oa Inflammation Drives Endothelial-to-Mesenchymal Transition and Interstitial Calcification in 3D in Vitro Culture

Calcific aortic stenosis is a serious pathology that accounts for 43% of those suffering from heart valve disease. Valve disease initially presents with inflammation and endothelial dysfunction, which can lead to an endothelial-to-mesenchymal (EndMT) shift in the endothelial layer of the valve. It is not yet well understood how valve endothelial cells (VEC) regulate valve interstitial cells (VIC) in inflammatory or osteogenic environments. We here present a 3D culture system that models cellular responses and interactions when exposed to different environmental conditions. Porcine aortic VEC and/or VIC were cultured in mechanically constrained 3D type I collagen hydrogels for up to 14 days. Early disease studies introduced different dosages of TNFα to VEC on gel surfaces for up to 48 hours. EndMT was assessed via real time PCR and cell invasion within the matrix. Apoptosis and proliferation in VEC via TUNEL and anti-BrdU IHC staining were also evaluated in early inflammatory conditions. Later early-stage disease studies introduced osteogenic and inflammatory environments to 3D VIC/VEC gels through the addition of osteogenic differentiation factors into the culture media (OGM), or 30 ng/ml TNFα. Calcium deposition within the matrix was measured via Alizarin Red staining. Real time PCR was used to evaluate the expression of calcification-related genes such as osteocalcin and runx-2. Early inflammatory conditions as induced by addition of TNFα for 48 hours stimulated EndMT-like VEC activation, including matrix invasion and upregulation of αSMA and snail. Addition of TNFα increases VEC proliferation in a dose dependent manner. TNFα does not significantly increase apoptosis, even at the highest dosage levels. VIC gels cultured in OGM for 14 days significantly calcified and expressed osteocalcin and runx-2. Co-culture with VEC inhibited both matrix calcium deposition and osteoblastic differentiation. When 30 ng/ml TNFα was added to VIC 3D cultures, TNFa induced increased matrix calcium deposition in 14 days, which was then mitigated by co-culture with VEC. The inhibitory effect of VEC on VIC calcification in OGM was blocked when TNFα was introduced to the culture. These results demonstrate that inflammatory microenvironments induce calcification in valve interstitial cells. There is a protective role for the valve endothelium in VIC calcification, which could be inhibited by EndMT induced by the inflammatory conditions found in diseased valves. We found that apoptosis is not a driving factor in inflammatory activation of VEC or osteogenic differentiation of VIC. Targeting TNFα and EndMT signaling could be important therapeutic strategies. More generally, targeting VEC is a promising approach for early treatment of valve disease. This co-culture 3D system is a powerful tool to elucidate mechanisms of valve disease.