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)导致肢体远端组织血流量减少。在一些患者中， 如果血流灌注不足得不到有效治疗，就会出现严重的肢体缺血。血流量的恢复 通过动脉形成的侧支动脉绕过闭塞(S)可能最终代表着一种 这些患者的治疗选择。然而，我们对动脉形成的了解仍然不完全，这是有限的。 我们将治疗性动脉生成转化为临床的能力。在这项建议中，我们的目标是更好地了解 切应力介导内皮细胞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而扩张的能力有影响。 这一建议符合我们最近的发现，在小鼠股动脉结扎(FAL) 模型中，暴露于逆流的侧支节段显著放大和持续 动脉形成。在试点研究中，我们确定内皮细胞(ECs)暴露于仿生的非 反向血流(即轻度动脉形成)波形显示全基因组DNA高甲基化和增强 DNA甲基转移酶1(DNMT1)的表达与内皮细胞在仿生逆流中的比较 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的EC是否对 血流动力学刺激(即，升高的剪应力大小+/-反向流动)由 DNMT1的表达和随后的DNA甲基化水平。我们将击倒并过度表达DNMT1在 ECS暴露在这些波形下，并检测促动脉形成的EC反应。在《目标2》中，我们将 确定因暴露于高剪应力而引起的EC DNMT1表达增强， 通过增加切应力“设定点”来降低动脉形成能力。这一目标是由研究推动的。 显示暴露于血流增加而没有反向流动的侧支节段表现出不足 动脉形成，其管腔仍然太窄，不能完全恢复切应力到其原始值。 然而，用5-AZA抑制DNA甲基化可以使这些片段放大并恢复剪应力。 这导致了我们的假设，侧支动脉的动脉形成能力暴露在增加的剪切力- 应激通过增加EC DNMT1的表达和随后的DNA超甲基化而减少。在这里，我们将 首先通过育种获得可诱导的EC特异性DNMT1基因敲除小鼠。DNMT1基因将被切除 在FAL手术时或FAL后2周，当侧支直径已经 达到它们的稳定状态。动脉形成将与对照组小鼠进行比较，以及在FAL手术中进行比较 后肢通过比较反向和非反向血流侧支节段。最终，如果我们的假设是 我们相信它可能会有实质性的临床影响，因为它会揭示EC DNA 高甲基化可能是内源性和/或治疗性动脉形成能力的主要限制因素 在动脉闭塞的情况下恢复远端血流灌注(S)。