Protein synthesis in collagen lattice-cultured fibroblasts is controlled at the ribosomal level.

Fibroblasts cultivated in three-dimensional lattices exhibit a large decrease of protein synthesis, mainly through transcriptional control. However, no previous work was devoted to a potential ribosomal regulation. We evaluated ribosomal ribonucleic acid (RNA) in monolayer- and collagen lattice-cultured fibroblasts. After one week of culture, total RNA was 60% lower in lattice-cultured fibroblasts than in monolayer-cultured cells. The decrease was identical for 18 S and 28 S rRNA subfractions. The half-life of RNA was much shorter in collagen lattice-cultured fibroblasts than in monolayers. These results suggest that protein synthesis in lattice-cultured fibroblasts is partly regulated at the ribosomal level.

Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells.

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.

In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.

Adhesion and activation of human neutrophils onto collagen chains separated by electrophoresis.

After separation of the various alpha chains of the collagens by SDS-PAGE, the binding of polymorphonuclear neutrophils (PMN) to these chains was detected by a double-antibody technique and the activation of PMN by nitro blue tetrazolium. All of the alpha chains tested were able to bind PMNs. The alpha 1 chain of type I collagen activated the PMN when it had not been treated with pepsin. Pepsinized types II and VI collagens did not activate PMN. The pepsinized alpha 1(III) chains and all three alpha chains from pepsinized type V collagen were able to activate PMN.

[Synthesis of collagen in culture of endothelial cells of the human umbilical vein. Effects of cyclosporine A]. / Synthèse de collagène par les cellules endothéliales de veine ombilicale humaine en culture. Effets de la ciclosporine A.

Interstitial renal fibrosis and gingival hypertrophy are frequent side-effects of cyclosporin A which have been attributed to a dysfunction of extracellular matrix synthesis. Endothelial cells might participate in the matrix accumulation observed. We studied the effects of increasing concentrations of cyclosporin A on protein synthesis by human umbilical vein endothelial cells. Collagen synthesis decreased significantly to 800 ng/ml in both medium and cell layer. The percentage of hydroxylation of its proline residues decreased significantly as from 400 ng/ml. The main proteins, analysed by SDS-PAGE, were thrombospondin, fibronectin and the alpha 1 and alpha 2 chains of type IV collagen. These fractions did not show any change after 24 hours exposure to 200 ng/ml of cyclosporin A. These results demonstrate an inhibitory effect of cyclosporin A on collagen synthesis by human umbilical vein endothelial cells. Consequently, matrix accumulation by increased collagen synthesis in cyclosporin A treated patient may not be directly related to the drug effect on endothelial cells.

Accumulation of glycosaminoglycans in radiation-induced muscular fibrosis.

The content and biosynthesis of glycosaminoglycans (GAGs) were studied in the pig thigh muscle after acute local gamma-irradiation. Seven months following irradiation, the muscular tissue next to the irradiation cone was replaced by severe mutilating fibrosis delimited by an intermediary perifbrotic zone. Fibrosis, perifibrotic tissue and normal muscle, were sampled and incubated with [3H]glucosamine and [35S]sulphate, and GAGs were isolated following pronase digestion. Results showed a parallel increase of collagen and GAG content in perifibrotic and fibrotic tissues. Sulphated GAGs, heparan sulphate and dermatan sulphate were preferentially accumulated in fibrotic tissue, while the hyaluronic acid content increased only slightly. Synthesis of sulphated GAGs was more elevated in fibrotic tissue than in perifibrotic zone as compared with normal muscle. Seven months after irradiation well-developed fibrotic tissue continued to synthesize and to accumulate extracellular matrix macromolecules, indicating the invasive aspect of post-irradiation fibrosis.

Long-term overproduction of collagen in radiation-induced fibrosis.

Collagen metabolism was investigated in the fibrotic tissue which developed in pig thigh muscle 6 to 15 months after acute gamma irradiation. During this period, total collagen deposits in the fibrotic tissue increased 10-fold compared to the healthy muscle tissue. These deposits were composed mainly of type I and III collagen, and the type I/type III ratio was lower in the fibrotic than in the muscle tissue. Small pieces of both fibrotic and muscle tissue were incubated with [14C]proline. The [14C]hydroxyproline content of the fibrotic tissue reflected large concomitant increases in the synthesis of total collagen, mainly of types I and III, which rose 14- and 17-fold, respectively. Similarly, the level of type I and type III procollagen mRNAs rose 9- and 5-fold, respectively, in the fibrotic tissue versus the muscle tissue. These results suggest that procollagen gene transcription or RNA maturation in the cell nuclei is activated in the fibrotic tissue. The possibility that such activation is due to the long-term inflammatory state of this tissue is discussed.

Human type VI collagen: isolation and characterization of alpha 1 and alpha 2 chains by two-dimensional preparative gel electrophoresis.

Two 140 kDa collagenous glycoproteins were isolated from 5 M guanidinium chloride extracts of human uterine leiomyoma by two-dimensional preparative gel electrophoresis. The glycoproteins represented the major concanavalin A binding fraction of the extract and were also present in adult human skin. On two-dimensional gel electrophoresis the glycoproteins appeared as elongated spots, indicating variations of their isoelectric points from 5 to 6. These glycoproteins were disulfide-bonded components of high molecular mass protein and, after reduction, became sensitive to collagenase treatment that generated peptides corresponding in size to those of the noncollagenous domains of type VI collagen. Antisera raised against these purified glycoproteins reacted with either pepsin-derived alpha 1(VI) or pepsin-derived alpha 2(VI) chains but not with alpha 3(VI) chain of human type VI collagen. Reciprocally, these glycoproteins reacted with monoclonal antibodies against type VI collagen. These results indicate that the glycoproteins represent the integral alpha 1 and alpha 2 chains of type VI collagen. The globular domains of alpha 1(VI) and alpha 2(VI) chains remaining after collagenase treatment appeared on two-dimensional gel electrophoresis as elongated spots, suggesting that the noncollagenous portions determine the well known microheterogeneity of the molecule. The differences in isoelectric points between and within alpha chains may facilitate the formation of microfibrillar network.

Comparative study of the precipitation of low density lipoproteins by aortic proteodermatan sulphate and heparin.

The aortic proteoglycans and heparin were shown to form insoluble complexes with human low density lipoproteins (LDL). The effect of temperature, polyethylene glycol and ionic strength on the formation of complexes between porcine aortic proteodermatan sulphate (PDS) and LDL has been studied by laser nephelometry and comparisons made with heparin LDL complexes. Turbidity was a nonlinear function of the quantity of LDL precipitated by PDS. The turbidity of aggregates was constant at temperatures between 2 degrees C and 30 degrees C but increased with temperature above 30 degrees C up to 50 degrees C. The formation of insoluble complexes decreased rapidly with increasing NaCl concentration. Polyethylene glycol enhanced the turbidity at 20 degrees C but not at 37 degrees C. It also increased the resistance of complexes to dissociation by increasing ionic strength. The turbidity of heparin--LDL complexes was linearly correlated with the quantity of precipitated LDL. The heparin-LDL aggregates were less sensitive to modification of temperature and ionic strength than the PDS-LDL aggregates. These results suggest that ionic interactions are weaker in PDS-LDL complexes than in the heparin-LDL complexes. Non-coulombic interactions and/or temperature dependent conformational changes may be involved in the stabilization of supramolecular PDS-LDL aggregates. No such interactions or changes appear to be involved in complex formation between heparin and LDL.

Effects of thyroid-stimulating hormone and phorbol ester on glycosaminoglycan synthesis in porcine thyroid epithelial cells in primary culture.

The effects of thyroid-stimulating hormone (TSH) and a tumor promoter: 12-0-tetradecanoyl-phorbol-13-acetate on glycosaminoglycan (GAG) synthesis were studied in porcine thyroid epithelial cells in primary culture. TSH is known to involve cyclic AMP mechanism and phorbol ester to act by protein kinase C pathway. Chronic treatment of cells with TSH increased the synthesis of heparan sulphate associated with the cell layer and hyaluronic acid in the culture medium. Phorbol ester increased the radioactivity of total GAGs in the culture medium but had no effect on GAGs associated with the cell layer. It inhibited the positive effect of TSH on heparan sulphate synthesis. These results suggest that in thyroid epithelial cells the synthesis of the GAGs associated with the cell layer and those secreted into the culture medium are regulated by different intracellular mechanisms.

Fibronectin and glycosaminoglycan synthesis by fibrotic pig fibroblasts in primary culture.

Synthesis of fibronectin and glycosamingoglycans (GAGs) was studied in fibroblasts from pigs with post-irradiation subcutaneous fibrosis. Fibrosis was developed in the femoral muscle by local gamma irradiation with a dose of 60 Gy. Normal fibroblasts were obtained from the healthy skin of the same animal. To measure GAG and fibronectin synthesis fibrotic and normal fibroblasts were labeled with 3H-glucosamine, 35S-sulfate and 35S-methionine. Fibrotic fibroblasts synthesized 2.5 times as much fibronectin as normal skin fibroblasts but total protein synthesis did not change. Parallel enhanced secretion of hyaluronic acid and dermatan sulfate into the cell culture medium were also observed. GAGs from the pericellular layer of trypsin-digested fibrotic fibroblasts exhibited increased 3H incorporation, but reduced 35S-sulfate incorporation. The largest reduction in the latter was observed for heparan sulfate. These results indicate that the fibroblasts from the well developed fibrotic tissue maintain enhanced synthesis of matrix macromolecules in primary cultures. Structural and/or metabolic changes in secreted GAGs, combined with the stimulation of tissue repair by growth factors may be responsible for the excessive deposition of collagen in post-irradiation fibrosis.

A parallel between two techniques of extraction of connective tissue macromolecules.

A method of extraction of the collagen and noncollagen proteins from deep dermis of young adult rabbits using a 0.1 M tartaric acid solution was set up. The tartaric acid extraction, together with the preliminary neutral salt extraction, solubilized 95% of the total collagen and 98% of the noncollagen proteins, far more than the 6 M guanidinium Cl solution used for comparison. Elastin was not extracted. Studies on the fibrillation of the extracted collagen in neutral solution at 25 degrees C or on the results of pepsin digestion in acidic solution at +4 degrees C showed that the tartaric acid-extracted collagen was in a nondenatured form, whereas that extracted by guanidinium Cl was largely denatured. Polyacrylamide gel electrophoresis (PAGE) indicated that most of the collagen was of type I and that many noncollagen proteins were present, mostly in the molecular weight range of 40 kDa. Bidimensional PAGE gave a reproducible pattern of these noncollagen proteins, showing that several additional proteins were present in tartaric acid extracts and not in guanidinium chloride extracts.

Modification of collagen and noncollagenous proteins in radiation-induced muscular fibrosis.

Six months after acute local gamma irradiation of the pig skin and adjacent muscle, the muscular tissue is replaced by a large mutilating and proliferative fibrosis deliminated by a perifibrotic inflammatory zone. The content and biosynthesis of collagen and noncollagenous proteins were studied in both fibrotic and perifibrotic zones after incubation of the biopsies with [14C]proline or [35S]methionine for 24 hr. Cells of perifibrotic and fibrotic regions synthesize about 10 times more proteins than those in the nonirradiated muscle. When compared to normal muscle tissue, our results indicate an important increase in collagen content and biosynthesis in fibrotic tissue. The increase in collagen biosynthesis in the irradiated tissue is more pronounced for type III collagen than for type I collagen. Biosynthesis of type III and type I collagens increases 20- and 10-fold, respectively, compared to the normal muscle. Type I to III collagen ratio in irradiated tissue decreases from 2.3 in normal tissue to 1.1 in fibrotic tissue. Histological examination of the biopsies as well as the protein pattern by polyacrylamide gel electrophoresis show striking differences in the perifibrotic and fibrotic areas as compared to the normal muscular tissue with a progressive disappearance of the myotubes replaced by a dense sclerotic tissue. The results indicate that the perifibrotic inflammatory area is engaged in a remodeling process and that the fibrotic tissue remains active in the neosynthesis of the extracellular matrix macromolecules with a high proportion of type III collagen. This high biosynthetic activity of the irradiated tissue may explain the pseudosarcomatous character of the radiation-induced lesions.

Parallel increase of plasma fibronectin and perchlorosoluble serum glycoproteins in radiation-induced lung damage.

A study of the behavior of plasma fibronectin during a radiation-induced inflammatory lung reaction was carried out. Rats were exposed to a single homogenous thoracic irradiation of 15 Gy from a 60Co gamma source. Perchlorosoluble glycoproteins were determined (as sialic acid) as well as plasma fibronectin at 2, 8, 27, 60 and 120 days after irradiation. A parallel increase of the perchlorosoluble glycoproteins and plasma fibronectin was found. Morphological and histological studies of the irradiated lungs showed a predominating endothelial cell injury in the lung microcapillaries. The parallel increase of plasma fibronectin and perchlorosoluble serum glycoproteins suggests that plasma fibronectin can be considered as an acute phase reactant. Its early increase may be the result of the inflammatory reaction and endothelial injury induced by the radiation.

Proteoglycans from pig aorta. Comparative study of their interactions with lipoproteins.

Different proteoglycans (PGs) were isolated from pig aorta for aggregation studies with hyaluronic acid and human low-density lipoproteins (LDL). Extraction of the intima-media with 4M-guanidinium chloride and digestion of the residue with collagenase solubilized 91% of aortic hexuronic acid content. From the guanidinium chloride extract two PGs were isolated by ion-exchange and gel-permeation chromatography: proteochondroitin sulphate (PGI) with a protein-core apparent Mr of 250 000 and proteodermatan-chondroitin sulphate (PGII) with a protein-core apparent Mr of 55 000. Only PGI forms high-Mr aggregates with hyaluronic acid. From the collagenase digest two other PGs were isolated: proteoheparan sulphate and proteochondroitin sulphate (PGIII and PGIV respectively). PGIV had a smaller hydrodynamic size than PGI. PGI and PGII formed insoluble complexes with human LDL in the presence of Ca2+. PGIII or PGIV did not form precipitates with the LDL. PGI and PGII, but neither PGIII nor PGIV, were bound to LDL-Sepharose. The main peaks of PGI and PGII were eluted from LDL-Sepharose with 60 mM- and 90 mM-NaCl respectively. The results indicate that aortic PGs have different interacting potentials with lipoproteins, depending on their Mr and their glycosaminoglycan composition.

