| Part Ⅰ Endothelial PDGF-BB modulates pericytefibroblast transition through the PDGFRβ pathway to promote fibrotic scarring after spinal cord injuryBackground:Fibrotic scar formation is a characteristic pathology of spinal cord injury(SCI)in the injured core,which has been widely regarded as the main barrier to axonal regeneration resulting in permanent functional recovery failure.Pericyte exhibits a fibroblast phenotype,continuously expressing platelet-derived growth factor receptorβ(PDGFRβ)and secreting extracellular matrix to form fibrotic scar.However,the mechanism of pericyte-fibroblast transition after SCI remains elusive.Methods:A mouse spinal cord contusion model was established,and the dynamic changes of fibrotic scar area and microvessel density before and after SCI were detected by immunofluorescence staining.To confirm the process of pericyte-fibroblast transition,we analyzed the expression change of pericyte marker and fibroblast markers using immunofluorescence staining after SCI.Then,the distribution and cellular origin of PDGF-BB were examined with immunofluorescence staining after SCI.Pericyte-fibroblast transition induced by MECs was detected with immunocytochemistry and Western blotting after PDGF-BB knockdown and inhibiting PDGF-BB/PDGFRβsignaling in vitro.Intrathecal injection of imatinib in mice was used to selectively inhibit PDGF-BB/PDGFRβsignaling.Basso mouse scale(BMS)score and footprint analysis were performed to assess the motor function recovery.Subsequently,axonal regeneration,fibrotic scarring,fibroblast population,the proliferation and apoptosis of fibroblasts,pericyte coverage,microvessel leakage,and the inflammatory response were assessed with immunofluorescence staining.Results:PDGFRβ~+ pericytes detach from the blood vessel wall and transition into fibroblasts to form fibrotic scar after SCI.The microvessel density returns to normal spinal cord level at 7 dpi,and the area of fibrous scars peaks at 7 dpi.PDGF-BB is mainly distributed in the periphery of the injured core,and microvascular endothelial cells(MECs)are one of the sources of PDGF-BB in the acute phase.The expression level of PDGF-BB is significantly increased after MECs phagocytic myelin debris(P<0.05).Myelin debris-induced endotheliao conditioned medium promotes pericyte-fibroblast transition and secretes extracellular matrix,and imatinib reverses microvascular endothelial-induced pericyte-fibroblast transformation by inhibiting PDGF-BB/PDGFRβsignaling in vitro.Pharmacologically inhibiting the PDGF-BB/PDGFRβpathway promotes motor function recovery and axonal regeneration(P<0.01)and inhibits fibrotic scar formation(P<0.01).After fibrotic scar formation,inhibiting the PDGFRβreceptor inhibits fibroblast proliferation and promotes fibroblast apoptosis(P<0.001).Imatinib does not alter pericyte coverage on microvessels in the lesion core(P>0.05),while microvessel leakage is significantly decreased by reducing inflammation(P<0.001).Conclusion:The crosstalk between MECs and pericytes promotes pericyte-fibroblast transition through the PDGF-BB/PDGFRβsignaling pathway after SCI.Further research results suggest that blocking the PDGF-BB/PDGFRβsignaling pathway contributes to functional recovery and axonal regeneration after SCI and provides a potential target for the treatment of SCI.Part Ⅱ Myelin debris impairs tight junctions and promotes the migration of microvascular endothelial cells after spinal cord injuryBackground: Persistent demyelination generates a large amount of myelin debris at the injury site after SCI,resulting in a severe inflammatory response and dysfunction.Microvascular endothelial cells(MECs),as amateur phagocytes,can engulf and degrade myelin debris,which promotes inflammation and angiogenesis.Tight junctions(TJs)maintain the integrity of the blood-spinal cord barrier,and the migration of MECs contributes to angiogenesis.However,the role of myelin debris engulfment by MECs on TJs and migration is unclear.Methods: A mouse spinal cord contusion model was established and the MECs b End.3 were treated with different concentrations of myelin debris.The engulfment of myelin debris by MECs in vivo and in vitro was detected by immunofluorescence and oil red O staining.The expression of TJs and connexin across MECs before and after SCI was detected by immunofluorescence.The effects of myelin debris engulfment on TJs,gap junction and integrin expression,and endothelial-mesenchymal transition were examined by immunocytochemistry and Western blotting analysis.Immunofluorescence was used to detect the leakage of microvessel fibrinogen before and after SCI,and a single-layer b End.3 cell leakage model was constructed to analyze the leakage of endothelial cells after myelin debris engulfment.Western blotting assay was performed to detect the expression level of aquaporin 4,a leakage marker.Scratch assays and migration assays were used to observe the effect of myelin debris engulfment on the migration of MECs,and to detect.The PI3K/AKT and ERK signaling pathways were blocked with specific inhibitors of LY294002 and U0126,respectively,to determine whether the PI3K/AKT and ERK signaling pathways mediate the role of myelin debris in promoting microvascular endothelial cell migration.Results: MECs were involved in cleaning myelin debris after SCI and in vitro.Compared with the uninjured spinal cord,the percentage of microvessel colocalization with TJs(ZO-1 and claudin-5)and gap junction connexin 43 decreased significantly(P<0.05).The expression levels of TJs proteins(ZO-1,occludin,and claudin-5),integrin β1,and gap junction connexin 43 decreased significantly in a dose-dependent manner after engulfment of myelin debris by MECs(P<0.05).There was obvious fibrinogen leakage of microvessel after SCI(P<0.01).The permeability and leakage of MECs were significantly increased(P<0.05),and the expression level of the leakage marker aquaporin 4 was significantly increased after engulfment of myelin debris(P<0.05).Myelin debris promoted endothelial-mesenchymal transformation and migration of MECs in a dose-dependent manner(P<0.05).Inhibiting the PI3K/AKT and ERK signaling pathways reversed the migration of MECs promoted by myelin debris engulfment(P<0.05).Conclusion: MECs engulf myelin debris and destroy TJs and gap junction,resulting in increased endothelial permeability and leakage.Myelin debris promotes microvascular endothelial cell migration through PI3K/AKT and ERK signaling pathways,and accelerating myelin debris clearance may help maintain the integrity of the blood-spinal cord barrier,thereby promoting the recovery of motor and sensory function after SCI. |
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