Number Of Stress Fibers Research Articles

Protein tyrosine phosphatase sigma and zeta1 regulate cardiac fibroblast contractile function through TGF-beta dependent and independent pathways

Abstract Background Heart failure is a frequently fatal cardiovascular disease with limited therapeutic options. To improve our understanding of signaling pathways in affected cells is therefore important. Protein tyrosine phosphatase sigma (PTPRσ) and zeta 1 (PTPRz1) have been shown to be involved in cardiac development and disease [1,2]. Both phosphatases are upregulated in patients with end stage heart failure. We demonstrated that PTPRz1 inhibits while PTPRσ is required for migration of neonatal cardiac fibroblasts (nCF) [3]. Purpose To further evaluate the influence of PTPRσ and PTPRz1 on fibroblast phenotype with regard to stress fiber formation and collagen contraction and their interaction with the TGFβ pathway. Methods nCF were transfected with either scrambled-, PTPRσ-, PTPRz1-, or both siRNAs. mRNA lysates were harvested 24h after transfection with subsequent rt-PCR analysis for the phosphatases. Transfected cells were seeded and stained with labeled phalloidin to measure the formation of stress fibers or in collagen gel matrices to evaluate their contractile capacities. To evaluate the nuclear translocation of SMAD3 immunohistochemical colocalization of SMAD3 and DAPI was quantified. Data were analyzed by Kruskal-Wallis and Mann-Whitney-U or ANOVA followed by t-test with Welsh correction using GraphPad prism. Results PTPRz1 and -σ knockdown (kd) was highly efficient (>85%). PTPRz1 mRNA was upregulated by 48% after PTPRσ kd (p < 0.05, n=4) but not vice versa and not after combined kd. Compared to control, nCF showed a 26% increase in the number of stress fibers in response to the PTPRz1 kd (p = 0.057, n=3-4) and an increase of 29% after PTPRσ/-z1 kd (p < 0.05, n=4). Collagen gels were contracted to about 70% of their surface by TGFβ in control (p<0.05, n=4) or any kd condition. They were also contracted by 24,5% after PTPRz1 knockdown (p < 0.01, n=4), whereas the additional kd of PTPRσ obliterated the effect. Nuclear translocation of SMAD3 was roughly doubled upon TGFβ stimulation in controls and all kd conditions. In unstimulated nCF, PTPRσ kd induced a 28%, PTPRz1 kd a 45%, and their combination a 114% increase of nuclei positive for SMAD3 (p < 0.05). Conclusion SMAD3 nuclear translocation is additively induced by PTPRz1 and PTPRσ kd independent of TGFβ. Stress fiber formation, however, seems to be inhibited by PTPRz1 independent of PTPRσ, while PTPRz1-kd-induced collagen contraction does depend on the presence of PTPRσ. PTPRσ and PTPRz1 thus have separate, overlapping and antagonistic effects that are TGFβ dependent and independent. The elucidation of the involvement of the phosphatases in fibroblast signaling and function could improve our understanding of adverse remodeling during heart failure and deliver novel options for therapeutic interventions.

High Concentrations of Genistein Decrease Cell Viability Depending on Oxidative Stress and Inflammation in Colon Cancer Cell Lines.

Genistein could play a crucial role in modulating three closely linked physiological processes altered during cancer: oxidative stress, mitochondrial biogenesis, and inflammation. However, genistein’s role in colorectal cancer remains unclear. We aimed to determine genistein’s effects in two colon cancer cells: HT29 and SW620, primary and metastatic cancer cells, respectively. After genistein treatment for 48 h, cell viability and hydrogen peroxide (H2O2) production were studied. The cell cycle was studied by flow cytometry, mRNA and protein levels were analyzed by RT-qPCR and Western blot, respectively, and finally, cytoskeleton remodeling and NF-κB translocation were determined by confocal microscopy. Genistein 100 µM decreased cell viability and produced G2/M arrest, increased H2O2, and produced filopodia in SW620 cells. In HT29 cells, genistein produced an increase of cell death, H2O2 production, and in the number of stress fibers. In HT29 cells, mitochondrial biogenesis was increased, however, in SW620 cells, it was decreased. Finally, the expression of inflammation-related genes increased in both cell lines, being greater in SW620 cells, where NF-κB translocation to the nucleus was higher. These results indicate that high concentrations of genistein could increase oxidative stress and inflammation in colon cancer cells and, ultimately, decrease cell viability.

