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J Thyberg

Publications and source records attributed to J Thyberg.

At least 91 records · Page 5Linked to original sources
PubMed1987

Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype.

Isolated arterial smooth-muscle cells (SMCs) cultured in medium containing whole blood serum or plasma-derived serum undergo modulation from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and cessation of the ability to contract. Instead, an extensive rough endoplasmic reticulum and a large Golgi complex are formed and, if properly stimulated, the cells start to proliferate actively and to produce extracellular-matrix components. In vivo, a similar change in the differentiated properties of SMCs appears to be an early key event in atherogenesis. The purpose of the present investigation was to try to identify plasma components that promote the modulation of the smooth-muscle phenotype. SMCs were enzymatically isolated from rat aorta and cultured in a defined, serum-free medium. The phenotypic state of the cells was determined by transmission electron microscopy, and their growth status was followed by 3H-thymidine autoradiography and cell counting. Under these conditions, Cohn fractions I (fibrinogen) and V (albumin) were found to partially support cell attachment and transition from the contractile to the synthetic phenotype, whereas fractions II-III and IV (globulins) were inactive in this respect. Analysis on adsorptive columns of gelatin Sepharose 4B indicated that Cohn fraction I, but not fraction V, contained fibronectin, an adhesive protein that is present in plasma and binds to fibrinogen. When seeded on a substrate of plasma fibronectin, the cells attached with high efficiency and modulated into the synthetic phenotype at a rate similar to that observed in serum-containing medium. In the absence of exogenous mitogens, the structural transformation of the cells was not accompanied by a proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The calcium antagonist nisoldipine and the calmodulin antagonist W-7 synergistically inhibit initiation of DNA synthesis in cultured arterial smooth muscle cells.

Cultured arterial smooth muscle cells go through a transition from a contractile to a synthetic phenotype. Morphologically, the transition includes a reduction in size of the myofilament bundles and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Functionally, it leads to loss of contractility, onset of cellular proliferation, and secretion of extracellular matrix components. This change in differentiated characteristics under in vitro conditions has attracted attention because of its resemblance to the modification of the smooth muscle cells that occurs in vivo during atherogenesis. Here, transmission electron microscopy and [3H]-thymidine autoradiography were used to study the role of calcium ions in the control of phenotypic properties and growth of cultivated rat aortic smooth muscle cells. The calcium antagonist nisoldipine was found to lack distinct effect on the structural reorganization of the cells, but showed a moderate prohibitory effect on the start of DNA synthesis early in primary culture. In growth-arrested secondary cultures, nisoldipine inhibited induction of DNA synthesis by serum or platelet-derived growth factor (PDGF). The agent's effect was inversely related to the concentration of calcium ions in the extracellular medium and was partially counteracted by the calcium agonist BAY K 8644. In contrast, W-7, an antagonist of the calcium-binding protein calmodulin, potentiated the effect of nisoldipine and, at higher concentrations, inhibited induction of DNA synthesis in itself. The results suggest that the mitogenic stimulation of arterial smooth muscle cells involves a flux of calcium ions through the plasma membrane and requires participation of calmodulin.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Leukotrienes stimulate initiation of DNA synthesis in cultured arterial smooth muscle cells.

The effects of leukotrienes on initiation of DNA synthesis in growth-arrested arterial smooth muscle cells cultivated in a defined, serum-free medium were studied. The results showed that LTB4, LTC4 and LTD4 were all stimulatory with a distinct effect already at 0.01 pM and a maximal effect at 10 pM; in contrast, LTE4 lacked effect. 5S,12S-DHETE, an isomer of LTB4, was inactive in itself but blocked the effect of LTB4. Treatment of the cells with indomethacin or acetylsalicylic acid, two cyclooxygenase inhibitors, blocked induction of DNA synthesis by LTB4, indicating that the effect of this compound was mediated by a cyclooxygenase product. Up to 10 pM, the leukotrienes stimulated initiation of DNA synthesis with similar potency to platelet-derived growth factor (PDGF). The maximum labelling index obtained with PDGF was, however, about twice that obtained with the leukotrienes. At suboptimal concentrations of PDGF, the leukotrienes had an additive effect. The prereplicative lag phase was 16-20 h with LTB4, 12-16 h with LTC4, and 8-12 h with PDGF. In metabolic experiments no signs of synthesis of leukotrienes were detected by cells stimulated with the calcium ionophore A23187, arachidonic acid, or LTA4 for 10-30 min, neither was any apparent degradation of LTB4 observed. On the other hand, LTC4 was transformed into LTD4 and LTE4. Taken together, the results indicate that leukotrienes are able to stimulate quiescent arterial smooth muscle cells to enter the cell cycle and to replicate their DNA. In vivo, stimulation of cell growth by leukotrienes could add to other established functions of these substances in tissue repair, inflammation, and atherogenesis.

