Endothelial Cell Dynamics in Low Flow

内皮细胞排列在所有血管的表面，是强大的机械感觉机器。 这包括感测和响应血液动力学输入。一个关键但研究不足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，内皮细胞可以经历深刻的运动活动。 然而，这种活动的细节和血流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。 在超低流量条件下可发生高度非典型的内皮反应。包括跨管腔桥接。 内皮细胞的向内移位与传统研究的向外出芽形成新血管明显不同。 低流量相关的内皮易位事件的库尚未建立，分子级联反应是未知的。 该计划的目标是阐明低流量条件下网络重塑内皮细胞易位事件的基础级联的新基础科学知识。 目的： 1)确定内皮细胞对低切应力的移位反应。 2)阐明直接通过内皮细胞进行血流介导的易位决定的分子控制。 3)确定细胞曲率对血流介导的内皮细胞行为的作用。 这些目标将使用体外流动系统和跨学科的相互作用来解决，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内衬的微流体室进行分析。微通道将由微加工模具形成的聚二甲基硅氧烷制成。 精确的流动模式将使用微粒子成像测速仪进行量化。将通过原位标记和实时成像来寻找和量化内皮极化、迁移、向内移位和壁分层。 将通过免疫染色、转录组分析和使用内皮细胞中的基因敲低/过表达和阻断抗体递送来评价机械转导谱和介导该过程的推定机械传感器。 由于内皮细胞存在于弯曲的形貌上，因此将在使用粘性指状图案生成的通道中评估该关键物理输入。曲率对流量调节基因表达和运动行为的影响将被量化。 总的来说，这项研究将提供关于关键物理线索和内皮细胞在低流量状态下易位时相互作用的基本新信息。

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.