Amino acid metabolism of experimental granulation tissue in vitro

1. The intracellular volume in granulation tissue was about 15% of the total urea space. 2. The experimental granuloma has a greater ability to retain amino acids during the proliferation phase than later during the synthesis of collagen. 3. The synthesis of collagen and other proteins by granulation tissue is related to the concentrations of proline and glutamic acid in the medium. 4. The rate of synthesis of proline from glutamic acid in granulation-tissue slices is greatest during collagen synthesis. It is enhanced by lactate. 5. Extracellular cations influence the synthesis of collagen and ouabain is inhibitory. Synthesis of other proteins is less sensitive in this respect. 6. It is suggested that the synthesis of collagen is related to the supply of certain amino acids, especially proline, and hence to the redox balance, and also to the function of the cell wall.     

Endogenous factors affecting arachidonic acid metabolism: I. Biosynthesis of prostacyclin and prostaglandins by carrageenin granulomas of rats

Arachidonic acid was converted by incubated slices of the rat carrageenin granuloma to prostacyclin (PGI₂), prostaglandins (PGs) E₂ and F_2∞ as detected by bioassay and radiochemical assay. PGI₂ was the major product of arachidonic acid metabolism in the granuloma slices. PGI₂ and PGE₂ formation was dependent on the concentration of the substrate and on the age of the granuloma. Slices obtained from 5-day old granulomas produced significantly more PGI₂ than slices prepared from 3-day old or 8- to 9-day old granulomas while PGE₂ generation was not dependent on the stage of the development of the granuloma. Homogenates of granuloma tissue hardly converted arachidonic acid to PGI₂ at all. This was probably due to the presence of an non- dialysable and heat labile material which, when partially isolated, inhibited PGI₂ production by bovine aortic microsomes.     

Fibroblast contraction of collagen lattices in vitro: Inhibition by chronic inflammatory cell mediators

Fibroblast-populated collagen lattices (FPCL), prepared in petri dishes with serum-containing culture medium and incubated at 37°C, undergo progressive and symmetric contraction (reduction in size) over a period of days. The in vitro contraction process requires viable cells with intact cytoskeletal elements, is associated with cell elongation, and is believed to represent a fibroblast function which also occurs in vivo during wound healing and tissue fibrosis. We report that soluble mediators elaborated by chronic inflammatory cells cultured in vitro, when added to FPCL, inhibit lattice contraction. Granulomas, isolated from the liver of Schistosoma mansoni-infected mice, secrete a factor(s) with an estimated molecular weight between 13,700 and 43,000 daltons (gel filtration: Sephadex G-200) and pl = 6 (preparative isoelectrofocusing in granular gel) which inhibits lattice contraction but is not toxic to fibroblasts. Supernatants (cell-free conditioned culture medium) of cultured macrophages isolated from these granulomas also contain this activity. The contraction inhibitory activity present in granuloma culture supernatants is abrogated by the addition of indomethacin to the lattices, while the addition of prostaglandin E₂ (PGE₂) alone to lattices inhibits contraction. Furthermore, culture supernatants interfere with fibroblast elongation in lattices. We propose that the ability of fibroblasts to contract collagen lattices in vitro and a fibrotic mass in vivo may be regulated by soluble products of chronic inflammatory cells, including macrophages. This process may be mediated by fibroblast-derived prostaglandins which alter cytoskeletal functions and has implications for understanding regulation of tissue fibrogenesis in a variety of diseases.     

Subcellular targets of the soluble SiO2-liberated macrophage factors in experimental granulation tissue.

The supernatant from SiO2-treated macrophages increased the incorporated radioactivity of collagen in the incubated experimental granulation tissue slices, especially in the rough endoplasmic reticulum fraction, markedly over that in the respective control preparation. The effect was observed after a 20 min incubation and increases linearly at least up to 3 h. The amount of RNA and phospholipids in the rough endoplasmic reticulum, calculated per protein, increased simultaneously in the incubated slices. The incorporation of the radioactive precursors into DNA in the slices and in the nuclei from proliferating granulation tissue was enhanced significantly by the soluble fraction from SiO2-treated macrophages. This effect could be seen after a 40 min incubation in the slices and after 5 min in the nuclei, when the incorporated radioactivity in DNA began to decrease in the control experiments.     

