Influence of bleomycin on the crosslinking of dermal collagen in rat

The effect of bleomycin on dermal collagen crosslinking was studied in male albino rats. The skin samples were taken on the 28th day in both groups. Results showed that the percent reversibilities of neutral salt soluble collagen gels and the solubility of insoluble collagen in KCNS, urea or pronase were decreased in bleomycin treated animals whereas the aldehyde content was significantly increased in bleomycin treated animals compared to controls. The electrophoretic pattern of neutral salt soluble collagen on SDS-polyacrylamide gels revealed a marked decrease of alpha/beta ratio in bleomycin treated group. These results indicated that both the intra and inter molecular crosslinks of collagen were increased in the bleomycin treated group.     

Isolation and partial characterization of bovine gingival AB collagen

Summary Limited proteolysis with pepsin solubilized 25% of the insoluble gingival matrix as mainly soluble collagenous material. Fractional salt precipication at neutral pH resulted in the separation of types III and I at 1.8 and 2.6 M NaCl, respectively. In addition, a collagenous fraction accounting for 2% of the solubilized collagen and precipitating at 4.5 M NaCl was shown to be identical with type V collagen. Isolation and partial characterization of the constituent-α-chains of the 4.5 M PPT by gel filtration, ion exchange and hydroxylapatite chromatography as well as disc electrophoresis showed that gingival type V collagen contains αA and αB chains in a ratio αB/αA of 1.73–1.8. Electron microscopic examination of ATP-precipitates showed that this collagen type gave only one kind of SLS aggregates with asymmetric band pattern characteristically different from that of type I collagen. The data provide evidence that gingival AB collagen is a heteropolymer in which the αA and αB chains are assembled in the same macromolecule in a 1∶2 ratio.     

In vivo labeling studies on the biosynthesis and degradation of collagen in experimental myocardial infarction.

The biosynthesis and degradation of myocardial collagen was studied in myocardium infarcted rats after a single intraperitoneal injection of 3H-proline. The incorporation of tritiated proline into collagen as 3H-hydroxyproline was regarded as a measure of collagen synthesis. The total content as well as total activity of hydroxyproline were measured in the whole homogenate, neutral salt soluble fraction, insoluble fraction and in urine collected at different time intervals and specific activities were calculated. Both collagen anabolism and catabolism were found to be affected in infarcted rat hearts. Degradation of existing collagen at the earlier stages of myocardial infarction and a simultaneous new collagen synthesis and deposition as insoluble form later took place.     

A neutral proteinase from human gingival fibroblasts active against the c-terminal cross-linking region of type I collagen

An enzyme that was capable of releasing small fragments containing hydroxynorleucine from the c-terminal extra-helical region of the alpha 1 chain of reduced (tritiated) soluble type I collagen was found, along with collagenase, in medium that had been conditioned by the culture of human gingival fibroblasts (Gin-1). The enzyme was present in a latent form or forms and could be activated by treatment with either trypsin or p-hydroxymercuribenzoate. It was maximally active at neutral pH and inhibited by EDTA. It is suggested that this enzyme, acting within a region of the molecule which is of major importance in stabilizing fibrillar collagen through intermolecular cross-linking, could potentially play an important role in collagen turnover in vivo.     

The solubilization of collagen and protein–polysaccharides from the developing cartilage of lathyrytic chicks

1. The solubilization of collagen and protein-polysaccharides from the developing cartilage of normal and lathyritic chicks was studied by using mild extraction procedures. One-third of the protein-polysaccharides could be solubilized in salt solutions at neutral pH from normal cartilage, whereas 95-100% could be extracted from the cartilage of animals that were severely lathyritic. Likewise, whereas in normal animals the collagen of cartilage was essentially insoluble in salt solutions at neutral pH, in lathyritic animals it was almost completely soluble. 2. The increased solubility of the collagen of cartilage from lathyritic animals enabled sufficient material to be collected so that the pure alpha1 chains of the collagen were isolated by repeated reconstitution, precipitation and CM-cellulose column chromatography. The purified alpha1 component was characterized by its relatively high content of hydroxylysine (14 residues/1000 amino acids). 3. About 37% of the collagen from the cartilage of normal chick embryos could be extracted as the gelatin at pH7.4 in lithium chloride solution. This was accompanied by the extraction of approx. 14% of the protein-polysaccharide content. 4. The protein-polysaccharides and the collagen from normal animals could be extracted from the cartilage relatively independently of one another under mild conditions. These same components obtained from lathyritic animals easily separated from one another after solubilization. This provided evidence that the two components are probably not covalently cross-linked. 5. The collagen of cartilage extracted as a gelatin from normal animals contained a high proportion of alpha chains compared with beta dimers, similar to the lathyritic collagen of cartilage and other tissues, and similar to the gelatin extracted from normal chick bone.     

