| BackgroundAnkylosing Spondylitis(AS)is a chronic autoimmune disease with significant genetic correlation.At present,there is no recognized specific marker for AS,and its pathogenesis remains unclear.According to the epidemiological investigation,the prevalence of AS in China is about 0.3%,and the number of patients is large.AS usually begins in young and middle-aged people aged 20 to 40 years old with insidious onset.It is characterized by chronic progressive inflammation of the sacroiliac joint as the initial symptoms and main features.Inflammatory lesions often spread upward to the axial skeleton and downward to the peripheral joints,and in the late stage can lead to the fibrous or bony fusion of soft tissues around the spine or hip joint,even causing severe physical deformity and disability,which brings great pain to the patient and his family.Although the pathogenesis of ankylosing spondylitis has been studied extensively and deeply in the world in the past two decades,there is still no unified understanding of the pathogenesis of inflammation and ossification and the relationship between them.At the same time,there has been no breakthrough in the clinical treatment of AS.Although the Tumour necrosis factor-?(TNF-?)inhibitor is recognized as a biological agent with the best efficacy,it can relieve the clinical symptoms in some people in a short term,but it is not ideal for effectively preventing patients with advanced ossifying rigidity.Once the lesion site of AS involves the spine and peripheral joints,the quality of life of patients will be greatly reduced.Therefore,an in-depth understanding of the microscopic structure of the immune environment of these parts and the biological behavior between cells in the pathological environment is particularly important for the understanding of the pathogenesis of AS,and also provides valuable targets for drug development.To date,opinions on the relationship between AS inflammation and ossification have been mixed.The cellular components in the local pathological microenvironment of AS ligaments and their functions have not been reported.Therefore,single-cell transcriptome sequencing was used in this study to analyze the composition and interaction forms of various cell subsets in the microenvironment of AS spine ligament attachment points.Furthermore,the molecular mechanism of the interaction between inflammatory cells represented by macrophages and ligament-derived stem cells with osteogenic differentiation potential was explored in vivo and in vitro to explore the relationship between inflammation and ossification in AS.ObjectivesPart ?: Single-cell transcriptomic analysis of spinal ligaments in ankylosing spondylitis was performed to identify the types of cellular interactions associated with inflammation and ossification,and to validate the expression levels of key molecules at the lesion site.Part ?: To explore the mechanism by which macrophages affect the process of osteogenic differentiation and cell migration of ligament-derived stem cells in the spinal ligament microenvironment of ankylosing spondylitis.Part ?: To verify the mechanism of VEGFA targeting VEGFR2 receptor-mediated ectopic osteogenesis in vivo and in vitro.Methods and MaterialsPart ?:(1)The same sites of the lumbar supraspinous ligament,interspinous ligament,and ligamentum flavum were collected from 5 AS patients with spinal orthopedic and 3 normal controls with spinal fractures.Single-cell suspension was prepared,and single-cell transcriptome sequencing was performed on the collected cells based on the BD Rhapsody technique under complete quality control.(2)The results of single-cell transcriptomics were processed using bioinformatics methods,focusing on exploring the composition of cell subpopulations in the pathological microenvironment at the attachment site of the spinal ligament,and focusing on the analysis of the relationship between cells related to AS inflammation and ossification phenotypes and target signal molecules between them.The expression of target molecules in the target cells of the disease group and the control group was analyzed for differences.(3)Serum and monocytes of AS and normal subjects were collected,and ligament tissues of patients with lumbar fracture were collected.The expression of target molecule VEGFA was detected by ELISA,Western Blot,RT-q PCR,and immunohistochemistry(IHC).Part ?:(1)The tissue sections of the ligaments at the lumbar attachment points of patients with AS and lumbar spine fractures were selected and immunofluorescence(IF)method was used to label macrophages and VEGFA,and their expression and correlation was analyzed.(2)Human peripheral blood mononuclear cells were induced in the M2 direction,and CD14 and CD206 were selected as marker genes to identify them by flow cytometry;CD73,CD90,CD105,CD14,CD34,and CD45 were selected as marker genes to identify ligament mesenchymal stem cells using flow cytometric.(3)Different concentrations of TNF-?(0,5,20ng/ml)were designed to interfere with M2 macrophages in AS,and the expression of VEGFA was detected by RT-q PCR and ELISA.