Cytoskeletal Disruption Research Articles

Cbl-b promotes cell detachment via ubiquitination of focal adhesion kinase.

Cancer cell detachment from the primary tumor site represents the first stage of metastasis. Previous studies have identified that cell detachment is triggered by cytoskeletal disruption, which may induce a wide variety of cellular changes. Focal adhesion kinase (FAK) exhibits crucial cellular functions, including regulation of the cytoskeleton. These observations have provided exciting insights into the effect of FAK in cell detachment; however, the involvement of FAK in cell detachment remains controversial. The aim of the present study was to evaluate the effect of FAK and its function in the process of cell detachment. The results revealed that FAK expression was downregulated following trypsin treatment in human gastric, lung, colon and breast cancer cell lines, as well as a human gastric epithelial cell line. Knockdown of FAK enhanced cell detachment in gastric cancer MGC803 cells, indicating that FAK inhibits cell detachment. Further investigation revealed that trypsin induced monoubiquitination of FAK. In addition, the lysosome inhibitor, NH4Cl, decreased trypsin-induced degradation of FAK. Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, was involved in this process, which interacted with FAK, as demonstrated by co-precipitation experiments, and promoted trypsin-induced ubiquitin-lysosome degradation of FAK. These results indicate that Cbl-b promotes cell detachment via ubiquitination of FAK. These findings provide novel insights regarding the effect of FAK and Cbl-b in the process of cancer cell detachment.

Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.

Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke.

Pathogen-inspired glycolipid probes reveal membrane heterogeneity and dynamics

Event Abstract Back to Event Pathogen-inspired glycolipid probes reveal membrane heterogeneity and dynamics Rachel Kraut1, 2*, Sarah Lehnert1, 3, Umit H. Yildiz3, Manoj Manna1, 4, Natalie Haustein1, Jagadish Sankaran1, 5, Artur Matysik6, Kamila Oglecka3, Bo Liedberg3 and Thorsten Wohland4 1 Nanyang Technological University, School of Biological Sciences, Singapore 2 TU Dresden, Biotec, Germany, Germany 3 Nanyang Technological University, School of Materials Science and Engineering, Singapore 4 National University of Singapore, Department of Chemistry, Centre for BioImaging Sciences, Singapore 5 National University of Singapore, Mechanobiology Institute, Singapore 6 Nanyang Technological University, Singapore Centre on Environmental Life Sciences Engineering, Singapore Many pathogens and bacterial toxins use glycosphingolipids to invade cells. We take advantage of this phenomenon to design toxin-derived peptides that interact with glycosphingolipid-containing domains at the plasma membrane. The peptide probes are able to trace the diffusion dynamics of domains in live cells, and their compositional heterogeneity in synthetic membrane systems such as supported lipid bilayers. We compared the diffusion dynamics of CtxB and the novel ganglioside-binding probes SBD from Alzheimer’s Aß, and TeNT46 from Tetanus in live cells using TIRF-Fluorescence Correlation Spectroscopy (FCS). All three probes displayed slow, bimodal diffusion typical for ordered domains, but their lipid preferences differed. FCS on cells and Imaging Surface Plasmon Resonance on hybrid bilayers showed that cholesterol and sphingomyelin affected binding and diffusion of SBD, TeNT46, and CtxB differently, and that toxin-bound domains are immobilized without the probe necessarily being bound by cholesterol. A new parameter, ΔCCF (difference in Cross-Correlation Function), showed that the probes' anisotropic diffusion coefficient distributions—i.e. heterogeneity—like the diffusion itself, was sensitive to sphingolipid and cytoskeletal disruption. However, ΔCCF detected heterogeneity to which conventional FCS is blind, revealing two distinguishable levels of membrane organization: short-range diffusion detectable by FCS, vs. longer-scale heterogeneity detectable by ΔCCF. This work may be of long-term interest for the design anti-pathogen decoys, for example using vesicular engineered lipid targets. Keywords: Glycosphingolipids, membrane dynamics, cell invasion, Membrane heterogeneity, pathogen-inspired glycolipid Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Symposium 12: Glycolipids and the Regulation of Complex Networks in Neural Membranes Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kraut R, Lehnert S, Yildiz UH, Manna M, Haustein N, Sankaran J, Matysik A, Oglecka K, Liedberg B and Wohland T (2016). Pathogen-inspired glycolipid probes reveal membrane heterogeneity and dynamics. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00052 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jul 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Rachel Kraut, Nanyang Technological University, School of Biological Sciences, Singapore, Singapore, rachel.kraut@biotec.tu-dresden.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rachel Kraut Sarah Lehnert Umit H Yildiz Manoj Manna Natalie Haustein Jagadish Sankaran Artur Matysik Kamila Oglecka Bo Liedberg Thorsten Wohland Google Rachel Kraut Sarah Lehnert Umit H Yildiz Manoj Manna Natalie Haustein Jagadish Sankaran Artur Matysik Kamila Oglecka Bo Liedberg Thorsten Wohland Google Scholar Rachel Kraut Sarah Lehnert Umit H Yildiz Manoj Manna Natalie Haustein Jagadish Sankaran Artur Matysik Kamila Oglecka Bo Liedberg Thorsten Wohland PubMed Rachel Kraut Sarah Lehnert Umit H Yildiz Manoj Manna Natalie Haustein Jagadish Sankaran Artur Matysik Kamila Oglecka Bo Liedberg Thorsten Wohland Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Endothelium-derived intermedin/adrenomedullin-2 protects human ventricular cardiomyocytes from ischaemia-reoxygenation injury predominantly via the AM1 receptor

