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C Franzblau

Publications and source records attributed to C Franzblau.

At least 37 records · Page 2Linked to original sources
PubMed1992

Long-term neonatal rat aortic smooth muscle cell cultures: a model for the tunica media of a blood vessel.

Cultured neonatal rat aortic smooth muscle cells have been used to study the synthesis and accumulation of extracellular matrix components in many laboratories. These cells are capable of accumulating large amounts of insoluble elastin in the extracellular matrix and can be maintained in culture for long periods of time without subcultivation. This study examined the elastin and collagen contents of such cells in culture for 5, 21 and 43 weeks. The percent elastin and collagen observed in the 43-week cultures were strikingly similar to that seen in the intact neonatal rat aorta. It should be noted that the percent collagen varied significantly between 5 weeks and 43 weeks, whereas that for elastin remained relatively constant throughout the same time course. Histological examination demonstrated that the elastin fibers in the extracellular matrix of the cultures were arranged in a pattern similar to the elastic lamellae of the aortic tunica media. Data presented here suggest that these cells in culture mimic the donor tissue from which they were derived with respect to elastin and collagen content as well as elastic fiber arrangement, and possibly represent an organotypic culture of the medial layer of a blood vessel.

Animals↗
PubMed1992

In-situ hybridization of tropoelastin mRNA during the development of the multilayered neonatal rat aortic smooth muscle cell culture.

Cultured neonatal rat aortic smooth muscle cells are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In this study, the ability of individual cells to synthesize tropoelastin was examined by in-situ hybridization methods. One-micron semi-thin epoxy resin-embedded transverse sections of cells cultured 1, 2, 3 and 4 weeks showed an increase with time in both the number of cells with hybridization signal and the signal intensity; tropoelastin mRNA hybridization signal intensity decreased thereafter up to 8 weeks in culture. In longitudinal sections through the early cultures (1-week), we observed mitotic cells with no detectable hybridization signal, and non-mitotic cells with either no, little or high signal intensity. These data suggest that mitotic cells do not synthesize tropoelastin, and that there is a strong correlation between the hybridization signal intensity and the rate of tropoelastin synthesis. These data also suggest in-situ hybridization methods can detect which cell(s) contain tropoelastin mRNA, their location in the multilayer, and variations in signal intensity. We conclude it is possible to correlate hybridization signal intensity with variations of tropoelastin mRNA levels within individual cells of the cultured smooth muscle cell multilayer.

Animals↗
PubMed1992

Elastin synthesis and accumulation in irradiated smooth muscle cell cultures.

Multilayer cultures of neonatal rat aortic smooth muscle cells, which were actively synthesizing elastin, were exposed to gamma-radiation. Elastin synthesis and accumulation was measured as a function of time after irradiation and compared to control (non-irradiated) cultures. Cells exposed to 50 Gy ceased to divide but continued to synthesize and accumulate elastin. The culture morphology suggested that the irradiated cells accumulated an extensive extracellular matrix between their cell layers. Interestingly, the amount of elastin accumulated in the irradiated cultures was nearly the same as in the controls despite the difference in cell number in the two cultures. Thus, on a per cell basis, the elastin accumulation was greater in the irradiated cultures than in the controls.

Animals↗
PubMed1991

The effect of beta-aminopropionitrile on elastin gene expression in smooth muscle cell cultures.

When beta-aminopropionitrile (BAPN) is added to neonatal rat aortic smooth muscle cell cultures there is a decrease in insoluble elastin accumulation with a concomitant increase in tropoelastin and tropoelastin fragments in the culture medium. The experiments described here examine the biological significance of this fragmentation. BAPN, as well as purified tropoelastin fragments isolated from spent medium of cells grown in the presence of BAPN, were added to cultures. A decrease in elastin mRNA was observed in cultures grown in the presence of BAPN and also in those cultures to which the purified tropoelastin moieties were added. These studies indicate that the inhibition of lysyl oxidase by BAPN prevents elastin crosslinking which results in an increase in tropoelastin moieties, thus leading to a down regulation of the steady state levels of elastin mRNA.

Aminopropionitrile↗
PubMed1991

Recombinant interleukin-1 beta inhibits elastin formation by a neonatal rat lung fibroblast subtype.

