title: Influence of glucocorticoid bioavailability on atherogenesis
title: The importance of glucocorticoid delivery in the development of atherosclerosis
titleType: AlternativeTitle
Creators: name: MacLeod, Clare Elizabeth
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: atherosclerosis
CBG
plaque development
glucocorticoids
neutrophil elastase

description: Elevated circulating glucocorticoids are causally linked to increased cardiovascular disease. Atherosclerosis is a key cardiovascular pathology, yet the contribution of glucocorticoids to underlying atherogenesis is unclear. Corticosteroid binding globulin (CBG) regulates glucocorticoid action by controlling systemic bioavailability, with only unbound ‘free’ glucocorticoids able to enter tissue and exert biological effects. Cleavage of CBG by neutrophil elastase (NE) is proposed to enhance bioavailability by reducing its affinity for glucocorticoids, elevating local ‘free’ glucocorticoid levels. Strikingly, Apoe-/- mice lacking NE display reduced atherosclerosis. This thesis addresses the hypothesis that CBG mediates atherogenesis by facilitating glucocorticoid action at sites of plaque development. First, the role of glucocorticoids and their regulation by CBG in atherogenesis was established in an adenovirus-induced murine model of atherosclerosis. Male C57Bl6/J mice were injected with recombinant adeno-associated viral vector serotype 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and fed a Western diet (21% fat; 0.21% cholesterol) for up to 18 weeks. Control mice were fed the same Western diet in the absence of AAV8-PCSK9. Brachiocephalic lesion development in AAV8-PCSK9- treated mice was accompanied by reduced hepatic Serpina6 (CBG) mRNA levels, in parallel with reduced circulating CBG binding capacity. Moreover, AAV8-PCSK9 treatment increased circulating NE protein levels, suggesting the ability of CBG to bind glucocorticoids may also be reduced in this model because of increased cleavage. Surprisingly, AAV8-PCSK9 treatment did not alter circulating total glucocorticoid levels. These data provide the first evidence of CBG dysregulation in this model of atherosclerosis. The increase in circulating NE levels in the AAV8-PCSK9 overexpression model of atherosclerosis resembles that seen in Apoe⁻/⁻ mice, which are resistant to atherosclerotic plaque development in the absence of NE. This suggests that NE may also play a role in atherogenesis in the PCSK9 overexpression model. To test this, male NE deficient mice (Ela⁻/⁻) and WT littermates were administered AAV8-PCSK9 and fed Western diet for 18 weeks. In contrast to published data from Apoe⁻/⁻/Ela-⁻/⁻ double-knockout mice, NE deficiency did not alter brachiocephalic lesion incidence, size, or composition (collagen, smooth muscle cell, and macrophage content), suggesting that NE does not play an important role in the development of lesions in this model of atherosclerosis. Additionally, deletion of NE did not alter total or free glucocorticoid levels in the circulation, indicating that NE-mediated CBG cleavage does not occur in the systemic circulation. The importance of CBG itself in regulating atherogenesis was explored next. To test the hypothesis that deletion of CBG will reduce lesion size through reduced glucocorticoid action, AAV8-PCSK9 was administered to mice lacking CBG (Cbg⁻/⁻) mice and wild type littermates which then received Western diet for 18 weeks. Total and free glucocorticoid levels were decreased in Cbg-/- mice. There was an apparent decrease in incidence of lesion development in the brachiocephalic artery of Cbg⁻/⁻ mice compared with WT controls; however, this did not achieve statistical significance (p=0.1312). This suggests that although CBG regulates the bioavailability of glucocorticoids this does not play an important role in the development of brachiocephalic lesions in the AAV8-PCSK9 model. Furthermore, method development of mass-spectrometry imaging (MSI) was able to detect and quantify local corticosterone in an aortic root lesion (n=1). In conclusion, this thesis presents data demonstrating the first glucocorticoid profiling of mice who develop inducible-atherosclerosis via AAV8-induced over-expression of PCSK9. Despite the reported role of NE in lesion development in the Apoe⁻/⁻ model, this thesis provides evidence that manipulation of CBG, either genetically or indirectly via NE, does not alter the development of lesions in this model. This suggests that the choice of model when investigating factors that influence atherogenesis should be given careful consideration. Future work may involve investigating the effect of NE and CBG deletion on alternative sites of lesion development, e.g., aortic root. Additionally, while this work does not support a role for changes in systemic glucocorticoid bioavailability in mediating plaque developing, advancement in steroid quantification by MSI may allow assessment of local glucocorticoid levels within various regions of atherosclerotic plaques to determine local glucocorticoid action.