Phenotypic features of stromal cells in normal, premalignant and malignant conditions.
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Actins↗
Biomedical subjects
G Gabbiani
Publications and source records attributed to G Gabbiani.
The biology of the myofibroblast.
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Animals↗
Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features.
It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.
Actins↗
The mammalian anti-alpha-smooth muscle actin monoclonal antibody recognizes an alpha-actin-like protein in planaria (Dugesia lugubris s.l.).
The presence of an alpha-smooth muscle (alpha-sm) actin-like protein in planaria (Dugesia lugubris s.l.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti alpha-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the alpha-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of alpha-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the alpha-sm actin can be considered to be a marker for myoid differentiation. The suggestion that alpha-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.
Actins↗
Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung.
Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.
Actins↗
Heparin induces alpha-smooth muscle actin expression in cultured fibroblasts and in granulation tissue myofibroblasts.
BACKGROUND: Heparin increases alpha-smooth muscle actin expression in smooth muscle cells in vivo and in vitro. It has been recently suggested that alpha-smooth muscle actin expression in fibroblasts is a marker of myofibroblastic differentiation. We have examined the effect of heparin and of four nonanticoagulant heparin derivatives on alpha-smooth muscle actin expression by fibroblasts in vitro and in vivo. EXPERIMENTAL DESIGN: For in vitro experiments, heparin was added for 7 days to different fibroblastic cultures. We studied cell proliferation and alpha-smooth muscle actin protein and mRNA expression. For in vivo studies, osmotic minipumps filled with NaCl or tumor necrosis factor-alpha without or with nonanticoagulant heparin were implanted subcutaneously. After 14 days, newly accumulated connective tissues around the pumps were processed for immunofluorescence and electron microscopic and biochemical studies. RESULTS: In vitro, heparin inhibited proliferation and increased the expression of alpha-smooth muscle actin protein and mRNA. Analysis of [3H]thymidine incorporation in synchronized cells suggested that heparin produces a selection of alpha-smooth muscle actin expressing cells. In vivo, the local application of tumor necrosis factor-alpha resulted in formation of a typical granulation tissue: immunofluorescence showed that accumulated fibroblastic cells express alpha-smooth muscle actin only in the presence of heparin derivatives. In tumor necrosis factor-alpha treated animals, electron microscopic examination established the presence of myofibroblasts, but alpha-smooth muscle actin was expressed in microfilament bundles only in the presence of heparin derivatives. CONCLUSIONS: These results show that heparin and its nonanticoagulant derivatives influence the expression of alpha-smooth muscle actin in fibroblastic cells both in vitro and in vivo and that this effect is probably related to the selection of a particular cell subpopulation. They suggest a possible role for heparin during the formation and evolution of granulation tissue.
Actins↗
Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture.
Myofibroblasts are mesenchymal cells that are prominent in liver injury. The origin of myofibroblasts in liver is debated, although morphologic evidence to date has suggested that these cells are derived from lipocytes (fat-storing cells, Ito cells). In the present study, we have utilized smooth muscle alpha actin antibody--a marker of myofibroblasts and smooth muscle cells--to study lipocytes in situ in normal and fibrotic rat liver as well as during their 'activation' in culture. Dual immunofluorescence studies on tissue sections from normal liver identified lipocytes as perisinusoidal desmin-positive, smooth muscle alpha actin-negative cells. In bile duct obstructed fibrotic liver, desmin-positive cells were numerous in areas of fibrosis and these cells also exhibited smooth muscle alpha actin. In carbon tetrachloride-induced fibrosis, cells expressing both desmin and smooth muscle alpha actin were present in fibrotic bands and in regenerating nodules. These results suggested that lipocytes had acquired characteristics of myofibroblasts during liver injury. To further address this issue we examined lipocytes immediately after isolation and also in primary culture. In freshly isolated lipocytes from normal liver, smooth muscle alpha actin was absent. In contrast, freshly isolated lipocytes from CCl4-treated animals expressed this smooth muscle marker immediately after isolation. In primary culture on plastic, lipocytes from normal liver began to express smooth muscle alpha actin coincident with culture-induced activation; at 14 days, smooth muscle alpha actin was identified in all cells. Electron microscopy demonstrated a highly developed array of microfilament bundles characteristic of actin filaments. Immunoblot of culture-activated lipocytes using the smooth muscle alpha actin antibody demonstrated the expected 42 kD protein (corresponding to the molecular size of smooth muscle alpha actin). Although smooth muscle alpha actin was readily detectable in culture-activated cells, it was not expressed in cells in which a quiescent phenotype was preserved by maintenance in culture on a laminin-rich gel. These findings demonstrate that the acquisition by lipocytes of a smooth muscle marker accompanies their 'activation', and are consistent with the hypothesis that lipocytes transform to myofibroblasts during liver injury.
