Models and mechanisms of sexual dimorphism in cardiovascular calcification

Abstract

Cardiovascular calcification is the active process of calcification of the cardiovascular soft tissue. It is currently predicted to be present in over 70 % of the population aged 65 years and over. One of the most common types of cardiovascular calcification occurs in the aortic valve and is named calcific aortic valve disease (CAVD). In later stages, CAVD can cause stenosis, blood regurgitation and significant cardiac dysfunction and morbidity. Males are at greater risk of developing aortic calcification and androgens are a risk factor in this condition. There is no effective pharmaceutical treatment for CAVD. The mechanisms underlying male predisposition to aortic valve calcification have yet to be elucidated and this is hampered by the lack of appropriate animal models; particularly valve-injury models which develop stenosis and calcification.

This project aimed to examine the sexual dimorphism in cardiovascular calcification by deducing what molecular mechanisms sex hormones influence both in valve interstitial cells and vascular smooth muscle cells and by examining sex differences in in vivo murine models of calcification.

Culturing valve interstitial cells in either a ‘pro-activated’ or ‘pro-quiescent’ state did not change the deposition of in vitro calcium. In both valve interstitial cells (VICs) (p<0.01), and vascular smooth muscle cells (VSMCs) (p<0.01), calcification was enhanced (4.8-fold in VSMC, 15.7-fold in VICs) with testosterone treatment (concentration and length of exposure) whereas estrogen (concentration and length of exposure) had no effect. Proteomics analysis of calcified male rat VICs treated with testosterone found 398 differentially expressed proteins compared to control cells. This included differential expression of proteins associated with metabolism and cellular transport.

Next, the sex differences in the ApolipoproteinE null (ApoE-/-) western diet murine model of cardiovascular calcification were analysed. During the 12 weeks of western diet treatment, neither male nor female mice developed aortic stenosis or regurgitation. Increased microcalcification (p<0.05) in the hearts of female mice was seen with PET/CT imaging. Additionally, there were larger atherosclerotic plaques (p<0.01) in the aortae of female compared to male mice. There was a higher prevalence of macrocalcification in the aortic root plaques of females, but no valve calcification was observed. However, circulating cholesterol and LDL were significantly lower in females (p<0.05) compared to males. Female mice also displayed reduced tibial trabecular bone volume (p<0.001) and trabecular number (p<0.001).

Due to the sexual dimorphism in the ApoE-/- murine model not reflecting the sexual dimorphism in humans, a new, surgical model of valvular calcification was developed, the wire injury model. A pilot study was first undertaken which revealed that the surgery and imaging could be performed with a low mortality rate. The subsequent ‘mild injury’ study was modified so both male and female mice were included, and the post-operative time was lengthened to 8 weeks. Males had a higher mortality rate and females gained significantly more body weight post-operativity than males (p<0.05). After the wire injury, females had significantly higher blood velocity across the aortic valve than males (p<0.05), although this did not change after the surgery. There was also no evidence of sexually dimorphic responses to the wire injury in other cardiovascular parameters (measured by echocardiography) or evidence of regurgitation. Valve cusps displayed thickening (from 30 μM to 1402.8 μM), fibrosis and stained positive for Mac2. The protocol was then developed to include further injury and extend the postoperative period again. Finally, this ‘moderate’ injury study displayed no aortic regurgitation and no change to aortic velocity post-surgery.

To conclude, sex hormones may drive the sexual dimorphism seen in cardiovascular calcification, but further development of translational in vivo models is required to fully elucidate the underpinning mechanisms.