The effect of shear stress reduction on endothelial cells: A microfluidic study of the actin cytoskeleton.

Reduced blood flow, as occurring in ischemia or resulting from exposure to microgravity such as encountered in space flights, induces a decrease in the level of shear stress sensed by endothelial cells forming the inner part of blood vessels. In the present study, we use a microvasculature-on-a-chip device in order to investigate in vitro the effect of such a reduction in shear stress on shear-adapted endothelial cells. We find that, within 1 h of exposition to reduced wall shear stress, human umbilical vein endothelial cells undergo reorganization of their actin skeleton with a decrease in the number of stress fibers and actin being recruited into the cells' peripheral band, indicating a fairly fast change in the cells' phenotype due to altered flow.

Abstract 068: Interplay Between Nox5 and Endoplasmic Reticulum Stress Regulates Vascular Signalling in Human Hypertension

Nox5, a major ROS-generating oxidase in human vessels, regulates vascular contraction. We demonstrated an ER-perinuclear Nox5 localization and questioned the role of ER stress in Nox5 regulation. Vascular smooth muscle cells (VSMC) were isolated from small arteries from subcutaneous fat from normotensive (NT) and hypertensive (HT) subjects. Nox5 compartmentalization (cell fractionation); ROS generation (chemiluminescence); peroxiredoxin/DJ-1 oxidation, activation of ER stress and contractile signalling (IRE1α, Src, PKC, MLC phosphorylation; immunoblotting) and actin cytoskeleton organization (phalloidin staining) were assessed. In hypertension, ROS levels (139±27% vs NT, p<0.05), oxidation of peroxiredoxin (870.4±188.7% vs NT, p<0.05) and DJ-1 (125±34% vs NT, p<0.05) were increased. IRE1α phosphorylation was increased in the HT group (58±21% vs NT, p<0.05). ER stress inhibition (4-PBA, 1mM) reduced ROS levels in HT subjects (20±6% vs NT, p<0.05), suggesting association between ER and oxidative stress. Nox5 expression was increased in the HT group (103±23% vs NT, p<0.05) in a compartment specific manner: Nox5 levels were reduced in plasma membrane (45±7% vs NT, p<0.05), but increased in the ER/nuclear fraction (46±13% vs NT, p<0.05). IRE1 inhibition (STF083010, 60μM) decreased Nox5 expression in the HT group (65±5% vs Ctl, p<0.05), while induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression in cells from both groups (p<0.05). To investigate the role of Nox5 on contractile signalling, cells were treated with mellitin (100nM), a Nox5 inhibitor. ROS generation and phosphorylation of c-Src, PKC and MLC 20 induced by Ang II were reduced by mellitin in both groups (p<0.05 vs Ctl). In contrast, silencing of p22phox increased ROS and activation of c-Src, PKC and MLC 20 in both groups, an effect blocked by mellitin (p<0.05 vs Ctl). VSMC from hypertensive subjects had increased number of stress fibres, an effect attenuated by mellitin. Our findings demonstrate that Nox5 is upregulated in a compartment specific manner and is regulated by ER stress in hypertension. Nox5 upregulation influences pro-contractile signalling and cytoskeleton reorganization in VSMC, processes that contribute to vascular dysfunction in hypertension.

