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.     

Build-up of collagen and hyaluronic acid polyelectrolyte multilayers

We have used a novel polyelectrolyte multilayer (PEM) coating consisting of the polyelectrolytes collagen and hyaluronic acid. The build-up by the layer-by-layer deposition technique is outlined by ex situ and in situ ellipsometric measurements. When collagen was added, the thickness of the PEM was increased, and the refractive index was decreased. Corresponding but opposite effects were noted when hyaluronic acid was added. These changes are considered to be explained by a diffusion mechanism. It was also found that the PEM layers were unstable at physiological pH. However, by cross-linking using N-(3-di-methylaminopropyl)-N'-ethylcarbodiimide together with N-hydroxysuccinimide, a stable PEM layer resulted. These tissue friendly PEM layers are expected to have a great impact in the design of artificial extracellular matrixes. Also, the insertion of fluorescence labels demonstrates the potential for incorporation of other functionalities.     

Synthesis and degradation test of hyaluronic acid hydrogels

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). (1)H NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS(3)), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests.     

Ascorbate deficiency results in decreased collagen production: under-hydroxylation of proline leads to increased intracellular degradation

Collagen production by cultured human lung fibroblasts was examined when the cells were made deficient in ascorbate. Cells grown in the absence of ascorbate produced 30% less collagen during a 6-h labeling period than cells incubated with as little as 1 microgram/ml ascorbate during the labeling period. Cells grown without ascorbate produced under-hydroxylated collagen which was subject to increased intracellular degradation from a basal level of 16% to an enhanced level of 49% of all newly synthesized collagen. The likely mechanism for increased intracellular degradation is the inability of under-hydroxylated collagen to assume a triple-helical conformation causing it to be susceptible to intracellular degradation. Measurement of collagen production by enzyme linked immunoassay (ELISA) using antibodies directed against triple-helical determinants of collagen showed that both types I and III collagens were affected. In contrast, another connective tissue component, fibronectin, was not affected. Analysis by ELISA showed a greater decrease in collagen production than did analysis by the collagenase method, suggesting that some non-helical collagen chains (detected by collagenase but not by ELISA) were secreted in the absence of ascorbate. These results provide a mechanism to account, in part, for the deficiency of collagen in connective tissues which occurs in a state of ascorbate deficiency.     

In vivo degradation of oxidized, regenerated cellulose

Oxidized, regenerated cellulose (ORC) was surgically implanted on the uterine horns of rabbits, and its biodegradation was studied in vivo. Samples of peritoneal lavages, serum, and urine were collected during the degradation process and analyzed for carbohydrate components utilizing high-performance liquid chromatography with pulsed amperometric detection (h.p.l.c.-p.a.d.). Degradation was rapid, and oligomeric products were evident primarily in the peritoneal fluid from the implantation site, with no apparent accumulation in either the serum or the urine. The size distribution and the amount of the oligomeric products decreased after day one, and by day four peritoneal lavages were essentially free of oligomers. The structure of the products formed was consistent with the lability of the polymer in solution, and the kinetics of degradation paralleled the results of the previously reported in vitro studies. Rabbit peritoneal macrophages, when incubated with ORC in vitro were observed to readily ingest and hydrolyze the polymeric material. A mechanism of degradation consisting of chemical depolymerization, followed by enzymatic hydrolysis mediated by glycosidases endogenous to peritoneal macrophages, is proposed.     

Reduced collagen and ascorbic acid concentrations and increased proteolytic susceptibility with prelabor fetal membrane rupture in women

Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.     

Effect of estradiol-17 on collagen biosynthesis, degradation and re-utilization in vivo

The effects of estradiol-17beta on total body collagen metabolism were studied in adult female guinea pigs in which all collagen had been prelabelled by the chronic administration of -Lproline during the period of rapid growth. Estradiol-17beta produced a decrease in collagen biosynthesis in skin in the presence of normal or increased collagen degredation. Total carbon-14 label was unchanged in skin and in the granuloma. Estradiol-17beta did not inhibit collagen biosynthesis in the granuloma; the content of soluable collagen was reduced. Total uterine weight, collagen and noncollagenous protein were increased by estradiol-17beta, and the total carbon-14 label was markedly increased. The wet weight and the dry, defatted weight of metaphyseal bone were increased by estradiol and the specific activity of hydroxyproline in collagen and proline in the noncollagenous protein was increased.     

Biosynthesis of collagen crosslinks. II. In vivo labelling and stability of lung collagen in rats

Rat lung collagen was labelled in vivo by a single intraperitoneal injection of [3H]lysine at several key timepoints in lung development: days 11 (alveolar proliferation), 26 (start of equilibrated growth), 42 (end of equilibrated growth), and 100 (adult lung structure present). The rates of deposition of labelled hydroxylysine and the difunctional, Schiff base-derived crosslinks hydroxylysinonorleucine (HLNL) and dihydroxylysinonorleucine (DHLNL) were quantified. We also measured total lung content of the trifunctional, mature crosslink hydroxypyridinium (OHP) in these same animals. While the relative rates of accumulation of labelled collagen [3H]hydroxylysine differed by a factor of about 6 at the different times of injection of labelled precursor, quantitative and qualitative patterns of collagen crosslinking were very similar at all of the lung developmental stages studied. Furthermore, there was little or no breakdown of the lung collagen pool as defined by the presence of labelled crosslinks; changes in lung DHLNL content could be completely accounted for by its maturation to OHP, regardless of the age of the rats when injected with the radioactive precursor. We conclude that mature, crosslinked collagen in the lungs of rats, which is obligatorily an extracellular pool, is not being degraded at a measurable rate. Therefore, studies of others that have shown apparent high rates of breakdown of newly synthesized collagen in lungs of whole animals using different methods are probably not reflective of the metabolic fate of total lung collagen, and may indicate that degradation of normal lung collagen occurs predominantly or exclusively intracellularly.     

