Objective Aortic valve disease, including calcification affects more than 2% of the human population and is caused by complex interactions between multiple risk factors including genetic mutations, the environment and biomechanics. At present, there are no effective treatments other than surgery and this is due to the limited understanding of the mechanisms that underlie the condition. Previous work has shown that valve interstitial cells (VICs) within the aortic valve cusps differentiate towards an osteoblast-like cell and deposit bone-like matrix that leads to leaflet stiffening and calcific aortic valve stenosis. However the mechanisms that promote pathological phenotypes in VICs are unknown. Approach and Results Using a combination of in vitro and in vivo tools with mouse, porcine and human tissue, we show that in VICs, reduced Sox9 expression and nuclear localization precedes the onset of calcification. In vitro, Sox9 nuclear export and calcific nodule formation is prevented by valve endothelial cells (VECs). While in vivo, loss of Tgfβ1 in the endothelium leads to reduced Sox9 expression and calcific aortic valve disease. Conclusions Together, these findings suggest that reduced nuclear localization of Sox9 in VICs is an early indicator of calcification and therefore pharmacological targeting to prevent nuclear export could serve as a novel therapeutic tool in the prevention of calcification and stenosis.

Collagen XIV is a fibril-associated collagen with an interrupted triple helix (FACIT). Previous studies have shown that this collagen type regulates early stages of fibrillogenesis in connective tissues of high mechanical demand. Mice null for Collagen XIV are viable, however formation of the interstitial collagen network is defective in tendons and skin leading to reduced biomechanical function. The assembly of a tightly regulated collagen network is also required in the heart, not only for structural support but also for controlling cellular processes. Collagen XIV is highly expressed in the embryonic heart, notably within the cardiac interstitium of the developing myocardium, however its role has not been elucidated. To test this, we examined cardiac phenotypes in embryonic and adult mice devoid of Collagen XIV. From as early as E11.5, Col14a1−/− mice exhibit significant perturbations in mRNA levels of many other collagen types and remodeling enzymes (MMPs, TIMPs) within the ventricular myocardium. By post natal stages, collagen fibril organization is in disarray and the adult heart displays defects in ventricular morphogenesis. In addition to the extracellular matrix, Col14a1−/− mice exhibit increased cardiomyocyte proliferation at post natal, but not E11.5 stages, leading to increased cell number, yet cell size is decreased by 3 months of age. In contrast to myocytes, the number of cardiac fibroblasts is reduced after birth associated with increased apoptosis. As a result of these molecular and cellular changes during embryonic development and post natal maturation, cardiac function is diminished in Col14a1−/− mice from 3 months of age; associated with dilation in the absence of hypertrophy, and reduced ejection fraction. Further, Col14a1 deficiency leads to a greater increase in left ventricular wall thickening in response to pathological pressure overload compared to wild type animals. Collectively, these studies identify a new role for type XIV collagen in the formation of the cardiac interstitium during embryonic development, and highlight the importance of the collagen network for myocardial cell survival, and function of the working myocardium after birth.