Cultured Rat Lung Fibroblasts Research Articles

Microtubules as a Critical Target for Arsenic Toxicity in Lung Cells in Vitro and in Vivo

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades.

THE CALCITRIOL ANALOGUE EB1089 IMPAIRS ALVEOLARIZATION AND INDUCES LOCALIZED REGIONS OF INCREASED FIBROBLAST DENSITY IN NEONATAL RAT LUNG

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, or calcitriol), is a potent mitogen for fibroblasts cultured from rat lungs at postnatal day 4 (P4), during the peak of septation (P3 to P7). In light of the key role of fibroblasts in alveolar septation, the authors conducted studies to measure the extent to which 1,25-(OH)2D3 affects lung maturation in vivo, as well as its ability to influence the stimulatory activity of all-trans retinoic acid (RA). To identify a calcitriol analogue with maximal mitogenic activity and low systemic toxicity, two compounds with reduced calcemic activity (EB1089 and CB1093) and a superagonist (MC1288) were evaluated in neonatal rat lung fibroblast cultures. All 3 analogues were more potent mitogens than 1,25-(OH)2D3 itself (MC1288 ∼ CB1093 > EB1089 > 1,25-(OH)2D3). In addition, each was more effective than 1,25-(OH)2D3(EB1089 > CB1093 > MC1288 > 1,25-(OH)2D3) in the activation of a vitamin D response element from the platelet-derived growth factor (PDGF)-A gene, whose expression is essential for normal alveolarization. Daily administration of EB1089 to rats 4 to 12 days of age caused an increase in mean alveolar chord length (P <.0001), and also elicited prominent regions of fibroblast hypercellularity, as defined in terms of a vimentin-positive, factor VIII–negative phenotype. EB1089 and RA each induced the expression of 2 important lung structural proteins, collagen and elastin. Regions of fibroblast hypercellularity induced by EB1089 were strongly positive for expression of the alveolarization-relevant growth factors, PDGF-AA and vascular endothelial growth factor (VEGF). These studies demonstrate that 1,25-(OH)2D3 disrupts the overall alveolarization process in the neonatal lung, although it stimulates expression of some proteins associated with lung morphogenesis.

Down-regulation of expression of rat pyruvate dehydrogenase E1α gene by self-complementary adeno-associated virus-mediated small interfering RNA delivery

Mutations in the E1α subunit gene (PDHA1) of the pyruvate dehydrogenase complex (PDC) are common causes of congenital lactic acidosis. An animal model of E1α deficiency could provide insight into the pathological consequences of mutations and serve to test potential therapies. Small interfering RNAs (siRNAs) were designed to cleave the messenger RNA (mRNA) of the E1α subunit and were tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co-transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1α mRNA. Quantitative PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 h and up to 65% by 56 h, compared to negative and positive controls. Since oligonucleotide-mediated siRNA delivery provided only transient suppression, we next selected two siRNA candidates and generated self-complementary, double-stranded adeno-associated virus (scAAV) vectors (serotypes 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53–80% 72 h after infection and 54–70% 10 days after infection in RLF cultures. The expression of E1α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days after infection in RLF cultures. Thus, scAAV siRNA-mediated knockdown of PDHA1 gene expression provides a strategy that may be applied to create a useful animal model of PDC deficiency.

325. In Vitro Gene Knockdown of Rat Pyruvate Dehydrogenase E1 α mRNA by Self- Complementary Adeno-Associated Virus-Mediated Small Interfering RNA Delivery

