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Results for “Phenotypic modulation”

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At least 19 recordsLinked to original sources

PubMed1990

Allylamine-induced phenotypic modulation of aortic smooth muscle cells.

Subchronic exposure of Sprague-Dawley rats for 20 days to allylamine resulted in a modulation of phenotypic expression of smooth muscle cells in vitro as characterized by alterations in cell morphology, ultrastructure, contractile function and synthetic/proliferative capabilities. Smooth muscle cells isolated from control animals were elongated and spindle-shaped at confluence, and contained a dense network of myofilaments. In contrast, cells isolated from treated animals were rounded and contained numerous ribosomes. Only cells obtained from control animals contracted in response to noradrenalin exposure (10 microM) in vitro. Smooth muscle cells obtained from allylamine-treated rats exhibited a 114% increase in 3H-thymidine uptake and a 204% increase in 3H-proline incorporation into collagen at confluence in comparison to controls. Serially passaged cells isolated from treated animals showed a similar enhancement (100%) in 3H-thymidine uptake. These results suggest that allylamine modulates aortic smooth muscle cells in vivo from a contractile to a more synthetic phenotype.

Allylamine↗

PubMed1991

Chronic treatment with P2-purinergic receptor agonists induces phenotypic modulation of the HL-60 and U937 human myelogenous leukemia cell lines.

In previous studies we have demonstrated that extracellular ATP (and UTP), acting through P2-purinergic receptors, can stimulate the inositol phospholipid signaling system in neutrophils and monocytes, as well as in neutrophil/monocyte progenitor cells. In this study we have examined the ability of extracellular nucleotides to modulate the phenotype of myelomonocytic progenitor cells. As model systems, we utilized the established HL-60 promyelocytic and U937 promonocytic human cell lines which were cultured in the continuous presence of nucleotides known to be potent agonists for P2-purinergic receptors. When cultured for 5 days with ATP gamma S (a phosphatase resistant analog of ATP) plus 10% fetal bovine serum, both HL-60 cells and U937 cells expressed several (but not all) phenotypic characteristics of differentiated phagocytes. In HL-60 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides, (2) a reduction in cell size with a decreased nuclear/cytoplasmic ratio, (3) a sharply reduced rate of proliferation, (4) a reduction in the percentage of cells expressing surface transferrin receptors, and (5) an increase in the percentage of cells expressing the type 1 complement receptor (CR1). In U937 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides and platelet activating factor, (2) a reduced rate of proliferation, (3) a reduction in the percentage of cells expressing surface transferrin receptors, and (4) increases in the percentage of cells expressing both type 1 (CR1) and type 3 (CR3) complement receptors. During the first 12-24 hr after exposure to ATP gamma S, HL-60 cells showed no obvious changes in morphology, viability, or the levels of beta-actin mRNA, but did show (1) a 4-fold increase in chemotactic peptide-induced Ca2+ mobilization, and (2) a greater than 90% decrease in c-myc mRNA levels. Significantly, when HL-60 cells were treated under serum-free conditions, the ability of ATP to enhance expression of functional FMLP receptors could be dissociated from the inhibitory effects of adenine nucleotides on cell proliferation observed in serum containing media. Moreover, treatment of serum-free HL-60 cultures with UTP, another P2-purinergic receptor agonist, also resulted in enhanced expression of functional FMLP receptors.

Actins↗

PubMed1990

Collagen synthesis by cultured arterial smooth muscle cells during spontaneous phenotypic modulation.

