ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Differential role for rapid proteostasis in Rho GTPase-mediated control of quiescent endothelial integrity.

Endothelial monolayer permeability is regulated by actin dynamics and vesicular traffic. Recently, ubiquitination was also implicated in the integrity of quiescent endothelium, as it differentially controls the localization and stability of adhesion and signaling proteins. However, the more general effect of fast protein turnover on endothelial integrity is not clear. Here, we found that inhibition of E1 ubiquitin ligases induces a rapid, reversible loss of integrity in quiescent, primary human endothelial monolayers, accompanied by increased F-actin stress fibers and the formation of intercellular gaps. Concomitantly, total protein and activity of the actin-regulating GTPase RhoB, but not its close homolog RhoA, increase ∼10-fold in 5 to 8 h. We determined that the depletion of RhoB, but not of RhoA, the inhibition of actin contractility, and the inhibition of protein synthesis all significantly rescue the loss of cell-cell contact induced by E1 ligase inhibition. Collectively, our data suggest that in quiescent human endothelial cells, the continuous and fast turnover of short-lived proteins that negatively regulate cell-cell contact is essential to preserve monolayer integrity.

Extravasation of biodegradable microspheres in the rat brain.

Drug development for neurological diseases is greatly impeded by the presence of the blood-brain barrier (BBB). We and others previously reported on extravasation of micrometer-sized particles from the cerebral microcirculation - across the BBB - into the brain tissue over the course of several weeks. This mechanism could potentially be used for sustained parenchymal drug delivery after extravasation of biodegradable microspheres. As a first step toward this goal, we set out to evaluate the extravasation potential in the rat brain of three classes of biodegradable microspheres with drug-carrying potential, having a median diameter of 13 µm (80% within 8-18 µm) and polyethylene glycol concentrations of 0%, 24% and 36%. Extravasation, capillary recanalization and tissue damage were determined in a rat cerebral microembolization model at day 14 after microsphere injection. Microspheres of all three classes had the potential to extravasate from the vessel into the brain parenchyma, with microspheres without polyethylene glycol extravasating the fastest. Microembolization with biodegradable microspheres led to impaired local capillary perfusion, which was substantially restored after bead extravasation. We did not observe overt tissue damage after microembolization with any microsphere: we found very limited BBB disruption (IgG extravasation), no microgliosis (Iba1 staining) and no large neuronal infarctions (NeuN staining). In conclusion, biodegradable microspheres with different polymer compositions can extravasate into the brain parenchyma while causing minimal tissue damage.

Epigenetic Modification of the von Willebrand Factor Promoter Drives Platelet Aggregation on the Pulmonary Endothelium in Chronic Thromboembolic Pulmonary Hypertension.

Rationale: von Willebrand factor (vWF) mediates platelet adhesion during thrombosis. While chronic thromboembolic pulmonary hypertension (CTEPH) is associated with increased plasma levels of vWF, the role of this protein in CTEPH has remained enigmatic. Objectives: To identify the role of vWF in CTEPH. Methods: CTEPH-specific patient plasma and pulmonary endarterectomy material from patients with CTEPH were used to study the relationship between inflammation, vWF expression, and pulmonary thrombosis. Cell culture findings were validated in human tissue, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Measurements and Main Results: vWF is increased in plasma and the pulmonary endothelium of CTEPH patients. In vitro, the increase in vWF gene expression and the higher release of vWF protein upon endothelial activation resulted in elevated platelet adhesion to CTEPH endothelium. Proteomic analysis revealed that nuclear factor (NF)-κB2 was significantly increased in CTEPH. We demonstrate reduced histone tri-methylation and increased histone acetylation of the vWF promoter in CTEPH endothelium, facilitating binding of NF-κB2 to the vWF promoter and driving vWF transcription. Genetic interference of NFκB2 normalized the high vWF RNA expression levels and reversed the prothrombotic phenotype observed in CTEPH-pulmonary artery endothelial cells. Conclusions: Epigenetic regulation of the vWF promoter contributes to the creation of a local environment that favors in situ thrombosis in the pulmonary arteries. It reveals a direct molecular link between inflammatory pathways and platelet adhesion in the pulmonary vascular wall, emphasizing a possible role of in situ thrombosis in the development or progression of CTEPH.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

The interplay of Rac1 activity, ubiquitination and GDI binding and its consequences for endothelial cell spreading.

