The University of Georgia College of Veterinary Medicine

eorgia Veterinary Scholars Program

GVSP Summer 2008 Scholars

Georgia Veterinary Scholar	Faculty Mentor
	No photo available
Leah Patipa University of Georgia Class of 2010	Dr. Michel Vandenplas

Modulation of Hyaluronidase Activity in Equine Tissues and Synovial Fluid

Patipa, Leah*. Figueiredo, Monica; Salter, Caroline; Vandenplas, Michel Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia Athens, GA

Degradation of the extracellular matrix, caused by activation of enzymes such as proteases, occurs during certain debilitating equine diseases such as laminitis and osteoarthritis. There is currently very little information regarding degradation of complex sugars such as hyaluronan within the extracellular matrix. Changes in hyaluronidase activity can occur indirectly through altered gene expression or directly through regulatory mechanisms at the enzyme level. We have validated quantitative RT-PCR assays to detect changes in HYAL1 and HYAL2 gene expression together with that for CD44, the major cellular hyaluronan receptor. To monitor changes in hyaluronidase activity we are establishing Native and SDS-PAGE
zymography assays. SybrGreen RT-qPCR assays showed no change in HYAL1 and HYAL2 gene expression during the early phases of experimentally-induced laminitis. In contrast a modest increase in CD44 expression was observed in Obel grade I laminitis samples. This may make more hyaluronan available for degradation by cellular hyaluronidase. Native- PAGE zymography allows for detection of hyaluronidase activity but does not
allow for discrimination between hyaluronidase isoforms. SDS-PAGE zymography allows for size separation of hyaluronidase isoforms but the negatively charged detergent inhibits hyaluronidase activity by >95%. Using guanidine salt denaturation after removal of the SDS with isopropanol we are able to weakly reconstitute hyaluronidase activity after SDS-PAGE zymography, thus revealing the hyaluronidase isoforms. Currently biological samples (e.g. synovial fluid and membranes) are being collected from healthy horses and horses with osteoarthritis so that we can monitor changes in hyalurondiase activity.