Toxicological effects of an organophosphorus pesticide (dimethoate) on urinary collagen metabolites in normal and high protein diets fed female albino rats

The effect of an organophosphorus pesticide (dimethoate) on the urinary excretion of hydroxyproline (total, nondialysable, dialysable and free fractions) and hydroxylysylglycosides, glucosylgalactosyl hydroxylysine and galactosehydroxylysine was investigated in two groups of female albino rats fed with normal and high protein diets. In comparison to controls, dimethoate treated animals were found to excrete significantly decreased amounts of urinary hydroxyproline fractions from 7th day onwards. The excretion of total hydroxylysylglycoside in urine parallels the excretion of hydroxyproline. The urinary output of both glu-gal-hyl and gal-hyl was also appreciably lower from dimethoate treated animals. The normal ratio of glu-gal-hyl and gal-hyl found in the urine of dimethoate treated animals was discussed in light of decreased turn over of collagen in both bone and skin. The effect of dimethoate in rats fed with high protein diet was comparatively less than those fed with normal diet.     

Influence of bleomycin on the metabolism of dermal collagen in albino rats

The metabolism of collagen in male rats by treatment with bleomycin was studied following the injection of [3H]proline and the determination of specific and total activity of [3H]hydroxyproline in skin collagen fractions and urine. In the case of the bleomycin-treated animals, there was found to be an increase in the neutral salt soluble collagen content with no change in insoluble collagen content as compared to the control group. The specific and total radioactivity of [3H]hydroxyproline in soluble and insoluble collagen fractions was also increased. Examination of [3H]hydroxyproline activity in soluble and insoluble collagen showed that the conversion of soluble to insoluble collagen was improved by the bleomycin-treated group. It was found that this was accompanied by a decrease in urinary excretion of total hydroxyproline and [3H]hydroxyproline during the first 12 hr after the administration of [3H]proline. Therefore, the results of the present investigation clearly indicate that the maturation of soluble to insoluble collagen is promoted and accompanied by a decrease in the catabolism of soluble collagen in the bleomycin-treated animals. In addition, administration of bleomycin increased the synthesis of collagen.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

Aging and cross-linking of skin collagen

This report represents a clear demonstration of a cross-link in collagen whose abundance is related to chronological aging of an organism. Recently its structure was identified as histidinohydroxylysinonorleucine. Quantification of the cross-link in various aged samples of bovine and human skin indicate that it rapidly increases from birth through maturation. Subsequently, a steady increase occurs with aging, approaching 1 mole/mole of collagen. This compound seems to be related to the relative proportions of soluble to insoluble collagen from skin in neutral salt, dilute acid, and denaturing aqueous solvents (higher concentration in the insoluble portion). It is absent from other major collagenous tissues such as dentin, bone and tendon.     

Impaired collagen maturity in vitamins B2 and B6 deficiency--probable molecular basis of skin lesions

Solubility of collagen was increased and the proportion of insoluble collagen was reduced in the skin of both riboflavin as well as pyridoxine-deficient rats. Collagen content of the skin, and aldehyde concentration of salt-soluble collagen were also lower in the deficient groups. The alpha:beta subunit ratio of salt-soluble collagen was higher in riboflavin deficiency. In food-restricted weight-matched control groups, similar changes in collagen solubility, but of lesser magnitude were observed. Both food restriction and riboflavin deficiency decreased plasma PLP concentration. Increase in the solubility of collagen, decrease in the aldehyde content of soluble collagen and increase in the alpha:beta subunit ratio of soluble collagen, suggest that the maturation of collagen may be affected in pyridoxine or riboflavin deficiency. These molecular events may be etiologically related to the pathogenesis of the skin lesions in vitamin B2 or B6 deficiency.     

