Biology subjects

Zapotoczny, B.

Publications and source records attributed to Zapotoczny, B..

6 recordsLinked to original sources

bioRxiv2026

Differential effects of fenofibrate and fenofibric acid on the regulation of liver endothelial permeability

Background and AimsFenofibrate is widely prescribed for hyperlipidaemia and has been associated with rare but severe cases of drug-induced liver injury (DILI), yet its effects on liver sinusoidal endothelial cells (LSECs) remain to be investigated. LSECs maintain a highly permeable specialized sinusoidal barrier characterized by transcellular pores (fenestrations), regulating the bidirectional transfer of circulating compounds to and from the hepatocytes. As drug-induced alterations in fenestration architecture could influence xenobiotic access to hepatocytes, these changes may modulate pathways associated with DILI. Understanding the effects of fenofibrate on LSEC ultrastructure may therefore provide insights into previously underexplored endothelial contributions to hepatic drug responses. MethodsBoth fenofibrate and its active metabolite, fenofibric acid, were evaluated for their effects on LSEC ultrastructure, mechanical properties, and functional markers. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) and were used to quantify fenestration architecture. AFM was additionally used to measure cellular mechanical properties, which were interpreted in the context of fluorescence-based quantification of cytoskeletal organization. Gene expression, viability, and cytotoxicity were assessed using PCR-based and biochemical assays. ResultsFenofibrate reduced fenestration number and porosity at both tested concentration (10, and 25 {micro}M). It also decreased the apparent Youngs modulus of LSECs, accompanied by changes in tubulin and actin architecture, without detectable cytotoxicity. In contrast, treatment with fenofibric acid did not result in significant structural or mechanical effects on LSECs, even at higher concentrations. ConclusionsTogether, these data identify LSECs as a drug-responsive hepatic cell type for fenofibrate, suggesting that LSECs could represent an underrecognized contributor to the complex, multifactorial processes underlying DILI. This work provides a framework for evaluating endothelial contributions to fenofibrate-associated liver effects in more complex models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/718907v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f9ec6forg.highwire.dtl.DTLVardef@11174a1org.highwire.dtl.DTLVardef@1000e2borg.highwire.dtl.DTLVardef@a23b00_HPS_FORMAT_FIGEXP M_FIG Fenofibrate reduces LSEC fenestrations and metabolic activity at higher concentrations, while its metabolite, fenofibric acid, does not affect LSEC, regardless of its concentration. C_FIG

cell biology↗

bioRxiv2025

Offsetting ROS-mediated arrest of endothelial fenestration dynamics permits long-term optical super-resolution- and AFM-imaging

Advances in cell biology demand methods that resolve the structure and dynamics of subcellular organelles in living cells. Live-cell super-resolution fluorescence microscopy meets this need but is constrained by phototoxicity, which disturbs cellular function and biases interpretation. Liver sinusoidal endothelial cells (LSECs), with their physiologically critical and highly dynamic fenestrations, represent a particularly challenging model system. We show that photoactivation-generated reactive oxygen species (ROS) are the primary cause of fenestration arrest during fluorescence imaging. Using three-dimensional structured illumination microscopy (3D SR-SIM), we systematically evaluated fluorophores and ROS scavengers to optimize imaging conditions. A combination of BioTracker staining and CO2-independent medium supplemented with N-acetylcysteine (NAC) preserved fenestration dynamics without altering fenestration number or size. Complementary atomic force microscopy (AFM) confirmed ROS-dependent impairment of fenestration dynamics and revealed nanomechanical changes upon illumination. These findings establish the mechanism underlying imaging-induced artefacts in LSECs and provide a broadly applicable strategy to extend live-cell super-resolution microscopy.

cell biology↗

bioRxiv2024

Hydrogen peroxide damage to scavenging function and ultrastructure of liver sinusoidal endothelial cells is prevented by n-acetyl-cysteine but not GSH

