Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell aging

Abstract

Acute local gamma-irradiation of porcine skin induces, as in human skin, extensive mutilating sclerosis, characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of extracellular matrix macromolecules. Collagen synthesis, content, and types were studied by measuring the incorporation of the radiolabeled precursor [3H]-proline into confluent primary cultures and subcultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. A significant increase in collagen biosynthesis and deposition, with a preferential enhancement of type III collagen, was observed in primary cultures of fibrotic fibroblasts when compared to those of normal dermis. However, the percentage (36%) of neosynthesized collagen in relation to the total neosynthesized non-collagenous and collagenous proteins remained unchanged. In subcultures of normal cells, collagen synthesis and content remained constant until about the 10th passage and then progressively declined until death of the fibroblasts, at around the 15th passage. During the senescence phase, normal fibroblasts acquired the capacity to synthesize higher levels of non-collagenous proteins. On the contrary, collagen synthesis and content diminished in subcultures of fibrosis-induced fibroblasts from the second passage, and then progressively declined as a function of aging; particularly the ratio of type III to type I collagen returned to normal values from the second passage. Moreover, these fibroblasts did not become senescent, persisted in synthesizing low levels of collagen, and acquired later (around the 40th passage) a higher capacity to synthesize non-collagenous proteins. These results demonstrate that primary cultures of porcine fibroblasts obtained from normal or radiation-induced fibrotic dermis reproduce the in vivo situation. The results strongly suggest that: 1) the modification of collagen synthesis, content, and types observed in primary cultures of fibroblasts might be due to factors causing long-lasting changes in phenotypic expression and/in stimulation of the expansion of some fibroblast clones engaged in the accelerated synthesis of extracellular matrix macromolecules, such as collagen or non-collagenous proteins. 2) the rapid decrease in this active capacity to synthesize and accumulate collagen, observed in subcultures of fibrosis-induced fibroblasts, might be related to the loss of some activation factors or to the dedifferentiation of the cells. 3) the switch from collagen to non-collagenous protein synthesis during later subcultures of fibroblasts obtained from radiation-induced fibrosis, might be due to molecular modification at the transcriptional or DNA level.