Mutations in the E1 α subunit (PDHA1) of the gene pyruvate dehydrogenase complex (PDC) are one of the most common causes of congenital lactic acidosis. An animal model of the E1 α deficiency would provide insight into the pathologic role of PDC mutations in genetic mitochondrial diseases. Small interfering RNAs (siRNAs) designed to cleave the messenger RNA (mRNA) of the E1 α subunit, were constructed and tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co- transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1 α mRNA. Real time PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 hours, and up to 65% by 56 hours, compared to negative and positive controls. Since oligonucleotide- mediated siRNA delivery provides only transient suppression, we next selected two siRNA candidates and generated self- complementary, double-stranded adeno-associated virus (scAAV) vectors (serotype 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53-80% 72 hours post-infection and 54-70% 10 days post-infection in RLF cultures. The expression of E1 α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days post-infection in RLF cultures. Thus, AAV scAAV siRNA-mediated gene knockdown of PDHA1 provides strategy that may be applicable for creating a useful animal model of PDC deficiency. Mutations in the E1 α subunit (PDHA1) of the gene pyruvate dehydrogenase complex (PDC) are one of the most common causes of congenital lactic acidosis. An animal model of the E1 α deficiency would provide insight into the pathologic role of PDC mutations in genetic mitochondrial diseases. Small interfering RNAs (siRNAs) designed to cleave the messenger RNA (mRNA) of the E1 α subunit, were constructed and tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co- transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1 α mRNA. Real time PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 hours, and up to 65% by 56 hours, compared to negative and positive controls. Since oligonucleotide- mediated siRNA delivery provides only transient suppression, we next selected two siRNA candidates and generated self- complementary, double-stranded adeno-associated virus (scAAV) vectors (serotype 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53-80% 72 hours post-infection and 54-70% 10 days post-infection in RLF cultures. The expression of E1 α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days post-infection in RLF cultures. Thus, AAV scAAV siRNA-mediated gene knockdown of PDHA1 provides strategy that may be applicable for creating a useful animal model of PDC deficiency.

Evidence for the involvement of TGF-β and PDGF in the regulation of prolyl 4-hydroxylase and lysyloxidase in cultured rat lung fibroblasts

Lung fibrosis is the end-point of numerous lung disorders induced by a pneumonia or by a variety of different noxes, one of which is the cytostatic drug bleomycin (BLM). Fibrosis is characterized by excessive extracellular matrix accumulation. Macrophage-fibroblast interactions are suggested to play an important role in the development of this disease. The present study was addressed to investigate one possible pathway of this interaction, the influence of soluble mediators produced by BLM-stimulated macrophages on lung fibroblast collagen synthesis and modification. Conditioned media (CM) of BLM-exposed macrophages of the cell line NR8383 submitted to rat lung fibroblast cultures increased the activity of prolyl 4-hydroxylase (P4H) in fibroblasts in a dose dependent manner. CM of stimulated macrophages increased the collagen concentration in fibroblast culture supernatant. The level of mRNAs specific for the alpha-subunit of P4H and that for alpha1(I) collagen were found to be increased by about two-fold, that for lysyloxidase (LO) by about 2.5-fold in fibroblasts cultured in CM of stimulated macrophages. Pre-incubation of CM of BLM-exposed macrophages with neutralizing antibodies against TGF-beta or against PDGF resulted in a partial reversal of the increasing effect of the CM on P4H- and LO-activities in fibroblasts. Both growth factors, TGF-beta and PDGF, added to fibroblast cultures led to significant increases of P4H activity in the treated cells. We conclude that TGF-beta and PDGF produced by stimulated macrophages are involved in the regulation of the expression of alpha1(I) collagen, of P4H-alpha-subunit and LO in lung fibroblasts. The results indicate that this is not a direct effect but involves the action of a so far unidentified mediator responsible for autocrine stimulation of collagen production.

Ontogeny and localization of TGF-beta type I receptor expression during lung development.

Transforming growth factor (TGF)-beta is a family of multifunctional cytokines controlling cell growth, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-beta are mediated by type I (TbetaR-I) and II (TbetaR-II) receptors. Our previous studies show that the expression of TbetaR-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of TbetaR-I during lung development. The expression level of TbetaR-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. TbetaR-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of TbetaR-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. TbetaR-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense TbetaR-I signal was observed in the epithelial lining of the developing bronchi. In gestational day 16 lungs, the expression of TbetaR-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of TbetaR-I expression. In postnatal lungs, TbetaR-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of TbetaR-I in cultured rat lung cells. TbetaR-I was expressed by cultured rat lung fibroblasts, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of TbetaR-I that is different from that of TbetaR-II during lung development. TbetaR-I, TbetaR-II, and TGF-beta isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for TbetaR-I in mediating TGF-beta signal transduction during lung development.

Fibroblast tropoelastin and alpha-smooth-muscle actin expression are repressed by particulate-activated macrophage-derived tumor necrosis factor-alpha in experimental silicosis.

