Dose-dependent effect of nocodazole on endothelial cell cytoskeleton

The endothelium lining the inner surface of blood vessels fulfils an important barrier function and specifically, it controls vascular membrane permeability as well as nutrient and metabolite exchange in circulating blood and tissue fluids. Disturbances in vascular endothelium barrier function (vascular endothelium dysfunction) are coupled to cytoskeleton rearrangements, actomyosin contractility, and as a consequence, formation of paracellular gaps between endothelial cells. Microtubules constitute the first effector link in the reaction cascade resulting in vascular endothelium dysfunction. Increased vascular permeability associated with many human diseases is also manifested as a side effect in anticancer mitosis-blocking therapy. The aim of this study was to examine the possibility of preventing side effects of mitostatic drugs in patients with vascular endothelium dysfunction and to establish effective doses able to disrupt the microtubular network without interfering with the endothelial barrier function. Previously, it was found that the population of endothelial cell microtubules is heterogeneous. Along with dynamic microtubules, cell cytoplasm contains a certain amount of post-translationally modified microtubules that are less active and less susceptible to external influences than dynamic microtubules. We have shown that the area occupied with stable microtubules is relatively large (approx. one third of the total cell area). We assume that it can account for a higher resistance of the endothelial monolayer to factors responsible for vascular endothelium dysfunction. This hypothesis was validated in this study, in which nocodazole was used to induce vascular endothelium dysfunction in lung endothelial cells. The effect of nocodazole on endothelial cell cytoskeleton was found to be dose-dependent. Nocodazole in micromolar concentrations not only irreversibly changed the barrier function, but also upset the viability of endothelial cells and induced their death. Nanomolar concentrations of nocodazole also increased the permeability of the endothelial monolayer; this effect was reversible at the drug concentration ranging from 100 to 200 nM. At 100 nM, nocodazole induced partial disruption of the microtubule network near the cell margin without any appreciable effect on acetylated microtubules and actin filaments. At 200 nM, nocodazole exerted a pronounced effect on the system of dynamic (but not acetylated) microtubules and increased the population of actin filaments in the central region of the cell. Our data suggest that disruption of peripheral microtubules triggers a cascade of reactions culminating in endothelial barrier dysfunction; however, the existence of a large population of microtubules resistant to nanomolar concentrations of the drug provides higher viability of endothelial cells and restores their functional activity.     

Perturbation of host cell cytoskeleton by cranberry proanthocyanidins and their effect on enteric infections

Cranberry-derived compounds, including a fraction known as proanthocyanidins (PACs) exhibit anti-microbial, anti-infective, and anti-adhesive properties against a number of disease-causing organisms. In this study, the effect of cranberry proanthocyanidins (CPACs) on the infection of epithelial cells by two enteric bacterial pathogens, enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium was investigated. Immunofluorescence data showed that actin pedestal formation, required for infection by enteropathogenic Escherichia coli (EPEC), was disrupted in the presence of CPACs. In addition, invasion of HeLa cells by Salmonella Typhimurium was significantly reduced, as verified by gentamicin protection assay and immunofluorescence. CPACs had no effect on bacterial growth, nor any detectable effect on the production of bacterial effector proteins of the type III secretion system. Furthermore, CPACs did not affect the viability of host cells. Interestingly, we found that CPACs had a potent and dose-dependent effect on the host cell cytoskeleton that was evident even in uninfected cells. CPACs inhibited the phagocytosis of inert particles by a macrophage cell line, providing further evidence that actin-mediated host cell functions are disrupted in the presence of cranberry CPACs. Thus, although CPAC treatment inhibited Salmonella invasion and EPEC pedestal formation, our results suggest that this is likely primarily because of the perturbation of the host cell cytoskeleton by CPACs rather than an effect on bacterial virulence itself. These findings have significant implications for the interpretation of experiments on the effects of CPACs on bacteria-host cell interactions.     

The red cell membrane and its cytoskeleton.

