Localization and Function of Sulfated Proteoglycans in the Equine Digital Laminae

Abstract

s Vol 30, No 2 (2010) 99 TAKE HOME MESSAGE Genetic and biochemical evidence for large polysulfated proteoglycans in lamellae, but not liver and lung tissue, show that lamellae are functionally specialized to resist compressive forces. INTRODUCTION Our published and on-going studies show ADAMTS-4 (aggrecanase) gene and protein expression is elevated in the lamellae of laminitic horses. ADAMTS-4 cleaves aggrecan and versican, the core proteins of large polysulfated proteoglycans that in complex with hyaluronan form hydrated gels and provide resistance to compressive forces. Here we investigate ADAMTS-4 substrates in lamellae. MATERIALS & METHODS cDNA-specific probes and relative quantitative real time PCR were used to detect the expression levels of genes encoding polysulfated proteoglycans. Ct values were analyzed using the DDCt method. Protein and glycosaminoglycan moieties in lamellar extracts were detected after SDSPAGE electrophoresis by Western blotting. RESULTS Genes encoding aggrecan, versican, chondroitin synthase and hyaluronan synthase were found to be consitutively expressed in normal digital lamellae but not, or to a much lesser extent in lungs and liver. Aggrecan core protein was detected in detergent (0.5%NP-40) lysates of lamellae, whereas keratanand chondroitinsulfate aggrecan proteoglycans were detected in guanidine hydrochloride extracts of lamellar proteoglycans. DISCUSSION AND CLINICAL RELEVANCE Gene expression and product analyses indicate that the equine digital lamellae are functionally specialized to resist compressive forces. Consequently, degradation of this material by elevated ADAMTS-4 may compromise lamellar function. CONCLUSION The presence of large polysulfated proteoglycans and elevated ADAMTS-4 in laminitic tissue suggest parallels between the pathophysiology of laminitis and osteoarthritis. Localization and Function of Sulfated Proteoglycans in the Equine Digital Laminae Erica Pawlak, Le Wang, Dominique Alfandari, Phillip J. Johnson, James K. Belknap, and Samuel J. Black, Department of Veterinary and Animal Science, University of Massachusetts Amherst, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri Columbia, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University TAKE HOME MESSAGE Polysulfatedproteoglycansof the lamellar extracellularmatrix (ECM) are similar to those of articular cartilage and hence may maintain hydration and resistance to compression. INTRODUCTION We have previously shown that ADAMTS-4 (aggrecanase), which is implicated in the pathophysiology of osteoarthritis through cleavage of large polysulfated proteoglycans, is elevated in experimentally induced and naturally acquired equine laminitis. To understand the role played by ADAMTS-4 in equine laminitis, it is necessary to localize substrates within the digital lamellae and to determine their contribution to lamellar structure and function. MATERIALS AND METHODS Aggrecan, chondroitin-6-sulfate, keratan sulfate, laminin, and hyaluran were detected via immunofluorescence in 10mm sections of lamellae. Specificity of antibodies was confirmed by Western blotting and by peptide competition. RESULTS Aggrecan and hyaluran appear to be largely concentrated in the secondary epidermal lamellae, while keratan sulfate and chondroitin sulfate display varying and unique distributions throughout the tissue. DISCUSSION Proteoglycan components of the lamellar ECM are similar to those of articular cartilage, consistent with the possibility that they protect the lamellae against general and localized compression resulting from weight bearing and motion-associated rotation of the third phalanx. CLINICAL RELEVANCE We propose that degradation of lamellar ECM large polysulfated proteoglycans by ADAMTS-4 reduces lamellar resistance to compression, thus exposing tensile elements of the ECM to excessive stretch and contributing to failure. CONCLUSION Lamellar proteoglycans are similar to those of articular cartilage and hence, likely to play a key role in maintaining lamellar integrity.