Biomedical subjects

K L Dreher

Publications and source records attributed to K L Dreher.

At least 19 recordsLinked to original sources

PubMed2000

Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism.

Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 microg/cm(2) ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.

Animals↗

PubMed2000

In vivo and in vitro correlation of pulmonary MAP kinase activation following metallic exposure.

Residual oil fly ash (ROFA) is a particulate pollutant produced in the combustion of fuel oil. Exposure to ROFA is associated with adverse respiratory effects in humans, induces lung inflammation in animals, and induces inflammatory mediator expression in cultured human airway epithelial cells (HAEC). ROFA has a high content of transition metals, including vanadium, a potent tyrosine phosphatase inhibitor that we have previously shown to disregulate phosphotyrosine metabolism and activate mitogen-activated protein kinase (MAPK) signaling cascades in HAEC. In order to study MAPK activation in response to in vivo metal exposure, we used immunohistochemical methods to detect levels of phosphorylated protein tyrosines (P-Tyr) and the MAPKs ERK1/2, JNK, and P38 in lung sections from rats intratracheally exposed to ROFA. After a 1-h exposure to 500 microg ROFA, rat lungs showed no histological changes and no significant increases in immunostaining for either P-Tyr or phospho-(P-) MAPKs compared to saline-instilled controls. At 4 h of exposure, there was mild and variable inflammation in the lung, which was accompanied by an increase in specific immunostaining for P-Tyr and P-MAPKs in airway and alveolar epithelial cells and resident macrophages. By 24 h of exposure, there was a pronounced inflammatory response to ROFA instillation and a marked increase in levels of P-Tyr and P-MAPKs present within the alveolar epithelium and in the inflammatory cells, while the airway epithelium showed a continued increase in the expression of P-ERK1/2. By comparison, HAEC cultures exposed to 100 microg/ml ROFA for 20 min resulted in marked increases in P-Tyr and P-MAPKs, which persisted after 24 h of exposure. P-Tyr levels continued to accumulate for up to 24 h in HAEC exposed to ROFA. These results demonstrate in vivo activation in cell signaling pathways in response to pulmonary exposure to particulate matter, and support the relevance of in vitro studies in the identification of mechanisms of lung injury induced by pollutant inhalation.

Air Pollutants↗

PubMed2000

Induction of pulmonary matrilysin expression by combustion and ambient air particles.

The molecular mechanism(s) by which chemically complex air pollution particles mediate their adverse health effects is not known. We have examined the ability of combustion and ambient air particles to induce pulmonary matrilysin expression due to the well-documented role of matrix metalloproteinases in tissue injury and repair responses. Rats were exposed to saline, residual oil fly ash (2.5 mg/rat), or ambient air particles (2.5 mg/rat) via intratracheal instillation and examined 3-72 h after exposure. Saline-exposed animals had low levels of matrilysin mRNA, whereas the animals exposed to either complex particle showed an early induction of pulmonary matrilysin gene expression as well as of the 19-kDa activated form of matrilysin. Immunocytochemistry and in situ hybridization analyses identified the alveolar macrophages and monocytes as primary sources of air pollution particle-induced matrilysin expression. Matrilysin gene induction and protein activation by combustion and ambient air particles correlated with the early histopathological changes produced by these particles. These results demonstrate the ability of combustion and ambient air particles to induce pulmonary matrilysin expression and suggest a role for this matrix metalloproteinase in the initiation of lung injury produced by these particles.

Air Pollutants↗

PubMed1999

Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression.