Gel-filtration (Sephadex G-75) analysis of hepatic cytosol reveals both qualitative and quantitative sex differences in oestrogen-binding proteins. The elution profile of [³H]oestradiol-labelled cytosol shows four species of oestrogen-binding proteins (peaks I, II, IV and V) common to both sexes. The amount of [³H]oestradiol binding in peak I is equivalent in both males and females and corresponds quantitatively to the specific oestrogen receptor. The amount of binding in the remaining three peaks is greater in males than females. In addition, an oestrogen-binding protein (peak III) is present that is unique to male cytosol. Proteinase-inhibition studies demonstrate that the observed multiplicity of oestrogen-binding proteins is not an artefact of proteolytic breakdown. Sex differences in oestrogen-binding proteins are absent in immature male and female animals; the oestrogen-binding protein profile in immature rats resembles that of an adult female. Gonadectomy of adult animals does not affect the oestrogen-binding-protein profile. In contrast, neonatal (day 1) castration results in partial feminization of the characteristic oestrogen-binding protein profile seen in the adult male; the appearance of Peak III is suppressed and marked decreases in the amount of oestradiol binding occurs in the remaining peaks. Hypophysectomy of adult animals results in near abolishment of the observed sex differences; the male oestrogen-binding protein profile is partially feminized and the female profile is partially masculinized, as characterized by the appearance of [³H]oestradiol binding in the region of peak III and increased amounts of binding in peaks IV and V. The present studies demonstrate a multiplicity of oestrogen-binding proteins in liver cytosol and raise the possibility that the presence of some of these proteins may be imprinted at birth through the hypothalamic–pituitary axis, by a mechanism requiring neonatal androgen exposure.     

An analysis of the mechanics of guard cell motion

This paper presents a mechanical analysis of the cellular deformations which occur during the opening and closing of stomata. The aperture of the stomatal pore is shown to be a result of opposing pressures of the guard and adjacent epidermal cells. The analysis indicates that the epidermal cells have a mechanical advantage over the guard cells. With no mechanical advantage, an equal reduction in the turgor pressure of both guard and epidermal cells would have a neglible effect upon stomatal aperture. However, due to the mechanical advantage of the surrounding cells, the stomatal aperture increases with equal reductions in turgor, until the adjacent epidermal cells become flaccid. The minimum diffusion resistance of the pore occurs at this point. Further reductions in guard cell turgor lead to closure of the pore. The analysis further demonstrates how the shape, size, wall thickness and material properties of the guard cell walls influence their behavior.     

Cinobufacini induced MDA-MB-231 cell apoptosis-associated cell cycle arrest and cytoskeleton function

Cinobufacini is a traditional Chinese anti-tumor drug and widely used in clinic experiences. But little is known about its effect on the cells. In this study, the effects of cinobufacini on breast cancer MDA-MB-231 cell were evaluated by CCK-8 assay, and the data showed cinobufacini could inhibit the MDA-MB-231 cells growth effectively in dose-dependent and time-dependent manners. Cell apoptosis and cell cycle were detected by flow cytometry analysis. After the cells being treated with 50 μg/mL cinobufacini for 48 h, the early apoptosis percentage (20.45 ± 1.46%) is much higher than the normal group (7.73 ± 1.21%). The cell cycle data indicated that cinobufacini caused a cell cycle arrest at S phase. What's more, cinobufacini can affect the disruption of cytoskeleton, and these alterations changed the cell-surface ultrastructure and the cell morphology which were detected by atomic force microscopy (AFM) at nanoscale level. It indicated that the cell membrane structure and cytoskeleton networks were destroyed and the cell tails were narrowed after the cell being treated with cinobufacini. The present study is to provide valuable new insights to understand the mechanism of the drug in anti-tumor process. Furthermore, the knowledge concerning the signaling of cell cycle is potentially important to clinical utility.     

Multiscale modeling and mechanics of filamentous actin cytoskeleton

The adaptive structure and functional changes of the actin cytoskeleton are induced by its mechanical behavior at various temporal and spatial scales. In particular, the mechanical behaviors at different scales play important roles in the mechanical functions of various cells, and these multiscale phenomena require clarification. To establish a milestone toward achieving multiscale modeling and simulation, this paper reviews mathematical analyses and simulation methods applied to the mechanics of the filamentous actin cytoskeleton. The actin cytoskeleton demonstrates characteristic behaviors at every temporal and spatial scale, and mathematical models and simulation methods can be applied to each level of actin cytoskeletal structure ranging from the molecular to the network level. This paper considers studies on mathematical models and simulation methods based on the molecular dynamics, coarse-graining, and continuum dynamics approaches. Every temporal and spatial scale of actin cytoskeletal structure is considered, and it is expected that discrete and continuum dynamics ranging from functional expression at the molecular level to macroscopic functional expression at the whole cell level will be developed and applied to multiscale modeling and simulation.     

