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

The Ability of Nitric Oxide to Lower Intraocular Pressure Is Dependent on Guanylyl Cyclase

Purpose While nitric oxide (NO) donors are emerging as treatments for glaucoma, the mechanism by which NO lowers intraocular pressure (IOP) is unclear. NO activates the enzyme guanylyl cyclase (GC) to produce cyclic guanosine monophosphate. We studied the ocular effects of inhaled and topically applied NO gas in mice and lambs, respectively. Methods: IOP and aqueous humor (AqH) outflow were measured in WT and GC-1α subunit null (GC-1−/−) mice. Mice breathed 40 parts per million (ppm) NO in O2 or control gas (N2/O2). We also studied the effect of ocular NO gas exposure (80, 250, 500, and 1000 ppm) on IOP in anesthetized lambs. NO metabolites were measured in AqH and plasma. Results: In awake WT mice, breathing NO for 40 minutes lowered IOP from 14.4 ± 1.9 mm Hg to 10.9 ± 1.0 mm Hg (n = 11, P < 0.001). Comparable results were obtained in anesthetized WT mice (n = 10, P < 0.001). In awake or anesthetized GC-1−/− mice, IOP did not change under similar experimental conditions (P ≥ 0.08, n = 20). Breathing NO increased in vivo outflow facility in WT but not GC-1−/− mice (+13.7 ± 14.6% vs. −12.1 ± 9.4%, n = 4 each, P < 0.05). In lambs, ocular exposure to NO lowered IOP in a dose-dependent manner (−0.43 mm Hg/ppm NO; n = 5 with 40 total measurements; P = 0.04) without producing corneal pathology or altering pulmonary and systemic hemodynamics. After ocular NO exposure, NO metabolites were increased in AqH (n = 8, P < 0.001) but not in plasma. Conclusions: Breathing NO reduced IOP and increased outflow facility in a GC-dependent manner in mice. Exposure of ovine eyes to NO lowers IOP

Autologous transfusion of stored red blood cells increases pulmonary artery pressure

RATIONALE: Transfusion of erythrocytes stored for prolonged periods is associated with increased mortality. Erythrocytes undergo hemolysis during storage and after transfusion. Plasma hemoglobin scavenges endogenous nitric oxide leading to systemic and pulmonary vasoconstriction.OBJECTIVES: We hypothesized that transfusion of autologous blood stored for 40 days would increase the pulmonary artery pressure in volunteers with endothelial dysfunction (impaired endothelial production of nitric oxide). We also tested whether breathing nitric oxide before and during transfusion could prevent the increase of pulmonary artery pressure.METHODS: Fourteen obese adults with endothelial dysfunction were enrolled in a randomized crossover study of transfusing autologous, leukoreduced blood stored for either 3 or 40 days. Volunteers were transfused with 3-day blood, 40-day blood, and 40-day blood while breathing 80 ppm nitric oxide.MEASUREMENTS AND MAIN RESULTS: The age of volunteers was 41 ± 4 years (mean ± SEM), and their body mass index was 33.4 ± 1.3 kg/m(2). Plasma hemoglobin concentrations increased after transfusion with 40-day and 40-day plus nitric oxide blood but not after transfusing 3-day blood. Mean pulmonary artery pressure, estimated by transthoracic echocardiography, increased after transfusing 40-day blood (18 ± 2 to 23 ± 2 mm Hg; P &lt; 0.05) but did not change after transfusing 3-day blood (17 ± 2 to 18 ± 2 mm Hg; P = 0.5). Breathing nitric oxide decreased pulmonary artery pressure in volunteers transfused with 40-day blood (17 ± 2 to 12 ± 1 mm Hg; P &lt; 0.05).CONCLUSIONS: Transfusion of autologous leukoreduced blood stored for 40 days was associated with increased plasma hemoglobin levels and increased pulmonary artery pressure. Breathing nitric oxide prevents the increase of pulmonary artery pressure produced by transfusing stored blood. Clinical trial registered with www.clinicaltrials.gov (NCT 01529502).<br/

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify an characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy

Acta Antiqua

Background: Transfusion of human blood stored for more than 2 weeks is associated with increased mortality and morbidity. During storage, packed erythrocytes progressively release hemoglobin, which avidly binds nitric oxide. We hypothesized that the nitric oxide mediated hyperemic response after ischemia would be reduced after transfusion of packed erythrocytes stored for 40 days.Methods and results: We conducted a crossover randomized interventional study, enrolling 10 healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced packed erythrocytes, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index measurements were performed before and 10 min, 1 h, 2 h, and 4 h after transfusion. The reactive hyperemia index during the first 4 h after transfusion of 40-day compared with 3-day stored packed erythrocytes was unchanged. Plasma hemoglobin and bilirubin concentrations were higher after transfusion of 40-day than after 3-day stored packed erythrocytes (P = 0.02, [95% CI difference 10-114 mg/l] and 0.001, [95% CI difference 0.6-1.5 mg/dl], respectively). Plasma concentrations of potassium, lactate dehydrogenase, haptoglobin, and cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored packed erythrocytes, but not after transfusion of 3-day stored packed erythrocytes (P = 0.01, [95% CI difference 0.446-0.66 ? M]). Conclusions: Transfusion of autologous packed erythrocytes stored for 40 days is associated with increased hemolysis, an unchanged reactive hyperemia index, and increased concentrations of plasma nitrite