Hyaluronidase-4 is produced by mast cells and can cleave serglycin chondroitin sulfate chains into lower molecular weight forms.

Mast cells represent a heterogeneous cell population that is well-known for the production of heparin and the release of histamine upon activation. Serglycin is a proteoglycan that within mast cell α-granules is predominantly decorated with the glycosaminoglycans heparin or chondroitin sulfate (CS) and has a known role in granule homeostasis. Heparanase is a heparin-degrading enzyme, is present within the α-granules, and contributes to granule homeostasis, but an equivalent CS-degrading enzyme has not been reported previously. In this study, using several approaches, including epitope-specific antibodies, immunohistochemistry, and EM analyses, we demonstrate that human HMC-1 mast cells produce the CS-degrading enzymes hyaluronidase-1 (HYAL1) and HYAL4. We observed that treating the two model CS proteoglycans aggrecan and serglycin with HYAL1 and HYAL4 in vitro cleaves the CS chains into lower molecular weight forms with nonreducing end oligosaccharide structures similar to CS stub neoepitopes generated after digestion with the bacterial lyase chondroitinase ABC. We found that these structures are associated with both the CS linkage region and with structures more distal toward the nonreducing end of the CS chain. Furthermore, we noted that HYAL4 cleaves CS chains into lower molecular weight forms that range in length from tetra- to dodecasaccharides. These results provide first evidence that mast cells produce HYAL4 and that this enzyme may play a specific role in maintaining α-granule homeostasis in these cells by cleaving CS glycosaminoglycan chains attached to serglycin.

Perlecan and vascular endothelial growth factor-encoding DNA-loaded chitosan scaffolds promote angiogenesis and wound healing.

The repair of dermal wounds, particularly in the diabetic population, poses a significant healthcare burden. The impaired wound healing of diabetic wounds is attributed to low levels of endogenous growth factors, including vascular endothelial growth factor (VEGF), that normally stimulate multiple phases of wound healing. In this study, chitosan scaffolds were prepared via freeze drying and loaded with plasmid DNA encoding perlecan domain I and VEGF189 and analyzed in vivo for their ability to promote dermal wound healing. The plasmid DNA encoding perlecan domain I and VEGF189 loaded scaffolds promoted dermal wound healing in normal and diabetic rats. This treatment resulted in an increase in the number of blood vessels and sub-epithelial connective tissue matrix components within the wound beds compared to wounds treated with chitosan scaffolds containing control DNA or wounded controls. These results suggest that chitosan scaffolds containing plasmid DNA encoding VEGF189 and perlecan domain I have the potential to induce angiogenesis and wound healing.

The localisation of inflammatory cells and expression of associated proteoglycans in response to implanted chitosan.

Implantation of a foreign material almost certainly results in the formation of a fibrous capsule around the implant however, mechanistic events leading to its formation are largely unexplored. Mast cells are an inflammatory cell type known to play a role in the response to material implants, through the release of pro-inflammatory proteases and cytokines from their α-granules following activation. This study examined the in vivo and in vitro response of mast cells to chitosan, through detection of markers known to be produced by mast cells or involved with the inflammatory response. Mast cells, identified as Leder stained positive cells, were shown to be present in response to material implants. Additionally, the mast cell receptor, c-kit, along with collagen, serglycin, perlecan and chondroitin sulphate were detected within the fibrous capsules, where distribution varied between material implants. In conjunction, rat mast cells (RBL-2H3) were shown to be activated following exposure to chitosan as indicated by the release of ß-hexosaminidase. Proteoglycan and glycosaminoglycans produced by the cells showed similar expression and localisation when in contact with chitosan to when chemically activated. These data support the role that mast cells play in the inflammatory host response to chitosan implants, where mediators released from their α-granules impact on the formation of a fibrous capsule by supporting the production and organisation of collagen fibres.