Biomedical subjects

R F Diegelmann

Publications and source records attributed to R F Diegelmann.

At least 55 records · Page 3Linked to original sources

PubMed1990

A microassay to quantitate collagen synthesis by cells in culture.

A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [3H]proline. After incubation, the plates were heated to 90 degrees C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble 3H-labeled collagen-derived peptides and the remaining insoluble 3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and non-collagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.

Cells, Cultured↗

PubMed1990

Transforming growth factor beta 1 selectively augments collagen synthesis by human intestinal smooth muscle cells.

Intestinal smooth muscle cells play a major role in the stricture formation that complicates chronic intestinal inflammation, by proliferating and producing collagen. Transforming growth factor beta 1 has been identified as an important inflammatory mediator in the fibrotic response of human tissue to inflammation. To determine whether this mediator might be involved in intestinal fibrosis, the effect of transforming growth factor beta 1 on collagen production and proliferation by human intestinal smooth muscle cells was studied in vitro. Cells in the second passage were grown to subconfluence in medium containing 10% Nu-Serum (Collaborative Research Inc., Bedford, MA), after which the concentration of Nu-Serum was decreased. Forty-eight hours later, transforming growth factor beta 1 was added to the culture medium to achieve concentrations of 1-500 pmol/L. After 24 hours exposure to the transforming growth factor beta 1, cellular collagen synthesis was determined by the uptake of [3H]proline into collagenase-sensitive protein. Transforming growth factor beta 1 caused a 100% increase in collagen production and a 40% increase in noncollagen protein production per cell, reflecting an increase in relative collagen synthesis of 58%. This effect was maximal at a concentration of 10 pmol/L. Epidermal growth factor, by comparison, had no significant effect on relative collagen synthesis. Transforming growth factor beta 1 caused a significant increase in the uptake of methylaminoisobutyric acid, a nonmetabolized amino acid analog, into the cells at 10 pmol/L. However, this effect was small (20% increase) compared with the effect on the uptake of proline into collagen (100% increase) at this concentration. When cell proliferation was examined by the uptake of [3H]thymidine, transforming growth factor beta 1 had no effect, whereas epidermal growth factor (1000 pmol/L) caused a 94% increase. Transforming growth factor beta 1 selectively augments collagen production by human intestinal smooth muscle cells in vitro. This effect is potent and is not related to effects on either cell proliferation or amino acid uptake. These data suggest that transforming growth factor beta 1 has an important role as an inflammatory mediator in the pathogenesis of intestinal fibrosis.

Cells, Cultured↗

PubMed1990

Fetal skin organ culture.

The fetal response to cutaneous injury has been investigated in a variety of models; most have studied the differences between fetal and adult healing mechanisms in vivo and in cell culture. Further disclosure of the cellular and biochemical events requires a model that can be manipulated to study single factors influencing fetal tissue repair without the complex interactions that occur in vivo, but in a system that more closely approximates normal skin than cell culture models. This paper presents a method for the organ culture of fetal skin and its advantages as a model to help elucidate fetal healing mechanisms. Skin sections (1 x 1 cm) excised from the backs of fetuses of New Zealand white rabbits on day 27 gestation (term = 31 days) were placed eccentrically in 65-mm culture dishes and fed daily with 2.5 mL of DMEM containing 10% fetal calf serum, antibiotics, and 10 mM ascorbic acid. A separate group, treated similarly, received 4-mm punch wounds to assess the in vitro response to wounding. The specimens were incubated at 37 degrees C in humidified room air on a rocker platform to provide alternate exposure of the skin to air and medium. Gross observation at 3 weeks showed cells extending into the central wound, indicating that viable cells were proliferating and/or migrating from the tissue. Skin was examined histologically and was viable over the 3-week period studied. Organ culture, by maintaining tissue in the natural extracellular matrix, allows cell-to-cell contact and communication to be maintained while allowing controlled environmental manipulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Protamine selectively inhibits collagen synthesis by human intestinal smooth muscle cells and other mesenchymal cells.

Collagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration-dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly-L-lysine decreased collagen production to a lesser degree than protamine. Poly-L-arginine was toxic to the cells. Poly-L-glutamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.

Ascorbic Acid↗

PubMed1989

Cyclic nucleotides regulate collagen production by human intestinal smooth muscle cells.

