Effect of hypoxia on the cardiovascular sphingolipid system

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

Requirement for sphingosine kinase 1 in mediating phase 1 of the hypotensive response to anandamide in the anaesthetised mouse

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids

High fat diet attenuates the anticontractile activity of aortic PVAT via a mechanism involving AMPK and reduced adiponectin secretion

Background and aim: Perivascular adipose tissue (PVAT) positively regulates vascular function through production of factors such as adiponectin but this effect is attenuated in obesity. The enzyme AMP-activated protein kinase (AMPK) is present in PVAT and is implicated in mediating the vascular effects of adiponectin. In this study, we investigated the effect of an obesogenic high fat diet (HFD) on aortic PVAT and whether any changes involved AMPK. Methods: Wild type Sv129 (WT) and AMPKα1 knockout (KO) mice aged 8 weeks were fed normal diet (ND) or HFD (42% kcal fat) for 12 weeks. Adiponectin production by PVAT was assessed by ELISA and AMPK expression studied using immunoblotting. Macrophages in PVAT were identified using immunohistochemistry and markers of M1 and M2 macrophage subtypes evaluated using real time-qPCR. Vascular responses were measured in endothelium-denuded aortic rings with or without attached PVAT. Carotid wire injury was performed and PVAT inflammation studied 7 days later. Key results: Aortic PVAT from KO and WT mice was morphologically indistinct but KO PVAT had more infiltrating macrophages. HFD caused an increased infiltration of macrophages in WT mice with increased expression of the M1 macrophage markers Nos2 and Il1b and the M2 marker Chil3. In WT mice, HFD reduced the anticontractile effect of PVAT as well as reducing adiponectin secretion and AMPK phosphorylation. PVAT from KO mice on ND had significantly reduced adiponectin secretion and no anticontractile effect and feeding HFD did not alter this. Wire injury induced macrophage infiltration of PVAT but did not cause further infiltration in KO mice. Conclusions: High-fat diet causes an inflammatory infiltrate, reduced AMPK phosphorylation and attenuates the anticontractile effect of murine aortic PVAT. Mice lacking AMPKα1 phenocopy many of the changes in wild-type aortic PVAT after HFD, suggesting that AMPK may protect the vessel against deleterious changes in response to HFD

High Fat Diet Attenuates the Anticontractile Activity of Aortic PVAT via a Mechanism Involving AMPK and Reduced Adiponectin Secretion

Background and aim: Perivascular adipose tissue (PVAT) positively regulates vascular function through production of factors such as adiponectin but this effect is attenuated in obesity. The enzyme AMP-activated protein kinase (AMPK) is present in PVAT and is implicated in mediating the vascular effects of adiponectin. In this study, we investigated the effect of an obesogenic high fat diet (HFD) on aortic PVAT and whether any changes involved AMPK.Methods: Wild type Sv129 (WT) and AMPKα1 knockout (KO) mice aged 8 weeks were fed normal diet (ND) or HFD (42% kcal fat) for 12 weeks. Adiponectin production by PVAT was assessed by ELISA and AMPK expression studied using immunoblotting. Macrophages in PVAT were identified using immunohistochemistry and markers of M1 and M2 macrophage subtypes evaluated using real time-qPCR. Vascular responses were measured in endothelium-denuded aortic rings with or without attached PVAT. Carotid wire injury was performed and PVAT inflammation studied 7 days later.Key results: Aortic PVAT from KO and WT mice was morphologically indistinct but KO PVAT had more infiltrating macrophages. HFD caused an increased infiltration of macrophages in WT mice with increased expression of the M1 macrophage markers Nos2 and Il1b and the M2 marker Chil3. In WT mice, HFD reduced the anticontractile effect of PVAT as well as reducing adiponectin secretion and AMPK phosphorylation. PVAT from KO mice on ND had significantly reduced adiponectin secretion and no anticontractile effect and feeding HFD did not alter this. Wire injury induced macrophage infiltration of PVAT but did not cause further infiltration in KO mice.Conclusions: High-fat diet causes an inflammatory infiltrate, reduced AMPK phosphorylation and attenuates the anticontractile effect of murine aortic PVAT. Mice lacking AMPKα1 phenocopy many of the changes in wild-type aortic PVAT after HFD, suggesting that AMPK may protect the vessel against deleterious changes in response to HFD