Application of intermedin/adrenomedullin-2 (IMD/AM-2) protects cultured human cardiac vascular cells and fibroblasts from oxidative stress and simulated ischaemia-reoxygenation injury (I-R), predominantly via adrenomedullin AM1 receptor involvement; similar protection had not been investigated previously in human cardiomyocytes (HCM). Expression of IMD, AM and their receptor components was studied in HCM. Receptor subtype involvement in protection by exogenous IMD against injury by simulated I-R was investigated using receptor component-specific siRNAs. Direct protection by endogenous IMD against HCM injury, both as an autocrine factor produced in HCM themselves and as a paracrine factor released from HCMEC co-cultured with HCM, was investigated using peptide-specific siRNA for IMD. IMD, AM and their receptor components (CLR, RAMPs1-3) were expressed in HCM. IMD 1nmolL−1, applied either throughout ischaemia (3h) and re-oxygenation (1h) or during re-oxygenation (1h) alone, attenuated HCM injury (P<0.05); cell viabilities were 59% and 61% respectively vs. 39% in absence of IMD. Cytoskeletal disruption, protein carbonyl formation and caspase activity followed similar patterns. Pre-treatment (4 days) of HCM with CLR and RAMP2 siRNAs attenuated (P<0.05) protection by exogenous IMD. Pre-treatment of HCMEC with IMD (and AM) siRNA augmented (P<0.05) I-R injury: cell viabilities were 22% (and 32%) vs. 39% untreated HCMEC. Pre-treatment of HCM with IMD (and AM) siRNA did not augment HCM injury: cell viabilities were 37% (and 39%) vs. 39% untreated HCM. Co-culture with HCMEC conferred protection from injury on HCM; such protection was attenuated when HCMEC were pre-treated with IMD (but not AM) siRNA before co-culture. Although IMD is present in HCM, IMD derived from HCMEC and acting in a paracrine manner, predominantly via AM1 receptors, makes a marked contribution to cardiomyocyte protection by the endogenous peptide against acute I-R injury.

Functional implications of axon initial segment cytoskeletal disruption in stroke.

Axon initial segment (AIS) is the proximal part of the axon, which is not covered with a myelin sheath and possesses a distinctive, specialized assembly of voltage-gated ion channels and associated proteins. AIS plays critical roles in synaptic integration and action potential generation in central neurons. Recent evidence shows that stroke causes rapid, irreversible calpain-mediated proteolysis of the AIS cytoskeleton of neurons surrounding the ischemic necrotic core. A better understanding of the molecular mechanisms underlying this "non-lethal" neuronal damage might provide new therapeutic strategies for improving stroke outcome. Here, we present a brief overview of the structure and function of the AIS. We then discuss possible mechanisms underlying stroke-induced AIS damage, including the roles of calpains and possible sources of Ca(2+) ions, which are necessary for the activation of calpains. Finally, we discuss the potential functional implications of the loss of the AIS cytoskeleton and ion channel clusters for neuronal excitability.

PPARγ regulated CIDEA affects pro-apoptotic responses in glioblastoma.

Refractoriness of glioblastoma multiforme (GBM) to current treatment paradigms has necessitated identification of new targets to better the existing therapeutic strategies. One such target is peroxisome proliferator-activated receptor gamma (PPARγ) – a transcription factor involved in regulation of lipid metabolism and inflammation. Expression of PPARγ, a known regulator of cell death-inducing DFFA-like effector (CIDEA), is modulated by hypoxia inducible factor (HIF-1α). While the involvement of CIDEA in lipid metabolism is known, its role in malignancies remains largely unknown. An elevated PPARγ and low CIDEA level was observed in GBM tumors as compared with surrounding non-neoplastic tissue. As reciprocal relation exists between PPAR and HIF-1α: and as HIF-1α is a key component in glioma progression, their role in regulating CIDEA expression in glioblastoma was investigated. Although HIF-1α inhibition had no effect on CIDEA expression, pharmacological inhibition of PPARγ elevated CIDEA levels. PPARγ mediated upregulation of CIDEA was accompanied by decreased recruitment of NFκB and SP1 to their predicted binding sites on CIDEA promoter. Ectopic expression of CIDEA triggered apoptosis, activated JNK, decreased HIF-1α activation and increased PPARγ levels in glioma cells. While CIDEA overexpression induced actin cytoskeletal disruption, cell cycle arrest, release of pro-inflammatory cytokine IL-6 in a JNK-dependent manner; CIDEA mediated apoptotic cell death, decreased STAT3 phosphorylation and increased p53 acetylation was JNK independent. This study highlights for the first time the existence of (i) PPARγ-CIDEA regulatory loop in glioma and (ii) novel function of CIDEA as regulator of glioma cell survival.

Characterizing Cellular Biophysical Responses to Stress by Relating Density, Deformability, and Size

Cellular physical properties are important indicators of specific cell states. Although changes in individual biophysical parameters, such as cell size, density, and deformability, during cellular processes have been investigated in great detail, relatively little is known about how they are related. Here, we use a suspended microchannel resonator (SMR) to measure single-cell density, volume, and passage time through a narrow constriction of populations of cells subjected to a variety of environmental stresses. Osmotic stress significantly affects density and volume, as previously shown. In contrast to density and volume, the effect of an osmotic challenge on passage time is relatively small. Deformability, as determined by comparing passage times for cells with similar volume, exhibits a strong dependence on osmolarity, indicating that passage time alone does not always provide a meaningful proxy for deformability. Finally, we find that protein synthesis inhibition, cell-cycle arrest, protein kinase inhibition, and cytoskeletal disruption result in unexpected relationships among deformability, density, and volume. Taken together, our results suggest that by measuring multiple biophysical parameters, one can detect unique characteristics that more specifically reflect cellular behaviors.

Cytoskeletal Components Define Protein Location to Membrane Microdomains*

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases.

Endothelial Thermotolerance Impairs Nanoparticle Transport in Tumors.