Actins↗
The cytoskeleton of arterial smooth muscle cells during human and experimental atheromatosis.
Smooth muscle cells (SMCs) implicated in the human atheromatous process show dedifferentiated features characterized by typical changes of cytoskeletal elements. In the normal media, quiescent SMCs express predominantly the alpha-smooth muscle (SM) actin isoform. In the human atheromatous plaque, and in rat experimental intimal thickening 15 days after balloon-induced endothelial injury, a decrease of the alpha-SM actin isoform and a predominance of the beta-cytoplasmic actin isoform develop. Proliferating SMCs in vivo assume fetal phenotypic features which are also observed in cultured SMCs. Thus, the study of cytoskeletal changes allows a better definition of SMC phenotype. Furthermore, in vitro SMCs may represent a useful experimental model to study phenotypic modifications of SMCs during the pathological process. Cytokines and growth factors, released by cells present in the atheromatous plaque, and extracellular components of the arterial wall, such as heparin, modulate the expression of alpha-SM actin in cultured SMCs and represent good candidates to exert important regulatory actions in vivo. A better understanding of the mechanisms leading to cytoskeletal modifications may help in the clarification of the mechanisms playing a role in the development of arterial pathological events.
Actins↗
Reversible expression of sm alpha-actin protein and sm alpha-actin mRNA in cloned cerebral endothelial cells.
The expression of smooth muscle (sm) alpha-actin was studied in cloned capillary cerebral endothelial cells of two phenotypes. Type I cells were cultured in medium containing 10% FCS, heparin and ECGS (or alpha-ECGF) and stained positive for a specific endothelial cell marker (Bandeiraea simplicifolia). Depletion of heparin and ECGS resulted in a smooth muscle-like appearance after 2-3 days. Cells of this phenotype, (type II) stained positive for the endothelial cell marker and for sm alpha-actin. In contrast to type I cells, type II cells expressed sm alpha-actin protein and mRNA as evidenced by Immunoblots and Northern blots. This phenotypic switch was shown to be reversible and so was the expression of sm alpha-actin.
Actins↗
Expression of alpha-smooth-muscle actin in stromal cells of the uterine cervix during epithelial neoplastic changes.
A total of 55 formalin-fixed and paraffin-embedded specimens of normal, inflamed and neoplastic uterine cervix have been studied in order to correlate the epithelial changes with the expression of alpha-smooth-muscle actin in stromal cells. This actin isoform is typical of smooth-muscle cells, but appears also temporarily in fibroblasts during wound healing and permanently during fibrocontractive diseases and stromal reaction to epithelial tumors. While positive stromal cells were absent in normal and inflamed cervix, they accumulated in relation to neoplastic tissues. The number of alpha-smooth-muscle actin positive cells and the intensity of stain were related to the increasing grading of cervical intra-epithelial neoplasia. Our results suggest that alpha-smooth-muscle actin is a marker of stromal-cell reaction to the development of neoplastic lesions. The evaluation of alpha-smooth-muscle actin in stromal cells of the uterine cervix may be a useful adjunct to diagnostic criteria of cervical intra-epithelial neoplasia and may help understanding of the mechanisms of mesenchymal-epithelial interactions during neoplasia.