Animals↗
PubMed1986

Uterine smooth muscle cells in primary culture. Alterations in fine structure, cytoskeletal organization and growth characteristics.

Smooth muscle cells (SMC) were enzymatically isolated from the myometrium of adult rat and human uteri and grown in primary culture. Cell fine structure and cytoskeletal organization were followed by transmission electron microscopy and cytochemical demonstration of actin filaments, microtubules and intermediate filaments, and initiation of DNA synthesis was investigated by thymidine autoradiography. During the first few days in culture the cells spread out on the substrate and went through a morphological transformation including loss of myofilaments followed by formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Actin filaments aggregated in stress fibers spanning the entire length of the cells and microtubules and intermediate filaments formed a radiating system originating in the juxtanuclear region. In vivo, the SMC contained intermediate filaments reactive for desmin, but as early as the first day of culture expressed vimentin as well. For five days at least, all cells remained positive for both proteins, but the staining for desmin decreased while that for vimentin increased. This structural modification was accompanied by initiation of DNA synthesis, with a peak on day 3 (45-55% labeled nuclei). Subconfluent, growth-arrested primary cultures responded weakly to purified platelet-derived growth factor and serum, and in secondary cultures no response to the mitogenic stimulation was obtained. The observations indicate that uterine SMC cultivated in vitro undergo a transformation from contractile to synthetic phenotype, similar to the transformation described previously for arterial SMC under the same conditions. The proliferative potential of the uterine cells is, however, markedly lower. The findings support the notions that the transition into synthetic phenotype is a necessary but not sufficient requirement for initiation of DNA synthesis in SMC and that visceral and vascular SMC represent separate differentiation pathways.

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↗
PubMed1986

Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF) and produce a PDGF-like protein.

It is shown here that the myogenic cell line L6J1, primary skeletal myoblasts, and primary adult arterial smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF). It is further demonstrated that conditioned media from L6J1 cultures contain material that (i) competes with 125I-labeled PDGF for binding to human fibroblasts, (ii) is specifically precipitated by antibodies against PDGF, and (iii) has a relative molecular mass comparable to that of PDGF and, after reduction, its constituent subunit chains. The secretion of PDGF-receptor-competing activity was at a maximum in exponentially growing cultures but remained at a high level also after the cells had become confluent, stopped dividing, and fused to form multinucleate myotubes. Similarly, it was previously demonstrated that adult rat arterial smooth muscle cells in primary culture produce a mitogenic protein with immunological and structural properties similar to PDGF. In accordance with these findings, it was recently shown that secretion of PDGF-like mitogens by a number of human tumor cell lines correlates with expression of the gene for the A chain rather than the B chain of PDGF. The results suggest that production of homodimers of PDGF A chains may stimulate proliferation of skeletal myoblasts and arterial smooth muscle cells in an autocrine or paracrine manner. This could fulfill important functions during myogenesis in the embryo as well as in tissue repair and atherogenesis in the adult.

Animals↗
PubMed1986

Phenotype modulation in primary cultures of smooth-muscle cells from rat aorta. Synthesis of collagen and elastin.

Early in primary culture, arterial smooth-muscle cells undergo a transition from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and of contractility. At the same time, an extensive rough endoplasmic reticulum and a large Golgi complex are formed, and active synthesis of DNA, RNA and proteins commences. In the present study, chemical and immunocytochemical methods were used to investigate the production of extracellular-matrix proteins in relation to this change in phenotypic properties. The results showed that the phase of rapid cellular proliferation that follows the structural modulation of smooth-muscle cells is associated with high rates of collagen and elastin synthesis, as measured by the incorporation of 3H-proline into 3H-hydroxyproline and 3H-valylproline, respectively. SDS-polyacrylamide gel electrophoresis and fluorography indicated that type-I collagen is the main collagen species synthesized by these cells. Smaller amounts of type-V collagen and (although not definitively identified) type-III collagen were also detected. Indirect immunofluorescence and immunoelectron microscopy demonstrated that smooth-muscle cells surround themselves with an incomplete basement membrane, containing laminin and type-IV collagen, and thin fibrils of type-I collagen. Adjacent to these fibrils, aggregates of amorphous, elastin-like material were also found. Our observations confirm and extend earlier notions of a close similarity between the behaviour of arterial smooth-muscle cells during in vitro cultivation and during the early stages of the formation of atherosclerotic lesions.