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions.The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10^?6 M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.     

Effect of hyperbarism on collagenous protein metabolism in granulation tissue.

Rats with subcutaneously implanted polyurethane sponges were exposed 6 hours daily for 7 days to high ambient atmospheric pressures (1.5, 2, 2.5 and 3 ATA). Another group was exposed 4 hours daily for 4 weeks to 3 ATA before inducing granulation tissue formation. 14C-proline was administered 16 hours before terminating the experiment. Free hydroxyproline, soluble and insoluble collagen and total noncollagenous protein were isolated from the 7-day granuloma and the amount and radioactivity of 14C-hydroxyproline and 14C-proline were determined. Seven days' graduated hyperbarism did not affect collagen synthesis; the maturation of collagen to insoluble forms was inhibited at 2 and 2.5 ATA, but not at 3 ATA. Stimulated degradation of collagen (free hydroxyproline) was observed at 2, 2.5 and 3 ATA. In animals subjected to long-term exposure at 3 ATA pressure, the collagen in the granuloma matured to insoluble forms more quickly. Biochemical changes were correlated with changes in the fine structure of the granulation tissue. The appearance of the fibroblast proteosynthetic apparatus was not influenced by hyperbarism. Progressive spherical transformation, fusion of mitochondria and lysosomal activation in the pericapillary fibroblasts occurred at 2, 2.5 and 3 ATA. In short-term experiment, the formation of cytosegresomes and cellular necrosis also contributed to the effect at 3 ATA, which is thus already a toxic pressure for granulation tissue.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

The regulatory influence of prostaglandins on protein synthesis in the human non pregnant cervix

Cervical connective tissue was obtained from non pregnant fertile women undergoing hysterectomy. Tissue specimens were mechanically chopped into 1 mm thick slices which were preincubated in Krebs-Ringer bicarbonate buffer containing PGE₂ or PGF_2α (300 ng/ml). After 60 min the slices were transferred into fresh buffer with the corresponding PG-concentration and ³H-labelled proline or hydroxyproline. Following incubation (60 min) the protein bound radioactivity was determined and related either to the dry weight or to the protein content of each slice. The two methods used did not show any qualitative differences. The experiments showed that PG:s had a marked effect on protein synthesis in the cervical tissue. During the follicular phase incubation with both types of PG:s was followed by a decreased incorporation indicating decreased net synthesis of collagen while there was an increased incorporation and hence increased synthesis in the luteal phase. There was no significant influence on the distribution of the model amino acid ¹⁴C-AIB in the presence of PG:s neither in the follicular nor in the luteal phase. The present data point to an acute effect of PGE₂ and PGF_2α on cervical collagen metabolism and indicate furthermore that the process is steroid hormone dependent. It is concluded that these substances may exert their effect by modulating the incorporation of amino acids into protein.     

Objective Assessment of Endogenous Collagen In Vivo during Tissue Repair by Laser Induced Fluorescence

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm² He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.     

Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Prostaglandin E2 elevation of cyclic-AMP in granuloma macrophages at various stages of inflammation: Relevance to anti-inflammatory and immunomodulatory functions

In the carrageenin-induced granuloma of rats the inflammatory tissue growth and macrophage invasion on the one hand and the cyclic-AMP content of the macrophages on the other, display opposite directional changes. Macrophages, isolated from this tissue at different stages of inflammation, were used to examine the effect of prostaglandin E₂ on intracellular levels of c-AMP. It appears that during infiltration of the macrophages into the inflammatory tissue, the sensitivity of adenylate cyclase to activation by PGE₂ increases. Arguments are presented that these observations made , are in direct relevance to the previously described anti-inflammatory effect of PGE on granuloma tissue .     

