(D-153) Endothelial cells with low fibronectin leucine rich transmembrane protein 2 (FLRT2) expression exhibit enhanced retention within vascular grafts

Introduction: Surgical intervention is often required to treat severe coronary artery disease. Currently, autologous vessel grafts are the only option for bypass graft procedures as small-diameter (< 6mm) synthetic vascular grafts fail due to unacceptable patency rates with reports of ~60% and ~14% patency after 1- and 3-years of implantation, respectively.¹ This is mainly because synthetic grafts lack a stable endothelial cell (EC) monolayer to prevent platelet activation, thrombosis, and neointimal hyperplasia, which can cause loss of patency. Many studies have examined methods to improve EC adhesion to biomaterials, but most seeded ECs are lost after implantation when exposed to fluid shear stress (SS) from flowing blood. Our lab previously identified a subpopulation of ECs that are more likely to resist detachment when exposed to fluid SS. We hypothesize that differential gene expression is responsible for the observation that some ECs adhere more strongly compared to others. The goal of this study is to use RNA-sequencing (RNA-seq) to examine the gene expression of the adherent subpopulation ECs compared to the whole population and to develop a method to select for the highly adherent ECs, in order to improve retention on a vascular graft.

Materials and Methods: Primary human umbilical vein endothelial cells (HUVECs) were exposed to 0, 5, 10, or 30 min of 15 dyn/cm² fluid SS with a parallel plate flow chamber (PPFC) to obtain the whole population (0 min SS) and adherent subpopulations (5, 10, or 30 min SS). RNA from the adherent ECs at each time point was extracted and used for RNA-seq. RNA was also transcribed into cDNA for RT-qPCR. HUVECs were sorted based on expression of fibronectin leucine rich transmembrane protein 2 (FLRT2) using fluorescence activated cell sorting (FACS) into low, intermediate, and high groups. Sorted HUVECs were seeded on 35mm dishes overnight. The cells were subjected to 30 min of 15 dyn/cm² SS with a PPFC and cell retention was quantified using nuclei staining and fluorescence microscopy. For vascular graft experiments, HUVECs were sorted into low and high FLRT2 expression groups and seeded onto vascular graft samples (DEGRAPOL DP15 grafts fabricated by electrospinning) overnight. Grafts were mounted in a custom bioreactor chamber and exposed to 30 min of 10 dyn/cm² fluid SS with a pulsatile blood pump. Cell retention was quantified as described above. N=5-6 samples per group. Results are reported as the mean ± standard error of mean.

Results: RNA-sequencing identified 2017, 1431, and 1255 differentially expressed genes (DEGs) in the adherent ECs isolated after 5, 10, or 30 min of 15 dyn/cm² fluid SS, respectively, when compared to the whole population (p< 0.05). From the DEGs, FLRT2 was significantly downregulated in all 3 adherent subpopulations. RT-qPCR confirmed the relative fold change of FLRT2 mRNA was 0.31±0.07, 0.47±0.09, and 0.45±0.10 for the 5-, 10-, and 30-min SS samples, respectively, compared to 1.00±0.15 for the 0 min SS (p< 0.001). The retention of the HUVECs sorted for low FLRT2 was 67.7±4.3% on tissue culture plates, which was significantly greater than 35.1±9.5% for the high and 31.6±8.0% for the intermediate FLRT2 groups (p< 0.0001). Retention studies on vascular graft samples confirmed that HUVECs with low expression of FLRT2 had a significantly greater average retention of 59.2±7.4% when exposed to 30 min of 10 dyn/cm² SS compared to 24.5±6.1% for HUVECS with high FLRT2 expression (p< 0.001).


Conclusions: We demonstrate that we can select for the more adherent ECs in a population. Using FACS, we sort ECs based on expression of FLRT2 and show that the ECs expressing low FLRT2 have increased retention when exposed to fluid SS in vitro. To our knowledge, this is the first study to successfully predict which ECs within a population are more likely to remain adherent within a vascular graft and provides proof-of-concept for a novel approach to seed a small-diameter vascular graft with only the highly adherent ECs in a population.

Discussion: The mechanism by which downregulation of FLRT2 allows for enhanced EC retention under fluid SS is still largely unknown. Our study showed that we can selectively sort for the more adherent ECs in a population based on low FLRT2 expression, which was the goal of our study. While the retention of low FLRT2-expressing HUVECs was around 63%, it is remains unknown what percentage of cell retention is needed to achieve long-term graft patency. The biological response including thrombosis and neointimal hyperplasia will need to be studied in an animal model in the future.

Acknowledgements (Optional) : We gladly thank Ab Medica S.p.a. for donating the DEGRAPOL DP15. This work was supported by NIH R01HL157642

References (Optional) : 1. Chard RB, Johnson DC, Nunn GR, Cartmill TBJTJot, surgery c. Aorta-coronary bypass grafting with polytetrafluoroethylene conduits: early and late outcome in eight patients. 1987;94:132-134.