Title: Disrupting Endothelial Cell Biomechanics through Connexin 43 Inhibition
Abstract: Endothelial cells are understood to bear and transmit intercellular stresses through their cell-cell junctions. However, identification of the specific cell-cell junction molecule responsible for intercellular stress generation remains an unanswered question. Thus far, the ability of tight junctions and adherens junctions to transmit intercellular stresses has been actively investigated, but the role of gap junctions is currently unknown. Gap junctions are formed by connecting two neighboring connexons, which are each made from individual connexins. Although endothelial cells primarily express Connexin 43, Connexin 40 and Connexin 37 for this study we focused on connexin43 as this is among the most abundant gap junction proteins expressed by endothelial cells. In this study, we seeded endothelial cells onto a soft polyacrylamide gel (stiffness= 1.2 KPa) and inhibited gap junction activity with the molecule 2’,5’-dihydroxychalcone, a well established Connexin43 (Cx43) inhibitor. We repeated our experiments under fluid flow in a parallel plate flow chamber to mimic arterial blood flow at a fluid shear stress of 1Pa. Intercellular stresses were measured in both static and fluid flow conditions. Our results reveal Cx43 inhibition to reduce intercellular stresses when compared to the untreated condition. The results we present here reveal for the first time the role gap junctions play in endothelial biomechanical force generation.