Actins↗
The striated body: a new structure in the sperm head of the rabbit spermatozoon.
The striated body, a new structure lying in the perinuclear substance of rabbit spermatozoa, is described. It is composed of protein cords displaying a perpendicular periodic arrangement of dark and light bands. It appears in late spermatids, immediately prior to spermiation and is still present in epididymal spermatozoa, mostly, but not exclusively close to the perforatorium. The nature of this component has been analysed using a series of cytochemical techniques, as well as various antibodies and immunocytochemistry. The absence of RNA and of DNA favours a nonviral origin for this structure. The striated body is devoid of actin, differing in that respect from the rest of the perinuclear substance.
Animals↗
Locally applied GM-CSF induces the accumulation of alpha-smooth muscle actin containing myofibroblasts.
We have examined the histological and cytoskeletal changes in rat connective tissues induced by subcutaneous perfusion with cytokines. Granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha (IL-1-alpha), transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) produced a significant fibroblast accumulation, neovascular development and a weak to moderate leukocyte infiltration, while interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) induced intense mononucleated leukocyte infiltration. Immunofluorescence staining showed that accumulated fibroblastic cells were positive for alpha-smooth muscle (SM) actin (but negative for the desmin and muscle myosin) only in GM-CSF-treated tissues. Electron microscopic examination established that a significant proportion of fibroblastic cell in GM-CSF-, IL-1-alpha- or TGF-beta-treated animals were typical myofibroblasts. Only in GM-CSF-treated animals did microfilament bundles of myofibroblasts contain alpha-SM actin, when examined by immuno electron microscopy. Our results suggest that locally applied cytokines induce the formation of distinct granulation tissues. In particular, GM-CSF stimulates alpha-SM actin synthesis in myofibroblasts, illustrating an unexpected extra-hematopoietic in vivo effect of this factor.
Actins↗
Smooth muscle alpha-actin is a marker for hair follicle dermis in vivo and in vitro.
We have examined the expression of smooth muscle alpha-actin in hair follicles in situ, and in hair follicle dermal cells in culture by means of immunohistochemistry. Smooth muscle alpha-actin was present in the dermal sheath component of rat vibrissa, rat pelage and human follicles. Dermal papilla cells within all types of follicles did not express the antigen. However, in culture a large percentage of both hair dermal papilla and dermal sheath cells were stained by this antibody. The same cells were negative when tested with an antibody to desmin. Overall, explant-derived skin fibroblasts had relatively low numbers of positively marked cells, but those from skin regions of high hair-follicle density displayed more smooth muscle alpha-actin expression than fibroblasts from areas with fewer follicles. 2-D SDS-PAGE confirmed that, unlike fibroblasts, cultured papilla cells contained significant quantities of the alpha-actin isoform. The rapid switching on of smooth muscle alpha-actin expression by dermal papilla cells in early culture, contrasts with the behaviour of smooth muscle cells in vitro, and has implications for control of expression of the antigen in normal adult systems. The very high percentage of positively marked cultured papilla and sheath cells also provides a novel marker of cells from follicle dermis, and reinforces the idea that they represent a specialized cell population, contributing to the heterogeneity of fibroblast cell types in the skin dermis, and possibly acting as a source of myofibroblasts during wound healing.
Actins↗
Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies.
BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.
Animals↗
Phenotypic features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening. Biochemical and morphologic studies.