The delivery of diagnostic and therapeutic agents to solid tumors is limited by physical transport barriers within tumors, and such restrictions directly contribute to decreased therapeutic efficacy and the emergence of drug resistance. Nanomaterials designed to perturb the local tumor environment with precise spatiotemporal control have demonstrated potential to enhance drug delivery in preclinical models. Here, we investigated the ability of one class of heat-generating nanomaterials called plasmonic nanoantennae to enhance tumor transport in a xenograft model of ovarian cancer. We observed a temperature-dependent increase in the transport of diagnostic nanoparticles into tumors. However, a transient, reversible reduction in this enhanced transport was seen upon reexposure to heating, consistent with the development of vascular thermotolerance. Harnessing these observations, we designed an improved treatment protocol combining plasmonic nanoantennae with diffusion-limited chemotherapies. Using a microfluidic endothelial model and genetic tools to inhibit the heat-shock response, we found that the ability of thermal preconditioning to limit heat-induced cytoskeletal disruption is an important component of vascular thermotolerance. This work, therefore, highlights the clinical relevance of cellular adaptations to nanomaterials and identifies molecular pathways whose modulation could improve the exposure of tumors to therapeutic agents.

Fine-tuning of NFκB by glycogen synthase kinase 3β directs the fate of glomerular podocytes upon injury

NFκB is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3β and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide or adriamycin elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L and B7-1. Broad range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3β by lithium or TDZD-8, mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3β was sufficient and essential for RelA/p65 phosphorylation specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with lipopolysaccharide or adriamycin, concomitant with suppression of podocytopathic mediators but retained Bcl-xL in glomerulus. Broad range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3β dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy.

Tributyltin induces disruption of microfilament in HL7702 cells via MAPK-mediated hyperphosphorylation of VASP.

Tributyltin (TBT) has been widely used for various industrial purposes, and it has toxic effects on multiple organs and tissues. Previous studies have found that TBT could induce cytoskeletal disruption, especially of the actin filaments. However, the underlying mechanisms remain unclear. The aim of the present study was to determine whether TBT could induce microfilament disruption using HL7702 cells and then to assess for the total levels of various microfilament-associated proteins; finally, the involvement of the MAPK pathway was investigated. The results showed that after TBT treatment, F-actin began to depolymerize and lost its characteristic filamentous structure. The protein levels of Ezrin and Cofilin remained unchanged, the actin-related protein (ARP) 2/3 levels decreased slightly, and the vasodilator-stimulated phosphoprotein (VASP) decreased dramatically. However, the phosphorylation levels of VASP increased 2.5-fold, and the ratio of phosphorylated-VASP/unphosphorylated-VASP increased 31-fold. The mitogen-activated protein kinases (MAPKs) ERK and JNK were discovered to be activated. Inhibition of ERK and JNK not only largely diminished the TBT-induced hyperphosphorylation of VASP but also recovered the cellular morphology and rescued the cells from death. In summary, this study demonstrates that TBT-induced disruption of actin filaments is caused by the hyperphosphorylation of VASP through MAPK pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1530-1538, 2016.

Involvement of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK cells

The outbreak of cyanobacterial blooms induces the production and release of microcystins (MCs) into water, representing a health hazard to aquatic organisms and even humans. Some recent studies have suggested that kidney is another important target organ of MCs except liver, however, the potential toxicity mechanisms are still unclear. In this study, we first investigated the collaborative effect of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK (Ctenopharyngodon idellus kidney) cells in vitro. CIK cells were treated with 0, 1, 10, and 100μg/L microcystin-LR (MC-LR) for 24 and 48h. Cell viability was increased by MC-LR in 1μg/L group, while decreased in 100μg/L group at 48h. Cell cycle assay showed that 1 and 10μg/L MC-LR induced cell cycle through G1 into S and G2/M phases, while 100μg/L MC–LR reduced G2/M phase population. MC-LR markedly induced apoptosis in 10 and 100μg/L groups. Elevated reactive oxygen species (ROS) production, increased malondialdehyde (MDA) contents, decreased glutathione (GSH) levels, and modulated antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) were observed in CIK cells exposed to MC-LR. These alterations were more pronounced at higher doses (10 and 100μg/L), indicating that oxidative stress was induced by MC-LR. Laser scanning confocal microscope observation showed aggregation and collapse of microfilaments (MFs) and microtubules (MTs) in CIK cells, and even loss of some cytoskeleton structure. Moreover, transcriptional changes of cytoskeletal genes (β-actin, lc3a, and keratin) were also determined, which have a high probability with cytoskeleton structure damage. Our data suggest that oxidative stress and cytoskeletal disruption may interact with each other and jointly lead to apoptosis and renal toxicity induced by MCs.

Mechanisms of microcystin-LR-induced cytoskeletal disruption in animal cells.

Microcystin-LR (MC-LR), a potent hepatotoxin produced by certain bloom-forming cyanobacteria, covalently binds to serine/threonine protein phosphatases and acts as an efficient inhibitor of this group of enzymes. MC-LR induces oxidative stress and the unfolded protein response in multiple cell types, leading to apoptosis through the mitochondrial and endoplasmic reticulum pathways. Histologic lesions of acute MC-LR toxicosis exhibit membrane blebbing, cell rounding and dissociation, indicating that this toxin may exert hepatotoxic effects by causing cytoskeletal disruption. Both invivo and invitro studies have revealed that exposure of human, mouse, or rat hepatocytes to MC-LR induces the rearrangement or collapse of the three components of the cytoskeleton. In addition, multiple cytoskeletal and cytoskeleton-associated proteins have been found to be affected by MC-LR. This review summarizes the increasing information in the literature pertaining to the molecular mechanisms of MC-LR-induced cytoskeletal disruption and may increase our understanding of its toxicity.

Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage.

Cardiomyocyte mitochondrial oxidative stress and cytoskeletal breakdown in the heart with a primary volume overload.

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. Because mitochondria are both a source and target of ROS, we hypothesized that the mitochondrially targeted antioxidant mitoubiquinone (MitoQ) will improve cardiomyocyte damage and LV dysfunction in VO. Isolated cardiomyocytes from Sprague-Dawley rats were exposed to stretch in vitro and VO of aortocaval fistula (ACF) in vivo. ACF rats were treated with and without MitoQ. Isolated cardiomyocytes were analyzed after 3 h of cyclical stretch or 8 wk of ACF with MitoSox red or 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate to measure ROS and with tetramethylrhodamine to measure mitochondrial membrane potential. Transmission electron microscopy and immunohistochemistry were used for cardiomyocyte structural assessment. In vitro cyclical stretch and 8-wk ACF resulted in increased cardiomyocyte mitochondrial ROS production and decreased mitochondrial membrane potential, which were significantly improved by MitoQ. ACF had extensive loss of desmin and β₂-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering identified by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not affect LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF.

Remodeling of Caveolae Mediates Stretch-Induced Increase of L-Type Calcium Current in Rat Mesenteric Artery