Quantitative and qualitative changes in collagen synthetic activity by rabbit arterial smooth muscle cells were monitored during spontaneous phenotypic modulation from days 2-15 of culture. The cultured smooth muscle cells transformed into a synthetic phenotype, reaching a maximum of 94.6% on day 4, and then gradually returned to a contractile phenotype accounting for 59.3% on day 15 of culture. The maximum collagen synthesis was found on day 7 when the cells were in early quiescent phase and showed a 91.7% synthetic phenotype. With an increasing proportion of cells in a contractile state, total collagen synthesis per cell decreased in parallel with the reduction in total protein synthesis. Synthesis of type I collagen was predominant, and the proportion of type I + III collagen was over 85% during the entire period of culture. Synthetic activity of type IV collagen, however, was relatively increased, and reached 3.8 +/- 0.4% at day 15 in comparison with 0.8 +/- 0.1% in the late logarithmic growth phase on day 4. This significant increment of type IV collagen in vitro seems to be correlated with the phenotypic modulation of cultured smooth muscle cells into a contractile phenotype.

Animals↗

PubMed1991

The role of vascular smooth-muscle cells in atherogenesis: phenotypic modulation of the medial smooth-muscle cells in the aortic bifurcation.

To elucidate the mechanism of migration of vascular smooth-muscle cells (SMCs) from media to intima, we have investigated the phenotypic modulation of the medial SMC at bifurcation of the celiac artery in 5 children and 3 young persons using a transmission electron microscope. We counted the number of separated SMCs from the elastic layers, although the medial SMCs are fundamentally attached to the elastic fibers, and are still contractile in their phenotypes. Both proximal and distal portions revealed that SMCs in the media were more or less present in the separated state and were ultrastructurally consistent with the synthetic state of SMC in the proximal area and the contractile state in distal areas. In order to migrate from media to intima, medial SMC should separate from the elastic layers and transform their phenotypes. In this paper, we examined the relationship between vascular SMCs and elastic layers in the media and ascertain that it was responsible for the contribution to the subsequent phenotypic modulation and their migration of medial SMCs.

Adolescent↗

PubMed1986

Surface proteins of Bordetella pertussis: comparison of virulent and avirulent strains and effects of phenotypic modulation.

The surface proteins of several Bordetella strains and their modulated derivatives were examined by surface radioiodination, cell fractionation, and Western blotting. A surface protein with a high Mr, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis and Bordetella parapertussis cells and was absent in avirulent B. pertussis strains. The electrophoretic profiles of lipopolysaccharide and the 40,000-Mr anion-selective porin were not determinants which correlated with phase variation or phenotypic modulation. At least three envelope proteins (91,000, 32,000, and 30,000 molecular weight) were found only in virulent B. pertussis strains and were absent or diminished in the avirulent phase and most phenotypically modulated strains. Two transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits.

Antibodies, Monoclonal↗

PubMed1988

In vitro and ex vivo regulation of vascular smooth muscle cell growth and phenotypic modulation by sulphated polysaccharides.

Heparin and pentosan polysulphate (PPS), a semi-synthetic sulphated polysaccharide, affected the phenotypic modulation of primary or subcultured rabbit smooth muscle cells (SMC) and at the same time inhibited their proliferation in culture. PPS was 5 fold more potent than heparin. Ex vivo, after subcutaneous administration of PPS or heparin (8 mg/kg/day for 13 days), SMC isolated from treated rabbits were growth-inhibited. At the same time, they reversed promptly to the contractile state as evidenced by immunofluorescent detection of intracellular myosin or electron microscopy. This ex vivo inhibitory effect was related to the dose and the duration of treatment, and was lost if 6 hours elapsed between the final dose and removal of the aorta. Such an effect was also observed after oral treatment with PPS (200 mg/kg/day for 7 days).

Animals↗

PubMed1990

Phenotype modulation in primary cultures of rat aortic smooth muscle cells. Effects of drugs that interfere with the functions of the vacuolar system and the cytoskeleton.

The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.

Animals↗

PubMed1988

Phenotypic modulation of hepatocarcinogenesis and reduction in N-nitrosomorpholine-induced hemangiosarcoma and adrenal lesion development in Sprague-Dawley rats by dehydroepiandrosterone.

