The Effects of Heparan Sulfate Infusion on Endothelial and Organ Injury in a Rat Pneumosepsis Model.

Septic shock is characterized by endothelial dysfunction, leading to tissue edema and organ failure. Heparan sulfate (HS) is essential for vascular barrier integrity, possibly via albumin as a carrier. We hypothesized that supplementing fluid resuscitation with HS would improve endothelial barrier function, thereby reducing organ edema and injury in a rat pneumosepsis model. Following intratracheal inoculation with Streptococcus pneumoniae, Sprague Dawley rats were randomized to resuscitation with a fixed volume of either Ringer's Lactate (RL, standard of care), RL supplemented with 7 mg/kg HS, 5% human albumin, or 5% human albumin supplemented with 7 mg/kg HS (n = 11 per group). Controls were sham inoculated animals. Five hours after the start of resuscitation, animals were sacrificed. To assess endothelial permeability, 70 kD FITC-labelled dextran was administered before sacrifice. Blood samples were taken to assess markers of endothelial and organ injury. Organs were harvested to quantify pulmonary FITC-dextran leakage, organ edema, and for histology. Inoculation resulted in sepsis, with increased lactate levels, pulmonary FITC-dextran leakage, pulmonary edema, and pulmonary histologic injury scores compared to healthy controls. RL supplemented with HS did not reduce median pulmonary FITC-dextran leakage compared to RL alone (95.1 CI [62.0-105.3] vs. 87.1 CI [68.9-139.3] µg/mL, p = 0.76). Similarly, albumin supplemented with HS did not reduce pulmonary FITC-dextran leakage compared to albumin (120.0 [93.8-141.2] vs. 116.2 [61.7 vs. 160.8] µg/mL, p = 0.86). No differences were found in organ injury between groups. Heparan sulfate, as an add-on therapy to RL or albumin resuscitation, did not reduce organ or endothelial injury in a rat pneumosepsis model. Higher doses of heparan sulfate may decrease organ and endothelial injury induced by shock.

The effects of resuscitation with different plasma products on endothelial permeability and organ injury in a rat pneumosepsis model.

BACKGROUND: Endothelial injury and permeability are a hallmark of sepsis. Initial resuscitation of septic patients with crystalloids is associated with aggravation of endothelial permeability, which may be related either to low protein content or to volume. We investigated whether initial resuscitation with different types of plasma or albumin decreases endothelial dysfunction and organ injury in a pneumosepsis rat model compared to the same volume of crystalloids. STUDY DESIGN AND METHODS: Sprague-Dawley rats were intratracheally inoculated with Streptococcus pneumoniae. Twenty-four hours after inoculation, animals were randomized to 2 control groups and 5 intervention groups (n = 11 per group) to receive resuscitation with a fixed volume (8 mL/kg for 1 h) of either Ringer's Lactate, 5% human albumin, fresh frozen plasma derived from syngeneic donor rats (rFFP), human-derived plasma (hFFP) or human-derived solvent detergent plasma (SDP). Controls were non-resuscitated (n = 11) and healthy animals. Animals were sacrificed 5 h after start of resuscitation (T = 5). Pulmonary FITC-dextran leakage as a reflection of endothelial permeability was used as the primary outcome. RESULTS: Inoculation with S. Pneumoniae resulted in sepsis, increased median lactate levels (1.6-2.8 mM, p < 0.01), pulmonary FITC-dextran leakage (52-134 µg mL-1, p < 0.05) and lung injury scores (0.7-6.9, p < 0.001) compared to healthy controls. Compared to animals receiving no resuscitation, animals resuscitated with rFFP had reduced pulmonary FITC leakage (134 vs 58 µg/mL, p = 0.011). However, there were no differences in any other markers of organ or endothelial injury. Resuscitation using different human plasma products or 5% albumin showed no differences in any outcome. CONCLUSIONS: Resuscitation with plasma did not reduce endothelial and organ injury when compared to an equal resuscitation volume of crystalloids. Rat-derived FFP may decrease pulmonary leakage induced by shock.

