In vivo uncoupling of arterial calcification and inflammation in androgen-treated apolipoprotein e-null mice

Calcification is a clinically-relevant feature of advanced atherosclerotic lesions but its pathogenesis remains unclear. Vascular calcification has typically been associated with plaque growth and inflammation however divergent effects of common therapies on plaque progression and lesion calcification suggest the existence of independent regulatory mechanisms. In this study we examined relationships between plaque growth, inflammation and calcification, using a mouse model of atherosclerosis where we previously observed uncoupling of plaque growth and calcification in response to androgen treatment. 34-week old male and female ApoE-null mice were treated with testosterone (T) or dihydrotestosterone (DHT) by subdermal implant for 8 weeks. Plaque and calcified area were quantified in sections from the aortic sinus. Protein expression was examined using immunohistochemistry. Androgens had null or inhibitory effects on plaque growth (P < 0.05) that were gender-specific. Suppression of plaque growth was associated with concomitant increases in serum HDL (1.7-fold, P < 0.01) and modulation of NF-kappaB, Ikappa-Balpha and VCAM-1 expression. T, but not DHT, treatment increased calcification despite declines in total plaque area in both male and female mice (2.5-fold, P < 0.05). Increased calcification was not associated with NF-kappaB activity but showed an inverse relationship with macrophage presence and other inflammatory markers that were suppressed by T action, including IL-6 (2-fold, P < 0.05) and downstream regulated genes STAT3 (2 to 5-fold, P < 0.05) and MCP-1 (2.5-fold, P < 0.01). Increases in calcification were associated with a reduction in AP-1 activity (c-JUN expression decreased 2-fold, P < 0.05) and proliferation (PCNA expressing cells decreased 2-fold, P < 0.05). Vascular calcification can be independently regulated in response to androgen treatment in ApoE-null mice. Uncoupling of calcification from plaque growth appears to occur via modulation of separate pathways defined by AP-1 or NF-kappaB, respectively. Suppression of c-JUN/AP-1 activity may represent an important dominant pathway controlling lesion calcification independently of inflammation and lesion growth.