Dietary administration of 0.25% dehydroepiandrosterone (DHEA) during and subsequent to 7 weeks treatment with N-nitrosomorpholine (NNM) resulted in significantly reduced development of adrenal cortical lesions and hemangiosarcomas in the liver. In addition, distinct phenotypic modulation of hepatocellular tumours was observed after combined hormone and carcinogen treatment. Thus the neoplasms were characterized by a higher degree of differentiation, lower mitotic rate and reduced potential for metastasis as compared to tumours observed after NNM alone. The data clearly indicate that DHEA exerts an inhibitory effect on both adrenal and hepatocarcinogenesis similar to that earlier reported for neoplasia in lung, thyroid, colon and skin.

Adenoma↗

PubMed1992

Phenotypic modulation of perisinusoidal cells following acute liver injury: a quantitative analysis.

Expression of the alpha-(smooth muscle) isoform of actin (alpha-SMA) by non-parenchymal cells in rat liver was studied following induction of acute liver injury using a single sublethal dose of carbon tetrachloride (CCl4). In normal liver, alpha-SMA immunoreactivity was identified in the smooth muscle cells of hepatic arteries and in the walls of portal and hepatic vein branches. Occasional alpha-SMA-containing stellate shaped cells were found in acinar zone 3 but most perisinusoidal cells (PSCs) did not express this protein. In CCl4-treated animals, there was an increase in the number of immunoreactive cells in perivenular zones, reaching a peak at day 3 following exposure to the toxin. These cells were morphologically identical to desmin-positive PSCs and the kinetics of the responses of alpha-SMA-positive and desmin-positive cells were similar. In en face labelling experiments, evidence of co-expression of alpha-SMA and desmin by non-parenchymal cells was obtained, although some desmin-positive PSCs did not appear to express alpha-SMA. These results suggest that PSCs rapidly undergo phenotypic modulation in response to acute liver injury with acquisition of alpha-SMA expression. It is proposed that these phenotypic changes coincide with functional alterations, such activated 'myofibroblast-like' cells being responsible for the enhanced matrix protein synthesis necessary for tissue repair.

Actins↗

PubMed1991

Phenotypic modulation in lipocytes in experimental liver fibrosis.

The presence of a-smooth muscle actin (smA)-positive cells has recently been reported in the fibrotic liver. Lipocytes have been considered to play important roles in hepatic fibrosis. However, the relation of the a-smA-positive cells and lipocytes has not been determined. The biological implication of a-smA expression remains unknown. To study these questions, we carried out double immunofluorescent staining of a-smA and desmin (a marker for lipocytes), or a-smA and collagen, and double immunohistochemical staining of a-smA and 5-bromo-2'-deoxyuridine (BrdUrd) in carbon tetrachloride-induced fibrotic rat livers. In normal and control livers, a-smA-positive cells were not seen in the lobules, whereas scattered desmin-positive cells were present. With the development of hepatic fibrosis, a-smA was expressed only in a portion of desmin-positive cells located predominantly around collagen bundles. A number of a-smA-positive cells in the lobules were labelled with BrdUrd. These results suggest phenotypic modulation in lipocytes and differentiation of lipocytes towards myofibroblast-like cells, since a-smA is expressed with desmin in myofibroblasts in scar tissue. The expression of a-smA may be related to events of the fibrotic process, such as tissue contraction or fibrogenesis per se.

Actins↗

PubMed1989

Altered synthesis of collagen types in cultured arterial smooth muscle cells during phenotypic modulation by dimethyl sulfoxide.

