Tetrahydrobiopterin: Nitric Oxide and Pulmonary Vascular Control

GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate limiting step for the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS). NOS, once active, generates nitric oxide (NO) an essential mediator of normal vascular tone and function. Suboptimal levels of BH4 may render NOS inefficient and may additionally result in the generation of deleterious superoxide anions (O2-). GTP-CH1 is itself inhibited by BH4, an effect mediated via the protein GTP-CH1 Feedback Regulatory Protein (GFRP). There is mounting evidence correlating a dysfunction of the BH4/NOS pathway with the vascular endothelial dysfunction observed e.g. atherosclerosis, but little evidence exists associating a dysfunction of this pathway with pulmonary hypertension (PH). I have shown that GTP-CH1 protein expression is developmentally regulated in the porcine lung in the first few weeks of life, correlating with functional effects of supplemented BH4 on isolated pulmonary arteries. GTP-CH1 protein expression is unchanged in animals exposed to hypobaric exposure (a model of persistent pulmonary hypertension of the newborn (PPHN)) when compared to their age matched controls and GTP-CH1 localisation appeared unchanged both developmentally and in PPHN. Thus GTP-CH1 levels do not appear to be implicated in the profound endothelial dysfunction observed in animals with PPHN. However, studies performed on the GTP-CH1 deficient mouse mutant (hph-1) indicate that these mice exhibit the structural characteristics of a pulmonary hypertensive phenotype when compared to the C57BL/6xCBA wild types. Finally, GFRP over expression in isolated endothelial cells attenuates inducible NOS (iNOS) mediated NO generation following cytokine stimulation, an effect likely to be mediated via direct inhibition of BH4 biosynthesis. In summary, these studies have demonstrated that regulation of BH4 via GTP-CH1 or GFRP, affects the NOS pathway both in vitro and in vivo and that sub optimal levels of BH4 may contribute towards the development of PH

In vivo pharmacological activity and biodistribution of S-nitrosophytochelatins after intravenous and intranasal administration in mice

AbstractS-nitrosophytochelatins (SNOPCs) are novel analogues of S-nitrosoglutathione (GSNO) with the advantage of carrying varying ratios of S-nitrosothiol (SNO) moieties per molecule. Our aim was to investigate the in vivo pharmacological potency and biodistribution of these new GSNO analogues after intravenous (i.v.) and intranasal (i.n.) administration in mice. SNOPCs with either two or six SNO groups and GSNO were synthesized and characterized for purity. Compounds were administered i.v. or i.n. at 1 μmol NO/kg body weight to CD-1 mice. Blood pressure was measured and biodistribution studies of total nitrate and nitrite species (NOx) and phytochelatins were performed after i.v. administration. At equivalent doses of NO, it was observed that SNOPC-6 generated a rapid and significantly greater reduction in blood pressure (∼60% reduction compared to saline) whereas GSNO and SNOPC-2 only achieved a 30–35% decrease. The reduction in blood pressure was transient and recovered to baseline levels within ∼2 min for all compounds. NOx species were transiently elevated (over 5 min) in the plasma, lung, heart and liver. Interestingly, a size-dependent phytochelatin accumulation was observed in several tissues including the heart, lungs, kidney, brain and liver. Biodistribution profiles of NOx were also obtained after i.n. administration, showing significant lung retention of NOx over 15 min with minor systemic increases observed from 5 to 15 min. In summary, this study has revealed interesting in vivo pharmacological properties of SNOPCs, with regard to their dramatic hypotensive effects and differing biodistribution patterns following two different routes of administration

Clinical Applications of Attractor Reconstruction Analysis

This poster was presented at the Science and Engineering Impact Showcase, King's College London, 4th April 2017

Mathematical Modelling of Heart Rate Changes in the Mouse

The CVS is composed of numerous interacting and dynamically regulated physiological subsystems which each generate measurable periodic components such that the CVS can itself be presented as a system of weakly coupled oscillators. The interactions between these oscillators generate a chaotic blood pressure waveform signal, where periods of apparent rhythmicity are punctuated by asynchronous behaviour. It is this variability which seems to characterise the normal state. We used a standard experimental data set for the purposes of analysis and modelling. Arterial blood pressure waveform data was collected from conscious mice instrumented with radiotelemetry devices over $24$ hours, at a $100$ Hz and $1$ kHz time base. During a $24$ hour period, these mice display diurnal variation leading to changes in the cardiovascular waveform. We undertook preliminary analysis of our data using Fourier transforms and subsequently applied a series of both linear and nonlinear mathematical approaches in parallel. We provide a minimalistic linear and nonlinear coupled oscillator model and employed spectral and Hilbert analysis as well as a phase plane analysis. This provides a route to a three way synergistic investigation of the original blood pressure data by a combination of physiological experiments, data analysis viz. Fourier and Hilbert transforms and attractor reconstructions, and numerical solutions of linear and nonlinear coupled oscillator models. We believe that a minimal model of coupled oscillator models that quantitatively describes the complex physiological data could be developed via such a method. Further investigations of each of these techniques will be explored in separate publications

Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis

AbstractIschemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1+/− and DDAH2−/− deficient mice by in vivo fluorescein angiography, immunohistochemistry and electroretinography revealed normal neurovascular function compared with wildtype control mice. Finally, DDAH1+/− and DDAH2−/− deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1+/− exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis

Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1β production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage