The DDAH/ADMA pathway is a critical regulator of NO signalling in vascular homeostasis

NO is an important regulator of cardiovascular remodelling and function. ADMA, an endogenous L-arginine analogue, reduces NO production by inhibiting the activity of NOS. ADMA levels in turn, are regulated by DDAH, which metabolises ADMA. High levels of ADMA and dysregulated DDAH activity are risk factors for cardiovascular disease and morbidity. To investigate this link, the DDAH I null mouse has been recently generated and has a lethal phenotype. Studies on vascular function in the DDAH I heterozygous knockout mouse, which is viable, demonstrates a causal link between reduced DDAH I activity, increased ADMA levels and reduced NO signalling and vascular dysfunction. In another study, detailed in vitro analyses reveal that the DDAH/ADMA pathway critically regulates endothelial cell motility and angiogenesis and establishes some of the molecular mechanisms involved. These studies highlight the importance of DDAH and ADMA in regulating NO dependent vascular homeostasis.Key words: asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), nitric oxide (NO), angiogenesis, endothelial, motilityNO is generated from L-arginine by NOS; a process which is competitively inhibited by the arginine analogues ADMA and L-NMMA. These endogenous factors are products of proteolytic degradation of methylated proteins. ADMA and L-NMMA are metabolised by DDAH I and II, thereby enhancing NO generation. Of relevance to vascular biology, dysfunctional DDAH activity and ADMA accumulation are risk factors for cardiovascular disorders, including hypertension, artherosclerosis, diabetes, insulin resistance, hypercholesterolemia and homocysteinemia (reviewed in ref. ¹).The DDAH I null mouse was generated recently by Leiper et al.² to facilitate investigation of the role of the DDAH/ADMA pathway in the pathology of cardiovascular disorders. While the absence of DDAH I causes a lethal phenotype, heterozygotes (HT) did not display any obvious abnormalities. However, ADMA levels were raised in tissues and plasma, in association with raised blood pressure and systemic vascular resistance, and reduced cardiac output and heart rate. Synthetic DDAH I inhibitors were designed by the authors and were shown by crystallography to bind to the active site of the enzyme and induce local distortions at this region. Confirming that loss of DDAH I was responsible for ADMA accumulation, these inhibitors enhanced ADMA levels in wildtype mice, and resulted in cardiovascular changes similar to those seen in the HT background. Inhibitor treatment also promoted ADMA release from wildtype blood vessels maintained ex vivo, indicating that the DDAH/ADMA pathway is directly responsible for maintaining cardiovascular function in this model.Evidence was also presented for a causal link between ADMA metabolism and reduced NO levels. In an ex vivo model, aortic rings from HT mice displayed enhanced phenylephrine-induced contraction and reduced acetylcholine-induced relaxation, while DDAH I inhibitors induced similar responses in aortic rings from wildtype mice; indicative of reduced levels of endothelial-derived NO. Further demonstrating an ADMA/NO-dependent mechanism, exogenous L-arginine restored a normal response to these vasomodulators in the HT model (by competing with ADMA for interaction with NOS). Similarly, cultured endothelial cells from HT vessels produced more ADMA and less NO than cells from wildtype vessels, and DDAH I inhibitors induced a similar phenotype in wildtype endothelial cells. The significance of DDAH I/ADMA and NO in vascular disease was tested in a disease model. Endotoxic shock was induced in rats by intravenous infusion of LPS, which induces excess NO production, resulting in systemic hypotension. After blood pressure had fallen by 20%, infusion of a DDAH I inhibitor was able to rapidly stabilise blood pressure, in accordance with inhibition of NO production through reduced ADMA metabolism. Thus, when DDAH I is reduced, ADMA is increased and endogenous NO inhibited, resulting in altered vascular function.Another related study investigated a mechanistic understanding of the role of ADMA/DDAH/NO in angiogenesis.³ The authors demonstrated that ADMA regulates endothelial cell motility and phenotype by inhibiting NO-dependent changes in activity of Rho-GTPases; key mediators of cytoskeletal dynamics and motility. Treatment of pulmonary artery endothelial cells with ADMA enhanced stress fibres and focal adhesion formation in conjunction with increased activity of RhoA in pull-down assays. In accordance with these observations, motility, tracked by time-lapse microscopy, was inhibited by ADMA treatment, and ADMA effects were reversed by a Rho kinase inhibitor (Y-27632) or by adenoviral-mediated gene transfer of a dominant negative RhoA mutant. RhoA activity is mediated by PKG, which mediates RhoA-Ser188 phosphorylation, preventing RhoA localization to the membrane and inhibiting its activity.⁴ In further support of a RhoA-dependent mechanism, ADMA reduced phosphorylation at RhoA-Ser188, while a PKG activator was also able to revert ADMA effects on motility. Further, a non-phosphorylatable mutant of RhoA, Ala188RhoA, or a specific PKG inhibitor, each inhibited cell motility to a similar level as ADMA treatment alone. Inhibition of NO production and endothelial cell motility by ADMA was also reversed by a NO donor, SNAP, or by DDAH I or II overexpression via adenovirus-mediated gene transfer. Thus, reduction of NO/PKG levels by ADMA reduces RhoA phosphorylation at Ser188 resulting in enhancement of RhoA activity and inhibition of cell motility.The significance of these molecular mechanisms to angiogenesis was demonstrated using endothelial cells and aortic ring explants from HT DDAH I and wildtype mice. HT endothelial cells, which secrete more ADMA and produce less NO than their wildtype counterparts, exhibit enhanced RhoA activity and stress fibre formation in conjunction with reduced motility. Reduced sprouting from ex vivo aortic rings was also observed in the HT model, which was mimicked by addition of exogenous ADMA in the wildtype background. These data demonstrate that in vivo, DDAH/ADMA levels are likely to play a key role in control of endothelial cell motility and angiogenesis by regulating NO production.     

Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering

The conformational associative properties of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100 kGy. The conformational change in lambda-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and lambda-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, iota-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan>kappa-carrageenan>iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation.     

A dye-binding assay for measurement of the binding of Cu(II) to proteins

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to α-synuclein of ∼1 × 10⁹ M⁻¹ was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).     

Developmentally acquired PKA localisation in mouse oocytes and embryos

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development.Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.     

Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions

Cell migration plays a critical role in a wide variety of physiological and pathological phenomena as well as in scaffold-based tissue engineering. Cell migration behavior is known to be governed by biochemical stimuli and cellular interactions. Biophysical processes associated with interactions between the cell and its surrounding extracellular matrix may also play a significant role in regulating migration. Although biophysical properties of two-dimensional substrates have been shown to significantly influence cell migration, elucidating factors governing migration in a three-dimensional environment is a relatively new avenue of research. Here, we investigate the effect of the three-dimensional microstructure, specifically the pore size and Young's modulus, of collagen-glycosaminoglycan scaffolds on the migratory behavior of individual mouse fibroblasts. We observe that the fibroblast migration, characterized by motile fraction as well as locomotion speed, decreases as scaffold pore size increases across a range from 90 to 150 μm. Directly testing the effects of varying strut Young's modulus on cell motility showed a biphasic relationship between cell speed and strut modulus and also indicated that mechanical factors were not responsible for the observed effect of scaffold pore size on cell motility. Instead, in-depth analysis of cell locomotion paths revealed that the distribution of junction points between scaffold struts strongly modulates motility. Strut junction interactions affect local directional persistence as well as cell speed at and away from the junctions, providing a new biophysical mechanism for the governance of cell motility by the extracellular microstructure.     

Lower uterine artery blood flow and higher endothelin relative to nitric oxide metabolite levels are associated with reductions in birth weight at high altitude

Reduced uteroplacental blood flow is hypothesized to play a key role in altitude-associated fetal growth restriction. It is unknown whether reduced blood flow is a cause or consequence of reduced fetal size. We asked whether determinants of uteroplacental blood flow were altered prior to reduced fetal growth and whether vasoactive and/or angiogenic factors were involved. Women residing at low (LA; 1,600 m, n = 18) or high altitude (HA; 3,100 m, n = 25) were studied during pregnancy (20, 30, and 36 wk) and 4 mo postpartum (PP) using Doppler ultrasound. In each study, endothelin (ET-1), nitric oxide metabolites (NO(x)), soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PlGF) levels were quantified. At HA, birth weights were lower (P < 0.01) and small-for-gestational age was more common (P < 0.05) compared with LA. HA was associated with lower uterine artery (UA) diameter (P < 0.01) and blood flow (P < 0.05). Altitude did not affect ET-1, sFlt-1 or PlGF; however, ET-1/NO(x) was greater and NO(x) lower during pregnancy and PP at HA vs. LA. ET-1/NO(x) was negatively associated with birth weight (20 wk, P < 0.01; 36 wk, P = 0.05) at LA and HA combined. At HA, UA blood flow (30 wk) was positively associated with birth weight (dagger). UA blood flow and ET-1/NO(x) levels accounted for 45% (20 wk) and 32% (30 wk) of birth weight variation at LA and HA combined, primarily attributed to effects at HA. We concluded that elevated ET-1/NO(x) and altered determinants of uteroplacental blood flow occur prior to altitude-associated reductions in fetal growth, and therefore, they are likely a cause rather than a consequence of smaller fetal size.     

Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

Background  

Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis.