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This study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the collagen gels. HFF cultured in Dulbecco's modified Eagle's (DME) medium supplemented with bovine serum albumin (BSA) and either endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices. HFF-mediated collagen contraction was inhibited by prostaglandins E1 or E2, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils.

The dose-dependent effects of heavy metals on cell proliferation, collagen synthesis, and non-collagen protein synthesis were studied in early passage cultures of human synovial cells exposed to 1-100 microM concentration of gold, silver, mercury, cadmium or lead for 5 days. The incorporation of [3H]thymidine into trichloroacetic acid insoluble material was inhibited 50% by each of the heavy metals at concentrations between 1 and 10 microM. Gold, lead and mercury (10 microM) decreased the DNA content of the cultures by less than 15%; silver (10 microM) and cadmium (10 microM) resulted in decreased DNA content, which was attributed to cytotoxicity. A dose-dependent inhibition of [3H]proline incorporation into bacterial collagenase resistant (non-collagen) protein was observed after incubation with 10 microM mercury, lead and silver. During incubations with 10 microM gold and cadmium, collagenase resistant protein accumulation increased. All the heavy metals except for gold inhibited collagen accumulation to a greater extent than non-collagen protein accumulation. Gold (10 microM) stimulated the amount of collagen produced per cell, and the percentage of collagen to total protein was increased 50%. The rate of collagen accumulation in medium decreased during incubation with 10 microM silver, mercury, cadmium and lead. The stimulation of collagen synthesis may be a unique property of gold related to the therapeutic indices of gold, compared to other heavy metals, in rheumatoid arthritis.

Serum galactosylhydroxylysyl glucosyltransferase (S-Glu-Gal-Hyl-Tase), liver galactosylhydroxylysyl glucosyltransferase (L-Glu-Gal-Hyl-Tase), liver hydroxylysyl galactosyltransferase (L-Gal-Hyl-Tase), and liver prolyl hydroxylase (L-PH) activities were measured in rats during the development of CCl4-induced cirrhosis (0.2 ml of 33% CCl4 in light mineral oil two times weekly for 10 weeks followed by 6 weeks of no treatment). Serum and liver markers of collagen synthesis increased in a time-dependent manner reaching maximum activity at 6 weeks (S-Glu-Gal-Hyl-Tase, two times; L-PH, two times). These enzyme levels returned to normal during the 4-week recovery period. In a separate 4-week experiment, colchicine (10 micrograms/rat/day) was administered with CCl4. Colchicine prevented the increase in S-Glu-Gal-Hyl-Tase, L-Glu-Gal-Hyl-Tase, and L-Gal-Hyl-Tase induced by CCl4 and resulted in a smaller increase in L-PH. These results demonstrate that S-Glu-Gal-Hyl-Tase elevation occurs following CCl4 because of increased liver collagen synthetic activity and the hepatocellular injury produced by CCl4.

Serum galactosylhydroxylysyl glucosyltransferase (S-GGT), liver galactosylhydroxylysyl glucosyltransferase (L-GGT), liver hydroxylysyl galactosyltransferase (L-GGal-T) and liver prolyl hydroxylase (L-PH) activities were measured in CF1 female mice each week for 10 weeks after infection with Schistosoma mansoni (50 cercariae per mouse). None of the enzyme activities measured were significantly increased in the first six weeks following the infection. However, the activities increased thereafter reaching a maximum of seven-fold for S-GGT, five-fold for L-GGT, three-fold for L-GGal-T and ten-fold for L-PH compared with control animals. The increase in S-GGT activity was positively correlated with L-GGT (r = 0.788), L-GGal-T (r = 0.774) and L-PH (r = 0.800). The present data suggest that during the first six weeks of the development of murine hepatic schistosomiasis mansoni infection, few eggs bombard the liver. The biochemical changes during this period are mild. During weeks 7 to 10, which is the active fibrotic stage, the liver biochemical markers of collagen synthesis increase rapidly and lead to fibrotic morphological features that are the chronic irreversible hepatic changes characteristic of this infection. The data reported here indicate that the aggressive fibrotic changes in liver during weeks 7 to 10 of murine schistosomiasis infection are predicted by S-GGT activity.

