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Biomedical subjects

M Absher

Publications and source records attributed to M Absher.

At least 19 recordsLinked to original sources
PubMed1998

Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

Actins↗
PubMed1996

Tamoxifen induces TGF-beta 1 activity and apoptosis of human MCF-7 breast cancer cells in vitro.

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-beta 1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-beta was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-beta.

Apoptosis↗
PubMed1995

Glucocorticoids coordinately regulate type I collagen pro alpha 1 promoter activity through both the glucocorticoid and transforming growth factor beta response elements: a novel mechanism of glucocorticoid regulation of eukaryotic genes.

Glucocorticoids have previously have shown to decrease Type I collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type I procollagen gene expression. These latter studies have included uridine incorporation into pro alpha 1 (I) and pro alpha 2 (I) mRNAs and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking region of the pro alpha 1 (I) collagen gene and the reporter gene, chloramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibroblasts that dexamethasone down regulates the promoter activity of the pro alpha 1 (I) collagen gene. The glucocorticoid-mediated down-regulation of procollagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whole ColCat 3.6 plasmid did not eliminate the effect. The possibility existed that another cis-element in the 5' flanking region of the pro alpha 1 (I) collagen gene was also required for the collagen glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate in a decrease of transforming growth factor-beta (TGF-beta) secretion into the media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid receptor binding to the GRE and TGF-beta activator protein to the TGF-beta element which were brought back to control values by coordinate exogenous TGF-beta treatment. Thus the interaction of these TGF-beta molecules with cellular membrane receptors and subsequent transduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expression through both the GRE and TGF-beta elements. Depression of procollagen gene expression by glucocorticoids through the TGF-beta element is mediated by decreased TGF-beta secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the basis for a novel mechanism of glucocorticoid-mediator regulation of eukaryotic genes containing the TGF-beta element.

Animals↗
PubMed1995

Glucocorticoid-induced down regulation of transforming growth factor-beta 1 in adult rat lung fibroblasts.

Transforming growth factor-beta 1 mRNA and transforming growth factor beta activity are decreased with exposure of normal adult rat lung fibroblasts to dexamethasone. Dexamethasone caused a decrease in transforming growth factor-beta 1 mRNA within 2 hours, which was sustained at least over a 24-hour period. The decrease in transforming growth factor-beta 1 mRNA was dose related. Dexamethasone treatment of rat lung fibroblasts also resulted in a decrease of transforming growth factor beta activity as determined by the mink lung cell growth inhibition assay. These data indicate that glucocorticoids may regulate collagen synthesis at least in part through the mediation of transforming growth factor-beta 1 in rat lung fibroblasts.

Animals↗
PubMed1995

Norepinephrine and ANG II stimulate secretion of TGF-beta by neonatal rat cardiac fibroblasts in vitro.

Transforming growth factor-beta (TGF-beta) is a ubiquitous growth-regulating protein that is capable of influencing the growth and function of heart cells in vitro. To better understand the role TGF-beta might play as a paracrine mediator of cardiac hypertrophy, the expression, secretion, and growth effects of TGF-beta were examined. Neonatal cardiac fibroblasts in vitro secreted latent TGF-beta 1 and TGF-beta 2 as high as 15 ng/10(6) cells. Angiotensin II (ANG II) and norepinephrine (NE) each augmented up to threefold the expression and secretion of latent TGF-beta 1 and TGF-beta 2 and also induced a shift in isoform predominance from beta 1 to beta 2. Each agent individually produced hypertrophic growth of neonatal cardiocytes and hyperplastic growth of cardiac fibroblasts. Paradoxically, the combination of NE and ANG II at intermediate and high concentrations resulted in less TGF-beta secretion (compared with either agent alone) and in hypertrophic growth of fibroblasts. These results suggest that the growth-promoting effects of ANG II and NE may in part be mediated via a paracrine stimulation of TGF-beta secretion.

Angiotensin II↗
PubMed1995

Bronchoalveolar lavage fluid from immature rats with hyperoxia-induced airway remodeling is mitogenic for airway smooth muscle.

We previously demonstrated that hyperoxia-exposed immature rats develop airway smooth muscle layer thickening; this remodeling appears partially attributable to smooth muscle hyperplasia. In this study, we tested the hypothesis that excess mitogenic activity for airway smooth muscle cells is present within the lungs of hyperoxia-exposed immature rats. We assessed the proliferative effect of bronchoalveolar lavage (BAL) fluid from air- and O2-exposed animals on cultured rat tracheal smooth muscle cells. BAL fluids from air- or O2-exposed immature rats increased DNA synthesis ([3H]-thymidine incorporation at 24 h of incubation) and cell number (compared with DMEM-treated control cells, at 2 days of incubation), but BAL fluid from O2-exposed animals had significantly greater mitogenic effects. This excess mitogenic activity was lipid inextractable and was ablated by trypsin digestion, indicating that at least one polypeptide growth factor was responsible; molecular sieve fractionation demonstrated a molecular weight of > 10 kD. Because platelet-derived growth factor (PDGF) has been identified in other models of hyperoxia exposure, we tested the further hypothesis that PDGF contributes to the observed excess mitogenic activity. Addition of neutralizing anti-PDGF antibodies to BAL-stimulated smooth muscle cultures did not reduce BAL fluid-induced mitogenesis. These data indicate that the lungs of O2-exposed rats contain excess mitogenic activity for airway smooth muscle, attributable to non-PDGF polypeptide growth factors. It is conceivable that this abnormal mitogenic activity contributes to O2-induced airway smooth muscle remodeling observed in immature rats in vivo.

