Resumen de Pathogenetic studies on hereditary cutaneous mucinosis of shar pei dogs
Despite cutaneous mucinosis of shar pei could be considered a valuable and interesting biological and medical model for understanding human disorders and for providing new insights into the normal and abnormal control of hyaluronic acid (HA) metabolism, no data regarding the mechanisms involved are available.
Hence, the objectives of these thesis were to study the cellular and molecular pathomecanisms underlying the hereditary primary cutaneous mucinosis of shar pei and to assess an objective method to define the severity of the disease.
In this thesis, mucinous material was better defined histologically and histochemically. With haematoxylin and eosin dermal collagen fibers were demonstrated to be separated by a pink-grey substance that accumulated especially in the upper dermis and in most severe cases it led to the formation of lakes or superficial dermal vesicles. By Alcian Blue at pH 2.5 plus PAS, collagen fibers appeared scattered between a network of basophilic material that bound strongly by the hyaluronan-binding protein as demonstrated histochemically. These results showed that cutaneous mucinosis is the consequence of an abnormal HA deposition and therefore the term hereditary cutaneous hyaluronosis (HCH) could be useful to better define this entity.
Furthermore, in order to find a correlation between HA accumulation and the expression of its main receptor CD44, an immunohistochemical study was also performed. CD44 was showed to be expressed in the skin especially in keratinocytes of basal and spinous layers, but no correlation was established between the degree of dermal mucin deposition and CD44 expression. Therefore, abnormalities in CD44 expression are not to be in the origin of or associated with the cutaneous accumulation of HA in the skin of shar pei dogs affected by HCH.
The study was then focused on one of the routes in the HA metabolism. Such as demonstrated by an ELISA-like assay, shar pei dogs affected by HCH had high levels of HA in the serum, very probably consequence of the lymphatic drainage of HA produced in the skin.
Next studies were performed to understand the pathogenesis of this cutaneous entity. An in vitro model consisting of primary cultures of dermal fibroblasts was established. An ultrastructural study focused on the morphology of fibroblast cells was first conducted by confocal laser microscopy. Fibroblasts of shar pei showed to actively synthesize and secrete HA in comparison with fibroblasts of control dogs. Indeed, by using a biotinylated hyaluronan binding protein, a green fluorescence corresponding to HA was detected both intracellularly and extracellularly.
Furthermore, in order to understand if a defect of HA synthesis or catabolism, or both, could be the origin of HA dermal accumulation, the enzymes involved in HA metabolism were investigated by RT-PCR directly from the primary fibroblast cultures. Fibroblasts from shar pei with HCH showed to over-express HAS2, the main HA synthesizing enzyme, but no increased transcription of HYAL1 and HYAL2, the enzymes involved in HA degradation, was detected. Therefore, a dysregulation of the expression of HAS2 can be considered the origin of HCH in shar pei dogs.
To further confirm the key role of dermal fibroblasts and HA in the pathogenesis of HCH, a morphological study of fibroblast cells was performed using different microscopic techniques. By optical microscopy, the actively synthesizing fibroblasts from shar peis adopted a rounded form differently from the elongated form of fibroblasts from controls. By time lapse on living cells, HA fluorescence disappeared after 70 minutes of digestion with hyaluronidase. By transmission electron microscopy, fibroblasts from shar pei revealed a high number of intracellular dense vesicles corresponding to lysosomes whereas in controls few lysosomal vesicles were detected. By scanning electron microscopy numerous slender cell membrane protrusions, resulting from ongoing HA synthesis, were demonstrated in shar pei whereas in controls these microvillous showed to be less diffusely distributed. These findings demonstrated that shar pei dermal fibroblasts constitute a unique and useful model to investigate hyaluronic acid synthesis and metabolism.
Finally, in absence of an objective method to define the degree of HCH, an ultrasonographic study together with the measurement of skin folds by plicometer instrument was conducted and results were statistically related with the age and the gender of selected animals. Moreover, in order to demonstrate if ultrasound could be a valid alternative to other more invasive methods of investigation as histology, results were correlated with histometric measurements and histological results. Shar pei skin thickness was showed to be higher than controls and a clear correspondence was demonstrated between ultrasound results and histometric analysis. On the contrary, no correlation was found between ultrasound results and biophysical parameters. In conclusion, ultrasonography can be considered a useful and accurate method to assess the degree of HCH in shar pei dogs.