Hepatic prolyl hydroxylase activity and collagen synthesis were measured in patients with alcoholic liver disease to determine the feasibility of using the enzyme prolyl hydroxylase as a marker of hepatic fibrogenesis. Alcoholic patients with liver histopathology consistent with normal, steatosis, alcoholic hepatitis, early cirrhosis, or advanced cirrhosis were analysed for liver prolyl hydroxylase activity and in vitro collagen synthesis. Prolyl hydroxylase activity and the rate of in vitro collagen synthesis were correlated when these parameters were measured in samples of the same liver biopsy. Mean prolyl hydroxylase activity was significantly raised in all groups of alcoholic patients with alcoholic liver disease, except those with steatosis, when compared with alcoholic patients with normal morphology. Alcoholic patients with early cirrhosis had enzyme activity (mean +/- SE: 1.367 +/-0.162 mU/mg protein) significantly raised over all other groups. Mean enzyme activity was less raised (0.985 +/- 0.097 mU/mg protein) in patients with advanced cirrhosis. The percentage of collagen synthesis in patients with early or advanced cirrhosis was also raised compared with alcoholic patients with normal morphology. Prolyl hydroxylase activity and the rate of collagen synthesis are significantly correlated (r=0.62). These findings suggest that hepatic prolyl hydroxylase activity is a useful indicator of hepatic fibrogenesis and its measurement on available liver biopsy tissue should be a potent diagnostic tool reflecting active fibrogenesis and predicting progression of alcoholic liverdisease.

These studies, conducted on early passage synovial cell mono-layers (derived from explant cultures of tissue obtained from patients with rheumatoid arthritis), have established that gold sodium thiomalate (GST) exposure results in dose-dependent changes in cell proliferation and protein synthesis as a consequence of the cellular accumulation of gold. The amount of gold found in the cell layer is correlated with the degree of inhibition of [3H]thymidine incorporation. Gold remains in the cell layer of treated cells after they have been subcultured twice in the absence of GST. Exposure of cells to a concentration of GST of 100 muM for 4 days results in 50% inhibition of [3H]thymidine incorporation. This antiproliferative effect is reversible at concentrations of 10 muM GST or less. Only partial recovery is observed after exposure to higher concentrations of GST which may be related to retained gold. The amount of collagen and noncollagen protein synthesized per cell increases at concentrations of GST of 10 muM and below but decreases with concentrations above 10 muM. A dose-dependent decrease in protein synthesized per flask and a decrease in the commitment to synthesize collagen relative to total protein synthesis follows exposure to GST in excess of 10 muM for 20 days which recovers partially after synovial cells are grown in GST-free medium for 10 days. An observed decrease in the percentage of type III collagen synthesized by synovial cells after GST exposure was not observed in cells grown in GST-free medium for 5 days after exposure, indicating that this effect of collagen synthesis is reversible. The reversible biochemical changes resulting from the exposure of cultured human synovial cells to GST are discussed as a mechanism of action of this drug on the proliferative synovitis that characterizes diseases such as rheumatoid arthritis.

Synthesis of collagen in the vasculature and other tissues of normotensive rats is markedly reduced by reserpine. Hypophysectomy produces similar effects. The effects of reserpine on collagen biosynthesis may be mediated through the hypothalamus.

Human mononuclear cell supernatants were obtained by incubating 3 X 10(6) cells per ml of Dulbecco's modified Eagles medium at 37 degrees C for 24 h or 48 h, either in presence or absence of phytohaemagglutinin. After removal of intact cells, the supernatants were dialysed and diluted (1:1, 1:2, 1:5, 1:10) again using the above medium. The diluted supernatants, containing a final concentration of 10% (v/v) human platelet-factor poor serum, were found to stimulate the proliferation of human synovial cells in culture and to increase both the total amount of collagen and the percentage of Type III collagen synthesized by these cells. Incubation of the mononuclear cells in presence of phytohaemagglutinin appeared to further enhance the stimulatory effects of the supernatants upon the synovial cells. Since activated mononuclear leucocytes such as lymphocytes and macrophages are present in rheumatoid synovia, this study suggests that factors released from activated mononuclear leucocytes may play an important role in the proliferation of rheumatoid synovial tissue and development of the pannus.

Collagen synthesis is increased in the aortas, mesenteric arteries, and to a lesser extent, in the hearts of rats either made hypertensive with desoxycorticosterone acetate-salt or that are spontaneously hypertensive. Several markers of collagen biosynthesis were shown to be increased, including prolyl hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase), prolyl hydroxylase-related antigen, total collagen content, and the incorporation of [(3)H]proline into total protein and into collagen. The antihypertensive agents chlorothiazide and reserpine, when administered before the onset of hypertension in the rats treated with desoxycorticosterone acetate-salt, prevented or diminished the increase in collagen biosynthesis. When reserpine was given after the onset of hypertension, prolyl hydroxylase activity was decreased concomitant with the decrease in blood pressure. Treatment with reserpine is particularly effective in diminishing arterial prolyl hydroxylase activity.

