Vimentin Filaments Research Articles

GRIP1 is Involved in the Interaction of Vimentin Filaments with Focal Adhesions in Endothelial Cells

GRIP1 is Involved in the Interaction of Vimentin Filaments with Focal Adhesions in Endothelial Cells

Abstract A070: Time-Dependent Oxidation and Aggregation of Vimentin upon Binding by β-lap Induced Immunogenic Cell Death

Abstract β-lapachone (β-lap) has been demonstrated as an NAD(P)H:quinone oxidoreductase1 (NQO1) bioactivatable compound. It initiated a futile redox cycle catalyzed by NQO1, resulting in reactive oxygen species (ROS) generation, with consumption of the reductive NAD(P)H. After a 4-h pulse of sufficiently lethal dose of β-lap, high levels of ROS diffused into nuclei to induce tumor-selective DNA damage and programmed necrosis in cancers overexpressing NQO1. Recently, our experiments showed that prolonged low dose β-lap administration exerted significant and NQO1 independent antitumor efficacy in mouse models with no overt signs of toxicity. Thus, we synthesized a photoactive β-lap probe to identify the molecular target of β-lap. Vimentin, a major constituent of the intermediate filament family of proteins, was identified as the direct binding target of β-lap. Vimentin was highly upregulated in multiple cancers including PDAC, and was a binding partner of β-lap with a dissociation constant at micromolar levels. Ligand binding assays in Panc1 cells employing β-lap as a specific competitor showed that vimentin was bound by β-lap-probe in a β-lap-competitive manner, and further confirmed that the in vivobinding target of β-lap was vimentin. Moreover, it was manifested that endogenous vimentin mRNA levels directly correlated with sensitivity to prolonged exposure of β-lap in a panel of pancreatic cancer cell lines, and knockout of vimentin in Panc1 cells markedly decreased lethality after prolonged exposure of β-lap. Mechanistically, we revealed that β-lap bound to vimentin and induced disulfide bonds that participated in disulfide cross-linking between a pair of vimentin dimers in a concentration and time dependent manner, forming vimentin oligomers of approximately 150 kDa. Prolonged exposure of β-lap promoted the disruption of filament architecture and vimentin degradation, ultimately resulting in cell death. In addition, the cell death by targeting vimentin exhibited a more inflammatory condition and enhanced DC activation. Together, our findings unveiled an unknown molecular target for β-lap and provided valuable insights into its mechanism of action. Targeting vimentin also presented a new strategy that inhibited the tumor growth and synergized with immunotherapy to disrupt immune evasion and reduce immune suppression against pancreatic cancers. Citation Format: Feng Qian, Lei Dou, Kaixin Wang, Yi Xia, Zhidong Chen, Yumeng Zu, Shuang Xi. Time-Dependent Oxidation and Aggregation of Vimentin upon Binding by β-lap Induced Immunogenic Cell Death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A070.

A novel pipeline employing deep multi-attention channels network for the autonomous detection of metastasizing cells through fluorescence microscopy

Metastasis driven by cancer cell migration is the leading cause of cancer-related deaths. It involves significant changes in the organization of the cytoskeleton, which includes the actin microfilaments and the vimentin intermediate filaments. Understanding how these filament change cells from normal to invasive offers insights that can be used to improve cancer diagnosis and therapy. We have developed a computational, transparent, large-scale and imaging-based pipeline, that can distinguish between normal human cells and their isogenically matched, oncogenically transformed, invasive and metastasizing counterparts, based on the spatial organization of actin and vimentin filaments in the cell cytoplasm. Due to the intricacy of these subcellular structures, manual annotation is not trivial to automate. We used established deep learning methods and our new multi-attention channel architecture. To ensure a high level of interpretability of the network, which is crucial for the application area, we developed an interpretable global explainable approach correlating the weighted geometric mean of the total cell images and their local GradCam scores. The methods offer detailed, objective and measurable understanding of how different components of the cytoskeleton contribute to metastasis, insights that can be used for future development of novel diagnostic tools, such as a nanometer level, vimentin filament-based biomarker for digital pathology, and for new treatments that significantly can increase patient survival.

The impairment of the assembly of vimentin filaments leads to suppression of the formation and maturation of focal contacts and an alteration of the type of cellular protrusions

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by the formation of lamellipodia and/or filopodia, and membrane blebs lay at the basis of amoeboid migration. Changing the conditions of migration can lead to an alteration of the character of cell movement, for example, inhibition of Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility type. The ability of cells to switch from one type of motility to another is called migration plasticity. The cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether the organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryonic fibroblasts REF52 with normal VIF organization, with VIF knockout (REF–/–) and with a mutation inhibiting the assembly of full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase of number of cells with blebs. In fibroblasts with short fragments of vimentin the number of cells forming blebs both spontaneously and in presence of CK-666 increased significantly. Disruption of the VIF organization did not lead to the significant changes in microtubules network or myosin light chain phosphorylation, but caused a significant reduction of the focal contact system. The most pronounced and statistically significant decrease in both the size and number of focal adhesions were observed in REF117. We believe that the regulation of membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that the organization of VIFs determines the type of cell protrusions, which in turn determines the character of cell movement. A novel role of VIF as a regulator of membrane blebbing, essential for the manifestation of migratory plasticity, is shown.

