Biology subjects

van Steen, A. C. I.

Publications and source records attributed to van Steen, A. C. I..

3 recordsLinked to original sources

bioRxiv2022

Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM1

Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration hotspot regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photo-convertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4th Ig-like dimerization domain are not involved, indicating that intracellular signalling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

immunology↗

bioRxiv2021

Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF- caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of all different steps of the leukocyte transmigration cascade including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay. SummaryA functional hydrogel-based blood-vessel-on-a-chip model is used to study the complete leukocyte transendothelial migration process in real time. T-lymphocytes and neutrophils exhibit distinct migration dynamics in the extravascular matrix after transendothelial migration, which can be altered using a chemotactic gradient.

cell biology↗

bioRxiv2021

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

cell biology↗