Detection of Glycan Shedding in the Blood: New Class of Multiple Sclerosis Biomarkers?

Abstract

IntroductionMultiple sclerosis (MS) is a devastating autoimmune disease, afflicting people in the prime of their lives. Presently, after initial clinical presentation, there are no reliable markers for whether a patient will develop MS, or whether their prognosis will be aggressive or relapsing–remitting. Furthermore, many MS patients do not respond to treatment. Thus, markers for diagnosis, prognosis, and treatment-responsiveness are lacking for a disease, where a precision medicine approach would be valuable. The glycocalyx (GLX) is the carbohydrate-rich outer surface of the blood vessel wall and is the first interaction between the blood and the vessel. We hypothesized that cleavage of the GLX may be an early stage predictor of immune attack, blood–brain barrier (BBB) breakdown, and disease severity in MS.MethodsTwo experimental models of MS, experimental autoimmune encephalitis (EAE), were included in this study. EAE was induced in C57BL/6J mice and Lewis rats, which were monitored for weight loss and clinical presentation in comparison to healthy controls. Plasma samples were obtained longitudinally from mice until peak disease severity and at peak disease severity in rats. Soluble GLX-associated glycosaminoglycans (GAG) and proteoglycans (PG) were detected in plasma samples.ResultsAll animals receiving EAE emulsion developed fulminant EAE (100% penetrance). Increased plasma levels of chondroitin sulfate were detected before the onset of clinical symptoms and remained elevated at peak disease severity. Hyaluronic acid was increased at the height of the disease, whereas heparan sulfate was transiently increased during early stages only. By contrast, syndecans 1, 3, and 4 were detected in EAE samples as well as healthy controls, with no significant differences between the two groups.DiscussionIn this study, we present data supporting the shedding of the GLX as a new class of biomarker for MS. In particular, soluble, sugar-based GLX components are associated with disease severity in two models of MS, molecules that would not be detected in proteomics-based screens of MS patient samples. Patient studies are presently underway.

Increased capillary stalling is associated with endothelial glycocalyx loss in subcortical vascular dementia

“…Another previous study showed enhanced leukocyte adhesion in the retina of knockout mice for syndecan 1, a component of the EG 27 . In addition, several studies have shown degradation of the EG in diseases characterized by decreased CBF, such as malaria infection, multiple sclerosis, and stroke 28,29,30 . Based on these previous studies and our results, we suggest that the loss of the EG in capillaries, caused by the pathological progression of SVaD, may induce greater leukocyte adhesion to the vascular endothelium and, thereby, promote capillary stalling.…”

The blood–brain barrier in health and disease: Important unanswered questions

“…While small dyes such as fluorescein (376 daltons) and Alexa Fluor (643 daltons) permeate the glycocalyx, dextrans (40–150 kD) penetrate <60% of its volume (Kutuzov et al, 2018). In disease, glycocalyx degradation is associated with more severe BBB leakage in models of multiple sclerosis (MS) and cardiac arrest (DellaValle et al, 2018; Zhu et al, 2018).…”

Multiple Soluble Components of the Glycocalyx Are Increased in Patient Plasma After Ischemic Stroke

“…Two microlitres of plasma was dotted in duplicate on nylon membranes (Hybond N + , Amersham, RPN203B) similar to. 5 Membranes were blocked in blocking buffer (5% skim milk+TBS-0.05%Tween20) and incubated overnight at 4°C in blocking buffer+primary antibody, washed (TBS-0.05%Tween20), incubated with blocking buffer+secondary antibody, washed, incubated with SuperSignal Femto Reagent (Thermo Scientific), imaged with CCD camera (LAS 4000, GE), analyzed with ImageJ 6 for raw integrated density, and transformed by a factor of 10 −5 . Primary antibody targets: glycosaminoglycans (GAGs): chondroitin sulfate (CS, Sigma), heparan sulfate (HS, Millipore), keratan sulfate (KS, US-Biologicals), and hyaluronic acid (HA, Bio-Rad); and proteoglycans (PGs): CD44 (DAKO), syndecan (Syn)-1 (R&D), 2 (R&D), 3 (R&D), 4 (Santa Cruz), glypican-1 (R&D), and BiGlycan (Abcam).…”