Particulate matter (PM) metal content and bioavailability have been hypothesized to play a role in the health effects epidemiologically associated with PM exposure, in particular that associated with emission source PM. Using rat tracheal epithelial cells in primary culture, the present study compared and contrasted the acute airway epithelial effects of an emission source particle, residual oil fly ash (ROFA), with that of its principal constitutive transition metals, namely iron, nickel, and vanadium. Over a 24-h period, exposure to ROFA, vanadium, or nickel plus vanadium, but not to iron or nickel, resulted in increased epithelial permeability, decreased cellular glutathione, cell detachment, and lytic cell injury. Treatment of vanadium-exposed cells with buthionine sulfoximine further increased cytotoxicity. Conversely, treatment with the radical scavenger dimethylthiourea inhibited the effects in a dose-dependent manner. RT-PCR analysis of RNA isolated from ROFA-exposed rat tracheal epithelial cells demonstrated significant macrophage inflammatory protein-2 and interleukin-6 gene expression as early as 6 h after exposure, whereas gene expression of inducible nitric oxide synthase was maximally increased 24 h postexposure. Again, vanadium (not nickel) appeared to be mediating the effects of ROFA on gene expression. Treatment with dimethylthiourea inhibited both ROFA- and vanadium-induced gene expression in a dose-dependent manner. Corresponding effects were observed in interleukin-6 and macrophage inflammatory protein-2 synthesis. In summary, generation of an oxidative stress was critical to induction of the ROFA- or vanadium-induced effects on airway epithelial gene expression, cytokine production, and cytotoxicity.

Animals↗

PubMed1998

Cytokine mediation of ozone-induced pulmonary adaptation.

Previous studies have shown that a single exposure of animals to ozone (O3) can induce protection or adaptation to the acute injurious effects of a subsequent O3 challenge. Although a number of mechanisms have been proposed to account for this response, none appear to be fully explanatory. We examined the role interleukin (IL)-6 may play in the induction of adaptation to O3-induced pulmonary injury. A statistically significant 29-fold increase in bronchoalveolar lavage fluid IL-6 levels was observed in rats exposed to 0.5 ppm O3 during nighttime hours when compared with daytime hours even though similar kinetics of inflammation were induced by each exposure. Animals receiving an initial nighttime O3 exposure showed a lesser degree of inflammation following a subsequent O3 exposure when compared with animals which received an initial daytime exposure. Rats pretreated with IL-6 both intratracheally and intraperitoneally and subsequently exposed to O3 showed a lesser degree of cellular inflammation when compared with respective controls. Pretreatment of rats with anti-IL-6-receptor antibodies (ra) prior to the nighttime O3 exposure completely abrogated the O3-induced cellular adaptive response without effecting the inflammatory response induced by the initial nighttime O3 exposure. In fact, administration of anti-IL-6ra augmented the neutrophil influx following the second O3 exposure. Anti-IL-6ra treatment did not alter the pulmonary edema adaptive response, suggesting that the O3-induced cellular and edema adaptive responses are regulated by different mechanisms. Our data indicate that mobilization of pulmonary antioxidants does not play a role in the IL-6-mediated early cellular adaptive response and suggest that IL-6 is an essential mediator of the O3-induced cellular adaptive response.

Adaptation, Physiological↗

PubMed1998

Induction of the lung myofibroblast PDGF receptor system by urban ambient particles from Mexico City.

Platelet-derived growth factor (PDGF) and its receptor system regulate mesenchymal cell proliferation. We recently reported that emission-source fly-ash particles and asbestos fibers induce the PDGF alpha-receptor through a macrophage-dependent pathway, and upregulation of this receptor greatly enhances the mitogenic response of lung myofibroblasts to PDGF (Lindroos and colleagues, Am. J. Respir. Cell Mol. Biol. 1997;16:283-292). In the present study we investigated the effect of particulate matter <= 10 micrometers in size (PM10) from the southern, central, and northern regions of Mexico City on PDGF receptor induction and compared these urban, ambient particles with Mt. St. Helen's volcanic ash particles as a negative control. All Mexico City PM10 samples, but not volcanic ash, stimulated rat alveolar macrophages to secrete a soluble, upregulatory factor(s) for the PDGF alpha-receptor on early passage rat lung myofibroblasts. The macrophage-derived upregulatory activity was blocked by the interleukin (IL)-1 receptor antagonist. The ability of PM10 to stimulate IL-1beta release was blocked in part by a recombinant endotoxin neutralizing protein (rENP). Lipopolysaccharide/endotoxin (LPS) and vanadium, both constituents that were present within these PM10 samples, also stimulated macrophages to secrete factor(s) that upregulated PDGF-Ralpha on lung myofibroblasts. Direct exposure of myofibroblasts to PM10 also elicited upregulation of the PDGF alpha-receptor, and this effect was blocked by rENP and mimicked by LPS, but not vanadium. These findings suggest that PM10 particles induce expression of the PDGF receptor system through macrophage-dependent and -independent mechanisms involving endotoxin and metals.