Signaling by the Rho GTPase Rac1 is key to the regulation of cytoskeletal dynamics, cell spreading and adhesion. It is widely accepted that the inactive form of Rac1 is bound by Rho GDI, which prevents Rac1 activation and Rac1-effector interactions. In addition, GDI-bound Rac1 is protected from proteasomal degradation, in line with data showing that Rac1 ubiquitination occurs exclusively when Rac1 is activated. We set out to investigate how Rac1 activity, GDI binding and ubiquitination are linked. We introduced single amino acid mutations in Rac1 which differentially altered Rac1 activity, and compared whether the level of Rac1 activity relates to Rac1 ubiquitination and GDI binding. Results show that Rac1 ubiquitination and the active Rac1 morphology is proportionally increased with Rac1 activity. Similarly, we introduced lysine-to-arginine mutations in constitutively active Rac1 to inhibit site-specific ubiquitination and analyze this effect on Rac1 signaling output and ubiquitination. These data show that the K16R mutation inhibits GTP binding, and consequently Rac1 activation, signaling and-ubiquitination, while the K147R mutation does not block Rac1 signaling, but does inhibits its ubiquitination. In both sets of mutants, no direct correlation was observed between GDI binding and Rac1 activity or -ubiquitination. Taken together, our data show that a strong, positive correlation exists between Rac1 activity and its level of ubiquitination, but also that GDI dissociation does not predispose Rac1 to ubiquitination.

Regulation of Rho GTPases in the Vasculature by Cullin3-Based E3 Ligase Complexes.

Cullin3-based ubiquitin E3 ligases induce ubiquitination of substrates leading to their proteasomal or lysosomal degradation. BTB proteins serve as adaptors by binding to Cullin3 and recruiting substrate proteins, which enables specific recognition of a broad spectrum of targets. Hence, Cullin3 and its adaptors are involved in myriad cellular processes and organ functions. Cullin3-based ubiquitin E3 ligase complexes target small GTPases of the Rho subfamily, which are key regulators of cytoskeletal dynamics and cell adhesion. In this mini review, we discuss recent insights in Cullin3-mediated regulation of Rho GTPases and their impact on cellular function and disease. Intriguingly, upstream regulators of Rho GTPases are targeted by Cullin3 complexes as well. Thus, Rho GTPase signaling is regulated by Cullin3 on multiple levels. In addition, we address current knowledge of Cullin3 in regulating vascular function, focusing on its prominent role in endothelial barrier function, angiogenesis and the regulation of blood pressure.

Preservation of renal endothelial integrity and reduction of renal edema by aprotinin does not preserve renal perfusion and function following experimental cardiopulmonary bypass.

