The Behavior of (14C)-D-Penicillamine in Collagen Metabolism

The most important effect of penicillamine on collagen metabolism is the reduction of collagen crosslinking. However, even after long time application of penicillamine, collagen is crosslinked to a certain degree. After intravenous injection of a trace dose of (¹⁴C) labelled D-penicillamine it can be determined that this substance is rapidly bound to neutral salt soluble, acetic acid soluble and urea soluble collagen fractions and to a lesser extent to insoluble collagen as well. The amount of penicillamine which binds to any of the collagen fractions depends on the turnover rate. When different tissues are compared, penicillamine seems to have the greatest affinity to tissues with a high collagen turnover. Further studies of neutral salt soluble collagen by CM-cellulose chroma-tography revealed a stable linkage of penicillamine to collagen alpha chains.     

Collagen Synthesis by Lathyrogen-Treated 3T6 Fibroblasts

The lathyrogen, beta-aminopropionitrile (BAPN), in ascorbate-free medium increased the synthesis of both collagenous and non-collagenous proteins in 3T6 fibroblasts. Cell layer collagen was underhydroxylated and more easily extracted with neutral salt in treated cultures. The ratios of proline: hydroxyproline in sequentially extracted collagen in the cell layer of both BAPN and control cultures showed virtually no difference in the acid soluble and insoluble fractions; however, the BAPN salt-soluble fraction was definitely underhydroxylated when compared to control cultures. The data suggest that the salt-soluble macromolecule represents, in addition to a cross-linking defect, an underhydroxylated intracellular collagen precursor.     

Salt-soluble collagen and elastin in the human aorta and pulmonary artery

Collagen and elastin, the major structural components of blood vessels, have a very low turnover. In disease, this rate may be increased and an elevation of the tissue concentration of the soluble degradation fragments might be anticipated. In this preliminary study the concentration of extractable collagen and elastin in the aorta and pulmonary artery of eight human subjects postmortem was determined. The proportion of pulmonary artery collagen and elastin that was soluble was generally either equal to or greater than that in the abdominal aorta. The fraction of collagen that was salt extractable was larger than the soluble elastin fraction.     

Isolation and partial characterization of collagen fibrils, fibers and fiber-bundles from insoluble calf dermis

Collagen fibrils, fibers and fiber bundles have been isolated from insoluble calf skin chips by disaggregation with a neutral salt buffer followed by differential centrifugation. The ratio of type I collagen/type III collagen was relatively constant, regardless of the size of the fibrous materials. On the other hand, the ratios of glycosaminoglycans/collagen and non-collagenous glycoproteins/collagen were significantly higher in the thinner fiber fractions. However, the ratio of hyaluronic acid to dermatan sulfate was unchanged. The role of the non-collanenous constituents in collagen fibrillogenesis in vivo is discussed.     

Cell Death and Proliferation and Its Relation to Collagen Degradation in Uterine Involution of Rat

Collagen concentration, procollagenase localization, and their association with cell proliferation and apoptosis during postpartum involution, were investigated biochemically and histochemically in postpartum day 1, 3, 5, and 7 rat uterine tissues. In control animals, uterine wet weight, soluble protein, and collagen decreased rapidly during days 1 to 3 postpartum, and the DNA concentration in the uterine horn rapidly decreased, as noted by others. Simultaneously, both apoptosis and cell proliferation were observed in these tissues. These processes were highest in smooth muscle cells on day 3 postpartum. Procollagenase was found in the cell cytoplasm through days 1 to 3 postpartum, was highest on the third day postpartum, and appeared to gradually diminish by day 5 postpartum. Disorganization of collagen fibers was observed, under polarized microscopy by a strong birefringence of collagen fibers of the circular smooth muscle cell layers. However, this disorganization of the uterine collagen diminished progressively from day 3 to day 7. Treatment with estradiol or a combination of estradiol and progesterone suppressed cellular turnover and attenuated the changes in DNA, total amino acids, and collagen on day 3 postpartum. In this study, cellular turnover and biochemical and morphological changes appeared to be closely associated. Gonadal steroid hormones appear to influence these changes and retard uterine involution. This study suggests that a dynamic turnover of the cellular population takes place during uterine involution. It is possible that other factors, in addition to steroid hormones, contribute to uterine involution. It is to be postulated that these factors either are themselves decreased or, alternatively, may increase the inhibition of other unknown factors by an indirect mechanism.     

