Summary
Cultured fibroblasts derived from normal subjects and juvenile diabetics attach in the absence of serum to plastic culture dishes and secrete macromolecules, including collagenous components, hyaluronic acid, and proteoglycans into the medium and onto the plastic surface where they form a microexudate carpet. Most diabetic fibroblasts examined did not spread as well as normal cells during a 4-hr interval after the initial attachment. There were no significant differences between normal and diabetic cells with respect to proline and lysine incorporation and lysine hydroxylation. The percentage glycosylation of hydroxylysine was marginally higher in the media proteins of diabetic cells, but glycosylation in both normal and diabetic cells was elevated over that typically observed in human skin collagen.
Collagenous components were estimated to constitute approximately 15–20% of the microexudate carpet fraction in both normal and diabetic cell strains. Diabetic fibroblasts exhibited a marginally lower ratio of heparan sulfate to chondroitin sulfate in the cell surface to matrix microexudate carpet fraction (trypsinate) than did normal fibroblasts. The hyaluronate and chondroitin sulfate contents of this fraction of diabetic cells were not significantly different from those of normal cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Spiro, R. G. 1976. Search for a biochemical basis of diabetic microangiopathy. Diabetologia 12: 1–14.
Beisswenger, P. J., and R. G. Spiro. 1970. Human glomerular basement membrane: chemical alteration indiabetes mellitus. Science 168: 596–598.
McMillian, D. E. 1975. Deterioration of the microcirculation in diabetes. Diabetes 24: 944–957.
Siperstein, M. D. 1970. The relationship of carbohydrate derangements to the microangiopathy of diabetes. In: E. Cerasi and R. Luft (Eds.), Pathogenesis ofdiabetes mellitus. Nobel Symposium, Vol. 13. Wiley, New York, pp. 81–102.
Raskin, P., J. F. Marks, H. Burns, M. E. Plumer, and M. D. Siperstein. 1975. Capillary basement membrane width in diabetic children. Am. J. Med. 58: 365–372.
Vracko, R., and E. P. Benditt. 1974. Manifestations ofdiabetes mellitus—their possible relationships to an underlying cell defect. Am. J. Pathol. 75: 204–221.
Vracko, R., and E. P. Benditt. 1975. Restricted replicative life-span of diabetic fibroblastsin vitro: its relation to microangiopathy. Fed. Proc. 34: 68–70.
Goldstein, S., J. W. Littlefield, and J. S. Soeldner. 1969.Diabetes mellitus and aging: diminished plating efficiency of cultured human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 64: 155–160.
Goldstein, S., E. J. Moerman, J. S. Soeldner, R. E. Gleason, and D. M. Barnett. 1978. Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic and normal donors. Science 199: 781–782.
Goldstein, S., S. Niewiarowski, and D. P. Singal. 1975. Pathological implications of cell agingin vitro. Fed. Proc. 34: 56–63.
Martin, G. M., C. A. Sprague, and C. J. Epstein. 1970. Replicative life-span of cultivated human cells: effects of donor's age, tissue and genotype. Lab. Invest. 23: 86–92.
Vracko, R. 1974. Basal lamina layering indiabetes mellitus. Evidence for accelerated rate of cell death and cell regeneration. Diabetes 23: 94–104.
Mellan, W. J., and V. J. Cristofalo. 1972. Human diploid cell cultures: their usefulness in the study of genetic variations in metabolism. In: G. H. Rothblat and V. J. Cristofalo (Eds.),Growth Nutrition and Metabolism of Cells in Culture. Academic Press, New York. Vol. 1, pp. 327–369.
Lembach, K. J., R. E. Branson, P. B. Hewgley, and L. W. Cunningham. 1977. The synthesis of macromolecular 3-hydroxyproline by attaching and confluent cultures of human fibroblasts. Eur. J. Biochem. 72: 379–383.
Schwartz, C. E., C. G. Hellerqvist, and L. W. Cunningham. 1978. A collagenous component of the microexudate carpet secreted by attaching human fibroblasts. Ann. N.Y. Acad. Sci. 312: 450–452.
