Expression Of Intermediate Filament Proteins Research Articles

#1464 Alterations of podocytes in focal segmental glomerulosclerosis: a pilot study

Abstract Background and Aims The molecular features of idiopathic podocytopathies remain unexplored, preventing the development of effective therapies to control the mechanisms of disease initiation and progression. This includes idiopathic focal segmental glomerulosclerosis (FSGS), one of the most common forms of immune glomerulopathies (IG) and the cause of renal failure and replacement therapy. In FSGS, podocytes undergo pronounced cytoskeletal alterations. Along with a decrease in Wilms tumor protein (WT1) and α-SMA expression, there is evidence for increased expression of intermediate filament proteins (vimentin, desmin, nestin) and Wnt/β-catenin signaling upregulation in IG. Decrease in WT1 is associated with upregulation in Wnt/β-catenin and p53 to promote podocyte dysfunction and albuminuria. Upregulation desmin increases the mechanical stability of cells, allowing podocytes to undergo morphological changes on the tensile glomerular capillary wall. This pilot study was aimed to evaluate the significance of WT1, β-catenin, and desmin glomerular expression in early primary FSGS. Method We reviewed the histopathological and clinical data of patients with biopsy proven FSGS (N = 14, experimental group), IgA nephropathy (N = 12, positive control), without AKI, infectious diseases, heart failure, respiratory insufficiency and cancer pathology. Renal surgery specimens from patients without IG (N = 12) were studied as negative control. Glomerulosclerosis was assessed as percentage of all glomeruli. Mesangial lesions were measured semi-quantitatively in scores (0-3). Glomerular WT1, β-catenin, and desmin expression was assessed by IHC-P with quantitative morphometry of IHC-staining area (open software QuPath, Orbit Image Analysis). Molecule's co-expression was analyzed by IF-p and confocal microscopy (Zeiss LSM 710; study conducted in the Center for Collective Use “Confocal microscopy” by Pavlov Institute of Physiology Russian Academy of Sciences). Results We examined young and middle-aged patients with CKD stages 1-3 and moderate morphological changes in the glomeruli. Gender, age, eGFR and the percentage of glomeruli with segmental sclerosis were not significantly different between IG groups (all p for interaction >0.05). Patients with FSGS have lower levels of serum albumin (p < 0.001) and higher levels of proteinuria (p < 0.001) as compared to IgAN. Glomeruli from IgAN patients showed more mesangial cell proliferation (p = 0.001), expansion of the mesangial matrix (p = 0.05) and global glomerular sclerosis (p = 0.003) as compared to FSGS. An immunomorphological study showed lower WT1 expression in both IG groups compared to the negative control. In the IgAN, lower WT1 expression was associated with higher BC area (Fig. 1A, D). BC expression did not differ between FSGS and negative control (Fig. 1B,D). In the combined IG group, BC directly correlated with global (r = 0.63, p = 0.001) and segmental (r = 0.63, p = 0.001) glomerular sclerosis. In FSGS, a trend towards to increase in the area of desmin IHC-staining was detected, however, no significant differences between groups were found (Fig. 1C,D). BC and desmin were co-localized with the podocyte marker WT1 (Fig. 2). BC and desmin were also expressed in WT1-negative glomerular cells (Fig. 2). Differential molecular expression patterns in glomeruli have been identified. In IgAN, co-expressing WT1 and BC (orange) was dominate in glomerular cells (Fig. 2A); the proportion of IHC expression of WT1 and BC in glomerular cells was directly correlated (r = 0.63, p = 0.031). In FSGS, WT1+ cells predominantly co-expressed desmin (magenta) but not BC; some glomerular cells were only desmin-positive (Fig. 2B). BC was expressed to a lesser extent compared to IgAN, and in some cells it was co-expressed with desmin (Fig. 2B). Conclusion In FSGS, an increase in desmin expression could be an early feature of pathogenesis before significant decrease in the number of WT1+ podocytes and changes in β-catenin expression. Activation of desmin likely characterizes damage and decrease in the number of functioning podocytes.

