The glycocalyx is crucial for normal endothelial function and microvascular responses to intraluminal blood flow. The glycocalyx tethers and concentrates the extracellular superoxide dismutase (ecSOD) which scavenge reactive oxygen species (ROS). We have previously found that ROS activated matrix metalloproteinases (MMPs) enzymes resulting in glycocalyx shedding in endothelium. We sought to test the hypothesis that shear stress protects against ROS induced reduction in glycocalyx by reducing MMP expression and restoring glycocalyx structure in human microvascular endothelium. Human adipose microvascular endothelial cells (HAMECs) were cultured in a custom built flow chamber designed for a wide surface area and subjected to no (NSS), low (LSS; 5 dynes/cm2), or high (HSS; 20 dynes/cm2) shear stress for 8 hours. Experiments were performed in the presence and absence of exogenous [hydrogen peroxide (H2O2, 2X10-4 mol/L) and endogenous ROS [buthionine sulfoximine (BSO; 10-3 mol/L)] following shear stress treatment (n=5). H2O2 and BSO reduced the glycocalyx of HAMECs which was measured by fluorescence detection of the cell surface density of heparan sulfate glycosaminoglycans with fluorescently labeled wheat germ agglutinin WGA (65.7% reduction in intensity; n=5). Both H2O2 and BSO increased the mRNA and protein expression of MMPs (2-3 fold; MMP1, MMP2 and MMP9) and ADAMs (3-5 fold; ADAM10 and ADAM17). In addition, ROS generation reduced the protein levels of tissue inhibitor of matrix metalloproteinases (TIMP) -1 and -3. There was a reduction in protein levels of syndecan-1 and ecSOD in the total cell lysate and increased in levels of syndecan-1 ectodomain and ecSOD in cell culture media determined detected by immunoprecipitation and Western blotting following ROS. The MMP inhibitor, marimastat (50µmol/L) effectively restored syndecan-1 and ecSOD on the endothelial cell surface. HAMECs exposed to LSS, and HSS demonstrated an improved glycocalyx density and increased cell surface syndecan-1 and ecSOD to the control levels following ROS generation. HSS reduced the ROS induction of ADAM10 (by 30% in LSS and 50% in HSS) and normalized its mRNA and protein expression to basal levels. Similarly, ROS induction of MMP9 mRNA levels was reduced (LSS: 55% reduction; HSS: 80% reduction). Protein expression of MMP9 was reduced in HAMECs following LSS and HSS. HSS and LSS significantly increased TIMP1 and TIMP3 mRNA (TIMP1 ratio of LSS vs. NSS=2.1±0.1; ratio of HSS to NSS=2.5±0.3, p<0.0001). These data indicate that shear stress maintains the expression of microvascular cell endothelial surface core proteins syndecan-1, ecSOD, and the glycocalyx. Taken together, these data support the conclusion that shear stress protects the expression of enzymes that regulate glycocalyx structure (MMPs and TIMPs) preventing protein shedding from endothelial cell surface.