Animals↗
PubMed1986

In vitro effects of sodium lauryl sulfate on DNA synthesis and fine structure of human thymocytes.

Sodium lauryl sulfate, a model substance in testing skin irritability was examined with regard to its effects on DNA synthesis and fine structure of human thymocytes incubated in vitro. At 5 h, incorporation of 3H-thymidine into DNA was inhibited with a discernable effect at 3 X 10(-4) M in serum-free medium and at 6 X 10(-4) M in medium to containing 15% serum. Under the former conditions, the effect on DNA synthesis was accompanied by disintegration of the cells as observed electron-microscopically. In contrast, no clear effects on cell morphology were seen in the latter case, in spite of a strongly depressed replicative activity. After 48 h of culture in medium containing 15% serum, there was a marked decrease in cell number due to spontaneous cell death and, consequently, a low rate of DNA synthesis. Sodium lauryl sulfate suppressed the incorporation of 3H-thymidine into DNA at 1.2 X 10(-3) M. No clear alterations in cell morphology were observed at 3 X 10(-4) M, whereas signs of irreversible cell damage, including pyknotic nuclei, were seen at 6 X 10(-4) M and complete disruption of most cells occurred at 1.2 X 10(-3) M. The results show that sodium lauryl sulfate inhibits DNA synthesis in human thymocytes and that there is a partial discrepancy between this inhibition and the adverse effects on the structural organization of the cell. It is proposed that the irritant effect of sodium lauryl sulfate in vivo is due to the breaking up of lymphoid or other cells and a consequent release of material that gives rise to an inflammatory response.

Animals↗
PubMed1986

Pathways of endocytosis in cultured macrophages. Electron microscopic autoradiographic tracing of iodinated plasma membrane proteins.

Lactoperoxidase-mediated iodination at 4 degrees C--an established method for covalent labelling of plasma membrane proteins--and quantitative electron microscopic autoradiography were used to follow the pathways of endocytosis in mouse macrophages in vitro. Directly after the labelling, the autoradiographic grains were concentrated to the cell surface. After warming to 37 degrees C, radioactive material was rapidly internalized into cytoplasmic vesicles and subsequently transferred to lysosomes as well as to the Golgi complex. Maximum grain density (% grains/% volume) over the vesicles was observed after 15 min, over the lysosomes after 30 to 45 min and over the Golgi complex after 30 and 90 min. Throughout the experimental period (120 min), the vesicles showed the largest fraction of intracellular grains, but higher grain densities occurred in lysosomes as well as in stacked Golgi cisternae and Golgi-associated vesicles. In spite of the internalization process, the labelling of the cell surface came to a steady state already after 30 min and at all intervals more than 50% of the autoradiographic grains were localized to this compartment. About 25% of the cell-associated radioactivity was lost rapidly with a half-life of 20 to 25 min and the remaining 75% slowly with a half-life of 7 to 9 h. The results indicate that membrane internalized by endocytosis partly follows a route to the lysosomes and that, additionally, there exists a route to and through the Golgi complex. They further support earlier notions of a bidirectional traffic between the surface and interior of the cell and suggest that recycling of membrane components may take place from endocytic vesicles, lysosomes, as well as the Golgi complex.

Animals↗
PubMed1985

Adult human arterial smooth muscle cells in primary culture. Modulation from contractile to synthetic phenotype.

Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [3H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1-2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3-4 days. It was accompanied by initiation of DNA replication and mitosis. The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis.

Adult↗
PubMed1985

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages. Double-labeling experiments with horseradish peroxidase and ferritin.

A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0-45 min at 37 degrees C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5-10 min of exposure and after 25-30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.