Calcium-independent cytoskeleton disassembly induced by BAPTA.

In living organisms, Ca2+ signalling is central to cell physiology. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) has been widely used as a probe to test the role of calcium in a large variety of cell functions. Here we show that in most cell types BAPTA has a potent actin and microtubule depolymerizing activity and that this activity is completely independent of Ca2+ chelation. Thus, the depolymerizing effect of BAPTA is shared by a derivative (D-BAPTA) showing a dramatically reduced calcium chelating activity. Because the extraordinary depolymerizing activity of BAPTA could be due to a general depletion of cell fuel molecules such as ATP, we tested the effects of BAPTA on cellular ATP levels and on mitochondrial function. We find that BAPTA depletes ATP pools and affects mitochondrial respiration in vitro as well as mitochondrial shape and distribution in cells. However, these effects are unrelated to the Ca2+ chelating properties of BAPTA and do not account for the depolymerizing effect of BAPTA on the cell cytoskeleton. We propose that D-BAPTA should be systematically introduced in calcium signalling experiments, as controls for the known and unknown calcium independent effects of BAPTA. Additionally, the concomitant depolymerizing effect of BAPTA on both tubulin and actin assemblies is intriguing and may lead to the identification of a new control mechanism for cytoskeleton assembly.     

Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line

Background

Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells.

Methods

Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1) cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers.

Results

Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21''s ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65.

Conclusion

Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells. Thus, it is worth noting that in p53 null or defective tumors, targeting in down-regulation of p65 may well be useful, leading to the potentiality of chemotherapeutic drugs.     

Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures

Endotoxins (lipopolysaccharide, LPS) from Gram-negative bacteria are considered as the agents responsible for the induction of endotoxic shock, producing severe cellular metabolic dishomeostasis. Cytotoxic lesions, as well as functional and metabolic disturbances, occur mainly in the liver, which is one of the target organs and exerts an LPS clearance function. In an attempt to approach the molecular basis of endotoxic shock, and to propose an experimental model, we have focused this study on cytoskeleton (microtubules and microfilaments) alterations induced by different doses of endotoxin in different target liver cells. Microfilaments and microtubules were studied by immunofluorescence and different microscopy techniques (optic fluorescence microscopy and confocal laser scanning microscopy) in order to improve the cytoskeleton study resolution. Parenchymal and sinusoidal cell morphology, severely damaged by the LPS action, is related to a disturbance on the cytoskeletal organisation, even more evident in a particular proliferating rat liver cell culture. The most relevant changes are seen in the microtubule patterns in all liver cells tested, which could be related, depending on cell type, either to a direct LPS action or to [Ca+2]i dishomeostasis as well as free radical and cytokine (IL-1beta and TNF-alpha) production. Due to their features, proliferating rat liver cell cultures are used as a sensitive cell model to understand the effect of LPS on cytoskeleton organisation.     

Macrovacuolation induced by cytochalasin: its relation to the cytoskeleton; morphological and cytochemical observations

At higher doses of cytochalasin (e.g. 3 micrograms/ml for 3-20 hr), cells of the rat fibroblastoid line, Hmf, undergo extreme retraction, arborization, and subsequent rounding, and develop big cystic vacuoles. These vacuoles are always closely invested by microfilamentous masses, the CD-induced derivatives of the actin-based cytoskeleton, which aggregate in the endoplasm. Vacuolation is progressive (e.g. 12% cells at 6 hr; greater than 80% at 18 hr), related to total dose (concentration X time); and to congener (CD greater than CB). Vacuole membranes have the same dimension (85 A), surface marker 5'-nucleotidase, and junctional specializations as those found at the cell surface; they lack the membrane markers associated with endomembrane systems (e.g. AcPase, TPPase, IDPase) and are not lysosomal. Vacuoles represent internalized plasma membrane; they apparently result from retention in the endoplasm, and fusion, of pinocytotic vesicles originating at the cell surface. Vacuole membrane is always in intimate relation to the actin-based microfilament aggregates that surround the vacuoles, and actin-membrane linker proteins fodrin and vinculin are localized at the vacuole boundaries. Vacuoles and their enveloping actin-filament aggregates are surrounded by arrays of vimentin-based intermediate filaments. A new membranous compartment with characteristics of plasma membrane is thus formed within the cell under the influence of CD. Rounding brought about by other means causes no vacuolization. Macrovacuolation, like the other changes caused by CD, is completely reversible on restoration of cells to normal medium.     

