Mechanism Of LOXL2 In Modulating Ligament Vascularization To Promote Ossification Of The Posterior Longitudinal Ligament

Ⅰ.BackgroundOssification of the posterior longitudinal ligament(OPLL)can be defined as a degenerative hyperostosis disease of the spine.The posterior longitudinal ligament(PLL)starts from the occiput and terminates at the sacrum.The PLL runs along the posterior side of vertebral bodies and intervertebral discs and is located in the anterior of the dural sac and spinal cord.If the PLL is ossified,the ossification can protrude into the spinal canal,causing severe neurological symptoms like paresthesia,disturbances of motility and autonomic disorders lowering the quality of life.OPLL is a unique form of heterotopic ossification that typically occurs in the cervical spine and predominantly affects males.Unlike other heterotopic ossification diseases,OPLL is not associated with trauma,systemic inflammatory diseases,burns,or long-term immobilization.Our previous study found that18%of patients suffering from degenerative cervical myelopathy(DCM)had OPLL based on 3D-CT findings.Epidemiological studies have shown that OPLL is more prevalent in East Asia,with an incidence rate ranging from 1.9%to 4.3%in Japan.In contrast,the incidence of OPLL is lower in North America and continental Europe,ranging from 0.1%to1.7%.During the initiation and progression of OPLL,the disorder is thought to develop through endochondral ossification at the histological level.The avascular ligamentous tissue is progressively transformed and replaced by highly vascularized bone tissue.Numerous genes and non-coding RNAs(nc RNAs)that regulate the osteogenic differentiation of ligament cells have been identified;however,the formation of ossification encompasses various other biological processes beyond osteogenic differentiation.The infiltration of blood vessels plays a crucial role in the disease process.Blood vessel formation involves two primary processes:vasculogenesis and angiogenesis.Vasculogenesis refers to the de novo creation of blood vessels,during which progenitor or stem cells differentiate,migrate,and assemble into a rudimentary vascular network.On the other hand,angiogenesis involves the development of new blood vessels branching out from pre-existing ones.Mesenchymal stromal cells(MSCs)play a critical role in angiogenesis through paracrine signaling.They secrete pro-angiogenic factors like vascular endothelial growth factor(VEGF),platelet-derived growth factor(PDGF),and fibroblast growth factor(FGF).These factors encourage endothelial cell proliferation,migration,and tube formation,facilitating the generation of new blood vessels.Additionally,under certain stimulations,MSCs can differentiate into endothelial cells and myocytes,participating in blood vessel formation.Despite these findings,the role of resident stem cells in blood vessel formation and the prospects for therapeutic intervention,particularly in bone formation,remain incompletely understood.II.Objectives1.Clarify the molecular characteristics as well as cellular components and evolution during the OPLL process at single-cell resolution and verify it in clinical samples.2.Clarify the role of new blood vessels in OPLL and verify whether ligament cells can be transformed into new blood vessels through vasculogenesis.3.Study the molecular mechanism of ligament vasculogenesis mediated by the LOXL2signaling pathway in the occurrence and development of cervical posterior longitudinal ligament ossification.4.Study the effect of specific small molecule inhibitors on inhibiting neovascularization and ossification progression in vitro and in vivo to provide effective targets for precise intervention in OPLL.Ⅲ.MethodsPart I:(1)Conduct a study on the histological characteristics of OPLL using safranin fast green,H&E staining,and immunohistochemistry,coupled with imaging analysis.(2)Obtain tissue samples from both OPLL and non-OPLL sources for transcriptomic sequencing.Analyze differentially expressed genes,screen hub genes,and perform enrichment analyses such as GO,KEGG,and GSEA to preliminarily explore the molecular characteristics of OPLL.(3)Utilize ligament cell and ligament hypertrophy sample sequencing data from public databases to investigate potential pathogenic mechanisms related to OPLL.(4)Generate single-cell suspensions from OPLL and non-OPLL samples.Perform single-cell sequencing using the 10×Genomics platform.Group cells in the ligament microenvironment based on characteristic genes displayed in sequencing results.(5)Employ RNA Velocity,Monocle3,etc.,to construct ligament cell differentiation trajectories and study the evolution of OPLL at single-cell resolution.(6)Verify sequencing and bioinformatics analysis results using immunofluorescence staining,Western Blot,and other methods.(7)Compare the vasculogenesis(endothelial differentiation)ability and characteristics of ligament cells with human umbilical vein endothelial cells and mesenchymal stem cells through tube formation experiments,q PCR,and VEGFA in vitro stimulation.(8)Investigate the in vivo vasculogenesis ability of ligament cells by injecting Matrigel with or without ligament cells under the skin of nude mice.(9)Conduct a preliminary study on regulators of ligament cell endothelial differentiation using VEGFA,PDGF-BB,imatinib,and bevacizumab.Part II:(1)Construct and transfect LOXL2 overexpression plasmids and knockdown si RNA into ligament cells.Conduct tube formation experiments and study the impact of LOXL2 enzyme activity inhibition on ligament cell vasculogenesis with pan-LOX inhibitor.(2)Utilize immunofluorescence staining,q PCR,Elisa,and Western Blot to examine the effects of LOXL2 overexpression on the transcription factor HIF1A and downstream signaling pathways.(3)Evaluate the role of VEGFA in OPLL by detecting the serum VEGFA concentration of patients and constructing a ROC curve for diagnostic biomarker assessment.(4)Perform endothelial cell Transwell experiments using ligament cell-conditioned medium that knocks down or overexpresses LOXL2 to reveal the impact of LOXL2 on endothelial cell migration ability.(5)Study the impact of LOXL2 enzymatic activity on ligament cell vasculogenesis and downstream signaling pathways by LOXL2 point mutation plasmid(OE-LOXL2 Y689F)and comparing results with the wild-type plasmid in various experiments.(6)Use the HIF1A pathway inhibitor sorafenib and the VEGFA monoclonal antibody bevacizumab in in vitro tube formation experiments to verify their potential to inhibit the enhancement of vascular differentiation caused by LOXL2 overexpression.(7)Intervene in the osteogenic differentiation of ligament cells using selective inhibitors of LOXL2 enzyme activity.Evaluate the impact of LOXL2 enzyme activity on osteogenic differentiation,combining with alizarin red staining.(8)Further clarify downstream inhibitors of the LOXL2 pathway through ALP staining experiments and study the effect of sorafenib on the osteogenic differentiation of ligament cells.(9)Use alizarin red staining to investigate whether sorafenib can inhibit the enhancement of osteogenic differentiation caused by overexpression of LOXL2.Part III:(1)Construct a BMP-2-induced mouse ossification model and intervene with specific LOXL2 enzyme activity inhibitors and sorafenib.Use micro CT,H&E,safranin-fast green,and immunohistochemistry/fluorescence to study the inhibitory effect of inhibitors on ossification progression,osteogenic differentiation,and neovascularization.(2)Perform micro CT scanning on mouse spines to study the effects of sorafenib on physiological bone structure.Take tissue sections from liver and spleen for Masson staining and Sirius red staining to detect potential liver and kidney toxicity of sorafenib.(3)Construct an enpp1gene knockout mouse with a spontaneous posterior longitudinal ligament ossification model.Orally administer sorafenib and use micro CT,H&E staining,safranin-fast green staining,and immunohistochemistry/fluorescence to study the inhibitory effect of sorafenib on the ossification process,osteogenesis,and neovascularization.Use von Frey pain meter and thermal pain experiments to explore the possible role of sorafenib in protecting neurological function.IV.ResultsPart I:This segment of the study provides a comprehensive analysis of the pathological characteristics,cell differentiation,and vasculogenesis mechanisms associated with cervical OPLL.Integration of imaging and histological staining reveals distinctive features of endochondral ossification in OPLL.Transcriptomic analysis of tissue and cells identifies numerous differentially expressed genes,including key factors crucial for vasculogenesis and bone formation,such as osteogenic markers SP7,BGLAP,and