Lasse Ryhänen

Decrease in Liver Collagen Accumulation in Carbon Tetrachloride-Injured and Normal Growing Rats Upon Administration of Zinc

Several attempts have been made to develop antifibrotic drugs for human use, but their success has been limited. The present data suggest that peroral zinc treatment has a direct and selective inhibitory effect on carbon tetrachloride-induced collagen accumulation in rat liver. Zinc did not normalize the carbon tetrachloride-induced increases in either liver relative weight, liver total protein content, fat accumulation, or the standard liver function tests, but it did efficiently inhibit liver collagen accumulation. It also reduced skin and liver collagen content and urinary hydroxyproline excretion in normal growing animals, indicating that the inhibition is not limited to the fibroproliferative inflammation associated with carbon tetrachloride injury. Neither inhibition of polysomal protein synthesis nor increased degradation of mature collagen fibers was found to play any major role in the effect of zinc. Instead, a plausible mechanism is inhibition of proline hydroxylation.

Assay of protocollagen lysyl hydroxylase activity in the skin of human subjects and changes in the activity with age

Abstract A method is described for the assay of protocollagen lysyl hydroxylase activity in human skin specimens of 100 to 300 mg wet weight. With the procedure reported here, about 90% of the enzyme activity could be extracted from the skin, and results for six skin samples analyzed separately from the same human subject agreed within a variation coefficient of 13%. The lysyl hydroxylase in human skin resembled that from chick embryos in requiring ascorbate, α-ketoglutarate, ferrous iron and atmospheric oxygen. In human skin the activity was highest in foetuses, and higher in infants than in adult subjects. The mean value in young foetuses was about 40 times, and the mean value in two infants of 2 and 4 months of age about 8 times that found in adult subjects.

Hydroxylation of lysyl residues in native and denatured protocollagen by protocollagen lysyl hydroxylase in vitro

Abstract [ Lys - 14 C] Protocollagen was isolated from embryonic tendon cells, and examined as a substrate for protocollagen lysyl hydroxylase. The K m for denatured protocollagen was found to be about 10 −18 M, which expressed in terms of molar concentration of the peptide chain. This value is substantially lower than the K m for any of the synthetic peptides tested previously as a substrate for lysyl hydroxylase. The effect of helical conformation of protocollagen on hydroxylysine formation was studied by incubating native and heat-denatured protocollagen with lysyl hydroxylase at 17° C. An inhibiting effect of helical conformation was found in that lysyl residues in native protocollagen were hydroxylated at a markedly lower rate than those in denatured protocollagen. In addition, the relationship between the enzyme concentration and the synthesis of hydroxylysine with native protocollagen deviated from linearity, and the values seemed to be approaching a limit of less than one hydroxylysyl residue per α-chain. The data support th earlier suggestion that formation of a triple-helical structure may be one of the critical factors limiting the hydroxylation of lysyl residues during collagen biosynthesis. To study whether the amino-terminal peptide extensions of protocollagen had any effect on hydroxylation of lysyl residues, the extensions were cleaved off by limited pepsin digestion. The results of hydroxylation experiments were found to be similar whether protocollagen or pepsin-modified protocollagen was used as the substrate.

Lysyl hydroxylase. Further purification and characterization of the enzume from chick embryos and chick embryo cartilage

A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.

The effect of malotilate on type III and type IV collagen, laminin and fibronectin metabolism in dimethylnitrosamine-induced liver fibrosis in the rat

BACKGROUND/AIMS Dimethylnitrosamine-induced liver damage was used as an experimental model to study the effect of malotilate on liver fibrosis. METHODS Deposition of type III and IV collagens, laminin and fibronectin were studied from liver section by immunohistochemical techniques using specific antibodies. Serum concentrations of aminoterminal propeptide of type III procollagen, and aminoterminal and carboxyterminal domains of type IV collagen were determined by radioimmunoassays from both malotilate-treated and untreated animals with dimethylnitrosamine injury. RESULTS A significant elevation of all three serum parameters was observed after 3 weeks of hepatic injury in animals without malotilate treatment, and a constant increase was noted in the amounts of hepatic type III and IV collagens, laminin and fibronectin. Malotilate prevented increases in serum markers of type III and IV collagen synthesis as well as accumulation of the collagens, laminin and fibronectin in the liver. CONCLUSIONS The results suggest that serum marker determinations can be used to monitor changes in type III and IV collagen synthesis in the liver. The data indicate that malotilate has a preventive effect in dimethylnitrosamine-induced experimental hepatic fibrosis.

Developmental chanegs in protocollagen lysyl hydroxylase activity in the chick embryo

Abstract Developmental changes in protocollagen lysyl hydroxylase activity were studied in whole chick embryos and in several tissues of chick embryos. In whole chick embryos, the enzyme activity increased between the 7th and 15th day of development, and decreased thereafter. In bone and skin the enzyme activities likewise had maximum values on the 15th day of development, whereas in lung, kidney, heart, spleen and liver the enzyme activities showed relatively small changes between the 9th and 21st day of development. Comparison of lysyl hydroxylase activities in several bones and other tissues of 15-day-old embryos indicated that the enzyme activity in all bones studied was considerably higher than that in skin. Howerever, values even higher than those in bone were found in leg tendon. The lowest enzyme activities were measured in heart, spleen and liver. Attempts to influence the development of lysyl hydroxylase activity by administration of lactate or ascorbate on to the chorioallantoic membrane caused no significant changes in the enzyme activity. The chanegs in lysyl hydroxylase activity observed in the present study agree with previous data on changes in prolyl hydroxylase activity and in collagen biosynthesis in the developing chick embryo. It seems possible that changes in lysyl hydroxylase activity like those in prolyly hydroxylase activity may correlate with changes in collagen biosynthesis in several situations.

