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.

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.

Stabilization of cell-cell junctions by active vitamin D ameliorates uraemia-induced loss of human endothelial barrier function.

Background: Uraemia induces endothelial cell (EC) injury and impaired repair capacity, for which the underlying mechanism remains unclear. Active vitamin D (VD) may promote endothelial repair, however, the mechanism that mediates the effects of VD in chronic kidney disease are poorly understood. Thus, we investigated uraemia-induced endothelial damage and the protection against such damage by active VD. Methods: We applied electric cell-substrate impedance sensing (ECIS) to study real-time responses of human ECs exposed to pooled uraemic and non-uraemic plasma with or without the addition of active VD. The effects of indoxyl sulphate and p-cresol were tested in non-uraemic plasma. Structural changes for vascular endothelial (VE)-cadherin and F-actin were assessed by immunostaining and quantified. Results: The exposure of ECs to uraemic media significantly decreased endothelial barrier function after 24 h. Cell migration after electrical wounding and recovery of the barrier after thrombin-induced loss of integrity were significantly impaired in uraemic-medium stimulated cells and cells exposed to indoxyl sulphate and p-cresol. This effect on ECIS was dependent on loss of cell-cell interaction. Mechanistically, we found that EC, exposed to uraemic media, displayed disrupted VE-cadherin interactions and F-actin reorganization. VD supplementation rescued both endothelial barrier function and cell-cell interactions in ECs exposed to uraemic media. These events were associated with an increment of VE-cadherin at intercellular junctions. Conclusions: Our data demonstrate a potentially clinically relevant mechanism for uraemia-induced endothelial damage. Furthermore, active VD rescued the uraemic medium-induced loss of cell-cell adhesion, revealing a novel role of active VD in preservation of endothelial integrity during uraemia.

Rac1 as a Potential Pharmacodynamic Biomarker for Thiopurine Therapy in Inflammatory Bowel Disease.

BACKGROUND: Azathioprine and mercaptopurine (MP) are effective in treating patients with inflammatory bowel disease (IBD). Immunosuppressive effects of thiopurines involve T-cell apoptosis after inhibition of GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). This study aimed to assess whether expression and activity of Rac1 or phosphorylated ezrin-radixin-moesin (pERM) in patients with IBD could provide a useful biomarker for the pharmacodynamic thiopurine effect and might be related to clinical effectiveness. METHODS: This was a 2-stage study: stage 1 concerned a cross-sectional cohort of patients with IBD clinically in remission and treated with (n = 10) or without stable weight-based thiopurine therapy (n = 11) and healthy controls (n = 6); stage 2 concerned a prospective study regarding IBD patients with clinically active disease who initiated MP therapy (n = 11) compared with healthy controls (n = 11). Expression and activity of Rac1 and ERM and pERM were determined. RESULTS: The median Rac1 expression was statistically significantly reduced by thiopurine maintenance therapy {0.54 [interquartile range (IQR) 0.47-0.88] versus 0.80 arbitrary units [IQR 0.64-1.46]} compared with patients without immunosuppressive therapy (P = 0.042), but not Rac1 activity and pERM. In responders to MP therapy (n = 6), both median active Rac1 [93 (IQR 81-151) to 76 ng Rac1/mg protein (IQR 62-98)] and Rac1 expression [16.2 (8.8-29.4) to 1.5 arbitrary units (0.9-5.3)] decreased (P = 0.028). In nonresponders (n = 3), Rac1 expression and activity increased. CONCLUSIONS: IBD patients treated with thiopurines had a lower expression of Rac1 compared with those not treated with thiopurine. Effective MP therapy led to decreasing concentrations of Rac1-GTP and Rac1 expression. Therefore, Rac1-GTP and expression of Rac1, but not phosphorylation of ERM, form potentially pharmacodynamic markers of therapeutic thiopurine effectiveness in patients with IBD.

Effective treatment of edema and endothelial barrier dysfunction with imatinib.

