Effect of hypoxia on the cardiovascular sphingolipid system

Alganga, Husam. S.F. (2017) Effect of hypoxia on the cardiovascular sphingolipid system. PhD thesis, University of Glasgow.

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Sphingosine kinase 1 (SK1) catalyses the synthesis of the important bioactive sphingolipid sphingosine-1-phosphate (S1P), that has an important role in vascular tone regulation and cardioprotection against ischaemia/reperfusion injury. The work presented in this thesis describes the influence of short periods of hypoxia on expression of SK1 in vascular endothelium and how this may regulate vascular function. The aims were achieved by using wire myography to study vascular function and confocal microscopy for the studies of expression and distribution of SK1 under normoxic and hypoxic conditions.
In the first study, it was found that exposure of isolated rat coronary artery to a short period of hypoxia increases SK1 expression and ser225 phosphorylation. It was also demonstrated that the hypoxia-induced increase in SK1 expression was reduced by pre-treatment with cycloheximide, a protein synthesis inhibitor, SKi, a non-selective SK inhibitor and PF543, a selective SK1 inhibitor. However, pre-treatment with proteasomal and/or lysosomal inhibitors did not increase SK1 expression under normoxia or hypoxia. Similarity, SK1 expression was also increased in aortic endothelium following exposure to short-term hypoxia and this effect was also inhibited by cycloheximide, SKi and PF543. Collectively, these data suggest that hypoxia increases SK1 synthesis in coronary and aortic endothelium. Moreover, the SKi-induced reduction in SK1 expression in coronary endothelium was reversed by proteasomal and/or lysosomal inhibitors, indicating that SKi stimulates both proteasomal and lysosomal degradation of SK1 under normoxia and hypoxia. In chapter two, it was demonstrated that S1P and CYM5541, an S1P3 agonist, induced dose-dependent relaxation in endothelium-intact aortic rings, whereas the S1P1 agonist SEW2871 was without effect. The S1P stimulated relaxation was significantly enhanced in endothelium-intact aortic rings subjected to short-term hypoxia and this effect was entirely endothelium-dependent. Interestingly, the vasorelaxation response to S1P was inhibited by pre-treatment with SKi and PF543 but not ROMe, a selective SK2 inhibitor under both normoxia and hypoxia. A nitric oxide synthase inhibitor also inhibited the S1P-induced relaxation in aortic rings. Moreover, the enhanced relaxation response to S1P due to hypoxia was maintained in aortae obtained from spontaneously hypertensive Wistar Kyoto rats. These findings suggest that the vasorelaxation response to S1P under normoxia and the enhanced response under hypoxia are mediated by SK1 and NO.
In chapter five, it was found that hypoxia did not change the SK1b expression in HUVECs and pre-treatment with SKi or cycloheximide exerted no effect under both normoxia and hypoxia. However, proteasomal and/or lysosomal inhibitors increased SK1 expression under hypoxic conditions. In heart tissue, no significant difference was seen in expression of SK1 following exposure to hypoxia. However, SK1 expression was reduced by pre-treatment with SK inhibitors and cycloheximide under normoxia but not hypoxia. SK1a was identified in heart tissue, which is more sensitive to the degradation-induced by SK inhibitors than SK1b.
In summary, the results of this study imply that short-term hypoxia induces an increase in SK1 expression in coronary and aortic vascular endothelium. The increased SK1 induced by hypoxia appears to mediate the enhanced vasorelaxation response to S1P in endothelium-intact aortae. In HUVECs and heart tissue, it is likely that hypoxia induces resistance of SK1 to SK inhibitor-induced downregulation through a compensatory increase in SK1 expression.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: Kennedy, Dr. Simon and Pyne, Prof. Susan
Date of Award: 2017
Depositing User: Dr Husam Alganga
Unique ID: glathesis:2017-8517
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Oct 2017 11:57
Last Modified: 15 Nov 2017 14:18
URI: https://theses.gla.ac.uk/id/eprint/8517
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