Primary adhered neutrophils increase JNK1-MARCKSL1-mediated filopodia to promote secondary neutrophil transmigration.

During inflammation, leukocytes extravasate the vasculature to areas of inflammation in a process termed transendothelial migration. Previous research has shown that transendothelial migration hotspots exist, areas in the vasculature that are preferred by leukocytes to cross. Several factors that contribute to hotspot-mediated transmigration have been proposed already, but whether one leukocyte transmigration hotspot can be used subsequently by a second wave of leukocytes and thereby can increase the efficiency of leukocyte transmigration is not well understood. Here, we show that primary neutrophil adhesion to the endothelium triggers endothelial transmigration hotspots, allowing secondary neutrophils to cross the endothelium more efficiently. Mechanistically, we show that primary neutrophil adhesion increases the number of endothelial apical filopodia, resulting in an increase in the number of adherent secondary neutrophils. Using fluorescence resonance energy transfer (FRET)-based biosensors, we found that neutrophil adhesion did not trigger the activity of the small GTPase Cdc42. We used kinase translocation reporters to study the activity of mitogen-activated protein (MAP) kinases and Akt in endothelial cells on a single-cell level with a high temporal resolution during the process of leukocyte transmigration and found that c-Jun N-terminal kinase (JNK) is rapidly activated upon neutrophil adhesion, whereas extracellular regulated kinase (ERK), p38, and Akt are not. Additionally, we show that short-term chemical inhibition of endothelial JNK successfully prevents the adhesion of neutrophils to the endothelium. Furthermore, we show that neutrophil-induced endothelial JNK1 but not JNK2 increases the formation of filopodia and thereby the adhesion of secondary neutrophils. JNK1 needs its downstream substrate MARCKSL1 to trigger additional apical filopodia and consequently neutrophil adhesion. Overall, our data show that primary neutrophils can trigger the endothelial transmigration hotspot by activating JNK1 and MARCKSL1 to induce filopodia that trigger more neutrophils to transmigrate at the endothelial hotspot area.

Long non-coding RNA LASSIE regulates shear stress sensing and endothelial barrier function.

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton.