The Mechanism Of Cyclic(RGDyk) Peptide Inhibiting Osteogenesis And Angiogenesis To Suppress Ossification Of The Posterior Longitudinal Ligament

ObjectiveOssification of the posterior longitudinal ligament(OPLL)is an ectopic ossification disease that occurs on the posterior longitudinal ligament and gradually progresses to compress the spinal cord and nerve roots,resulting in severe symptoms of cervical stenosis and neurological dysfunction,including sensory and motor impairments and sphincter dysfunction.Without intervention,most patients eventually develop limb paralysis or even quadriplegia.OPLL is more prevalent in East Asian countries,with a prevalence of approximately 3.0%,but is less common in European and American countries,with a prevalence of approximately 0.1%-1.7%.Difference in the incidence of OPLL among ethnic groups suggests that the disease may be genetic,which is currently the widely accepted view in the field.OPLL is considered a muti-gene hereditary disease with familial aggregation,which is also related to external environmental factors and metabolic processes in the body.Currently,the only treatment for OPLL that causes spinal or nerve compression is surgery,but patients often face risks such as intraoperative spinal nerve injury,limited postoperative symptom improvement,continued OPLL progression,and multiple surgical complications.Since Key first reported OPLL in 1830,the pathogenesis of OPLL has not been elucidated,and effective preventive or curative measures are lacking clinically.Studies have shown that the abnormal osteogenic phenotype transformation of OPLL cells in the posterior longitudinal ligament,is an important mechanism for the progression of OPLL.Fibroblasts are the main components of OPLL cells.A large number of literature reports indicate that fibroblasts from different tissues or organs exhibit stem cell characteristics and can differentiate into bone,fat,cartilage,and other tissues.Endothelial cells(ECs)can secrete various cytokines to promote osteoblast formation and are crucial for maintaining OPLL cells niches.ECs participate in vascular formation and provide osteoblasts with the nutrients necessary for ectopic bone tissue formation,thus promoting the pathological process of OPLL together with OPLL cells.Currently,domestic and foreign scholars generally believe that OPLL is initiated by multiple factors such as genetics,environment,and mechanical stress,which initiate bone formation in the local posterior longitudinal ligament and form ectopic ossification.Mechanical stress plays an important role in bone growth and development,and OPLL most commonly affects the C5 segment,which may be related to the concentration of stress on the cervical spine in this area.However,there is little research on the molecular mechanisms of abnormal stress-induced changes in the cell microenvironment and their mediation of OPLL pathogenesis.Integrin is a transmembrane glycoprotein receptor widely expressed on cell surfaces.It is composed ofα and β subunits that form non-covalent bonds.18 α subunits and 8 β subunits have been discovered,which can combine to form 24 known integrin heterodimers.Integrins play a role in transmitting intracellular signals to the extracellular matrix or extracellular signals to the inside of the cell by binding to cytoskeletal proteins or extracellular ligands.Moreover,the integrin family of molecules has emerged as a research hotspot due to their functions in promoting cell proliferation,migration,osteogenic differentiation,and angiogenesis.Therefore,we conducted research on the integrin family of molecules,and the results showed that integrin αvβ3,we used c(RGDyk)peptide,which is a highly efficient,high-affinity,and selective integrin αvβ3 inhibitor,to investigate its effect and specific mechanism on osteogenic differentiation of OPLL cells and angiogenesis of ECs.Therefore,this study aims to clarify the therapeutic value of integrin αvβ3 as a target for OPLL and provide a theoretical basis for the search for new treatment strategies for OPLL in clinical practice.MethodPart 1:(1)Cultivate primary OPLL cells and normal posterior longitudinal ligament cells,extract cellular RNA,and screen for differentially expressed integrin subtypes;(2)Use Wb and immunohistochemistry to validate differentially expressed integrin subtypes;(3)Verify the role of angiogenesis in the pathological process of OPLL using immunofluorescence;(4)Cultivate primary OPLL cells through osteogenic,adipogenic,and chondrogenic differentiation systems,and verify the differentiation results using corresponding cell staining,and use flow cytometry to detect surface markers of OPLL cells.Part 2:(1)Use immunofluorescence staining to verify the expression levels of integrinαVβ3 in OPLL cells and ECs selectively inhibited by the c(RGDyk)peptide;(2)Use q PCR,Wb,ALP,and ARS staining to detect the effect of c(RGDyk)peptide on osteogenic differentiation of OPLL cells;(3)Verify the effect of c(RGDyk)peptide on ECs angiogenesis through cell migration and tube formation experiments;(4)Use micro-CT,immunohistochemistry,and immunofluorescence experiments to verify the effect of c(RGDyk)peptide on ectopic bone formation in vivo,explore the therapeutic effect of c(RGDyk)peptide on OPLL,and verify that integrin αVβ3 may be a key molecule in the pathogenesis of OPLL.Part 3: Use Wb to detect the specific molecular mechanism of integrin αVβ3 promoting osteogenic differentiation of OPLL cells and angiogenesis of ECs.ResultsPart 1: Under the optical microscope,primary OPLL cells and PLL cells showed spindle,polygonal,and fibroblast-like morphologies.OPLL cells can differentiate into osteoblasts,adipocytes,and chondrocytes through the osteogenic,adipogenic,and chondrogenic differentiation systems,and the expression of stem cell-specific markers on their surfaces indicated their multipotency.By extracting RNA and detecting the αv,α3,α5,α6,β1,β3,and β5 integrin subtypes in stem cells,it was found that OPLL cells expressed differentially expressed αv and β3 subtypes.Furthermore,Western blot and immunohistochemistry confirmed that integrin αvβ3 showed the most significant upregulation in OPLL tissues.Immunofluorescence results indicated that angiogenesis also played an important role in the pathological bone formation process of OPLL.Therefore,integrin αvβ3 may mediate OPLL pathogenesis by facilitating bone and blood vessel formation.Part 2: Integrin αVβ3 was activated in OPLL cells and expressed significantly higher levels under osteogenic differentiation conditions.However,as the concentration of c(RGDyk)peptide varied,the expression of integrin αVβ3 decreased significantly.IntegrinαVβ3 was also highly expressed in well-growing endothelial cells(ECs),but its expression decreased with the effects of c(RGDyk)peptide.Therefore,c(RGDyk)peptide selectively inhibited the expression of integrin αVβ3.c(RGDyk)peptide inhibited OPLL cell osteogenic differentiation,as demonstrated by significantly reduced ALP and ARS staining intensity and downregulated expression of osteogenic-related genes after osteogenic differentiation culture of OPLL cells.The peptide also inhibited ECs migration,as evidenced by the significantly lower migration ability of well-growing ECs in the c(RGDyk)peptidestimulated group and lower tube formation ability of ECs cultured in pure 3D Matrigel than those in the c(RGDyk)peptide-added 3D Matrigel group.Furthermore,c(RGDyk)peptide inhibited the formation of ectopic bone in vivo,as evidenced by the reduced immunohistochemical staining of OCN,CD31,and integrin αVβ3,as well as lower bone volume fraction and bone density of the bone matrix.Part 3: Integrin αVβ3 could upregulate the expression of the key transcription factor RUNX2 in osteogenic differentiation of OPLL cells through the downstream FAK/ERK pathway and promote their osteogenic differentiation.c(RGDyk)peptide could selectively inhibit the expression of integrin αVβ3 in OPLL cells,thereby inhibiting their osteogenic differentiation.In addition,the cross-talk between integrin αVβ3 and VEGFR2 signaling pathways could activate the downstream FAK/ERK pathway and promote the migration of ECs.However,c(RGDyk)peptide inhibited the FAK/ERK-dependent migration of ECs by blocking the close relationship between integrin αVβ3 and VEGFR2 signaling pathways.ConclusionThis study confirms the critical role of integrin αVβ3 in the formation of OPLL.The c(RGDyk)peptide can selectively inhibit the expression of integrin αVβ3 and regulate the FAK/ERK signaling pathway,thereby suppressing osteogenesis and angiogenesis.Therefore,integrin αVβ3 may be a potential therapeutic target for OPLL,and c(RGDyk)peptide may become a novel therapeutic strategy for OPLL.