Background: Voltage-dependent L-type Ca2+ channel (VDCCL) of vascular smooth muscle is facilitated by mechanical stimuli, which is thought to contribute to the myogenic contraction of resistant arteries. However, the molecular mechanisms underlying the VDCCL activation is controversial.Aim and Methods: We examined the hypothesis that the remodeling of caveolae and its downstream signaling contributes to the facilitation of VDCCL by mechanical stretch in rat mesenteric arterial smooth muscle cells using patch-clamp technique and biochemical analysis.Results: Hypotonic membrane stretch (HMS) reversibly increased VDCCL current. HMS also induced caveolar reorganization as well as the phosphorylation of caveolin-1. Methyl-b cyclodextrin (MβCD) also reorganized caveolae and increased VDCCL. Subsequent application of HMS did not further increase the VDCCL, indicating stretch and cyclodextrin increased VDCCL via common pathway. Both the HMS and MβCD phosphorylated c-Jun N-terminal protein kinase (JNK), of which inhibition prevented the HMS-induced VDCCL facilitation. Caveolin-1 knockdown with siRNA blocked HMS-induced JNK phosphorylation and VDCCL activation. High KCl (70 mM)-induced [Ca2+]i increase was markedly facilitated by HMS and this facilitation was significantly attenuated by caveolin-1 knockdown. Finally actin cytoskeletal disruption with cytochalasin-D blocked HMS-induced phosphorylations of caveolin-1 and JNK and facilitation of VDCCL and [Ca2+]i increase.Conclusion: These results indicate hypotonic stretch activates VDCCL by actin-cytoskeleton-dependent caveolar reorganization. Phosphorylation of caveolin-1 seems to be closely associated with the caveolar remodeling. Activation of JNK is induced by caveolar remodeling and transduces this signal to VDCCL activation. These results define a novel function for caveolae, cavelolin-1 and JNK in stretch-induced activation of VDCCL.

C-39: The impact of cryopreservation on fitness of cell therapy products in human clinical trials – the MSC paradigm

Marrow-derived mesenchymal stromal cells (MSCs) from either autologous or from random donor origin are being investigated in a large number of clinical trials as a cellular pharmaceutical for treatment of immune disorders and in regenerative medicine applications. Most typically, a polyclonal pool of culture expanded cells is cryopreserved after serial passaging and banked for later use. Human subjects subsequently receive an intravenous transfusion of MSCs which were retrieved from cryostorage no more than a few hours beforehand. It is assumed that viable, immediate post-thaw MSCs deploy the same biochemical, homing and immune modulatory features as their pre-freeze counterparts. We here propose that this assumption may be erroneous and may provide, in part, an explanation for discrepancy between pre-clinical models of MSC effectiveness and negative outcome of a large prospective randomized Phase III clinical trial (NCT00366145) of random donor MSCs for treatment of steroid-resistant acute GvHD. We show that MSC functional fitness as a transfusional product can be significantly altered due to post thaw heat shock response and cytoskeletal disruption.

Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte-neuron interaction.

Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 μM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers βIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection.

Macrophage induced gelsolin in response to Group B Streptococcus (GBS) infection.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences. We have shown that interaction of macrophages with GBS causes macrophage calpain activation, cytoskeletal disruption and apoptosis, consequences of intracellular calcium increase induced by membrane permeability alterations provoked by GBS-β-haemolysin. Open question remains about what effect calcium influx has on other calcium-sensing proteins such as gelsolin, involved in cytoskeleton modulation and apoptosis. Therefore we analysed the effect of GBS-III-COH31:macrophage interaction on gelsolin expression. Here we demonstrate that an early macrophage response to GBS-III-COH31 is a very strong gelsolin increase, which occurs in a time- and infection-ratio-dependent manner. This is not due to transcriptional events, translation events, protein turnover alterations, or protein-kinase activation, but to calcium influx, calpain activation and caspase-3 degradation. In fact, EGTA and PD150606 (calpain inhibitor) prevented gelsolin increase while BAF (caspase inhibitor) enhanced it. Since gelsolin increase is induced by highly β-haemolytic GBS-III-NEM316 and GBS-V-10/84, but not by weakly β-haemolytic GBS, or GBS-III-COH31 in conditions suppressing β-haemolysin expression/activity and the presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor), GBS-β-haemolysin is solely responsible for gelsolin increase causing, through membrane permeability defects, calcium influx and calpain activation. Early gelsolin increase could represent a macrophage response to antagonize apoptosis since gelsolin knockdown increases macrophage susceptibility to GBS-induced apoptosis. This response seems to be GBS specific because macrophage apoptosis by Staurosporine or Cycloeximide does not induce gelsolin.