BACKGROUND: Acute kidney injury is a severe complication following cardiopulmonary bypass (CPB) and is associated with capillary leakage and microcirculatory perfusion disturbances. CPB-induced thrombin release results in capillary hyperpermeability via activation of protease-activated receptor 1 (PAR1). We investigated whether aprotinin, which is thought to prevent thrombin from activating PAR1, preserves renal endothelial structure, reduces renal edema and preserves renal perfusion and reduces renal injury following CPB. METHODS: Rats were subjected to CPB after treatment with 33.000 KIU/kg aprotinin (n = 15) or PBS (n = 15) as control. A secondary dose of 33.000 KIU/kg aprotinin was given 60 min after initiation of CPB. Cremaster and renal microcirculatory perfusion were assessed using intravital microscopy and contrast echography before CPB and 10 and 60 min after weaning from CPB. Renal edema was determined by wet/dry weight ratio and renal endothelial structure by electron microscopy. Renal PAR1 gene and protein expression and markers of renal injury were determined. RESULTS: CPB reduced cremaster microcirculatory perfusion by 2.5-fold (15 (10-16) to 6 (2-10) perfused microvessels, p < 0.0001) and renal perfusion by 1.6-fold (202 (67-599) to 129 (31-292) au/sec, p = 0.03) in control animals. Both did not restore 60 min post-CPB. This was paralleled by increased plasma creatinine (p < 0.01), neutrophil gelatinase-associated lipocalin (NGAL; p = 0.003) and kidney injury molecule-1 (KIM-1; p < 0.01). Aprotinin treatment preserved cremaster microcirculatory perfusion following CPB (12 (7-15) vs. 6 (2-10) perfused microvessels, p = 0.002), but not renal perfusion (96 (35-313) vs. 129 (31-292) au/s, p > 0.9) compared to untreated rats. Aprotinin treatment reduced endothelial gap formation (0.5 ± 0.5 vs. 3.1 ± 1.4 gaps, p < 0.0001), kidney wet/dry weight ratio (4.6 ± 0.2 vs. 4.4 ± 0.2, p = 0.046), and fluid requirements (3.9 ± 3.3 vs. 7.5 ± 3.0 ml, p = 0.006) compared to untreated rats. In addition, aprotinin treatment reduced tubulointerstitial neutrophil influx by 1.7-fold compared to untreated rats (30.7 ± 22.1 vs. 53.2 ± 17.2 neutrophil influx/section, p = 0.009). No differences were observed in renal PAR1 expression and plasma creatinine, NGAL or KIM-1 between groups. CONCLUSIONS: Aprotinin did not improve renal perfusion nor reduce renal injury during the first hour following experimental CPB despite preservation of renal endothelial integrity and reduction of renal edema.

Patient-Specific 3-Dimensional Model of Smooth Muscle Cell and Extracellular Matrix Dysfunction for the Study of Aortic Aneurysms.

INTRODUCTION: Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological therapy exists. Smooth muscle cells (SMCs) dysfunction and extracellular matrix (ECM) degradation have been proposed as underlying causes. We investigated SMC spatial organization and SMC-ECM interactions in our novel 3-dimensional (3D) vascular model. We validated our model for future use by comparing it to existing 2-dimensional (2D) cell culture. Our model can be used for translational studies of SMC and their role in AAA pathophysiology. MATERIALS AND METHODS: SMC isolated from the medial layer of were the aortic wall of controls and AAA patients seeded on electrospun poly-lactide-co-glycolide scaffolds and cultured for 5 weeks, after which endothelial cells (EC) are added. Cell morphology, orientation, mechanical properties and ECM production were quantified for validation and comparison between controls and patients. RESULTS: We show that cultured SMC proliferate into multiple layers after 5 weeks in culture and produce ECM proteins, mimicking their behavior in the medial aortic layer. EC attach to multilayered SMC, mimicking layer interactions. The novel SMC model exhibits viscoelastic properties comparable to biological vessels; cytoskeletal organization increases during the 5 weeks in culture; increased cytoskeletal alignment and decreased ECM production indicate different organization of AAA patients' cells compared with control. CONCLUSION: We present a valuable preclinical model of AAA constructed with patient specific cells with applications in both translational research and therapeutic developments. We observed SMC spatial reorganization in a time course of 5 weeks in our robust, patient-specific model of SMC-EC organization and ECM production.

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.

Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells.

The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.

Microembolus clearance through angiophagy is an auxiliary mechanism preserving tissue perfusion in the rat brain.

Considering its intolerance to ischemia, it is of critical importance for the brain to efficiently process microvascular occlusions and maintain tissue perfusion. In addition to collateral microvascular flow and enzymatic degradation of emboli, the endothelium has the potential to engulf microparticles and thereby recanalize the vessel, through a process called angiophagy. Here, we set out to study the dynamics of angiophagy in relation to cytoskeletal remodeling in vitro and reperfusion in vivo. We show that polystyrene microspheres and fibrin clots are actively taken up by (brain) endothelial cells in vitro, and chart the dynamics of the actin cytoskeleton during this process using live cell imaging. Whereas microspheres were taken up through the formation of a cup structure by the apical endothelial membrane, fibrin clots were completely engulfed by the cells, marked by dense F-actin accumulation surrounding the clot. Both microspheres and fibrin clots were retained in the endothelial cells. Notably, fibrin clots were not degraded intracellularly. Using an in vivo microembolization rat model, in which microparticles are injected into the common carotid artery, we found that microspheres are transported by the endothelium from the microvasculature into the brain parenchyma. Microembolization with microspheres caused temporal opening of the blood-brain barrier and vascular nonperfusion, followed by microsphere extravasation and restoration of vessel perfusion over time. Taken together, angiophagy is accompanied by active cytoskeletal remodeling of the endothelium, and is an effective mechanism to restore perfusion of the occluded microvasculature in vivo.