Increased aortic and liver prolyl hydroxylase activity has been suggested as an early biochemical indicator of the fibrotic changes which occur in rabbits with injury induced arteriosclerosis. Daily administration of epinephrine (0.025-0.050 mg/kg, i.v.) and thyroxine (0.050 mg/kg, i.p.) to rabbits for 3 weeks produced aortic fibrous plaques with a 4-fold increase in aortic prolyl hydroxylase and also a 5-fold increase in liver prolyl hydroxylase. Histopathologically, the livers of these rabbits show subcapsular areas of necrosis. When total prolyl hydroxylase related antigen was measured. the increase in liver prolyl hydroxylase activity accounted for only a small portion of the total prolyl hydroxylase antigen. However, in the aorta a majority of the increase in antigen is due to the increased amount of enzyme. DNA content per aorta was unchanged and RNA content increased in the aortic tissue of the arteriosclerotic rabbits. However DNA and RNA levels increased 60% in the livers of arteriosclerotic rabbits. In vitro incorporation of radioactively labeled proline into collagenase digestable protein was at least 2-fold greater in aorta and liver minces from arteriosclerotic rabbits. Michaelis--Menten kinetic parameters were obtained for the liver prolyl hydroxylase purified by affinity chromatography from arteriosclerotic rabbits. The Km for the enzyme from treated animals was not significantly different from control. However, the Vmax of the enzyme purified from diseased liver was 4-fold greater when compared to controls.

Synovial cells derived from patients with either rheumatoid arthritis, or simple joint-trauma were grown in tissue culture. The rheumatoid osteoarthritic and non-arthritic synovial cells in cultured all had similar levels of prolyl hydroxylase activity. Following a 3 hour incubation with ascorbate (10(-4)M), prolyl hydroxylase activity was elevated to a similar extent in all synovial cell cultures examined. The activation of prolyl hydroxylase by ascorbate (10(-4)M) was accompanied by increased radioactive hydroxyproline formation and secretion into the media. Increased amounts of collagenase degradable radioactive protein were also secreted into the media, but no changes in total collagen synthesis (media plus cell layer) were observed as a result of ascorbate supplementation using this assay system.

This rapid, sensitive method of determining collagenase (EC 3.4.24.7) activity incorporates several advantages of previous methods. Soluble [14C]acetylated collagen is prepared as the enzyme substrate and collagen-cleavage products are separated from noncleaved collagen by precipitation with dioxane/methanol. The assay is more reproducible than previous methods and has a lower detection limit, 15 mU of enzyme activity. We used the method in a competitive substrate assay with isolated extracellular hepatic matrix from cirrhotic and normal rat liver. Purified collagenase was consistently bound to normal rat matrix to a greater extent than to cirrhotic matrix, suggesting that in hepatic fibrosis the extracellular matrix is not as susceptible to collagenase degradation as that in normal liver.

The activities of prolyl hydroxylase (Pro-OHase), galactosylhydroxylysyl glucosyltransferase (Glu-Gal-Hyl-Tase), and hydroxylysyl galactosyltransferase (Gal-Hyl-Tase) were assayed in lung tissues of hamsters with bleomycin-induced experimental pulmonary fibrosis. Serum Glu-Gal-Hyl-Tase and aspartate transaminase (Asp-NH2-Tase) were measured in the same animals. Lung fibrosis was induced by intratracheal bleomycin instillation, and the enzyme activities were assayed 2, 3, and 4 weeks after bleomycin administration. The activities of the three lung enzymes increased significantly after bleomycin instillation. However, no difference in the values of serum Glu-Gal-Hyl-Tase or Asp-NH2-Tase were observed. Histologic examination of lung sections indicated progressive fibrotic foci. These results thus indicate that the activities of collagen processing enzymes are elevated in the fibrotic lung tissues as a reflection of the increased rate of collagen synthesis during the period of active fibrogenesis, but unlike liver fibrosis, the elevation of tissue Glu-Gal-Hyl-Tase is not predicted by a corresponding increase in serum levels of this enzyme.