Animals↗
PubMed1994

Hyperoxia inhibits proliferation of cultured rat tracheal smooth muscle cells.

Exposure of the lung to elevated oxygen leads to structural and cellular injury followed by extensive tissue remodeling. In vitro models utilizing isolated cells exposed to hyperoxic conditions or exogenously added oxidants may be injurious or stimulatory depending on the specific cell type and level and duration of exposure. In the present study, proliferation of cultured rat tracheal smooth muscle cells was inhibited by oxygen concentrations of 40 and 70% compared with a "normoxic" concentration of 21%. Exposure to 70% oxygen had a hypertrophic effect on the cells, as indicated by increased cellular protein content, whereas cells exposed to 21% oxygen did not show increased protein content. Exogenously added oxidant, H2O2, resulted in complete inhibition of growth of tracheal smooth muscle cells at concentrations > 3 microM. Much higher concentrations of H2O2 were required to inhibit proliferation of vascular smooth muscle cells and rat lung fibroblasts. The heightened sensitivity of airway smooth muscle cells to oxygen and oxidants may be an important factor in the early events following hyperoxia-induced lung injury.

Animals↗
PubMed1994

Automated procedures for the quantitation of protein.

An entirely automated 96-well microplate-based system to perform procedures for the measurement of protein is described. This single instrument system utilizes a series of computer-controlled mechanical subsystems to move the plate, control incubations and dispense samples or reagents in order to perform the assay. This system allows the investigator to reproducibly perform these protein assays on large numbers of biological samples with minimal effort.

Autoanalysis↗
PubMed1993

Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

Amino Acid Sequence↗
PubMed1993

Auto-induction of transforming growth factor-beta in human lung fibroblasts.

The type beta transforming growth factors (TGF-beta s) are a family of potent cytokines with diverse effects on proliferation, differentiation, turnover of extracellular matrix components, oncogene expression, and other aspects of cellular phenotype. Unlike lung fibroblasts of certain species, unstimulated human lung fibroblast lines produce little or no TGF-beta in culture. However, TGF-beta has been reported to autoregulate its own production in certain human tumor cells and in rodent cell lines. To test whether this phenomenon is operative in fibroblasts from normal human lung tissue, confluent cultures of IMR90 normal fetal lung fibroblasts were exposed to TGF-beta. Cultures were exposed briefly to purified TGF-beta 1 under serum-free conditions and secretion of newly synthesized TGF-beta over the ensuing 72 h was determined by immunoblotting and bioassays made specific with the use of neutralizing antibodies. Steady-state levels of mRNA for TGF-beta 1 were detected by Northern and slot blot hybridization analysis of total cellular RNA. The 2.5 kb TGF-beta 1 mRNA species rose within 1.5 h of exposure of IMR90 cells to TGF-beta 1 and reached maximal levels after 16 h. Increased levels of TGF-beta were detected in conditioned medium 9 h after the start of the exposure. Thereafter, TGF-beta continued to accumulate at an elevated rate (90 +/- 7 versus < or = 15 pg/10(6) cells/h in uninduced cells) for up to 72 h. As little as 1 ng/ml TGF-beta 1 auto-induced TGF-beta secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Variable expression of platelet-derived growth factor family proteins in acute lung injury.

During acute lung injury, there is an outpouring of growth factors into the alveolar space that drive local repair and fibrosis. During the remodeling that follows the instillation of bleomycin via the trachea into the adult rat, at least two platelet-derived growth factor (PDGF)-like peptides are released sequentially into lung lining fluid. Groups of four to five animals were killed at 3, 6, 15, and 26 days after exposure to bleomycin and lungs lavaged with isotonic saline. PDGF-like peptides in epithelial lining fluid (ELF) were partially purified by cation exchange chromatography and concentrated. Isolated peptides were analyzed by immunoblotting to determine their molecular weight and immunologic identity. Western blots were probed with polyclonal antibodies to PDGF-BB and PDGF-AA. PDGF-like peptides of two distinct size classes (38-40 kD and 29 kD) were present in alveolar fluid from all rats with lung injury induced by bleomycin. No PDGF-like peptides were found in comparably prepared ELF from control animals. The 38-40 kD peptide was detected only with anti-PDGF-BB antibody; the 29 kD peptide was detected only with anti-PDGF-AA antibody. The presence of these two peptides varied independently with time after exposure to bleomycin. The 38-40 kD peptide was at peak levels at 3 to 6 days. In contrast, the 29 kD peptide was present at all times following injury but with far less variation over time. In parallel with these immunoassays for PDGF-like molecules, there was abundant growth-promoting activity for fibroblasts present in concentrated ELF during the course of injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Spontaneous production of PDGF A-chain homodimer by rat lung fibroblasts in vitro.

