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

内皮 DNA 甲基化、动脉生成能力和剪切应力“设定点”。

• 批准号: 9311466
• 负责人: Richard J. Price
• 金额: $ 20.03万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311466
• 关键词: Affect; Arteries; Atherosclerosis; Automobile Driving; Biological Assay; Biomimetics; Blood; Blood Vessels; Blood capillaries; Blood flow; Breeding; Bypass; C57BL/6 Mouse; Caliber; Cell Adhesion Molecules; Cell Proliferation; Cells; Clinic; Clinical; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; Distal; Endothelial Cells; Endothelium; Epigenetic Process; Exhibits; Exposure to; Flowmetry; Future; Generations; Genes; Genome; Growth; Hindlimb; Hypermethylation; Intercellular adhesion molecule 1; Intermittent Claudication; Ischemia; Knockout Mice; Lasers; Lead; Leg; Ligation; Limb structure; LoxP-flanked allele; Measures; Mediating; Modeling; Modification; Mus; Nature; Operative Surgical Procedures; Patients; Perfusion; Peripheral arterial disease; Physiological; Pilot Projects; Positioning Attribute; Recovery; Recruitment Activity; Research; Role; Running; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Stimulus; Tamoxifen; Testing; Therapeutic; Time; Tissues; Translating; Work; angiogenesis; artery occlusion; base; bisulfite sequencing; capillary; cell type; chemokine; density; discount; epigenetic regulation; expectation; fascinate; femoral artery; genome-wide; hemodynamics; inhibitor/antagonist; insight; knock-down; monocyte; mouse Cre recombinase; novel therapeutics; overexpression; pressure; response; restoration; shear stress

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).

外周动脉疾病 (PAD) 会导致远端肢体组织血流量减少。在一些患者中， 如果灌注不足得不到有效治疗，就会发生严重的肢体缺血。血流恢复 通过绕过闭塞的侧支动脉的动脉生长最终可能代表 这些患者的治疗选择。然而，我们对动脉发生的了解仍然不完整，因此限制了我们的研究。 我们将治疗性动脉生成转化为临床的能力。在本提案中，我们旨在更好地了解 剪切应力介导的内皮细胞 DNA 甲基转移酶 (胞嘧啶-5) 1 (DNMT1) 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。 这一提议从逻辑上遵循我们最近的发现，即在小鼠股动脉结扎（FAL）中 模型中，暴露于逆流的侧支部分表现出显着的放大和持续 动脉生成。在试点研究中，我们确定内皮细胞（EC）暴露于仿生非 逆流（即轻度动脉生成）波形表现出全基因组 DNA 高甲基化并增强 与暴露于仿生逆流的 EC 相比，DNA 甲基转移酶 1 (DNMT1) 的表达 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 血流动力学刺激（即升高的剪切应力大小+/-反向流动方向）通过以下方式调节 DNMT1 表达和随后的 DNA 甲基化水平。我们将敲低并过表达 DNMT1 EC 暴露于这些波形并测定促动脉 EC 反应。在目标 2 中，我们将 确定是否通过暴露于升高的剪切应力而引起 EC DNMT1 表达增强， 通过增加剪切应力“设定点”来损害动脉生成能力。这个目标是由研究推动的 表明暴露于增加流量而没有反向流动方向的侧支段表现出不足 动脉生成，使得它们的管腔仍然太窄而无法将剪切应力完全恢复到其原始值。 然而，用 5-AZA 抑制 DNA 甲基化可以使这些片段扩大并恢复剪切应力。 这使我们得出这样的假设：侧支动脉的动脉生成能力暴露于增加的剪切力下。 EC DNMT1 表达增加和随后的 DNA 高甲基化可减轻应激。在这里，我们将 首先通过育种产生可诱导的 EC 特异性 DNMT1 敲除小鼠。 DNMT1基因将被切除 来自 EC，无论是在 FAL 手术时还是 FAL 后 2 周，此时侧支直径已经 达到稳定状态。动脉生成将与对照小鼠以及 FAL 操作小鼠进行比较 通过比较反向和非反向流动侧支节段来观察后肢。最终，如果我们的假设是 经过验证，我们相信它可能会产生重大的临床影响，因为它会揭示 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 在动脉闭塞的情况下恢复远端灌注。