Background Glycosaminoglycans (GAGs) are polysaccharide chains that contribute to the formation of a mesh-like layer on the surface of the endothelium: the glycocalyx. The integrity of this layer is vital for endothelial cell homeostasis and its disruption leads to endothelial dysfunction. Endothelial damage associated with certain systematic vasculitides has recently been linked to autosomal recessive loss-of-function mutations in the ADA2 gene. Direct effects of the ADA2 protein on the endothelium are unclear, although ADA2 is known to have a GAG binding site. We isolated blood outgrowth endothelial cells (BOECs) in order to study the repair potential of ADA2 on the endothelial glycocalyx, in healthy volunteers. Methods BOECs were isolated and cultured from healthy donors. Firstly, peripheral blood mononuclear cells (PBMCs) were extracted from whole blood, cultured in EBM-2 media on collagen-coated plates, for 7-21 days, until colonies formed with characteristic cobbled-shaped morphology. BOECs were confirmed, using flow cytometry, to have classical endothelial cell surface marker expression (CD31, CD144, low CD34). BOECs (n=3) were cultured on chamber slides for 2-3 days until confluent and then incubated with GAG degradative enzymes for 2 hours; confirmed by staining. Cells were then treated with either ADA2 (10 U/L) or untreated and allowed to recover for a further 18 hours. The samples were then fixed, blocked and stained with primary antibodies specific for the GAGs; Heparan sulfate (HS) and Chrondroitin sulfate (CS). The slides were stained with FITC secondary antibodies and Hoechst nuclear stain. Samples were analysed by confocal microscopy, and data acquired over 3 random fields was evaluated using Image J software. Results 100% of BOECs expressed both HS and CS, with GAG coverage over the entire cell. Following enzymatic treatment, HS and CS expression was removed. Incubation with ADA2 for 18 hours restored the original GAG coverage. Over this time, untreated BOECs (media alone), expressed significantly less GAGs than with ADA2 (p<0.001) and were restored to only 52% (+/- 3.6% SEM) of their initial coverage. It took a further 18 hours before untreated BOECs recovered their full glycocalyx expression. Conclusions Glycocalyx removal precedes endothelial damage. This is the first time that the promotion of glycocalyx and GAG repair by an exogenous enzyme, ADA2, has been reported. ADA2 accelerated BOEC glycocalyx recovery following GAG degradation and BOECs widely expressed the two most commonly expressed GAGs. This data provides a suggested mechanism by which ADA2 protects against endothelial damage. Therefore, in ADA2-deficient patients, this may contribute to the endothelial damage and ensuing inflammation and vasculitis that is observed.