Cardiovascular disease (CVD) is the major cause of morbidity and mortality in developed countries. Men have a higher incidence and an earlier onset of CVD than age-matched women. It has been shown that young women have lower CVD incidence than young men and the increase in CVD rates in older (post-menopausal) women when estrogen levels decline suggests that estrogens are cardioprotective. This theory has been extensively investigated demonstrating a favourable reduction in LDL, reducing expression of cell adhesion molecules and promoting vasodilatation by inducing nitric oxide (NO) production in endothelial cells. However, not many studies have explored the alternate theory that male sex hormones, androgens (T & DHT), may promote CVD. Investigators examining male androgens in vivo have shown inconsistent results, with both adverse and protective effects on atherosclerosis formation. This study aimed to elucidate the role of androgens and their effects on the formation and progression of atherosclerotic plaque in males and females.
Chapter 3 describes the establishment of the en face Oil Red O for quantitating atherosclerotic plaque. The Oil Red O technique was employed to compare the reliability and the consistency of the plaque area to the already established H&E staining method. This chapter focuses on the en face Oil Red O staining technique to see if the new method could give a faster, more efficient and reliable alternative to the very labour intensive H&E staining. From this study the en face Oil Red O staining technique was a more time and labour efficient method that is a reliable method to the already establish H&E staining when investigating atherosclerotic plaque levels.
Chapter 4 examines the effect of exogenously administered male sex hormones, testosterone (T) and dihydrotestosterone (DHT), on atherosclerosis in male and female ApoE-/- mice. The aim of this experiment was to examine if the androgens had a dose- and or gender- specific effect on the plaque formation in the animals. The experimental design involved the use of three different doses of high (1cm implant), medium (0.5 cm implant) and low (0.25 cm implant) T or DHT and the mice were treated for a period of 16-weeks. The results showed that T had gender- and dose- specific effects. Briefly, there was no effect of T treatment on plaque formation in females. However, in males, the high dose of T decreased plaque but at the low dose T had the opposite effect by increasing plaque levels. The DHT results differed to the T results indicating different pathways of action. DHT treatment in the females demonstrated atheroprotective effects at the high and medium dose. The males had no effect at the high dose of DHT but the low dose was atherogenic.
Chapter 5 aimed to explore the effect of DHT treatment in cells involved in early atherogenic processes using male and female HUVEC and MDM and to ascertain if AR cofactors are hormonal regulated. The results showed that there was no hormonal regulation in the MDM by DHT. Alternatively, in HUVEC there was a significant up-regulation of ARA24, ARA54, ARA160 and SRC-1 mRNA levels with DHT treatment and a down-regulation of p300 and NCoR1 mRNA levels. In particular, this chapter also investigated protein expression of the cofactor SRC-1 in HUVEC, which was increased in males with DHT treatment. SRC-1 protein levels were also examined with in vivo studies. Different doses of T and DHT were administered to male and female ApoE-/- mice. The 0.5 cm and 1 cm dose of T in the male showed an increase in the SRC-1 protein level and DHT, at the 0.25 cm and 0.5 cm implant increased SRC-1 protein levels. SRC-1 levels in female animals did not change with any dose of DHT, whereas the 0.25 cm T showed an increase in SRC-1 and 0.5 cm T implant decreased SRC-1 protein levels. This study demonstrates that AR cofactors can be hormonally regulated by androgens at the mRNA and protein level.
Overall, male sex hormones have an effect on atherosclerosis formation. Androgens have important gender- and dose- specific effects on plaque formation, and can hormonally regulate AR cofactors at the mRNA and protein level. The results of this thesis have produced more questions than it has answered. This study requires further investigation into the benefits and consequences of using androgens, examining the underlying molecular pathways that may be involved with the AR cofactors and the use of exogenous androgen treatment for both men and women.