Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains

We have previously demonstrated that or-smooth muscle (alpha -SM) actin is predominantly distributed in the central region and beta -non-muscle (beta -NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha -actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta -NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and ol-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha -SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha -actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sar-comere-like units with alpha -actinin-rich dense bodies analogous to Z-lines, are the contractile vimentin structures of cultured SMCs that link to the network of vimentin-containing intermediate alpha -actinin filaments through the dense bodies and dense plaques.

Circulating bone marrow cells can contribute to neointimal formation

To examine the source of smooth muscle-like cells during vascular healing, C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) nucleated bone marrow cells from congenic (Ly 5.1) male donors. Successful repopulation (88.4 +/- 4.9%) by donor marrow was demonstrated in the female mice by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody after 4 weeks. The arteries of the female mice were then subjected to two types of insult: (1) The iliac artery was scratch-injured by 5 passes of a probe causing severe medial damage. After 4 weeks, the arterial lumen was obliterated by a cell-rich neointima, with cells containing a smooth muscle actin present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y-chromosome-specific probe. (2) In an organized arterial thrombus formed by inserting an 8-0 silk suture into the left common carotid artery, donor cells staining with alpha smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage, Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair. Copyright (C) 2001 S. Karger AG, Basel.

Promoter studies of a polydnavirus gene from Cotesia rubecula (Hym : Braconidae)

The Cotesia rubecula polydnavirus gene, CrV1, is expressed in a highly transient fashion. Within four hours after egg deposition and virus infection, tissues of the host caterpillar, Pieris rapae, express high levels of the transcript. Twelve hours after infection no transcripts are visible. We have previously shown that the CrV1 secreted protein is mainly produced in host haemocytes. In haemocytes, immune functions such as phagocytosis and cell spreading are abolished by destabilization of the cell cytoskeleton. To test whether the observed down-regulation of CrV1 transcripts is mediated by transcriptional control or by other factors, such as the disruption of cytoskeleton in CrV1-inactivated cells, we cloned the promoter and the 3' untranslated region of the CrV1 gene to study CrV1 expression. The promoter region of the CrV1 gene was cloned into baculovirus expression systems along with the CAT reporter gene. Molecular analyses showed that the CAT gene under the control of CrV1 promoter is expressed as early as 2 h post infection and continues until late phase of infection suggesting that down-regulation of CrV1 expression in host haemocytes is perhaps mediated by post-transcriptional mechanisms.

Neointimal formation by circulating bone marrow cells

The origin of smooth muscle cells involved in vascular healing was examined. Eighteen C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) bone nucleated marrow cells from congenic (Ly 5.1) male donors. Successful repopulation by donor marrow was demonstrated after 4 weeks by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody. The iliac artery of six of the chimeric mice was scratch-injured by five passes of a probe, causing severe medial damage. After 4 weeks the arterial lumen was obliterated by a cell-rich neointima, with alpha-smooth muscle actin-containing cells present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y chromosome-specific probe. An organized arterial thrombus was formed in the remaining 12 chimeric mice by inserting an 8.0 silk suture into the left common carotid artery. Donor cells staining with alpha-smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton break-down. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

Reasons for performing study: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. Objectives: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. Methods: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. Results: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. Conclusions: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. Potential relevance: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Effectiveness of Thalidomide and Tamoxifen in Preventing Neointimal Hyperplasia in Experimental Vascular Injury in Rats

Introduction. Chronic allograft vasculopathy is an important cause of graft loss. Considering the inflammatory response in the development of chronic vascular lesions, therapeutic approaches to target the inflammatory process may be useful. We sought to investigate the possible protective effects on balloon catheter-induced vascular injury of thalidomide and tamoxifen, 2 drugs with powerful anti-inflammatory, immunomodulatory, and antifibrotic effects, using an animal model that mimics the morphologic features of chronic allograft vasculopathy. Methods. Male Wistar rats subjected to balloon catheter carotid injury (INJ) were treated with thalidomide (100 mg/kg), or tamoxifen (10 mg/kg), or vehicle. Contralateral right carotid arteries were used as uninjured controls. Morphometric and immunohistochemical analyses were performed at 14 days postinjury. Results. Injured carotid arteries showed marked neointimal hyperplasia, which was significantly inhibited among animals treated with thalidomide or tamoxifen: neointimal/media ratios of 1.4 +/- 0.4 versus 0.2 +/- 0.1 versus 0.4 +/- 0.2, for INJ, INJ + Thalid, and INJ + Tamox; respectively (P < .001). The endothelial cell loss was significantly less pronounced among animals subjected to carotid balloon injury that were treated with thalidomide (24 +/- 14 vs 1 +/- 1 cells per section in INJ, respectively (P < .05). Therapy with either thalidomide or tamoxifen effectively maintained alpha-smooth muscle actin expression in the media, similar to uninjured arteries. In this setting, tamoxifen was additionally effective to prevent the migration of myofibroblasts in to the intima. Conclusion. Thalidomide and tamoxifen were effective to reduce neointimal hyperplasia secondary to vascular damage. The vasculoprotective effects of thalidomide were more pronounced to preserve endothelial cells, whereas tamoxifen inhibited smooth muscle cell migration and proliferation. A possible beneficial effect of combined therapy with thalidomide plus tamoxifen should be addressed in future studies.

