HUMAN CORNEAL FIBRILLOGENESIS - COLLAGEN V STRUCTURAL-ANALYSIS AND FIBRILLAR ASSEMBLY BY STROMAL FIBROBLASTS IN CULTURE

Purpose. The stroma of the developing cornea is a highly organized ext racellular matrix formed essentially by uniform, small-diameter collag en fibrils with constant interfibrillar spacing. Unlike the fibrilloge nesis of chicken cornea, the assembly and maturation of human corneal fibrils have been poorly investigated. In the current study, the autho rs aimed to ascertain the heterotypic organization (collagens I and V) of the human corneal fibrils at the supramolecular level. To gain mor e insight into the molecular structure of collagen V, its cellular sou rce, and its role in fibrillogenesis, the authors used cultured human corneal fibroblasts. Methods. The structure of human corneal stroma af ter brief homogenization of the tissue was analyzed by immunogold labe ling using specific polyclonal antibodies and rotary shadowing. Bioche mical, electron microscopic, and immunolabeling approaches were used t o investigate the collagen fibril formation and the extracellular matr ix synthesis using human corneal fibroblasts grown in culture as a mod el system. Results, The authors showed that in human corneal stroma, c ollagen I is distributed uniformly along the striated fibrils, in cont rast to collagen V, which could be identified only at sites at which t he fibrils partially were disrupted. Rotary shadowing observations of the homogenate revealed that collagen VI, a major component of the hum an cornea, was associated closely with the collagen fibril surface. Co rneal fibroblasts synthesize and deposit a collagenous matrix with fib rils resembling those of the human cornea in appearance and collagen c omposition. Biochemical data indicate that a high concentration (20% t o 30%) of collagen V is synthesized by stromal fibroblasts and that co llagen V molecules are processed similarly to matrix forms in which th e extension peptides are retained on the molecules. Conclusions, The h eterotypic nature (collagens I and V) of human corneal fibrils was det ermined. Results indicate that human corneal fibroblasts synthesize th e major collagen types in human cornea (collagens I, V, and VI) and ex press all the posttranslational equipment for correct collagen molecul ar assembly and processing in a manner that closely resembles the situ ation in situ, offering the opportunity for more detailed study of thi s process, which is essential for optical transparency.