Overexpression of cytochrome P450 epoxygenases prevents development of hypertension in spontaneously hypertensive rats by enhancing atrial natriuretic peptide

Abstract

Cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) exert well recognized vasodilatory, diuretic, and tubular fluid-electrolyte transport actions that are predictive of a hypotensive effect. The study sought to determine the improvement of hypertension and cardiac function by overexpressing P450 epoxygenases in vivo. Long-term expression of CYP102 F87V or CYP2J2 in spontaneously hypertensive rats (SHR) was mediated by using a type 8 recombinant adeno-associated virus (rAAV8) vector. Hemodynamics was measured by a Millar Instruments, Inc. (Houston, TX) microtransducer catheter, and atrial natriuretic peptide (ANP) mRNA levels were tested by real-time polymerase chain reaction. Results showed that urinary excretion of 14,15-EET was increased at 2 and 6 months after injection with rAAV-CYP102 F87V and rAAV-CYP2J2 compared with controls (p < 0.05). During the course of the 6-month study, systolic blood pressure significantly decreased in P450 epoxygenase-treated rats, but the CYP2J2-specific inhibitor C26 blocked rAAV-CYP2J2-induced hypotension and the increase in EET production. Cardiac output was improved by P450 epoxygenase expression at 6 months (p < 0.05). Furthermore, cardiac collagen content was reduced in P450 epoxygenase-treated rats. ANP mRNA levels were up-regulated 6- to 14-fold in the myocardium, and ANP expression was significantly increased in both myocardium and plasma in P450 epoxygenase-treated rats. However, epidermal growth factor (EGF) receptor antagonist 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG-1478) significantly attenuated the increase in the EET-induced expression of ANP in vitro. These data indicate that overexpression of P450 epoxygenases attenuates the development of hypertension and improves cardiac function in SHR, and that these effects may be mediated, at least in part, by ANP via activating EGF receptor.

Fig. 1.

P450 epoxygenase overexpression mediated by rAAV and quantitative analysis of total 14,15-DHET and 14,15-EET in urine and different tissues. A, Western blot analysis shows elevated levels of CYP2J2 in heart, kidney, liver, and aorta and CYP102 F87V and GFP in heart tissue 6 months after treatment of animals with rAAV-CYP2J2, rAAV-GFP, and rAAV-CYP102 F87V, respectively. B and C, total 14,15-DHET and 14,15-EET levels in urine of rats 2 and 6 months after injection of rAAV vectors; n = 6/group, *, p < 0.05 compared with control. D and E, total 14,15-DHET and 14,15-EET levels in heart, kidney, and aorta of rats 6 months after injection of rAAV vectors; n = 5/group, *, p < 0.05 compared with control.

Fig. 2.

The effects of rAAV-CYP2J2 and rAAV-CYP102 F87V treatments on blood pressure and cardiac function in SHR. A, systolic blood pressure measured by tail-cuff method; *, p < 0.05 compared with control group. B, carotid artery pressure measured invasively in rats 6 months after injections; *, p < 0.05 compared with control group. C, carotid artery pressure was measured invasively in rats 2 months after oral administration of C26; *, p < 0.05 compared with rAAV-CYP2J2 group. D and E, quantitative analysis of 14,15-EET and 14,15-DHET in urine of rats 2 months after injection of rAAV-CYP2J2 and C26 administration; n = 5/group, *, p < 0.05 compared with rAAV-CYP2J2 group. F and G, dP/dt_max and CO measured invasively in rats 6 months after injections; *, p < 0.05 compared with control group.

Fig. 3.

The effects of rAAV-CYP2J2 and rAAV-CYP102 F87V treatments on aortic contractility. A, Ea index. B, reactivity of aortic rings to NE. C, reactivity of aortic rings to ACh. Contractility in response to NE decreased and dilation in response to ACh increased in aortic rings from rAAV-CYP102 F87V- and rAAV-CYP2J2-treated rats; *, p < 0.05 compared with controls.

Fig. 4.

The effects of rAAV-CYP102 F87V and rAAV-CYP2J2 treatments on heart remodeling. Ratio of whole heart weight to body weight (A) and microscope images of cross-section of ventricle (B) to show size of cardiomyocytes (top) and comparison of cardiomyocyte diameter (bottom), which suggest gene treatments prevent the hypertension-induced myocardial hypertrophy. C, heart collagen content assessed by Sirius red staining: representative photomicrographs (Sirius red staining for collagen content) of heart sections from the various treatment groups. D, quantification of collagen-positive areas showed reduced collagen deposition in hearts from rAAV-CYP2J2- and rAAV-CYP102 F87V-treated rats; *, p < 0.05 compared with controls.

Fig. 5.

The effects of rAAV-CYP102 F87V and rAAV-CYP2J2 treatments on secretion of ANP and expression of ANP. A, serum ANP in four groups; *, p < 0.05 compared with control groups. B and C, relative mRNA copy number of ANP relative to glyceraldehyde-3-phosphate dehydrogenase in ventricular and atrial myocardium, respectively, determined by real-time quantitative PCR. ANP mRNA levels were much greater in myocardium of the rAAV-CYP102 F87V and rAAV-CYP2J2 groups; *, p < 0.01 compared with control groups. D, urinary cGMP levels; *, p < 0.05 compared with control groups. E and F, Western blot of ANP expression in myocardium and relative density of ANP band against β-actin, showing that ANP level is significantly up-regulated in myocardium in rAAV-CYP102 F87V- and rAAV-CYP2J2-treated rats; *, p < 0.01 compared with controls.

Fig. 6.

Effects of epoxygenase overexpression or EET on expression of ANP. A and B, immunohistochemistry staining for ANP in ventricular myocardium and atria myocardium, respectively, reveals more positive cells and stronger overall staining in rAAV-CYP102 F87V- and rAAV-CYP2J2-treated groups (top) and quantification of ANP-positive cells (bottom); *, p < 0.05 compared with controls. C, ANP concentration in media of cultured cardiomyocytes incubated with synthetic EETs; *, p < 0.05 compared with control. D, intracellular cGMP levels in cultured cardiomyocytes; *, p < 0.05 compared with control. E, representative Western blot and relative density of ANP band: effects of inhibitors of MMP (1,10-phenanthroline), HBEGF (CRM-197), and EGFR (AG-1478) on ANP expression in cultured cardiomyocytes. F, relative density of ANP band against β-actin; #, p < 0.05 compared with control (CON); *, p < 0.01 compared with EET treatment.