Abstract 499: A Novel <i>En Face Ex Vivo</i> Live Imaging Approach For The Visualization Of Intimal Myeloid Cell Dynamics

Populations of CD11c + intimal myeloid cells (MCs) are known to reside in regions of the vasculature predisposed to atherosclerosis, such as the lesser curvature of the ascending arch. In Ldlr-/- mice, these cells have been shown to take up lipid and contribute to the formation of a nascent lesion under conditions of hypercholesterolemia. The role of resident CD11c+ cells in normal, healthy aortas has only recently been investigated. One function for these MCs may be the clearance of pathogens from the subendothelial space via reverse transendothelial migration (RTM). RTM can be triggered by systemic stimulation of cytokine and toll-like receptors (such as toll-like receptor 4 by lipopolysaccharide [LPS]), and induces a reduction in the number of resident intimal MCs in the lesser curvature. Our investigation of intimal RTM suggests that this cell loss is due to egress rather than cell death and is dependent on CCL19-CCR7 signaling, however we have yet to directly visualize this process. We endeavour to visualize resident intimal MC dynamics, including RTM, using ex vivo live imaging of the aortic intima. Ascending aortic arches were harvested from mice with fluorescent intimal myeloid cells (CD11c-eYFP mice or LyzM Cre -TdTomato reporter mice), stained with an endothelial marker (VE-Cadherin), and mounted en face in an imaging system that allows for constant perfusion of buffer across the tissue. Using a confocal microscope, images of the lesser curvature were continually acquired over a period of several hours following different stimuli to visualize cell behaviour. Intimal MCs were observed extending and retracting their dendrites under conditions of fluid flow, as well as undergoing cell division. Following the addition of the inflammatory stimulus LPS, cells can be seen migrating across and disappearing from the field of view. Thus, this method allows for direct visualization of intimal myeloid cell behaviour following perturbation by either physical or pharmacological stimuli. This approach, in tandem with pulsed perfusion of CD45-targeted antibodies, could provide a valuable toolkit to study immune cell egress from the intima of the lesser curvature of the aortic arch, under homeostasis and during the progression of atherosclerosis.

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