Cyclic Equiaxial Stretch Induces Osteogenic Differentiation and Augments 3D Lesion Height in 3D Cocultured Aortic Valve Cells

Calcific aortic valve disease is an active process characterized by compromised endothelial integrity and obstructive calcific lesions, whose emergence is poorly understood. Valves experience an equiaxial stretch in regions susceptible to calcific lesion formation, but mechanobiological mechanisms are not tested. Here, we analyze how cyclic strain regulates interstitial (PAVIC) and endothelial (PAVEC) interactions in 3D environments. Equiaxial cyclic strain was applied to 3D cultures of PAVEC, PAVIC, or PAVEC-PAVIC cocultures over 7 days in control or osteogenic media (OGM) conditions. Cell phenotype and tissue remodeling were quantitatively compared to mechanically strained (anchored) but nonstretched controls. Cyclic stretch shifted PAVIC from myofibroblastic to osteogenic phenotype with OGM, while in PAVEC, the cyclic stretch increased apoptosis. Intriguingly, we determined that PAVEC-PAVIC in coculture with OGM develop raised 3D calcified lesions (∼25-50% of gel thickness) similar to lesions seen in vivo. Lesions contained radially reoriented collagen fibers with similarly aligned PAVIC, increased local PAVEC density, and decreased PAVEC cell area. The cyclic stretch synergistically increased the lesion number and height but not the projected area. The cyclic stretch enhanced osteogenic differentiation (Runx2 and OPN) but not myofibroblastic differentiation (aSMA) in cocultures. It significantly increased VE-cadherin and eNOS and reduced VCAM1, but with OGM, the eNOS expression reduced. Finally, we determined that ROCK inhibition abolished 3D lesion formation and myofibroblastic and osteoblastic differentiation, supporting the idea that these integrated behaviors were mechanobiologically mediated by cell migration and/or contractility. Our results identify that the 3D cyclic stretch induces emergent PAVEC-PAVIC interactions not capturable in less complex environments that together control 3D calcific lesion morphology.