RUNX2,along with LOXL2 and MMP1 genes linked to ECM decomposition and vasculogenesis or angiogenesis.Functional enrichment analysis underscores the regulatory role of ECM remodeling,angiogenesis,and bone development pathways in OPLL.LOXL2 emerges as a significant hub gene,upregulated in ossified ligament samples and enriched in various biological processes.Single-cell sequencing uncovers PRG4~+stem cells in ligament tissue,demonstrating their potential to differentiate into osteoblasts,chondrocytes,and endothelial cells.In vivo and in vitro experiments confirm ligament cells’ability to differentiate into functional vascular structures,driven by PDGF-BB and VEGFA as key regulatory factors.Part II:Overexpression of LOXL2 in ligament cells promotes vascular structure formation on Matrigel in vitro.Inhibitors of LOXL2 enzyme activity fail to block the enhanced vasculogenesis caused by LOXL2 overexpression.LOXL2 overexpression correlates with increased expression of the transcription factor HIF1A and its downstream VEGFA-C and PDGFB.Knocking down LOXL2 inhibits capillary structure formation and expression of these factors.Ligament cell conditioned medium with LOXL2 overexpression promotes endothelial cell migration.Serum VEGFA concentrations suggest VEGFA as a potential diagnostic marker for OPLL.Studies on LOXL2 point mutation plasmid(OE-LOXL2 Y689F)indicate that LOXL2 enzyme activity is not essential for regulating endothelial differentiation,whereas VEGFA monoclonal antibodies or VEGFR inhibitors can reverse endothelial and osteogenic differentiation promoted by LOXL2.Part III:Sorafenib,a multi-target tyrosine kinase inhibitor,significantly slows down the ossification process in the BMP-2-induced ectopic ossification model in mice when orally administered at 50 mg/kg.It reduces bone volume,total volume,and bone surface,hindering mature bone formation and increasing the deposition of cartilage matrix components.Sorafenib downregulates SP7~+and RUNX2~+osteoblast expression and inhibits neovascularization.Toxicological experiments reveal that while sorafenib effectively inhibits ossification,it has no negative impact on the normal bone structure of the spine and shows no toxic effects on the liver and kidneys.In enpp1 knockout mice with a spontaneous ligament ossification model,sorafenib significantly reduces the degree of spinal ligament ossification,improves neurological symptoms,and disrupts the coupling between vasculogenesis and ossification,effectively inhibiting the progression of spinal ligament ossification.V.ConclusionIn conclusion,the pathological progression of OPLL aligns with the characteristics of endochondral ossification.Tissue and cell sequencing data reveal precise cross-regulatory effects among pathways related to blood vessels,bone,and extracellular matrix metabolism during ossification.Bioinformatics analysis highlights the potential crucial role of LOXL2in the course of OPLL.Ligament tissue’s PRG4~+stem cells demonstrate the unique ability to directly differentiate into blood vessels,regulated by VEGFA and PDGF-BB.LOXL2,independent of its enzymatic activity,may influence the vasculogenesis of ligament cells by regulating HIF1A and its downstream factors.VEGFA,a key player in the HIF1A pathway,emerges as a potential biomarker for OPLL.The small molecule inhibitor sorafenib,intervening in the LOXL2/HIF1A pathway,demonstrates significant effects in both in vitro and in vivo experiments,reversing endothelial and osteogenic differentiation mediated by LOXL2.In vivo,sorafenib effectively inhibits ectopic bone tissue formation,reducing ossification and associated neovascularization without impacting normal spinal structure or organ tissues such as the liver and kidneys.This study unveils the innovative finding that ligament cells with mesenchymal stem cell characteristics actively participate in and regulate the occurrence and development of OPLL by differentiating toward blood vessels,presenting a novel mechanism in OPLL pathogenesis.By delving into the neovascularization perspective,this study illuminates a novel mechanism and potential intervention targets involving LOXL2 and its downstream signaling pathways in OPLL.These insights contribute valuable information for the diagnosis and treatment of cervical OPLL,offering novel perspectives for future research and clinical applications.