Double-Hit-Induced Leukocyte Extravasation Driven by Endothelial Adherens Junction Destabilization.

During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

Depletion of Arg/Abl2 improves endothelial cell adhesion and prevents vascular leak during inflammation.

Endothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak.

The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries and veins.

OBJECTIVE: Vascular endothelial-cadherin- and integrin-based cell adhesions are crucial for endothelial barrier function. Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins. APPROACH AND RESULTS: We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells. CONCLUSIONS: Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristic.

Imaging and quantitative analysis of integrin-dependent cell-matrix adhesions.

Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP). For complete details on the use and execution of this protocol, please refer to Margadant et al. (2012),1 van der Bijl et al. (2020),2 Amado-Azevedo et al. (2021).3.

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3.

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34⁺ HSPCs. Human CD34⁺ HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34⁺ HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34⁺ HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34⁺ HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34⁺ HSPCs.

Endothelial cells of pulmonary origin display unique sensitivity to the bacterial endotoxin lipopolysaccharide.

Acute respiratory distress syndrome (ARDS) is a major clinical problem without available therapies. Known risks for ARDS include severe sepsis, SARS-CoV-2, gram-negative bacteria, trauma, pancreatitis, and blood transfusion. During ARDS, blood fluids and inflammatory cells enter the alveoli, preventing oxygen exchange from air into blood vessels. Reduced pulmonary endothelial barrier function, resulting in leakage of plasma from blood vessels, is one of the major determinants in ARDS. It is, however, unknown why systemic inflammation particularly targets the pulmonary endothelium, as endothelial cells (ECs) line all vessels in the vascular system of the body. In this study, we examined ECs of pulmonary, umbilical, renal, pancreatic, and cardiac origin for upregulation of adhesion molecules, ability to facilitate neutrophil (PMN) trans-endothelial migration (TEM) and for endothelial barrier function, in response to the gram-negative bacterial endotoxin LPS. Interestingly, we found that upon LPS stimulation, pulmonary ECs showed increased levels of adhesion molecules, facilitated more PMN-TEM and significantly perturbed the endothelial barrier, compared to other types of ECs. These observations could partly be explained by a higher expression of the adhesion molecule ICAM-1 on the pulmonary endothelial surface compared to other ECs. Moreover, we identified an increased expression of Cadherin-13 in pulmonary ECs, for which we demonstrated that it aids PMN-TEM in pulmonary ECs stimulated with LPS. We conclude that pulmonary ECs are uniquely sensitive to LPS, and intrinsically different, compared to ECs from other vascular beds. This may add to our understanding of the development of ARDS upon systemic inflammation.

Fast in vitro protocol for the visualization and quantitative high-throughput analysis of sprouting angiogenesis by confocal microscopy.

We describe an optimized, cost-effective, reproducible, and robust protocol to study sprouting angiogenesis in glass-bottom 96-well plates by confocal microscopy, ideal for screening of drug or shRNA libraries. Effective and stable knockdown of gene expression in primary endothelial cells is achieved by lentiviral transduction. Dynamic behavior of individual cells and fluorescent proteins is analyzed by time-lapse imaging, while competitive advantages in tip cell formation are assessed using mixtures of differentially labeled cell populations. Finally, we present a macro for high-throughput analysis. For complete information on the use and execution of this protocol, please refer to van der Bijl et al. (2020) and Kempers et al. (2021).

Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases.

Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases.

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions.

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.

Microtubule dynamics and Rac-1 signaling independently regulate barrier function in lung epithelial cells.

Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of beta-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.