Vascular Glycobiology of Oxidative Stress and Endothelial Dysfunction, a Potential Therapeutic Target

The integrity of endothelial glycocalyx is crucial for normal endothelial function and endothelial response to shear stress. Endothelial glycocalyx also tethers and concentrates the extracellular superoxide dismutase isoform 3 (SOD3) which protects the endothelium against the oxidative damage of reactive oxygen species (ROS). Degradation of endothelial glycocalyx results in endothelial dysfunction and vascular remodeling. The proteolytic enzymes Matrix Metalloproteinases (MMPs) and Disintegrin Metalloproteinases (ADAMs) are capable of disrupting endothelial cell surface proteins such as syndecans resulting in derangement of endothelial glycocalyx. We experimentally induced oxidative stress in human adipose microvascular endothelial cells (HAMECs) in the presence or absence of exogenous hydrogen peroxide (H2O2) and endogenous oxidative stress inducer buthionine sulfoximine (BSO) and MMP inhibitors. We found that either H2O2 or BSO, increased MMPs (MMP1, MMP2, and MMP9) and ADAMs (ADAM10 and ADAM17). Consequently, there was a reduction in protein levels of syndecan-1 and SOD3 in the total cell lysate and increases in levels of syndecan-1 ectodomain and SOD3 in cell culture media detected by immunoprecipitation and Western blotting. The upregulation of MMPs was accompanied by a decline in the mRNA and protein levels of their inhibitors, TIMPs. Interestingly, we found that the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) normalized MMP expression and restored TIMP 1 and TIMP 3 after H2O2 or BSO. Pharmacological inhibition of MMPs using marimastat and lisinopril mitigated the effect of oxidative stress on the glycocalyx. Using immunofluorescent labeled wheat germ agglutinin, we found that lisinopril and marimastat effectively restored the cell surface density of heparan sulfate glycosaminoglycans (HS-GAGs), syndecans and SOD3 on the endothelial cell surface. Lastly, we found that lisinopril decreased HDAC mRNA expression induced by H2O2 suggesting an epigenetic mechanism of action for lisinopril on endothelial responses to oxidative stress. In conclusion, our findings suggest that oxidative stress induced loss of the endothelial glycocalyx involves epigenetic regulation of MMPs that results in the shedding of SOD3 and syndecan-1. These data shed light on a therapeutically targetable epigenetic regulatory mechanism by which oxidative stress may induce vascular remodeling and endothelial dysfunction.