Synthesis and degradation of hyaluronic acid by bacteria of Streptococcus genus

Modern data on metabolism of hyaluronic acid by bacteria from Streptococcus genus are presented. Several species of bacteria forming capsule from hyaluronic acid, which is analogous to glycosaminoglycan of vertebrates, are considered. Different aspects of hyaluronic acid synthesis are described: biochemical synthesis pathway, genetic basis, regulation of expression of genes belonging to hyaluronic acid synthesis operon. Biological role and physiologic importance of hyaluronic acid for bacteria, including its role in overcoming immune barrier by pathogenic species, are discussed. Process of depolymerization of hyaluronic acid in presence of hyaluronatlyases secreted by certain streptococci is considered. Characteristic of streptococcal enzyme hyaluronatlyase, its mechanism of catalytic effect, and biological function are presented.     

Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.     

Complementary roles of intracellular and pericellular collagen degradation pathways in vivo

Collagen degradation is essential for cell migration, proliferation, and differentiation. Two key turnover pathways have been described for collagen: intracellular cathepsin-mediated degradation and pericellular collagenase-mediated degradation. However, the functional relationship between these two pathways is unclear and even controversial. Here we show that intracellular and pericellular collagen turnover pathways have complementary roles in vivo. Individual deficits in intracellular collagen degradation (urokinase plasminogen activator receptor-associated protein/Endo180 ablation) or pericellular collagen degradation (membrane type 1-matrix metalloproteinase ablation) were compatible with development and survival. Their combined deficits, however, synergized to cause postnatal death by severely impairing bone formation. Interestingly, this was mechanistically linked to the proliferative failure and poor survival of cartilage- and bone-forming cells within their collagen-rich microenvironment. These findings have important implications for the use of pharmacological inhibitors of collagenase activity to prevent connective tissue destruction in a variety of diseases.     

Streptococcal hyaluronic acid: proposed mechanisms of degradation and loss of synthesis during stationary phase.

Streptococcal hyaluronic acid was found to distribute into two discrete sizes. Cellular hyaluronic acid from strain D181 had an average molecular weight of 10 X 10(6), whereas the average molecular weight of extracellular hyaluronic acid from the same strain was 2 X 10(6). Cellular streptococcal hyaluronic acid was purified to homogeneity. Proteases were unable to cleave the purified cellular polymer, indicating that a peptide was not involved in cross-linking five extracellular hyaluronate polymers to form a cell-bound complex. Lipids apparently are not part of the cellular hyaluronic acid because phosphorus and glycerol were not detected by radioisotopic techniques, and denaturing conditions did not change the size of the polymer. Membranes obtained from various strains of group A and C streptococci cleaved the cellular form of the hyaluronate polymer demonstrating the presence of a membrane-bound hyaluronidase-like activity. By contrast, this activity was not found in the extracellular products of the strains studied. Furthermore, membranes derived from streptococci at the stationary phase of growth no longer had the capacity to synthesize hyaluronic acid. The loss of this property appeared to be due to changes in the structure of the membrane.     

Role of fibroblast migration in collagen fiber formation during fetal and adult dermal wound healing

Adult dermal wounds, in contrast to fetal wounds, heal with the formation of scar tissue. A crucial factor in determining the degree of scarring is the ratio of types I and III collagen, which regulates the diameter of the combined fibers. We developed a reaction-diffusion model which focuses on the control of collagen synthesis by different isoforms of the polypeptide transforming growth factor-β (TGFβ). We used the model to investigate the current controversy as to whether the fibroblasts migrate into the wound from the surrounding unwounded dermis or from the underlying subcutaneous tissue. Numerical simulations of a spatially independent, temporal model led to a value of the collagen ratio consistent with that of healthy tissue for the fetus, but corresponding to scarring in the adult. We investigated the effect of topical application of TGFβ and show that addition of isoform 3 reduces scar tissue formation, in agreement with the experiment. However, numerical solutions of the reaction-diffusion system do not exhibit this sensitivity to growth factor application. Mathematically, this corresponds to the observation that behind healing wavefront solutions, a particular healed state is always selected independent of transients, even though there is a continuum of possible positive steady states. We explain this phenomenon using a caricature system of equations, which reflects the key qualitative features of the full model but has a much simpler mathematical form. Biologically, our results suggest that the migration into a wound of fibroblasts and TGFβ from the surrounding dermis alone cannot account for the essential features of the healing process, and that fibroblasts entering from the underlying subcutaneous tissue are crucial to the healing process.     

The effect of synovial fluid proteins in the degradation of hyaluronic acid induced by ascorbic acid

The degradation of hyaluronic acid induced by ascorbic acid and the effect of synovial fluid proteins, such as ceruloplasmin, transferrin, and albumin, were investigated on the basis of the elution volume and the molecular weight of hyaluronic acid using high-performance gel permeation chromatography. Hyaluronic acid was degraded to less than one-third of the original molecular weight in the range of the physiological concentrations of ascorbic acid. Synovial fluid proteins protected against the ascorbate-dependent degradation of hyaluronic acid at their physiological concentrations. It is suggested that the inhibitory activity of ceruloplasmin mainly depends on the ferroxidase activity and that of transferrin is probably due to iron binding property.     

In vivo genotoxicity evaluation of dimethylarsinic acid in MutaMouse.

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, MutaMouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into MutaMice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the MutaMouse.     

Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver

In a recent communication it was shown that intravenously injected radioactively labelled hyaluronic acid was preferentially taken up by the liver and degraded. We now report that uptake occurs in the liver endothelial cells and that these cells degrade the polysaccharide in vitro into low-molecular weight (LMW) products.