Reactive oxygen species (ROS) are prevalent in the liver during intoxication, infection, inflammation, and ageing. Changes in liver sinusoidal endothelial cells (LSECs) are associated with various liver diseases. We investigated how oxidative stress induced by H2O2 affects isolated rat LSECs at different concentrations (0.5-1000{micro}M) and exposure times (10-120 min). Our findings show that H2O2 exposure affects several LSEC functions in a dose- and time-dependent manner: (1) cell viability, reducing potential, and scavenging function decreased as H2O2 concentration and exposure time increased; (2) intracellular ROS levels rose with higher H2O2 concentrations; (3) fenestrations exhibited a dynamic response, initially closing but partially reopening at H2O2 concentrations above 100{micro}M after about 1 h; (4) scavenging function was affected after just 10 min of exposure, with the impact being irreversible and primarily affecting degradation rather than receptor-mediated uptake; (5) the tubulin network was disrupted in high H2O2 concentration while the actin cytoskeleton appears to remain largely intact. Finally, we found that reducing agents and thiol donors such as N-Acetyl Cysteine (NAC) and Glutathione (GSH) could protect cells from ROS-induced damage but could not reverse existing damage. Pretreatment with NAC, but not GSH, reduced the negative effects of ROS exposure suggesting that LSEC does not store an excess amount of GSH but rather can readily produce it in the occurrence of oxidative stress conditions. The observed thresholds in dose and time-dependent changes as well as the treatments with NAC/GSH confirm the existence of ROS depleting system in LSEC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/609175v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1f25779org.highwire.dtl.DTLVardef@14cda58org.highwire.dtl.DTLVardef@92dea4org.highwire.dtl.DTLVardef@56e97c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIROS by H2O2 irreversibly depletes LSEC endocytic/scavenging function in vitro C_LIO_LIH2O2 exposure causes dynamic, dose-dependent defenestration of LSEC within 0.5 h C_LIO_LIPartial refenestration can occur after about 1h of exposure to H2O2 C_LIO_LINAC/GSH mitigate H2O2-induced ROS effects in LSEC C_LIO_LILSEC do not store excess GSH but produce GSH when exposed to oxidative stress C_LI

cell biology↗

bioRxiv2024

Tubulin-Targeted Therapy in Melanoma Increases Cell Invasive Potential by Activation of Actomyosin Cytoskeleton - an in vitro study

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells, in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines - WM35 and WM266-4 - representing the less and more invasive melanomas. We investigated mechanisms of cellular processes regulating the activation of actomyosin being the effect of colchicine treatment. Additionally, we investigated biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632), and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy and novel method based on constriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased cancer cell invasiveness. We emphasized a cellular pathway of Rho-ROCK-dependent actomyosin contraction to be responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy. HighlightsO_LIColchicine strongly increased (trans)migration in the subpopulation of melanoma cells. C_LIO_LIColchicine disrupted tubulin but compensated for the activation of the actomyosin network. C_LIO_LIRho-ROCK-targeted or myosin-targeted therapies vanished the side-effect of colchicine. C_LIO_LIIncreased actin polymerisation correlated with the transit time in the constriction channel. C_LIO_LIElastic modulus variations reflected observed alterations in the actomyosin network. C_LI

biophysics↗

bioRxiv2023

Super-resolution histology of paraffin-embedded samples via photonic chip-based microscopy

Fluorescence-based super-resolution optical microscopy (SRM) techniques allow the visualization of biological structures beyond the diffraction limit of conventional microscopes. Despite its successful adoption in cell biology, the integration of SRM into the field of histology has been deferred due to several obstacles. These include limited imaging throughput, high cost, and the need for complex sample preparation. Additionally, the refractive index heterogeneity and high labeling density of commonly available formalin-fixed paraffin-embedded (FFPE) tissue samples pose major challenges to applying existing super-resolution microscopy methods. Here, we demonstrate that photonic chip-based microscopy alleviates several of these challenges and opens avenues for super-resolution imaging of FFPE tissue sections. By illuminating samples through a high refractive-index waveguide material, the photonic chip-based platform enables ultra-thin optical sectioning via evanescent field excitation, which reduces signal scattering and enhances both the signal-to-noise ratio and the contrast. Furthermore, the photonic chip provides decoupled illumination and collection light paths, allowing for total internal reflection fluorescence (TIRF) imaging over large and scalable fields of view. By exploiting the spatiotemporal signal emission via MUSICAL, a fluorescence fluctuation-based super-resolution microscopy (FF-SRM) algorithm, we demonstrate the versatility of this novel microscopy method in achieving superior contrast super-resolution images of diverse FFPE tissue sections derived from human colon, prostate, and placenta. The photonic chip is compatible with routine histological workflows and allows multimodal analysis such as correlative light-electron microscopy (CLEM), offering a promising tool for the adoption of super-resolution imaging of FFPE sections in both research and clinical settings.

pathology↗

bioRxiv2022

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD)

The biological processes underlying ischemic stroke, although complex, are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1h of OGD and, with longer OGD duration and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.

biophysics↗