Calcific aortic valve disease (CAVD) is a chronic disorder characterized by an abnormal mineralization of the leaflets, which is accelerated in bicuspid aortic valve (BAV). It is suspected that mechanical strain may promote/enhance mineralization of the aortic valve. However, the effect of mechanical strain and the involved pathways during mineralization of the aortic valve remains largely unknown. Valve interstitial cells (VICs) were isolated and studied under strain conditions. Human bicuspid aortic valves were examined as a model relevant to increase mechanical strain. Cyclic strain increased mineralization of VICs by several-fold. Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analyses revealed that mechanical strain promoted the formation of mineralized spheroid microparticles, which coalesced into larger structure at the surface of apoptotic VICs. Apoptosis and mineralization were closely associated with expression of ENPP1. Inhibition of ENPP1 greatly reduced mineralization of VIC cultures. Through several lines of evidence we showed that mechanical strain promoted the export of ENPP1-containing vesicles to the plasma membrane through a RhoA/ROCK pathway. Studies conducted in human BAV revealed the presence of spheroid mineralized structures along with the expression of ENPP1 in areas of high mechanical strain. Mechanical strain promotes the production and accumulation of spheroid mineralized microparticles by VICs, which may represent one important underlying mechanism involved in aortic valve mineralization. RhoA/ROCK-mediated export of ENPP1 to the plasma membrane promotes strain-induced mineralization of VICs.Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Química Geral e Inorgânica, Instituto de Química de Araraquara, Araraquara, R. Professor Francisco Degni, S/N, Quitandinha, CEP 14800-900, SP, BrasilUniversidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Química Geral e Inorgânica, Instituto de Química de Araraquara, Araraquara, R. Professor Francisco Degni, S/N, Quitandinha, CEP 14800-900, SP, BrasilLaboratoire d'Études Moléculaires des Valvulopathies (LEMV), Groupe de Recherche en Valvulopathies (GRV), Quebec Heart and Lung Institute/Research Center, Department of Surgery, Laval University, Quebec, CanadaDepartment of Physics, Laval University, Québec, CanadaDepartment of Medicine, Laval University, Québec, Canada