Endothelial Cell Dynamics in Low Flow

低流量下的内皮细胞动力学

• 批准号: RGPIN-2020-05646
• 负责人: Pickering, JGeoffrey
• 金额: $ 3.06万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=764590
• 关键词: Endothelial; Cell; Dynamics; Low; Flow

Endothelial cells line the surface of all blood vessels and are powerful mechanosensory machines. This includes sensing and responding to hemodynamic inputs. A critical but understudied flow environment is the developing vascular plexus that is perfused by ultra-low flow. During this phase, endothelial cells can undergo profound locomotive activity. However, the details of this activity and the hemodynamic controls are poorly understood. The long-term goal of our research program is to understand the interplay between flow and endothelial cell phenotype.  Highly atypical endothelial responses can occur under ultra-low flow conditions. including bridging across the lumen. Inward translocation of endothelial cells differs markedly from the traditionally studied outward sprouting to form a new vessel. The repertoire of low-flow-associated endothelial translocation events is not established and the molecular cascades are unknown. The goal of this program is to elucidate new basic science knowledge on the cascades that underlie the network-reshaping endothelial cell translocation events in conditions of low flow. Objectives: 1) To determine the repertoire of endothelial cell translocation responses to low shear stresses. 2) To elucidate molecular controls that direct flow-mediated translocation decisions by endothelial cells. 3) To determine the role of cellular curvature on flow-mediated endothelial cell behaviour. These Aims will be addressed using in vitro flow systems and cross-disciplinary interactions, and will be integrated with training of young natural scientists. To elucidate the flow conditions that drive specific endothelial morphological or motile phenotypes we will undertake analyses using endothelial cell-lined microfluidic chambers. Micro-channels will be fabricated from polydimethylsiloxane formed from micro-machined molds. The precise flow patterns will be quantified using micro-particle imaging velocimetry. Endothelial polarization, migration, inward translocation, and wall delamination will be sought and quantified by in situ labeling and real-time imaging. The mechano-transduction profiles and putative mechanosensors mediating the processes will be evaluated by immunostaining, transcriptome profiling, and using gene knock-down/overexpression in endothelial cells and blocking antibody delivery. Because endothelial cells exist on a curved topography, this key physical input will be evaluated in channels generated using viscous finger patterning. The impact of curvature on flow-regulated gene expression and locomotive behaviour will be quantified. Collectively, this research will provide fundamental new information on the interplay between key physical cues and endothelial cells as they translocate under low-flow states.

内皮细胞排列在所有血管的表面，是强大的机械感觉机器。这包括感知和响应血流动力学输入。一个关键但尚未得到充分研究的流动环境是由超低流量灌注的发育中的血管丛。在这个阶段，内皮细胞可以进行深刻的运动活动。然而，这种活动的细节和血液动力学控制知之甚少。我们研究计划的长期目标是了解血流和内皮细胞表型之间的相互作用。高度非典型的内皮反应可在超低流量条件下发生。包括在管腔间架桥。内皮细胞向内移位与传统研究的向外发芽形成新血管有明显不同。低流量相关的内皮易位事件尚未确定，分子级联也是未知的。该计划的目标是阐明在低流量条件下网络重塑内皮细胞易位事件的级联的新基础科学知识。目的：1)确定内皮细胞对低剪切应力的易位反应。2)阐明内皮细胞直接流介导的易位决定的分子控制。3)确定细胞曲率在血流介导的内皮细胞行为中的作用。这些目标将通过体外流动系统和跨学科互动来实现，并将与年轻自然科学家的培训相结合。为了阐明驱动特定内皮形态或运动表型的流动条件，我们将使用内皮细胞衬里的微流体室进行分析。微通道将由微机械模具形成的聚二甲基硅氧烷制成。精确的流动模式将被量化使用微粒成像测速。内皮极化、迁移、向内移位和壁层脱层将通过原位标记和实时成像来寻找和量化。将通过免疫染色、转录组分析、内皮细胞基因敲除/过表达和阻断抗体递送来评估机械转导谱和介导该过程的假定机械传感器。由于内皮细胞存在于弯曲的地形上，这种关键的物理输入将在使用粘性手指图形生成的通道中进行评估。曲率对流动调节基因表达和运动行为的影响将被量化。总的来说，这项研究将提供关键物理信号与内皮细胞在低流量状态下转运之间相互作用的基本新信息。