise in BP in the prevention study. Olmesartan-treated SHRSP rats demonstrated reduced superoxide (O2-) and hydrogen peroxide (H2O2) production in both reversal and prevention studies. Minor effects by both drug treatments were observed for the rGstm gene family in the prevention but not the reversal study. Both drug treatments did not influence vascular of renal rGstm1 expression in either reversal or prevention studies. Improvement in BP did not improve rGstm1 gene expression. The rGstm1 was not responsive to pharmacological interventions due to strain-dependent genetic abnormalities. Functional polymorphisms in two key enzymes involved in ROS and NO balance were investigated for association with CAD and vascular compliance as single polymorphism and as haplotypes. There was an association between CYBA A-930G with CAD, with the A allele being recessive. There was also an association between NOS3 G894T with CAD, only when G allele was dominant. The T-786C of NOS3 was associated with small artery compliance index (C2), in both CAD and control groups

Transgenic overexpression of glutathione S-transferase μ-type 1 reduces hypertension and oxidative stress in the stroke-prone spontaneously hypertensive rat

Background: Combined congenic breeding and microarray gene expression profiling previously identified glutathione S-transferase m-type 1 (Gstm1) as a positional and functional candidate gene for blood pressure (BP) regulation in the stroke-prone spontaneously hypertensive (SHRSP) rat. Renal Gstm1 expression in SHRSP rats is significantly reduced when compared with normotensive Wistar Kyoto (WKY) rats. As Gstm1 plays an important role in the secondary defence against oxidative stress, significantly lower expression levels may be functionally relevant in the development of hypertension. The aim of this study was to investigate the role of Gstm1 in BP regulation and oxidative stress by transgenic overexpression of the Gstm1 gene. Method: Two independent Gstm1 transgenic SHRSP lines were generated by microinjecting SHRSP embryos with a linear construct controlled by the EF-1a promoter encoding WKY Gstm1 cDNA [SHRSP-Tg(Gstm1)1WKY and SHRSPTg(Gstm1)2WKY]. Results: Transgenic rats exhibit significantly reduced BP and pulse pressure when compared with SHRSP [systolic: SHRSP 205.2 3.7 mmHg vs. SHRSP-Tg(Gstm1)1WKY 175.5 1.6 mmHg and SHRSP-Tg(Gstm1)2WKY 172 3.2 mmHg, P< 0.001; pulse pressure: SHRSP 58.4 0.73 mmHg vs. SHRSP-Tg(Gstm1)1WKY 52.7 0.19 mmHg and SHRSP-Tg(Gstm1)2WKY 40.75 0.53 mmHg, P< 0.001]. Total renal and aortic Gstm1 expression in transgenic animals was significantly increased compared with SHRSP [renal relative quantification (RQ): SHRSP-Tg(Gstm1)1WKY 1.95 vs. SHRSP 1.0, P< 0.01; aorta RQ: SHRSP-Tg(Gstm1)1WKY 2.8 vs. SHRSP 1.0, P< 0.05]. Renal lipid peroxidation (malondialdehyde: protein) and oxidized : reduced glutathione ratio levels were significantly reduced in both transgenic lines when compared with SHRSP [malondialdehyde: SHRSP 0.04 0.009mmol/l vs. SHRSP-Tg(Gstm1)1WKY 0.024 0.002mmol/l and SHRSPTg(Gstm1)2WKY 0.021 0.002mmol/l; (oxidized : reduced glutathione ratio): SHRSP 5.19 2.26mmol/l vs. SHRSPTg(Gstm1)1WKY 0.17 0.111mmol/l and SHRSPTg(Gstm1)2WKY 0.471 0.223mmol/l]. Transgenic SHRSP rats containing the WKY Gstm1 gene demonstrate significantly lower BP, reduced oxidative stress and improved levels of renal Gstm1 expression. Conclusion: These data support the hypothesis that reduced renal Gstm1 plays a role in the development of hypertension