Laurel T. Tainsh

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 α1 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 α1–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 α1 and β1 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.

Weight Loss, Saline Loading, and the Natriuretic Peptide System

Background In epidemiologic studies, obesity has been associated with reduced natriuretic peptide (NP) concentrations. Reduced NP production could impair the ability of obese individuals to respond to salt loads, increasing the risk of hypertension and other disorders. We hypothesized that weight loss enhances NP production before and after salt loading. Methods and Results We enrolled 15 obese individuals (mean BMI 45±5.4 kg/m2) undergoing gastric bypass surgery. Before and 6 months after surgery, subjects were admitted to the clinical research center and administered a large‐volume intravenous saline challenge. Echocardiography and serial blood sampling were performed. From the pre‐operative visit to 6 months after surgery, subjects had a mean BMI decrease of 27%. At the 6‐month visit, N‐terminal pro‐atrial NP (Nt‐proANP) levels were 40% higher before, during, and after the saline infusion, compared with levels measured at the same time points during the pre‐operative visit (P<0.001). The rise in Nt‐pro‐ANP induced by the saline infusion (≈50%) was similar both before and after surgery (saline, P<0.001; interaction, P=0.2). Similar results were obtained for BNP and Nt‐proBNP; resting concentrations increased by 50% and 31%, respectively, after gastric bypass surgery. The increase in NP concentrations after surgery was accompanied by significant decreases in mean arterial pressure (P=0.004) and heart rate (P<0.001), and an increase in mitral annular diastolic velocity (P=0.02). Conclusion In obese individuals, weight loss is associated with a substantial increase in the “setpoint” of circulating NP concentrations. Higher NP concentrations could contribute to an enhanced ability to handle salt loads after weight loss.

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

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 CYP2J2, 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 Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, 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 Cyp2j gene products regulate the pulmonary vascular response to hypoxia.

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 α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) 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 sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-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 sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)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 sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.

WEIGHT LOSS, SALINE LOADING, AND THE NATRIURETIC PEPTIDE SYSTEM

In epidemiologic studies, obesity has been associated with reduced natriuretic peptide (NP) concentrations leading to the hypothesis that obesity is a state of relative “NP deficiency”. However, data from prior studies have been restricted to resting NP concentrations, which may not reflect how

Genetic mapping of a modifier locus affecting hypertension in soluble guanylate cyclase α1 deficient mice

Background We previously reported that male mice deficient in the a1 subunit of the NO receptor soluble guanylate cyclase (sGCa1 -/mice), an important nitric oxide (NO) receptor, are hypertensive [1]. The phenotype depends on the genetic background: sGCa1 -/mice on a 129S6 (S6) background (sGCa1 ) but not on a C57BL/6 (B6) background (sGCa1 ) develop hypertension [2]. These findings suggest that hypertension associated with sGCa1-deficiency is modulated by genetic factors. We aimed to identify modifier genes underlying the hypertension in sGCa1 -/-S6 mice.

sGCα1-deficient mice: a novel murine model of spontaneous primary open angle glaucoma

Background Primary open angle glaucoma (POAG) is a progressive eye disease that leads to blindness due to the irreversible loss of retinal ganglion cells and degeneration of the optic nerve (ON). As of yet, there is no cure for glaucoma. Although available therapies delay disease progression, they offer incomplete protection. Elevated intraocular pressure (IOP) is an important risk factor for POAG. However, the exact molecular mechanisms that trigger increased IOP and glaucomatous optic neuropathy remain incompletely understood. While a few spontaneous murine models of glaucoma exist, none are models of POAG, the most prevalent form of glaucoma. Impaired nitric oxide (NO) signaling has been implicated in the development of glaucoma. To further test the hypothesis that impaired NO/cGMP signaling contributes to the pathogenesis of POAG, we tested whether mice lacking the a1 subunit of the NO receptor soluble guanylate cyclase (sGCa1 -/mice) develop POAG.