(4)Osteogenic differentiation of AS and control lumbar ligament stem cells were conducted simultaneously.Alkaline phosphatase(ALP)staining and activity quantification were performed on the 7th day of induced differentiation,and alizarin red staining and calcium nodule quantification were performed on the 21 st day,then their osteogenic differentiation ability was compared.(5)We selected the culture supernatant of M2 macrophages stimulated by the optimal concentration of TNF-?(20ng/ml)in the above experiment and divided the experiment into control group(osteogenesis induction only),TNF-? stimulation group,and TNF-? stimulation + VEGF inhibitor group.Intervention on AS ligament stem cells were conducted in the process of osteogenic differentiation and culture,and ALP staining and activity was detected after 7 days of culture;RT-q PCR detection of the expression of three osteogenic indicators ALP,Runx2,COL1A1 was conducted to reflect the osteogenic differentiation of the three groups of cells.(6)We designed VEGFA stimulation experiments of different concentrations to intervene AS ligament stem cells in the process of osteogenic differentiation induction.ALP staining and activity quantification were performed on 3 and 7 days;Alizarin red staining and calcium were performed on 9 and 14 days,and nodules were quantified;RT-q PCR was used to detect the expression of osteogenic indicators to test their ability to promote bone formation.(7)The VEGFA receptor-related enriched genes were analyzed in AS ligament stem cells through GO analysis,and the GO terms and associated gene networks obtained by Clue GO were visualized,and the intracellular signal molecules and related pathways activated after VEGFA stimulation of AS ligament stem cells were deeply explored.(8)Exploration of the molecular mechanism of VEGFA regulating the osteogenic differentiation of AS ligament stem cells: detection of fluorescent colocalization of VEGFA and its functional target receptors(VEGFR1,R2,R3)in AS ligament slices,and their co-localization;stem cells from 3 cases of AS group and 3cases of control group ligament were induced to osteogenic at the same time under VEGFA stimulation,and Western Blot was used to detect the degree of activation of each receptor;different time gradients for VEGFA were designed to stimulate AS ligament stem cells in the process of osteogenic differentiation experiment,and the omics analysis results of the activation pathway was verified through Western Blot;through the inhibition test of the target pathway and target receptor,combined with ALP staining and activity detection,alizarin red staining and calcium nodule quantitative osteogenic ability detection,the above molecular mechanism of VEGFA-mediated osteogenesis was further verified.(9)We divided the experiment into control group(adding an equal amount of DMSO solvent),simple VEGFA stimulation,simple Ki8751(receptor VEGFR2 inhibitor)intervention,and VEGFA stimulation group after Ki8751 intervention,and detected the migration of VEGFA on AS ligament stem cells through the migration chamber to detect the impact of ability.Part ?:(1)We selected and constructed the U937 cell line that was stably interfered with VEGFA and designed different groups,and verified the interaction of macrophages through VEGFA-mediated osteogenic differentiation of ligament stem cells through co-culture with AS lumbar ligament stem cells.(2)We constructed the type of proteoglycan-induced arthritis(PGIA)model as AS mouse model induced by human cartilage proteoglycan and separated the spine and surrounding tissues and make tissue sections,and applied small animal X-ray,hematoxylin-eosin staining(HE staining),Safranin O-Fast Green staining to take pictures of pathological staining of the spine for testing the success of the model construction.(3)The expression of VEGFA in the spinal joint disc and surrounding tissues and serum of AS mouse model and normal control group were detected by IHC staining and ELISA.(4)Ki8751 intervention(VEGFR2inhibitor)experiment on AS mouse model: the experiment was divided into 3 groups,namely the negative control group(inhibitor solvent injection),the early inhibitor intervention group(intervention starting at week 0 of the experimental process),and the intermediate inhibitor intervention group(intervention starting at 10 weeks of the experimental process),and aimed to explore the effect of inhibiting VEGF receptor signal in AS mouse animal model on spine heterotopic ossification and destruction,and compared the effects of different intervention time courses on the efficacy.(5)We selected osteoblast-related transcription factors(Osterix)as osteoblast marker molecules and selected CD44 and CD90 as stem cell marker molecules;performed pathology on osteoblasts and mesenchymal stem cells of AS model spinal tissue slices through IHC and IF staining respectively;compared the number of the two kinds of cells around the joint disc between the model group and the intervention group injected with Ki8751 to test the role of the VEGFR2 signal axis in the migration and osteogenic differentiation of stem cells in the mouse spine.ResultsPart ?:(1)Through single-cell transcriptomics sequencing and analysis,a total of 26 cell subpopulations were identified in the AS and control group(lumbar fracture patients)at the lumbar ligament attachment points.There was a strong correlation between M2 macrophage subpopulation and the stem cell subpopulation,and they were all characterized by high differentiation in AS.Bioinformatics analysis showed that M2macrophages(subgroup 10)in AS may further promote the osteogenic differentiation of stem cell subgroup(subgroup 0)by overexpressing VEGFA.