The effect of cyclic nucleotides on collagen production by human intestinal smooth muscle cells was examined in vitro. Cholera toxin and isobutylmethylxanthine, agents that elevate cyclic adenosine monophosphate, caused selective inhibition of collagen production when cells were exposed to these agents for 24-72 h. Exposure for 6 h inhibited noncollagen protein synthesis without effects on collagen production. Forskolin similarly inhibited collagen production, decreasing relative collagen synthesis 40% at 10 microM and 60% at 100 microM. After 48 h of exposure to cholera toxin and isobutylmethylxanthine, levels of cyclic adenosine monophosphate had increased in a concentration-dependent manner. The effect of cyclic nucleotide analogues was also examined. Dibutyryl cyclic adenosine monophosphate inhibited collagen production, whereas dibutyryl cyclic guanosine monophosphate increased collagen production by 65%. This effect was maximal at a concentration of 10 microM. These observations suggest that cyclic adenosine monophosphate has a significant effect on collagen production by human intestinal smooth muscle cells in vitro and may play a role in the modulation of collagen production by these cells in vivo.

1-Methyl-3-isobutylxanthine↗

PubMed1989

Analysis of transforming growth factor beta receptor binding in embryonic, fetal, and adult rabbit fibroblasts.

Adult wound repair traits including inflammation, fibroplasia, and collagen deposition are not seen at fetal wound sites. This observation raised questions about regulatory mechanisms extant in fetal healing. Transforming growth factor beta (TGF-beta) is an important regulatory polypeptide known to orchestrate fibroplasia and collagen synthesis during adult wound repair. Previous studies have suggested that the wounded rabbit fetus is capable of responding with these adult characteristics if provided with exogenous TGF-beta. In order to test whether the observed in vivo effects of TGF-beta in the rabbit fetus might be due to a direct effect on the fibroblast, TGF-beta receptor binding characteristics of early passage cultured embryonic (14 days' gestation), fetal (24 days' gestation), and adult rabbit fibroblasts were studied by flow cytometry. Experiments were carried out using fluorescein-conjugated TGF-beta (F-TGF-beta) with analysis on an EPICS V flow cytometer. F-TGF-beta was incubated with each of the three fibroblast types at 37 degrees C after which time the cells were washed twice and analyzed with a minimum of 10(5) cells for each data point. F-TGF-beta bound rapidly and reversibly to the embryonic, fetal, and adult fibroblasts with saturation being achieved at 1 nmol/L for fetal and adult cells, and 8 nmol/L in the embryonic fibroblasts. Saturating concentrations of F-TGF-beta yielded mean channel numbers (a function of relative amounts of F-TGF-beta-bound) of 172, 114, and 97 for embryonic, fetal, and adult cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Characterization and quantitation of wound matrix in the fetal rabbit.

Fetal response to injury has been characterized by the deposition of a matrix that is not primarily collagen. This study was designed to identify this matrix, in order to better understand the fetal mechanism of tissue repair. Silastic/polyvinyl alcohol sponge (PVA) wound implants were placed paravertebrally in 24-day gestation (31 days = term) fetal (n = 65) and adult (n = 43) rabbits and then harvested from one hour to 6 days post-wounding. Histologic analysis of the fetal wound matrix deposited in the PVA implants suggested the presence of glycosaminoglycans (GAG) rather than the collagen found in adult wound implants. To further analyze the fetal wound matrix, the GAG content was quantitated using an Alcian Blue dye-binding assay. Results showed significantly increased (p less than 0.05) GAG deposition on days 2-6 in the fetal wound compared to the adult wound. Fetal GAG levels were approximately three times those of the adult during this period. Separation of individual GAG species by cellulose acetate electrophoresis demonstrated that the GAG matrix of the fetal wound was composed predominantly of hyaluronic acid. This finding was confirmed by selective enzymatic digestion of separated GAG species using highly specific polysaccharidases. These observations of hyaluronic acid deposition in the fetal wound may be ascribed an important physiologic role by providing a more fluid and malleable matrix rather than a restrictive matrix composed of collagen. This new evidence coupled with earlier findings of the lack of an acute inflammatory response in the fetus further supports the hypothesis that the fetal response to injury is significantly different from the adult response.

Alcian Blue↗

PubMed1989

Analysis of fibrogenic processes in denervated tissues of spinal cord injury patients.

