| Background:Epoxyeicosatrienoic acids (EETs), which are cytochrome P450 (CYP) epoxygenase derived metabolites of arachidonic acid(AA), display many of the characteristics of endothelium-derived hyperpolarizing factor (EDHF) and cause hyper polarization of underlying vascular smooth muscle cells via activation of Ca2+ activated K+ channels. So EETs are thought to be potential candidates for EDHF. It was demonstrated that Arachidonic acid cytochrome P450 epoxygenase metabolites, EETs, have potent vasodilatory and diuretic feature, and therefore have hypotensive effect. No in vivo studies, however, were performed to support it. In the current study, we investigated this hypothesis via overexpressing CYP epoxygense genes in spontaneously hypertensive rats (SHR).Methods and Results:1) The recombinant adeno-associated virus vector (rAAV) was utilized to mediate long-term transfection of CYP2J2 genes in adult SHR, and animal systolic blood pressure was monitored using arterial caudilis indirect manometric method and main stable hydrolysis metabolic product of 14, 15-EET, 14, 15-DHET, in urine was measured to reflect the production of EETs. Results showed that at 2 months the urinary excretion of 14, 15-DHET increased by 11-folds in the rAAV-2J2 groups compared with rAAV-GFP-treated group. 2) The systolic blood pressure in rAAV-2J2-treated group decreased from 173.0±2.8mHg to 163.5±5.8mmHg ( p﹤0.01) at month 2, and it is 165.0±4.7 mmHg at month 6 after gene injection. In contract, in rAAV-GFP treated control animals, the systolic blood pressure was kept higher (from 174±3.9mmHg to 176.7±5.3mmHg) at month 2 and increased to 195.2±20.8mmHg at month 6 (~30mmHg higher than that in rAAV-2J2-treated group, p<0.001). 3) Before the rats were sacrificed at month 6, the blood pressure form carotid artery was detected with Pressure-Volume System. The artery blood pressure of 2J2-treated group was decreased to 191.0±25.1 mmHg (vs. 226.8±25.8mmHg, p<0.01). At the same time, the cardiac functions were measured. The results showed that maximum intra-cardiac pressure was 202.1±30.0 mmHg in 2J2-treated group, significantly lower than control (vs. 241.2±18.2 mmHg , p﹤0.01). The end-systolic pressure in treated group was 199.0±33.3mmHg, and that in the control group was 237.6±14.2 mmHg. Other valuable indexes for evaluating the myocardial contractility, dp/dt max and dp/dt min, were also measured. Both the dp/dt max and the absolute value of dp/dt min were decreased from 17005.5±1850.8mmHg/s and 16930.8±2080.0mmHg/s in control group to 16930.8±2080.0mmHg/s and 12258.1±4155.6mmHg/s in 2J2-treated group, respectively. However, the cardiac output (CO) in treatment animals was significantly higher than control (26870.76431.3μL/min vs. 19826.1±2906.0μL/min, p<0.05). 4) Recorded arterial elastance (Ea) index in the rAAV-2J2 agroup was 1.5±0.4 (vs.2.6±0.4, p﹤0.001), which demonstrated that the overexpression of the CYP2J2 gene attenuates the arterial sclerosis. The responsiveness to norepinephrine (NE) and acetylcholine (Ach) of the thoracic aorta rings was checked and it was demonstrated 2J2 gene could attenuate the reactivity of the rings to NE, and enhance the diastole respond to Ach. 5) Interestingly, we found the atrial natriuretic peptide (ANP) mRNA were up-regulated 14 folds in myocardium of the rAAV-2J2 group; furthermore, the C-type receptor's (NPR-C) mRNA of ANP was increased in heart, aorta, kidney and lung tissues. However, the expression levels of some other vasoactive substances such as endothelin-1 and its receptors, adrenomedullin and its receptors have no change detectable. 6) Finally, we cultured the primary myocardium cell to study effect of EETs on ANP level, and we also found addition of exogenous EETs up-regulated the protein of ANP and had a dose-effect relationship.Conclusion:For first time our study indicates transfection of rAAV-mediated 2J2 genes could decrease the blood pressure and prevent development of hypertension in SHR. Its mechanisms are including: 1) CYP epoxygenase and its metabolites EETs mediate negative inotropic effect on the cardiac contractility., which is one of their mechanism on blood-pressure regulation. On the same time, the reduction in cardiac contractility didn't cause to decrease in the cardiac output. In contrast, cardiac output was increased. The negative inotropic effect with increasing CO would be valuable for the 2J2-transgene application to clinic. 2) Except for the direct effects of their metabolites EETs on the myocardium and vessels, CYP2J2 may provide beneficial effects via up-regulating expression of ANP, which can depress the cardiac contractility and produce vasodilatation and natriuresis. The signal mechanism may also play an important role in regulation of blood pressure. 3) CYP2J2 gene could attenuate the reactivity of the rings to norepinephrine, and enhance the diastole respond to acetylcholine. The effect would benefit the regulation of CYP2J2 gene on blood pressure. 4) The overexpression of CYP2J2 gene can decline the Ea of the aorta and induce a larger increase in aorta compliance. The function of CYP2J2 gene's attenuating the arterial sclerosis and improving its relaxation should partly attribute to ANP and NPR-C, which were up-regulated by CYP2J2 gene.
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