Secreted Phosphoprotein 1 Research Articles

Suppression of DNMT2/3 by proinflammatory cytokines inhibits CtBP1/2-dependent genes to promote the occurrence of atrophic nonunion

Failure of bone healing after fracture often results in nonunion, but the underlying mechanism of nonunion pathogenesis is poorly understood. Herein, we provide evidence to clarify that the inflammatory microenvironment of atrophic nonunion (AN) mice suppresses the expression levels of DNA methyltransferases 2 (DNMT2) and 3A (DNMT3a), preventing the methylation of CpG islands on the promoters of C-terminal binding protein 1/2 (CtBP1/2) and resulting in their overexpression. Increased CtBP1/2 acts as transcriptional corepressors that, along with histone acetyltransferase p300 and Runt-related transcription factor 2 (Runx2), suppress the expression levels of six genes involved in bone healing: BGLAP (bone gamma-carboxyglutamate protein), ALPL (alkaline phosphatase), SPP1 (secreted phosphoprotein 1), COL1A1 (collagen 1a1), IBSP (integrin binding sialoprotein), and MMP13 (matrix metallopeptidase 13). We also observe a similar phenomenon in osteoblast cells treated with proinflammatory cytokines or treated with a DNMT inhibitor (5-azacytidine). Forced expression of DNMT2/3a or blockage of CtBP1/2 with their inhibitors can reverse the expression levels of BGLAP/ALPL/SPP1/COL1A1/IBSP/MMP13 in the presence of proinflammatory cytokines. Administration of CtBP1/2 inhibitors in fractured mice can prevent the incidence of AN. Thus, we demonstrate that the downregulation of bone healing genes dependent on proinflammatory cytokines/DNMT2/3a/CtBP1/2-p300-Runx2 axis signaling plays a critical role in the pathogenesis of AN. Disruption of this signaling may represent a new therapeutic strategy to prevent AN incidence after bone fracture.

TMIC-39. PERIVASCULAR CONTAINMENT OF T CELLS IN IDH-MUTANT ASTROCYTOMA ASSOCIATES WITH HIGH ABUNDANCE OF GEMISTOCYTIC TUMOR CELLS AND IMMUNE STIMULATORY MICROGLIA

Abstract Isocitrate dehydrogenase mutant (IDHmt) astrocytoma is considered a T cell-deficient tumor, yet little is known regarding the underlying mechanism that drives T cell exclusion. To this end, we have systematically characterized the IDHmt astrocytoma immune microenvironment. We used multiplex immunofluorescence stainings (n=75; 1293 regions), bulk (n=152) and single nucleus RNA-sequencing (snRNAseq) (n=8) alongside spatial proteomics and RNA sequencing (n=12; 72 regions of interest) on samples from one patient cohort from the Erasmus Medical Center in the Netherlands, enabling integrative spatially resolved multi-omics analysis. We identified three spatially distinct T cell distributions in IDHmt astrocytoma: perivascular accumulation of T cells (T cell cuffs); presence of T cells in the tumor stroma; and absence of T cells. T cells were most frequently present in cuffs. Notably, T cell cuffs were characterized by a high abundance of a histologically distinct tumor cell type called gemistocytes. Conversely, we find gemistocyte high regions to be enriched for T cell cuffs, B cells and tumor associated macrophages (TAMs). Gemistocyte high tumors have an increased expression of immune cell migration and activation programs. SnRNAseq demonstrates that these programs originate from TAMs and T cells. Interestingly, we find that gemistocytic tumor cells form a transcriptionally distinct tumor cell sub-cluster of the astrocyte-like tumor cell state that expresses CD44. Spatial transcriptomics consequently confirmed that gemistocytes co-occurred with a population of immune stimulatory microglia that expresses the CD44-ligand Secreted Phosphoprotein 1 (SPP1), the chemo-attractants CCL3, CCL4 and CCL4L2, and markers for reactive microglia. In summary, we systematically uncovered a previously unrecognized cellular network between T cells, gemistocytic tumor cells and a subpopulation of immune-stimulatory microglia. This spatially confined population of TAMs, together with gemistocytic tumor cells, harbors signatures associated with astrogliosis, a CNS-specific mechanism of T cell exclusion, and thus may explain the perivascular confinement of T cells.

In silico molecular docking studies of 2, 4 and 2, 6-di-tert-butylphenol against NAGK and SPP1 proteins to recuperate rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that primarily affects the synovial joints. It is connected to progressive disability, early death and socioeconomic burdens. So, there is an urgent need to develop novel drugs that effectively treat patients at each stage of disease progression. N-acetylglucosamine kinase (NAGK) and secreted phosphoprotein 1(SPP1) are two attractive drug targets for RA. NAGK protein converts endogenous N-acetyl-D-glucosamine (GlcNAc) into GlcNAc 6-phosphate. GlcNAc has suppressive effects on experimental RA in mouse models. SPP1 stimulates the synthesis of a key proinflammatory cytokine implicated in the pathogenesis of RA. In order to design new drug candidates, crystal structure of human NAGK (PDB Id: 2CH5) was retrieved from the protein data bank. Amino acid sequence of SPP1 (Uniprot Id: P10451) was retrieved from uniprot database and modeled by the Phyre2 server. Computed SPP1 model energy was minimized using the YASARA energy minimization server and validated by the Ramachandran plot and the ProSA-web error-detection tool. 2, 4-Di-tert-butylphenol and 2, 6-Di-Tert-butylphenol are known compound isolates from bark extract of Schleichera oleosa having anti-arthritic and anti-inflammatory activities as accessed by PASS online server. These compounds were docked against active site of NAGK and SPP1 proteins using MTiAutoDock server. The binding affinity of these compounds against NAGK and SPP1 proteins ranges from -6.12 to -7.17 kcal/mol. The findings of this study will open the way for the development of herbal remedies for rheumatoid arthritis based on the Schleichera oleosa plant, potentially leading to the development of novel drugs.

Exosomes derived from mesenchymal stromal cells exert a therapeutic effect on hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway

ObjectiveHypoxic pulmonary hypertension (HPH) is a progressive and life-threatening disease characterized by perivascular inflammation, pulmonary vascular remodeling, and occlusion. Mesenchymal stromal cell-derived exosomes (MSC-exo) have emerged as potential therapeutic agents due to their role in cell communication and the transportation of bioactive molecules. In this study, we aimed to investigate the therapeutic effects of MSC-exo against HPH and elucidate the underlying molecular mechanism. MethodsExosomes were isolated from conditioned media of human bone mesenchymal stromal cells using ultracentrifugation and characterized through western blotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). An HPH animal model was established in male SD rats, and MSC-exo or phosphate-buffered saline (PBS) were administered via the tail vein for three weeks. Subsequently, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. Lung tissues from HPH rats and normal rats underwent high-throughput sequencing and transcriptomic analysis. Gene Ontology (GO) analysis was employed to identify upregulated differentially expressed genes. Additionally, rat pulmonary artery smooth muscle cells (PASMC) exposed to platelet-derived growth factor-BB (PDGF-BB) were used to simulate HPH-related pathological behavior. In vitro cellular models were established to examine the molecular mechanism of MSC-exo in HPH. ResultsMSC-exo administration protected rats from hypoxia-induced increases in RVSP, RVHI, and pulmonary vascular remodeling. Additionally, MSC-exo alleviated PDGF-BB-induced proliferation and migration of PASMC. Transcriptomic analysis revealed 267 upregulated genes in lung tissues of HPH rats compared to control rats. Gene Ontology analysis indicated significant differences in pathways associated with Yes Associated Protein 1 (YAP1), a key regulator of cell proliferation and organ size. RT-qPCR and western blot analysis confirmed significantly increased expression of YAP1 in HPH lung tissues and PASMC, which was inhibited by MSC-exo treatment. Furthermore, analysis of datasets demonstrated that Secreted Phosphoprotein 1 (SPP1), also known as Osteopontin (OPN), is a downstream binding protein of YAP1 and can be upregulated by PDGF-BB. MSC-exo treatment reduced the expression of both YAP1 and SPP1. Lentivirus-mediated knockdown of YAP1 inhibited PDGF-BB-induced PASMC proliferation, migration, and SPP1 protein levels. ConclusionOur findings demonstrate that MSC-exo exert a therapeutic effect against hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway. The inhibition of YAP1 and downstream SPP1 expression by MSC-exo may contribute to the attenuation of pulmonary vascular remodeling and PASMC proliferation and migration. These results suggest that MSC-exo could serve as a potential therapeutic strategy for the treatment of HPH. Further investigations are warranted to explore the clinical applicability of MSC-exo-based therapies in HPH patients.

