Investigating the endothelial glycocalyx in ischaemic stroke

Abstract

Ischaemic stroke is a leading cause of death and disability worldwide and yet has only two clinically approved treatments. The endothelial glycocalyx is an important mediator of blood-brain barrier permeability and leukocyte adhesion and extravasation and has been shown to be remodelled in ischaemic stroke. However, previous studies evaluating the endothelial glycocalyx in stroke primarily use indirect methods. Furthermore, the leukocyte glycocalyx is possibly a key mediator of post-stroke inflammation but is massively understudied. The aim of this project was to use novel, direct and multi-modal methodologies to advance our understanding of glycocalyx remodelling in stroke. Here we use bulk RNA-sequencing, mass spectrometry (ToFSIMS), machine learning analysis of lectin staining, and flow cytometry for the first time to characterise glycocalyx remodelling in the photothrombotic model of stroke in mice. We show that the transcriptome of glycocalyx biosynthesis and degradation genes was altered bidirectionally at both 4 h and 24 h, and HS biosynthesis was inhibited at 4 h. ToF-SIMS revealed that overall, the abundance of GAGs in the brain did not change in response to stroke but the ionic composition of the glycocalyx was altered. We reveal that lectin staining of the glycocalyx in situ was altered in blood vessels at 4 h and 24 h post-stroke. Flow cytometric analysis of lectin, HS, HA, syndecan-1, and heparanase on seven distinct cell types (endothelial cells and leukocytes) showed that there were bidirectional changes to cell surface glycocalyx components which were cell-type specific but that heparanase was released universally post-stroke. Finally, inhibition of heparanase had a minimally beneficial effect on markers of inflammation in young, healthy-weight animals and comorbid animals, while matrix metalloproteinase-9 inhibition significantly improved behavioural outcome but no other measures of stroke pathophysiology. Together these results confirm previous work that the endothelial glycocalyx is remodelled in ischaemic stroke as well as contributing new knowledge defining changes to the transcriptome, ionic composition and to specific components at the cell surface. We also identified for the first time changes to the leukocyte glycocalyx in ischaemic stroke. Together, this work highlights the complexity of glycocalyx remodelling and could reveal new targets to improve therapeutic care and outcome for stroke patients.

Date of Award	31 Dec 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ingo Schiessl (Supervisor), Stuart Allan (Supervisor), Catherine Lawrence (Supervisor) & Douglas Dyer (Supervisor)