Lawrence B. Sandberg

Elastin Structure, Biosynthesis, and Relation to Disease States

Elastin, a component of connective tissue, is present in virtually every organ of the body. Its role is unique and undoubtedly important, even though it may make up only a small percentage of a tis...

Quantitation of elastin production in cultured vascular smooth muscle cells by a sensitive and specific enzyme-linked immunoassay.

An enzyme-linked immunoassay (ELISA) procedure has been developed to quantitate the amount of elastin produced by cultured porcine aortic smooth muscle cells. ELISA was used to determine both titer and specificity of antisera raised in rabbits against porcine aortic alpha-elastin conjugated with key-hole limpet hemocyanin. Under optimum conditions (1: 3000 dilution of antiserum, 20 ng alpha-elastin per assay well), sensitivity averaged 60 ng/ml). Specificity was confirmed by immunoprecipitation of [125I]-tropoelastin, radial immunodiffusion, Western blotting and lack of cross-reactivity with serum proteins or collagen. Extensive cross-reactivity was found with both human alpha-elastin and porcine beta-elastin, while porcine tropoelastin was able to compete with 80% of the alpha-elastin determinants. Affinity of anti-porcine antisera for sheep alpha- elastin was significantly lower. When the ELISA was made specific for tropoelastin by coating wells with 60 ng of this antigen, a time-dependent and serum-dependent rate of production of tropoelastin was observed in the culture medium of primary and secondary cultures of smooth muscle cells. Comparison of elastin production in cultures of porcine smooth muscle cells suggests that porcine aortic elastin production varies as a function of cell density and phase of growth.

The Antigenicity of Soluble Porcine Elasjins: I. Measurement of Antibody by A Radioevemunoassay

Immunization of rabbits with either porcine tropoelastin, lung alpha-elastin, or aortic alpha-elastin resulted in the production of antibodies against the respective antigens. The assay of antibody activity with a radioimmunoassay indicated a high degree of cross reactivity between these three soluble elastin preparations. Through the use of competitive protein binding experiments it was possible to detect antigen-specific differences between tropoelastin and the two alpha-elastins. No antigenic differences were observed between lung or aortic alpha-elastin by any of the assay procedures uded in this investigation. Absorption of the various antisera with either insolubilized tropoelastin, lung elastin or aortic elastin allowed the preparation of non-crossreactive antibodies and provided further evidence for the antigenic differences between tropoelastin and the soluble alpha-elastins. The implications of the findings, as they relate to our current understanding of the molecular and structural arrangement of elastin and the elastin precursor, are discussed.

[10] Isolation and characterization of insoluble and soluble elastins☆

Publisher Summary This chapter discusses isolation and characterization of insoluble and soluble elastins. Elastin demonstrates remarkable functionality, correlating with macroscopic and microscopic structure and with molecular composition. Its ubiquitousness necessitates our understanding of these relationships, because tissue-specific extraction methods are necessary for its purification. Lack of agreement on criteria of purity stems from its complex supramolecular organization and intricate relationships with other macromolecules primarily of connective tissues. Purity is assessed by absence of collagen and collagen-like features (methionine, hydroxylysine, high hydroxyproline), absence of carbohydrate, presence of high content of non polar amino acids (glycine, alanine, proline, valine), and low content of polar amino acids (aspartate, glutamate, lysine, histidine, arginine).this chapter also provides assessment of results of various major methods. Immunologic methods such as radio immunoprecipitation and rocket immunoelectrophoresis may prove to be at least as sensitive in determining the amount of material present. However, because of the insoluble nature of mature elastin, many of these methods are not useful unless elastin is present in a degraded soluble form. Cross-link analysis may also be helpful for quantitation if hydrolyzed samples can be used.

In vitro studies of elastin metabolism.

Neonatal pig and rat aortas were studied for their ability to synthesize elastin in an in vitro situation. Smooth muscle cells from the rat aorta produced excellent multilayered cultures and produced soluble elastin (tropoelastin), insoluble elastin, and small amounts of collagen. BAPN proved to be toxic to these cells, adversely affecting the level of extracellular protein production. Tissue minces from pig aorta continued to synthesize elastin for two hours after removal. However, a 24 hour study indicated that elastin synthesis had almost completely shut down and that collagen synthesis continued in an apparently normal fashion. It is concluded that in vitro elastin synthesis is an extremely sensitive process easily altered by culture conditions and the addition of extraneous substances such as BAPN, and also highly influenced by the past history of the smooth muscle cells involved.

General Discussion on Turnover and Elastolysis

Dr. Yu: I would like to ask Dr. Robert and Dr. Hornebeck to clarify their studies of human aorta which apparently breaks down continuously. I hope this only occurs in the diseased aorta. Do you think a normal full grown animal aorta displays this kind of turnover through the action of elastolytic enzymes?

Lysyl Oxidase Activity in Lungs of Copper-Deficient Hamsters

Lysyl oxidase, the enzyme responsible for mediating crosslink formation in collagen and elastin, requires copper for its activity. In this study, lysyl oxidase activity and insoluble elastin content were unchanged in lungs from copper-deficient hamsters compared to controls. The lack of dramatic diminution in lysyl oxidase activity in animals who demonstrate significant structural alterations in the lung suggests that other mechanisms in addition to inhibition of crosslink formation are operative in this model.

General Discussion on Morphology of Elastin and Elastic Tissue

Dr. Ross: I would like to make a statement about the work that Dr. Cotta-Pereira showed because I am guilty of having used the term histoalchemy for oxytalan and elaunin staining procedures. As a matter of fact, I even used it in a paper we wrote several years ago. In a sense, it is histoalchemy, but I think what his work has shown, confirmed in part by Dr. Keith’s presentation, is that these stains now may have a very useful purpose even though they don’t tell us anything about the chemistry of the fibers. The concordance that Dr. Cotta-Pereira beautifully showed between oxytalan, those microfibrils with a small percentage of elastin; and the elastic fiber, principally elastin; permit us to use these relatively simple approaches at the light microscope level, to get some notion as to what’s residing where, since he’s now shown this in a number of different systems. And I think that represents an important contribution in the sense that it now will permit us to use these stains and go back and look at a number of tissues with some sense of confidence about what’s there. It doesn’t answer the question, obviously, why the stains work the way they do; but it does say something about what’s there. It also poses a number of fascinating questions. His observations in the skin suggest that this may be at least part of the reason for dermal-epidermal adherence, since he would suggest that these bundles of microfibrils which stain for oxytalan run from a series of connections in the deep dermis via elastic fibers and those containing small amounts of elastin, right up to the basal lamina. People have been looking for these modes of connection and this, in fact, may provide such connective modes. So I think these have opened a series of observations that will permit us to ask a number of questions that we’ve not really been able to ask, simply because we haven’t been thinking in those terms before.

The Isolation and Amino Acid Sequence of Some Thrombin Produced Porcine Tropoelastin Peptides

The amino acid sequence of porcine aortic tropoelastin has been studied extensively by Foster, et al. (1). They used trypsin cleavage to determine about 55% of the total sequence. It has been very difficult to obtain the remaining sequence or to place the peptides in order, both because of the nature of the cleavage and the hydrophobicity of the peptides.