Collagen and related research最新文献

Cartilage-derived growth factor (CDGF), a protein closely related to basic fibroblast growth factor, is known to have both mitogenic and chemokinetic properties in microvascular endothelial cells (MVEC). Becaue of the angiogenic properties of CDGF and its rate in accelerating wound repair, the capacity of this factor to stimulate both proliferation and matrix synthesis was compared in distinct populations of vascular endothelial cells: MVEC from bovine adrenal cortex and macrovascular endothelial cells from the bovine aorta (BAEC). No significant differences in the responses to mitogenic stimulation using CDGF (5-100 units/ml) were observed using MVEC and BAEC. Only rapidly dividing MVEC, however, showed significant increases in collagen secretion in the presence of CDGF. The differential responsiveness of these two cell populations to a defined growth factor underscores the phenotypic diversity of endothelium.

Colchicine has been reported to disrupt microtubules and thereby inhibit collagen secretion. Because of this “Canti-collagen” activity, colchicine has been suggested for use in the treatment of hepatic fibrosis. Using biochemical and immunohistochemical techniques, our laboratory has identified the hepatocyte as one possible source of collagen in the liver. The present study examined the direct effect of colchicine on collagen secretion by hepatocytes in culture. Parenchymal cells were isolated from the livers of adult rats four days following a two-thirds hepatectomy. Total collagen and the fraction secreted into the medium were quantitated as incorporation of [³H]proline into bacterial collagenase-sensitive protein. Treatment of the hepatocytes with 100[,M colchicine (2-3 hours) resulted in a substantial inhibition of collagen secretion. However, upon longer exposure of the hepatocytes to the drug (24 hours and 8 days), the inhibitory effect on collagen secretion was abolished. The anti-protein secretion activity of the colchicine in the conditioned medium removed from the hepatocytes was still present as verified by a 2.5 hour fibroblast-collagen secretion bioassay. The secretion of the plasma proteins albumin, fibrinogen and the third component of complement was not altered by the presence of colchicine. We conclude that the hepatocyte is a highly efficient secretory cell, and is not entirely dependent upon microtubules as organelles for protein secretion. Therefore, to the extent that hepatocytes may contribute to hepatic fibrosis, the therapeutic use of colchicine to block collagen secretion might be expected to have only limited effectiveness.

The carboxy terminal sequence of sheep, bovine and human tropoelastin (GFPGGACLGKA/SCGRKRK) has been inferred in earlier studies from sequencing of cloned complementary and genomic DNA. However, this putative carboxy terminal sequence was not found previously in peptides recovered from tryptic digests of tropoelastin. In order to determine whether the amino acid sequence described above is found in insoluble elastin, antibodies were raised against the chemically synthesized peptides with the appropriate sequences and the antibodies were shown to react with peptides derived from human, bovine, porcine, dog and hamster insoluble elastins. These results strongly suggest that the sequence (GFPGGACLGKA/SCGRKRK) at the carboxy terminus of tropoelastin is found in the elastins of many species.

Tropoelastin was examined in bovine lung, aortic and ligament tissues using both organ culture and cell-free translation systems. The bovine tissues synthesized two tropoelastin polypeptides of approximately 70,000 and 68,000 daltons. Two polypeptides were also seen amongst the translation products directed by mRNAs isolated from each of the individual tissues. Both proteins were shown to be tropoelastins directly by immunoprecipitation with specific antibody and limited NH₂-terminal sequence analyses and indirectly by two-dimensional gel electrophoresis. The finding of two forms of tropoelastin is similar to that previously reported in chick tissues although the apparent molecular weights of the tropoelastins differ between the two species. Another interesting observation is that the proportion of the two tropoelastins differs amongst the three fetal tissues examined. This situation is similar to the differences seen in the ratio of tropoelastin a and b between embryonic chick lung and aortic tissues.

A comparative study has been undertaken to ascertain the effects of different tissue pretreatment procedures on the recovery of the major genetic types of collagen from human placenta. Essentially the same recovery of types I, III, IV and V collagen was obtained from placenta which was directly processed, from placenta which was stored at -70ΰC after washing, and from dried acetone extracts of this tissue. Each collagen type isolated from the treated tissue preparations displayed properties consistent with those exhibited by its counterpart obtained from fresh tissue which was directly processed. Furthermore, it was observed that while the amount of types I and III collagen recovered was directly proportional to the level of pepsin employed, the recovery of types IV and V collagen was inversely related to this isolation parameter. These results establish that human placenta can be either stored frozen or as a dry acetone extract without affecting either the yield or the profile of the different genetic types of collagen recovered and that the amounts of different genetic types of collagen recovered can be modulated by the amount of pepsin employed in the isolation.

Skin and aortic samples from two patients who died by lethal perinatal Osteogenesis Imperfecta (O. I.) were studied by optical and electron microscopy and compared with similar samples from two normal human fetuses and one newborn child. No significant abnormalities were observed in the dermis of O. I. patients apart from small differences in the diameter of reticular collagen fibrils. On the contrary, in the aortas of both patients collagen fibrils were significantly smaller than in the controls; moreover, elastin lamellae were deeply altered and consisted of roundish aggregates of elastin, massively permeated by cytochemically recognizable glycosaminoglycans. As identical features were described in experimental lathyrism by using inhibitors of the enzyme lysyl oxidase (Pasquali Ronchetti et al., 1984), the conclusion is reached that in the two cases of lethal perinatal O. I. examined, a severe lysyl oxidase deficiency could account for the observed ultrastructural abnormalities of elastin and that, besides defects of collagen type I, additional alterations of cellular metabolism might be responsible for the clinical heterogeneity of the diesease.

An antiserum to rabbit bone stromelysin (proteoglycanase) was raised in sheep and characterized as specific, recognizing the enzyme from both different tissue sources and different species. This antiserum and a specific antiserum to rabbit bone collagenase were used in the study of metalloproteinase production by rabbit articular chondrocytes stimulated with either interleukin 1 or mononuclear cell-conditioned medium. It was shown by electroimmunoblotting that the apparently co-ordinate (mole:mole) induction of collagenase and stromelysin activity with time correlated in either case with an increase in enzyme protein. The stimulated production of both enzymes could be modified in parallel by a variety of compounds. Immunohistochemical studies confirmed that although most cells were producing both metalloproteinases simultaneously, some chondrocytes produced detectable levels of only one. The data are discussed in relation to the mechanisms of breakdown in connective tissues.

The localization of collagenase inhibitor in bovine dentin was investigated by immunohistochemistry and immunoelectron microscopy using a specific rabbit anitserum raised against the inhibitor purified from culture medium of bovine unerupted third molar pulps. Immunoreactive collagenase inhibitor was mainly localized as an amorphous or granular accumulation along the wall of dentinal tubules in. decalcified bovine dentin. A part of the immunoreactive inhibitor appeared as two periodic bands on the collagen fibrils surrounding the dentinal tubules. Those two, a mayor and a minor band, corresponded to D-periodic fibril bands V-VII and I, respectively. As those two bands are located on both borders between overlap and hole zones of the collagen fibril, C- and N-terminal non-helical peptides and eight regions which are nD (n = 1-4) apart from each non-helical peptide along the triple helix are all equally possible binding sites of the collagenase inhibitor on the collagen molecule. It is interesting that the major band is located not exactly at, but fairly close to, the locus of collagenolytic cleavage, which lies between D-periodic fibril bands IV and V.