Abstract
The endothelial glycocalyx (eGCX) is a protective barrier on the luminal surface of endothelial cells. The syndecan family of proteoglycans constitute integral components of the eGCX and can act as receptors for many cell signaling pathways. Under inflammatory conditions, the eGCX can be cleaved and shed by proteases such as matrix metalloproteinases and thrombin. We analyzed the ability of thrombin to cleave syndecan core proteins from the eGCX of microvessels in human lung samples using transmission electron microscopy (TEM) and immunogold staining for syndecan‐4 ectodomain (SDC4ecto). The concentration of SDC4ecto in supernatant from human lung slices treated with thrombin was assessed via enzyme‐linked immunosorbent assay and western blotting. The effect of thrombin‐cleaved recombinant SDC4ecto fragments on endothelial barrier resistance was investigated using electric cell‐substrate impedance sensing assay. TEM immunogold staining of human lung demonstrated decreased expression of SDC4ecto within the eGCX following thrombin treatment. Similarly, the concentration of SDC4ecto in human lung slice supernatant was reduced following thrombin treatment, consistent with western blot analysis showing thrombin‐induced cleavage of both recombinant and endogenous full‐length SDC4ecto. In human umbilical vein endothelial cell monolayers, recombinant SDC4ecto treated with thrombin generated a significant decrease in barrier resistance, and this effect was sensitive to a pharmacological inhibitor of Rho kinase, suggesting the involvement of the RhoA pathway in SDC4ecto signaling. In conclusion, these findings suggest that thrombin causes endothelial barrier injury by cleaving SDC4ecto from the eGCX in human lung vessels. Furthermore, an autocrine mechanism may exist whereby thrombin‐cleaved SDC4ecto can act as a signaling molecule to promote endothelial hyperpermeability.Support or Funding InformationNIH Grant HL070752 & GM097270This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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