Effect of colchicine on atherosclerosis. I. Clinical and biological studies.

The effect of colchicine was studied in 51 hypertensive subjects with several other vascular risk factors. Colchicine was administered for 3-4 months in a daily dose of 1 mg per os. The treatment did not change the lipid content in the blood and in skin biopsies, and had no effect on systolic and diastolic blood pressure. On the contrary, colchicine treatment significantly improved the conjunctival biomicroscopy score, the duration of the dicrotic wave and the peripheral resistance index. The results show the improvement of the microcirculatory parameters (elasticity of arteries) without changes of serum and tissue lipid parameters in the patients treated with colchicine.

Effect of colchicine on atherosclerosis. II. Biochemical studies on skin biopsies from patients treated perorally with colchicine.

The biosynthesis of proteins and glycosaminoglycans (GAGs) was determined in skin biopsies from atherosclerotic patients treated perorally for 3 months with 1 mg/day colchicine. The biopsies were incubated with 3H-glucosamine and 14C-proline for 5 h and subsequently digested with pronase. In an aliquot of the pronase digest, the specific radioactivity of 14C-proline and 14C-hydroxyproline were determined. The 3H-glucosamine-labeled GAGs were identified by specific enzymic assay and quantified after electrophoretic separation. 3 months treatment with colchicine did not modify the total amounts of proline and hydroxyproline in skin proteins, but diminished the amount of the GAGs as expressed by uronic acid content. Colchicine treatment decreased also the specific radioactivity of proline and hydroxyproline, which reflects a decrease of total protein and collagen synthesis. The incorporation of 3H-glucosamine in the 3H-GAGs was also decreased, mainly in hyaluronic acid. These results suggest that peroral administration of colchicine modifies the synthesis of extracellular matrix proteins and polysaccharides by skin fibroblasts.

Effect of colchicine on atherosclerosis. III. Study of dermal elastic fibers by quantitative histochemistry, automated image analysis.

Computerized automatic-image analytical procedure was applied on dermal biopsies stained for elastin by a new procedure giving a completely white background and staining only the elastic fiber system. In arteriosclerotic hypertensive patients, a 3-4 months' treatment with 1 mg colchicine per day resulted in a significant (60-80%, p less than 0.01) increase of the dermal elastic fiber density both in the superficial papillary dermis and in the deep dermis. This result shows that the age-dependent increase of elastic fibers can be influenced by pharmacological means. The inhibition by colchicine of the synthesis and secretion of the fibroblast-derived metalloelastase-type protease could be a plausible explanation of this finding.

The effect of procyanidolic oligomers on the composition of normal and hypercholesterolemic rabbit aortas.

Rabbits were fed with normal (group 1 and 2) and cholesterol rich diets (group 3 and 4) concomitantly to a daily peroral administration of 50 mg/kg procyanidolic oligomers (PCO) to groups 2 and 4. After 10 weeks, the cholesterol content of the blood serum and the excised aortic intima-media were significantly higher in groups 3 and 4 than in groups 1 and 2. The DNA, hydroxyproline, uronic acid contents were similar in aortic dry weight basis in all four groups. The intima-media samples were extracted successively with 0.15 M NaCl, 0.02 M sodium phosphate pH 7.4 (NaCl extract) and with 4 M guanidinium chloride, 0.05 M sodium acetate pH 5.8 prior (G1 extract) and following (G2 extract) hydrolysis of the collagen with collagenase. The cholesterol contents of G1 extracts were higher in groups 2 and 4 than in groups 1 and 3. The cholesterol content of aortic elastin increased with cholesterol feeding (group 3). With simultaneous administration of cholesterol and PCO the cholesterol content of aortic elastin in group 4 was significantly lower than in group 3. The uronic acid contents increased in G1 extracts and in the collagenase digest with PCO treatment of both normal and hypercholesterolemic rabbits. The ratio of dermatan-sulphate to chondroitin-sulphate decreased with hypercholesterolemia (group 3) and with PCO (group 2 and 4). The parallelism between increased cholesterol and uronic acid contents and modified glycosaminoglycan composition in G1 extract, indicate that the interaction of cholesterol with macromolecules of the aorta can be modulated by PCO. This drug modifies the extractibility of aortic cholesterol and glycosaminoglycans and reduces the association of cholesterol to elastin.