DNA Methyltransferase 1–Dependent DNA Hypermethylation Constrains Arteriogenesis by Augmenting Shear Stress Set Point

Abstract

BackgroundArteriogenesis is initiated by increased shear stress and is thought to continue until shear stress is returned to its original “set point.” However, the molecular mechanism(s) through which shear stress set point is established by endothelial cells (ECs) are largely unstudied. Here, we tested the hypothesis that DNA methyltransferase 1 (DNMT1)–dependent EC DNA methylation affects arteriogenic capacity via adjustments to shear stress set point.Methods and ResultsIn femoral artery ligation–operated C57BL/6 mice, collateral artery segments exposed to increased shear stress without a change in flow direction (ie, nonreversed flow) exhibited global DNA hypermethylation (increased 5‐methylcytosine staining intensity) and constrained arteriogenesis (30% less diameter growth) when compared with segments exposed to both an increase in shear stress and reversed‐flow direction. In vitro, ECs exposed to a flow waveform biomimetic of nonreversed collateral segments in vivo exhibited a 40% increase in DNMT1 expression, genome‐wide hypermethylation of gene promoters, and a DNMT1‐dependent 60% reduction in proarteriogenic monocyte adhesion compared with ECs exposed to a biomimetic reversed‐flow waveform. These results led us to test whether DNMT1 regulates arteriogenic capacity in vivo. In femoral artery ligation–operated mice, DNMT1 inhibition rescued arteriogenic capacity and returned shear stress back to its original set point in nonreversed collateral segments.ConclusionsIncreased shear stress without a change in flow direction initiates arteriogenic growth; however, it also elicits DNMT1‐dependent EC DNA hypermethylation. In turn, this diminishes mechanosensing, augments shear stress set point, and constrains the ultimate arteriogenic capacity of the vessel. This epigenetic effect could impact both endogenous collateralization and treatment of arterial occlusive diseases.

Epigenetic Regulation of Endothelial Cell Function by Nucleic Acid Methylation in Cardiac Homeostasis and Disease

“…Interestingly, and consistent with the previously described study, increased expression of DNMT1 observed under unidirectional flow resulted in reduced monocyte adhesion to ECs which was reversed by treatment with 5azadC in both in vitro and in vivo models [72]. Notably, when ECs maintained under shear stress were exposed to reversed flow, DNMT1 expression remained unaltered whilst monocyte adhesion to ECs was increased, prompting the authors to propose that hypermethylation under shear stress with unilateral flow may limit the arteriogenic capacity of ECs, thereby preventing hypertrophy/hyperplasia which may lead to impaired perfusion and tissue hypoxia [72]. In addition to DNMT1, other DNMT enzymes have been implicated in regulating EC function in response to altered blood flow.…”

“…Similarly, elevated DNMT1 levels in response to perturbed blood flow were attenuated with 5aza treatment [71]. Other studies have also reported increased DNMT1mediated DNA methylation in the context of both increased shear stress (induced in vivo by vascular occlusion or in vitro using cone and plate flow apparatus) and unidirectional flow, associated with global hypermethylation of genes associated with cellular metabolism, nucleic acid turnover and transcription [72]. Interestingly, and consistent with the previously described study, increased expression of DNMT1 observed under unidirectional flow resulted in reduced monocyte adhesion to ECs which was reversed by treatment with 5azadC in both in vitro and in vivo models [72].…”

“…Other studies have also reported increased DNMT1mediated DNA methylation in the context of both increased shear stress (induced in vivo by vascular occlusion or in vitro using cone and plate flow apparatus) and unidirectional flow, associated with global hypermethylation of genes associated with cellular metabolism, nucleic acid turnover and transcription [72]. Interestingly, and consistent with the previously described study, increased expression of DNMT1 observed under unidirectional flow resulted in reduced monocyte adhesion to ECs which was reversed by treatment with 5azadC in both in vitro and in vivo models [72]. Notably, when ECs maintained under shear stress were exposed to reversed flow, DNMT1 expression remained unaltered whilst monocyte adhesion to ECs was increased, prompting the authors to propose that hypermethylation under shear stress with unilateral flow may limit the arteriogenic capacity of ECs, thereby preventing hypertrophy/hyperplasia which may lead to impaired perfusion and tissue hypoxia [72].…”

MicroRNA-146a Regulates Perfusion Recovery in Response to Arterial Occlusion via Arteriogenesis

“…Further supporting this hypothesis, the ERASE trial found that a combination therapy of revascularization and exercise improved functional outcomes of PAD patients compared to just exercise alone (Fakhry et al, 2015 ). Overexpression of miR-93 (Ganta et al, 2017 ) or miR-199a inhibition (Heuslein et al, 2017 ) have both been shown to be capable of inducing arteriogenesis and improving in the ischemic downstream tissue, demonstrating such miR-based approaches are indeed possible.…”

“…We used a previously detailed FAL scheme (Meisner et al, 2012 ; Heuslein et al, 2015b , 2016 , 2017 ) that produces consistent arteriogenesis in the collateral arteries of the gracilis adductor muscles (Chappell et al, 2008 ; Distasi et al, 2009 ; Nickerson et al, 2009a ; Dai and Faber, 2010 ; Meisner et al, 2013 , 2014 ; Heuslein et al, 2015b , 2016 , 2017 ), along with minimal heterogeneity in the baseline collateral structure with known changes in flow direction from baseline. Male mice, 10–12 weeks of age, were anesthetized (i.p.…”