Activated protein C and thrombin participate in the regulation of astrocyte functions

Protein C anticoagulant system is a multifunctional cofactor-dependent system. In addition to anticoagulant function, activated protein C (APC) also exhibits neuroprotective activity in hypoxia and stroke, but there are no data on potential effects of APC on astrocytes. In the present work we have studied the influence of APC and thrombin on rat astrocytes in primary culture. It was found that thrombin at concentrations above 10 nM (1 U/mL) induced significant activation in the cultured astrocytes resulting in reactive astrogliosis. The cultures exposed to thrombin for 24 h demonstrated a significant increase in proliferation and the S100b protein expression. Thrombin at high concentrations produced visible changes in the cytoskeleton of astrocytes, in particular, an increase in the number of stress fibers in the cultured cells. Moreover, thrombin apparently affected astrocyte migration. Thus, the treatment of serum-starved astrocytes with thrombin resulted in changes in cell monolayer uniformity and formation of “free fields”. APC prevented thrombin-induced proliferation of astrocytes and the S100b protein expression, reducing the parameters under study to the control values. In addition, APC reduced thrombin-induced disorganization of fibrils and formation of “free fields”. The results have demonstrated a new aspect of the protective effect of APC, which suppresses astrocyte activation induced by the proinflammatory effect of thrombin. It suggests a potential application of APC as a regulator of astrogliosis in pathological brain conditions.

Effects of 5-Fluorouracil in Nuclear and Cellular Morphology, Proliferation, Cell Cycle, Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

The mechanical properties of a cell, as determined by its actin cytoskeleton, are important for nanoneedle insertion into a living cell

Previously, we reported that a nanoneedle of 200 nm diameter manipulated by an atomic force microscope apparatus could be inserted into a living cell. The insertion probabilities varied according to cell type. However, the nanoneedle was never successfully inserted into artificial liposomes. In the current study, we found that the stress fibers and actin filaments comprising the plasmalemmal undercoat are important, determining factors as to whether a nanoneedle can be successfully inserted into a cell. Depolymerization of microtubules increased both the number of stress fibers and insertion efficiency in NRK cells. These results indicate that the insertion efficiency of a nanoneedle (200 nm in diameter) into a cell with a smaller actin meshwork in its plasmalemmal undercoat is enhanced and the formation of stress fibers obviously contributes to this incremental enhancement. These facts are not only important as technical information to improve the efficiency of cell manipulation but also as observations of the mechanical properties of the native cell cortex.

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 μm thick type I collagen matrices. After 24 h, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24 h of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. Following 24 h exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho-kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture.

IpaA Targets β1 Integrins and Rho to Promote Actin Cytoskeleton Rearrangements Necessary for Shigella Entry

Shigella invasion into the colonic epithelium involves many steps including the formation of large membrane protrusions by the epithelial cells that facilitate bacterial engulfment. IpaA, a Shigella protein secreted into target cells upon cell contact induces a loss of actin stress fibers in cells and promotes the reorganization of actin at the site of entry. The mechanism for this is not known but is thought to involve recruitment of the focal adhesion protein vinculin to IpaA. Here we have examined the mechanism for the effects of IpaA on the actin cytoskeleton. We show that IpaA-induced loss of actin stress fibers and cell rounding do not require vinculin expression or an intact vinculin binding site on IpaA. Rather, we find that cells expressing IpaA exhibited elevated Rho activity and increased myosin light chain phosphorylation. In addition, IpaA decreases integrin affinity for extracellular matrix ligands by interfering with talin recruitment to the integrin cytoplasmic tail. The combination of these two effects, namely weakened adhesion and increased contractility, account for the loss of actin stress fibers and cell rounding observed in cells exposed to IpaA.

Expression of a naturally occurring constitutively active variant of the epidermal growth factor receptor in mouse fibroblasts increases motility

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor receptor (EGFR) in many cancers is associated with progression of superficial to invasive forms of the disease. The naturally occuring type III mutant epidermal growth factor receptor (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the epidermal growth factor receptor. EGFRvIII is expressed frequently by a number of human solid tumours including those of the lung, breast, prostate, brain and ovary. Our study was designed to investigate the effect of EGFRvIII expression on cell motility and compare it to that of ligand-activated EGFR using transfected fibroblasts. We show here using time-lapse video recording that expression of EGFRvIII greatly enhances the motility of fibroblasts independently of ligand stimulation. In addition, expression of EGFRvIII caused a marked increase in the number of cellular protrusions (lamellipodia) and a reduction in the number of stress fibers and focal adhesions. The EGFR tyrosine kinase inhibitor, AG1478, and the MEK inhibitor, U0126, blocked these cellular effects of EGFRvIII. Two cell lines expressing different levels of EGFR were used for comparison. The low-expressing cell line responded to EGF treatment by increasing motility in a manner very similar to the motility induced by EGFRvIII. In contrast, the high-expressing cell line responded to EGF by detachment from the extracellular matrix and decreased motility. Cellular detachment was correlated to a high phosphorylation of PLC-gamma, whereas increased motility was correlated to a high level of ERK phosphorylation. Overall these results indicate that tumor-associated EGFR mutations might be critical for tumor cell motility, invasion and thus progression of disease.

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.

Myosin light chain kinase inhibitors can block invasion and adhesion of human pancreatic cancer cell lines.

Invasion and metastasis of pancreatic cancer (PC) require cell motility and adhesion, which depend on the activity of cytoskeleton. A cytoskeletal component indispensable for these processes is myosin II, the cytoplasmic analogue of smooth and skeletal muscle myosin. Because the activity of myosin II is accelerated by phosphorylation of myosin II on its regulatory light chain (RLC) by myosin light chain kinase (MLCK), we used two specific MLCK inhibitors, ML-7 and ML-9, for suppression of motility and adhesion of PC cell lines. Both drugs were potent inhibitors, as measured by in vitro motility assay and adhesion assay. When treated with the same concentration of ML-7, the PC cells were rounded up, and the number of stress fibers was reduced markedly. The in vitro migration and adhesion of PC cells were inhibited by ML-7 and ML-9 in a dose-dependent manner, supporting a specific and competitive inhibition of MLCK by these drugs. The inhibition occurred at nontoxic concentrations. These results highlight the importance of myosin II in the invasion and metastasis of PC cells and suggest the possibility that blocking of myosin II activity by a specific MLCK inhibitor may be a therapeutic strategy for preventing the invasion and metastasis of PC.

Flow-Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Objective: Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. Methods: Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-µm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 dyn/cm2). Results: On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8 ± 0.3 (mean ± SE, n = 36) (static) to 0.9 ± 0.5 per cell in perfused microchannels. Perfusion with 100 µM histamine prevented this response to flow (5.5 ± 0.8 per cell, n = 12). Stress fibers were initially aligned with the long axis of the microchannel at an angle of 9 ± 0.7° (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19 ± 4°; this was prevented by perfusion with histamine (5.6 ± 1°). The number of stress fibers per cell, for cells grown on flat surfaces (8.1 ± 0.3, static, n = 36) was significantly greater than for cells on curved surfaces, and did not change with flow (8.1 ± 0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation (with respect to the longitudinal axis of the channel) was 42 ± 1.4° (static) and did not change with flow (38 ± 4.2°). Conclusions: Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

Flow‐Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-microm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 (dyn/cm2). On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8+/-0.3 (mean +/- SE, n = 36) (static) to 0.9+/-0.5 per cell in perfused microchannels. Perfusion with 100 microM histamine prevented this response to flow (5.5+/-0.8 per cell, n = 12). Stress fibers were initially aligned with tile long axis of the microchannel at an angle of 9+/-0.7 degrees (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19+/-4 degrees this was prevented by perfusion with histamine (5.6+/-1 degrees). The number of stress fibers per cell, for cells grown on flat surfaces (8.1+/-0.3, static, n = 36) was significantly greater than cells on curved surfaces, and did not change with flow (8.1+/-0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation with respect to the longitudinal axis of the channel) was 42+/-1.4 degrees (static) and did not change with flow (38+/-4.2 degrees). Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