Air Pollutants↗

PubMed1997

Soluble transition metals mediate residual oil fly ash induced acute lung injury.

Identification of constituents responsible for the pulmonary toxicity of fugitive combustion emission source particles may provide insight into the adverse health effects associated with exposure to these particles as well as ambient air particulate pollution. Herein, we describe results of studies conducted to identify constituents responsible for the acute lung injury induced by residual oil fly ash (ROFA) and to assess physical-chemical factors that influence the pulmonary toxicity of these constituents. Biochemical and cellular analyses performed on bronchoalveolar lavage fluid obtained from rats following intratracheal instillation of ROFA suspension demonstrated the presence of severe inflammation, an indicator of pulmonary injury, which included recruitment of neutrophils, eosinophils, and monocytes into the airway. A leachate prepared from ROFA, containing predominantly Fe, Ni, V, Ca, Mg, and sulfate, produced similar lung injury to that induced by ROFA suspension. Depletion of Fe, Ni, and V from the ROFA leachate abrogated its pulmonary toxicity. Correspondingly, minimal lung injury was observed in animals exposed to saline-washed ROFA particles. A surrogate transition metal sulfate solution containing Fe, V, and Ni largely reproduced the lung injury induced by ROFA. Metal interactions and pH were found to influence the severity and kinetics of lung injury induced by ROFA and soluble transition metals. These findings provide direct evidence for the role of soluble transition metals in the pulmonary injury induced by the combustion emission source particulate, ROFA.

Acute Disease↗

PubMed1997

Metal and sulfate composition of residual oil fly ash determines airway hyperreactivity and lung injury in rats.

The biological effects of particulate matter (PM) deposition in the airways may depend on aqueousleachable chemical constituents of the particles. The effects of two residual oil fly ash (ROFA) PM samples of equivalent diameters but different metal and sulfate contents on pulmonary responses in Sprague-Dawley rats were investigated. ROFA sample 1 (R1) had approximately twice as much saline-leachable sulfate, nickel, and vanadium, and 40 times as much iron as ROFA sample 2 (R2), while R2 had a 31-fold higher zinc content. Four groups of rats were intratracheally instilled with a suspension of 2.5 mg R2 in 0.3 ml saline (R2), the supernatant of R2 (R2s), the supernatant of 2.5 mg R1 (R1s), or saline only. By 4 days after instillation, 4 of 24 rats treated with R2s or R2 had died, compared with non treated with R1s or saline, and pathological indices were greater in both R2 groups compared with the R1s group. In surviving rats, baseline pulmonary function parameters and airway hyperreactivity to acetylcholine challenge were significantly worse in R2 and R2s groups than in the R1s group. Numbers of bronchoalveolar lavage neutrophils, but not other inflammatory cells or biochemical parameters of lung injury, were greater in both R2 groups compared with the R1s group. These results reinforce the hypothesis that the composition of soluble metals and sulfate leached from ROFA, an emission source particle, is critical in the development of airway hyperreactivity and lung injury.

Acetylcholine↗

PubMed1997

In vivo evidence of free radical formation in the rat lung after exposure to an emission source air pollution particle.