The effect of recombinant interleukin-1 beta (rIL-1 beta) on elastin accumulation by lipid-laden interstitial cells (LIC) derived from neonatal rat lung was examined. The LIC, a fibroblast subtype, synthesized large amounts of elastin which was deposited into the extracellular matrix. This elastin was alkali-resistant and had an amino acid composition typical of adult rat elastin. Treatment of lipid-laden interstitial cell cultures with rIL-1 beta at 100 pg/ml caused a dramatic decrease in elastin accumulation as assessed by hot alkali treatment and transmission electron micrographs of the cell cultures. Tropoelastin formation was selectively decreased by rIL-1 beta relative to other proteins. Steady state levels of elastin mRNA were slightly decreased by rIL-1 beta at 5 pg/ml and markedly decreased by rIL-1 beta at 50 pg/ml or greater. The addition of indomethacin had no effect on rIL-1 beta-induced decreases in elastin mRNA levels. Inhibiting protein synthesis with cycloheximide blocked the effect of rIL-1 beta on elastin mRNA levels. The level of alpha 1(I) collagen mRNA was decreased by rIL-1 beta, but only at concentrations higher than that needed to induce a decrease in elastin mRNA. These data indicate that rIL-1 beta decreased steady state levels for elastin mRNA and elastin accumulation and can selectively regulate the accumulation of elastin and collagen.

Animals↗
PubMed1991

A controlled precursor add-back model of elastogenesis in smooth muscle cell cultures.

Neonatal rat aortic smooth muscle cell cultures are capable of synthesizing and accumulating relatively large amounts of insoluble elastin in the extracellular matrix. There are two major soluble elastin molecules in these cultures, one of 77 kDa (protropoelastin) and the other of 71 kDa (tropoelastin). We examined the ability of the cell culture system to insolubilize exogenously added soluble elastin precursor moieties into the elastin matrix. To accomplish this, cultures were allowed to develop an enriched elastic fiber matrix for approximately two weeks in first passage. This accumulated matrix then served as the "substrate" for the exogenously added precursor elastin molecules. Culture-derived radioactive soluble elastin was added to the "substrate" cultures and the presence of radioactivity in the insoluble elastin as well as in the lysine-derived crosslinks unique to elastin (desmosines) was measured. When purified [3H]-valine radiolabeled protropoelastin was used, more than 15% of the radioactivity added was detected in the alkali-resistant insoluble elastin within 24 hours. After an initial 4-hour incubation of the cells with [3H]-lysine-labelled soluble elastin, most of the radioactivity in the insoluble elastin was associated with the lysine and only a negligible amount was detected in the desmosines. However, during a 16-day chase period, the ratio of radioactive desmosines to lysine increased dramatically, suggesting that not only insolubilization, but crosslinking occurs as well. The add-back system described herein should provide a means to probe the molecular properties of protropoelastin and increase our understanding of the mechanisms of elastic fiber formation.

Animals↗
PubMed1991

Measurement of urinary desmosine by isotope dilution and high performance liquid chromatography. Correlation between elastase-induced air-space enlargement in the hamster and elevation of urinary desmosine.

The accuracy of methods employed to measure the elastin-specific crosslinks, desmosine (DES) and isodesmosine (IDES), has been called into question because contaminants in the urine may cause elevated values. In the present study urine samples were spiked with a known amount of [14C]DES and refluxed in 6 N HCl. Sephadex G-15 chromatography of the hydrolyzed urine employed to remove contaminants. DES and IDES were quantified by high performance liquid chromatography (HPLC) as well as by amino acid analysis. The amount of isotope recovered was used to determine losses during the overall procedure and the isotope dilution to calculate the amounts of endogenous DES and IDES originally present in the urine. Because similar values were obtained by both methods, the more rapid HPLC method was used for all succeeding analyses. In one experiment, the DES amounts in urine collected from hamsters for 3 days after intratracheal treatment with human neutrophil elastase (300 micrograms) or porcine pancreatic elastase (300 micrograms) were 0.212 +/- 0.012 (mean +/- SEM, two measurements on a single pool) and 0.816 +/- 0.005 (two measurements) microgram per hamster per day, respectively. Urine from control hamsters had a mean value of 0.074 +/- 0.008 (eight measurements) microgram per hamster per day. The HNE- and PPE-treated hamsters had mean linear intercept values of 119 and 159% of control values, respectively, giving a positive correlation between increase in airspace size and elevation of urinary DES.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

In vivo fibroplasia of a porous polymer in the cornea.

We have developed a melt-blown fibrous construction of polybutylene/polypropylene in which we previously demonstrated keratocyte ingrowth and collagen synthesis in vitro. In the present studies, we evaluated this material in vivo in interlamellar corneal pockets for periods of up to six months. By day 42, the porous interstices of the disc were heavily populated with keratocytes. Extracellular matrix deposition occurred and there was a 5000-fold increase in total protein and a 1000-fold increase in total collagen over background. The cells within the disc continued to be synthetically active for the six months of our study. Discs remained in corneas for periods of up to one year without any extrusion. This material has great promise as a porous peripheral component of a keratoprosthetic device.