Liver proline oxidase activity and collagen synthesis in rats with cirrhosis induced by carbon tetrachloride

In rats treated with CCl₄ for 7 weeks, liver proline oxidase activity was drastically reduced 24 h after the initial administration of the toxic agent and remained low throughout the treatment period. This was accompanied by a larger accumulation of added proline in the incubation medium and a lesser release of ¹⁴CO₂ from [¹⁴C]proline during incubation.Collagen synthesis by liver slices of CCl₄-treated rats increased in proportion to proline concentration, a plateau being reached at 0.48 mM proline. The plateau did not occur within the range studied with liver slices of normal liver.Increased collagen synthesis in vitro was accompanied by increased deposition of collagen in vivo only during the first 3 weeks of CCl₄ treatment. No further increase in liver collagen content occurred thereafter. Discontinuance of CCl₄-administration was followed by a return to normal of proline oxidase activity and in vitro collagen synthesis within 2 weeks. Nevertheless, collagen content remained elevated.The results suggest that proline oxidase activity, together with the previously shown increased formation of proline from precursor amino acids, may control the amount of proline available for collagen biosynthesis; and that the rate of degradation of collagen, perhaps by collagenase, may determine the levels of collagen remaining after discontinuance of CCl₄-administration.     

Epidermal growth factor increases collagen production in granulation tissue by stimulation of fibroblast proliferation and not by activation of procollagen genes.

The effects of epidermal growth factor (EGF) on granulation-tissue formation and collagen-gene expression were studied in experimental sponge-induced granulomas in rats. After daily administration of 5 micrograms of EGF into the sponge, total RNA was extracted from the ingrown granulation tissue at days 4 and 7 and analysed by Northern hybridization for the contents of mRNAs for types I and III procollagens. EGF treatment increased procollagen mRNA, particularly at day 4. To determine whether this elevation was due to increased proliferation of collagen-producing fibroblasts or to activation of collagen-gene expression in these cells, fibroblast cultures were started from granulation tissue and treated with EGF. These experiments confirmed that EGF is a potent mitogen for granuloma fibroblasts in a dose-dependent manner. The effect of EGF treatment on radioactive hydroxyproline production in cultured cells was inhibitory. The decreased rate of collagen synthesis was also indicated by decreased amounts of procollagen mRNAs. The results suggest that the stimulation of wound healing and collagen production by EGF is due to increased fibroblast proliferation, and not to increased expression of type I and III procollagen genes.     

Collagen polymorphism in experimental granulation tissue.

Dermal granulation tissues produced either in response to an acute inflammation by turpentine injection, or to a chronic inflammation by sponge implantation, have been shown to contain a high proportion of Type III collagen in marked contrast to the small amount normally present in mature skin. Although the acute granuloma is rapidly resorbed the synthesis of Type III is maintained in long-term sponge implants. The results demonstrate a reversion to embryonic collagen in proliferating granulation tissue, a fact of considerable importance in understanding wound healing.     

Collagen metabolism and phenotype after prostaglandin E2 treatment of granuloma: direct and macrophage-modulated effects

Local administration of PGE₂ (2 μg) to polyether sponges, implanted s.c. in rats, inhibited hydroxyproline and total protein accumulation, without altering relative amounts of collagen, when administered early during granuloma development. In contrast, while DNA as well as total protein accumulation was inhibited by local PGE₂ treatment of established granuloma, hydroxyproline accumulation and the relative amounts of collagen were enhanced. This PGE₂-induced collagen enhancement was associated with an increased type III : type I collagen ratio, possibly due to differential intracellular breakdown of newly synthesized collagen. The solubility of granuloma collagen was unaffected by PGE₂. Impregnation of sponges with carrageenan before implantation, thereby giving macrophage-dominated granuloma, did not affect the changes in protein and DNA induced by later treatment with PGE₂, but did reverse the PGE₂-induced accumulation of hydroxyproline. This latter effect probably reflects macrophage-mediated, PGE₂ enhancement of collagenolytic activity.     

