Ascorbate-induced fibroblast cell matrix: reaction of antibodies to procollagen I and III and fibronectin in an axial periodic fashion.

Abstract

Fibronectin and procollagen types I and III are constituents of the extracellular matrix of human fibroblasts. Ultrastructural immunocytochemistry using the peroxidase anti-peroxidase method showed fibronectin and procollagen antibodies reacting in continuous fashion on 10 nm diameter extracellular fibrils on human fibroblasts. Intracellular localization showed an intense accumulation of procollagen within cells cultured under routine conditions. This accumulation appeared almost as if there were a blockade in secretion of procollagen under routine culture conditions. Cells treated with ascorbic acid do not have the dense intracellular accumulation of procollagens seen with the apparent blockade of secretion in cells cultured under routine conditions. Ascorbate treated cells also have a more pronounced extracellular accumulation of matrix fibronectin and procollagen constituents. At the electromicroscopic level a new 40 nm diameter fibril is formed after ascorbic acid treatment of human fibroblasts. Antibody to fibronectin and procollagen I and III are seen binding to the 40 nm diameter fibrils in a periodic or stuttered appearance. The fibronectin and procollagen antibodies react with a 70 nm axial repeat along these 40 nm fibrils formed after ascorbate treatment. These studies suggest that under routine culture conditions "precursor" fibrils of fibronectin and procollagen are formed. Ascorbic acid treatment leads to enhanced matrix formation. Ultrastructural studies clearly show antibodies to fibronectin bind to fibronectin on native collagen fibrils formed by human fibroblasts cultured with ascrobic acid. Lastly there is an asymmetric or 70 nm axial periodic distribution of fibronectin along these definitive or mature collagen fibrils formed after ascorbic acid treatment