|dc.description.abstract||Cardiovascular disease (CVD) is the leading cause of death worldwide in adult men and women. Atherosclerotic cardiovascular disease (ASCVD) has been highlighted as the main cause of death among CVDs, due to development and progression of plaques resulting in major adverse cardiovascular events (MACE). The multifactorial processes resulting in the generation of plaques have been established, and anti-atherogenic effects of 17β-estradiol (E2) shown. Yet, a greater prevalence and mortality was recorded in some postmenopausal women using hormone replacement therapy (HRT). Recent evidence has shown the adverse or lack of effect of E2 treatment in ASCVD models depending on timing post menopause. The occurrence of MACE is correlated with complex, high-risk plaques, but the direct effects of E2 on plaque progression and development are yet to be elucidated. Key signalling factors that modulate vascular cell proliferation and inflammation underlie plaque advancement, including the nodal multifunctional calcium/calmodulin dependent protein kinase II (CaMKII). There is a sex-related difference in the activation profile of CaMKII in cardiac pathologies, yet its role in plaque progression, which leads to ASCVD, is unknown. Therefore this thesis set out to address the potential mechanisms driving the E2-induced advancement of atherosclerotic plaques using the murine model of atherosclerosis, Apolipoprotein E- deficient (ApoE-/-) mice. The association of CaMKII with E2-induced effects and the specific role of CaMKII in the pathogenesis of atherosclerosis were investigated with a focus on plaque calcification.
To determine the effect of exogenous E2 on plaque advancement, female ApoE-/- mice with intermediate (25 weeks) and advanced (45 weeks) plaques were treated twice a week for 8 weeks with subcutaneous injections of E2. E2 treatment caused a significant increase in calcification of intermediate plaques with no effect on plaque size. This increase in calcification was associated with a significant decrease in total CaMKII expression within intermediate plaques, with no effect on phosphorylated CaMKII (pCaMKII), an indicator of CaMKII activity. E2 treatment had no effect on plaque size, calcification or CaMKII expression and phosphorylation in ApoE-/- mice with advanced plaques.
To investigate the role of CaMKII in the typical progression of intermediate plaques, 25 week-old female ApoE-/- mice were treated with intraperitoneal injections of KN-93, a potent CaMKII inhibitor. CaMKII inhibition resulted in an observed decrease in calcification, with no effect on plaque size. This was associated with no significant changes in pCaMKII and the calcification marker, osteocalcin.
Finally, the role of CaMKII in the progression of early plaques was investigated in 20 week-old male and female ApoE-/- mice. Plaques in female ApoE-/- mice were more progressed than those observed in male ApoE-/- mice and there was no evidence of calcification in male ApoE-/- mice. CaMKII inhibition in female ApoE-/- mice showed a trend towards decreased calcification with no effect on plaque size, pCaMKII or the calcification markers osteocalcin and RUNX2. CaMKII inhibition in male mice showed a trend towards increased plaque formation with no effect on the protein parameters measured.
The findings from this thesis identify CaMKII as an instigator in the advancement of atherosclerotic plaques through increased calcification. Additionally, this thesis establishes the differential, CaMKII-mediated effects of E2 on plaques dependent on stage of progression. This thesis provides novel evidence for a possible mechanism through which E2 may be mediating adverse effects in ASCVD.||