Grant: Discovery Grants Program - Individual

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.