Muscle-specific regulation of tropomyosin gene expression and myofibrillogenesis differs among muscle systems examined at metamorphosis of the gastropod Haliotis rufescens

The spatial and temporal association of muscle-specific tropomyosin gene expression, and myofibril assembly and degradation during metamorphosis is analyzed in the gastropod mollusc. Haliotis rufescens. Metamorphosis of tile planktonic larva to the benthic juvenile includes rearrangement and atrophy of specific larval muscles, and biogenesis of the new juvenile muscle system. The major muscle of the larva - the larval retractor muscle - reorganizes at metamorphosis, with two suites of cells having different fates. The ventral cells degenerate, while the dorsal cells become part of the developing juvenile mantle musculature. Prior to these changes in myofibrillar structure, tropomyosin mRNA prevalence declines until undetectable in the ventral cells, while increasing markedly in the dorsal cells. In the foot muscle and right shell muscle, tropomyosin mRNA levels remain relatively stable, even trough myofibril content increases. In a population of median mesoderm cells destined to form de novo the major muscle of the juvenile and adult (the columellar muscle), tropomyosin expression is initiated at 45 h after induction of metamorphosis. Myofibrillar filamentous actin is not detected in these cells until about 7 days later. Given that patterns of tropomyosin mRNA accumulation in relation to myofibril assembly and disassembly differ significantly among the four major muscle systems examined, we suggest that different regulatory mechanisms, probably operating at both transcriptional and post-transcriptional levels, control the biogenesis and atrophy of different larval and postlarval muscles at metamorphosis.

Transcriptome changes induced by docetaxel in human mammary cell lines expressing different levels of ERBB2

The taxane docetaxel is currently the most effective chemotherapeutic drug for the treatment of advanced breast cancer. However, a considerable proportion of breast cancer patients do not respond positively to docetaxel. The mechanisms of docetaxel resistance are poorly understood. Overexpression of ERBB2 occurs in 15-30% of breast tumors and is associated with chemoresistance to a variety of anticancer drugs. In the present study, we sought to identify genes involved in ERBB2-mediated chemoresistance to docetaxel. We generated SAGE libraries from two human mammary cell lines expressing basal (HB4a) and high (C5.2) levels of ERBB2 before and after intensive exposure to docetaxel and identified potential ERBB2 target genes implicated in a variety of cellular processes including cell proliferation, cell adhesion, apoptosis and cytoskeleton organization. Comparison of the transcriptome of the cell lines before and after docetaxel exposure revealed substantially different expression patterns. Twenty-one differentially expressed genes between HB4a and C5.2 cell lines, before and after docetaxel treatment, were further analyzed by qPCR. The alterations in the expression patterns in HB4a and C5.2 cell lines in response to docetaxel treatment observed by SAGE analysis were confirmed by qPCR for the majority of the genes analyzed. Our study provides a comprehensive view of the expression changes induced in two human mammary cells expressing different levels of ERBB2 in response to docetaxel that could contribute to the elucidation of the mechanisms involved in ERBB2-mediated chemoresistance in breast cancer.

Expression of recombinant human follicle stimulating hormone mRNA in Chinese hamster ovary cells under different promoters

Levels of recombinant human follicle stimulating hormone (r-hFSH) mRNA expressed under butyrate and zinc treatment were compared in two CHO-K1 derived cell lines. In King cells under the metallothionein promoter, butyrate induced the increase in both r-hFSH productivity (q(FSH)) and mRNA levels proportionally. In the presence of 1 mM butyrate and 40 mu M zinc, a 4-fold increase in q(FSH) and mRNA levels was achieved as compared to zinc (40) alone; this wasa approximately 6 times higher than in serum free medium. In Darren cells under the beta-actin promotor butyrate induced an increase in q(SFH) but not in mRNA levels.