Soluble Factors in Pisum Extracts Which Moderate Pisum beta-Glucan Synthetase Activity

Homogenates of growing regions of the pea (Pisum sativum L.) epicotyl contain soluble factors (130,000g supernatant) which alter pea β-glucan synthetase activity, as assayed using the substrate UDP-glucose and either particulate fractions or tissue slices as source of enzyme. A heat-stable dialyzable component is present which enhances as much as 3-fold the synthesis of alkali-soluble and -insoluble products from millimolar levels of substrate. A heat-labile nondialyzable component is also present which suppresses synthesis. This component dominates (the net effect of total crude extract) when low (μm) levels of substrate are employed. Methylation analysis shows that both components primarily affect the proportion of β-1,4 rather than β-1,3 linkages which are synthesized. The enhancing factor increases V_max of the synthetase system and only activates in the presence of high levels of substrate. The suppressing factor appears to inactivate the synthetase, since losses of product or substrate are not significant during brief incubation with extract, the factor acts progressively with time with a pH optimum, and it destroys activity during preincubation with particles or slices. It co-precipitates with a protease (gelatinase) at between 20% and 40%-saturated (NH₄)₂SO₄, and it co-fractionates with a major component of total protease on Sephadex gel columns (G-200) with an elution volume corresponding to molecular weight 65,000. The concentrations of these factors are such that they could be natural moderators of synthetase activity in vivo if the two were ever brought in contact, and the inactivator could account for the lability of β1,4-glucan synthetase which occurs upon tissue homogenization.     

Reiteration frequency of procollagen genes in the guinea pig genome: Collagen genes are not amplified during granuloma fibroblasts differentiation

Procollagen mRNA was purified from collagen synthesizing polysomes obtained from an experimental guinea pig granuloma, and iodinated in vitro. The procollagen ¹²⁵I-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot $\text{1}\text{2}$ 800–900 mol · s · l^?1 for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11–13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplification in tissues highly specialized in the synthesis of collagen such as granuloma.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.     

Metabolic phases during the development of granulation tissue

1. The metabolism of incubated slices of sponge-induced granulation tissue, harvested 4-90 days after the implantation, was studied with special reference to the capacity of collagen synthesis and to the energy metabolism. Data are also given on the nucleic acid contents during the observation period. Three metabolic phases were evident. 2. The viability of the slices for the synthesis of collagen was studied in various conditions. Freezing and homogenization destroyed the capacity of the tissue to incorporate proline into collagen. 3. Consumption of oxygen reached the maximum at 30-40 days. There was evidence that the pentose phosphate cycle was important, especially during the phases of the proliferation and the involution. The formation of lactic acid was maximal at about 20 days. 4. The capacity to incorporate proline into collagen hydroxyproline in vitro was limited to a relatively short period at 10-30 days. 5. The synthesis of collagen was dependent on the supply of oxygen and glucose, which latter could be replaced in the incubation medium by other monosaccharides but not by the metabolites of glucose or tricarboxylic acid-cycle intermediates.     

Effect of Ethylene on the Increase in Catalase Activity through Microbody Development in Wounded Sweet Potato Root Tissue

Catalase activity increases when slices of sweet potato roottissue are incubated in air. The increase is due to de novosynthesis of the enzyme protein and probably also to activationof a precursor protein [Esaka et al. (1983) Plant & CellPhysiol. 24: 615]. The activity-increase was partly depressedwhen tissue slices were incubated in ethylenecontaining air,while the immunologically determined amount of catalase proteindid not increase, rather it decreased, under the same conditions.We propose that ethylene inhibits the de novo synthesis of catalaseprotein but not the activation of precursor protein. Catalasefrom tissue slices incubated in ethylene-containing air migratedfaster on a polyacrylamide gel than that from intact tissueor tissue slices incubated in air. When either polyacrylamideor an SDS-polyacrylamide gel applied with crude extract fromtissue slices incubated in ethylene-containing air underwentimmunological blotting, the blots were much fainter than thosefor intact tissue. In addition, microbody membrane fractionfrom incubated tissue slices contained a significant amountof catalase which was sedimented at the bottom of a sucrosedensity gradient (20–70%) and was not solubilized by highconcentrations of lubrol PX. The fraction showed an exceptionallyhigh catalase activity per unit amount of immunoreactive proteinto anti-catalase antibody. We propose that ethylene causes somemodification of catalase protein which facilitates the formationof aggregates or cores. ¹Present address: Laboratory of Food Technology, Faculty ofApplied Biological Science, Hiroshima University, Fukuyama,Hiroshima 720, Japan. ²Present address: Terumo Co. Ltd., Omiya, Fujinomiya, Shizuoka418, Japan. (Received October 16, 1982; Accepted February 24, 1983)