Synthetic activity of collagen types was examined in cultured arterial smooth muscle cells during modulation from synthetic to contractile phenotype by treatment with dimethyl sulfoxide (DMSO). Smooth muscle cells of rabbit thoracic aorta cultured with a 1% supplement of DMSO for 8 days (DMSO group) predominantly exhibited cellular features of the contractile type with abundant microfilaments and a distinct basement membrane. Cultured cells in the DMSO group or in controls during stationary or subconfluent phase were labeled with [3H]proline for 24 h, and the samples including the cell layer and medium were analyzed. The incorporation of proline into bacterial collagenase-digestible fractions was increased slightly in the DMSO group. Type analysis of the collagenous protein by SDS-PAGE and subsequent fluorography disclosed a markedly increased ratio of type IV/I collagen and a slightly increased type V/I collagen ratio, as compared with those of controls. A decrease of type III collagen production in DMSO-treated cells probably due to their lower cell density was also recognized. From these biochemical and morphological observations, it is suggested that increased synthesis of minor collagen types, particularly type IV collagen, is closely associated with smooth muscle phenotypic expression following DMSO treatment. Similar cellular events may occur in smooth muscle cells migrating into the intima during the process of arteriosclerosis in vivo.

Animals↗

PubMed1986

Effects of nicotine on phenotypic modulation and initiation of DNA synthesis in cultured arterial smooth muscle cells.

During the early stages of atherogenesis, as well as during in vitro cultivation, smooth muscle cells modulate from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex; it leads to decreased contractility and the commencement of cell growth and secretion of extracellular matrix components. In this paper, the effects of nicotine on adult rat arterial smooth muscle cells cultivated in vitro were studied by transmission electron microscopy and 3H-thymidine autoradiography. The results show that the drug speeded the initial rate of transition of the cells from contractile to synthetic phenotype in primary culture. Further, it stimulated the initiation of DNA synthesis in growth-arrested secondary cultures. Its effect was independent of other mitogens and additive to that of serum. The influences of nicotine, both on the modulation of the smooth muscle phenotype and the initiation of DNA synthesis, occurred at concentrations lower than those obtained in the blood after smoking and could contribute to the role of smoking as a risk factor for atherosclerosis.

Arteries↗

PubMed1992

Identification of the phenotypic modulation of rabbit arterial smooth muscle cells in primary culture by flow cytometry.

In atherosclerotic lesions, smooth muscle cells (SMC) change from a contractile to a synthetic phenotype. The in vivo and in vitro phenotypic transformations of SMC have been confirmed by transmission electron microscopy (TEM), but the relationship between this change and the cell cycle is still unknown. We demonstrated the structural modulation of rabbit arterial SMC in primary culture by TEM and immunocytochemistry and simultaneously studied changes in two-dimensional histograms of the relative DNA and RNA contents by flow cytometry. During the first day of primary culture, the cells exhibited the contractile phenotype and were composed of a population in the G0 phase characterized by low contents of DNA and RNA. On the second day of culture, some of the cells (18.2%) had started but not completed the transition into the synthetic phenotype and a cell population in the G1A phase with an RNA content above the G0 level appeared in almost the same proportion. This cell population could be categorized as an "intermediate" type. Moreover, after 3 days when about three-quarters of the cells had undergone structural transition, the same proportion of cells had entered into the cycling phase, while some cells still remained in the G0 and G1A phases. Thus, cell cycle analysis by flow cytometry corresponded well with the observations obtained by TEM and immunocytochemistry. These results show that flow cytometry can rapidly and relatively conveniently monitor the process of phenotypic modulation in SMC and is a useful method for the analysis of such transitions.

Animals↗

PubMed1986

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor.

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.

Animals↗

PubMed1992

Prevention of smooth muscle cell phenotypic modulation in vein grafts: a histomorphometric study.

