Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure

Abstract

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx — a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells — functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans (GAGs), a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis (PIT) mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine (NAC)+GAG oligosaccharides as compared to NAC administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys139 (6- kDa linker), and Lys107 and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part, is attributed to the activation of proHPSE. Since proHPSE, but not HPSE, localizes outside cells by binding with HS proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

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“…The resulting dried tissue samples were treated with actinase E (100 mg/mL) in 720 µL of 50 mM Tris acetate buffer (pH 8.0) at 45°C for 48 hours. Microscale isolation of GAGs was performed according to the method of Ko et al [39]. Brie y, the ltered extracts were puri ed by centrifugation in a Vivapure Q mini H spin column (Sartorius Stedim Biotech GmbH, Göttingen, Germany), and then columns were washed three times with 500 µL of 16% NaCl and eluted with 0.2 M NaCl.…”