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

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

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

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

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

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

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

(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

A Triazole Disulfide Compound Increases the Affinity of Hemoglobin for Oxygen and Reduces the Sickling of Human Sickle Cells

Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt “sickled” shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4′-di­(1,2,3-triazolyl)­disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy-forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with β-Cys93, which inhibits the salt-bridge formation between β-Asp94 and β-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen, and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the <i>P</i>₅₀ of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb β-Cys93, may provide new treatment options for patients with sickle cell disease

Retinal nerve fiber layer (RNFL) thinning and glaucomatous optic neuropathy in sGCα₁^−/− mice. A

<p>: Quantitative analysis, assessed by SD-OCT (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060156#pone.0060156.s001" target="_blank">fig. S1</a>), of total retinal thickness (<b>left panel</b>) and RNFL thickness, in young (6-week-old, <b>middle panel</b>) and old (70-week-old, <b>right panel</b>) wild-type (WT, <i>n</i> = 19 and 13, respectively) and soluble guanylate cyclase α₁-deficient (sGCα₁^−/−) mice (<i>n</i> = 15 and 14, respectively; *<i>P</i> = 1.2×10⁻²). <b>B</b>: Representative whole-mount retinas from age-matched young (20-week-old) and old (56-week-old) WT and sGCα₁^−/− mice, reacted with antibodies directed against SMI32, staining retinal nerve fibers yellow. Scale bars: 500 μm. <b>C</b>: Representative confocal images, taken at a similar distance from the optic nerve, of flat-mounted retinas isolated from age-matched 52-week-old WT and sGCα₁^−/− mice that were reacted with antibodies directed against βIII Tubulin, and quantitative analysis of the number of RGCs/high-powered field (<i>n</i> = 8 and 7, respectively; *<i>P</i> = 3.6×10⁻²). A retinal ganglion cell (red) is indicated by an arrow. Scale bars: 20 μm. <b>D</b>: Representative cross sections through the optic nerve of 52-week-old WT and sGCα₁^−/− mice stained with paraphenylenediamine, and quantitative analysis of the calculated number of axons/optic nerve (ON). The arrow indicates an injured area in the optic nerve, characterized by the absence of well-formed myelinated axons (<i>n</i> = 7 and 6, respectively; *<i>P</i> = 4.9×10⁻²). Scale bars: 25 μm.</p