Vimentin regulates mitochondrial ROS production and inflammatory responses of neutrophils.

The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.

Oxidative stress elicits the remodeling of vimentin filaments into biomolecular condensates

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

Mechanobiology in Metabolic Dysfunction-Associated Steatotic Liver Disease and Obesity.

With the ongoing obesity epidemic, the prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is expected to rise and necessitates a greater understanding of how the disease proceeds from benign excess lipid in hepatocytes to liver fibrosis and eventually to liver cancer. MASLD is caused, at least in part, by hepatocytes' storage of free fatty acids (FAs) that dysfunctional adipocytes are no longer able to store, and therefore, MASLD is a disease that involves both the liver and adipose tissues. The disease progression is not only facilitated by biochemical signals, but also by mechanical cues such as the increase in stiffness often seen with fibrotic fatty livers. The change in stiffness and accumulation of excess lipid droplets impact the ability of a cell to mechanosense and mechanotranduce, which perpetuates the disease. A mechanosensitive protein that is largely unexplored and could serve as a potential therapeutic target is the intermediate filament vimentin. In this review, we briefly summarize the recent research on hepatocyte and adipocyte mechanobiology and provide a synopsis of studies on the varied, and sometimes contradictory, roles of vimentin. This review is intended to benefit and encourage future studies on hepatocyte and adipocyte mechanobiology in the context of MASLD and obesity.

Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity.

The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.

Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM.

Single-particle tracking demonstrates that individual filaments in bundles of vimentin intermediate filaments are transported in the cytoplasm by motor proteins along microtubules. Furthermore, using 3D FIB-SEM the authors showed that vimentin filament bundles are loosely packed and coaligned with microtubules. Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a ~50 μm3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

Silibinin attenuates TGF-β2-induced fibrogenic changes in human trabecular meshwork cells by targeting JAK2/STAT3 and PI3K/AKT signaling pathways

Transforming growth factor-β2 (TGF-β2) induced fibrogenic changes in human trabecular meshwork (HTM) cells have been implicated in trabecular meshwork (TM) damage and intraocular pressure (IOP) elevation in primary open-angle glaucoma (POAG) patients. Silibinin (SIL) exhibited anti-fibrotic properties in various organs and tissues. This study aimed to assess the effects of SIL on the TGF-β2-treated HTM cells and to elucidate the underlying mechanisms. Our study found that SIL effectively inhibited HTM cell proliferation, attenuated TGF-β2-induced cell migration, and mitigated TGF-β2-induced reorganization of both actin and vimentin filaments. Moreover, SIL suppressed the expressions of fibronectin (FN), collagen type I alpha 1 chain (COL1A1), and alpha-smooth muscle actin (α-SMA) in the TGF-β2-treated HTM cells. RNA sequencing indicated that SIL interfered with the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) signaling pathway, extracellular matrix (ECM)-receptor interaction, and focal adhesion in the TGF-β2-treated HTM cells. Western blotting demonstrated SIL inhibited the activation of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) and the downstream PI3K/AKT signaling pathways induced by TGF-β2, potentially contributing to its inhibitory effects on ECM protein production in the TGF-β2-treated HTM cells. Our study demonstrated the ability of SIL to inhibit TGF-β2-induced fibrogenic changes in HTM cells. SIL could be a potential IOP-lowering agent by reducing the fibrotic changes in the TM tissue of POAG patients, which warrants further investigation through additional animal and clinical studies.

Discrete-to-continuum models of pre-stressed cytoskeletal filament networks

We introduce a mathematical model for the mechanical behaviour of the eukaryotic cell cytoskeleton. This discrete model involves a regular array of pre-stressed protein filaments that exhibit resistance to enthalpic stretching, joined at cross-links to form a network. Assuming that the inter-cross-link distance is much shorter than the length scale of the cell, we upscale the discrete force balance to form a continuum system of governing equations and deduce the corresponding macroscopic stress tensor. We use these discrete and continuum models to analyse the imposed displacement of a bead placed in the domain, characterizing the cell rheology through the force–displacement curve. We further derive an analytical approximation to the stress and strain fields in the limit of small bead radius, predicting the net force required to generate a given deformation and elucidating the dependency on the microscale properties of the filaments. We apply these models to networks of the intermediate filament vimentin and demonstrate good agreement between predictions of the discrete, continuum and analytical approaches. In particular, our model predicts that the network stiffness increases sublinearly with the filament pre-stress and scales logarithmically with the bead size.

