Phenotypic comparison of tendon, corneal and skin fibroblast populations.

Abstract

Fibroblasts are connective tissue cells that are responsible for the synthesis and turnover of extracellular components, including collagens, proteoglycans and glycosaminoglycans, and adhesive proteins such as fibronectin or laminin. The ability of fibroblasts to regulate their extracellular environment is largely dependent upon the action of matrix metalloproteinases (MMPs), key proteolytic enzymes synthesised and secreted by fibroblasts. Fibroblasts regulate the synthesis of their surrounding extracellular matrix (ECM), which in turn has a profound effect on how cues are presented to and perceived by the cells themselves. Fibroblasts are capable of synthesising diverse connective tissues and exhibit differences in growth characteristics, metabolism and morphology based upon their tissue of localisation. This thesis investigates the hypothesis that fibroblasts isolated from diverse connective tissues are phenotypically distinct. To test this, primary skin, comeal and tendon fibroblasts were subjected to shear stress produced by a parallel plate flow chamber. Prior to stimulation, the three fibroblast cell lines maintained discrete morphological differences based upon their tissue of origin. Upon stimulation, skin fibroblasts exhibited a larger cell area and all cell lines became increasingly rounded. Furthermore, comeal and skin fibroblasts demonstrated an increased number of focal adhesions per cell, while tendon fibroblasts exhibited a decrease in the number of focal adhesions with stimulation. All three cell lines demonstrated an increase in gelatinase (MMP-2 and MMP-9) activity, though each maintained cell line-specific regulation of gelatinase activity. Microarray analysis, validated by semi-quantitative RT-PCR and Western blotting, indicated that each cell line maintained unique, tissue-specific transcriptional and translational responses. Genes involved in these differential responses were functionally diverse and shown to be both up- and down-regulated with stimulation. Furthermore, levels of encoded proteins from four genes of interest - lumican, dyxin, Crpl and neogenin - altered with stimulation, though their expression did not correlate with mRNA levels in all cases. The data presented here provide unequivocal evidence that tendon, comeal and skin fibroblasts are morphologically and phenotypically distinct. Furthermore, this investigation provides an invaluable resource for further study of the factors that control fibroblast heterogeneity and may provide avenues for the manipulation and improvement of tissue engineered prostheses and implants for reconstructive surgery.