Exposure to air pollution particles can be associated with increased human morbidity and mortality. The mechanism(s) of lung injury remains unknown. We tested the hypothesis that lung exposure to oil fly ash (an emission source air pollution particle) causes in vivo free radical production. Electron spin resonance (ESR) in conjunction with the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN) was used to detect radical adducts. Rats were instilled with 500 micrograms of either oil fly ash or saline. Twenty-four hours later, ESR spectroscopy of the chloroform extract from lungs of animals exposed to the oil fly ash gave a spectrum consistent with a carbon-centered radical adduct (hyperfine coupling constants alpha N = 15.0 G and alpha H beta = 2.5 G), while those spectra from lungs instilled with saline revealed a much weaker signal. This signal was reproduced by instilling animals with the soluble fraction of the oil fly ash, which contains soluble metal compounds. The same signal was observed after instillation of either a mixture of vanadium, nickel, and iron sulfates or VOSO4 alone. We conclude that, after instillation of an air pollution particle in the rat, ESR analysis of lung tissue demonstrates in vivo free radical production. This generation of free radicals appears to be associated with soluble metals in the oil fly ash.

Air Pollution↗

PubMed1997

Genetic variability in combustion particle-induced chronic lung injury.

Occupational exposure to anthropogenic particles is associated with lung injury in humans. We hypothesized that residual oil fly ash (ROFA), an emission source particulate, may induce acute lung injury and fibrosis in sensitive rat strains and that fibronectin (Fn) gene expression will correspond to the development of fibrosis. Male Sprague-Dawley (SD), Wistar (WIS), and Fischer 344 (F-344) rats (60 days old) were exposed to saline or ROFA (8.3 mg/kg) by intratracheal instillation and examined for up to 12 wk. Histology indicated focal areas of lung damage showing inflammatory cell infiltration as well as alveolar, airway, and interstitial thickening in all three rat strains during 1-7 days postexposure. Trichrome staining of the lung sections indicated a sporadic incidence of focal alveolar fibrosis at 1, 3, and 12 wk in SD rats, whereas WIS and F-344 rats showed only a modest increase in trichrome staining in the septal areas. Of all Fn mRNA isoforms examined by polymerase chain reaction, only EIIIA(+) was upregulated during 6 h-1 wk in ROFA-exposed SD and WIS rats but not in F-344 rats. In situ hybridization analysis in SD rats revealed Fn mRNA expression by macrophage and alveolar and airway epithelium and within fibrotic areas. Immunohistochemical analysis revealed increased presence of Fn EIIIA(+) protein in the areas of fibrotic injury and basally to the airway epithelium. In summary, Fn EIIIA(+) increases early in the course of particle-induced lung injury and remodeling, which may or may not result in discernible alveolar fibrosis. There is a rat strain variation in ROFA-induced fibrosis and associated Fn EIIIA(+) expression.

Air Pollutants↗

PubMed1997

Epithelial injury induced by exposure to residual oil fly-ash particles: role of reactive oxygen species?

Exposure of animals to airborne particulates is associated with pulmonary injury and inflammation. In the studies described here, primary cultures of rat tracheal epithelial (RTE) cells were exposed to suspensions of residual oil fly ash (ROFA). ROFA exposure resulted in progressive cytotoxicity whereby the amount of lactate dehydrogenase (LDH) released was significantly greater at 24 h than at 6 h after exposure. In a dose-dependent manner, exposure to 5, 10, or 20 microg/cm2 of ROFA for 24 h resulted in cytotoxicity and detachment of cells from the collagen matrix, along with altered permeability of the RTE cell layer. ROFA exposure caused cellular glutathione levels to decrease, producing a condition of oxidative stress in the RTE cells. Treatment of RTE cells with buthionine sulfoxamine, an inhibitor of gamma-glutamyl cysteine synthetase, was found to augment ROFA-induced cytotoxicity. Treatment with dimethylthiourea (DMTU) inhibited ROFA-induced LDH release and permeability changes in a dose-dependent manner. Treatment with the nitric oxide synthase inhibitor NG-monomethyl-D-arginine (D-NMA) for 24 h was without effect. In rats intratracheally instilled with ROFA (500 microg/rat), intraperitoneal administration of DMTU (500 mg/kg) significantly ameliorated the degree of pulmonary neutrophilic inflammation present at 24 h. Overall, these in vitro findings suggest that ROFA-induced RTE cell injury may be mediated by hydroxyl-radical-like reactive oxygen species (i.e., species scavenged by DMTU) that are generated via non-nitric oxide pathways. The delay in induction of maximal RTE cell injury may reflect the time necessary to produce an oxidative burden by depleting antioxidant defenses such as cellular glutathione.

