Signaling Microdomains within the Myoendothelial Junction

Abstract

Communication between endothelial and smooth muscle cells is critical to maintain homeostatic vascular tone and blood pressure. In resistance arteries, the cells make direct cytoplasmic contact via cellular extensions that project through the thin extracellular matrix of the internal elastic lamina that separates them. These structures are termed myoendothelial junctions (MEJ). There are a number of proteins and signaling processes localized to this part of the arterial wall that regulate bi-directional communication between the endothelium and smooth muscle. In particular, a number of proteins localized to the MEJ mediate calcium signaling and there is localized calcium release from the endoplasmic reticulum via the inositol (1, 4, 5) trisphosphate receptor that can influence vascular tone and negative feedback. There is a detailed description of the creation and isolation of in vitro MEJ using the vascular cell co-culture technique, which has allowed for deeper exploration of signaling molecules and processes occurring at the MEJ. Smooth muscle cells and endothelial cells grow on opposite sides of a filter inset, and the MEJ form within the holes of the filter. This model can be used to immunofluorescently label proteins or the cellular fractions can be individually isolated to investigate protein expression and phosphorylation via western blotting. Using this unique model, we show the in vitro MEJ has a unique lipid composition that is rich in diacylglycerol and phosphatidylserine. This likely facilitates localization of signaling molecules such as protein kinase C and selective activation of endothelial nitric oxide synthase. Additionally, the multifunctional protein calreticulin is highly expressed at the MEJ of resistance arteries and endothelial deletion of calreticulin results in impaired MEJ calcium signaling, thus vasoconstriction to phenylephrine and blood pressure. Our data suggests that EC monolayer Calr could be iii functionally different than MEJ-localized Calr, which may be located outside the ER.Further work will need to be done in order to clarify and confirm MEJ Calr function and how it affects calcium signaling at the MEJ specifically in response to phenylephrine. In summary, the MEJ is an important signaling microdomain strategically located in the wall of resistance arteries to mediate heterocellular communication.