90th Annual Meeting Abstracts
Endothelial Cells with Eph B4 Heterozygous Knockdown: A Novel Cellular Model of Vein Graft Adaptation
*Amanda Feigel, MD, *Akihito Muto, MD, PhD, *Tiffany Fancher, MD, *Mariangela Rivera, BS, *Aaron Feinstein, BA, *Susun Kim, *Yuka Kondo, MD, PhD, *Alan Dardik, MD, PhD
Yale University, New Haven, CT
Objective: Vein graft adaptation (VGA) is characterized by thickening of the vein wall, a complex process involving cell proliferation and migration. Previously published, VGA is also characterized by loss of the venous determinant Eph-B4. To study molecular mechanisms that mediate VGA, we developed a novel in vitro cellular model of VGA.
Design: Before-after trial
Setting: Basic science laboratory
Interventions: Ephrin B2/Fc
Main Outcome Measures: Endothelial cells (EC) were isolated from both wild type (WT) and Eph-B4 heterozygous knock-out (Eph-B4-KO) mice. Cell proliferation was directly counted and confirmed with proliferating cellular nuclear antigen staining (PCNA). qPCR was used to evaluate basal mRNA expression and Western blot was used to evaluate protein levels. Under basal and Ephrin-B2/Fc-stimulated conditions angiogenic tube formation was measured by tube formation assay, cell migration was evaluated using a Boyden chamber, and nitric oxide (NO) production was evaluated fluorometrically.
Results: Under basal conditions Eph-B4-KO cells proliferate 53% more slowly than WT EC (p=.012), and 43% more slowly when stimulated with Ephrin-B2/Fc. PCNA staining confirmed 60% fewer proliferating cells in Eph-B4-KO cells under basal conditions. Basal levels of Akt were 84% higher, and VEGF-A was 36% lower in Eph-B4-KO cells compared to WT EC. qPCR confirmed 49% less VEGF-A mRNA expression in Eph-B4-KO cells. Eph-B4-KO cells had greater tube formation and NO production under both basal and stimulated conditions. Eph-B4-KO cells had 62% reduced cell migration in response to Ephrin-B2/Fc (p=.008).
Conclusions: EC isolated from Eph-B4-KO mice is a novel in vitro cellular model of VGA. This model suggests that reduced Eph-B4 expression during VGA may be mediated by upstream signals such as VEGF-A as well as downstream pathways such as Akt.