Affinity-optimizing enhancer variants disrupt development.

Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.

Nitric oxide regulates tissue transglutaminase localization and function in the vasculature.

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro L-arginine methyl ester (L-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by L-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.

A physiologic and biochemical profile of clinically rejected lungs on a normothermic ex vivo lung perfusion platform.

BACKGROUND: Although ex vivo lung perfusion (EVLP) is increasingly being used to evaluate and manipulate potential donor lungs before lung transplantation (LTx), data on the biochemistry of lungs during EVLP are limited. In this study, we examined the physiology and biochemistry of human lungs on an EVLP circuit. METHODS: We recovered unallocated double lungs in standard fashion and split them into single lungs. All lungs received a nebulized arginase inhibitor, 2-S-amino-6-boronohexanoic acid (ABH), at either the onset (n = 6) or after 3 h (n = 8) of EVLP. Serial biochemical analysis included levels of arginase, endogenous nitric oxide synthase (eNOS), cyclic guanosine monophosphate, and reactive oxygen species. We considered lungs transplantable if they sustained a PaO2:FiO2 ≥ 350 in addition to stable pulmonary function during EVLP. RESULTS: We recovered a total of 14 single lungs. We deemed three single lungs from different donors to be transplantable after EVLP. These lungs had superior oxygenation, lower carbon dioxide, and more stable pulmonary artery pressures. Transplantable lungs had higher baseline levels of eNOS and higher final levels of cyclic guanosine monophosphate than non-transplantable lungs. Early ABH administration was associated with a transient increase in dynamic compliance. CONCLUSIONS: In this biochemical characterization of lungs deemed unsuitable for LTx, early levels of eNOS and late levels of cyclic guanosine monophosphate appear to be associated with improved allograft function during EVLP. In addition, nebulized ABH is associated with a significant increase in dynamic compliance. These data suggest that biochemical markers during EVLP may predict acceptable allograft function, and that this platform can be used to biochemically manipulate donor lungs before LTx.

Inhaled hydrogen sulfide improves graft function in an experimental model of lung transplantation.

OBJECTIVES: Ischemia/reperfusion injury (IRI) is a common complication of lung transplantation (LTx). Hydrogen sulfide (H(2)S) is a novel agent previously shown to slow metabolism and scavenge reactive oxygen species, potentially mitigating IRI. We hypothesized that pretreatment with inhaled H(2)S would improve graft function in an ex vivo model of LTx. METHODS: Rabbits (n = 10) were ventilated for 2 h prior to heart-lung bloc procurement. The treatment group (n = 5) inhaled room air (21% O(2)) supplemented with 150 ppm H(2)S while the control group (n = 5) inhaled room air alone. Both groups were gradually cooled to 34°C. All heart-lung blocs were then recovered and cold-stored in low-potassium dextran solution for 18 h. Following storage, the blocs were reperfused with donor rabbit blood in an ex vivo apparatus. Serial clinical parameters were assessed and serial tissue biochemistry was examined. RESULTS: Prior to heart-lung bloc procurement, rabbits pretreated with H(2)S exhibited similar oxygenation (P = 0.1), ventilation (P = 0.7), and heart rate (P = 0.5); however, treated rabbits exhibited consistently higher mean arterial blood pressures (P = 0.01). During reperfusion, lungs pretreated with H(2)S had better oxygenation (P < 0.01) and ventilation (P = 0.02), as well as lower pulmonary artery pressures (P < 0.01). Reactive oxygen species levels were lower in treated lungs during reperfusion (P = 0.01). Additionally, prior to reperfusion, treated lungs demonstrated more preserved mitochondrial cytochrome c oxidase activity (P = 0.01). CONCLUSIONS: To our knowledge, this study represents the first reported therapeutic use of inhaled H(2)S in an experimental model of LTx. After prolonged ischemia, lungs pretreated with inhaled H(2)S exhibited improved graft function during reperfusion. Donor pretreatment with inhaled H(2)S represents a potentially novel adjunct to conventional preservation techniques and merits further exploration.

Hydrogen sulfide decreases reactive oxygen in a model of lung transplantation.

BACKGROUND: Ischemia-reperfusion injury is a common complication after lung transplantation. Ischemia-reperfusion injury is thought to be mediated by reactive oxygen species (ROS). Hydrogen sulfide (H(2)S) is a novel agent that has been previously shown to scavenge ROS and slow metabolism. We evaluated the effect of infused H(2)S on the presence of ROS after reperfusion in an ex vivo model of lung transplantation. METHODS: Heart-Lung blocks were recovered from New Zealand white rabbits (n = 12) and cold stored in Perfadex solution for 18 h. After storage, the heart-lung blocks were reperfused ex vivo with donor rabbit blood. In the treatment group (n = 7), a bolus of sodium H(2)S was added at the beginning of reperfusion (100 µg/kg) and continuously infused throughout the 2-h experiment (1 mg/kg/h). The vehicle group (n = 5) received an equivalent volume of saline. Serial airway and pulmonary artery pressures and arterial and venous blood gases were measured. RESULTS: Oxygenation and pulmonary artery pressures were similar between the 2 groups. However, treatment with H(2)S resulted in a dramatic reduction in the presence of ROS after 2 h of reperfusion (4,851 ± 2,139 versus 235 ± 462 related fluorescence units/mg protein; P = 0.003). A trend was seen toward increased levels of cyclic guanosine monophosphate in the H(2)S-treated group (3.08 ± 1.69 versus 1.73 ± 1.41 fmol/mg tissue; P = .23). CONCLUSIONS: After prolonged ischemia, infusion of H(2)S during reperfusion was associated with a significant decrease in the presence of ROS, a suspected mediator of ischemia-reperfusion injury. To our knowledge, the present study represents the first reported therapeutic use of H(2)S in an experimental model of lung transplantation.

S-nitrosation of arginase 1 requires direct interaction with inducible nitric oxide synthase.

Arginase constrains endothelial nitric oxide synthase activity by competing for the common substrate, L -Arginine. We have recently shown that inducible nitric oxide synthase (NOS2) S-nitrosates and activates arginase 1 (Arg1) leading to age-associated vascular dysfunction. Here, we demonstrate that a direct interaction of Arg1 with NOS2 is necessary for its S-nitrosation. The specific domain of NOS2 that mediates this interaction is identified. Disruption of this interaction in human aortic endothelial cells prevents Arg1 S-nitrosation and activation. Thus, disruption of NOS2-Arg1 interaction may represent a therapeutic strategy to attenuate age related vascular endothelial dysfunction.