Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major health emergency since its emergence in late 2019. Endothelial dysfunction is a hallmark of COVID-19, leading to severe illness, i.e. coagulopathy, multi-organ failure. FX06, a fibrin-derived peptide naturally occurring in the human body, formerly known as Bβ15-42, is a promising therapeutic candidate for endothelial complications like capillary leakage in COVID-19 and other forms of acute respiratory disorders. The aim of this project is to investigate whether FX06 can attenuate COVID-19 cytokine-triggered inflammatory processes in vitro.
To mimic the inflammatory status of COVID-19, a human pulmonary microvascular endothelial cell line (ECs) - HULEC-5a, was treated with a cytokine cocktail comprised of ten different cytokines or chemokines at concentrations found in serum profiles of COVID-19 patients with severe illness, further referred to as the severe cytokine cocktail. ECs were treated with the severe cytokine cocktail for 24 h, in the absence or presence of FX06 for 2 h.
The severe cytokine cocktail enhanced peripheral blood mononuclear cell (PBMC)-endothelial adhesion and monolayer transmigration. This deleterious effect was significantly reduced by FX06. FX06 was also shown to mitigate the cytotoxic activity of allogeneic CD8+ T cells, which increased upon cytokine treatment. FX06 restored continuous vascular endothelial (VE)-cadherin/CD144 distribution on the EC surface and reversed morphological changes mediated by the severe cytokine cocktail, such as the elongation of F-actin stress fibers. FX06 reduced capillary-like structure formation of the severe cytokine cocktail treated-ECs, indicating FX06 down-regulated the pro-inflammatory angiogenic activity caused by the severe cytokine cocktail. Additionally, FX06 might assist in maintaining the normal barrier function of ECs by altering the surface expression of Syndecan-1 (SDC1/CD138). Proteomics and phosphoproteomics analyses demonstrated that FX06 in the presence of the severe cytokine cocktail inactivated RhoGTPase, which was confirmed by western blotting that FX06 attenuated RhoA, a member of RhoGTPase, enhanced by the severe cytokine cocktail and down-regulated the expression of the phosphorylated downstream protein, ROCK1.
Overall, FX06 shows promising potential in normalizing ECs and reducing vascular leakage to protect the endothelium against the proinflammatory effect of COVID-19-triggered cytokines.
Copyright © 2025 Wang, Lebedev, Li, Rao, Wu, Doyle, Wynne, Blanco, Mysior, Simpson, Scholz, Wülfroth, Zacharowski, Kolch, Zhernovkov and Eissner.

Capillary leakage is a hallmark of severe dengue, yet its precise mechanisms remain elusive. Emerging evidence highlights the dengue virus's targeting of mechanically active endothelial cells as a key contributor to dengue shock syndrome. The viral nonstructural protein 1 (NS1) has been identified as a central player, disrupting endothelial integrity and inducing vascular hyperpermeability independently of pro-inflammatory cytokines. This study provides a direct assessment of NS1-induced endothelial pathology by combining single-cell force spectroscopy and a microvessel-on-a-chip platform. We demonstrate that NS1 significantly alters endothelial cell mechanics, reducing cell stiffness and compromising junctional integrity, thereby directly linking these mechanical alterations to vascular dysfunction. These findings establish a framework for understanding the mechano-pathology of dengue and offer a platform for developing targeted therapeutic strategies to mitigate severe disease outcomes.

Endothelial cell (EC) dysfunction in response to circulating plasma factors is a known causal factor in many systemic diseases. However, no appropriate assay is available to investigate this causality ex vivo. In liver cirrhosis, systemic inflammation is identified as central mechanism in progression from compensated to decompensated cirrhosis (DC), but the role of ECs therein is unknown. We aimed to develop a novel ex vivo assay for assessing EC responses to patient-derived plasma (PDP) and assess the potential of this assay in a cohort of liver cirrhosis patients.
Image-based morphological profiling was utilized to assess the impact of PDP on cultured ECs. Endothelial cell (EC) monolayers were exposed to 25% stabilized PDP (20 compensated cirrhoses, 20 DCs, and 20 healthy controls (HCs). Single-cell morphological profiles were extracted by automated image-analysis following staining of multiple cellular components and high-content imaging. Patient profiles were created by dimension reduction and cell-to-patient data aggregation, followed by multivariate-analysis to stratify patients and identify discriminating features.
Patient-derived plasma (PDP) exposure induced profound changes in EC morphology, displaying clear differences between controls and DC patients. Compensated cirrhosis patients showed overlap with healthy controls and DC patients. Supervised analysis showed Child-Pugh (CP) class could be predicted from EC morphology. Most importantly, CP-C patients displayed distinct EC phenotypes, in which mitochondrial changes were most discriminative.
Morphological profiling presents a viable tool to assess the endothelium ex vivo. We demonstrated that the EC phenotype corresponds with disease severity in liver cirrhosis. Moreover, our results suggest the presence of mitochondrial dysfunction in ECs of CP-C patient.
© 2024 The Authors.

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.
© 2024. The Author(s).

Autophagy is a cellular process that degrades damaged cytoplasmic components and regulates cell death. The homeostasis of endothelial cells (ECs) is crucial for the preservation of glomerular structure and function in aging. Here, we investigated the precise mechanisms of endothelial autophagy in renal aging. The genetic deletion of Atg7 in the ECs of Atg7flox/flox;Tie2-Cre mice accelerated aging-related glomerulopathy and tubulointerstitial fibrosis. The EC-specific Atg7 deletion in aging mice induced the detachment of EC with the disruption of glomerular basement membrane (GBM) assembly and increased podocyte loss resulting in microalbuminuria. A Transwell co-culture system of ECs and kidney organoids showed that the iron and oxidative stress induce the disruption of the endothelial barrier and increase vascular permeability, which was accelerated by the inhibition of autophagy. This resulted in the leakage of iron through the endothelial barrier into kidney organoids and increased oxidative stress, which led to ferroptotic cell death. The ferritin accumulation was increased in the kidneys of the EC-specific Atg7-deficient aging mice and upregulated the NLRP3 inflammasome signaling pathway. The pharmacologic inhibition of ferroptosis with liproxstatin-1 recovered the disrupted endothelial barrier and reversed the decreased expression of GPX4, as well as NLRP3 and IL-1β, in endothelial autophagy-deficient aged mice, which attenuated aging-related renal injury including the apoptosis of renal cells, abnormal structures of GBM, and tubulointerstitial fibrosis. Our data showed that endothelial autophagy is essential for the maintenance of the endothelial barrier during renal aging and the impairment of endothelial autophagy accelerates renal senescence by ferroptosis and NLRP3 inflammasome signaling pathways. These processes may be attractive therapeutic targets to reduce cellular injury from renal aging.