Divergent tumor and immune cell reprogramming underlying immunotherapy response and immune-related adverse events in lung squamous cell carcinoma

BackgroundLung squamous cell carcinoma (LUSC) remains a leading cause of cancer-related deaths with few therapeutic strategies. Immune checkpoint inhibitors (ICIs) have demonstrated promising efficacy in patients with LUSC. However, ICIs...

Associations of cortical SPP1 and ITGAX with cognition and common neuropathologies in older adults.

The secreted phosphoprotein 1 (SPP1) gene expressed by CD11c+ cells is known to be associated with microglia activation and neuroinflammatory diseases. As most studies rely on mouse models, we investigated these genes and proteins in the cortical brain tissue of older adults and their role in Alzheimer's disease (AD) and related disorders. We leveraged protein measurements, single-nuclei, and RNASeq data from the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) of over 1200 samples for association analysis. Expression ofSPP1and its encoded protein osteopontin were associated with faster cognitive decline and greater odds of common neuropathologies. At single-cell resolution, integrin subunit alpha X (ITGAX) was highly expressed in microglia, where specific subpopulations were associated with AD and cerebral amyloid angiopathy. The study provides evidence of SPP1andITGAXassociation with cognitive decline and common neuropathologies identifying a microglial subset associated with disease.

Role of SPP1 in the diagnosis of gastrointestinal cancer.

Recently, the incidence rate of digestive system tumors has increased in China and these tumors occur in a younger population. The present study aimed to determine the expression levels and potential clinical value of secreted phosphoprotein 1 (SPP1) in gastrointestinal cancer. The microarray datasets GSE104836, GSE189830 and GSE103236, obtained from the gene expression omnibus database, were analyzed to determine differentially expressed genes in patients with colorectal cancer (CRC), gastric cancer (GC) and esophageal cancer (EC). A total of 42 patients with CRC, GC or EC and 21 healthy controls were recruited to obtain blood and tissues samples. SPP1 expression levels were detected using reverse transcription-quantitative PCR. Moreover, levels of significance of SPP1 in patients with CRC, GC and EC were analyzed using receiver operating characteristic analysis. Potential correlations between SPP1 and carcinoembryonic antigen (CEA) were assessed using Pearson's correlation coefficient. SPP1 was significantly upregulated in the serum, plasma and tissue of patients with CRC, GC or EC. In addition, the area under the curve of SPP1 was >0.5 in the plasma, serum and cancer tissue of patients with early and late CRC, GC or EC. The present study further demonstrated that the specificity and sensitivity of SPP1 was higher in patients with late CRC, GC or EC compared with patients with early CRC, GC or EC. Moreover, SPP1 and CEA were significantly positively correlated in serum of patients with CRC, GC or EC. In conclusion, the current study demonstrated that SPP1 exhibited significant diagnostic value for gastrointestinal tumors, which suggested that SPP1 may exhibit potential as a diagnostic marker of CRC, GC and EC. The present study provided a novel theoretical basis for the role of SPP1 as a diagnostic marker of digestive system tumors.

Secreted phosphoprotein 1 exacerbates renal ischemia-reperfusion injury by inhibiting PI3K/AKT signaling pathway

BackgroundRenal ischemia-reperfusion injury (IRI) is a prevalent reason for acute kidney injury and a key clinical issue for patients under anesthesia and about to have surgery. We aim to investigate the Secreted phosphoprotein 1 (SPP1) role in renal IRI and the underlying mechanisms. MethodsUsing Gene Expression Omnibus (GEO) database helped in analyzing the SPP1 expression in renal IRI. We established two models, a mouse renal ischemia-reperfusion (I/R) besides a hypoxia-reoxygenation (H/R) HK-2 cell. Renal tubular lesions were measured using H&E staining. Depending on the TUNEL assay, immunohistochemistry, qRT-PCR, as well as western blot, we applied the assessment of apoptosis and apoptosis-associated protein levels. At the same time, a western blot was performed for assessing PI3K/AKT pathway-associated proteins. ResultsGEO data and experimental validation revealed elevated SPP1 content in the kidney tissues of renal I/R mice more than in sham mice. In vitro and in vivo studies revealed an increase in cell apoptosis due to SPP1 overexpression, but the opposite is true when SPP1 is silenced. SPP1 downregulation led to high p-PI3K and p-AKT protein levels, and the LY294002 application inhibited SPP1 inhibition-mediated anti-apoptotic effect ConclusionTaken together, SPP1 exacerbates renal IRI in vivo and in vitro via promoting programmed cell death by inhibiting PI3K/AKT signaling pathway.

