Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro.

Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.

Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization.

In situ hybridization was used to determine the morphologic distribution of tropoelastin and alpha 1(I) procollagen mRNA expression in elastic intralobar arteries from neonatal calves with hypoxic pulmonary hypertension induced by a 15-day exposure to a simulated altitude of 1500 m. In vessels from normotensive control animals, low levels of hybridizable tropoelastin mRNA were detected in smooth muscle cells (SMC) of the inner media associated with large elastic lamellae. Compared to control arteries, vessels from hypertensive animals demonstrated a markedly different pattern of hybridization. In these arteries, strong hybridization signals for tropoelastin mRNA were seen in SMC lying between the elastic lamellae of the outer media, and the density of labeling associated with these medial cells decreased progressively toward the lumen. Endothelial and adventitial cells in both control and hypertensive arteries were negative for tropoelastin mRNA. Type I procollagen mRNA was dispersed through the media of control arteries, and in hypertensive calves, the hybridization signal was more intense and was unevenly distributed through the media similarly to that for tropoelastin mRNA. Adventitial cells were strongly positive for procollagen mRNA, and the signal was equally intense for both control and hypertensive arteries. Cells that had no detectable tropoelastin mRNA were noted in the outer media of both control and hypertensive vessels. These cells occurred as broad circumferential bands in the normotensive artery and as nodular foci in the hypertensive artery. Immunocytochemical studies with antibodies to smooth muscle specific actin, desmin, and vimentin demonstrated that cells within these foci, as well as tropoelastin mRNA-positive cells, were SMC. These studies demonstrate that expression of tropoelastin and procollagen mRNA was differentially stimulated by pulmonary hypertension within specific regions and SMC populations of the vascular wall.

Microfibrillar protein from elastic tissue: a critical evaluation.

Many workers have claimed to have isolated proteins which have been derived from the microfibrillar components of elastic tissue. Virtually all of these preparations have been derived from extracts made with strong solutions of guanidinium chloride (GuHCl) under reducing conditions following Ross and Bornstein (1969). The products have ranged from heterogeneous mixtures of proteins to discrete glycoproteins. In no case has identity between an individual protein and the elastin-associated microfibrils been confirmed by immunoelectron microscopy. We have undertaken a detailed re-examination of the extractability of elastin-associated microfibrils and of the composition of the extracts from foetal bovine nuchal ligament. Finely homogenized samples were subjected to a series of extractions (including cyclical treatments with GuHCl and purified bacterial collagenase) in the presence of inhibitors of protease activity. Under these conditions it has been shown that--(i) microfibrils were removed progressively by GuHCl, throughout the extraction schedule, without the need for reduction; (ii) all remaining microfibrils were removed by reductive GuHCl extraction; (iii) the product from this reductive extraction consisted of a heterogeneous mixture of proteins including several glycoproteins; (iv) a major antigenic constituent of the mixture of proteins localized to elastin-associated microfibrils, as shown by immunoelectron microscopy. It is concluded that, while reductive GuHCl extracts do contain components with antigenic activity that is localized on elastin-associated microfibrils, they have many non-microfibrillar components. We stress that claims that a macromolecule is microfibrillar must be substantiated by immunoelectron microscopy.