VISTA Expression on Cancer-Associated Endothelium Selectively Prevents T-cell Extravasation.

Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, down regulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homolog, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers under physiologic flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity.

Intercellular adhesion molecule 2 regulates diapedesis hotspots by allowing neutrophil crawling against the direction of flow.

Intercellular adhesion molecules (ICAMs) are cell surface proteins that play a crucial role in the body's immune response and inflammatory processes. ICAM1 and ICAM2 are two ICAM family members expressed on the surface of various cell types, including endothelial cells. They mediate the interaction between immune cells and endothelial cells, which are critical for the trafficking of leukocytes across the blood vessel wall during inflammation. Although ICAM1 plays a prominent role in the leukocyte extravasation cascade, it is less clear if ICAM2 strengthens ICAM1 function or has a separate function in the cascade. With CRISPR-)Cas9 technology, endothelial cells were depleted for ICAM1,ICAM2, or both, and we found that neutrophils favored ICAM1 over ICAM2 to adhere to. However, the absence of only ICAM2 resulted in neutrophils that were unable to find the transmigration hotspot, i.e. the preferred exit site. Moreover, we found that ICAM2 deficiency prevented neutrophils to migrate against the flow. Due to this deficiency, we concluded that ICAM2 helps neutrophils find the preferred exit sites and thereby contributes to efficient leukocyte extravasation.

Serine phosphorylation of the RhoGEF Trio stabilizes endothelial cell-cell junctions.

The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.

Spatiotemporal regulation of Rho GTPase signaling during endothelial barrier remodeling.

The vasculature is characterized by a thin cell layer that comprises the inner wall of all blood vessels, the continuous endothelium. Endothelial cells can also be found in the eye's cornea. And even though cornea and vascular endothelial (VE) cells differ from each other in structure, they both function as barriers and express similar junctional proteins such as the adherens junction VE-cadherin and tight-junction member claudin-5. How these barriers are controlled to maintain the barrier and thereby its integrity is of major interest in the development of potential therapeutic targets. An important target of endothelial barrier remodeling is the actin cytoskeleton, which is centrally coordinated by Rho GTPases that are in turn regulated by Rho-regulatory proteins. In this review, we give a brief overview of how Rho-regulatory proteins themselves are spatiotemporally regulated during the process of endothelial barrier remodeling. Additionally, we propose a roadmap for the comprehensive dissection of the Rho GTPase signaling network in its entirety.

Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength.

The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

Endothelial ICAM-1 Adhesome Recruits CD44 for Optimal Transcellular Migration of Human CTLs.

The endothelial lining of blood vessels is covered with a thin polysaccharide coat called the glycocalyx. This layer of polysaccharides contains hyaluronan that forms a protective coat on the endothelial surface. Upon inflammation, leukocytes leave the circulation and enter inflamed tissue by crossing inflamed endothelial cells, mediated by adhesion molecules such as ICAM-1/CD54. To what extent the glycocalyx participates in the regulation of leukocyte transmigration is not clear. During extravasation, leukocyte integrins cluster ICAM-1, resulting in the recruitment of a number of intracellular proteins and subsequent downstream effects in the endothelial cells. For our studies, we used primary human endothelial and immune cells. With an unbiased proteomics approach, we identified the full ICAM-1 adhesome and identified 93 (to our knowledge) new subunits of the ICAM-1 adhesome. Interestingly, we found the glycoprotein CD44 as part of the glycocalyx to be recruited to clustered ICAM-1 specifically. Our data demonstrate that CD44 binds hyaluronan to the endothelial surface, where it locally concentrates and presents chemokines that are essential for leukocytes to cross the endothelial lining. Taken together, we discover a link between ICAM-1 clustering and hyaluronan-mediated chemokine presentation by recruiting hyaluronan to sites of leukocyte adhesion via CD44.

Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM-1.

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 photoconvertible 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, the 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 the loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, the 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 signaling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

A pulmonary endothelial amplification loop aggravates ex-vivo transfusion-related acute lung injury via increased toll-like receptor 4 and intra-cellular adhesion molecule-1 expression.