Synthesis of an elastin component of molecular weight about 70,000 by polysomes from chick embryo aortas☆

Abstract Polysomes were isolated from aortas of 17-day-old chick embryos, and the synthesis of the nascent polypeptide chains was completed in vitro. When a mixture of a labeled amino acids found in elastin was used, the major radioactive product obtained was of molecular weight about 70,000 and was similar to elastin by several criteria. The 70,000 molecular weight product was extractable in propanol-butanol, it was not labeled with [35S]methionine, and it was precipitated by antibodies against elastin. Polypeptides larger than 70,000 molecular weight were also synthesized but these larger polypeptides incorporated relatively small amounts of [14C]valine, and they appeared to represent proα chains of procollagen. The results suggest that the major gene product for elastin has a molecular weight of about 70,000.

Biochemical and morphological characterization of carbon tetrachloride-induced lung fibrosis in rats

The short-term and long-term lung CCl4 injuries in rats were studied by i.p. CCl4 for 2 or 5 weeks, respectively, and the lung injury in the third progression group receiving i.p. CCl4 for 2 weeks followed by 3 weeks without. Acute haemorrhagic interstitial pneumonia resulted from short-term injury; chronic interstitial pneumonia from long term injury, and residua of injury or advanced chronic interstitial pneumonia in the progression group. All groups also exhibited features for diffuse alveolar damage. Connective tissue stains revealed both interstitial and intra-alveolar fibrosis in short-term injury. Hydroxyproline content and the activities of prolyl hydroxylase and galactosyl-hydroxylysyl glucosultransferase were elevated. This suggests an early onset of pulmonary fibrosis. Immunohistochemistry revealed the interstitial accumulation of BM proteins. In contrast, increased type III pN-collagen could also be found in the intra-alveolar spaces. The degrees of both interstitial and intra-alveolar fibrosis, BM proteins and type III pN-collagen, and also hydroxyproline content were greater in long-term injury, while the progression group showed on average fewer fibrotic changes than did the long-term injury group, but more than the shortterm injury pointing to persistence or progression of the changes. Additionally, intra-alveolar crystallized haemoglobin was found following short-term injury. We conclude that CCl4-induced lung injury is an useful experimental model to study pulmonary fibrosis. The mechanism of CCl4 lung injury is not known but free radical-mediated lipid peroxidation is suggested.

Effects of divalent cations on collagen biosynthesis in isolated chick embryo tendon cells

Isolated chick embryo tendon cells were used in [14C]proline and [14C]lysine labelling experiments to investigate the effect of divalent cations on collagen biosynthesis with a special reference to prolyl hydroxylation and lysyl modifications. The following metals were studied by adding them to the incubation medium of the cells: Ca2+, Cd2+, Co2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+. Zn2+ caused a potent reductin in collagen prolyl hydroxylation with a concomitant increased cellular retention of collagenase-digestible material. These effects were detectable even at physiological concentrations. At the same concentrations of Zn2+, lysyl hydroxylation was considerably less inhibited than prolyl hydroxylation, and the extent of hydroxylysyl glycosylation was even increased. Co2+ was also an efficient inhibitor of collagen prolyl hydroxylation, but at concentrations ten times higher than those of Zn2+. In the presence of other metal ions, no or only up to 10% inhibition of prolyl hydroxylation was noted even at those concentrations at which [14C]proline incorporation into the protein was decreased. However, an increased cellular retention of collagen was detected in the presence of some metal ions. No reduction in lysyl hydroxylation was found in the presence of Ca2+ or Mg2+.

Improved metabolic control in patients with non-insulin-dependent diabetes mellitus is associated with a slower accumulation of glycation products in collagen.

Abstract. Twenty‐one patients with non‐insulin‐dependent diabetes in poor metabolic control were subjected to intensified therapy, in most cases with insulin, to investigate whether it is possible to slow down the accumulation of advanced glycosylation end products of collagen by improving glycaemic control. Fasting and mean daily blood glucose, serum fructo‐samine and glycohaemoglobin levels, as well as glycation of collagen were measured before and after 1.5 years of intensified therapy. All these parameters except for fructosamine correlated significantly with fasting blood glucose and glycohaemoglobin when measured before the insulin therapy was started, when the patients had had poor but stable metabolic control for a long period of time. After 1.5 years of intensified therapy the level of glycation of collagen did not significantly correlate with the fasting blood glucose or glycohaemoglobin levels, suggesting that the non‐enzymatic glycosylation of collagen reflects a longer period of metabolic control of diabetes than the glycohaemoglobin level. Intensified treatment improved previously poor metabolic control in patients with non‐insulin‐dependent diabetes, and this improvement was reflected in a decrease in fasting and mean daily blood glucose levels, serum fructosamine and glycohaemoglobin concentrations, and in the level of early products of glycation of collagen. The average content of advanced glycosylation end products of collagen, assayed in terms of collagen‐linked fluorescence did not decrease. However, they accumulated more slowly in the patient tercile with the greatest decrease in the level of fasting blood glucose than in the tercile with the smallest decrease, and even a decrease in fluorescence was observed in the patients with the greatest improvement in the metabolic control. Our findings suggest that the improvement of metabolic control in non‐insulin‐dependent diabetes is reflected in a slower accumulation of advanced glycosylation end products in collagen. If the slower accumulation of advanced glycosylation end products in collagen is translated into a slower development of the long‐term complications of diabetes remains to be studied.