BACKGROUND: Tissue edema and endothelial barrier dysfunction as observed in sepsis and acute lung injury carry high morbidity and mortality, but currently lack specific therapy. In a recent case report, we described fast resolution of pulmonary edema on treatment with the tyrosine kinase inhibitor imatinib through an unknown mechanism. Here, we explored the effect of imatinib on endothelial barrier dysfunction and edema formation. METHODS AND RESULTS: We evaluated the effect of imatinib on endothelial barrier function in vitro and in vivo. In human macro- and microvascular endothelial monolayers, imatinib attenuated endothelial barrier dysfunction induced by thrombin and histamine. Small interfering RNA knock-downs of the imatinib-sensitive kinases revealed that imatinib attenuates endothelial barrier dysfunction via inhibition of Abl-related gene kinase (Arg/Abl2), a previously unknown mediator of endothelial barrier dysfunction. Indeed, Arg was activated by endothelial stimulation with thrombin, histamine, and vascular endothelial growth factor. Imatinib limited Arg-mediated endothelial barrier dysfunction by enhancing Rac1 activity and enforcing adhesion of endothelial cells to the extracellular matrix. Using mouse models of vascular leakage as proof-of-concept, we found that pretreatment with imatinib protected against vascular endothelial growth factor-induced vascular leakage in the skin, and effectively prevented edema formation in the lungs. In a murine model of sepsis, imatinib treatment (6 hours and 18 hours after induction of sepsis) attenuated vascular leakage in the kidneys and the lungs (24 hours after induction of sepsis). CONCLUSIONS: Thus, imatinib prevents endothelial barrier dysfunction and edema formation via inhibition of Arg. These findings identify imatinib as a promising approach to permeability edema and indicate Arg as novel target for edema treatment.

The STOX1 genotype associated with pre-eclampsia leads to a reduction of trophoblast invasion by alpha-T-catenin upregulation.

By using complementary in vitro and ex vivo approaches, we show that the risk allele (Y153H) of the pre-eclampsia susceptibility gene STOX1 negatively regulates trophoblast invasion by upregulation of the cell-cell adhesion protein alpha-T-catenin (CTNNA3). This is effectuated at the crucial epithelial-mesenchymal transition of proliferative into invasive extravillous trophoblast. This STOX1-CTNNA3 interaction is direct and includes Akt-mediated phosphorylated control of nucleo-cytoplasmic shuttling and ubiquitin-mediated degradation as shared with the FOX multigene family. This, to our knowledge, is the first time a genotype associated with pre-eclampsia has been shown to directly limit first trimester extravillous trophoblast invasion, the earliest hallmark of pre-eclampsia.

Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces.

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

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.

FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB.

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference-based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

RhoA, RhoB and RhoC differentially regulate endothelial barrier function.

RhoGTPases are known regulators of intracellular actin dynamics that are important for maintaining endothelial barrier function. RhoA is most extensively studied as a key regulator of endothelial barrier function, however the function of the 2 highly homologous family-members (> 88%) RhoB and RhoC in endothelial barrier function is still poorly understood. This study aimed to determine whether RhoA, RhoB and RhoC have overlapping or distinct roles in barrier function and permeability in resting and activated endothelium. By using primary endothelial cells in combination with siRNA transfection to establish individual, double or triple knockdown of the RhoA/B/C RhoGTPases, we found that RhoB, but not RhoA or RhoC, is in resting endothelium a negative regulator of permeability. Loss of RhoB accounted for an accumulation of VE-cadherin at cell-cell contacts. Thrombin-induced loss of endothelial integrity is mediated primarily by RhoA and RhoB. Combined loss of RhoA/B showed decreased phosphorylation of Myosin Light Chain and increased expression of VE-cadherin at cell-cell contacts after thrombin stimulation. RhoC contributes to the Rac1-dependent restoration of endothelial barrier function. In summary, this study shows that these highly homologous RhoGTPases differentially control the dynamics of endothelial barrier function.

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.

Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells.

Free fatty acids may create a state of continuous and progressive damaging to the vascular wall manifested by endothelial dysfunction. In this study we determine the mechanisms by which fatty acids palmitate (C16:0) and oleate (C18:1) affect intracellular long chain acyl-CoA (LCAC) content, energy metabolism, cell survival and proliferation and activation of NF-kappaB in cultured endothelial cells. A 48-h exposure of human umbilical vein endothelial cells (HUVEC) to 0.5 mM palmitate or 0.5 mM oleate increased total long chain acyl-CoA (LCAC) content 1.7 and 2 fold, respectively and decreased ATP(total)/ADP(total) ratio by 26+/-5% (mean+/-SEM) and 15+/-2%, respectively, which was prevented by the acyl-CoA synthetase inhibitor triacsin C. Furthermore, palmitate inhibited cell proliferation by 34+/-5%, while oleate stimulated it by 12+/-2%. alpha-Tocopherol fully and triacsin C partially abolished the effect of palmitate on cell proliferation. Palmitate and oleate increased caspase-3 activity 3.2 and 1.4 fold, respectively. Palmitate-induced caspase-3 activation was prevented by triacsin C and slightly reduced by alpha-tocopherol and by the de novo ceramide synthesis inhibitor fumonisin B(1). Both fatty acids induced antioxidant-sensitive nuclear translocation of NF-kappaB after 72 h, but not after 48 h. In conclusion, we showed that fatty acids influence different aspects of HUVEC function resulting in amongst other activation of apoptotic and inflammatory pathways. Our results indicate that the effects depend on the fatty acid type and may be related to accumulation of LCAC.

