The anti-inflammatory effects of testosterone on atherosclerosis

Testosterone deficiency is prevalent in men with type 2 diabetes (T2D) and is associated with greatly elevated risk of cardiovascular mortality. Testosterone replacement therapy (TTh) has beneficial effects on surrogate markers and risk factors of atherosclerosis, including inflammation, cholesterol and insulin resistance, improving survival in men with T2D. The underlying mechanisms of this action remain poorly understood. Inflammation is a central feature to both T2D and atherosclerosis and is driven by monocyte/macrophages, placing these immune cells at the crossroads of disease pathology. The recruitment of immune cells to atherosclerosis-prone areas of the vasculature is influenced by many factors, including the inflammatory status of the circulating monocytes and activation of the vascular endothelium by numerous proatherogenic stimuli. Macrophages are involved in all stages of atherosclerotic plaque development and are central to disease progression through the expression of inflammatory cytokines, chemokines and lipid accumulation. Their interaction with vascular cells and recruited immune cells within the developing plaque can amplify the inflammatory activation of the vascular wall, lead to vascular remodeling, promote apoptosis within the atheroma and ultimately contributes to the destabilisation and rupture of the atherosclerotic lesion. Using combined in vivo and in vitro approaches, the aim of this thesis was to investigate whether testosterone treatment improves cardiovascular risk in men by reducing inflammation associated with the development and progression of atherosclerosis. The clinical study investigated the influence of testosterone therapy on monocyte inflammatory markers and atherosclerosis risk factors in a randomised double-blinded placebo-controlled clinical trial consisting of 65 men with poorly controlled T2D and hypogonadism. Orchidectomised ApoE-/- mouse as a model of testosterone deficiency and atherosclerosis, respectively, was used to assess the effects of androgen status and testosterone treatment on inflammatory mechanisms of atheroma formation. Additionally, in vitro studies investigated the influence of testosterone on inflammatory markers in cultured human monocytes and macrophages. Testosterone treatment in orchidectomised ApoE-/- mice significantly reduced lipid accumulation in the aortic root compared to testosterone deficient orchidectomised littermates, which was associated with reduced monocyte/macrophage infiltration and a localised reduction of ICAM-1 at the site of the atherosclerotic plaque. Furthermore, systemic circulating ICAM-1, indicative of disease extent, was also reduced with testosterone treatment. This may be due to a downregulation in pro-inflammatory cytokine TNFα, a potent inducer of ICAM-1, as the clinical study revealed that gene expression of this key inflammatory mediator was significantly reduced over time in monocytes from patients receiving TTh and significantly reduced compared to placebo-treated patient monocytes after six months. These findings importantly indicate for the first time that testosterone influences monocyte inflammatory activation in men with T2D by altering expression of TNFα as a potential mechanism to protect against atherosclerotic plaque development in hypogonadism. This effect of testosterone was independent of atherosclerosis risk factors including serum lipids, HbA1c and body composition as they were not altered following six months of TTh. Testosterone also did not significantly improve lesion stability as collagen content, smooth muscle migration, fibrous cap thickness, and MMP expression was not affected in the lesion of ApoE-/- mice. While these in vivo studies suggest anti-inflammatory actions of testosterone on mechanisms of atherogenesis and monocyte/macrophage function, in vitro testosterone treatment had no effect on the inflammatory profile of human monocytes and macrophages in response to pro-inflammatory stimulation or on macrophage subset phenotype thus leaving the specific cellular pathways of action to be elucidated. This thesis has demonstrated that testosterone reduces aortic atherosclerosis indicated by decreased lipid deposition and reduced monocyte/macrophage infiltration into atherosclerotic lesions. This is likely modulated through local anti-inflammatory actions at the vessel wall, as opposed to systemically, and potentially via a testosterone-induced reduction of monocyte inflammatory activation by TNFα in diabetic men. Risk factors for atherosclerosis were unaffected by testosterone treatment both in hypogonadal men with T2D and ApoE-/- mice again suggesting beneficial actions in these models may be confined to plaque localised effects. Overall, this indicates a local anti-atherogenic mechanism at the vessel wall by which testosterone may protect against the development and progression of atherosclerosis to reduce cardiovascular disease in men.