Unveiling the secrets of vimentin filament architecture relevant to human disease.

Unveiling the secrets of vimentin filament architecture relevant to human disease.

Vimentin filaments integrate low-complexity domains in a complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability.

Vesicular translocation of PARP-1 to cytoplasm causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.

ADP-ribosylation plays a significant role in various biological processes including genomic stability maintenance, transcriptional regulation, energy metabolism, and cell death. Using macrodomain pull-down assay with microglia lysates and MALDI-TOF-MS analysis, we identified vimentin as a major protein highly ADP-ribosylated by the poly(ADP-ribose) polymerases-1 (PARP-1) in response to LPS. ABT-888, a potent inhibitor of PARP-1/2 blocks the disassembly and ADP-ribosylation of vimentin. PARP-1 is a highly abundant nuclear protein. Its nuclear functions in repairing DNA damages induced by various stress signals, such as inflammatory stresses, have been well studied. In contrast, limited studies have been done on the cytoplasmic role(s) of PARP-1. Our study focuses on the cytoplasmic role of PARP-1 during microglia activation. Using immunofluorescence microscopy and Western blotting, we showed that a significant amount of PARP-1 is present in the cytosol of microglia cells stimulated and activated by LPS. Live cell imaging showed the translocation of nuclear PARP-1-EGFP to the cytoplasm in vesicular structures upon LPS stimulation. ABT-888 and U0126 can block this translocation. Immunofluorescence staining with various organelle marker antibodies revealed that PARP-1 vesicles show colocalization with Lamin A/C, suggesting they might be derived from the nuclear envelope through nuclear envelope budding. In conclusion, we demonstrated that PARP-1 is translocated from the nucleus to cytoplasm via vesicles upon LPS stimulation and that cytoplasmic PARP-1 causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.

Disruption of gonocyte development following neonatal exposure to di (2-ethylhexyl) phthalate

Pre- and/or post-natal administrations of di(2-ethylhexyl) phthalate (DEHP) in experimental animals cause alterations in the spermatogenesis. However, the mechanism by which DEHP affects fertility is unknown and could be through alterations in the survival and differentiation of the gonocytes. The aim of the present study was to evaluate the effect of a single administration of DEHP in newborn mice on gonocytic proliferation, differentiation and survival and its long-term effects on seminiferous epithelium and sperm quality. BALB/c mice distributed into Control and DEHP groups were used. Each animal in the DEHP group was given a single dose of 500 mg/Kg at birth. The animals were analyzed at 1, 2, 4, 6, 8, 10 and 70 days postpartum (dpp). Testicular tissues were processed for morphological analysis to determine the different types of gonocytes, differentiation index, seminiferous epithelial alterations, and immunoreactivity to Stra8, Pcna and Vimentin proteins. Long-term evaluation of the seminiferous epithelium and sperm quality were carried out at 70 dpp. The DEHP animal group presented gonocytic degeneration with delayed differentiation, causing a reduction in the population of spermatogonia (Stra8 +) in the cellular proliferation (Pcna+) and disorganization of Vimentin filaments. These events had long-term repercussions on the quality of the seminiferous epithelium and semen. Our study demonstrates that at birth, there is a period that the testes are extremely sensitive to DEHP exposure, which leads to gonocytic degeneration and delay in their differentiation. This situation can have long-term repercussions or permanent effects on the quality of the seminiferous epithelium and sperm parameters.

(113) A Novel 3D Cellular Model for Peyronie's Disease Treatment Screening: PD.SPHERE