This is a prospective study of the relationship between graft preparation technique and the subsequent morphologic fate of vein grafts. Paired vein grafts (optimal vs injury prepared) were placed in a canine model and removed over time. Vein grafts intentionally injured by warm saline storage demonstrated endothelial and smooth muscle cell damage. In the acute postimplantation period, platelet adhesion and white cell infiltration of the graft were present. By 7 days, the endothelium had "healed", but the underlying smooth muscle cells had modulated and were of the transitional or synthetic phenotype. This persisted at 30 days, but by 60 days the graft wall had remodeled to a contractile smooth muscle cell phenotype. Changes in the extracellular matrix were greatest at 30 days corresponding to changes in the smooth muscle cell phenotype. None of these injurious responses were noted in optimally prepared, papaverine treated vein grafts. The combined intima and media (lumen to adventitial edge) was measured at baseline and at graft excision with use of digitized graphic techniques. The intimal/medial thickness of injured vein graft walls was always greater than that of pair-matched optimally treated vein grafts (p less than 0.01 analysis of variance). Optimal preparation of vein grafts is effective in minimizing endothelial and smooth muscle cell injury at the time of arterial reconstruction. This preservation of endothelial and smooth muscle cell integrity prevents subsequent morphologic changes associated with the "arterialization response".

Analysis of Variance↗

PubMed1991

A standardised method of culturing aortic explants, suitable for the study of factors affecting the phenotypic modulation, migration and proliferation of aortic smooth muscle cells.

The study of factors affecting phenotypic change and growth of aortic smooth muscle cells (SMC) typically involves either the isolation of SMC by enzymatic dissociation or observation of outgrowth of cells from primary explants of vascular tissue. Explants provide a system in which the growth of cells can be investigated without dissociating them totally from their normal environment and avoids some of the problems of variability associated with enzymatic digestion. We describe here a standardised method for the preparation of medial explants of arterial tissue using a McIlwain tissue chopper, which is both fast and reproducible. Measurement was made of the percentage of explants showing outgrowth and of the distance migrated by cells at various times after plating explants singly into wells of a 96-well plate. Using this method, by 12 days after explanting, more than 95% of explants from normal rabbit aorta had shown outgrowth, in contrast to only 50% of explants prepared using a scalpel blade. Explants from atherosclerotic rabbit aorta showed a shorter lag phase before outgrowth commenced than explants from normal rabbit aorta of a similar age, but the subsequent rate of growth was the same. In contrast, when explants of normal rabbit aorta were grown in hyperlipidic rabbit serum, the lag phase was the same as for normal serum, but the subsequent rate of growth was greater. Explants from normal rabbit aorta treated with heparin showed an increased lag phase but reduced rate of growth. Treatment with heparinase decreased the lag phase and increased the rate of growth as did elastase.

Animals↗

PubMed1991

Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.

Cells, Cultured↗

PubMed1985

Phenotype modulation in primary cultures of arterial smooth-muscle cells: reorganization of the cytoskeleton and activation of synthetic activities.

During primary culture, arterial smooth-muscle cells (SMCs) undergo transition from a contractile to a synthetic phenotype. As a consequence, they lose the ability to contract and, instead, acquire the ability to synthesize DNA, divide and produce extracellular-matrix components. In the present study, we used cytochemical and electron-microscopic methods to study the organization of the cytoskeleton in primary cultures of adult rat and human arterial SMCs. Freshly isolated cells were all in contractile phenotype and stained intensely with NBD-phallacidin, a fluorescent marker for F-actin. Diffuse, positive staining was also obtained using indirect-immunofluorescence microscopy with antibodies against tubulin and vimentin, which are subunit proteins of microtubules and intermediate filaments, respectively. Fine structurally, the cytoplasm of these cells was mainly filled with microfilament bundles coalescing in dense bodies. After a few hours in culture, the SMCs attached to the substrate and started to extend processes in various directions. These stained with antibodies to tubulin and vimentin, but not with NBD-phallacidin. Within 1-3 days of culture, the cells spread out on the substrate and developed a system of actin-containing stress fibre bundles spanning their entire length, as well as a radiating system of microtubules and vimentin filaments, originating in the juxtanuclear region. Fine structurally, these changes corresponded to a marked decrease in the number of microfilaments, an increase in the number of microtubules and intermediate filaments, and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex. The morphological transformation of the cells was accompanied by the coordinated activation of DNA, RNA and protein synthesis.

Actins↗