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

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

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

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

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

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.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.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).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

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 LOA