Characterization of type I collagen from the skin of blue grenadier (Macruronus novaezelandiae)

The skin collagen of a fish, blue grenadier (Macruronus novaezelandiae), has been purified and characterized. The fish skin was readily soluble in dilute acetic acid, with no pepsin treatment needed. The collagen was purified by salt precipitation. Skin samples from fish of various ages showed that even in the oldest sample, more than 8 years of age, the collagen was still readily acid soluble. The purified collagen had a melting temperature of 22 degrees C; the shrinkage temperature for the skin was 48 degrees C. Its tissue distribution, examined by immunohistology, and its chemical properties indicated a close homology to mammalian type I collagen. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that three distinct alpha-chains were present. These were purified by ion-exchange chromatography on CM-cellulose and by gel permeation chromatography on Superose 6. The three purified alpha-chain fractions were examined by amino acid analysis and by SDS-PAGE of their cyanogen bromide fragments. These data indicated that the additional chain was genetically distinct, and most closely related to the alpha 1-chain, from which it was poorly resolved on SDS-PAGE.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosenic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylyse, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonezymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot acount for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia.

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosemic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylysine, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonenzymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated with DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot account for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

Partial purification and characterization of a neutral proteinase with collagen telopeptidase activity produced by human gingival fibroblasts

A neutral proteinase was purified 1930-fold from medium conditioned by the culture of human gingival fibroblasts that had been stimulated to secrete enzymes by concanavalin A. This enzyme had an apparent molecular weight of 35,000 (gel chromatography) and apparent isoelectric point of 4.3 (chromatofocusing). It was inhibited by chelating agents, serum, and nonactivated conditioned fibroblast medium, but not by phenylmethylsulphonyl fluoride or N-ethylmaleimide. This proteinase removes the C-telopeptide from the alpha 1 chain of type I collagen, an activity which could be important in the degradation of collagen in the extracellular matrix. It was also found to digest fibronectin but had no effect on proteodermatan sulphate under the conditions used. It appears to be unrelated to previously described fibroblast extracellular proteinases and we, therefore, tentatively propose the name fibroblast metalloproteinase IV.     

Identification of collagens synthesized by cultures of normal human corneal and keratoconus stromal cells

We have examined the collagens synthesized by cultures of normal human corneal stromal cells. Radioactively labeled products, accumulated in the culture medium during a 24-h labeling period, were treated with pepsin and analyzed by SDS-polyacrylamide gel electrophoresis. The cell layer collagen was characterized by 2.6 M and 4.4 M salt fractionation at neutral pH. CM-cellulose column chromatography, SDS-gel electrophoresis, and cyanogen bromide peptide mapping. Type I alpha 1 and alpha 2 chains were the predominant components in both the cell layer and the medium fractions of normal human stromal cultures; type III collagen was found mostly in the culture medium; and type V collagen was associated with the cell layer. Immunofluorescent techniques used to visualize collagen deposition in the cell layer confirmed the presence of these collagen types. Keratoconus is a disease characterized by thinning and scarring of the central cornea. Stromal cells grown from keratoconus corneas produced similar types of collagen (types I, III, and V) as normal human controls. Cells from keratoconus patients, however, contained more type V collagen in the cell layer than did normal cells. The difference was seen only in the 4.4 M salt precipitates. Since type V collagen is one component of cell surfaces, the primary defect in cultures from keratoconus corneas could involve cell membrane and cell surface components.     

Metabonomics analysis of urine and plasma from rats given long-term and low-dose dimethoate by ultra-performance liquid chromatography-mass spectrometry

This study assessed the effects of long-term, low-dose dimethoate administration to rats by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Dimethoate (0.04, 0.12, and 0.36mg/kg body weight/day) was administered daily to male Wistar rats through their drinking water for 24weeks. Significant changes in serum clinical chemistry were observed in the middle- and high-dose groups. UPLC-MS revealed evident separate clustering among the different dose groups using global metabolic profiling by supervised partial least squares-discriminant analysis. Metabonomic analysis showed alterations in a number of metabolites (12 from urine and 13 from plasma), such as l-tyrosine, dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), citric acid, uric acid, suberic acid, glycylproline, allantoin, isovalerylglutamic acid and kinds of lipids. The results suggest that long-term, low-dose exposure to dimethoate can cause disturbances in liver function, antioxidant and nervous systems, as well as the metabolisms of lipids, glucose, fatty acids, amino acids, and collagen in rats. DMTP and DMDTP, which had the most significant changes among all other studied biomarkers, were considered as early, sensitive biomarkers of exposure to dimethoate. The other aforementioned proposed toxicity biomarkers in metabonomic analysis may be useful in the risk assessment of the toxic effects of dimethoate. Metabonomics as a systems toxicology approach was able to provide comprehensive information on the dynamic process of dimethoate induced toxicity. In addition, the results indicate that metabonomic approach could detect systemic toxic effects at an earlier stage compared to clinical chemistry. The combination of metabonomics and clinical chemistry made the toxicity of dimethoate on rats more comprehensive.     

