Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber . Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin .When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth . Soluble elastin was not detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent . Also, soluble elastin production per cell declined with increasing population doubling or with age in culture .Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium . The addition of dexamethasone (0 .1 pM) and bleomycin (1 gg/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 ftg/ml) depressed production 50% and antagonized stimulation by dexamethasone . Ascorbate (50 l-tg/ml), soybean trypsin inhibitor (1 mg/ ml), insulin (100 pU/ml), and aminoacetonitrile (50 Ag/ml) had no effect, but cycloheximide at 10 -4 M completely inhibited soluble elastin production . In this report, we examine elastin synthesis by cells from bovine fetal ligamentum nuchae. This tissue, in large grazing mammals such as cattle and sheep, has the highest content of elastin of any tissue and the relative ease with which ligament elastin can be purified makes it the best tissue for studying the biochemical and physical properties ofelastin .

The effect of zinc deficiency on bone collagenase activity and collagen turnover was studied in the chick. Zinc deficiency symptoms, evident after 8 days on the low zinc diet, included tibia deformities and decreased alkaline phosphatase. Bone collagen metabolism was markedly altered, with a significant reduction in collagen synthesis and turnover. Half-turnover time for tibia collagen was 13 days in the control and 35 days in the zinc-deficient chicks. Tibia collagenase activity was reduced by 40-80% in the zinc-deficient as compared to the control chicks. Heparin markedly increased collagenase activity in the zinc-deficient tibias elevating activity to control levels. But commercially available heparin was found high in zinc content which may explain this effect entirely. These data show that zinc deficiency decreases bone collagen turnover and probably accounts for the leg deformities seen in zinc-deficient chicks.

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development . Undifferentiated (nonelastin-producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae . ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at -180 d of gestation) does not stimulate elastin production in undifferentiated cells . Matrixinduced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix . Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable . Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM . The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer . When grown on tissue culture plastic, ligament cells secrete >80% of newly synthesized tropoelastin into the culture medium . When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine .