Deletion of the Murine Cytochrome P450 <i>Cyp2j</i> Locus by Fused BAC-Mediated Recombination Identifies a Role for <i>Cyp2j</i> in the Pulmonary Vascular Response to Hypoxia

<div><p>Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human <i>CYP2J2</i>, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine <i>Cyp2j</i> locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to <i>CYP2J2</i>, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that <i>Cyp2j</i> gene products regulate the pulmonary vascular response to hypoxia.</p></div

Representative data for quantitation of <i>Cyp2j</i> gene expression.

<p>(A) Mouse <i>Cyp2j</i> gene expression in different tissues was measured using RT-MLPA. (B) Mouse <i>Cyp2j</i> gene expression in liver and kidney of wild-type (WT) and null (KO) mice measured using RT-MLPA is shown. (C) <i>CYP2J2</i> gene expression in human tissues. (D) Human <i>CYP2J2</i> mRNA levels in lung and heart of <i>Cyp2j</i>^+/+, <i>Cyp2j^−/−</i> , <i>Cyp2j^+/+</i> -<i>Tg</i> and <i>Cyp2j⁻</i>^/−-<i>Tg</i> mice quantified by RT-PCR are shown. The measurements were performed three times using pooled mouse RNA from three individual mice.</p

Gene expression by quantitative RT-PCR.

<p>(A, B, C) <i>Cyp2c44</i>, <i>Cyp2c38</i>, and <i>Cyp2c29</i> mRNA levels in lung and heart of <i>Cyp2j^+/+</i>, <i>Cyp2j^−/−</i> and <i>Cyp2j^−/− -Tg</i> mice. Experiments were run in triplicate. Mouse tissue RNAs were pooled from three individual mice.</p

(A) Percent increase in left lung pulmonary vascular resistance (LPVR) in response to left mainstem bronchial occlusion (LMBO) in <i>Cyp2j^+/+</i> and <i>Cyp2j^−/−</i> mice (n = 10 per group).

<p>(B) Continuous tracings of arterial oxygen partial pressure (PaO₂) measurements during LMBO in <i>Cyp2j^+/+</i> (n = 4) and <i>Cyp2j^−/−</i> (n = 3) mice; (C) Percent increase in LPVR in response to LMBO in <i>Cyp2j^+/+</i> (n = 6) and <i>Cyp2j^−/−-Tg</i> (n = 5) mice; (D) Percent increase in LPVR in response to LMBO in MS-PPOH or vehicle-treated <i>Cyp2j^+/+</i> mice (n = 5 per group); (E) Percent increase in LPVR in response to LMBO in L-NAME-treated <i>Cyp2j^+/+</i> (n = 5) and <i>Cyp2j^−/−</i> (n = 6) and untreated <i>Cyp2j^+/+</i> and <i>Cyp2j^−/−</i> mice (n = 10 per group); Data are means ± SEM. *P<0.05, **P<0.005.</p

11, 12- and 14, 15-EETs measurements in BALF (A, B) and the generation of EETs and DHETs by pulmonary microsomes (C) of <i>Cyp2j^+/+</i>, <i>Cyp2j^−/−</i> and <i>Cyp2j^−/−-Tg</i> mice.

<p>The black bar represents the DHET quantity before EET hydrolysis, and the grey bar represents the DHET quantity after EET hydrolysis in the samples. Triplicate measurements were performed for each mouse. Data are means ± SEM. n = 3 for each group in A, B; n = 4 for each group in C.</p

Creation of human <i>CYP2J2</i> transgenic mice.

<p>(A) Schematic diagram showing generation of the recombinant <i>hCYP2J2</i> BAC. Two targeting vectors were constructed to remove the sequences flanking <i>hCYP2J2</i> by homologous recombination in <i>E. coli</i>. Primers P15 to 22 were used to identify the recombinants in E. coli. PCR data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003950#pgen.1003950.s005" target="_blank">Figure S5</a>. Trpr∧r, trimethoprim resistance; Amp∧r, Ampicillin resistance; Hyg, Hygromycin. (B). Transgenic mice were identified by PCR and confirmed by DNA blotting. M, λ DNA marker/<i>Hin</i>d III; −, negative PCR control; +, positive PCR control to amplify BAC; 1 to 5, 8, founder mice.</p

Comparison of systemic hemodynamic measurements in conscious <i>Cyp2j^+/+</i> and <i>Cyp2j^−/−</i> mice.

<p>Data are means ± SEM. <i>Cyp2j^+/+</i> male (n = 7), <i>Cyp2j^+/+</i> female (n = 10), <i>Cyp2j^−/−</i> male (n = 7), and <i>Cyp2j^−/−</i> female (n = 9) mice. HR, heart rate; SBP, systolic blood pressure.</p

Comparison of systemic hemodynamic measurements in anesthetized <i>Cyp2j^+/+</i> and <i>Cyp2j^−/−</i> mice.

<p>Data are means ± SEM. HR, heart rate; LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure; CVP, central venous pressure; SVR, systemic vascular resistance; EF, ejection fraction; CO, cardiac output; SV, stroke volume; dP/dt_max, maximum rate of developed left ventricular pressure; dP/dt_min, minimum rate of developed left ventricular pressure; τ, time constant of isovolumic relaxation; SW, stroke work; Ea, arterial elastance; (n = 6 per group).</p