Platelet-derived growth factor (PDGF) is considered a decisive mediator of fibroblast growth and phenotype within the lung. The cellular sources of PDGF within the lung remain undefined. The ability of lung fibroblasts themselves to produce PDGF in vitro was therefore investigated. Northern and Western blot analyses revealed the expression of PDGF-A mRNA and secretion of A-chain containing proteins by fibroblasts derived from adult and fetal rat lung. PDGF-A gene or protein expression were below the limits of detection in two human lung fibroblast lines examined in a similar manner. PDGF-B transcripts or proteins were not detected in any lung fibroblast line examined. Conditioned medium (CM) was collected from these same lung fibroblast lines and tested for its ability to promote cell growth using human fetal lung fibroblasts as targets. Both adult and fetal rat lung fibroblasts were found to produce a potent and efficacious stimulus for cell growth. Growth-promoting activity in rat fibroblast-derived CM functioned as a "competence" factor and was partially inhibited by anti-PDGF antibody. Thus rat lung fibroblasts in vitro produce potent growth factors of which at least one appears to be PDGF-AA. Differences in the expression of PDGF-AA between rat and human lung fibroblasts exist. Growth factor-producing fibroblasts may play a role in lung repair and remodeling through production PDGF-AA in vivo.

Animals↗
PubMed1992

Bleomycin regulation of transforming growth factor-beta mRNA in rat lung fibroblasts.

Pulmonary fibrosis is a well-known toxic response to bleomycin treatment. Here we demonstrate the direct effects of bleomycin on lung fibroblasts that resulted in a marked increase of collagen synthesis as compared with total noncollagen protein synthesis. Bleomycin treatment of rat lung fibroblast cultures resulted in an increase of total cellular transforming growth factor-beta (TGF-beta) mRNA and increased secretion of TGF-beta protein into the conditioned media. beta 2-Microglobulin was measured as an mRNA that did not increase with bleomycin treatment. The bleomycin-induced increase of TGF-beta mRNA was decreased by cells cultured in the presence of either cycloheximide, an inhibitor of protein synthesis, or 2-mercapto-1-(beta-4-pyridethyl) benzimidazole, an inhibitor of RNA synthesis. To assess the mechanism underlying increased steady-state mRNA levels, the nuclear fraction was isolated from bleomycin-treated cells and the TGF-beta transcripts were determined. Transcription of TGF-beta mRNA was increased 12 h after bleomycin treatment, whereas the transcription of type I procollagen, type III procollagen, and beta-actin mRNAs were increased after 48 h of bleomycin treatment. beta 2-Microglobulin mRNA synthesis was not increased within this time frame. These results suggest bleomycin regulation of TGF-beta at both the mRNA and protein levels. Rats lung fibroblasts were separated by cell sorting into two subpopulations. One population of fibroblasts demonstrated increased procollagen type I mRNAs, whereas fibroblasts in the other population had increased procollagen type III mRNA. Following bleomycin treatment, TGF-beta mRNA was shown to be located more prominently in those fibroblasts that contain primarily collagen type I mRNAs.

Animals↗
PubMed1991

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix.

The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells.

Animals↗
PubMed1991

Cytokine signaling in lung: transforming growth factor-beta secretion by lung fibroblasts.

Control of growth and phenotypic expression of interstitial fibroblasts is a critical determinant of lung architecture and physiology during processes of growth and remodeling. We examined the ability of lung fibroblasts to produce transforming growth factor-beta (TGF-beta), a cytokine that is known to modulate proliferation and phenotypic expression of mesenchymal cells. Cultures of fibroblasts isolated from rat lungs spontaneously secrete TGF-beta as measured in the standard bioassay of anchorage-independent growth of normal rat kidney (NRK) cells in soft agar. Rat lung fibroblasts secrete TGF-beta in an inactive precursor form. Fibroblasts cultured from adult and fetal rat lungs produced comparable amounts of TGF-beta. The ability of lung fibroblast supernatant fluids to induce colony formation in soft agar could be completely neutralized by preincubation of samples with anti-TGF-beta immunoglobulin (Ig). Anti-platelet-derived growth factor IgG had no effect on anchorage-independent growth of NRK cells driven by rat fibroblast culture supernatant samples. These results indicate that TGF-beta does not require the presence of and interaction with secondary cytokines for its activity. In contrast to the results obtained with rat cells, neither human fetal nor adult lung fibroblasts secreted detectable amount of active TGF-beta or its inactive precursor. This was not due to the presence of TGF-beta inhibitors in fibroblast culture media, because the addition of purified porcine TGF-beta to conditioned medium from human lung fibroblast cultures yielded the expected increase in NRK cell growth in soft agar. These results point to differing cytokine control patterns in the lungs of the two species.

Animals↗