To test the hypothesis that altered collagen metabolism is a contributing factor in the apparent delayed wound healing in denervated regions of spinal cord injury (SCI) patients, a tissue implant (PVA) was used to directly measure collagen deposition. Sterile PVA implants were placed subcutaneously in the inner aspect of the upper arm above the cord injury (innervated) and in the inner aspect of the upper leg below the cord injury (denervated) of 20 spinal cord injury patients and compared to eight healthy volunteers. On day 14, the implants were removed and analyzed histologically by trichrome stain and biochemically for hydroxyproline as a measure of collagen deposition. No remarkable histologic differences were observed in the sponge material removed from the upper regions compared to the lower denervated regions of the spinal cord injury patients. Sponges from both areas were infiltrated with fibroblasts containing well-developed rough endoplasmic reticulum and large quantities of trichrome-positive collagen. Likewise, upper and lower histology of controls was identical and nondistinguishable from the corresponding sections obtained from the spinal cord injury patients. Quantitation of the hydroxyproline in the arms of the spinal cord injury patients (n = 20) showed 4.3 +/- 0.7 nmol hydroxyproline per milligram of sponge compared to 4.1 +/- 0.4 nmol/mg in the denervated regions of the lower limb. The hydroxyproline content in the arms of control volunteers was 5.2 +/- 0.7 nmol/mg compared to 3.9 +/- 0.8 nmol/mg in the leg (n = 8). These observations suggest that fibrogenic processes in denervated regions are not reduced significantly compared to innervated regions.

Adult↗

PubMed1988

Modulation of tendon fibroplasia by exogenous electric currents.

A chicken tendon explant model system has been developed to investigate the effects of extremely-low-frequency (ELF), low-amplitude, unipolar, square wave pulsed electric fields on fibroplasia in vitro. An electric field parameter set consisting of 1-Hz, 1-ms duration pulses, with a time-averaged current density of 7 mA/m2 (peak current density 7 A/m2) induced maximal (32%) increase in fibroblast proliferation in tendon explants exposed for 4 days. Exposure to the same field at an average current density of 1.8 mA/m2 had no effect on fibroblast proliferation, whereas exposure to current densities on greater than 10 mA/m2 inhibited proliferation and relative collagen synthesis, without affecting noncollagen protein synthesis. Fibroplasia was significantly increased in explants oriented parallel to applied electric fields having current densities of 3.5 or 7 mA/m2, but there was no detectable effect on explants oriented perpendicular to the same electric field. Fibroblast proliferation and relative collagen synthesis were inversely proportional to donor age for chickens in the 3- to 16-week age group used in this study. For these dependent variables (proliferation and relative collagen synthesis), there was no interaction between donor age and ELF electric field exposure.

Animals↗

PubMed1988

Collagen content and types in the intestinal strictures of Crohn's disease.

The collagen content and the relative amount of collagen types were quantitated in control intestine as well as in both inflamed and strictured intestine resected from patients with Crohn's disease. The major collagen type in control intestine was type I (68%), followed by types III (20%) and V (12%). In strictured intestine both collagen content and the relative amount of type V collagen were significantly increased compared with control intestine. Histologic studies demonstrated that in strictured specimens there was a striking proliferation of smooth muscle cells of the muscularis mucosae associated with an accumulation of collagen in the submucosa. The thickness of the muscularis propria was also increased. Immunohistochemical studies demonstrated small amounts of type V collagen in the submucosa of control bowel. In contrast, large amounts of type V collagen were seen in the fibrotic, expanded submucosa of strictured bowel, particularly in the areas where smooth muscle cells of the muscularis mucosae had proliferated. Intestinal strictures in Crohn's disease are therefore characterized by an accumulation of collagen, a proliferation of smooth muscle cells, and an increase in type V collagen, a collagen type produced in relatively large amounts by smooth muscle cells. These changes appear to result in both a loss of the normal compliance of the intestine and a thickening of the intestine wall, resulting ultimately in the intestinal obstruction so frequently seen in patients with Crohn's disease.

Amino Acids↗

PubMed1988

Transforming growth factor beta (TGF-beta) induces fibrosis in a fetal wound model.

The adult cellular response to tissue injury is characterized by acute inflammation followed eventually by fibroblast proliferation and collagen synthesis. Fetal tissue responses to injury differ markedly from those of the adult; an early acute inflammatory response is absent, few fibroblasts participate, and no collagen is deposited. The object of the present study was to analyze the effects of transforming growth factor beta (TGF-beta), an important regulatory molecule in adult healing events, on the fetal tissue response following wounding. Fetal cellular and extracellular matrix responses to injury were evaluated by placing subcutaneous wound implants containing TGF-beta (0.01 to 10 ng) in fetal rabbits at 24 days gestation (term = 31 days). Histologic responses one to seven days later were compared with fetal and adult control implants without TGF-beta. The histology of the adult implant was characterized by an early acute inflammatory response: by day 7 fibroblasts and collagen were predominant. In contrast, control implants removed from fetal rabbits had no histologic evidence of acute inflammation or fibroblast penetration and no collagen was deposited. When implants containing 1.0 ng TGF-beta were removed from fetal rabbits at seven days, a grossly fibrotic reaction was observed: histology confirmed marked fibroblast penetration with collagen deposition. Fetal implants containing 0.01 ng or 10 ng TGF-beta showed few fibroblasts but had increased numbers of inflammatory cells compared with controls. These observations demonstrate that the fetal response becomes adultlike with fibroblast proliferation and collagen accumulation when TGF-beta is added, thus documenting the responsiveness of the fetal system to adult repair signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Effect of beta-aminopropionitrile and ascorbate on fibroblast migration.