Lung elastin synthesis is normally confined to periods of development, is maximal during alveolarization, and declines to low levels in mature lung. We have previously described an elastogenic response in the adult rat lung associated with experimental granulomatous disease induced by silica instillation. Reinitiated tropoelastin expression was identified throughout the lung in fibroblasts expressing alpha-smooth-muscle actin, whereas fibroblasts within the granulomatous lesions failed to express both tropoelastin and alpha-smooth- muscle actin (Mariani and colleagues, Am. J. Pathol. 1995;147:988-1000). We hypothesized that inflammatory cells within the granulomatous lesions produce factors that alter fibroblast phenotype. We found that macrophages accumulating within granulomatous lesions of silicotic rat lungs produce and secrete tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine previously appreciated as a repressor of tropoelastin gene expression. In experimental cell systems, macrophages activated by particulates, either in vivo or in vitro, conditioned medium with a tropoelastin-repressing activity. This activity repressed both tropoelastin and alpha-smooth-muscle actin expression in primary cultures of rat lung fibroblasts in a time- dependent, transient manner. The particulate-activated macrophage-conditioned medium was found to contain TNF-alpha, which was both necessary and sufficient to induce these changes in lung fibroblast gene expression. These data indicate that macrophage-derived factors can modulate lung fibroblast tropoelastin expression in the diseased lung. Furthermore, the findings extend the association between expression by lung fibroblasts of tropoelastin and alpha-smooth-muscle actin.

A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1 (LGL1; GenBank accession no. AF109674) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1 is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Molecular and cellular basis of radiation fibrosis

Purpose: Recent data from the literature and the experimental work of the authors clearly indicate that TGF- beta 1 is a key modulator of cellular events, for example, induction of terminal differentiation, resulting in radiation-induced fibrosis. Therefore, the present study analysed which cellular processes induced by exogeneously added TGF- beta could be responsible for the induction, development and manifestation of the fibrotic phenotype in culture. Materials and methods: Rat lung fibroblast cultures (passage 1) were used. As a function of treatment with TGF- beta and/or anti-TGF beta -antibody, the clonogenic activity and differentiation pattern were analysed by colony-formation assays. Results: It could be demonstrated that treatment of rat lung progenitor fibroblasts with TGF- beta 1 resulted in a pronounced shift in the differentiation pattern, i.e. induction of post-mitotic fibrocytes. This TGF- beta 1-dependent terminal differentiation could be abolished by simultaneous treatment with a neutralizing antibody directed against TGF- beta 1. Conclusions: The data presented indicate that TGF- beta 1 is one major candidate mediating the accelerated terminal differentiation of progenitor fibroblasts to post-mitotic functional fibrocytes, which results in the fibrotic phenotype of this cell system.

Iron mobilization from crocidolite as enhancer of collagen content in rat lung fibroblasts

Asbestos exposure causes pulmonary fibrosis by mechanisms that remain uncertain. There is increasing evidence that iron from asbestos is responsible for many of its effects. In this paper, we investigated the effect of iron mobilized from crocidolite asbestos on collagen content in rat lung fibroblast cultures under serum-free conditions. Crocidolite (2, 4, 6 μg/cm 2 well) increased collagen content in a dose-dependent manner (+ 42 ± 8, + 92 ± 10, and + 129 ± 13% vs controls). This effect was specific for collagen, since it did not alter total protein content and was inhibited by the iron chelator deferoxamine (DFO). Preincubation of crocidolite with citrate (1 mM) for 48 hr resulted in iron mobilization (51 μM) and increased collagen production (> 3-fold) in treated cells. These effects occurred without the intervention of serum factors. The absence of cell damage, proliferation or lipid peroxidation leads to the supposition that iron from crocidolite per se may act as a profibrogenic agent. Although the in vivo participation of other cells and factors cannot be excluded, we conclude that iron released from crocidolite plays a role in collagen increase occurring during asbestosis.