Balloon catheter denudation of rat carotid artery that results in significant medial damage is followed by marked intimal smooth muscle cell (SMC) proliferation associated with limited endothelial regrowth. In this report we demonstrate that: (a) SMC of the carotid media, preceding their intimal proliferation, develop a cytoskeletal profile and morphology consistent with a de-differentiated SMC phenotype; and (b) both medial and intimal SMC subsequently revert to a cytoskeletal profile and morphology reflecting incomplete but significant re-differentiation toward normal SMC phenotype. Specifically, early after balloon injury, SMC of the media and those that have migrated into the intima contain decreased amounts of actin, desmin, and tropomyosin and increased amounts of vimentin; moreover, beta-actin becomes the dominant actin isoform, whereas alpha-actin decreases as compared with that found in normal medial SMC. Late after balloon injury, actin is still less abundant, however, desmin, tropomyosin, and vimentin return toward normal values and both medial and intimal SMC again show a predominance of alpha-actin, although the endothelium does not regenerate over the central surface of intimal thickening in this model. The SMC surface to volume ratio significantly decreases early after balloon injury, whereas it is not significantly different late after balloon injury as compared with that of SMC of the normal carotid media. We demonstrate, furthermore that: (c) adjacent luminal SMC are interconnected by gap junctions and develop focal tight junctions, a feature not reported previously to occur in smooth muscle; these cells however do not form any well defined membrane specialization with the leading edge of endothelium, supporting the view that presence of modified SMC on the luminal surface of chronically denuded vessels is not responsible for the cessation of endothelial regrowth.
Actins↗
Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.
It has been suggested that perisinusoidal liver cells (PSC) play a pivotal role in the pathogenesis of fibrocontractive changes. Using light and electron microscopic immunolocalization techniques, a series of 207 normal and pathologic human liver specimens were evaluated for the expression of alpha smooth muscle (SM) actin and desmin in this and other nonparenchymal cell types. In normal adult liver tissue, PSCs were practically devoid of desmin and exceptionally stained for alpha-SM actin, whereas this actin isoform frequently was encountered in PSCs from the embryonic to the adolescent period. A broad spectrum of pathologic conditions was accompanied by the presence of alpha-SM actin containing PSCs; these were detected preferentially in periportal or perivenular zones according to the predominant location of the underlying hepatocellular damage. The occurrence of this PSC phenotype generally was associated with fibrogenesis and was in some cases detected earlier than overt collagen accumulation. Fibrous bands subdividing liver tissue in cirrhosis and focal nodular hyperplasia, as well as desmoplastic reaction to malignant tumors, contained alpha-SM actin-rich cells admixed with variable proportions of cells coexpressing desmin. In end stages, this population was less numerous than in active fibrotic or cirrhotic processes. Using immunogold electron microscopy, alpha-SM actin was localized in microfilament bundles of typical PSCs. Our results are compatible with the assumption that the appearance of alpha-SM actin and desmin-expressing myofibroblasts results at least in part from a phenotypic modulation of PSCs.
Actins↗
ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin.
The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.
ADP Ribose Transferases↗
Modulation of gelsolin content in rat aortic smooth muscle cells during development, experimental intimal thickening and culture. An immunohistochemical and biochemical study.
Gelsolin is a Ca2+ and polyphosphoinositol-phospholipid regulated modulator of actin polymerization present in most mammalian cells and in plasma. Cytoplasmic gelsolin was first described in highly motile cells such as leukocytes, where actin polymerization is dynamic; however arterial smooth muscle cells (SMC), despite their stabilized actin bundles, express high levels of gelsolin. We have investigated gelsolin modulation in rat aortic SMC by immunohistochemistry, Western blotting and Northern hybridization using three models which are known to show modulation of actin isoform expression: development, aortic intimal thickening after experimental endothelial injury, and growth in culture. When related to the protein and mRNA content of adult aortic SMC, gelsolin is expressed about 50% in aortic SMC of five-day-old rats, 20-30% in SMC of intimal thickening 15 days after endothelial injury (when SMC are actively replicating) and in SMC growing in culture; in intimal thickening 60 days after injury (when SMC have returned to quiescence), the gelsolin content becomes similar to that of control SMC. The high level of gelsolin in smooth muscle (SM) tissues and the down regulation with proliferation and migration raises the question as to whether gelsolin in these cells has functions other than the dynamic control of actin filament length. The similar modulation patterns of gelsolin and alpha-SM actin suggest a preferential interaction between these two proteins.
Aging↗