Ascorbate and beta-aminopropionitrile (BAPN) have direct, but diverse affects on collagen matrix production. Ascorbate is necessary for the intracellular hydroxylation of prolyl and lysyl residues during collagen biosynthesis whereas BAPN inhibits the enzyme lysyl oxidase in the extracellular space thus preventing collagen crosslink formation. To study the influence of these two agents on fibroplasia, an in vitro model was used to analyze fibroblast migration, proliferation, and collagen synthesis. Biopsies of chicken tendon were covered with a fibrin clot to simulate an in vivo wound environment, and then they were exposed to either ascorbate or BAPN for up to 7 days. Fibroblast migration into the fibrin clot was measured using a Zeiss Mopp II planimeter, DNA synthesis by 125IUDR incorporation, and collagen synthesis by [3H]proline incorporation into collagenase-digestible protein. Tendon biopsies treated daily with fresh ascorbate (0.1 mM) had significantly greater fibroblast migration than controls without ascorbate (P less than 0.05). Cellular proliferation, collagen synthesis, and total protein synthesis were not significantly altered by ascorbate treatment. In contrast, BAPN inhibited fibroblast migration in a dose-dependent fashion without inhibiting proliferation (0.25 and 0.5 mM), collagen, and noncollagen protein synthesis. Therefore, the effect of BAPN on migration does not appear to be due to generalized cytotoxicity. These combined studies suggest that compounds such as ascorbate and BAPN which can modify collagen may also modify fibroblast migration.

Aminopropionitrile↗

PubMed1987

Heparin induces specific protein release from human intestinal smooth muscle cells.

Human intestinal smooth muscle cells have recently been identified as the major cell type responsible for stricture formation in Crohn's disease. Heparin, a sulfated glycosaminoglycan, has been shown to be a key modulator of vascular smooth muscle cell growth both in vivo and in vitro and to affect the release of proteins from these cells. Heparin has also been shown to affect the growth of human intestinal smooth muscle cells. In this report we demonstrate that heparin, in addition to its effects on proliferation, also has very specific effects on proteins released by these cells in vitro. Examination of the culture medium proteins of heparin-treated human intestinal cells revealed an increase in three proteins of molecular weight between 150-250 kd, an increase in a 37 kd protein and a decrease in synthesis of lower molecular weight (less than 20 kd) proteins. In substrate-attached material a transient effect on a 48 kd protein was observed. No effects on intracellular labeled proteins could be demonstrated. The 35S-methionine labeled protein profile of human intestinal smooth muscle cells exposed to heparin is similar to that observed in rat vascular smooth muscle cells yet distinct differences do exist. Extracellular processing does not account for the released proteins nor is de novo protein synthesis required suggesting that altered intracellular protein processing is the mechanism for the heparin-induced protein pattern. The release of specific proteins following exposure to heparin may reflect a significant influence of this glycosaminoglycan on the metabolism of smooth muscle cells in general and particularly in the human intestine.

Cells, Cultured↗

PubMed1987

Collection of leukocytes, fibroblasts, and collagen within an implantable reservoir tube during tissue repair.

An implantable chamber consisting of a small reservoir and a perforated segment of silicone tubing has been developed for the collection of leukocytes, fibroblasts, and collagen to analyze inflammatory components and fibroplasia during tissue repair. Using aseptic techniques, these sterile chambers were placed into subcutaneous pockets on the backs of Sprague-Dawley rats with cells obtained in daily aspirates for 14 d. Differential cell counts were made by using aliquots from the wound fluid. The aspirated cells represented the characteristic, sequential influx of neutrophils, inflammatory macrophages, lymphocytes, activated macrophages, and fibroblasts documented in other models of tissue repair. On d 14, the connective tissue within the lumen of the silicone tube was removed and analyzed for collagen synthesis by measuring 3H-proline incorporation into collagenase-sensitive protein. This new device for studying wound healing provides a convenient means to harvest cells, fluid, and tissue for cellular, humoral, and biochemical analyses of tissue repair.