Curcumin mitigates gentamicin induced-renal and cardiac toxicity via modulation of Keap1/Nrf2, NF-κB/iNOS and Bcl-2/BAX pathways

Gentamicin (GEN) is an aminoglycoside antibiotic used to treat gram-negative bacterial infections. Our study aimed to explore curcumin's (CMN) protective role against GEN-induced renal and cardiac toxicity. Rats were randomly classified into 4 equal groups; Control (cont), GEN (100 mg/kg b.wt, i.p.) for seven days, CMN (200 mg/kg b.wt, orally) for 21 days, and CMN + GEN groups. GEN caused renal and cardiac dysfunctions; increased urea, creatinine, uric acid, cystatin C, CK-MB, LDH, and troponin I serum levels. MDA level was elevated significantly while activities of SOD, CAT, and GSH level were reduced significantly in renal and cardiac tissues. GEN-intoxicated rats showed up-regulation of NF-κB, IL-1β, Keap1, HMOX1, and BAX with down-regulation of Nrf2, and Bcl-2 mRNA expression in renal and cardiac tissues. Also, GEN-induced up-regulation of renal mRNA expression of KIM-1, NGAL, and intermediate filament proteins [desmin, nestin, and vimentin] as well cardiac gene expression of cMyBP-C and H-FABP. GEN-induced toxicity was significantly attenuated by CMN co-treatment as CMN improved renal and cardiac biomarkers, reduced oxidative stress and inflammatory response, and reversed alterations in mRNA expression of all tested renal and cardiac genes. These outcomes indicated that CMN could protect renal and cardiac tissues against GEN-induced oxidative stress, inflammation, and apoptosis.

Disrupted neurogenesis, gliogenesis, and ependymogenesis in the Ccdc85c knockout rat for hydrocephalus model.

Coil-coiled domain containing 85c (Ccdc85c) is a causative gene for congenital hydrocephalus and subcortical heterotopia with frequent brain hemorrhage. We established Ccdc85c knockout (KO) rats and investigated the roles of CCDC85C and intermediate filament protein expression, including nestin, vimentin, GFAP, and cytokeratin AE1/AE3 during the lateral ventricle development in KO rats to evaluate the role of this gene. We found altered and ectopic expression of nestin and vimentin positive cells in the wall of the dorso-lateral ventricle in the KO rats during development from the age of postnatal day (P) 6, whereas both protein expression became faint in the wild-type rats. In the KO rats, there was a loss of cytokeratin expression on the surface of the dorso-lateral ventricle with ectopic expression and maldevelopment of ependymal cells. Our data also revealed disturbed GFAP expression at postnatal ages. These findings indicate that lack of CCDC85C disrupts the proper expression of intermediate filament proteins (nestin, vimentin, GFAP, and cytokeratin), and CCDC85C is necessary for normal neurogenesis, gliogenesis, and ependymogenesis.

Dynamic changes in whole genome DNA methylation, chromatin and gene expression during mouse lens differentiation

BackgroundCellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium.ResultsUsing whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6’s ability to interact with sites containing one or two methylated CpG dinucleotides.ConclusionsOur study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.

P.149 Differential expression of intermediate filament proteins: lamins A/C and desmin within and between adult skeletal muscles