Characterization of collagen precursors found in rat skin and rat bone.

Two genetic types of collagenous proteins, type I and type III, were isolated by extraction and differential salt precipitation from rat skin. The yield of collagen precursors was increased by injecting animals with colchicine 30 min before sacrifice to inhibit secretion of collagen. DEAE-cellulose chromatography was used to separate collagen from collagen precursors. Although these preparations contained more type I collagen than type III collagen, there were always more type III than type I precursors. The precursor chains of type I fractions were separated on CM-cellulose chromatography after denaturation. Three precursor forms were found for each collagen alpha chain, a complete chain (proalpha chain), and a precursor chain with only an amino-terminal (pNalpha chain) and carboxy-terminal extension (pCalpha chain). Species differences were demonstrated between rat collagen precursors and other species using rat calvaria (frontal and parietal) bones extracted with either 0.5 N acetic acid or neutral salt buffers containing protease inhibitors. Native rat procollagen elutes earlier than chicken or human procollagen on DEAE-cellulose chromatography and does not separate significantly from the pC collagen form. The collagenase resistant amino terminal peptides of rat pNalpha1 and pNalpha2 were the same size (16 000) but could be separated by DEAE-cellulose chromatography.     

Effect of Oxygen Free Radicals on Corneal Collagen

Corneal collagen was labeled in vivo by injection of ¹⁴C-proline into the anterior chamber of rabbit eyes. The isolated corneal collagen was incubated in iron-free phosphate buffered saline (pH 7.4) containing I mM axorbate and 0.1 mM CuSO₄ for either 1 hour or 3 hours at 37°. Addition of 2 volumes of 8 M urea-I mM dithiothreitol and heating for 1 min at 100° solubilized virtually all of the collagen in the control incubations but left a significant amount of insoluble collagen in specimens exposed to the hydroxyl radical generating system. This residue amounted to 19% and 38% of the initial radioactivity in samples incubated for 1 h and 3 h, respectively. The chromatographic profiles (gel filtration on CL-4B) of the soluble fraction showed an increase in both aggregation and degradation products of collagen in the 1 h incubation mixture, whereas after 3 h there was an increase only in degradation products. These observations suggest that additional crosslinking of the soluble collagen aggregates observed at 1 h may be responsible for their subsequent disappearance at 3 h, with concomitant increase of the insoluble fraction. Collagen degradation by OH may play a role in corneal ulceration, whereas hydroxyl radical-mediated crosslinking is consistent with age-dependent increases in insoluble collagen.     

FBJ virus-induced osteosarcoma contains type I, type I trimer, type III as well as type V collagens

The FBJ osteosarcoma (a virus-induced osteosarcoma named after its discoverers, Finkel, Biskis, and Jinkins) contains an extensive extracellular matrix. Collagens were extracted by digestion with pepsin in dilute acetic acid from tumors grown in lathyritic mice and fractionated by differential salt precipitation, yielding five fractions. Fraction 1 (precipitated at acidic 0.7 M and neutral 2.0 M NaCl) gave rise mainly to alpha 1(III) chain on phosphocellulose column chromatography. The alpha 1(III) chain was identified by its typical behavior on interrupted electrophoresis and analysis of the CNBr-cleaved peptides. The alpha 1(III) chain of the FBJ tumor had a high content of hydroxylysine and neutral saccharide. Fraction 2 (precipitated at acidic 0.7 M and neutral 4.5 M NaCl) yielded alpha 1(I) and alpha 2(I) chains on the phosphocellulose column from which alpha 1(I) was eluted as a broad peak, conceivably reflecting a high content of hydroxylysine and neutral saccharide. Fraction 4 (precipitated at acidic 1.2 M and neutral 4.5 M NaCl) yielded type V collagen, which also featured an exceptionally high content of neutral saccharide (Yamagata, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 1208-1214). The proportions of type I, type I trimer, type III, and type V collagens extracted by pepsin digestion from FBJ tumor were calculated to be 33, 29, 26, and 12%, respectively. The FBJ tumor is free from invasion by blood vessels, shows no deposition of calcium, and thus has the appearance of cartilage. But type II collagen, a specific gene product of cartilage, could not be identified in any of the fractions analyzed. Contrary to its appearance, collagen type analyses indicate that FBJ osteosarcoma is literally induced from osteogenic cells.