Methylglyoxal and methylglyoxal-arginine adducts do not directly inhibit endothelial nitric oxide synthase.

Increased formation of the reactive dicarbonyl compound methylglyoxal (MGO) and MGO-derived advanced glycation end products (AGEs) seems to be implicated in endothelial dysfunction and the development of diabetic vascular complications. MGO reacts with arginine residues in proteins to generate the major glycated adducts 5-hydro-5-methylimidazolone (MG-H1) and argpyrimidine (AP). We investigated whether the free forms of these adducts contribute to vascular cell dysfunction by inhibition of endothelial nitric oxide synthase (eNOS). MG-H1 and AP were synthesized and purified by reversed-phase chromatography, and the conversion of labeled L-arginine to L-citrulline was used to monitor eNOS activity. In contrast to the endogenous eNOS inhibitor asymmetric dimethylarginine (half maximal inhibitory concentration, approximately 5 micromol/L), pathophysiological concentrations of MGO and MG-H1 and AP did not inhibit eNOS activity. Although MGO-derived AGEs are implicated in the development of diabetic vascular complications, this study indicates that this is not mediated via direct inhibition of eNOS activity.

Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells.

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.

THSD1 preserves vascular integrity and protects against intraplaque haemorrhaging in ApoE-/- mice.

AIMS: Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS: Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION: THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically.

BACKGROUND: Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to 'classical' biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied. METHODS: In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces. RESULTS: Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011). CONCLUSION: In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion.

ROCK2 primes the endothelium for vascular hyperpermeability responses by raising baseline junctional tension.

Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response.

Opposing effects of the angiopoietins on the thrombin-induced permeability of human pulmonary microvascular endothelial cells.

BACKGROUND: Angiopoietin-2 (Ang-2) is associated with lung injury in ALI/ARDS. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1. METHODOLOGY/PRINCIPAL FINDINGS: Permeability was assessed by measuring macromolecule passage and transendothelial electrical resistance (TEER). Angiopoietins did not affect basal permeability. Nevertheless, they had opposing effects on the thrombin-induced permeability, in particular in the initial phase. Ang-2 enhanced the initial permeability increase (passage, P = 0.010; TEER, P = 0.021) in parallel with impairment of VE-cadherin organization without affecting VE-cadherin Tyr685 phosphorylation or increasing RhoA activity. Ang-2 also increased intercellular gap formation. Ang-1 preincubation increased Rac1 activity, enforced the VE-cadherin organization, reduced the initial thrombin-induced permeability (TEER, P = 0.027), while Rac1 activity simultaneously normalized, and reduced RhoA activity at 15 min thrombin exposure (P = 0.039), but not at earlier time points. The simultaneous presence of Ang-2 largely prevented the effect of Ang-1 on TEER and macromolecule passage. CONCLUSIONS/SIGNIFICANCE: Ang-1 attenuated thrombin-induced permeability, which involved initial Rac1 activation-enforced cell-cell junctions, and later RhoA inhibition. In addition to antagonizing Ang-1, Ang-2 had also a direct effect itself. Ang-2 sensitized the initial thrombin-induced permeability accompanied by destabilization of VE-cadherin junctions and increased gap formation, in the absence of increased RhoA activity.

The pre-eclampsia gene STOX1 controls a conserved pathway in placenta and brain upregulated in late-onset Alzheimer's disease.

Pre-eclampsia and late-onset Alzheimer's disease (LOAD) share no clinical features. In contrast to these clinical dissimilarities, striking parallels exist between the (epi)genetic features associated with pre-eclampsia and LOAD for the genes located on 10q22. The parallels in identity between the 10q22 genes involved and active in the organs (placenta, brain) primarily affected in the respective diseases led us to explore, if the pre-eclampsia susceptibility gene STOX1 is functionally involved in LOAD. We demonstrate that isoform A of STOX1 is abundantly expressed in the brain, correlates with severity of disease, and selectively transactivates LRRTM3 in neural cells with increased amyloid-beta protein precursor processing. Similar in vitro results were seen in trophoblast. Our data indicate that STOX1 controls a conserved pathway shared between placenta and brain with overexpression in LOAD.