The cell cytoskeleton is composed of three main components: microfilaments, microtubules, and intermediate filaments. Intermediate filaments consist of intermediate filament proteins that are widely distributed in cells and tissues, either in the cytoplasm or the nucleus. Desmin is a tissue-specific cytoplasmic intermediate filament protein, while A-type lamins including lamin A/C are ubiquitously expressed nuclear intermediate filament proteins. Both play major roles in skeletal muscle functions. Mutations in the <i>DES</i> gene, encoding Desmin, and <i>LMNA</i> gene, encoding A-type lamins, lead to a group of rare disorders called desminopathies and laminopathies, respectively. The most frequent pathologies are those involving skeletal muscles. Despite the universal expression of lamins A/C in almost all cells and tissues, different phenotypes associated with laminopathies reflect a high degree of tissue-specificity, the reasons for which are not completely understood. It has also been shown that specific muscles/muscle groups are affected earlier than others in these myopathies. We hypothesized that desmin and lamins A/C are not uniformly expressed in different skeletal muscles and or muscle parts. Accordingly, we have investigated the relative expression of desmin, lamin A & lamin C in 9 skeletal muscles, of which 7 were divided into belly and myotendinous junction (MTJ). Our preliminary results revealed that there is no significant difference in the relative expression of desmin and lamins A/C between bellies and MTJs within a single muscle. We also observed that the relative expression of lamins A & C is correlated with myogenic markers; MyoD and myogenin, while desmin is not. Strikingly, FDB muscle showed the highest expression of lamins A/C compared to other muscles. We are currently investigating desmin, lamin A and lamin C protein levels by western blot and immunohistochemistry and the effect of age and exercise on the expression of these intermediate filament proteins.

Reactive astrocytes as treatment targets in Alzheimer's disease-Systematic review of studies using the APPswePS1dE9 mouse model.

Astrocytes regulate synaptic communication and are essential for proper brain functioning. In Alzheimer's disease (AD) astrocytes become reactive, which is characterized by an increased expression of intermediate filament proteins and cellular hypertrophy. Reactive astrocytes are found in close association with amyloid‐beta (Aβ) deposits. Synaptic communication and neuronal network function could be directly modulated by reactive astrocytes, potentially contributing to cognitive decline in AD. In this review, we focus on reactive astrocytes as treatment targets in AD in the APPswePS1dE9 AD mouse model, a widely used model to study amyloidosis and gliosis. We first give an overview of the model; that is, how it was generated, which cells express the transgenes, and the effect of its genetic background on Aβ pathology. Subsequently, to determine whether modifying reactive astrocytes in AD could influence pathogenesis and cognition, we review studies using this mouse model in which interventions were directly targeted at reactive astrocytes or had an indirect effect on reactive astrocytes. Overall, studies specifically targeting astrocytes to reduce astrogliosis showed beneficial effects on cognition, which indicates that targeting astrocytes should be included in developing novel therapies for AD.

Moringa oleifera ethanolic extract attenuates tilmicosin-induced renal damage in male rats via suppression of oxidative stress, inflammatory injury, and intermediate filament proteins mRNA expression

Tilmicosin (Til) is a popular macrolide antibiotic, widely used in veterinary practice. The present study was designed to address the efficacy of Moringa oleifera ethanolic extract (MOE) in protecting against Tilmicosin (Til) - induced nephrotoxicity in Sprague Dawley rats. Animals were treated once with Til (75 mg/kg bw, subcutaneously), and/or MOE for 7 days (400 or 800 mg/kg bw, by oral gavage). Til-treatment was associated with significantly increased serum levels of creatinine, urea, sodium, potassium and GGT activity, as well as decreased total protein and albumin concentrations. Renal tissue hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels were elevated, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes were diminished. The levels of renal tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) and the mRNA expression of intermediate filament protein encoding genes (desmin, nestin and vimentin) in the kidney were up- regulated with histopathological alterations in renal glomeruli, tubules and interstitial tissue. These toxic effects were markedly ameliorated by co-treatment of MOE with Til, in a dose dependent manner. Taken together, these results indicate that MO at 800 mg/kg protects against Til-induced renal injury, likely by its potent antioxidant and anti-inflammatory properties, which make it suitable to be used as a protective supplement with Til therapy.

Intermediate filament protein expression and sugar moieties in normal canine placenta.

