Abstract 571: Non-reversed Increase In Shear Stress Magnitude Enhances Global Endothelial Cell Dna Methylation And Limits Extent of Collateral Artery Growth Following Femoral Arterial Ligation

Abstract

Femoral artery ligation (FAL) in the mouse leads to increased flow across all collateral arteries with some collateral segments experiencing increased flow and reversed flow direction. We have recently shown that these “reversed flow” segments exhibit remarkably permanent, amplified arteriogenesis compared to those exposed to “non-reversed” flow. As the role of epigenetics in arteriogenesis remains unknown, we sought to determine the effect of biomimetic shear stress waveforms on DNA methylation and DNA methyltransferase 1 (DNMT1) expression in endothelial cells. Next generation sequencing of bisulfite converted genomic DNA was used to generate global DNA methylation profiles of ECs exposed to shear stress waveforms biomimetic of our “reversed” and “non-reversed” conditions. We found that the “non-reversed” waveform increased gene promoter hypermethylation in-vitro. Furthermore, DNMT1 mRNA expression was increased 1.25-fold in non-reversed conditions compared to reversed conditions as determined by RT-PCR (p≤0.05, n=8). We then tested the hypothesis that increased DNMT1 expression and subsequent DNA hypermethylation, elicited by a “non-reversed” flow waveform, limits steady state, long-term arteriogenic capacity in-vivo. Whole mount vascular casting was used to determine the lumenal diameter of both reversed and non-reversed collateral artery segments from mice treated with 5-AZA, a preferential inhibitor of DNMT1, or vehicle control 14 days post-FAL for an additional 14 days. We found that lumenal diameter was significantly enhanced in non-reversed collateral segments of 5-AZA treated mice (70.9±7.9μm vs. 50.1±3.4μm, p≤0.05, n=6) while there was no difference in reversed segments. Together, we conclude that a non-reversed, increased shear stress magnitude results in a rapid increase DNMT1 expression and DNA hypermethylation, limiting the extent of arteriogenesis in non-reversed collateral artery segments of the mouse gracilis muscle. However, inhibition of DNMT1 with 5-AZA enables for enhanced collateral growth. Further interrogation of the central relationships between hemodynamics, epigenetics, and collateral artery growth may lead to durable therapeutic options for arterial occlusive diseases.