Hyaluronic acid cues for functional endothelialization of vascular constructs

Current vascular implant materials insufficiently recruit endothelial cells (ECs) to form a normally functional and confluent endothelium, a key challenge to reinstating vascular homeostasis at the surgical site. Recent studies indicate that hyaluronan (HA), a connective tissue GAG whose biological effects are often dictated by its fragment size, may inherently stimulate endothelialization. We previously showed that ECs respond poorly to large HA fragments (10 kDa < MW < 1 MDa), therefore, we currently sought to comprehensively study the effects of exogenous high molecular weight (>1000 kDa) and oligomeric (0.75-10 kDa) ranges on various phenotypic and functional aspects of cultured ECs. HA-1500 (1500 kDa) was enzymatically digested into oligomers under iteratively defined conditions, until a mixture (HA-o) containing a maximal yield of HA-6-mer and 12-mers (33.3 +/- 2.44% and 39.2 +/- 2.68% w/w, respectively) was obtained. The effects of HA-1500, HA-o and pure HA-6-mers on rat aortic ECs were compared. DNA and tube formation assays revealed HA-o and HA-6-mers to stimulate EC proliferation and EC tube formation (angiogenesis) much more than non-HA controls, while HA-1500 had a significant but more modest effect. Both HA-o and HA-6-mers attenuated platelet adhesion and activation on EC layers, while HA-1500 drastically inhibited the same relative to controls. However, flow cytometry and cytokine array studies found that HA-o incited increased expression levels of EC activation markers (ICAM-1, VCAM-1) and promoted the release of select inflammatory cytokines to a greater degree than HA-1500. These results suggest that HA-o and HA-1500 both provide benefits, although frequently of different kinds, to endothelial cell sustenance, proliferation and normal functionality. Thus, tissue engineering scaffolds containing both these cues in optimized ratios could potentially serve as excellent materials for vascular EC regeneration. This forms the basis of our ongoing studies.