Jacqueline L. Whatmore

Adiposity is a major determinant of plasma levels of the novel vasodilator hydrogen sulphide

Aims/hypothesisHydrogen sulphide is a recently identified endogenous endothelium-dependent vasodilator. Animal models of diabetes have shown that low plasma H2S levels are associated with marked endothelial dysfunction and insulin resistance. However, human studies on H2S and vascular function in health and disease are lacking.MethodsPlasma was obtained from male patients with type 2 diabetes (n = 11), overweight (n = 16) and lean (n = 11) volunteers. H2S levels were determined by zinc trap spectrophotometry. Anthropometric measurements (BMI/waist:hip ratio), lipid profile, systemic blood pressure, biochemical indices of diabetes (fasting glucose, insulin sensitivity, Hb1Ac) and microvascular function (minimum vascular resistance) were determined.ResultsMedian plasma H2S levels (25th, 75th percentiles) in age-matched lean, overweight and type 2 diabetes individuals were 38.9 (29.7, 45.1) µmol/l, 22.0 (18.6, 26.7) µmol/l and 10.5 (4.8, 22.0) µmol/l, respectively. Median plasma H2S levels were significantly lower in patients with type 2 diabetes compared with lean (p = 0.001, Mann–Whitney) and overweight participants (p = 0.008). Median plasma H2S levels in overweight participants were significantly lower than in lean controls (p = 0.003). Waist circumference was an independent predictor of plasma H2S (R2 = 0.423, standardised beta: −0.650, p < 0.001). This relationship was independent of diabetes, which only contributed a further 5% to the model (R2 = 0.477). Waist circumference or other measures of adiposity (waist:hip ratio/BMI) remained independent predictors of plasma H2S after adjustment for systolic blood pressure, microvascular function, insulin sensitivity, glycaemic control and lipid profile.Conclusions/interpretationPlasma H2S levels are reduced in overweight participants and patients with type 2 diabetes. Increasing adiposity is a major determinant of plasma H2S levels.

ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils

Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.

Increased blood vessel density and endothelial cell proliferation in multiple sclerosis cerebral white matter.

Multiple sclerosis (MS) is primarily considered an inflammatory demyelinating disease, however the role of vasculature in MS pathogenesis is now receiving much interest. MS lesions often develop along blood vessels and alterations in blood brain barrier structure and function, with associated changes in the basement membrane, are pathological features. Nevertheless, the possibility of angiogenesis occurring in MS has received little attention. In this study we used triple label enzyme immunohistochemistry to investigate blood vessel density and endothelial cell proliferation in MS samples (n=39) compared with control tissue to explore evidence of angiogenesis in MS. The results showed that in all MS samples examined blood vessel density increased compared with controls. The greatest increase was found in subacute lesions where numbers of positively stained vessels increased from 43.9+/-8.5% in controls to 84.2+/-13.3% (P=0.001). Furthermore, using an antibody against endoglin (CD105), a specific marker of proliferating endothelial cells, which are characteristic of angiogenesis, we have shown that vessels containing proliferating endothelial cells were more pronounced in all MS tissue examined (normal-appearing white matter, acute, subacute and chronic lesions, P>or=0.027) compared with control and this was greatest in the MS normal-appearing white matter (68.8+/-19.8% versus 10.58+/-6.4%, P=0.003). These findings suggest that angiogenesis may play a role in lesion progression, failure of repair and scar formation.

The synthesis and functional evaluation of a mitochondria-targeted hydrogen sulfide donor, (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)decyl) triphenylphosphonium bromide (AP39)

Synthesis and bioavailability of the endogenous gasomediator hydrogen sulfide (H2S) is perturbed in many disease states, including those involving mitochondrial dysfunction. There is intense interest in developing pharmacological agents to generate H2S. We have synthesised a novel H2S donor molecule coupled to a mitochondria-targeting moiety (triphenylphosphonium; TPP+) and compared the effectiveness of the compound against a standard non-TPP+ containing H2S donor (GYY4137) in the inhibition of oxidative stress-induced endothelial cell death. Our study suggests mitochondria-targeted H2S donors are useful pharmacological tools to study the mitochondrial physiology of H2S in health and disease.

Observation and characterisation of the glycocalyx of viable human endothelial cells using confocal laser scanning microscopy

This paper describes the use of confocal laser scanning microscopy (CLSM) to observe and characterise the fully hydrated glycocalyx of human umbilical vein endothelial cells (HUVECs). Viable HUVECs in primary culture were studied at room temperature in HEPES-buffered, phenol red- and serum-free CS-C cell culture medium. A fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC) (2 μg ml−1, 30 min) was used to detect N-acetylneuraminic (sialic) acid, which is a significant component of the endothelial glycocalyx. Single confocal sections, less than 1.3 μm thick, were collected at intervals of 0.5 μm, scanning through the entire z-axis of a series of cells. Cell-surface associated staining was observed, which enabled the glycocalyx thickness to be deduced as 2.5 ± 0.5 μm. This dimension is significantly greater than that measured by electron microscopy, for glutaraldehyde-fixed cells (0.10 ± 0.04 μm). The specificity of WGA-FITC staining was demonstrated by treatments with several enzymes, known to degrade glycocalyx (heparatinase, chondroitinase, hyaluronidase and neuraminidase), of which neuraminidase (1 U ml−1, 30–60 min) was the most effective, removing up to 78 ± 2% of WGA-FITC binding to HUVECs. Cell viability was assessed simultaneously with ethidium homodimer-1 staining and confirmed by standard colorimetric 3-[4,5]dimethylthiazol-2,5diphenyltetrazolium bromide (MTT) test. CLSM thus provides a useful approach for in situ visualisation and characterisation of the endothelial glycocalyx in viable preparations, revealing a thickness that is an order of magnitude greater than found in ex situ measurements on fixed cells.

