Senescence as a main mechanism of Ritonavir and Ritonavir-NO action against melanoma.

The main focus of this study is exploring the effect and mechanism of two HIV-protease inhibitors: Ritonavir and Ritonavir-nitric oxide (Ritonavir-NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC50 values of Ritonavir-NO were approximately two times lower than IC50 values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of ß-galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir-NO treatment only in metastatic B16F10 cells, ranking it as a late-response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton-as we observed diminished expression of vinculin, α-actin, and ß-tubulin. Permanent inhibition of S6 protein by Ritonavir-NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO-modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.

Functional response of osteoblasts in functionally gradient titanium alloy mesh arrays processed by 3D additive manufacturing.

We elucidate here the osteoblasts functions and cellular activity in 3D printed interconnected porous architecture of functionally gradient Ti-6Al-4V alloy mesh structures in terms of cell proliferation and growth, distribution of cell nuclei, synthesis of proteins (actin, vinculin, and fibronectin), and calcium deposition. Cell culture studies with pre-osteoblasts indicated that the interconnected porous architecture of functionally gradient mesh arrays was conducive to osteoblast functions. However, there were statistically significant differences in the cellular response depending on the pore size in the functionally gradient structure. The interconnected porous architecture contributed to the distribution of cells from the large pore size (G1) to the small pore size (G3), with consequent synthesis of extracellular matrix and calcium precipitation. The gradient mesh structure significantly impacted cell adhesion and influenced the proliferation stage, such that there was high distribution of cells on struts of the gradient mesh structure. Actin and vinculin showed a significant difference in normalized expression level of protein per cell, which was absent in the case of fibronectin. Osteoblasts present on mesh struts formed a confluent sheet, bridging the pores through numerous cytoplasmic extensions. The gradient mesh structure fabricated by electron beam melting was explored to obtain fundamental insights on cellular activity with respect to osteoblast functions.

Schwann cells promote endothelial cell migration.

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.

Mechanical changes in human dental pulp stem cells during early odontogenic differentiation.

INTRODUCTION: Cell adhesion and migration in bioactive scaffolds require actin cytoskeleton remodeling and focal adhesion formation. Additionally, human dental pulp stem cells (hDPSCs) undergo several changes in their mechanical properties during odontogenic differentiation. The effect of factors essential for odontogenesis on actin stress fiber elasticity and focal adhesion formation is not known. METHODS: Live hDPSCs cultured in odontogenic media were imaged for cytoskeleton changes using an atomic force microscope. The Young's modulus (kPa) of the cytoskeleton was recorded as a function of culture medium for 10 days. Focal adhesion formation was assessed using immunofluorescence. Cultured hDPSCs were incubated with a monoclonal vinculin antibody, and filamentous actins were visualized using 0.5 µmol/L phalloidin. RESULTS: Cytoskeletal elasticity significantly increased in response to odontogenic media. Both the number and physical size of focal adhesions in hDPSCs also increased. Up-regulation of vinculin expression was evident. The increase in the formation of focal adhesions was consistent with actin remodeling to stress fibers. CONCLUSIONS: Our findings suggest that hDPSCs firmly attach to the glass substrate in response to odontogenic media. Successful regeneration of pulp-dentin tissue using biomimetic scaffolds will likely require cell-extracellular matrix interactions influenced by biochemical induction factors.

Morphological adaptation and protein modulation of myotendinous junction following moderate aerobic training.

Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.

Identification of vinculin as a potential plasma marker for age-related macular degeneration.

PURPOSE: To identify plasma protein biomarkers for age-related macular degeneration (AMD) using a large-scale quantitative proteomic discovery procedure. METHODS: Plasma proteomes from 20 exudative AMD patients and 20 healthy control patients were comparatively profiled by four-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins existing at statistically different levels were validated by enzyme-linked immunosorbent assay (ELISA) and Western blotting in 233 case-controlled samples. Newly discovered plasma biomarkers were further confirmed using in vivo and in vitro experiments. RESULTS: Out of 320 proteins identified, vinculin, protein S100A9, triosephosphate isomerase, protein S100A8, protein Z-dependent protease inhibitor, C-X-C motif chemokine 7, and tenascin X showed significantly differential expression in AMD patient plasma compared to control plasma. Among these, the area under the curve (AUC) for vinculin was 0.871 for discriminating between exudative AMD and controls (n = 201) and 0.879 for discriminating between AMD and controls (n = 233). A proteogenomic combination model using vinculin and two known risk genotypes in ARMS2 and CFH genes additionally provided excellent discrimination of AMD from controls (AUC = 0.916). The plasma level of vinculin was not associated with any confounding clinical variables, such as age, smoking, and other comorbidities. Additionally, vinculin was strongly expressed in retinal pigment epithelial cells of human eyes, and its expression was elevated when exposed to oxidative stress in vitro. CONCLUSIONS: Vinculin was identified as a potential plasma biomarker for AMD. The early detection of AMD using novel plasma biomarkers with genetic modeling may enable timely treatment and vision preservation in the elderly.