Bosutinib prevents vascular leakage by reducing focal adhesion turnover and reinforcing junctional integrity.

Endothelial barrier dysfunction leads to edema and vascular leak, causing high morbidity and mortality. Previously, Abl kinase inhibition has been shown to protect against vascular leak. Using the distinct inhibitory profiles of clinically available Abl kinase inhibitors, we aimed to provide a mechanistic basis for novel treatment strategies against vascular leakage syndromes. We found that the inhibitor bosutinib most potently protected against inflammation-induced endothelial barrier disruption. In vivo, bosutinib prevented lipopolysaccharide (LPS)-induced alveolar protein extravasation in an acute lung injury mice model. Mechanistically, mitogen-activated protein 4 kinase 4 (MAP4K4) was identified as important novel mediator of endothelial permeability, which signaled via ezrin, radixin and moesin proteins to increase turnover of integrin-based focal adhesions. The combined inhibition of MAP4K4 and Abl-related gene (Arg, also known as ABL2) by bosutinib preserved adherens junction integrity and reduced turnover of focal adhesions, which synergistically act to stabilize the endothelial barrier during inflammation. We conclude that MAP4K4 is an important regulator of endothelial barrier integrity, increasing focal adhesion turnover and disruption of cell-cell junctions during inflammation. Because it inhibits both Arg and MAP4K4, use of the clinically available drug bosutinib might form a viable strategy against vascular leakage syndromes.

In vitro endothelial hyperpermeability occurs early following traumatic hemorrhagic shock.

BACKGROUND: Endothelial hyperpermeability is suggested to play a role in the development of microcirculatory perfusion disturbances and organ failure following hemorrhagic shock, but evidence is limited. OBJECTIVE: To study the effect of plasma from traumatic hemorrhagic shock patients on in vitro endothelial barrier function. METHODS: Plasma from traumatic hemorrhagic shock patients was obtained at the emergency department (ED), the intensive care unit (ICU), 24 h after ICU admission and from controls (n = 8). Sublingual microcirculatory perfusion was measured using incident dark field videomicroscopy at matching time points. Using electric cell-substrate impedance sensing, the effects of plasma exposure on in vitro endothelial barrier function of human endothelial cells were assessed. RESULTS: Plasma from traumatic hemorrhagic shock patients collected at ED admission induced a 19% loss of in vitro endothelial resistance compared to plasma from controls (p < 0.001). This loss was due to reduced cell-cell contacts (p < 0.01). Plasma withdrawn at later time points did not affect endothelial barrier function (p > 0.99). Interestingly, in vitro endothelial resistance showed a positive association with in vivo microcirculatory perfusion (r = 0.56, p < 0.01). CONCLUSIONS: Plasma from traumatic hemorrhagic shock patients obtained following ED admission, but not at later stages, induced in vitro endothelial hyperpermeability. This coincided with in vivo microcirculatory perfusion disturbances.

Most exposed: the endothelium in chronic kidney disease.

