Bioengineering Proteoglycan‐based Matrices For Blood Contacting Applications

Abstract

Proteoglycans and their glycosaminoglycan chains are functional components of the extracellular matrix that modulate interactions with other extracellular matrix molecules, as well as with cells and growth factors. Perlecan is an abundant proteoglycan in the vascular basement membrane and contains a C‐terminal domain V (DV) with one glycosaminoglycan attachment site and an α2β1 integrin binding site. Perlecan can only be obtained in low abundance from natural sources, thus alternative sources are needed for therapeutic applications. The aim of this research was to bioengineer DV and to incorporate it into biomaterials for blood‐contacting applications.Human DV was expressed in the human embryonic kidney cell line, HEK‐293 and purified by immunoaffinity chromatography and biochemically characterized with respect to protein core, glycosaminoglycan structure and yield. Human primary endothelial cell, smooth muscle cell and platelet adhesion were assessed.DV was produced as two populations decorated with either chondroitin sulfate or heparan sulfate. The level of heparan sulfate substitution was increased by culturing the cells with higher glucose concentrations in the medium while there was no effect on the level of chondroitin sulfate. DV was produced with a yield of 2 mg/L, which is a 2.5 fold increase in the yield of endothelial‐derived perlecan.The proteoglycan forms of DV were effective in promoting endothelial, but not smooth muscle cell or platelet adhesion. In the absence of glycosaminoglycans DV supported the adhesion of endothelial cells and platelets via integrin α2β1. However, while smooth muscle cells adhere to perlecan in the absence of glycosaminoglycan chains, these cells did not adhere to DV in the absence of glycosaminoglycans. This suggests that the proteoglycan forms of DV have potential roles in encouraging endothelialization of materials while inhibiting unwanted activities associated with platelet and smooth muscle cell adhesion. We have shown this to be the case for the full length perlecan when trialed in an ovine vascular graft model.We are currently developing methods to combine DV with various biomaterials including a silk fibroin substrate through various chemical crosslinking methods. Early work in this area suggests that we can fine tune how both the protein and glycosaminoglycan components of DV are presented to maintain the vascular cell‐selective activities of DV.Support or Funding InformationFunding provided by the Australian Research Council