Abstract Introduction Peyronie's disease (PD) is a common issue that affects approximately 10% of males. Currently, researchers primarily study the pathogenesis and potential treatments for PD using monolayer fibroblast cells and a limited number of animal models. However, these models do not accurately represent the complex cellular structure and interactions involved in PD pathogenesis. We have successfully developed a three-dimensional (3D) cellular model, called PD.SPHERE, using a ground-breaking 'micro-gravity' cell culture method. This model is cultivated from extracted human PD tissues and serves as a valuable tool for in vitro treatment screening for PD. However, we have yet to use this model to evaluate the effectiveness of available and potential treatments. Objective Our objective is to determine the efficacy of the novel 3D cellular PD model, PD.SPHERE, as a treatment screening tool. Methods To achieve this, we cultured fibroblasts isolated from human PD tissues using the 'micro-gravity' approach. In this method, cells were seeded in a non-adhesive 96-well plate, placed on an orbital shaker, and incubated at 37°C with 5% CO2 and 95% relative humidity. We confirmed the proper morphology and cellular composition through microscopic assays. This 3D model was then used to assess the effects of collagenase clostridium histolyticum (CCH) and the actinidin enzyme, obtained from kiwi fruits. The PD.SPHERE was treated with actinidin at 10 mg/ml for 48 hours. We subsequently quantified collagen content using a soluble collagen quantification kit and observed viability, morphology, and changes in cellular compositions of the spheroids through immunofluorescent techniques and confocal microscopy. Results Preliminary results from our model have indicated significant changes in cellular morphology. Treatment with actinidin led to a significant decrease in spheroid size and density compared to the control group (P<0.01). The ratio of vimentin and actin filaments to nuclei staining was slightly but significantly reduced (P<0.05) in the spheroids treated with actinidin at 10 mg/ml. The cellular viability of the treated group showed a slight decrease, as revealed by Sytox Green staining. The assessment of collagen contents in the treated spheroids is ongoing. Conclusions Preliminary results suggest that our PD.SPHERE model can be effectively used to assess morphological and cellular component changes in PD tissues after treatment. Actinidin appears to compromise the plasma membrane of fibroblasts in PD.SPHERE. Our model holds promise for personalized treatment planning and potential for developing high-throughput treatment screening platforms. It also demonstrates consistency and reproducibility while closely mimicking the morphology of a PD plaque. Further research is required to collect signature gene expression data, physiological and immunological information, such as collagen production, extracellular matrix organization, and cellular expression of tight junctions, chemokines, and cytokines. These additional measures will help verify our model's pathophysiological, immunological, and treatment response aspects. Disclosure No.

Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera

BackgroundEndothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines.MethodsMorphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ.ResultsThe four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of “spiking pattern” of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ.ConclusionsECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.

Differential distribution of intermediate filament proteins in the bovine and ovine tongues.

Intermediate filaments constitute the most heterogeneous class among the major classes of cytoskeletal proteins of mammalian cells. The 40 or more intermediate filament proteins have been classified into five types which show very specific rules of expression in specialized cell types. This study aimed to investigate the immunohistochemical distribution of cytokeratins (CKs) 8, 18, and 19 as well as the intermediate filaments vimentin, laminin, and desmin in bovine and ovine tongues. Immunohistochemical staining was performed for CKs 8, 18, 19, vimentin, laminin, and desmin. Our results revealed similar immunostaining intensity and distribution among various CKs, contrasting with distinct patterns for vimentin, laminin, and desmin. Immunoreactions were primarily localized in serous acini and ductal epithelium for cytokeratins, while vimentin and laminin were evident in connective tissue, endothelium, serous acini, and desmin in striated and smooth muscles. This study highlighted the absence of CKs 8, 18, 19, vimentin, and desmin in the lingual epithelium of bovine and ovine tongues. These findings enabled the classification of epithelial cells based on their specific cytokeratin patterns. Furthermore, vimentin was identified in mesodermal tissues and organs, desmin in muscle tissue, and laminin played crucial roles in basement membrane formation, nerve tissue regeneration, innervation of epithelial taste buds, and tissue separation and connection. Our findings provide essential insights into intermediate filament dynamics at the cellular and tissue levels. They serve as a foundation for future studies using systematic molecular biological techniques in this field.

Expression of oestrogen and progesterone receptor and intermediate filament proteins in the oviduct of mares with endometrosis

Equine endometrosis is one of the major causes of equine infertility, in the course of which periglandular fibrosis signs occur in the endometrium. As an oviduct is a key place of early embryo development, we hypothesized that loss of contact between mucosal cells and perturbation in hormonal action may coexist in the uterus and oviducts. Thus, this study aimed to investigate the expression of the intermediate filaments (vimentin and desmin) as well as the estrogen receptor alpha (ERα) and progesterone receptor A (PRA) in the stroma and epithelia of endometrium and oviduct in healthy mares and these with endometrosis. Out of 56 culled-in slaughterhouse mare endometrium samples were annotated in two groups (i) healthy endometrium (n=26) and endometrium with signs of moderate and severe endometrosis (n=20). The expression of vimentin, desmin, ERα, and PRA was investigated immunohistochemically and compared between the above groups as well as between the phase of the estrous cycle. The expression of both vimentin and desmin was higher in the endometrial stroma of endometrosis group compared to healthy endometrium. The expression of ERα was higher in the healthy endometrial stroma and luminal epithelium during the follicular phase than luteal, however, no differences were noted in endometrium of mares with endometrosis. Moreover, PRA expression was significantly higher in the stoma oviduct during the follicular phase than luteal. It seems possible that mucosal cells through their altered interactions may support the occurrence of moderate and severe endometrosis. In addition, the oviduct appears to be less sensitive to hormonal changes than the uterus. The changed expression of intermediate filaments and steroid hormones may be some of the leading factors which can contribute to the oviduct functional status in endometrosis.

Inhibition of Rho kinase (ROCK) impairs cytoskeletal contractility in human Müller glial cells without effects on cell viability, migration, and extracellular matrix production

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.