Question:

Endothelial cells (EC) are lining up the inner surface of the vasculature controlling barrier function and cellular adhesion. Under physiological situations the EC are decorated with a glycocalyx, composed of heparansulfates and proteoglycans anchored to the cell surface via linker proteins like syndecan I. It is known that under hypoxic conditions this glycocalyx is greatly reduced. This may have significant impact on the physiological function of the vascular endothelial lining.

Since hypoxia is often accompanied with no-flow conditions we tested here the hypothesis that loss of flow (low shear stress) induces the shedding of glycocalyx and moreover that during long-term exposure to flow EC are regaining their typical cell surface modification.

Methods:

Primary isolates of endothelial cells from human umbilical veins (HUVEC) were subjected either into normal static cell culture or subjected to laminar flow (15 dyn/cm²) for up to 5 days. The presence of the glycocalyx was traced by flow cytometry using FITC-labeled lectins from Ulex, Triticum, Concanavalin, Bandeirea,and Arachis. In order to test possible effects of a cellular glycocalyx coverage on cell attachment, we performed adhesion assays using endothelial colony forming cells (ECFC), HUVEC, and monocytes (MonoMac 6). Those adhesive cells were fluorescently labeled with calcein-AM and exposed to the acceptor HUVEC layer at 1.5 dyn/cm² for 20 min, followed by one short washing and fixation with formaldehyde. Optical fields (10 each) were photographed and adhered cells were counted.

Results:

Freshly isolated endothelial cells cultivated under static monolayer culture conditions show only limited lectin binding. However, if those cells were exposed to long-term flow they regained a glycocalyx coverage indicated by significant binding of the lectins from Ulex and Triticum. Concanavalin A was not altered and was used as unspecific control because it binds to mannose, which is lacking on EC anyway. Bandeirea and Arachis lectins were bound significantly less intense and may represent a mask of their binding sites by the growing carbohydrate side chains. Importantly, a stop-flow of only 4 hours significantly reduced lectin binding, indicating rapid loss of the glycocalix. The long-flow induced glycoalix formation corresponded to a significant lower adhesion of all cell types analyzed (ECFC, HUVEC, or MonoMac) to a flow-exposed HUVEC monolayer.

Conclusion:

Endothelial cells rapidly lose their physiological glycocalyx surface layer in static in vitro culture. However they might regain that surface modification during long-term flow-exposure for 5 days. The glycocalyx coverage prevents cells adhesion and might be important for proper barrier function of the vessel wall. Interestingly, although it takes days to rebuild the glycocalyx, it is destroyed within a few hours of no-flow.