Microvascular Endothelial Cells Can Exhibit Autophagy in Vivo: Role in Neutrophil Transendothelial Cell Migration?

Leukocyte‐endothelial cell interaction regulates immune cell trafficking and is a key feature of inflammatory responses as mediated by complex series of molecular and cellular events. In recent years numerous metabolic, catabolic and redox sensitive cellular pathways have also been implicated in the regulation of leukocyte trafficking. In particular, autophagy, a house keeping process aimed at the orderly degradation and recycling of defunct cellular components, has been linked to the development of numerous inflammatory conditions. As much of these works have focussed on the occurrence of autophagy in immune cells, the aim of the present study was to investigate the potential role of endothelial cell (EC) autophagy in neutrophil transendothelial cell migration (TEM). Autophagy functions at a low basal level in many cell types through the formation of autophagosomes (AP). These organelles can be identified by their association with the membrane‐bound lipid modified form of autophagy protein microtubule‐associated protein 1A/1B‐light chain 3 (LC3) through development of characteristic LC3‐punctae. To investigate autophagy in venular ECs in vivo, we established a high resolution confocal microscopy method to observe formation of LC3‐punctae in cremasteric postcapillary venules using GFP‐LC3 transgenic mice. Briefly, GFP‐LC3 mice were injected locally with fluorescently‐labelled non‐blocking anti‐PECAM‐1 antibody, in order to label ECs, prior to analysis of tissues by confocal microscopy. GFP‐LC3 punctae were quantified in 3D using Imaris Bitplane Software. With this approach, venular ECs were found to exhibit a low level of basal autophagy that was significantly enhanced (~75%) in response to the conventional autophagy stimulus of starvation. To investigate the potential involvement of this reaction in neutrophil trafficking, the effect of numerous inflammatory stimuli were tested in the GFP‐LC3 mice. These studies identified local LPS (300ng; 4 h reaction) as an effective inducer of LC3 punctae formation in venular ECs in vivo. Local application of the early autophagy inhibitor 3‐methyladenine (3‐MA) suppressed formation of LC3 punctae in response to both starvation and LPS, indicating its efficacy in inhibiting stimulated EC autophagy. Local 3‐MA however had no impact on LPS‐induced neutrophil extravasation, suggesting that inhibition of canonical autophagy in ECs does not affect acute and local neutrophil migration. On‐going studies are aimed at investigating the role of EC autophagy in regulation of neutrophil trafficking in other inflammatory reactions and through the use of EC‐specific autophagy deficient mice. Collectively the findings provide the first direct evidence for the occurrence of autophagy in microvascular endothelial cells in vivo and provide a platform for further exploration of this phenomenon in inflammatory conditions.