HIF-1α Expression Precedes Ephrin-B2 Expression During AVF Maturation

Abstract

Introduction: Arteriovenous fistulae (AVF) continue to show poor clinical results, with 50% 1-year patency. To understand AVF maturation, we previously reported a novel mouse AVF model recapitulating human AVF maturation. In order to determine if AVF have increased arterial identity, we examined Ephrin-B2 expression, an embryonic determinant of arteries. Since AVF creation may be associated with surgical injury and generation of reactive oxygen species, we also examined whether HIF-1α is expressed as well as its temporal relationship to Ephrin-B2 expression. Methods: Aortocaval fistulae were created in C57Bl/6 mice, and sham-operated mice were controls. Specimens of AVF or inferior vena cava were explanted up to day 42. Analysis was performed with Amplex Red for extracellular H2O2, and qPCR, immunohistochemistry, and Western blotting for Ephrin-B2 and HIF-1α. Results: AVF released more extracellular H2O2 compared to veins (n = 3; p = 0.007). AVF expressed increased numbers of Ephrin-B2 transcripts between days 7 and 21 postoperatively (n = 8; p < 0.05, ANOVA), and increased numbers of HIF-1α transcripts between days 3 and 21 (n = 8; p < 0.05, ANOVA). Western blot showed increased Ephrin-B2 (n = 3; p = 0.036) and HIF-1α (n = 3; p = 0.049) protein density compared to veins (postoperative day 3). Immunohistochemistry showed increased Ephrin-B2 and HIF-1α immunoreactivity in the AVF endothelium (n = 2; day 3). Conclusion: AVF have increased expression of both Ephrin-B2 and HIF-1α during early maturation. HIF-1α expression temporally precedes Ephrin-B2 expression, suggesting that HIF-1α may induce Ephrin-B2. These results suggest that clinical strategies to improve AVF outcomes could target the oxidative stress pathway.