Synthesis of crosslinked elastin by a mesothelial cell culture.

Synthesis of crosslinked elastin was measured in cultures of pleural mesothelial cells. Results indicate that mesothelial cells are a rich source of crosslinked elastin and may therefore be useful for in vitro studies of this connective tissue component.

Lysyl oxidase activity and synthesis of desmosines in cultured human aortic cells and skin fibroblasts: comparison of cell lines from control subjects and patients with the Marfan syndrome or other annulo-aortic ectasia.

The activity of lysyl oxidase, the cross-linking enzyme of elastin and collagen, was measured in culture media of human skin fibroblasts, human aortic medial smooth muscle cells (SMCs) and adventitial fibroblasts using [3H]lysine-labelled elastin substrate. In addition, biosynthesis of isodesmosine and desmosine, the cross-linking amino acids of elastin, was studied by metabolic labelling with [14C]lysine and subsequent amino acid chromatography of protein hydrolysates. Lysyl oxidase activity in culture media of skin fibroblasts and aortic smooth muscle cells increased with the growth of the cell population and was at the highest level in cultures of high cell density. Lysyl oxidase activity in the aortic cell cultures was about three times that of skin fibroblasts. Aortic smooth muscle cells synthesized at least 100 times more desmosines than skin or adventitial fibroblasts. No differences were observed in lysyl oxidase activity and synthesis of desmosines between aortic smooth muscle cells or skin fibroblasts from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and the corresponding controls.

Impairment of elastin resynthesis in the lungs of hamsters with experimental emphysema induced by sequential administration of elastase and trypsin.

The nonelastolytic proteases trypsin and chymotrypsin were administered to hamsters 24 hours after intratracheal injection of elastase. Severity of the disease, extent of degradation and resynthesis, new cross-link formation, and the levels of the enzyme lysyl oxidase, which mediates the cross-link formation, were compared with the same parameters measured in hamsters with experimental emphysema induced by elastase alone. Increases in mean linear intercept indicated that a more severe form of the disease was produced. Although elastin degradation after 1 week was similar in both groups, resynthesis of the elastin destroyed by the elastolytic insult was significantly impaired in the animals injected sequentially with elastase and trypsin or chymotrypsin. Formation of new elastin as monitored by 14C-lysine incorporation into the elastin specific cross-links desmosine and isodesmosine was reduced approximately 40%, although there was no significant difference in the levels of lysyl oxidase activity. It is suggested that the most likely mechanism compatible with the recorded observations involves destruction of the microfibrillar component of the elastic fiber by trypsin or chymotrypsin, resulting in the absence of the requisite template for resynthesis of the pulmonary elastin.

Cigarette smoke blocks cross-linking of elastin in vitro.

Water-soluble components of the gas phase of filtered cigarette smoke inhibit formation of covalent desmosine cross-links during conversion of tropoelastin to elastin in vitro. These same smoke components also suppress lysyl-oxidase-catalyzed oxidation of lysine epsilon-amino groups in tropoelastin (the chemical step preceding formation of all elastin cross-links, including desmosine) in a dose-dependent fashion. However, gas phase smoke does not block the oxidation of diaminopentane by lysyl oxidase. Thus, gas phase cigarette smoke may possess substrate-directed (rather than enzyme-directed) inhibitory components capable of interfering with elastin cross-linking in vitro. Similar effects occurring in smokers' lungs could impede elastin repair and contribute to the development of pulmonary emphysema.

Effects of extracellular matrix upon elastogenesis.

Bovine fetal ligamentum nuchae fibroblasts were cultured on tissue culture plastic or directly on killed ligament tissue. Soluble elastin production and formation of insoluble elastin where monitored by radioimmunoassay and incorporation of [14C] lysine into desmosine, respectively. Cells grown on tissue culture plastic synthesized elastin precursors but were unable to form insoluble elastin whereas cells grown on dead ligament not only formed insoluble elastin, but showed an increase in levels of immunoreactive soluble elastin in culture medium. Stimulation of elastin synthesis by a soluble factor originating in ligament tissue is unlikely since medium conditioned by viable or nonviable ligament tissue had no effect on ligament cells cultured on plastic. Thus, direct cell-matrix contact appears to be important in mediating elastin biosynthesis.

Elastin cross-linking in vitro. Studies on factors influencing the formation of desmosines by lysyl oxidase action on tropoelastin.

The formation of isodesmosine and desmosine in vitro by the action of lysyl oxidase on tropoelastin was studied. The synthesis of desmosines occurred in the absence of additional substances. The formation of desmosines was not affected by removal of molecular O2 from the reaction medium nor was it affected by the lack of proline hydroxylation in tropoelastin. However, there was virtually no desmosine formation at 15 degrees C, a temperature not conducive to coacervation, indicating that coacervation is an important prerequisite for cross-linking.

The smooth muscle cell. III. Elastin synthesis in arterial smooth muscle cell culture.

Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.

Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in vitro.

Direct evidence showing that a soluble form of elastin is the precursor of cross-linked elastin was obtained from pulse-chase experiments using chick embryo aortas and by demonstrating the conversion of soluble elastin into cross-linked elastin in a cell-free system. Acetic acid extracts of embryonic chick aorta pulse-labeled with [14C]lysine contain two radioactive proteins of molecular weights 74,000 and 138,000 which have been identified previously as soluble elastin and the pro-alpha chain of collagen, respectively. In pulse-chase experiments, the radioactivity incorporated in the soluble elastin during the pulse with [14C]lysine disappeared during a 24-hour chase with [12C]lysine and 89% of that which disappeared was accounted for in the desmosines of alkali-insoluble elastin. The disappearance of the radioactivity from the soluble fraction and its appearance in the desmosines of elastin were inhibited by beta-aminopropionitrile, a specific inhibitor of the cross-linking enzyme lysyl oxidase. In addition in vitro experiments, it was shown that the radioactivity in the desmosines of elastin can arise from that present in an acid-soluble precursor protein. This precursor protein is soluble elastin, as demonstrated by the formation of desmosines when a homogeneous preparation of soluble elastin was incubated with purified lysyl oxidase.