Gene delivery of cytochrome p450 epoxygenase ameliorates monocrotaline-induced pulmonary artery hypertension in rats

Abstract

Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure, and death. Endothelial dysfunction and inflammation were implicated in the pathogenesis of PAH. Epoxyeicosatrienoic acids (EETs), products of the cytochrome P450 epoxygenase metabolism of arachidonic acid, are potent vasodilators that possess anti-inflammatory and other protective properties in endothelial cells. We investigated whether gene delivery with the human cytochrome P450 epoxygenase 2J2 (CYP2J2) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Significant pulmonary hypertension developed 3 weeks after the administration of MCT, but gene therapy with CYP2J2 significantly attenuated the development of pulmonary hypertension and pulmonary vascular remodeling, without causing changes in systemic arterial pressure or heart rate. These effects were associated with increased pulmonary endothelial NO synthase (eNOS) expression and its activity, inhibition of inflammation in the lungs, and transforming growth factor (TGF)-β/type II bone morphogenetic protein receptor (BMPRII)-drosophila mothers against decapentaplegic proteins (Smads) signaling. Collectively, these data suggest that gene therapy with CYP2J2 may have potential as a novel therapeutic approach to this progressive and oftentimes lethal disorder.

Figure 1.

Expression of cytochrome P450 epoxygenase 2J2 (CYP2J2) and detection of 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) in urine and tissues, 2 weeks after gene delivery. (A) Representative immunoblotting results show increased CYP2J2 expression in heart, liver, lung, and aorta of pCB6-2J2–treated rats compared with empty pCB6 vector–treated rats. Experiments were repeated three times. (B) Urinary concentrations of 14,15-DHET according to ELISA in saline control rats (Control) and rats treated with monocrotaline (MCT), either alone (MCT) or together with pCB6 empty vector (MCT + pCB6) or pCB6-2J2 (MCT + 2J2). (C) Concentrations of 14,15-DHET according to ELISA in tissues of lung, liver, and kidney in various groups. Data shown are mean ± SEM of 6– 8 rats/group. *P < 0.05 versus control. ^#P < 0.05 versus MCT + pCB6.

Figure 2.

Reversal effects of CYP2J2 gene delivery on right ventricular systolic pressure (RVSP) (A), right ventricle/left ventricle + septum (RV/LV + S) (B), mean systemic arterial pressure (mSAP) (C), and heart rate (D), and effects of N^G-l-nitro-arginine methyl ester (l-NAME) on RVSP (E) and RV/LV + S (F). Hemodynamic measurements were performed 2 weeks after gene transfer, and results showed that CYP2J2 overexpression reversed the MCT-induced changes in RVSP and RVH, but had no effects on mSAP and heart rate. l-NAME attenuated the beneficial effect of CYP2J2 overexpression in the model of PAH induced by MCT. Data shown are mean ± SEM of 6–8 rats/group. *P < 0.05 versus control. ^#P < 0.05 versus MCT + pCB6.

Figure 3.

Representative photomicrographs of peripheral pulmonary arteries stained with elastic van Gieson stain (A; original magnification, ×400; scale bar, 20 μm). Quantification of percent medial thickness for vessels 25–50 μm (B) and 51–100 μm (C) in external diameter. CYP2J2 gene delivery attenuated hypertrophy of vessel walls in MCT rats. Data shown are mean ± SEM of 6–8 rats/group. *P < 0.05 versus control. ^#P < 0.05 versus MCT + pCB6.

Figure 4.

Concentrations of IL-6 in lung extracts and serum were determined by ELISA, 2 weeks after gene transfer. Treatment with CYP2J2 significantly inhibited the MCT-induced elevation of IL-6 in lung extracts (A) and in serum (B). Data shown are mean ± SEM of 6– 8 rats/group. *P < 0.05 versus control. ^#P < 0.05 versus MCT + pCB6.

Figure 5.

Activity of eNOS (A) and NOS (B) in lung tissue of rats, and Western blotting for eNOS and β-actin expression in MCT-treated rat lungs (C and D). A significant decrease occurred in eNOS activity (A) and expression (C and D) in rats treated with MCT, either alone or together with pCB6 empty vector. In contrast, in MCT-treated rats receiving pCB6-2J2, eNOS activity and protein levels were near normal. l-NAME significantly weakened the CYP2J2-induced increase in eNOS activity. Results are representative of 3–5 independent experiments. (D) Blots were scanned, and relative eNOS expression was normalized to β-actin . Data shown are mean ± SEM of 6–8 rats/group. *P < 0.05 versus control. ^#P < 0.05 MCT + CYP2J2 versus MCT + pCB6. ^&P < 0.05 MCT + CYP2J2 + l-NAME versus MCT + CYP2J2. ^{^}P < 0.05 MCT + CYP2J2 + l-NAME versus MCT + l-NAME. ^@P < 0.05 MCT + l-NAME versus MCT

Figure 6.

Protein expression of TGF-β/Smads signaling in MCT-treated rat lungs assessed by Western blotting. A significant increase occurred in TGF-β1 (A) and p-Smad2 (B) expression in rats treated with MCT, either alone or together with pCB6 empty vector. However, in MCT-treated rats receiving pCB6-2J2, TGF-β1 and p-Smad2 protein levels were increased. Results are representative of three independent experiments. Blots were scanned, and relative TGF-β1 (C) and p-Smad2 (D) were normalized to β-actin and Smad2, respectively. Data shown are mean ± SEM of 6–8 rats/group. *P < 0.05 versus control. ^#P < 0.05 versus MCT + pCB6.

Figure 7.

Bone morphogenetic protein (BMP) and platelet-derived growth factor receptor β (PDGFRβ) signaling in MCT-treated rat lungs assessed by Western blotting. A significant decrease occurred in BMPRII (A) and p-Smad1 (B) expression in rats treated with MCT, either alone or together with pCB6 empty vector. However, in MCT-treated rats receiving pCB6-2J2, concentrations of BMPRII and p-Smad1 protein were increased. Results are representative of three independent experiments. Blots were scanned, and relative BMPRII (C) and p-Smad1 (D) were normalized to β-actin and Smad1, respectively. (E) Concentrations of PDGFRβ and phosphorylated PDGFRβ were upregulated in MCT-treated animals, and treatment with CYP2J2 attenuated the upregulation. (F and G) Relative PDGFRβ and phosphorylated PDGFRβ, respectively, were normalized to β-actin. Data shown are mean ± SEM of 6– 8 rats/group. *P < 0.05 versus control. ^#P < 0.05 versus MCT + pCB6.