Air Pollution↗

PubMed1997

Bioavailable transition metals in particulate matter mediate cardiopulmonary injury in healthy and compromised animal models.

Many epidemiologic reports associate ambient levels of particulate matter (PM) with human mortality and morbidity, particularly in people with preexisting cardiopulmonary disease (e.g., chronic obstructive pulmonary disease, infection, asthma). Because much ambient PM is derived from combustion sources, we tested the hypothesis that the health effects of PM arise from anthropogenic PM that contains bioavailable transition metals. The PM samples studied derived from three emission sources (two oil and one coal fly ash) and four ambient airsheds (St. Louis, MO; Washington; Dusseldorf, Germany; and Ottawa, Canada). PM was administered to rats by intratracheal instillation in equimass or equimetal doses to address directly the influence of PM mass versus metal content on acute lung injury and inflammation. Our results indicated that the lung dose of bioavailable transition metal, not instilled PM mass, was the primary determinant of the acute inflammatory response for both the combustion source and ambient PM samples. Residual oil fly ash, a combustion PM rich in bioavailable metal, was evaluated in a rat model of cardiopulmonary disease (pulmonary vasculitis/hypertension) to ascertain whether the disease state augmented sensitivity to that PM. Significant mortality and enhanced airway responsiveness were observed. Analysis of the lavaged lung fluids suggested that the milieu of the inflamed lung amplified metal-mediated oxidant chemistry to jeopardize the compromised cardiopulmonary system. We propose that soluble metals from PM mediate the array of PM-associated injuries to the cardiopulmonary system of the healthy and at-risk compromised host.

Air Pollutants, Occupational↗

PubMed1997

The role of reactive oxygen and nitrogen species in the response of airway epithelium to particulates.

Epidemiologic and occupational studies indicate adverse health effects due to inhalation of particulate air pollutants, but precise biologic mechanisms responsible have yet to be fully established. The tracheobronchial epithelium forms the body's first physiologic barrier to such airborne pollutants, where ciliary movement functions to remove the offending substances caught in the overlying mucus layer. Resident and infiltrating phagocytic cells also function in this removal process. In this paper, we examine the role of reactive oxygen and nitrogen species (ROS/RNS) in the response of airway epithelium to particulates. Some particulates themselves can generate ROS, as can the epithelial cells, in response to appropriate stimulation. In addition, resident macrophages in the airways and the alveolar spaces can release ROS/RNS after phagocytosis of inhaled particles. These macrophages also release large amounts of tumor necrosis factor alpha (TNF-alpha), a cytokine that can generate responses within the airway epithelium dependent upon intracellular generation of ROS/RNS. As a result, signal transduction pathways are set in motion that may contribute to inflammation and other pathobiology in the airway. Such effects include increased expression of intercellular adhesion molecule 1, interleukin-6, cytosolic and inducible nitric oxide synthase, manganese superoxide dismutase, cytosolic phospholipase A2, and hypersecretion of mucus. Ultimately, ROS/RNS may play a role in the global response of the airway epithelium to particulate pollutants via activation of kinases and transcription factors common to many response genes. Thus, defense mechanisms involved in responding to offending particulates may result in a complex cascade of events that can contribute to airway pathology.

Air Pollutants↗

PubMed1995

Ozone-induced tissue injury and changes in antioxidant homeostasis in normal and ascorbate-deficient guinea pigs.

