Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the $α_1$ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase $α_1$–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the $α_1$ and $β_1$ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG

Genetic modifiers of hypertension in soluble guanylate cyclase α1–deficient mice

Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure–related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure–modifying mechanism in a setting of impaired NO/cGMP signaling

Atrial natriuretic peptide is negatively regulated by microRNA-425

Numerous common genetic variants have been linked to blood pressure, but no underlying mechanism has been elucidated. Population studies have revealed that the variant rs5068 (A/G) in the 3′ untranslated region of NPPA, the gene encoding atrial natriuretic peptide (ANP), is associated with blood pressure. We selected individuals on the basis of rs5068 genotype (AG vs. AA) and fed them a low- or high-salt diet for 1 week, after which they were challenged with an intravenous saline infusion. On both diets, before and after saline administration, ANP levels were up to 50% higher in AG individuals than in AA individuals, a difference comparable to the changes induced by high-salt diet or saline infusion. In contrast, B-type natriuretic peptide levels did not differ by rs5068 genotype. We identified a microRNA, miR-425, that is expressed in human atria and ventricles and is predicted to bind the sequence spanning rs5068 for the A, but not the G, allele. miR-425 silenced NPPA mRNA in an allele-specific manner, with the G allele conferring resistance to miR-425. This study identifies miR-425 as a regulator of ANP production, raising the possibility that miR-425 antagonists could be used to treat disorders of salt overload, including hypertension and heart failure

Genetic modifiers of hypertension in soluble guanylate cyclase α1–deficient mice

Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure–related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure–modifying mechanism in a setting of impaired NO/cGMP signaling

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

Androgen-sensitive hypertension associated with soluble guanylate cyclase-α₁ deficiency is mediated by 20-HETE

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the alpha(1)-subunit of the NO receptor soluble guanylate cyclase (sGC(alpha 1)(-/-) mice) display sex-and strain-specific hypertension: male but not female sGC(alpha 1)(-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex-and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGC(alpha 1)(-/-) S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy5,8,11,14- eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGC(alpha 1)(-/-) S6 than of male sGC(alpha 1)(-/-) B6 mice. Furthermore, treating male sGC(alpha 1)(-/-) S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background-and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGC(alpha 1)(-/-) deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGC(alpha 1)(-/-) deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling

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