Endothelial DNA Methylation, Arteriogenic Capacity, and Shear Stress "Set-Point."

外周动脉疾病（PAD）导致远端肢体组织中的血流减少。在一些患者中， 如果灌注不足得不到有效治疗，将发生严重的肢体缺血。恢复血流 通过旁路闭塞的侧支动脉的动脉生成性生长， 这些患者的治疗选择。然而，我们对动脉生成的认识仍然不完全， 我们将治疗性动脉生成转化为临床的能力。在本建议中，我们旨在更好地了解 切应力介导的内皮细胞DNA甲基转移酶（胞嘧啶-5）1（DNMT 1）变化的作用 表达对侧支血管通过动脉生成扩大的能力有影响。 这一建议符合我们最近的发现，即在小鼠股动脉结扎（FAL） 模型，侧支血管段暴露于反向流动表现出显着放大和持续 动脉生成在初步研究中，我们确定内皮细胞（EC）暴露于仿生非- 反向流动（即轻度动脉生成）波形表现出全基因组DNA超甲基化和增强的 与暴露于仿生逆流的EC相比，DNA甲基转移酶1（DNMT 1）表达 波形（即放大的动脉生成）。在目标1中，我们将确定促动脉生成EC是否对 血液动力学刺激（即升高的剪切应力大小+/-反向流动方向）通过以下方式调节 DNMT 1表达和随后的DNA甲基化水平。我们将敲低并过表达DNMT 1， 暴露于这些波形的EC和测定促动脉生成EC响应。在目标2中，我们将 确定通过暴露于升高的剪切应力引起的增强的EC DNMT 1表达， 通过增加剪切应力“设定点”来损害动脉生成能力。这一目标的动机是研究 显示了暴露于增加的流动而没有反向流动方向的侧支节段表现出不充分的 动脉形成，使得它们的管腔保持太窄而不能将剪切应力完全恢复到其原始值。 然而，用5-AZA抑制DNA甲基化允许这些片段扩大并恢复剪切应力。 这导致我们假设，侧支动脉的动脉生成能力暴露于增加的剪切- 应激通过增加的EC DNMT 1表达和随后的DNA超甲基化而减弱。在这里，我们将 首先通过育种产生可诱导的EC特异性DNMT 1敲除小鼠。DNMT 1基因将被切除 在FAL手术时或FAL后2周，当侧支直径已经 达到稳定状态。将动脉生成与对照小鼠以及FAL手术小鼠进行比较。 后肢通过比较反向和非反向流动侧支血管段。最终，如果我们的假设是 经过验证，我们相信它可能具有重大的临床影响，因为它将揭示EC DNA 高甲基化可能是内源性和/或治疗性动脉生成能力的主要限制因素， 以在动脉闭塞的情况下恢复远端灌注。

Peripheral arterial disease (PAD) causes reduced blood flow in distal limb tissue. In some patients, critical limb ischemia will develop if the perfusion deficit is not effectively treated. The restoration of blood flow via the arteriogenic growth of collateral arteries bypassing the occlusion(s) may eventually represent a therapeutic option for these patients. However, our still incomplete understanding of arteriogenesis has limited our ability to translate therapeutic arteriogenesis to the clinic. In this proposal, we aim to better understand the role that shear stress-mediated changes in endothelial cell DNA methyltransferase (cytosine-5) 1 (DNMT1) expression have on the capacity of collateral vessels to enlarge through arteriogenesis. This proposal follows logically from our recent discovery that, in the mouse femoral artery ligation (FAL) model, collateral segments exposed to reversed flow exhibit remarkably amplified and sustained arteriogenesis. In pilot studies, we determined that endothelial cells (ECs) exposed to a biomimetic non- reversed flow (i.e. mild arteriogenesis) waveform exhibit genome-wide DNA hypermethylation and enhanced DNA methyltransferase 1 (DNMT1) expression when compared to ECs exposed to a biomimetic reversed flow waveform (i.e. amplified arteriogenesis). In Aim 1, we will determine whether pro-arteriogenic EC responses to hemodynamic stimuli (i.e. elevated shear stress magnitude +/- reversed flow direction) are modulated by DNMT1 expression and subsequent DNA methylation levels. We will knockdown and overexpress DNMT1 in ECs exposed to these waveforms and assay for pro-arteriogenic EC responses. In Aim 2, we will then determine whether enhanced EC DNMT1 expression, elicited by exposure to elevated shear stress, compromises arteriogenic capacity by increasing shear stress “set-point”. This aim is motivated by studies showing that collateral segments exposed to increased flow without reversed flow direction exhibit insufficient arteriogenesis, such that their lumens remain too narrow to fully restore shear stress to its original value. However, inhibiting DNA methylation with 5-AZA allows these segments to enlarge and restore shear stress. This leads us to the hypothesis that the arteriogenic capacity of collateral arteries exposed to increased shear- stress is diminished by increased EC DNMT1 expression and subsequent DNA hypermethylation. Here, we will first generate inducible EC-specific DNMT1 knockout mice through breeding. The DNMT1 gene will be excised from ECs, either at the time of FAL surgery or 2 weeks after FAL, when collateral diameters have already reached their steady state. Arteriogenesis will be compared to control mice, as well as within FAL operated hindlimbs by comparing reversed and non-reversed flow collateral segments. Ultimately, if our hypothesis is verified, we believe it may have substantial clinical impact because it would reveal that EC DNA hypermethylation could be a major limiting factor in the ability of endogenous and/or therapeutic arteriogenesis to restore distal perfusion in the presence of arterial occlusion(s).