Acid Phosphatase↗
PubMed1985

The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation.

Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.

Actins↗
PubMed1985

Microtubules and the organization of the Golgi complex.

Electron microscopic and cytochemical studies indicate that microtubules play an important role in the organization of the Golgi complex in mammalian cells. During interphase microtubules form a radiating pattern in the cytoplasm, originating from the pericentriolar region (microtubule-organizing centre). The stacks of Golgi cisternae and the associated secretory vesicles and lysosomes are arranged in a circumscribed juxtanuclear area, usually centered around the centrioles, and show a defined orientation in relation to the rough endoplasmic reticulum. Exposure of cells to drugs such as colchicine, vinblastine and nocodazole leads to disassembly of microtubules and disorganization of the Golgi complex, most typically a dispersion of its stacks of cisternae throughout the cytoplasm. These alterations are accompanied by disturbances in the intracellular transport, processing and release of secretory products as well as inhibition of endocytosis. The observations suggest that microtubules are partly responsible for the maintenance and functioning of the Golgi complex, possibly by arranging its stacks of cisternae three-dimensionally within the cell and in relation to other organelles and ensuring a normal flow of material into and away from them. During mitosis, microtubules disassemble (prophase) and a mitotic spindle is built up (metaphase) to take care of the subsequent separation of the chromosomes (anaphase). The breaking up of the microtubular cytoskeleton is followed by vesiculation of the rough endoplasmic reticulum and partial atrophy, as well as dispersion of the stacks of Golgi cisternae. After completion of the nuclear division (telophase), the radiating microtubule pattern is re-established and the rough endoplasmic reticulum and the Golgi complex resume their normal interphase structure. This sequence of events is believed to fulfil the double function to provide tubulin units and space for construction of the mitotic spindle and to guarantee an approximately equal distribution of the rough endoplasmic reticulum and the Golgi complex on the two daughter cells.

Animals↗
PubMed1985

Arterial smooth muscle cells in primary culture produce a platelet-derived growth factor-like protein.

Adult rat arterial smooth muscle cells (SMC) in primary culture modulate from contractile to synthetic phenotype. This process includes partial loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex. It gives the cells the ability to initiate DNA synthesis and actively proliferate when stimulated with serum or isolated growth factors. After a few divisions, growth becomes partly independent of exogenous mitogens and does not cease until multiple cell layers have been formed. Here, it is demonstrated that serum-free conditioned medium from primary cultures of adult rat arterial SMC contains a factor that initiates DNA synthesis in growth-arrested secondary cultures of SMC. The mitogenic activity was neutralized by antibodies to platelet-derived growth factor (PDGF), and no mitogenic activity occurred in conditioned medium from cultures pretreated with actinomycin D, excluding release into the medium of PDGF adsorbed to the plastic vessels during the initial culture in serum-containing medium. Exposure of human fibroblasts to samples of the conditioned medium at 4 degrees C inhibited subsequent binding of 125I-labeled PDGF. It was further shown that the SMC of the primary cultures were able to initiate DNA synthesis in a chemically defined medium lacking PDGF and other growth factors. During the early, most active, and partly autonomous growth phase, the SMC had a low binding capacity for 125I-labeled PDGF and responded but little to stimulation with exogenous PDGF. Later on, with increasing cell density and decreasing growth rate, the ability to bind and respond to exogenous PDGF increased. Taken together, the observations suggest that modulation of SMC from contractile to synthetic phenotype is accompanied by production of a PDGF-like protein and autocrine or possibly by mitogen-independent initiation of DNA synthesis. Functionally, this may be important during wound healing and in the development of atherosclerotic lesions.

Animals↗
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↗
PubMed1985

Cation exchanger properties of isolated rat peritoneal mast cell granules.

The synthetic carboxylic cation exchanger resin Amberlite IRC-50 was charged with histamine by suspension in histamine-containing solution with admixture of [14C]histamine. Mast cell granules were isolated from mast cells suspended in isotonic sucrose. The release of histamine induced from the two materials by superfusion with isotonic NaCl and KCL solutions showed identical kinetics, in accordance with the view that the release of histamine is due to a cation exchange: Na+ (K+) in equilibrium Hi+ at carboxyl groups in the granule heparin-protein complex.

Animals↗
PubMed1985

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes.

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.

Animals↗