Proteomic analysis of the myoendothelial junction

Abstract

Myoendothelial junctions (MEJ) are cellular projections through the internal elastic lamina originating from the vascular smooth muscle cell (VSMC) or endothelial cell (EC) layers that may serve as a distinct signaling domain, however the proteins that make up this domain are unknown. Using a vascular cell co‐culture model (VCCC) (Isakson et al., 2005), we demonstrate for the first time isolation of proteins specifically localized to the MEJ in vitro. Proteins from VSMC, EC and MEJ fractions were labeled separately with Cy5, Cy3, and Cy2 dyes respectively and subject to 2D‐DIGE analysis. Proteins within the MEJ fraction that displayed a minimum 2.5 increase in fluorescence intensity when compared to EC or VSMC fractions as determined by DeCyder software were chosen for mass spectrometry analysis and were compared to the NCBI protein database. Of the proteins identified at the MEJ, plasminogen activator inhibitor‐1 (PAI‐1) was shown to be one of the most abundant. Preferential expression of PAI‐1 at the MEJ was confirmed in vitro using immunoblotting and immunocytochemistry on the VCCC, as well as in vivo using transmission electron microscopy in multiple vascular beds. Based on this data we conclude that proteomic analysis of MEJ fractions in vitro provides valuable insight into possible protein interactions at the MEJ in vivo. Supported by NIH RO1 088554 and an AHA SDG.