Zanna, M.J. Docampo, D. Fondevila, A.R.R. Carvalho, S. Cerrato, A. Bassols, L. Ferrer
This work has been accepted as free communication at 24th Annual Congress of the ESVD-ECVD, Florence 2010.
Abstract
Shar peis are affected by hereditary cutaneous hyaluronosis (HCH), a condition caused by an increased synthesis of hyaluronic acid (HA) by dermal fibroblasts secondary to high HA synthase 2 enzymatic activity. In this study, different microscopic techniques were used to better characterize at cellular level the process of HA synthesis in cultured dermal fibroblasts from shar peis with HCH and from control dogs. By optical microscopy, the actively synthesizing fibroblasts from shar peis adopted a rounded form different from elongated fibroblasts from control animals. By fluorescence microscopy, HA accumulated in diseased fibroblasts, not only extracellularly in a tangled network, but also intracellularly in dense particles at the edge of the cells and in a diffuse pattern. In fibroblasts from control dogs, HA fluorescence was less intense both extracellularly and intracellularly. This fluorescence disappeared after 70 min of digestion of living cells with hyaluronidase, as demonstrated by time-lapse microscopy. By transmission electron microscopy, fibroblasts from shar peis revealed a high number of intracellular dense vesicles corresponding to lysosomes, whereas in control dogs few lysosomes were detected. By scanning electron microscopy, numerous slender cell membrane protrusions, resulting from ongoing HA synthesis, were demonstrated in shar peis, whereas in control dogs these microvilli were less diffusely distributed. In shar peis, these observations were indicative of cells with a highly active HA metabolism. Altogether, these findings further confirmed the key role of dermal fibroblasts and HA in the pathogenesis of HCH, and they demonstrated that shar pei dermal fibroblasts constitute a useful model to investigate HA synthesis and metabolism.