Animals↗

PubMed1987

Collagen synthesis by human intestinal smooth muscle cells in culture.

Growth kinetics and collagen production were determined in smooth muscle cells isolated from human jejunum and maintained in cell culture. Collagen synthesis increased during the first 15 days in culture at a time when the rate of cell proliferation was maximal. When confluent, these cells produced significantly more collagen than human dermal fibroblasts cultured under identical conditions. The smooth muscle cells required daily replenishment of ascorbate for maximal collagen synthesis. The types of collagen produced by human intestinal smooth muscle cells in culture were the same as those collagens extracted from strictured human bowel (types I, III, and V). These findings suggest that collagen production by human intestinal smooth muscle cells has a role in the repair as well as the fibrosis of the gastrointestinal tract.

Ascorbic Acid↗

PubMed1987

Heparin modulates human intestinal smooth muscle cell proliferation, protein synthesis, and lattice contraction.

The effect of heparin on human intestinal smooth muscle cell proliferation, collagen and noncollagen protein synthesis, and collagen lattice contraction was studied in vitro. Proliferation of serum-stimulated cells was inhibited in a concentration-related fashion by continuous exposure to heparin. The inhibition of proliferation was reversible when heparin was removed from the culture medium. Collagen synthesis was inhibited by 24-h exposure to heparin, but only during that phase of culture (8-12 days) when collagen synthesis was maximal. Noncollagen protein synthesis was down-regulated by 24-h exposure to heparin at all phases of culture tested (5-21 days). Heparin also abolished the contraction of collagen lattices populated by human intestinal smooth muscle cells. These studies demonstrate that heparin plays a significant role in the modulation of human intestinal smooth muscle cell behavior in vitro and suggest that a similar role is played by heparinlike components of the extracellular matrix in vivo.

Cell Division↗

PubMed1987

Fetal response to injury in the rabbit.

Fetal, neonatal, and adult tissue response to a standardized injury was studied using subcutaneous wound implants, linear incisions, and punch wounds in New Zealand white rabbits. In the fetus, sutured incisions healed by primary intention without antecedent inflammation. However, neither contraction nor healing by secondary intention was seen in punch or unsutured wounds. Healing both by primary and secondary intention following inflammatory infiltration was observed uniformly in neonatal and adult rabbits. Wound implants were extensively infiltrated with collagen in the adults studied; however, no collagen was seen in fetal implants and collagen hydroxyproline content could not even be detected by high performance liquid chromatography techniques; rather, a matrix rich in hyaluronic acid was found. The fetal tissue response to injury differs from the adult, proceeding in the absence of a classical inflammatory stimulus and lacking contractile capabilities. The deposition of extracellular matrix rich in hyaluronic acid but devoid of collagen suggests that the fetal response to injury may be a process more closely resembling regeneration or growth rather than repair by scar deposition.

Animals↗

PubMed1987

Colchicine does not provide a sustained blockage of collagen and plasma protein secretion by rat hepatocytes.

Colchicine has been reported to disrupt microtubules and thereby inhibit collagen secretion. Because of this "anti-collagen" activity, colchicine has been suggested for use in the treatment of hepatic fibrosis. Using biochemical and immunohistochemical techniques, our laboratory has identified the hepatocyte as one possible source of collagen in the liver. The present study examined the direct effect of colchicine on collagen secretion by hepatocytes in culture. Parenchymal cells were isolated from the livers of adult rats four days following a two-thirds hepatectomy. Total collagen and the fraction secreted into the medium were quantitated as incorporation of [3H]-proline into bacterial collagenase-sensitive protein. Treatment of the hepatocytes with 100 microM colchicine (2-3 hours) resulted in a substantial inhibition of collagen secretion. However, upon longer exposure of the hepatocytes to the drug (24 hours and 8 days), the inhibitory effect on collagen secretion was abolished. The anti-protein secretion activity of the colchicine in the conditioned medium removed from the hepatocytes was still present as verified by a 2.5 hour fibroblast-collagen secretion bioassay. The secretion of the plasma proteins albumin, fibrinogen and the third component of complement was not altered by the presence of colchicine. We conclude that the hepatocyte is a highly efficient secretory cell, and is not entirely dependent upon microtubules as organelles for protein secretion. Therefore, to the extent that hepatocytes may contribute to the hepatic fibrosis, the therapeutic use of colchicine to block collagen secretion might be expected to have only limited effectiveness.

Animals↗