Fibroblastic rheumatism

Fibroblastic rheumatism (FR) was first described in 1980 by Chaouat et al., and there have been few cases reported to date. The cause remains unknown. We report the first Latin-American patient with FR, to our knowledge, who is also the patient with the most striking dermatological features described in the literature. The diagnosis was based on the presence of a number of typical features. Clinically, the patient presented skin nodules and polyarthropathy with flexion contractures of the fingers. The histological findings compressed fibroblastic proliferation, thickened collagen fibres, dermal fibrosis and a decreased number of elastic fibres. Immunoreactivity for beta-catenin, alpha-smooth muscle actin and the monoclonal antibody HHF-35 showed myofibroblastic differentiation. Treatment with prednisone slightly reduced the number of nodules but did not improve the rheumatological symptoms. This condition has shown a poor response to many treatments proposed by previous authors. Further study will be necessary to identify effective treatment.

A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor

The molecular mechanism by which polydnaviruses of endoparasitoid wasps disrupt cell-mediated encapsulation reactions of host insects is largely unknown. Here we show that a polydnavirus-encoded protein, produced from baculovirus and plasmid expression vectors, prevents cell surface exposure of lectin-binding sites and microparticle formation during immune stimulation of haemocytes. The inactivation of immune-related cellular processes by this protein was analysed using a specific lectin and annexin V and shown to be virtually identical to polydnavirus-mediated effects on haemocytes. Cytochalasin D application has similar effects on haemocytes, suggesting that the immune suppression by the polydnavirus protein is caused by the destabilization of actin filaments. Since the exposure of cell surface glycoproteins and the formation of microparticles are part of an immune response to foreign objects or microorganisms and a prerequisite for cell-mediated encapsulation of microorganisms and parasites, the virus-encoded protein may become an important tool for the inactivation of cellular immune reactions in insects and an essential component in understanding immune suppression in parasitized host insects.

Synergistic effect of ethanol and mitomycin C on corneal stroma

PURPOSE: To investigate the combined effects of ethanol and mitomycin C (MMC) application on the corneal stroma of rabbits that underwent photorefractive keratectomy (PRK). METHODS: Twenty-four rabbits (24 eyes) underwent PRK to correct -9.00 diopters of myopia. Twelve eyes had ethanol application before removing the epithelium and 12 eyes had the epithelium manually removed without ethanol, Eyes in both groups had topical MMC 0.02% application for 12 seconds immediately after excimer laser ablation. Twelve rabbits were sacrificed at two time points-4 hours and 4 weeks after surgery-and immunohistochemistry was performed with TUNEL assay, alpha-smooth muscle actin (alpha-SMA), and DAPI. RESULTS: More TUNEL-positive cells were observed in the ethanol-treated group compared to the mechanical debridement group at 4 hours after surgery (P<.01). No significant difference in alpha-SMA-positive cells was detected, between the two groups at 4 weeks after sugery. However, decreased keratocyte density in the anterior stroma was more pronounced in the ethanol-treated group compared to the mechanical debridement (P<.02). CONCLUSIONS: Ethanol application for epithelial removal during PRK seems to produce a synergistic effect with MMC, resulting in fewer keratocytes in the anterior stroma of rabbit corneas treated with MMC and ethanol than in corneas treated with MMC alone after PRK.

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p < 0.05), eosinophils counting (p < 0.001), iNOS-positive cells (p < 0.001), collagen and elastic fiber deposition (p < 0.05), actin density (p < 0.05) and 8-iso-PGF2 alpha expression (p < 0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p < 0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway. (C) 2008 Elsevier B.V. All rights reserved.

Different strains of mice present distinct lung tissue mechanics and extracellular matrix composition in a model of chronic allergic asthma

The impact of genetic factors on asthma is well recognized but poorly understood. We tested the hypothesis that different mouse strains present different lung tissue strip mechanics in a model of chronic allergic asthma and that these mechanical differences may be potentially related to changes of extracellular matrix composition and/or contractile elements in lung parenchyma. Oscillatory mechanics were analysed before and after acetylcholine (ACh) in C57BL/10, BALB/c, and A/J mice, subjected or not to ovalbumin sensitization and challenge. In controls, tissue elastance (E) and resistance (R), collagen and elastic fibres` content, and alpha-actin were higher in A/J compared to BALB/c mice, which, in turn, were more elevated than in C57BL/10. A similar response pattern was observed in ovalbumin-challenged animals irrespective of mouse strain. E and R augmented more in ovalbumin-challenged A/J [E: 22%, R: 18%] than C57BL/10 mice [E: 9.4%, R: 11 %] after ACh In conclusion, lung parenchyma remodelled differently yielding distinct in vitro mechanics according to mouse strain. (C) 2008 Elsevier B.V. All rights reserved.