Annexin I in fibrotic rat lung and cultured lung fibroblasts following irradiation

. Radiation-induced lung fibrosis is a result of collagen accumulation in the interstitium, partly due to increased collagen synthesis by fibroblasts. One feature of active collagen synthesis is increased membrane trafficking in the fibroblasts. A group of proteins called annexins is believed to play a regulatory role in membrane fusion and exocytosis. Therefore, increased annexin activity might be expected in the fibrotic lung. We tested this hypothesis by measuring annexin I levels, hydroxyproline content and ultrastructural changes in radiation-induced pulmonary fibrosis in rat. Three months after a single exposure to 30 Gy of X-rays to the right hemithorax, the right lung of the rat was atrophied and fibrotic with a concomitant increase in size of the shielded left lung. Electron micrographs revealed that the irradiated lung was ladened with interstitial collagen fibrils, with increased number of fibroblasts amongst them. Hydroxyproline concentration in the irradiated lung was nearly twice that in the sham-irradiated lung. Annexin I in the irradiated lung, on the other hand, was markedly reduced, and barely detectable on immunoblots. Since increased annexin I might precede enhanced collagen production, we also measured annexin I levels in rat lungs 3 days after 30 Gy irradiation and correlated that with hydroxyproline concentration. We found no appreciable difference in annexin I levels and hydroxyproline content between sham-irradiated and irradiated lungs at 3 days. To determine whether annexin I levels in cultured fibroblasts were altered by irradiation, we assayed annexin I in cultured rat lung fibroblasts 3 days after 0-10 Gy exposure, with concomitant measurement of 14C-proline incorporation. The annexin I level in fibroblasts irradiated with 10 Gy X-rays was 55% higher than in shamirradiated fibroblasts. However, incorporation of 14C-proline into collagenase-sensitive macromolecules in the culture medium and extracellular matrix was not different between these two groups of cells. These data demonstrate a radiation-induced increase in immunoreactive annexin I in cultured lung fibroblasts, but fail to support the hypothesis of a positive correlation between annexin I concentration and fibrosis in irradiated rat lung.

Serine proteinase inhibitors influence the stability of tropoelastin mRNA in neonatal rat lung fibroblast cultures

Elastin, an elastic extracellular structural protein, is a polymer comprised of soluble tropoelastin (TE) monomers that are joined by covalent cross-links and become insoluble. In cultured vascular smooth muscle cells, the steady-state level of TE mRNA is influenced by soluble elastin moieties in the culture medium, either TE or its fragmentation products. We have hypothesized that an enzyme-mediated proteolytic event may modulate the quantities of TE and its fragmentation products in the culture medium of mesenchymal cells, and thereby indirectly regulate the steady-state level of TE mRNA. Neonatal rat lung fibroblasts were cultured in the presence or absence of the serine proteinase inhibitor, aprotinin, and the quantities of soluble elastin and TE mRNA were analyzed. Exposures to aprotinin lasting up to 12 h increased the soluble elastin content of the culture medium. The increase in the soluble elastin content did not reflect an increase in TE mRNA, which diminished after exposures for 12 h or longer. The decrease in TE mRNA resulted from a decrease in its half-life, rather than a decrease in the rate of TE gene transcription. Aprotinin did not reduce TE mRNA in plasminogen-depleted cultures, but the effect of aprotinin was evident when purified plasminogen was added back to the cultures. Therefore, a serine proteinase, possibly plasmin, may participate in a feedback mechanism and modulate the quantity of TE in lung fibroblast cultures. This mechanism may help ensure that intracellular TE synthesis occurs in tandem with extracellular elastin deposition and cross-linking.

Tenascin in rat lung development: in situ localization and cellular sources.

Tenascin (TN) is a hexameric extracellular matrix glycoprotein that may play an important role during lung development. TN protein is temporally and spatially restricted during lung organogenesis. The temporo-spatial and cellular expression of TN mRNA in lung remains unclear. Localization of message expression of TN in rat lung tissue was first investigated by using in situ hybridization performed with an antisense RNA probe. TN mRNA was present primarily within the mesenchyme of day 16 gestational age fetal rat lung tissue, whereas immunoreactive TN protein was found along the basement membrane. In postnatal day 3 rat lung tissue, TN mRNA was detected along alveolar septal walls and was concentrated at secondary septal tips. Expression of TN message was consistent with localization of immunoreactive TN protein. Accumulation of TN mRNA in alveolar septal tips suggests that mesenchyme may be the major source of TN mRNA. To investigate the cellular source of TN in rat lung, we studied the expression of TN in cultured rat lung fibroblasts, endothelial cells, and alveolar epithelial cells. Two TN isoforms having molecular mass of 230 and 180 kDa were in conditioned medium and in cellular extracts of lung fibroblasts and endothelial cells. TN was secreted and deposited in the extracellular matrix closely associated with the surface of lung fibroblasts and endothelial cells. Lung alveolar epithelial cells showed undetectable or barely detectable amounts of TN. These studies demonstrated that TN isoforms are expressed not only by lung fibroblasts but also by lung endothelial cells. The unique spatial localization of TN mRNA during lung development and expression of TN by different lung cell types suggested TN may be involved in matrix organization and cell-cell interactions during lung development.