Non-metastatic 2 (NME2)-mediated suppression of lung cancer metastasis involves transcriptional regulation of key cell adhesion factor vinculin.

Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far, two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n=382), survival data (n=530) and lymph node metastases (n=100) in lung cancer patients identified non-metastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling. Importantly, we detected binding of NME2 in promoter of focal adhesion factor, vinculin. Reduced expression of NME2 led to enhanced transcription of vinculin. In comparison, NME1, a close homolog of NME2, did not bind to vinculin promoter nor regulate its expression. In line, enhanced metastasis of NME2-depleted lung cancer cells was found in zebrafish and nude mice tumor models. The metastatic potential of NME2-depleted cells was remarkably diminished upon selective RNA-i-mediated silencing of vinculin. Together, we demonstrate that reduced NME2 levels lead to transcriptional de-repression of vinculin and regulate lung cancer metastasis.

HAb18G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells.

Focal adhesions (FAs), integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147) is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC) cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001), length/width ratios (P<0.0001), and mean intensities (P<0.0001). CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t1/2) recovery times (22.7±3.3 s) of vinculin-mediated focal adhesions (P<0.0001). In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2). Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.

ROCK-2 is associated with focal adhesion maturation during myoblast migration.

Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell-ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK-2 during myoblast migration. In this study we report that ROCK inhibition with Y-27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y-27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK-2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK-2 containing adhesions in response to Y-27632. Taken together, our observations provide further proof that ROCK-2 regulates directional myoblast migration through focal adhesion formation and maturation.

MicroRNA-155 negatively affects blood-brain barrier function during neuroinflammation.

Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.

Mono-(2-ethylhexyl) phthalate (MEHP) promotes invasion and migration of human testicular embryonal carcinoma cells.

Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNA-binding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.

The chicken cornea as a model of wound healing and neuronal re-innervation.

PURPOSE: The cornea is the major refractive component of the eye and serves as a barrier to the external environment. Understanding how the cornea responds to injury is important to developing therapies to treat vision disorders that affect the integrity and refractive properties of the cornea. Thus, investigation of the wound healing responses of the cornea to injury in a cost-effective animal model is a valuable tool for research. This study characterizes the wound healing responses in the corneas of White Leghorn chicken. METHODS: Linear corneal wounds were induced in post-natal day 7 (P7) chicks and cellular proliferation, apoptosis and regulation of structural proteins were assessed using immunohistochemical techniques. We describe the time course of increased expression of different scar-related markers, including vimentin, vinculin, perlecan and smooth muscle actin. RESULTS: We find evidence for acute necrotic cell death in the corneal region immediately surrounding cite of incision, whereas we failed to find evidence of delayed cell death or apoptosis. We find that the neuronal re-innervation of SV2-positive axon terminals within the corneal stroma and epithelium occurs very quickly after the initial scarring insult. We describe an accumulation of cells within the stroma immediately underlying the scar, which results, at least in part, from the local proliferation of keratocytes. Further, we provide evidence for scar-induced accumulations of CD45-positive monocytes in injured corneas. CONCLUSIONS: We conclude that the chick cornea is an excellent model system in which to study wound healing, formation of scar tissue, and neuronal re-innervation of sensory endings.

Rho-Rho kinase pathway in the actomyosin contraction and cell-matrix adhesion in immortalized human trabecular meshwork cells.

PURPOSE: The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3). METHODS: Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK). RESULTS: Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. IC50 values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC50 values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK. CONCLUSIONS: Y-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes.

Imaging podosome dynamics and matrix degradation.

Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer.

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

Effects of mechanical stress on cell adhesion: a possible mechanism for morphological changes.

The transmission of mechanical forces to cells is followed among all by biological signals related to changes in the assembly or disassembly of integrins associated linker proteins, such as vinculin. We applied for 3 hours 2% cyclic mechanical strain at the frequency of 1 Hz to human fibroblasts cultured on a deformable substrate; substrate deformation resulted to modify the number, length and area of vinculin positive focal adhesion contacts when compared to not stretched cells. The mechanism behind these morphological changes is related to Akt and RhoA roles in focal adhesion assembly. In the case of Akt and Rho inhibition, focal contacts disassembled only in presence of stretching mechanical stress, highlighting the role of mechanical stress on focal adhesion maturation in terms of multimolecolar assembly which from focal complexes leads to fibrillar adhesion.

