Autoregulatory “Multitasking” at Endothelial Cell Junctions by Junction-Associated Intermittent Lamellipodia Controls Barrier Properties

Abstract

Vascular endothelial cell (EC) junctions are key structures controlling tissue homeostasis in physiology. In the last three decades, excellent studies have addressed many aspects of this complex and highly dynamic regulation, including cell signaling, remodeling processes of the proteins of tight junctions, adherens junctions, and gap junctions, the cytoskeleton, and post-transcriptional modifications, transcriptional activation, and gene silencing. In this dynamic process, vascular endothelial cadherin (VE-cadherin) provides the core structure of EC junctions mediating the physical adhesion of cells as well as the control of barrier function and monolayer integrity via remodeling processes, regulation of protein expression and post-translational modifications. In recent years, research teams have documented locally restricted dynamics of EC junctions in which actin-driven protrusions in plasma membranes play a central role. In this regard, our research group showed that the dynamics of VE-cadherin is driven by small (1–5 μm) actin-mediated protrusions in plasma membranes that, due to this specific function, were named “junction-associated intermittent lamellipodia” (JAIL). JAIL form at overlapping, adjacent cells, and exactly at this site new VE-cadherin interactions occur, leading to new VE-cadherin adhesion sites, a process that restores weak or lost VE-cadherin adhesion. Mechanistically, JAIL formation occurs locally restricted (1–5 μm) and underlies autoregulation in which the local VE-cadherin concentration is an important parameter. A decrease in the local concentration of VE-cadherin stimulates JAIL formation, whereas an increase in the concentration of VE-cadherin blocks it. JAIL mediated VE-cadherin remodeling at the subjunctional level have been shown to be of crucial importance in angiogenesis, wound healing, and changes in permeability during inflammation. The concept of subjunctional regulation of EC junctions is strongly supported by permeability assays, which can be employed to quantify actin-driven subjunctional changes. In this brief review, we summarize and discuss the current knowledge and concepts of subjunctional regulation in the endothelium.

High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers

“…J3 and J4 are linear with J4 having continuous CDH5 signal distributed over a larger area than J3. J5 junctions are visually reminiscent of the reticular junctions previously described (Millán et al, 2010) but could also appear as transient structures (Kim and Cooper, 2018; Seebach et al, 2020). J0 junctions might result from mis-segmented cells therefore in downstream analyses we considered cells with less than 20% J0.…”

High-content image analysis to study phenotypic heterogeneity in endothelial cell monolayers

“…Immunostaining and live imaging of EC monolayers has highlighted differences in cell morphology and junction patterns in response to stimuli or under distinct cell culture conditions (Abu Taha et al, 2014;Lampugnani et al, 1992;Seebach et al, 2016;Vestweber et al, 2009). We focussed on analysing proliferation, junctions, and NOTCH activation.…”

“…Previous studies on EC junction (Brezovjakova et al, 2019;Seebach et al, 2015;Wiseman et al, 2019) did not analyse the proportion of different junction pattern; our method classifies whole junctions' objects using an expert-trained ML algorithm (CPA with Fast Gentle Boosting, Appendix 1). In line with the literature in the field (Seebach et al, 2016), we classified junctions in a scale from 0 to 5 (Figure 5A). J0, J1 and J2 are discontinuous, highly jagged, or jagged junctions respectively.…”

“…J3 and J4 are linear with J4 having continuous CDH5 signal distributed over a larger area than J3. J5 junctions are visually reminiscent of the reticular junctions previously described (Millán et al, 2010) but could also appear as transient structures (Kim and Cooper, 2018;Seebach et al, 2020). J0 junctions might result from missegmented cells therefore in downstream analyses we considered cells with less than 20% J0.…”

The Anti-Colon Cancer Effects of Essential Oil of Curcuma phaeocaulis Through Tumour Vessel Normalisation

“…VE-cadherin also generates a dynamic balance between endothelial cell growth and perictyte coverage in blood vessels and helps maintain blood vessel homeostasis. Also, VE-cadherin regulates the growth rate of vascular endothelial cells by regulating angiogenic factors ( 37 , 38 ). Studies have shown that VE-cadherin knockout mice experience difficulties forming initial continuous blood vessels due to adhesion barriers between endothelial cells, causing death within 10 days of embryonic development ( 39 ).…”