It has been reported previously that ozone (O3) toxicity from acute (4 hr) exposure is enhanced by ascorbate (AH2) deficiency in guinea pigs. We hypothesized that lung injury from continuous 1-week O3 exposure would also be increased under conditions of AH2 deficiency because of (1) a diminished antioxidant pool to counteract the oxidant challenge, (2) impaired reparation of tissue injury, and/or (3) altered antioxidant redox homeostasis. Female Hartley guinea pigs (260-330 g) were made AH2 deficient by providing a diet similar to guinea pig chow, but having no AH2. The dietary regimen was started 1 week prior to exposure and was continued during exposure to O3 (0, 0.2, 0.4, or 0.8 ppm, 23 hr/day, 7 days) as well as 1 week post-exposure. Bronchoalveolar lavage (BAL) and tissue AH2 were measured in subgroups at the beginning of exposure (1 week on the AH2-deficient diet), at its termination and 1 week post-exposure. AH2 measured in ear tissue punches proved to be an easy and effective monitor for AH2 deficiency. One week on the AH2-deficient diet caused a 70-80% drop in ear, lung and liver AH2, while AH2 in BAL was decreased by 90%. Immediately after the exposure, total BAL protein and albumin (markers of lung permeability) were increased (approximately 50%) at 0.8 ppm with no difference between the dietary groups. O3 caused an increase in total BAL cells and neutrophils in a concentration-dependent manner with only a slight augmentation due to diet. Exposure to O3 caused an increase in lung and BAL AH2 in normal guinea pigs. Glutathione and uric acid were also increased in the lung and BAL after O3 exposure (40-570%) in both dietary groups, and the levels remained elevated during the recovery period. Lung alpha-tocopherol was not changed due to O3. A significant overall diet-related decrease was seen in AH2-deficient guinea pigs, immediately after the exposure and recovery. In summary, lung injury/inflammation following 1 week O3 exposure and recovery were minimally affected by AH2 deficiency. Antioxidants also appeared to increase in response to O3 exposure despite the deficiency in AH2.

Animals↗

PubMed1995

Temporal expression and cellular distribution of pulmonary fibronectin gene induction following exposure to an emission source particle.

This study examines the ability of an emission source particle, residual oil fly ash (ROFA), to influence pulmonary fibronectin (Fn) gene expression. Fn is an extracellular matrix (ECM) protein involved in a variety of cellular functions, including inflammation, cell proliferation, and fibrosis. Temporal expression and spatial distribution of Fn gene induction were assessed by in situ hybridization in rat lung during acute phase of lung injury occurring 6 to 72 h following intratracheal instillation of ROFA. Fn mRNA was not detected in rat lungs treated with either saline or at 6 h after ROFA treatment. However, Fn mRNA was induced in airway epithelial cells 24 h after ROFA instillation. Histopathology showed peribronchial inflammation and focal edema. Diffuse inflammation in alveolar region with limited expression of Fn mRNA was evident 48 h after ROFA exposure, occurring mainly in proliferating epithelial cells. Extensive Fn mRNA expression was seen in proliferating fibroblasts and in hyperplastic epithelial cells within incipient fibrotic lesions 72 h after exposure, while the intensity of expression in the airway epithelial cells was decreased. Therefore, Fn mRNA induction was associated with inflammatory and incipient fibrotic lesions, indicating its possible involvement in airway hyperreactivity and initiation of fibrogenesis.

Air Pollutants↗

PubMed1992

Biglycan and decorin gene expression in normal and fibrotic rat liver: cellular localization and regulatory factors.

The expression of genes encoding the core proteins of the novel small chondroitin/dermatan sulfate proteoglycans decorin and biglycan was studied in the livers of normal rats and in liver tissue during fibrogenesis induced by prolonged bile-duct ligation and thioacetamide poisoning. The cell types responsible for the expression of these transcripts and some key regulatory factors were identified. Both biglycan and decorin messenger RNAs were detected in normal liver tissue. Their relative abundance increased strongly during liver fibrogenesis, reaching highest levels in cirrhotic tissue 8 wk after common bile-duct ligation and after 12 wk of peroral thioacetamide administration, respectively. Specific proteoglycan transcripts were almost absent in hepatocytes from normal and regenerating liver, and only trace amounts were observed in freshly isolated and cultured Kupffer cells. Fat-storing cells clearly expressed both biglycan and decorin transcripts. The steady-state levels of their messenger RNAs increased threefold (biglycan) and fourfold (decorin) during primary culture. Myofibroblastlike cells (transformed fat-storing cells after the second passage) contained dramatically reduced levels of decorin messenger RNA and also lower levels of biglycan messenger RNA compared with primary cultures. These changes of core protein messenger RNA expression were not reflected by the synthesis rates of medium proteoglycans labeled with 35S as Na2SO4, in particular that of medium chondroitin sulfate. Transiently acidified (but not native) conditioned media from Kupffer cells and myofibroblastlike cells and transforming growth factor-beta 1 enhanced the relative abundances of biglycan and decorin messenger RNAs up to five times in primary-cultured fat-storing cells. Biglycan and decorin in myofibroblastlike cells did not respond to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Molecular characterization of vascular smooth muscle decorin: deduced core protein structure and regulation of gene expression.

