Role of Extracellular Vimentin in Cancer-Cell Functionality and Its Influence on Cell Monolayer Permeability Changes Induced by SARS-CoV-2 Receptor Binding Domain

The cytoskeletal protein vimentin is secreted under various physiological conditions. Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted into the extracellular space. While surface vimentin is involved in processes such as viral infections and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer cells, and its activation pathway has significant roles in general cellular functions. In this study, we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion, and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7 monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown that the receptor binding domain of SARS-CoV-2 spike protein can alter blood–brain barrier integrity. Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This defines an influence of extracellular vimentin in SARS-CoV-2 infections.

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin

Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

127 Plasma from Patients with Burn Injury Increases Endothelial Permeability In-Vitro

Introduction The contribution of endothelial injury to the pathogenesis of burn shock is not well characterized. Human umbilical endothelial cells (HUVECs) have been used to study endotheliopathy in myriad shock states. This work investigates the impact of burn patient plasma on the vascular endothelium and its barrier function. Methods HUVECs were seeded into the apical chambers of transwell plates and cultured over 5–7 days to a confluent monolayer which was confirmed by a transendothelial electrical resistance (TEER) of ≥30Ω. After IRB approval, plasma was collected from burn-injured patients 4 hours after admission. Demographic and injury characteristics were collected from the medical record. Plasma Syndecan-1 (SDC-1) was quantified by ELISA. HUVEC monolayers were exposed to 10% multi-donor pooled healthy human plasma (HHP) or burn patient plasma. Monolayers were subsequently incubated with FIT-C Dextran (40,000 kD). FIT-C diffusion through monolayers was measured in basal chamber supernatants. Monolayer permeability was measured with indices calculated by normalizing values to blank (transwell inserts) and HHP-treated monolayer FIT-C diffusion. HUVECs were also cultured on glass slides and exposed to HHP or burn patient plasma. Cells were fixed with 4% Paraformaldehyde and F-Actin was stained with Texas Red-Phalloidin. Intercellular gap area was calculated using imaging software. Differences between treatment conditions were analyzed with Welch’s t-test and one-way ANOVA, simple linear regression was used to characterize the relationship between plasma SDC-1 and permeability indices, significance was set at p < 0.05. Results Eight burn patient plasma samples were tested. Patients were mostly male (75%) with a mean age of 50±20 years and mean %TBSA burn of 37±34%. Five burn plasma samples significantly increased monolayer permeability. There were no significant differences between patient samples that increased permeability in age, TBSA, gender, or in-hospital mortality. Monolayer permeability indices increased between 7–15% (p< 0.05) among burn plasma treatment conditions (n=6) that increased permeability. There was a strong relationship between monolayer permeability index (%) and plasma SDC-1 (µg/mL) (p=0.03, R2=0.93). Morphological F-actin rearrangement was apparent on microscopy and intercellular gap area was increased in burn plasma treatment conditions (12% vs. 49%, p≤0.0007, n=6). Conclusions Plasma from burn patients induces endothelial damage that increases endothelial cell monolayer permeability. The endothelial biomarker SDC-1 is a reliable indicator of endothelial damage. F-actin rearrangement and an increase in intercellular gap area likely contributes to burn endotheliopathy.

SPAK-p38 MAPK signal pathway modulates claudin-18 and barrier function of alveolar epithelium after hyperoxic exposure

Background Hyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to barrier dysfunction. Previous study has showed that STE20/SPS1-related proline/alanine-rich kinase (SPAK) interferes with the intestinal barrier function in mice. The aim of the present study is to explore the association between SPAK and barrier function in the alveolar epithelium after hyperoxic exposure. Methods Hyperoxic acute lung injury (HALI) was induced by exposing mice to > 99% oxygen for 64 h. The mice were randomly allotted into four groups comprising two control groups and two hyperoxic groups with and without SPAK knockout. Mouse alveolar MLE-12 cells were cultured in control and hyperoxic conditions with or without SPAK knockdown. Transepithelial electric resistance and transwell monolayer permeability were measured for each group. In-cell western assay was used to screen the possible mechanism of p-SPAK being induced by hyperoxia. Results Compared with the control group, SPAK knockout mice had a lower protein level in the bronchoalveolar lavage fluid in HALI, which was correlated with a lower extent of TJ disruption according to transmission electron microscopy. Hyperoxia down-regulated claudin-18 in the alveolar epithelium, which was alleviated in SPAK knockout mice. In MLE-12 cells, hyperoxia up-regulated phosphorylated-SPAK by reactive oxygen species (ROS), which was inhibited by indomethacin. Compared with the control group, SPAK knockdown MLE-12 cells had higher transepithelial electrical resistance and lower transwell monolayer permeability after hyperoxic exposure. The expression of claudin-18 was suppressed by hyperoxia, and down-regulation of SPAK restored the expression of claudin-18. The process of SPAK suppressing the expression of claudin-18 and impairing the barrier function was mediated by p38 mitogen-activated protein kinase (MAPK). Conclusions Hyperoxia up-regulates the SPAK-p38 MAPK signal pathway by ROS, which disrupts the TJ of the alveolar epithelium by suppressing the expression of claudin-18. The down-regulation of SPAK attenuates this process and protects the alveolar epithelium against the barrier dysfunction induced by hyperoxia.