THE SYNTHESIS OF ELASTIN, COLLAGEN, AND GLYCOSAMINOGLYCANS BY HIGH-DENSITY PRIMARY CULTURES OF NEONATAL RAT AORTIC SMOOTH-MUSCLE - AN ULTRASTRUCTURAL AND BIOCHEMICAL-STUDY

  • 1 January 1982
    • journal article
    • research article
    • Vol. 27  (1) , 34-46
Abstract
Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 .times. 106 cells/25 cm2 Falcon flask) with adequate nutrient media and pH control, grow rapidly and form multilayers of cells with typical hill and valley organization. After 10 days growth, insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become large irregular refractile aggregates and later coalesced to form larger aggregates and small fibers. With light microscopy and EM, elastin was the predominant matrix protein formed, with the hill regions of cultures containing abundant elastin aggregates and some collagen. In 2-wk-old cultures, differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesizing organelles and myofilaments and were in close association with large often coalescing elastin aggregates compared to younger more superficial cells which contained more free polyribosomes, less myofilaments, and were associated with fewer and smaller elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies and lysosomes. Elastin aggregates and newly formed elastic fibers were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-wk culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulfate (32%), chondroitin 4-sulfate (43%), keratan and heparan sulfate (30%), with only a trace of hyaluronic acid. Primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthetize and deposit large amounts (mg) of insoluble elastin which aggregate and form elastic fibers in vitro.

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