Comparative study of isoflavone, quinoxaline and oxindole families of anti-angiogenic agents

A study designed to compare the effects on VEGF-induced angiogenesis of a number of known anti-angiogenic agents together with some novel derivatives thereof was undertaken. Thus the isoflavone biochanin A 1, indomethacin 2, the 3-arylquinoxaline SU1433 and its derivatives 3–6, the benzoic acid derivative 7, the oxindoles SU5416 8 and SU6668 11, together with their simple N-benzyl derivatives 9, 10, and 12 were selected for study. Using an in vitro assay the compounds were evaluated for their ability to inhibit VEGF-induced angiogenesis in HUVECs, and the cytotoxicity of representative compounds was also studied in tumour cell lines using 24-h exposure. The results indicate that the SU compounds, SU1433, SU5416 and SU6668, are more potent inhibitors of VEGF-induced angiogenesis than indomethacin or the naturally occurring biochanin A, presumably because they inhibit VEGF receptor signalling. Blocking one of the phenolic OH groups of SU1433 reduced anti-angiogenic activity, as did blocking the NH groups of SU5416 and SU6668. Cytotoxicity studies indicate that none of the compounds examined exhibited cytotoxicity at anti-angiogenic concentrations.

Synthesis of surface sphingomyelin in the plasma membrane recycling pathway of BHK cells

Sphingomyelin, which has been degraded at the BHK cell surface by exogenous sphingomyelinase, is converted back into sphingomyelin with kinetics similar to those of plasma membrane recycling. Resynthesis of sphingomyelin under these conditions proceeds at a rate about 4-fold higher than normal biosynthesis of sphingomyelin. Neither resynthesis of sphingomyelin nor its return to the surface is inhibited by brefeldin A (BFA), which is a potent blocker of vesicular transport through the Golgi but has no effect on plasma membrane recycling. However, resynthesis of plasma membrane sphingomyelin is greatly decreased in cells undergoing mitosis or energy depletion, where endocytosis is inhibited. We conclude that the main site of surface sphingomyelin synthesis in BHK cells could be in recycling endosomes and not in the Golgi apparatus as proposed previously. We also suggest a model pathway by which cholesterol may reach the plasma membrane via recycling endosomes.

ARF1‐regulated phospholipase D in human neutrophils is enhanced by PMA and MgATP

Human neutrophil PLD activity stimulated with GTP‐γ‐S was reconstituted with recombinant ARF1 in cytosol‐depleted cells. PMA‐pretreatment of intact cells greatly enhanced the subsequent reconstitution of the ARF1‐regulated PLD activity. This enhancement was only observed provided that the intact cells were pretreated with PMA, suggesting the stable recruitment of a cytosolic component, presumably protein kinase C, to the membranes. rARF1‐reconstituted PLD activity was not dependent on MGATP, but could be considerably enhanced by MgATP. Maximal effects of MgATP were seen at 1 mM. This enhancement by MgATP could not be attributed to protein kinase C. Neomycin was found to inhibit ARF1‐regulated PLD activity suggesting the requirement for polyphosphoinositides. We conclude: (i) that many of the observed effects of PMA may be dependent on the presence of the small GTP‐binding protein, ARF, and (ii) polyphosphoinositides are required for ARF1‐stimulated PLD activity.

Tenascin C induces a quiescent phenotype in cultured adult human astrocytes

Astrocytic scar formation occurs subsequent to brain and spinal cord injury and impedes repair. The exact mechanisms of scar formation have yet to be elucidated but it is known that astrocytes within the scar have a different antigenic phenotype from normal or reactive astrocytes. Astrocyte cell culture offers a suitable system to identify factors that induce the scar phenotype as well as factors that reverse this process and that may help identify therapeutic strategies to treat astrogliosis. However, when placed in standard culture conditions, astrocytes become activated/reactive and express molecules characteristic of scar tissue in vivo. In the present study, we made use of this phenomenon to identify culture conditions that change the activated phenotype of cultured astrocytes into one characteristic of normal quiescent astrocytes. In particular, we examined the effect of extracellular matrix (ECM) proteins found in the human brain, on the phenotype of human adult astrocytes. Significantly fewer astrocytes expressed scar properties when grown on tenascin‐C (TN‐C) than those cultured on other ECM proteins or poly‐L‐lysine‐coated dishes. TN‐C also significantly reduced the proliferation rate of the astrocytes in vitro. In addition, further manipulation of culture conditions induced partial astrocyte reactivation. Our findings suggest that astrocytes grown on TN‐C revert to a quiescent, nonactivated state that is partially reversible. This raises the possibility that therapeutic strategies aimed at manipulating TN‐C levels during CNS injury may help reduce astrocytic scarring.

The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro

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