Flow-Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Objective: Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. Methods: Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-µm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 dyn/cm2). Results: On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8 ± 0.3 (mean ± SE, n = 36) (static) to 0.9 ± 0.5 per cell in perfused microchannels. Perfusion with 100 µM histamine prevented this response to flow (5.5 ± 0.8 per cell, n = 12). Stress fibers were initially aligned with the long axis of the microchannel at an angle of 9 ± 0.7° (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19 ± 4°; this was prevented by perfusion with histamine (5.6 ± 1°). The number of stress fibers per cell, for cells grown on flat surfaces (8.1 ± 0.3, static, n = 36) was significantly greater than for cells on curved surfaces, and did not change with flow (8.1 ± 0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation (with respect to the longitudinal axis of the channel) was 42 ± 1.4° (static) and did not change with flow (38 ± 4.2°). Conclusions: Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro.

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of alpha-smooth muscle (alpha-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, alpha-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor beta (TGFbeta) in FBS and PCS was determined and the effect of neutralizing anti-TGFbeta antibody was evaluated. It was determined that FBS contained more TGFbeta than PCS, but that essentially all the TGFbeta was latent in both sera. However, results obtained with anti-TGFbeta antibody show that active TGFbeta is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFbeta or an enzyme that is able to activate latent serum TGFbeta. Alternatively, sera may contain two different forms of latent TGFbeta, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds.

Down regulation of talin alters cell adhesion and the processing of the alpha 5 beta 1 integrin.

The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of beta 1. We analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.

Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin.

The bacterial pathogen Clostridium difficle synthesizes two high-molecular-weight toxins (A and B), which exhibit toxic effects in vivo and in vitro. Here, we present evidence that the major intracellular targets of these two toxins are the Rho GTPases. Overexpression of RhoA, RhoB, or RhoC GTPases in transfected HeLa cells conferred an increased resistance to toxins A and B, indicating that these toxins cause their cytopathic effects primarily by affecting Rho proteins. In addition, toxin A and B treatment appeared to result in modification of Rho, since Rho isolated from toxin-treated cells had a decreased ability to be ADP-ribosylated by Clostridium botulinum C3 exoenzyme. In contrast, the lethal toxin (LT) of Clostridium sordellii, although structurally and immunologically related to C. difficile toxin B, appeared to induce cytopathic effects independently of the Rho GTPases. Overexpression of RhoA in transfected HeLa cells did not protect them from the effect of LT, and Rho isolated from lysates of LT-treated cells was not resistant to modification by C3. Immunofluorescence studies showed that LT treatment caused a cytopathic effect that was very different from those described for C. difficile toxins A and B, resulting in an increase in cortical F-actin, with a concomitant decrease in the number of stress fibers, and in the formation of numerous microvilli containing the actin-bundling protein fimbrin/plastin.

Shock wave induced endothelial damage— In situ analysis by confocal laser scanning microscopy

For more than a decade, extracorporal shock wave lithotripsy has been a standard clinical method for the treatment of urinary stones. However, side effects that are likely to be correlated to vessel damage can often be observed using noninvasive diagnostic techniques, e.g., magnetic resonance imaging. To avoid side effects it is useful to understand the interaction between shock waves and the vascular system. In particular, this is important in view of new applications like gallstone lithothripsy. In the present study, we exposed human umbilical vessels to electromagnetically generated ultrasound shock waves to analyze subsequent alterations of their endothelial layer. Following en face preparation and fluorescent staining, the endothelium was examined in a confocal laser scanning microscope. Endothelial cells of the shock wave exposed vessels revealed permeabilization of plasma membranes and mitochondrial alterations as potentially lethal damage. An increase in the number of stress fibres may indicate functional changes possibly influencing vessel wall permeability.