Varying amounts of acute static stretching and its effect on vertical jump performance

Numerous studies have shown that stretching routines can induce strength and force deficits, although the amount of stretching needed to cause these deficits remains unclear. Therefore, the purpose of the study was to examine the relationship between varying amounts of acute static stretching on jumping performance. By systematically increasing the amount of stretching, possible differences in jump height may be discovered, defining a line where acute static stretching becomes detrimental to performance. Ten collegiate athletes and 10 recreational athletes completed 3 different stretching treatments and 1 control treatment on different days in a within-treatment design. Stretching treatments consisted of 2, 4, or 6 sets of stretches, with each stretch held for 15 seconds with a 15-second rest. Stretches were done to the quadriceps, hamstrings, and plantar flexors. Upon arrival, each subject performed a 5-minute warm-up on a stationary upright cycle. After a brief rest period, participants performed 3 trials of a vertical jump test, followed by one of the treatment protocols. After another rest period, a second set of vertical jump trials was performed. Post-6 sets was significantly lower than Pre-6 sets (p < or = 0.05). Additionally, Post-6 sets was significantly lower than Pre-4 sets, Pre-2 sets, and Pre-control (p < or = 0.05). No other conditions were significantly different. In conclusion, 6 sets of stretches, or 90 seconds per muscle group, should not be performed before power activities such as jumping where optimal performance is desired.     

Cytoplasmic relocation of Daxx induced by Ro52 and FLASH

The RING-finger protein Ro52/TRIM21 is known to be an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. We recently showed that Ro52 is an E3 ubiquitin ligase and localizes to cytoplasmic bodies that are highly motile along the microtubule network. To expand our knowledge of Ro52, we searched partners co-operating with Ro52. We performed a yeast two-hybrid screening of a human brain cDNA library with Ro52 as bait. This screening identified several genes encoding Ro52-interacting proteins, including the apoptosis-related proteins, Daxx and FLASH. Further yeast two-hybrid assays revealed that Daxx binds to the B30.2 domain of Ro52 and that FLASH binds to coiled-coil domains of Ro52 through its death-effector domain-recruiting domain. These results suggest that Ro52, Daxx, and FLASH form heteromeric protein complexes. Indeed, this was supported by results of immunoprecipitation experiments in which we found that Daxx is co-immunoprecipitated with Ro52 in the presence of overexpressed FLASH. Importantly, our fluorescence microscopy revealed that, although Daxx is predominantly located in the nucleus, overexpression of both Ro52 and FLASH leads to relocation of Daxx into the cytoplasm. Thus, Ro52 seems to co-operate with FLASH to induce cytoplasmic localization of Daxx in cells.     

Apoptotic cell death induced by serum and its prevention by thiols

We recently reported that serum contains low molecular weight factors that inhibit growth and cause cell death in vitro. The present study focused on identifying components of basal media that counteract the toxic effects of serum. Amino acids L-cyst(e)ine and L-tryptophan were found to prevent serum-induced cell death of TIG-1 human fetal lung fibroblasts and other cell types. In addition to L-cysteine, other thiol-bearing and dithiol-cleaving compounds showed a similar ability to rescue the cells. Various inhibitors of protein or RNA synthesis also prevented the cell death. By contrast, nonthiol-containing reducing agents and super oxide dismutase (SOD), an active oxygen-eliminating enzyme, were ineffective. Thiol compounds appeared to exert a supportive level in TIG-1 cells cultured in FBS, whereas protein synthesis inhibitors did not alter the reduced intracellular thiol content. Fragmentation of DNA occurred prior to the plasma membrane breakdown of dying cells. Taken together, these data suggest that serum-induced cell death represents a form of apoptosis in which molecules containing thiol groups are active participants. © 1994 Wiley-Liss, Inc.     

The effect of ion channels on cell membrane motion

Cell membranes possess ion channels and pumps which allow them to pass electrical currents. In this paper it is shown that the ion channels and pumps should induce electric fields in the area surrounding the membrane. The magnitude of this field is computed. It is shown that when the channels and pumps are separated by a sufficient distance, then the electric fields which they induce will cause the solution which surrounds the membrane to move. This movement of the fluid should cause motion of the membrane. Thus it is demonstrated that under certain conditions the conductance of ions across cell membranes will give rise to cell membrane motion. It is suggested that this phenomenon may play a role in processes such as chemotaxis, cell division, and pinocytosis.     

Spectrin-level modeling of the cytoskeleton and optical tweezers stretching of the erythrocyte

We present a three-dimensional computational study of whole-cell equilibrium shape and deformation of human red blood cell (RBC) using spectrin-level energetics. Random network models consisting of degree-2, 3, ..., 9 junction complexes and spectrin links are used to populate spherical and biconcave surfaces and intermediate shapes, and coarse-grained molecular dynamics simulations are then performed with spectrin connectivities fixed. A sphere is first filled with cytosol and gradually deflated while preserving its total surface area, until cytosol volume consistent with the real RBC is reached. The equilibrium shape is determined through energy minimization by assuming that the spectrin tetramer links satisfy the worm-like chain free-energy model. Subsequently, direct stretching by optical tweezers of the initial equilibrium shape is simulated to extract the variation of axial and transverse diameters with the stretch force. At persistence length p = 7.5 nm for the spectrin tetramer molecule and corresponding in-plane shear modulus mu(0) approximately 8.3 microN/m, our models show reasonable agreement with recent experimental measurements on the large deformation of RBC with optical tweezers. We find that the choice of the reference state used for the in-plane elastic energy is critical for determining the equilibrium shape. If a position-independent material reference state such as a full sphere is used in defining the in-plane energy, then the bending modulus kappa needs to be at least a decade larger than the widely accepted value of 2 x 10(-19) J to stabilize the biconcave shape against the cup shape. We demonstrate through detailed computations that this paradox can be avoided by invoking the physical hypothesis that the spectrin network undergoes constant remodeling to always relax the in-plane shear elastic energy to zero at any macroscopic shape, at some slow characteristic timescale. We have devised and implemented a liquefied network structure evolution algorithm that relaxes shear stress everywhere in the network and generates cytoskeleton structures that mimic experimental observations.     

Effect of active recovery on acute strength deficits induced by passive stretching

We herein examined whether immediate muscular activity (active recovery) after stretching decreased stretch-induced strength deficits in human muscles. Our within-subject study included 8 subjects who were used as their own controls. For each subject, both legs were subjected to the same warm-up and stretching treatments, and then one leg was exposed to active recovery (experimental treatment) while the other was allowed to recover passively (control). Unilateral maximal voluntary contraction (MVC) of knee extensors was measured at baseline, poststretching, and postrecovery to monitor strength evolution. Our results revealed that the MVC strength at the baseline time point for control (590.8 +/- 104.2) and treated (602.2 +/- 112.7) legs decreased poststretching by 8.0 and 8.9%, respectively, and further decreased postrecovery by 1.3 and 1.2%, respectively. Maximal voluntary contraction strength tests demonstrated very good reliability, having intraclass coefficients of correlation ranging from 0.92-0.98. Mixed analysis of variance showed that the stretching program yielded significantly increased flexibility (p < 0.01) and significantly decreased MVC (p < 0.001) in both legs. The over-time variability between legs was marginal (1%), and no significant between-leg differences were observed. Indeed, the improvement in strength restoration due to active vs. passive recovery was -0.5 +/- 15 N, which was significantly lower (p < 0.01; 1-tailed t-test) than the amount of strength inhibition (32.6 N), estimated as 60% of the overall strength deficit (54.3 +/- 29.7 N). These results confirm that significant strength is lost poststretching but fail to show greater improvement in strength following active vs. passive recovery. Collectively, the present findings indicate that, contrary to the belief of many coaches, muscular exercises during the poststretching period are unlikely to minimize stretch-induced strength deficits.     

Permeabilization and lysis induced by bacteriocins and its effect on aldehyde formation by Lactococcus lactis

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.     