Animals↗
PubMed1990

The synthesis and role of integrin in corneal epithelial cells in culture.

Cell-substratum interaction and recognition is essential for cellular adherence and protein synthesis. To understand the mechanisms underlying diseases related to corneal erosion, it is necessary to examine the role integral proteins play in cellular adherence and spreading. In the current study, we used antibodies to evaluate the accumulation of integrin by corneal epithelial cells in tissue culture. We demonstrated that while the protein was incorporated into the cell layer at 6, 24, 48, and 72 hr, a far greater amount was secreted into the media. The total amount of integrin did not change with time, and the level of mRNA did not increase from 24 to 48 hr. During this time period, fibronectin was not detected. Antibodies to the fibronectin-binding receptor inhibited cellular spreading over a period of 18 hr. These results indicate that rabbit corneal epithelial cells do synthesize integrin and that integrin plays a critical role in cellular spreading.

Animals↗
PubMed1990

Monocyte activation by smooth muscle cell-derived matrices.

Mononuclear phagocytes adhere to and penetrate the vessel wall endothelium and contact the subendothelial space prior to the development of the atherosclerotic plaque. In an attempt to model the early events of plaque development we used an elastin-rich, multicomponent, cell-derived matrix from neonatal rat aortic smooth muscle cells as a substratum for monocytes. Using this model, we show that human monocyte morphology and metabolism are markedly altered by the matrix substratum. When a mixed mononuclear cell population is seeded on matrix or plastic, only monocytes adhere to the matrix surface. In contrast, lymphocytes as well as monocytes adhere to the plastic surface. The matrix-adherent monocytes develop large intracellular granules and form extensive clusters of individual cells. Metabolically, these cells develop sodium fluoride resistant non-specific esterase activity and their media contain more growth factor activity and PGE2. Although total protein synthesis is equivalent in both cultures, the matrix contact induces an increase in specific proteins in the media. We also show that a purified alpha-elastin substratum induces some, but not all, of the monocyte changes seen when using the matrix substratum. Using the alpha-elastin substratum, there is selective adhesion of monocytes and increased growth factor activity, however, the cells are morphologically different from the matrix-adherent cells. Thus, the use of the smooth muscle cell-derived matrix, in conjunction with purified matrix components, serves as a model that can provide insight into the mechanisms of monocyte adhesion and stimulation by the matrix environment that exists in vivo. Such mechanisms may be particularly important in atherogenesis.

Animals↗
PubMed1990

In vitro evaluation of fibroplasia in a porous polymer.

We evaluated a polybutylene/polypropylene blend in a blown microfiber configuration in vitro for potential use as the peripheral area of a keratoprosthetic device. Material properties such as ultimate tensile strength and ultimate elongation were measured. Stromal fibroblasts were seeded onto the material in vitro, and cell proliferation between uncoated and Type I collagen coated discs did not differ significantly. Fibroblasts could be seen migrating along the fibers and also traversing the fibers. The synthesis of connective proteins was examined. Laminin, fibronectin, and Type I collagen were detected by day 8. The experiments demonstrate that stromal fibroblasts can adhere onto the fibers, proliferate, and synthesize connective tissue proteins. Experiments are now being conducted to further evaluate the material in vivo.

Animals↗
PubMed1989

Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA).

A solid-phase, direct binding ELISA for serum amyloid A (SAA) proteins is described, in which noncovalent interactions of SAA with other plasma constituents are disrupted to permit direct coating of the wells of flexible polyvinyl chloride microtitration plates with an amount of SAA antigen proportional to its concentration in plasma. The wells are coated overnight at 60 degrees C with plasma diluted in 3 M potassium bromide and 0.1 M sodium bicarbonate. pH 9.6. The next day, any remaining sites on the wells are blocked by incubation for 1 h at ambient temperature with a 5% solution of dry milk solids and 0.05% Tween 20 in 0.02 M phosphate buffer, pH 7.4. The wells are rinsed and incubated for 90 min at 37 degrees C with polyclonal rabbit or rat anti-human SAA antiserum. Then, the wells are rinsed and incubated with goat anti-rabbit or rat IgG antiserum to which has been conjugated horseradish peroxidase. o-phenylenediamine and hydrogen peroxide substrates are added to the wells, color is allowed to develop, and sulfuric acid is added to stop the enzyme-catalyzed reaction. The amount of SAA coated to wells is quantified by absorbance at 490 nm. Four or more serial three-fold dilutions of plasma samples are assayed simultaneously on separate plates. Each plate contains a set of wells with identical concentrations of SAA standard protein diluted in decreasing concentrations of plasma proteins corresponding to the dilution of sample. The method can detect SAA concentrations in plasma samples ranging from 1 microgram/ml to greater than 1000 micrograms/ml. The method is suited to serial monitoring of SAA concentration in patients undergoing treatment for inflammatory conditions and to the quantitative analysis of large numbers of samples.

