Brain Microvascular Endothelial Derived Microparticles as a Non‐Invasive Diagnostic Tool for Multiple Sclerosis

Abstract

BackgroundMultiple sclerosis (MS), an autoimmune‐neurodegenerative disease, exhibits features of neurovascular inflammation including endothelial activation and shedding of microparticles (MPs). While the brain lacks ‘classical’ lymphatics, we previously reported expression of venous‐lymphatic endothelial markers (LYVE‐1, podoplanin, prox‐1, VEGFR‐3, VEGF‐C, VEGF‐D) in both brain tissue and sera, with altered expression of these proteins in MS. Based on differential expression of these markers in brain tissue, we propose that these markers may originate within brain microvascular endothelium and undergo changes in disease which can be analyzed diagnostically.ObjectivesTo validate the neurolymphatic biomarker matrix as a novel, non‐invasive and cost‐effective tool for diagnosing MS and discriminating MS subtypes.MethodsNeurolymphatic biomarker expression was determined in human cerebral microvascular endothelial cells (hCMECs) following 24–48 hours of TNF‐α (20 ng/ml) exposure +/− IFN‐γ (1000 U/ml) using western blotting. MPs from these cells were visualized using scanning electron microscopy. Human sera were analyzed by flow cytometry, western and dot blotting to characterize relative partitioning of neurolymphatic biomarkers in MP.ResultshCMECs released MPs that contain lymphatic and junctional markers as well as caveolin‐1. Plasma MPs were seen to liberate soluble (non‐particulate) neurolymphatic biomarkers upon storage, indicating that serum represents a more stable MP‐derived pool of these markers. MP flow analysis of intracellular actin revealed that permeabilization destabilizes phosphatidylserine‐Annexin V interaction which is used to designate MP identity, suggesting that flow cytometry of MPs may require more judicious interpretation. All neurolymphatic markers so far tested were able to distinguish MS from healthy control serum samples; FOXC2 expression can further distinguish subtypes of MS: RRMS from SPMS.ConclusionLymphatic biomarkers in plasma and sera are expressed by brain microvascular endothelial cells and exhibit changes in expression in response to inflammatory stimuli (cytokines). Such alterations parallel transfer of these markers into caveolin‐1 ‐enriched microparticles, which represent a circulating ‘snapshot’ of the vascular surface that can be analyzed by several immune approaches, particularly following storage‐dependent solubilization of these biomarkers.Support or Funding InformationSupported by the Annette Funicello Research Fund for Neurological Diseases and the Malcolm Feist Predoctoral Fellowship from Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center‐ShreveportThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.