Two overlapping clones containing sequences homologous to bovine, human and chicken decorin have been recovered from poly A+ RNA isolated from rat vascular smooth muscle cells (VSMC) using cDNA cloning and reverse transcription-polymerase chain reaction (PCR) methodologies. Results of nucleotide sequence analysis performed on these clones demonstrated that they encode the complete mature rat decorin protein expressed by VSMC. Within the coding region, rat decorin exhibits 76% nucleotide sequence homology to human and bovine decorin, and 69% homologous to chicken decorin indicating a significant level of conservation among these species. This level of conservation among species was also maintained at the protein level with rat decorin being 77% homologous to its human, bovine and chicken homologues. As previously observed its human homologue, rat decorin, is made up of seven, tandem, leucine-rich repeat sequences. Furthermore, within the core of these repeats was the consensus protein sequence NKISK which has been proposed to be the fibronectin binding region of decorin (G. Schmidt et al., Biochem. J. 280, 411-414 (1991)). The vast majority of amino acid substitutions within rat decorin were of the conservative type. The highest frequency of amino acid substitutions were found to be localized within a hypervariable region located near the amino terminus of the decorin core protein. Unlike rat biglycan, rat decorin mRNA levels were found to increase significantly in density-arrested VSMC cultures. In contrast to rat biglycan gene expression, no quantitative differences in rat decorin mRNA levels were observed between proliferating VSMC and VSMC made quiescent through serum depletion. Finally, specific extracellular matrix (ECM) proteins were able to regulate the expression of decorin at the mRNA level in a slightly different manner than previously observed for biglycan.

Amino Acid Sequence↗

PubMed1991

Expression of antisense transcripts encoding an extracellular matrix protein by stably transfected vascular smooth muscle cells.

The ability to selectively inhibit the expression of various extracellular matrix proteins would provide an approach towards elucidating not only the function of these molecules but allow some insight to be gained into the mechanisms by which these molecules influence various aspects of vascular smooth muscle cell behavior such as growth, migration, morphology, and extracellular matrix assembly. The present study explores the feasibility of employing an antisense RNA approach to study vascular smooth muscle extracellular matrix proteoglycan function. Vascular smooth muscle cells were transfected with a test plasmid designed to express an antisense transcript encoding an extracellular matrix protein. The test plasmid was constructed by inserting the cDNA encoding the majority of the rat chondrosarcoma extracellular matrix link protein in an antisense orientation relative to the rat metallothionein-I promoter. Several stably transfected cell lines were isolated and characterized with respects to link antisense transcript expression, morphology, growth, and smooth muscle cytoskeletal protein expression. Results from these studies have shown that vascular smooth muscle cells could be stably transfected and link antisense transcript could be expressed at high levels by these cells. In addition, the rat metallothionein-I promoter was found to be a strong inducible promoter in vascular smooth muscle cells. Cell biology characterization of the various transfected vascular smooth muscle cell lines generated in this study has shown that one type of transfected vascular smooth muscle cell line expressed cellular characteristics indicative of early passaged vascular smooth muscle cells while a second type of transfected cell line was morphologically similar to vascular smooth muscle cells recovered from neonatal rat aortas and mechanically injured arteries obtained from adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