Characterization of the mechanism involved in bleomycin-induced increased hyaluronan production in rat lung.

The molecular mechanisms behind the accumulation of hyaluronan during bleomycin-induced lung injury in rats were investigated. The stimulatory effects of bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM)-conditioned media on hyaluronan synthesis in normal rat lung fibroblast cultures were studied as well as the hyaluronan binding activity on AM. BALF obtained on days 1 and 5 after bleomycin instillation exhibited hyaluronan stimulatory activity similar to that of 10% fetal serum; the activity returned to control values on day 14 after bleomycin treatment. Conditioned media from cultures of AM obtained from bleomycin-treated rats exhibited stimulatory effects higher than that of media from AM of control rats and equal to or higher than that of 10% fetal calf serum. The stimulatory activity in BALF was significantly inhibited by neutralizing antibodies against transforming growth factor-beta; the activity in AM-conditioned media was only partially affected. Neutralizing antibodies against platelet-derived growth factor-BB or -AA had no such inhibiting effect. Interestingly, AM from bleomycin-treated rats exhibited low hyaluronan binding activity. [3H]Hyaluronan binding by AM on days 1 and 5 after bleomycin administration was about 2-fold and 4-fold lower, respectively, compared with that by AM derived from saline-treated rats. This decrease was normalized 14 days after bleomycin treatment. In conclusion, our results indicate that factors with high potential to stimulate hyaluronan synthesis in rat lung fibroblasts are accumulated in BALF from bleomycin-treated rats and that AM are likely to be one source of such stimulatory factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a series of tyrosine-derived polycarbonates as degradable biomaterials.

A series of four polycarbonates derived from the ethyl, butyl, hexyl, and octyl esters of desaminotyrosyl-tyrosine was prepared by condensation polymerization. The resulting polymers had weight average molecular weights ranging from 120,000-450,000, and their chemical structure was confirmed by elemental analysis, nuclear magnetic resonance, and Fourier transform infrared spectroscopy. The polycarbonates were evaluated as degradable biomaterials. Their surface properties were determined by electron spectroscopy for chemical analysis, attenuated total reflectance-Fourier transformed infrared spectroscopy, and contact angle measurement. The degree of surface hydrophobicity was related to the length of the alkyl ester pendent chain. The tensile properties were dependent on the chemical structure of the polymers: For thin, solvent cast film specimens, the tensile modulus varied from 1.2-1.6 GPa, and the strength at break from 60-220 MPa. The degradation of polymeric films was followed in vitro by measuring changes in mechanical strength for up to 40 weeks, and the decrease in molecular weight and changes in surface chemistry for up to 80 weeks. The length of the pendent chain affected the degradation behavior and strength retention; the polymers with short pendent chains were more readily hydrolyzable. For sterilization, ethylene oxide treatment was less destructive, as judged by molecular weight retention, than gamma-irradiation. Spin-cast films of all tested polycarbonates were not cytotoxic toward cultured rat lung fibroblasts. The cell response was influenced by the chemical structure of the polymer. The least hydrophobic polycarbonate (having a short ethyl ester pendent chain) was a more stimulating substrate for cell growth than the more hydrophobic polymers (carrying longer alkyl ester pendent chains).

Retinoic acid increases elastin in neonatal rat lung fibroblast cultures.

The factors that regulate elastin synthesis during pulmonary alveolar septal formation have not been identified. Because maximal alveolar elastin synthesis occurs over a relatively brief period (postnatal days 4-14 in the rat), we hypothesized that changes in the local concentrations of factors that regulate elastin synthesis may precede or accompany this period. Because pulmonary retinoid stores decline just before the fourth postnatal day, we also hypothesized that this decline could be accompanied by the utilization of retinoic acid, one of the most biologically active retinoids, in a regulatory process that increases elastin synthesis. If these hypotheses are correct, then retinoic acid should increase elastin synthesis by pulmonary cells. Therefore, cultures of neonatal rat lung fibroblasts were exposed to retinoic acid, and elastin production was quantitated. Retinoic acid produced a two- to threefold increase in the steady-state level of elastin mRNA, in soluble elastin, and in insoluble elastin. The transcriptional initiation rate of the elastin gene was 1.8-fold higher in nuclei that were isolated from retinoic acid-treated cells than in nuclei that were isolated from control cells. This indicates that the increase in steady-state elastin mRNA results, at least partially, from an increase in elastin transcription. Lung fibroblasts that were isolated from 8-day-old rats, but not cultured, contained retinoic acid. These findings suggest that retinoic acid is a potential regulator of elastin synthesis in developing pulmonary alveoli.