Accumulating evidence indicates that the pathological changes of the endothelium may contribute to the development of cardiovascular complications in chronic kidney disease (CKD). Non-traditional risk factors related to CKD are associated with the incidence of cardiovascular disease, but their role in uraemic endothelial dysfunction has often been disregarded. In this context, soluble α-Klotho and vitamin D are of importance to maintain endothelial integrity, but their concentrations decline in CKD, thereby contributing to the dysfunction of the endothelial lining. These hormonal disturbances are accompanied by an increment of circulating fibroblast growth factor-23 and phosphate, both exacerbating endothelial toxicities. Furthermore, impaired renal function leads to an increment of inflammatory mediators, reactive oxygen species and uraemic toxins that further aggravate the endothelial abnormalities and in turn also inhibit the regeneration of disrupted endothelial lining. Here, we highlight the distinct endothelial alterations mediated by the abovementioned non-traditional risk factors as demonstrated in experimental studies and connect these to pathological changes in CKD patients, which are driven by endothelial disturbances, other than atherosclerosis. In addition, we describe therapeutic strategies that may promote restoration of endothelial abnormalities by modulating imbalanced mineral homoeostasis and attenuate the impact of uraemic retention molecules, inflammatory mediators and reactive oxygen species. A clinical perspective on endothelial dysfunction in CKD may translate into reduced structural and functional abnormalities of the vessel wall in CKD, and ultimately improved cardiovascular disease.

Pharmacological interventions to reduce edema following cardiopulmonary bypass: A systematic review and meta-analysis.

PURPOSE: To compare the effectiveness of different types of pharmacological agents to reduce organ specific edema following cardiopulmonary bypass (CPB). METHODS: Pubmed, Embase.com and Cochrane were searched for studies administrating a pharmacological agent before CPB. Primary outcome was postoperative edema. RESULTS: Forty-four studies (clinical n = 6, preclinical n = 38) fulfilled eligibility criteria. Steroids were used in most clinical studies (n = 5, 83%) and reduced postoperative edema in 4 studies, however heterogeneity precluded meta-analysis. In preclinical studies, a total of 31 different drugs were tested of which 20 (65%) reduced edema in at least one organ. Particularly neutrophil inhibitors, and modulators of coagulation or endothelial barrier reduced pulmonary edema (SMD -2.77 [-3.93, -1.61]; -1.29 [-2.12, -0.46], -2.33 [-4.69, 0.03], respectively) compared to no treatment. Reducing renal (SMD -0.91 [CI -1.65 to -0.18]), intestinal (SMD -1.98 [CI -3.92 to -0.04]) or myocardial (SMD -1.95 [CI -3.91 to -0.01]) edema following CPB required specific modulators of endothelial barrier. CONCLUSION: Overall, neutrophil inhibitors and direct modulators of endothelial barrier (PAR1, Tie2 signaling) most effectively reduced edema following CPB, in particular pulmonary edema. Future research should focus on a combination of these strategies to reduce edema and assess the effect on organ function and outcome following CPB.

Ubiquitin-based modifications in endothelial cell-cell contact and inflammation.

Endothelial cell-cell contacts are essential for vascular integrity and physiology, protecting tissues and organs from edema and uncontrolled invasion of inflammatory cells. The vascular endothelial barrier is dynamic, but its integrity is preserved through a tight control at different levels. Inflammatory cytokines and G-protein-coupled receptor agonists, such as histamine, reduce endothelial integrity and increase vascular leakage. This is due to elevated myosin-based contractility, in conjunction with phosphorylation of proteins at cell-cell contacts. Conversely, reducing contractility stabilizes or even increases endothelial junctional integrity. Rho GTPases are key regulators of such cytoskeletal dynamics and endothelial cell-cell contacts. In addition to signaling-induced regulation, the expression of junctional proteins, such as occludin, claudins and vascular endothelial cadherin, also controls endothelial barrier function. There is increasing evidence that, in addition to protein phosphorylation, ubiquitylation (also known as ubiquitination) is an important and dynamic post-translational modification that regulates Rho GTPases, junctional proteins and, consequently, endothelial barrier function. In this Review, we discuss the emerging role of ubiquitylation and deubiquitylation events in endothelial integrity and inflammation. The picture that emerges is one of increasing complexity, which is both fascinating and promising given the clinical relevance of vascular integrity in the control of inflammation, and of tissue and organ damage.

CSN5 inhibition triggers inflammatory signaling and Rho/ROCK-dependent loss of endothelial integrity.

RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo. CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo. Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.