Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

ABO immune complexes (ABO-IC) formed by ABO-incompatible antigen-antibody interaction are associated with hemolysis and platelet destruction in patients transfused with ABO-nonidentical blood products. However, the effects of ABO-IC on endothelial cells (EC) are unclear. ABO-IC were formed in vitro from normal donor-derived plasma and serum. Human pulmonary artery EC (HPAEC) were cultured and treated with media, ABO-identical and –non-identical plasma, and ABO-IC. EC barrier integrity was evaluated using transendothelial electrical resistance (TEER), scanning electron microscopy (SEM), vascular endothelial (VE)-cadherin and phalloidin staining, and Rho-associated Kinase (ROCK) inhibitor treatment. TEER revealed significant/irreversible barrier disruption within 1–2 h of exposure to ABO non-identical plasma and ABO-IC; this occurred independently of EC ABO type. Treatment with ABO-IC resulted in decreased VE-cadherin staining and increased phalloidin staining in a time-dependent manner, suggesting that the resultant increased EC barrier permeability is secondary to actin stress fiber formation and loss of cell surface VE-cadherin. Inhibition of ROCK was effective in protecting against IC-induced barrier disruption even two hours after ABO-IC exposure. ABO-IC causes increased EC barrier permeability by decreasing cell surface VE-cadherin and promoting stress fiber formation, which is preventable by inhibiting ROCK activation to protect against EC contraction and gap formation.

MO015ANALYZING THE EFFECT OF MICROPLASTIC PARTICLES ON HUMAN PODOCYTES

Background and Aims Despite increasing use of plastic based products and their potential health risks on the humans, very little is known about their possible accumulation in the food chain and their further long-term effect on the human health. Recently, there are increasing reports related to the potential risk of polystyrene microplastics to the human respiratory system and human intestinal epithelia cell line. In this study, we assayed the primary effect of microplastic particles on the human kidney cells. To that aim, we used human podocytes cells and four different types of plastic particles including; polyvinyl chloride (PVC), polypropylene (PP), polyamide (PA) and tyre wear particles to evaluate the effects of microplastics on the viability and morphology of human podocytes in vitro. Method In this study, we applied different biological methods such as, cell viability test and phalloidin staining, to assay the toxicity of particles and their further effects on the actin cytoskeleton organization in human podocytes, respectively. Furthermore, Raman imaging is used to track particle attachment on the cells and to evaluate the possible changes in the cell compartment following the particle treatment. The particle uptake by the cells and changes in cellular biological features were visualized with the use of scanning electron microscopy (SEM). Results As a primary result, the cytotoxicity response of particle treatment was found to be dependent on the polymer type. As an example higher concentration of PP particle as compared to PVC, PA, and tyre wear caused a similar rate of cell mortality. Furthermore, the degree of particle attachment on the cells depended on their adhesion properties, which was higher in PA, PVC and tyre wear in comparison to PP particles. These particles remained attached to the cell surface even after two-three times of washing with PBS. Based on the phalloidin staining results, particle treatment induced cytoskeleton reorganization in podocytes in vitro. With the use of Raman imaging particle attachment was confirmed based on the fingerprint spectra related to each particle. Conclusion This study suggests that exposure duration and particle concentrations are two of the key factors to evaluate the toxicological effect of particles on podocytes as a highly-specialized epithelial cells in the kidney. It is supposed that two mechanisms can be related to the harmful effects of plastic particles on podocytes. First, particle attachment on the cell surface leading to limitation of nutrient uptake by the cells. Second, uptake of smaller size particles into the cells through phagocytosis. More studies are necessary to determine the direct effect of microplastics on human kidney cells.

A Crucial Role for Caspase 1 in Thrombo-Inflammatory Microparticle Release

Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell injury signals trigger the macrophages P2X7 receptor and thereby induce the release both proinflammatory IL-1β and prothrombotic MP. Prothrombotic MP carry tissue factor (TF) and high content of phosphatidylserine (PS), and can induce thrombosis causing major clinical complications in patients. We previously identified the P2X7 receptor as a crucial component of thrombosis in mice, but the mechanistic details of macrophage MP release in this thrombo-inflammatory pathway remain incompletely understood. The the generation of these MP requires thiol-disulfide exchange-dependent activation of the inflammasome and is accompanied by the release of various soluble proteins into the extracellular space. We hypothesized that the released proteome presents regulators and structural components of the MP generation pathway and employed proteomics to unveil their identity. Amongst the most abundant proteins were γ-actin and actin cytoskeleton associated proteins, including PS-binding proteins annexin 1 and annexin 5. Cytoskeletal remodeling processes leading to formation of filopodia downstream of P2X7 receptor activation were crucial for the biogenesis of thrombo-inflammatory MP, since pharmacological inhibitors of inflammasome activation, cytoskeletal remodeling and the thioredoxin system attenuated both, filopodia formation and the release of highly procoagulant MP. Confocal microscopy demonstrated raft dependent translocation of TF onto filopodia that was prevented by the same inhibitory strategies. Surprisingly, phalloidin-staining of non-permeabilized macrophages revealed that F-actin is exposed to the cell surface decorating the base of filopodia. Positive phalloidin-staining of thrombo-inflammatory MP further demonstrated that F-actin remained associated with the MP surface. Strikingly, blocking surface actin by incubation with high concentration of phalloidin prevented the release of PS-rich MP, demonstrating that exposure of F-actin during filopodia formation is functionally linked to the biogenesis of thrombo-inflammatory MP. As the underlying common mechanism for thiol-disulfide exchange-dependent cell surface actin exposure and MP release, we showed that blockade of the cysteine protease caspase 1, which mediates processing and release of IL-1β downstream of inflammasome activation is also required for the release of thrombo-inflammatory MP. Although caspase 1-mediated activation of caplain was required for the release of filamin A localized TF to the cell cortex, calpain was not involved in the release of thrombo-inflammatory MP release. The N-terminus of γ-actin harbors a recognition and cleavage motif for caspase 1. Residual γ-actin released from caspase 1-blocked macrophages showed decreased electrophoretic mobility, indicating prior cleavage of actin that becomes exposed on the cell surface. We show here that the proteome released during thrombo-inflammatory activation of macrophages includes critical players in the biogenesis of MP and may provide diagnostic fingerprints in complex biological samples. Our data demonstrate an entirely unexpected role for caspase 1 and surface exposure of polymerized actin in the severing of prothrombotic MP from filopodia and thus position this protease upstream of both IL-1β processing and thrombo-inflammatory MP in cardiovascular diseases. Disclosures No relevant conflicts of interest to declare.

Involvement of anion channel(s) in the modulation of the transient outward K+ channel in rat ventricular myocytes

The cardiac Ca2+-independent transient outward K+ current ( Ito), a major repolarizing ionic current, is markedly affected by Cl− substitution and anion channel blockers. We reexplored the mechanism of the action of anions on Ito by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs+ substitution for intracellular K+. Replacement of most of the extracellular Cl− with less permeant anions, aspartate (Asp−) and glutamate (Glu−), markedly suppressed the current. Removal of external Na+ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on Ito. In contrast, the permeant Cl− substitute Br− did not markedly affect the current, whereas F− substitution for Cl− induced a slight inhibition. The Ito elicited during Br− substitution for Cl− was also sensitive to blockade by 4-AP. The ability of Cl− substitutes to induce rightward shifts of the steady-state inactivation curve of Ito was in the following sequence: NO3− > Cl− ≈ Br− > gluconate− > Glu− > Asp−. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of Ito similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp− substitution for Cl− was without significant effect on the intensity. These results suggest that the Ito channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.