In the female dog, the placenta is considered zonal, endotheliochorial and labyrinthic. The distribution of the intermediate filaments as well as the surface glycoproteins in the canine placenta are still unknown. The aim of the present study was to provide this information for further understanding of pathological conditions in the bitch. Samples were obtained from normal uterine horns at the end of gestation. Tissues were routinely fixed and stained. Monoclonal antibodies against cytokeratins, vimentin and desmin were used for immunohistochemical staining. UEA-1, PNA, RCA-1, SBA, DBA, WGA and ConA were used for the lectin histochemical staining. A computer morphometrical analysis was made. Statistical analysis was then accomplished. The results showed the maximum immunohistochemical percentage for vimentin in the supraglandular connective tissue, for pancytokeratin in the spongy zone and for desmin in miometrium. SBA showed the highest staining percentage in the gland cells of the spongy zone, while ConA was the highest in the syncytiotrophoblastic cells and gland cells of the deep glandular zone. The results obtained indicate that the lectin binding pattern is partially different from other animal species. On the contrary, the intermediate filament data coincide with similar observations from other mammals.

Argan oil ameliorates sodium fluoride-induced renal damage via inhibiting oxidative damage, inflammation, and intermediate filament protein expression in male rats.

Fluoride is widely distributed in the environment and has been associated with the development of different health hazards in animals and humans. Argan oil (AO) is a natural vegetable oil with various beneficial pharmacological effects. This study was designed to investigate the potential protective effect of AO supplementation as pre-treatment or co-treatment on sodium fluoride (NaF)-induced nephrotoxicity in rats. Male Sprague Dawley rats (n = 50) were randomly assigned to one of five equal groups: control group, AO-treated group (6ml/kg b.wt.), NaF-treated group (20mg/kg b.wt.), pre-treated group, and co-treated group. All rats were daily administered by oral gavage for duration of 30days. The results showed that AO administration significantly improved renal function and antioxidant status and decreased the lipid peroxidation in NaF-treated rats. Additionally, AO normalized the renal levels of inflammatory markers and mRNA expression level of the intermediate filament protein genes, indicating NaF-induced podocyte damage was ameliorated. Histopathological evaluation of the kidney confirmed the before mentioned biochemical results. AO counteracted the nephrotoxic effects of NaF in rats particularly at co-exposure. These results concluded that AO administration exhibited a significant nephroprotective effect against renal injury induced by NaF in rats.

Intermediate filament protein expression pattern and inflammatory response changes in kidneys of rats receiving doxorubicin chemotherapy and quercetin

The aim of this study was to explore the potential effects of quercetin (QUR) on doxorubicin (DOX)-induced nephrotoxicity. Fifty male rats were assigned to five groups (10 rats each): a control group, a DOX-treated group (total dose, 15 mg/kg bw, intraperitoneally), a QUR-treated group (50 mg/kg bw/day, orally), a prophylaxis co-treated group, and a therapeutic co-treated group. Biochemical parameters and renal function were measured. Moreover, kidney tissues were homogenized for inflammatory marker evaluation and real-time qPCR analysis to determine the changes in intermediate filament protein mRNA levels (desmin, vimentin, connexin 43 and nestin). QUR exhibited a significant nephroprotective effect, particularly when it was administered prior to and simultaneously with DOX treatment (prophylaxis co-treated group). This role was biochemically demonstrated by the significant modulation of DOX-induced body weight loss, hypoproteinemia, and elevated serum creatinine and urea. Moreover, QUR attenuated the inflammatory response as shown by decreased renal nitric oxide, tumor necrosis factor-α production and myeloperoxidase activity elicited by DOX injection. These biochemical improvements were accompanied by a significant histopathological restoration of rat kidney tissue and successful down-regulation of the intermediate filament protein mRNA levels, indicating amelioration of DOX-induced podocyte injury. Taken together, these results conclusively demonstrated that QUR administration has a prophylactic effect on DOX-induced injury in the rat kidney.

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIMSA/SA) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIMSA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIMSA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIMSA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.