Unique contractile and structural protein expression in dog ileal inner circular smooth muscle.

This study was designed to test the hypothesis that there is heterogeneous expression of contractile and structural proteins between the smooth muscle cells (SMCs) in the inner and outer circular muscle (ICM and OCM) layers of the ileum. Immunohistochemical staining and quantitation of fresh frozen sections of the dog ileum was performed using protein specific antibodies. Smooth muscle (SM) SMA myosin heavy chain (MHC), alpha- and gamma-SM actin, and vinculin all show greater expression in the ICM relative to the OCM. SMB MHC and fibronectin show the opposite pattern, with greater expression in the OCM relative to the ICM. Differences in expression of these proteins are consistent with proposed differences in function of these muscle layers. Hypotheses regarding muscle tone and the coordination and regulation of peristalsis via these different muscle layers based on this data can now be made and tested.

Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions.

Structural characteristics of the tendinous cord-papillary muscle junction in healthy and hypertensive rats.

Although the myotendinous junction (MTJ) of skeletal striated muscle is well known, more detailed studies regarding the structure of the cardiac MTJ are scarce. The objective of the present study was to investigate the morphological characteristics of the MTJ in hearts of healthy and hypertensive (SHR) female rats using histological, ultrastructural and three-dimensional (SEM) methods, as well as to evaluate the expression of vinculin by immunofluorescence. In the two groups, light microscopy showed branching tendinous cords and collagen bundles penetrating the apex of the finger-like projections of the papillary muscle. SEM analysis revealed an enlarged apex of the papillary muscle in SHR which was not observed in healthy animals. The loss of force transmission appears to be compensated by the amplified connection between the papillary muscle and valvular collagen. A large number of intercalated disks close to the fiber apex, small amounts of an amorphous intercellular substance and numerous vesicles were observed in SHR. In these animals, the expression of vinculin was more marked showing a regular distribution and a pattern of transverse striations along the sarcolemma. The presence of this protein in transverse bands suggests that vinculin surrounds myofibrils in the region of the Z band. Vinculin staining was also more marked in the region of the tendinous cord-papillary muscle junctions of SHR compared to control animals. Vinculin was quantified by electrophoresis and higher amounts of this protein were observed in SHR compared to control animals.

Down-regulation of vinculin upon MK886-induced apoptosis in LN18 glioblastoma cells.

Glioblastomas are a type of malignant brain tumor and are among the most difficult cancers to treat. One strategy to treat aggressive cancers is the use of drugs that target multiple signaling pathways. MK886 is a drug known to inhibit both 5- lipoxygenase-activating-protein (FLAP) and peroxisome proliferator activated receptor-alpha (PPAR-alpha). The objectives of this study were to investigate the ability of MK886 to induce apoptotic cell death in LN18 glioblastoma cells and to characterize the cell death mechanisms. MK886 induced massive apoptotic LN18 cell death that was manifested by the release of nucleosomes, annexinV binding to phosphatidylserine in the absence of nuclear staining, and changes in the fluorescent intensity of Mito Tracker Deep Red 633 indicating changes in mitochondrial oxidative function and mass. The alteration of the mitochondrial function implied that MK886 induced apoptosis in LN18 cells via a mitochondrial pathway. The broad caspases inhibitor ZVAD-FMK inhibited MK886-induced nucleosome release, but not annexinV binding or MK886-altered mitochondrial function. Real time RT-PCR demonstrated that LN18 cells expressed significant levels of FLAP and PPAR- alpha mRNAs. A low level of arachidonate 5-lipoxygenase (ALOX-5) mRNA was detected, but little, if any, arachidonate 12- lipoxygenase (ALOX-12) mRNA was present. In addition, MK886-induced apoptosis in LN18 cells was accompanied by a decrease in the protein and mRNA levels of vinculin, but not other focal adhesion proteins. In summary, the data presented here indicate that disruption of the actin-vinculin-cell-cytoskeleton matrix of the LN18 glioblastoma is a component of the MK886 induced apoptosis. In addition, MK886 treated LN18 cells could provide one model in which to investigate drugs that target lipoxygenase and PPAR-alpha pathways in the chemotherapeutic treatment of glioblastomas.