BACKGROUND: Transfusion-Related Acute Lung Injury (TRALI) is a life-threatening complication of blood transfusions characterized by pulmonary endothelial cell damage and edema, with a high incidence in critically ill patients. The pathophysiology of TRALI is unresolved, but can generally be hypothesized to follow a 2-hit model in which the first hit is elicited by the underlying clinical condition of the patient (e.g., inflammation, which can be reflected by LPS in experimental models), and the second hit is delivered by the blood transfusion product (e.g., HLA class I antibodies). Here, we report a synergistic role for LPS and HLA class I antibody binding to pulmonary endothelium resulting in enhanced inflammatory responses. MATERIALS AND METHODS: Pulmonary endothelial cells were treated with PBS or low-dose LPS, exclusively or in combination with anti-HLA class I. Endothelial surface expression of HLA class I, TLR4, and inflammatory marker ICAM-1 were measured, and trans-endothelial migration (TEM) of neutrophils was investigated. RESULTS: LPS treatment of pulmonary endothelium enhanced HLA class I antibody binding, and combined LPS and HLA class I antibody binding enhanced TLR4 (LPS receptor) and ICAM-1 expression on the endothelial cell surface. Low-dose LPS and HLA antibody together also increased neutrophil TEM under physiological flow by on average 5-fold. CONCLUSION: We conclude that LPS and anti-HLA class I antibody have the ability to activate the pulmonary endothelium into a spiral of increasing inflammation, opening the opportunity to potentially block TLR4 to prevent or reduce the severity of TRALI in vivo.

Protocol to analyze transmigration of human cytotoxic T cells under physiological flow conditions in vitro.

This protocol presents an assay for transmigration analysis of human cytotoxic T cells (CTL) under physiological flow in vitro. We describe detailed analysis steps of human CTL behavior, from adhesion to diapedesis, using live cell imaging which cannot be achieved by in vivo imaging. The flow system is made of 2D plastic surfaces covered by an endothelial monolayer limiting the system but allows for quantitative analysis of CTL behavior with high modifiability. For complete details on the use and execution of this protocol, please refer to Schoppmeyer et al. (2022).

Prophylactic furosemide to prevent transfusion-associated circulatory overload: a randomized controlled study in rats.

Transfusion-associated circulatory overload (TACO) is the leading cause of transfusion related morbidity and mortality. The only treatment is empirical use of furosemide. Our aim was to investigate if furosemide can prevent TACO. A randomized controlled trial was performed using a previously validated two-hit rat model for TACO. Volume incompliance was induced (first hit) in anemic, anesthetized Lewis rats. Rats were randomized to placebo, low-dose (5 mg kg-1) or high-dose (15 mg kg-1) furosemide-administered prior to transfusion (second-hit) and divided over two doses. Primary outcome was change in left-ventricular end-diastolic pressure (∆LVEDP) pre- compared to post-transfusion. Secondary outcomes included changes in preload, afterload, contractility and systemic vascular resistance, as well as pulmonary outcomes. Furosemide treated animals had a significantly lower ∆LVEDP compared to placebo (p = 0.041), a dose-response effect was observed. ∆LVEDP in placebo was median + 8.7 mmHg (IQR 5.9-11), + 3.9 (2.8-5.6) in the low-dose and 1.9 (- 0.6 to 5.6) in the high-dose group. The effect of furosemide became apparent after 15 min. While urine output was significantly higher in furosemide treated animals (p = 0.03), there were no significant changes in preload, afterload, contractility or systemic vascular resistance. Furosemide rapidly and dose-dependently decreases the rise in hydrostatic pulmonary pressure following transfusion, essential for preventing TACO.

Differential Contribution of NF-κB Signaling Pathways to CD4+ Memory T Cell Induced Activation of Endothelial Cells.