肝臓星細胞(ビタミンA貯蔵細胞)の活性化に伴う中間径フィラメントの発現量と局在の変化

肝臓星細胞(ビタミンA貯蔵細胞)の活性化に伴う中間径フィラメントの発現量と局在の変化

Role of Retinal Pigment Epithelial Cell β-Catenin Signaling in Experimental Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy is caused by the contraction of fibrotic membranes on the epiretinal surface of the neurosensory retina, resulting in a traction retinal detachment and loss of visual acuity. Retinal pigment epithelial (RPE) cells play an important role in formation of such fibrotic, contractile membranes. We investigated the role of Wnt/β-catenin signaling, a pathway implicated in several fibrotic diseases, in RPE cells in proliferative vitreoretinopathy. In vitro culture of swine RPE sheets resulted in nuclear translocation of β-catenin in dedifferentiated RPE cells. FH535, a specific inhibitor of β-catenin signaling, reduced the outgrowth of cultured RPE sheets and prevented dedifferentiated RPE cell proliferation and migration. It also inhibited formation of contractile membranes by dedifferentiated RPE cells on collagen I matrices. Expression and function of the β-catenin signaling target connexin-43 were down-regulated by FH535, and functional blockade of connexins with carbenoxolone also prevented the in vitro formation of fibrotic, contractile membranes. Intravitreal injection of FH535 in swine also inhibited formation of dense, contractile membranes on the epiretinal surface and prevented development of traction retinal detachment. These findings demonstrate that β-catenin signaling is involved in formation of contractile membranes by dedifferentiated RPE cells and suggest that adjunctive treatment targeting this pathway could be useful in preventing proliferative vitreoretinopathy.

Intermediate Filament Proteins Expression and Carbohydrate Moieties in Trophoblast and Decidual Cells of Mature Cat Placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Mechanical Properties of Vimentin Intermediate Filament Networks

One marker of mesenchymal cells is the expression of vimentin intermediate filament (VIF) proteins, which assemble into networks. The cytoskeleton is typically comprised of these intermediate filament networks, actin, and microtubules. While some aspects of VIF networks have been characterized in bulk, their mechanical properties in situ have not been well defined. Here we use microrheological techniques to study VIF network mechanics in cells in which microtubules and actin have been removed so that VIF can be studied independently of the other cytoskeletal components. Simultaneous imaging of the network allows us to correlate network structure with its local mechanical properties.

HB‐EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis

Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.

Isoflurane affects the cytoskeleton but not survival, proliferation, or synaptogenic properties of rat astrocytes in vitro

More than half of the cells in the brain are glia and yet the impact of general anaesthetics on these cells is largely unexamined. We hypothesized that astroglia, which are strongly implicated in neuronal well-being and synapse formation and function, are vulnerable to adverse effects of isoflurane. Cultured rat astrocytes were treated with 1.4% isoflurane in air or air alone for 4 h. Viability, proliferation, and cytoskeleton were assessed by colorimetric assay, immunocytochemistry, or a migration assay at the end of treatment or 2 days later. Also, primary rat cortical neurones were treated for 4 days with conditioned medium from control [astrocyte-conditioned media (ACM)], or isoflurane-exposed astrocytes (Iso-ACM) and synaptic puncta were assessed by synapsin 1 and PSD-95 immunostaining. By several measures, isoflurane did not kill astrocytes. Nor, based on incorporation of a thymidine analogue, did it inhibit proliferation. Isoflurane had no effect on F-actin but reduced expression of α-tubulin and glial fibrillary acidic protein both during exposure (P<0.05 and P<0.001, respectively) and 2 days later (P<0.01), but did not impair astrocyte motility. ACM increased formation of PSD-95 but not synapsin 1 positive puncta in neuronal cultures, and Iso-ACM was equally effective. Isoflurane decreased expression of microtubule and intermediate filament proteins in astrocytes in vitro, but did not affect their viability, proliferation, motility, and ability to support synapses.

Differential Increases in the Expression of Intermediate Filament Proteins and Concomitant Morphological Changes of Transdifferentiating Rat Hepatic Stellate Cells Observed &lt;i&gt;In Vitro&lt;/i&gt;

The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.