Microvascular Endothelial Cells Engulf Myelin Debris And Promote Macrophage Recruitment And Fibrosis After Neural Injury

Demyelination after spinal cord injury(SCI)and multiple sclerosis(MS)causes partial or complete loss of sensory function or motor control without effective treatment strategies.Myelin debris generated immediately following SCI stimulates inflammatory response and inhibits axon regeneration.Therefore,engulfment and clearance of myelin debris is critical for functional recovery.Bone marrow-derived macrophages(BMDMf)have been known to engulf and clear myelin debris,however BMDMf are not significantly recruited to injury site until one week after SCI.It is therefore critical to identify other cell types,especially resident phagocytes during early stages of injury that play a role in myelin debris engulfment and the secondary injury.The main findings are as follows:1.Using mouse SCI and MS models we showed that myelin debris-containing microvessels were frequently observed in the injured region.Furthermore,neutral lipids can be detected in microvessels at the injured core after SCI.In vitro primary mouse brain microvascular endothelial cells(BMECs)and primary BMECs-induced microvessel-like structures on Matrigel confirmed that microvascular ECs could engulf myelin debris.ECs engulfment of myelin debris does not require LRP1,CR3 or Mac2receptors that are employed macrophages,however depends on IgG opsonization and Fc receptor for IgG.Thus,microvessels and the lining ECs in the demyelinating spinal cords are novel source for myelin debris engulfment and clearance using a distinct mechanism from macrophages.2.RNA-sequencing analysis showed that ECs after myelin engulfment upregulated vesicle-encoding genes related to lysosomes,autophagosomes and endosomes.We then showed that myelin debris was predominantly delivered to lysosomes for degradation to neutral lipid,through autophagy but not endosome pathway.Moreover,we showed that autophagy-lysosome pathway is required for myelin degradation into neural lipids by generating autophagy-deficient endothelial cell lines with Atg5 knockout via CRISPR-Cas9 approach,or by treating endothelial cells with lysosomal inhibitor chloroquine.We thus demonstrate that endothelial cells degrade the engulfed myelin debris into neutral lipids through autophagy-lysosome pathway.3.Microvessels are enlarged in the demyelinated region of mouse SCI or MS model,suggesting the presence of lesion-related factors that stimulate microvascular growth.We showed in myelin-ECs injection into normal spinal cord,and subcutaneous injection of matrigel plugs that contain myelin-ECs as well cell culture assay,that myelin debris promotes endothelial cell angiogenesis.The pro-angiogenic effect of myelin debris depends on autophagic degradation of myelin debris into neutral lipids and VEGF up-regulation.These results demonstrate that engulfment and autophagic processing of myelin debris promotes endothelial cells proliferation and angiogenesis.4.We found GFP~+BMDMf that infiltrates to injured site closely correlates well in number and localization with newly formed microvessels in demyelinating mouse model,suggesting macrophage recruitment is dependent on the newly formed microvessels.We showed that myelin debris engulfment by endothelial cells promotes macrophage adhesion onto endothelial monolayer and macrophage chemotaxis towards supernatant from myelin-ECs.Further analysis showed that myelin debris increases expression of adhesion molecule like VCAM-1 to promote macrophage adhesion,and increases expression of chemotaxis molecule like MCP-1 to stimulate macrophage chemotaxis,consistent with RNA-sequencing data.These results demonstrate that myelin debris stimulates endothelial cell inflammation and the inflamed microvessels could boost inflammatory responses.5.RNA sequencing identified the top upregulated genes implicated in fibrosis.In vivo data from two demyelinating models showed that a dense collagen I and fibronectin colocalize well with enlarged microvessels,suggesting that endothelial cells could be a novel source for fibrotic scar formation through endothelial production of pro-fibrotic components.Prolonged exposure of myelin debris induces high expression of collagen and fibronectin in endothelial cells.Further mechanistic analysis showed that myelin debris induced endothelial-to-mesenchymal transition(EndMT)via upregulation of TGF-βsignaling.Noteworthy,after 6 weeks of SCI and 15 days of EAE,microvessels showed co-localization between CD31 andα-SMA,indicative of in vivo EndMT.Our in vivo and in vitro data support that myelin debris stimulates endothelial production of collagen and fibronectin,probably via EndMT.Taken together,we show here a previously unidentified role for microvessels and their lining endothelial cells in engulfing myelin debris.We demonstrate that IgG opsonization of myelin debris is required for their effective engulfment by microvascular endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris.We further show that,following myelin uptake and autophagy-dependent processing,Microvascular endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating inflammation,angiogenesis,and fibrosis via EndMT.