Blotting, Western↗
PubMed1989

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells.

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.

Amino Acid Sequence↗
PubMed1989

Effect of the reducing environment on the accumulation of elastin and collagen in cultured smooth-muscle cells.

We show here that cultured neonatal-rabbit aortic smooth-muscle cells produce and accumulate significant amounts of insoluble elastin. When grown in the presence of ascorbic acid, the amount of insoluble elastin in these cultures decreases, whereas the accumulation of collagen increases. These changes have been attributed to increased hydroxylation of proline in elastin. The function of ascorbic acid in proline hydroxylation is thought to be that of a reductive cofactor that maintains the proper oxidation state of molecular iron in the enzyme complex. This study shows that both ascorbic and isoascorbic acids act similarly to modify the accumulation of elastin and collagen in culture. On the other hand, cultures grown in the presence of dithiothreitol, a reducing agent previously shown to act as a cofactor for prolyl hydroxylase, do not demonstrate altered elastin accumulation. These studies are consistent with the suggestion that there is a specific role for ascorbic acid in this cellular system that cannot be replaced by other reducing cofactors.

Amino Acids↗
PubMed1989

Modified hydroxyethylmethacrylate hydrogels as a modelling tool for the study of cell-substratum interactions.

The interactions between cells and their extracellular substratum environment are complex and difficult to study. Defined, synthetic substrata are valuable tools for experimentally determining the role of ionic and receptor-specific interactions between cells and their substrata. Hydrogels have been modified to contain stoichiometrically defined quantities of both positive and negative charge as well as specific proteins. These synthetic surfaces are water-rich matrices that possess hydroxyl groups, positive and negative ionized charges and native proteins, and can be considered as models of extracellular matrices on which an assessment of charge contribution and macromolecular content and specificity can be addressed with respect to cell-matrix interactions. This study shows that simple gels made of polyhydroxyethylmethacrylate do not support the spreading of cells but that the generation of copolymers by the addition of monomers that contain ionizable functional groups, will permit cell spreading. These simple modifications do not lead to cellular proliferation, yet when collagen is entrapped in the hydrogel substratum, proliferation occurs. The proliferative rate of cells grown on collagen-containing surfaces may be modified by altering the stoichiometry of the ionizable polymers used to make the surface. This study describes a synthetic, definable model for the study of cell-substratum interactions and control.

Acrylates↗
PubMed1988

Elastin mRNA levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures.

Insoluble elastin accumulation, elastin mRNA translational efficiencies, and elastin mRNA levels were evaluated in cultures of neonatal rat aortic smooth muscle cells grown for several days in consecutive passages. When the products of in vitro translation were immunoprecipitated with an anti-alpha-elastin antibody, a single 79,000-Da protein was obtained. Northern blot analysis also indicated an elastin mRNA species corresponding to approximately 4.2 kilobases. Insoluble elastin accumulation increased in cells cultured for 7-21 days in first through fourth passages, while with one exception, relative levels and translational activity of elastin mRNA decreased with time in culture. The data indicated that a simple relationship between elastin accumulation and elastin mRNA levels was not evident.

Animals↗
PubMed1988

Carbohydrate moieties of the basal lamina: their role in attachment and spreading of basal corneal epithelial cells.

Three lectins, Wheat germ agglutinin, succinyl Concanavalin A and Ricinus communis agglutinin were used to block specific sugar moieties in the basal lamina. Corneal epithelial basal cells were plated onto freshly denuded basal lamina. Attachment was studied by quantifying the adherence of prelabeled cells and by examining attachment sites using transmission electron microscopy. Spreading was examined using scanning electron microscopy. Attachment of the cells occurred within 15 min and spreading was apparent after 45 min. Both Wheat germ agglutinin and beta-N-acetylglucosaminidase inhibited cellular attachment. Succinyl Concanavalin A and Ricinus Communis agglutinin permitted attachment, but inhibited extensive cellular spreading. The results indicate that the attachment of basal cells is dependent on N-acetylglucosamine residues, and spreading is mediated by alpha methylmannoside, glucose, and galactose residues.

Animals↗