Identification of a subpopulation of highly adherent endothelial cells for seeding synthetic vascular grafts

Abstract

ObjectiveThere is an unmet clinical need for a bypass graft that can be used as an alternative to an autologous vessel graft for the treatment of severe coronary artery disease. Small-diameter (<6mm) synthetic vascular grafts are not suitable because of unacceptable patency rates. This mainly occurs without an endothelial cell (EC) monolayer to prevent platelet activation, thrombosis, and intimal hyperplasia. While numerous studies have explored methods to improve EC adhesion to biomaterials, there are still no reliable methods to endothelialize small-diameter grafts, as most seeded ECs are lost due to exposure to fluid shear stress (SS) after implantation. The goal of this work is to determine if EC loss is a random process or if it is possible to predict which cells are more likely to remain adherent. Approach and ResultsIn initial studies, we sorted ECs using fluid SS and identified a subpopulation of ECs that are more likely to resist detachment. We use RNA-sequencing (RNA-seq) to examine gene expression of adherent ECs compared to the whole population to identify targets for improving adhesion. Fibronectin leucine rich transmembrane protein 2 (FLRT2), which encodes protein FLRT2, emerged as a candidate due to its downregulation in the adherent ECs and known role in cell adhesion. Using fluorescence activated cell sorting (FACS), we sorted ECs based on FLRT2 expression levels and demonstrated that ECs expressing low levels of FLRT2 exhibit greater retention under fluid SS in vitro. ConclusionFor the first time, we show EC detachment is not an entirely random process and we predicted which ECs were more likely to remain adherent on a vascular graft upon exposure to fluid SS. This provides validation for the concept that we can seed a small-diameter vascular graft only with highly adherent ECs to maintain a stable endothelium and improve graft patency rates. Non-standard Abbreviations and Acronymsendothelial cell (EC), shear stress (SS), fibronectin leucine rich transmembrane protein 2 (FLRT2), tissue engineered vascular graft (TEVG), fluorescence activated cell sorting (FACS)