Collagen and mechanical strength in various organs of rats treated with D-penicillamine or amino-acetonitrile.

After oral treatment with D-penicillamine (D-Pc) or with aminoacetonitrile (AAn) for 10 days, mechanical and chemical parameters were studied simultaneously in various organs of Sprague Dawley rats. Tensile strength of skin strips and of tail tendons, breaking strength of femur bones and tensile strength of granuloma tissue (induced by implanted glass rods) were measured and calculated. In the same tissue the soluble collagen fractions and the insoluble collagen were determined. Total collagen and the ratio insoluble vs. soluble collagen were calculated. Tensile strength of skin, tendon and granuloma tissue were greatly reduced by D-Pc treatment but only minimally influenced by AAN treatment. On the other hand only AAN significantly reduced the breaking strength of bone. All these changes were closely correlated with the content of insoluble collagen in the respective tissues. The correlation coefficients to total collagen were similar but lower. The correlation coefficients between strength and the ratio insoluble vs. soluble collagen were generally still lower. Earlier findings in aged and corticoid treated rats, proving that insoluble collagen content determines mechanical strength of connnective and supporting tissue thus could be confirmed.     

Collagen and Mechanical Strength in Various Organs of Rats Treated With D-Penicillamine or Amino-Acetonttrile

After oral treatment with D-penicillamine (D-Pc) or with aminoacetonitrile (AAN) for 10 days, mechanical and chemical parameters were studied simultaneously in various organs of Sprague Dawley rats. Tensile strength of skin strips and of tail tendons, breaking strength of femur bones and tensile strength of granuloma tissue (induced by implanted glass rods) were measured and calculated. In the same tissue the soluble collagen fractions and the insoluble collagen were determined. Total collagen and the ratio insoluble vs. soluble collagen were calculated. Tensile strength of skin, tendon and granuloma tissue were greatly reduced by D-Pc treatment but only minimally influenced by AAN treatment. On the other hand only AAN significantly reduced the breaking strength of bone. All these changes were closely correlated with the content of insoluble collagen in the respective tissues. The correlation coefficients to total collagen were similar but lower. The correlation coefficients between strength and the ratio insoluble vs. soluble collagen were generally still lower. Earlier findings in aged and corticoid treated rats, proving that insoluble collagen content determines mechanical strength of connective and supporting tissue thus could be confirmed.     

Collagen-bound collagenase

The presence of collagenase bound to collagen extracted and purified from several animal and human sources by a standard procedure has been confirmed by different methods. Polyacrylamide (10%) gel electrophoresis at pH 8.1 of intact or "spontaneously"degraded neutral salt soluble collagen results in the separation of two components: the upper one says at the origin and represents collagen or collagen ragments, whereas the lower protein component contains no collagen, often preserves specific collagenolytic activity, and migrates as a single band in SDS/polyacrylamide electrophoresis. With lower polyacrylamide gel concentration the electrophoretic separation of the two components is less clear. Removal of the lower protein component from collagen solutions by two different methods (TCA-ethanol purification cycles and pepsin digestion) results in concomitant loss of their "spontaneous" instability. Eluates of the lower protein component stimulate the heterologous production of a monospecific antibody capable of inhibiting the collagenolytic activity of homologous crude collagenase preparations. It is suggested that collagen-bound collagenase is not an artifact of the extraction procedure but rather a physiological reality, probably corresponding in the living animal to the enzyme closely associated with extracellular collagen fibers, revealed by immunohistochemical methods.     