Endothelial cells (ECs) are important contributors to inflammation in immune-mediated inflammatory diseases (IMIDs). In this study, we examined whether CD4+ memory T (Tm) cells can drive EC inflammatory responses. Human Tm cells produced ligands that induced inflammatory responses in human umbilical vein EC as exemplified by increased expression of inflammatory mediators including chemokines and adhesion molecules. NF-κB, a key regulator of EC activation, was induced by Tm cell ligands. We dissected the relative contribution of canonical and non-canonical NF-κB signaling to Tm induced EC responses using pharmacological small molecule inhibitors of IKKß (iIKKß) or NF-κB inducing kinase (iNIK). RNA sequencing revealed substantial overlap in IKKß and NIK regulated genes (n=549) that were involved in inflammatory and immune responses, including cytokines (IL-1ß, IL-6, GM-CSF) and chemokines (CXCL5, CXCL1). NIK regulated genes were more restricted, as 332 genes were uniquely affected by iNIK versus 749 genes by iIKKß, the latter including genes involved in metabolism, proliferation and leukocyte adhesion (VCAM-1, ICAM-1). The functional importance of NIK and IKKß in EC activation was confirmed by transendothelial migration assays with neutrophils, demonstrating stronger inhibitory effects of iIKKß compared to iNIK. Importantly, iIKKß - and to some extent iNIK - potentiated the effects of currently employed therapies for IMIDs, like JAK inhibitors and anti-IL-17 antibodies, on EC inflammatory responses. These data demonstrate that inhibition of NF-κB signaling results in modulation of Tm cell-induced EC responses and highlight the potential of small molecule NF-κB inhibitors as a novel treatment strategy to target EC inflammatory responses in IMIDs.

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.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium.

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2-9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.

Rho GTPase signalling networks in cancer cell transendothelial migration.

Rho GTPases are small signalling G-proteins that are central regulators of cytoskeleton dynamics, and thereby regulate many cellular processes, including the shape, adhesion and migration of cells. As such, Rho GTPases are also essential for the invasive behaviour of cancer cells, and thus involved in several steps of the metastatic cascade, including the extravasation of cancer cells. Extravasation, the process by which cancer cells leave the circulation by transmigrating through the endothelium that lines capillary walls, is an essential step for metastasis towards distant organs. During extravasation, Rho GTPase signalling networks not only regulate the transmigration of cancer cells but also regulate the interactions between cancer and endothelial cells and are involved in the disruption of the endothelial barrier function, ultimately allowing cancer cells to extravasate into the underlying tissue and potentially form metastases. Thus, targeting Rho GTPase signalling networks in cancer may be an effective approach to inhibit extravasation and metastasis. In this review, the complex process of cancer cell extravasation will be discussed in detail. Additionally, the roles and regulation of Rho GTPase signalling networks during cancer cell extravasation will be discussed, both from a cancer cell and endothelial cell point of view.

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 human 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-α (also known as TNF) caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualization 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. This article has an associated First Person interview with the first authors of the paper.

The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium.

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.

Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor.

Rho GTPases are regulatory proteins, which orchestrate cell features such as morphology, polarity and movement. Therefore, probing Rho GTPase activity is key to understanding processes such as development and cell migration. Localization-based reporters for active Rho GTPases are attractive probes to study Rho GTPase-mediated processes in real time with subcellular resolution in living cells and tissue. Until now, relocation Rho biosensors (sensors that relocalize to the native location of active Rho GTPase) seem to have been only useful in certain organisms and have not been characterized well. In this paper, we systematically examined the contribution of the fluorescent protein and Rho-binding peptides on the performance of localization-based sensors. To test the performance, we compared relocation efficiency and specificity in cell-based assays. We identified several improved localization-based, genetically encoded fluorescent biosensors for detecting endogenous Rho activity. This enables a broader application of Rho relocation biosensors, which was demonstrated by using the improved biosensor to visualize Rho activity during several cellular processes, such as cell division, migration and G protein-coupled receptor signaling. Owing to the improved avidity of the new biosensors for Rho activity, cellular processes regulated by Rho can be better understood. This article has an associated First Person interview with the first author of the paper.