The effect of Chlamydia trachomatis infection on the host cell cytoskeleton and membrane compartments

Human epithelial cells and the McCoy cell line were infected with Chlamydia trachomatis, serotype E. The organization of the cytoplasm was then studied with probes which stained cytoskeletal components and membrane compartments. The major actin-containing stress fibre bundles were not associated with inclusions due to the peri-basal and peri-apical location of these bundles within the host cell. The cytokeratin network was distorted by the presence of inclusions so that a common basket of these intermediate filaments surrounded both nucleus and peri-nuclear inclusions. The microtubule network was similarly distorted, but the nucleus and inclusion were surrounded by separate rather than joint baskets of tubules. After reversible depolymerization by nocadazole the microtubules in amniotic epithelial cells began to reassemble at the peri-nuclear microtubule-organizing centre, so that independent microtubule networks were rapidly regenerated around the nucleus and inclusion. Mitochondria of amniotic epithelial cells were vitally stained with the fluorescent probe DiOC6 (3,3'-dihexyloxacarbocyanine iodide) after 48 h of infection and found to be widely distributed throughout the host cytoplasm. When the morphology of the Golgi complex was examined with C6-NBD-ceramide (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine) the main cisternae were retained in a juxta-nuclear position, although scattered stained structures were also present close to the cytoplasmic surface of the inclusion. These results demonstrate that the peri-nuclear position of inclusions is determined by the configuration of the cytoskeleton, and that normal host-cell architecture is maintained during infection, albeit in a distorted form.     

Biphasic effect of danazol on human vascular endothelial cell permeability and f-actin cytoskeleton dynamics

Breakdown of endothelial barrier function is a hallmark event across a variety of pathologies such as inflammation, cancer, and diabetes. It has also been appreciated that steroid hormones impart direct biological activity on endothelial cells at many levels. The purpose of this investigation was to explore the effect of the androgen-like steroid, danazol, on endothelial cell barrier function in vitro. Primary human endothelial cells exposed to 0.01-50 μM danazol were evaluated for changes in permeability. We found that danazol altered endothelial permeability in a biphasic manner in which nanomolar concentrations enhance barrier function while micromolar concentrations are detrimental. Monitoring of trans-endothelial electrical resistance demonstrated that these barrier enhancing effects were rapid (within 5 min) and lasted for over 24h. Analysis of intracellular f-actin organization showed that barrier enhancement also correlated with the formation of a submembranous cortical actin ring. Conversely, at higher danazol concentrations, contractile cell phenotypes were observed, represented by stress fiber formation. Competitive binding studies performed using steroid hormone receptor antagonists proved that this activity is the result of androgen and estrogen receptor ligation. These findings suggest that low dose danazol may provide a therapeutic window for diseases involving vascular leakage.     

mTOR pathway inhibition attenuates skeletal muscle growth induced by stretching

The present study has aimed to verify the influence of calcineurin and mTOR pathways in skeletal muscle longitudinal growth induced by stretching. Male Wistar rats were treated with cyclosporin-A or rapamycin for 10 days. To promote muscle stretching, casts were positioned so as completely to dorsiflex the plantar-flexor muscles at the ankle in one hind limb during the last 4 days of treatment with either cyclosporin-A or rapamycin. Thereafter, we determined soleus length, weight, protein content, and phenotype. In addition, NFATc1, Raptor, S6K1, 4E-BP1, iNOS, and nNOS gene expression in the soleus were determined by real-time polymerase chain reaction. Soleus length, weight, and protein content were significantly reduced by rapamycin treatment in animals submitted to stretching (P<0.05). In contrast, cyclosporin-A treatment did not alter these parameters. In all cyclosporin-A treated groups, there was a significant reduction in NFATc1 expression (P<0.001). Similarly, a significant reduction was noted in Raptor (P<0.001) and S6K1 (P<0.01) expression in all rapamycin-treated groups. No alteration was observed in 4E-BP1 gene expression among rapamycin-treated groups. Stretching increased gene expression of both NOS isoforms in skeletal muscle. Rapamycin treatment did not interfere with NOS gene expression (P<0.05). Cyclosporin-A treatment did not impair muscle growth induced by stretching but instead caused a marked slow-to-fast fiber shift in the soleus; this was attenuated by stretching. The data presented herein indicate that mTOR pathway is involved in skeletal muscle longitudinal growth. We gratefully acknowledge the financial support given by FAPESP.