Human hyaline cartilage--biochemical and immunochemical study

Hyaline cartilage from the knee or rib of newborns was divided in neutral salts into a soluble and insoluble part. The insoluble part was treated with collagenase and was separated into 2 peaks by gel chromatography on Sephadex G-200. The 1st peak revealed a low concentration of proteins, no hydroxyproline, and very high levels or uronates. In the 2nd peak, very high values of hydroxyproline and very low values of uronate were found. Thus the 1st peak contained only proteoglycans and no collagen, while the entire collagen was found in the 2nd peak. The individual fractions were inoculated into rabbits to obtain antibodies. Compared to the fraction containing macromolecular proteoglycans, antisera proved remarkably suitable particularly for analysis of human spleen, yet also of other organs. These anti-cartilage antisera visualized primarily spongy structures of pericapillary sheaths and circumferential reticulum of the periarterial lymphatic sheaths, but to a lesser extent also other extracellular structures of the spleen and of other organs.     

The role of soluble factors in bleomycin-induced pulmonary fibrosis.

Endotracheal administration of bleomycin causes pulmonary fibrosis characterized by increased collagen synthesis and deposition. Incubation of normal lung mince with neutral salt soluble extracts of lungs from normal and bleomycin-treated rats caused a dose-dependent inhibition of collagen and noncollagenous protein synthesis. Bleomycin-treated lung extracts, however, were significantly less effective in such inhibition when compared with normal lung extracts. This inhibitory activity was not diminished by dialysis in tubing with nominal molecular weight cutoff of 10,000 but was destroyed by heat (70 C) and trypsin digestion. This inhibitory activity could not be ascribed to residual serum or bleomycin in the lung extracts. Fractionation on Sephacryl S-200 (Pharmacia, Piscataway, NJ) showed inhibitory activity to be heterogeneous with Mr (apparent molecular weight) greater than 100,000. Extracts from spleen showed similar inhibitory activity but showed no difference in intensity between normal and bleomycin-treated spleen. These data suggest that loss or decrease in production of lung inhibitory regulatory factors is partly responsible for the noted increase in collagen production and deposition in bleomycin-induced pulmonary fibrosis.     

Collagen fibril diameter and its relation to collagen turnover in three soft connective tissues in the rat

Collagen fibril diameters were measured in electron micrographs of rat skin, gingiva and periodontal ligament. Gingiva was divided into two zones, termed elastin-containing gingiva and attached gingiva, depending on the presence or absence of elastic fibrils. The results revealed that skin had the largest fibrils, followed by elastin-containing gingiva, attached gingiva and periodontal ligament respectively. These differences in fibril diameter were highly significant. The observed trend in fibril diameter was the inverse of that documented for collagen turnover and collagen phagocytosis in the same tissues. A link between fibril diameter and collagen turnover is discussed.     

A stereologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover

The purpose of this study was to assess the importance of the phagocytic mechanism of collagen resorption in the normal turnover and remodelling of soft connective tissues. Collagen phagocytosis by fibroblasts in rat skin, attached gingiva, and periodontal ligament was quantitated using the methodology of electron microscopic stereology. Periodontal ligament contained five and 15 times as much phagocytosed collagen as attached gingiva and skin respectively. Also, for each tissue examined, a positive correlation was observed between the amount of collagen phagocytosed and the known rate of mature collagen turnover.     

Effects of Age and Diabetes Mellitus on Cyanogen Bromide Digestion of Human Dura Mater Collagen

To extend previous studies on the apparent acceleration of collagen aging in diabetes mellitus, dura collagen from human adults of different ages, with and without diabetes, was processed to yield soluble and insoluble fractions. Insoluble fractions were cleaved by cyanogen bromide. Release of peptides from insoluble collagen by cyanogen bromide decreased markedly with age and was much less from the collagen of diabetics than from nondiabetics of similar ages. The acrylamide gel profiles of peptides released were similar, but not identical, for samples of different ages and for samples from diabetics. It was concluded that age-dependent and diabetes-dependent cross-linking was widespread throughout helical regions of collagen molecules and that collagen throughout the body is altered in diabetes. Analyses of fragments of insoluble collagen are required to gain information on the chemistry of the cross-links that form with aging and diabetes.     

