Modulation of Endothelial Barrier Function

Berman, Rodney Simon (1994) Modulation of Endothelial Barrier Function. PhD thesis, University of Glasgow.

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Abstract

A. Effects of the hypoxanthine-xanthine oxidase system and homocysteine on endothelial barrier function 1. Endothelial barrier function was assessed by use of an in vitro model in which the transfer of trypan blue-labelled albumin was measured across monolayers of bovine aortic endothelial cells (BAEC) grown on polycarbonate membranes. 2. Addition of either hypoxanthine (0.2 mM) or xanthine oxidase (20 mU ml-1) alone to BAEC monolayers during a 90 minute incubation period was found to have no effect on the level of albumin transfer obtained, but a combination of both was found to significantly increase transfer. 3. The increase in albumin transfer induced by hypoxanthine and xanthine oxidase was abolished by catalase (3 U ml-1), significantly reduced by allopurinol (4 mM), but unaffected by superoxide dismutase (6000 U ml-1), the hydroxyl radical scavengers, mannitol (15 mM), dimethylthiourea (10 mM) and N-(2-mercaptopropionyl)- glycine (1 mM), overnight pretreatment with the iron chelator, deferoxamine (0.5 mM), ferric chloride (50muM) an inhibitor of nitric oxide synthase, N?-nitro-L-arginine (L-NOARG), or the antioxidant, dithiothreitol (3 mM). 4. Addition of xanthine (0.2 mM) in combination with xanthine oxidase (20 mU ml-1) generated a similar increase in albumin transfer across BAEC monolayers to that obtained using hypoxanthine in combination Modulation of Endothelial Barrier Function Summary with xanthine oxidase. The increase induced by xanthine and xanthine oxidase was similarly abolished by catalase (3 U ml-1). 5. Hydrogen peroxide (0.1-30 mM) itself induced an increase in albumin transfer across monolayers of BAEC, exhibiting a biphasic concentration-response curve with peaks at around 0.1-0.3 nM and 10-30 mM. The increase in albumin transfer induced by 0.1 mM was abolished by 0.3 U ml-1 catalase, whilst that induced by 10 mM hydrogen peroxide was abolished by 3000 U ml-1 catalase. 6. Homocysteine (0.5 and 1.5 mM) was found to have no effect on the level of albumin transfer across BAEC monolayers which was obtained when it was added alone. However, when it was added in combination with copper sulphate (5 and 50 muM) which catalyses its oxidation to homcystine, a significant increase in albumin transfer was observed. 7. The increase in albumin transfer induced by the combination of homocysteine (1.5 mM) and copper sulphate (50 muM) was abolished by catalase (1 U ml-1), but was unaffected by superoxide dismutase (6000 U ml-1), mannitol (15 mM), dimethylthiourea (1 mM) or overnight pretreatment with deferoxamine (0.5 mM). 8. The data suggest that the endothelial barrier dysfunction induced by the combination of hypoxanthine and xanthine oxidase is likely mediated solely by the actions of hydrogen peroxide and not by superoxide anion, hydroxyl radical, peroxynitrite anion, nitric oxide or hypochlorous acid. Also, it was shown that xanthine and hypoxanthine may both equally well be used as substrates for xanthine oxidase in order to induce endothelial barrier dysfunction. Modulation of Endothelial Barrier Function Summary These findings further indicate that the endothelial barrier dysfunction which is associated with ischaemia-reperfusion injury could well be mediated by the hypoxanthine-xanthine oxidase system which is known to be activated in this condition. 9. The data also indicate that endothelial barrier dysfunction is induced by the copper-catalysed oxidation of homocysteine, rather than by a direct action of homocysteine itself. This dysfunction is also likely mediated solely by hydrogen peroxide and not by superoxide anion or hydroxyl radical. This ability of homocysteine to induce endothelial barrier dysfunction in the presence of copper may contribute to the atherogenic actions of homocysteine observed in sufferers of homocystinuria. B. Effects of lipopolysaccharide (LPS) on endothelial barrier function 10. Following 24 hours' incubation, after which the transfer of albumin across monolayers of BAEC was measured, LPS (0.1-1000 ng ml-1) was found to induce a concentration-dependent increase in albumin transfer. 11. The increase in albumin transfer induced by LPS (30 ng ml-1) was found to develop with a biphasic time-course. An early, transient peak was observed which was maximal at around 2 hours. The level of albumin transfer then declined back towards basal levels before rising again, nearing a second maximum by 24 hours. 12. The increase in albumin transfer induced following 24 hours' incubation with LPS (30 ng ml-1) was abolished by polymixin B Modulation of Endothelial Barrier Function Summary (10 mug ml-1), enhanced by the nitric oxide synthase inhibitor, L-NMMA (2 mM), but unaffected by the nitric oxide synthase inhibitors, L-NOARG (100 muM) and L-NAME (500 muM), the cyclo-oxygenase inhibitor, flurbiprofen (30 muM), catalase (1000 U ml-1) or by a 20 hour pretreatment with the glucocorticoid, dexamethasone (1muM). 13. The increase in albumin transfer induced following 2 hours' incubation with LPS (30 ng ml-1) was abolished by polymixin B (10 mug ml-1), enhanced by L-NMMA (2 mM), but unaffected by L-NAME (500 muM) or by a 20 hour pretreatment with dexamethasone (1 muM).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Billy Martin
Keywords: Pharmacology, Physiology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-74834
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:56
Last Modified: 27 Sep 2019 15:56
URI: http://theses.gla.ac.uk/id/eprint/74834

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