Vaheri, A., K. Alitalo, K. Hedmon, J. Kesic-Oja, M. Kurkinen, and J. Wartiovaara. 1978. Fibronectin and the pericellular matrix of normal and transformed adherent cells. Ann. N.Y. Acad. Sci. 312: 343–353.
Kraemer, P. M. 1971. Heparan sulfates of cultured cells I. Membrane associated and cell-sap species in Chinese hamster cells. Biochemistry 10: 1437–1445.
Bornstein, P., and K. A. Piez. 1964. A biochemical study of human skin collagen and the relation between intra- and intermolecular cross-linking. J. Clin. Invest. 43: 1813–1823.
Schneider, E. L., E. J. Stanbridge, and C. J. Epstein. 1974. Incorporation of3H-uridine and3H-uracil into RNA. A simple technique for the detection of mycoplasma contamination of cultured cells. Exp. Cell Res. 84: 311–318.
Askenasi, R. S., and N. A. Kefalides. 1972. Simple chromatographic method for determination of14C-labeled lysine, hydroxylysine, and hydroxylysine glycosides. Anal. Biochem. 47: 67–72.
Lembach, K. J. 1976. Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor. J. Cell. Physiol. 89: 277–288.
Terry, A. H., and L. A. Culp. 1974. Substrate attached glycoproteins from normal and virus-transformed cells. Biochemistry 13: 414–425.
Saito, H., T. Yamagata, and S. Suzuki. 1968. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243: 1536–1542.
Bitter, T., and H. M. Muir. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4: 330–334.
King, J., and V. K. Laemmli. 1971. Polypeptides of the tail fibers of bacteriophage T4. J. Mol. Biol. 62: 465–477.
Maizel, J. V. 1971. Polyacrylamide gel electrophoresis of viral proteins. In: K. Maramorosch and H. Koprowski (Eds.), Methods in Virology. Vol. 5. Academic Press, New York, pp. 179–246.
Peterkofsky, B., and R. Diegelmann. 1971. Use of a mixture of proteinase-free collagenase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 10: 988–994.
Igarashi, Y., and Y. Yaoi. 1975. Growth-enhancing protein obtained from cell surface of cultured fibroblasts. Nature 254: 248–250.
Smith, B. D., P. H. Byers, and G. R. Martin. 1972. Production of procollagen by human fibroblasts in culture. Proc. Natl. Acad. Sci. U.S.A. 69: 3260–3262.
Church, R. L., and M. L. Tanzer. 1975. Isolation and amino acid composition of human procollagen [Pro α1(I)]2 Pro α2 from skin fibroblasts in culture. FEBS Letters 53: 105–109.
Miller, E. J. 1972. Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11: 4903–4909.
Kohn, R. R., and S. Hensse. 1977. Abnormal collagen in cultures of fibroblasts from human beings withdiabetes mellitus. Biochem. Biophys. Res. Commun. 76: 765–771.
Fessler, L. I., N. P. Morris, and J. H. Fessler. 1975. Procollagen: biological scission of amino acid carboxyl extension peptides. Proc. Natl. Acad. Sci. U.S.A. 72: 4905–4909.
Author information
Authors and Affiliations
Additional information
This work was supported by Grants AM 11821 and AM 19606 from the National Institutes of Health, United States Public Health Service, by a grant from the American Diabetes Association (Leon W. Cunningham, Principal Investigator) and by the Vanderbilt Diabetes Endocrinology Center grant, AM 17026. R. E. Branson was the recipient of a research fellowship of the Juvenile Diabetes Foundation and of a National Institutes of Health Postdoctoral Fellowship AM 05636.
Rights and permissions
About this article
Cite this article
Branson, R.E., Lembach, K.J. & Cunningham, L.W. Comparison of collagen and glycosaminoglycan synthesis in attaching control and diabetic human skin fibroblasts. In Vitro 16, 159–167 (1980). https://doi.org/10.1007/BF02831506
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02831506