Effects of age and diabetes mellitus on cyanogen bromide digestion of human dura mater collagen

To extend previous studies on the apparent acceleration of collagen aging in diabetes mellitus, dura collagen from human adults of different ages, with and without diabetes, was processed to yield soluble and insoluble fractions. Insoluble fractions were cleaved by cyanogen bromide. Release of peptides from insoluble collagen by cyanogen bromide decreased markedly with age and was much less from the collagen of diabetics than from nondiabetics of similar ages. The acrylamide gel profiles of peptides released were similar, but not identical, for samples of different ages and for samples from diabetics. It was concluded that age-dependent and diabetes-dependent cross-linking was widespread throughout helical regions of collagen molecules and that collagen throughout the body is altered in diabetes. Analyses of fragments of insoluble collagen are required to gain information on the chemistry of the cross-links that form with aging and diabetes.     

The structural basis of cell-mediated immunological reactions of collagen: Reactivity of separated α-chains of calf and rat collagen in cutaneous delayed hypersensitivity reactions

Denatured calf and rat skin collagen and α1- and α2-chains from these collagens were tested for their capacity to sensitize guinea-pigs for the cutaneous delayed hypersensitivity reaction and to elicit this reaction in sensitized animals. Alpha-chains from neutral salt extracted and from urea extracted collagen were also compared. All preparations were fully active as sensitizers as well as elicitors of the skin reaction. In spite of extensive cross-reactions, significant differences between α1- and α2-chains of a given species as well as between corresponding chains of different species could be detected. Differences between chains from neutral salt extracted collagen and from urea extracted collagen were also revealed.     

Introduction

The effects of various avocado oils on collagen metabolism in skin were studied in growing rats fed diets containing 10% (w/w) of the tested oils. Rats fed the unrefined avocado oil extracted with hexane from the intact fruit, its unsaponifiables or the avocado seed oil, showed significant increases in soluble collagen content in skin, though total collagen content was not affected. The increased soluble collagen content appears to be a consequence of the inhibition of lysyl oxidase activity. The active factor was found to be present in the unrefined avocado oil and probably originated from the avocado seed, since collagen metabolism was affected only by fractions which contained lipids fraction from the seed. In comparison rats fed the refined or unrefined soybean oils showed no effects.     

The effect of various avocado oils on skin collagen metabolism.

The effects of various avocado oils on collagen metabolism in skin were studied in growing rats fed diets containing 10% (w/w) of the tested oils. Rats fed the unrefined avocado oil extracted with hexane from the intact fruit, its unsaponifiables or the avocado seed oil, showed significant increases in soluble collagen content in skin, though total collagen content was not affected. The increased soluble collagen content appears to be a consequence of the inhibition of lysyl oxidase activity. The active factor was found to be present in the unrefined avocado oil and probably originated from the avocado seed, since collagen metabolism was affected only by fractions which contained lipids fraction from the seed. In comparison rats fed the refined or unrefined soybean oils showed no effects.     

Dynamics of collagen accumulation and activity of collagen-degrading enzymes in the liver of rats with carbon tetrachloride-induced hepatic fibrosis

Liver fibrosis in rats was induced by repeated subcutaneous injections of carbon tetrachloride. Total collagen, soluble and insoluble collagen fractions as well as type I and type III collagen content in the liver were subsequently measured over a 3-18 week period. Liver collagen was found to increase exponentially during this time. Insoluble collagen accumulated more rapidly than soluble forms, and the accumulation of type III collagen was relatively greater than type I collagen. Changes in specific liver enzymes were also observed. Collagenase, collagenolytic cathepsin and collagen peptidase activities all increased. Levels of collagen-degrading enzymes increased rapidly during the first weeks of fibrosis-induction, and were followed by a more gradual increase during the remainder of the study.     

Collagen fibril formation in the presence of dexamethasone disodium phosphate

Dexamethasone disodium phosphate was found to inhibit in vitro fibrillogenesis in a buffered collagen solution that otherwise formed in vivo like fibrils. A nonlinear relationship was observed between the steroid salt concentration and the kinetic parameter half transition time. Full fibril inhibition occurred at dexamethasone phosphate concentrations above 15 mM. At lower concentrations, sample buffers that also contained inorganic phosphate were not different from the control in activation energy of 224.3 +/- 29.3 kJ/mol (53.6 +/- 7.0 kcal/mol). The idea is advanced that the soluble steroid salt associates directly to the collagen and prevents the formation of lateral, hydrophobic interactions between adjacent collagen aggregates.