(2)The detection of VEGFA expression in each sample showed that it was highly expressed in the serum,monocytes,and lumbar ligament tissues of AS patients.Part ?:(1)After fluorescence labeling of lumbar ligament macrophages and VEGFA in AS and the control group,correlation analysis results showed that macrophages and VEGFA in AS lumbar ligament were increased compared with the control group,and the number of the two was positively correlated.(2)The results of flow cytometry showed that M2 macrophages and ligament stem cells met the requirements.(3)TNF-?stimulation experiments showed that it could promote the secretion of VEGFA of M2 macrophages of AS patients,and the secretion volume increased with the increase of the stimulation concentration.(4)Osteogenic induction experiments of ligament stem cells showed that the osteogenic differentiation ability of AS ligament mesenchymal stem cells was stronger than that of the control group.(5)The culture supernatant of M2 macrophages and VEGFA stimulation experiments in AS patients showed that VEGFA can promote the osteogenic differentiation ability of ligament stem cells and the expression of osteogenesis-related genes.(6)Through GO analysis of VEGFA receptor-related enriched genes in AS ligament stem cells,it was found that the activation of PI3K/Akt may be involved in the osteogenic differentiation of AS lumbar ligament stem cells.(7)Integrating the fluorescence co-localization of the VEGFA ligand receptor on AS lumbar ligament stem cells,stimulation experiment VEGFA receptor phosphorylation detection,and signal pathway detection,we found that VEGFA could activate the AS lumbar ligament stem cells in the process of osteogenic differentiation,phosphorylation of surface VEGFR2 receptor,and PI3K/Akt signaling pathway.(8)The above pathway inhibition test and receptor inhibition test showed that the VEGFA-mediated osteogenic effect of AS lumbar ligament stem cells could be reduced by inhibitors,suggesting that it may mediate the osteogenesis phenotype through the two factors.(9)The cell migration experiment confirmed that VEGFA could promote the migration of AS lumbar ligament stem cells,and inhibiting VEGFR2 could prevent their migration.Part ?:(1)The expression of VEGFA in the U937 cell line after lentiviral interference was detected by RT-q PCR and Western Blot.Compared with the U937 cell line in the non-interference group,the VEGFA expression in the U937 cell line was significantly lower,suggesting that the construction of VEGFA stably interfering U937 cell line was successful;co-culture experiments showed that under the stimulation of the inflammatory microenvironment represented by TNF-?,AS macrophages could secrete VEGFA in the form of M2 to stimulate the osteogenic differentiation of lumbar ligament stem cells.After interfering with VEGFA,the bone-promoting ability of macrophages decreased.(2)Small animal X-ray,HE staining,and Safranin-Fast Green staining showed: AS mice spinal vertebral space became narrow.Not only that,osteophyte formation,loss of intervertebral disc cartilage,vertebral body fusion,and the overall appearance of "bamboo-like changes" were discovered,indicating that the model was built successfully.(3)The test showed that VEGFA is highly expressed in the spinal joint disc and surrounding tissues and serum of AS mice.(4)We designed two VEGFR2inhibitors(Ki8751)interventions for AS model mice in the early and middle stages of the disease.The results showed that compared with the model group,the intervention group could reduce the spinal autoinflammatory ankylosis score of AS mice,and the effect of early intervention was better.(5)Comparing the stem cell fluorescence double-labeling and osteoblast immunohistochemistry results using and not using the inhibitor intervention,blocking the VEGFA/VEGFR2 signal axis of the AS mouse model could inhibit the migration and osteogenic differentiation of stem cells in the tissues around the spine.ConclusionsThis study demonstrated the cellular composition of the pathological microenvironment at the lumbar ligament attachment site in ankylosing spondylitis through three-part experiments,and focused on the interaction between macrophages and ligament mesenchymal stem cells at the lumbar ligament attachment site.This study revealed the molecular mechanism of macrophages expressing a large amount of VEGFA protein in the inflammatory microenvironment of AS,targeting VEGFR2 receptor on the surface of peripheral ligament mesenchymal stem cells through paracrine,and mediating its migration and osteogenic differentiation through activation of PI3K/Akt pathway.VEGFA and its receptors are expected to be potential therapeutic targets for the pathological heterotopic bone formation of AS,providing theoretical basis for the development and use of targeted anti-ossification drugs in clinical practice. |
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