Direct inhibition of platelet function by organic nitrates via nitric oxide formation

This study investigates the mechanisms of platelet inhibition by the nitrate esters isosorbide dinitrate, isoidide dinitrate, isomannide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate as compared to the spontaneous nitric oxide (NO)-donor linsidomine, the active metabolite of molsidomine. Nitrates and linsidomine dose-dependently inhibited aggregation, ATP secretion and thromboxane formation of washed human platelets at a rank order of potency, identical with that for stimulation of cyclic GMP in cultured rat lung fibroblasts. While linsidomine (0.1 mM) caused a 3-fold platelet cGMP elevation, there was a weak (≦ 30%) but significant cGMP stimulation by organic nitroesters, which was tightly correlated with inhibition of platelet aggregation ( r = 0.926, P = 0.008). Zaprinast (2 μM) potentiated, while methylene blue (1 μM) and oxyhemoglobin (10 μM) reversed the antiaggregatory effects. Linsidomine (0.5 μM−0.1 mM) dose-dependently released NO in a cell-free system. No spontaneous NO release was detected with organic nitroesters (0.1 mM). These data suggest that, to some extent, bioactivation of organic nitroesters occurs in platelets resulting in platelet inhibition via the NO/cGMP system.

Lipid peroxidation and disintegration of the cell membrane structure in cultures of rat lung fibroblasts treated with asbestos.

Rat lung fibroblasts were cultured for 24 and 48 h with UICC standard reference asbestos samples of amosite, crocidolite or Canadian chrysotile at various concentrations, and thiobarbituric acid-reactive substances (TBARS) in the media and the cells were measured. Tests by the trypan blue dye-exclusion method showed that cell viability was 80 +/- 5% (mean +/- SD) and 71 +/- 6% when cultured for 48 h in the presence of 500 micrograms ml-1 of amosite and crocidolite, respectively, but was not affected by chrysotile. In cultures for 24 and 48 h, both chrysotile and crocidolite at > 250 micrograms ml-1 significantly increased TBARS in the medium, whereas amosite did so at > 500 micrograms ml-1. TBARS in the cells was not increased significantly by chrysotile at any concentration in the cultures for 24 and 48 h, but crocidolite at > 250 micrograms ml-1 significantly increased TBARS in the cells when cultured for 24 or 48 h. Although amosite at all concentrations tested did not increase significantly TBARS in the cells of the 24-h culture, it did increase TBARS significantly in the cells when cultured for 48 h at a concentration of 500 micrograms ml-1 amosite. The increases of TBARS in media of the cultures with asbestos were accompanied by increases of lactate dehydrogenase (LDH) activity in the media, indicating the leakage of the enzyme from the cell into the media. The activity of LDH and the amount of TBARS showed a good correlation with each other, with a significant correlation coefficient value of 0.655.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergism between interleukin-1ß and interferon-γ, an inducer of nitric oxide synthase, in rat lung fibroblasts

An L-arginine-dependent pathway, by which L-arginine is metabolised to citruline and nitrogen oxides, has been recently identified in some cell types. In cultured rat lung fibroblasts the presence of L-arginine was necessary for the production of nitrite to be induced by rat recombinant interferon-γ and synergistically enhanced by lipopolysaccharide and interleukin-1β. Lipopolysaccharide and interleukin-1β did not induce nitrite biosynthesis by themselves. Biosynthesis was apparently dependent on tetrahydrobiopterin, since it could be blocked by diaminohydroxypyrimidine, an inhibitor of tetrahydrobiopterin synthesis. Dexamethasone blocked nitrite production by a receptor-mediated mechanism. These data indicate that rat lung fibroblasts express an L-arginine-dependent nitric oxide synthase which can be induced by some mediators of inflammation.

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.