MIF is a critical regulator of mononuclear phagocytic infiltration in hepatocellular carcinoma

Immunotherapy targeting tumor-associated macrophages (TAMs) is a promising approach to treating cancer. However, the limited drug targets and ambiguous mechanisms impede the development of clinical immunotherapy strategies. To elucidate the underlying processes involved in mononuclear phagocyte (MNP) infiltration and phenotypic changes in hepatocellular carcinoma (HCC), we integrated single-cell RNA-sequencing data from 100,030 cells derived from patients with HCC and healthy individuals and compared the phenotypes and origins of the MNPs in the tumor core, tumor periphery, adjacent normal tissue, and healthy liver samples. Using machine learning and multi-omics analyses, we identified 445 infiltration-associated genes and potential drug targets affecting this process. Through invitro experiments, we found that the expression of macrophage migration inhibitory factor (MIF) is the upstream regulator of secreted phosphoprotein 1 (SPP1) and promote migration in TAMs. Our findings also indicate that MIF promotes tumor metastasis and invasion and is a promising potential target for treating HCC.

Secreted phosphoprotein 1 regulates natural compound 3’,4’,5,7-tetrahydroxyflavone to inhibit mast cell-mediated allergic inflammation

BackgroundMast cells (MCs) are important effector cells in anaphylaxis and anaphylactic disease. 3’,4’,5,7-tetrahydroxyflavone (THF) presents in many medicinal plants and exerts a variety of pharmacological effects. In this study, we evaluated the effect of THF on C48/80-induced anaphylaxis and the mechanisms underlying its effects, including the role of secreted phosphoprotein 1 (SPP1), which has not been reported to IgE-independent MC activation.ResultsTHF inhibited C48/80-induced Ca2+ flow and degranulation via the PLCγ/PKC/IP3 pathway in vitro. RNA-seq showed that THF inhibited the expression of SPP1 and downstream molecules. SPP1 is involved in pseudo-anaphylaxis reactions. Silencing SPP1 affects the phosphorylation of AKT and P38. THF suppressed C48/80-induced paw edema, hypothermia and serum histamine, and chemokines release in vivo.ConclusionsOur results validated SPP1 is involved in IgE-independent MC activation anaphylactoid reactions. THF inhibited C48/80-mediated anaphylactoid reactions both in vivo and in vitro, suppressed calcium mobilization and inhibited SPP1-related pathways.

P-805 Gene expression of human endometrial organoids hormonally treated in vitro for 28 days

Abstract Study question Does gene expression of human endometrial organoids (EO) mimic the in vivo response to a 28- days substitutive hormonal treatment? Summary answer Gene expression profile of human EO exposed to 28-day hormone treatment showed correspondence with the phases of the menstrual cycle in vivo. What is known already The recent development of human EO demonstrated their translational potential for reproductive medicine. Current studies testing functional response to hormones of EO are based on short-term responsiveness to estrogen (E2), progesterone (P4) and differentiation factors up to 10 days. It is unknown how organoids can be used to faithfully reproduce and/or model a complete menstrual cycle of 28 days. High-throughput analyses on endometrium have identified specific markers available for characterizing differentiation and functional aspects related to secretory activity and receptivity to implantation. Study design, size, duration Experimental research was carried out including 4 endometrial biopsies donated by women with normal endometrium function, recruited at an academic center for assisted reproduction between 2022-06-15 to 2022-07-28. These endometrial biopsies were processed for organoid derivation and culture. The EO were hormonally treated in vitro and analyzed for gene expression. Participants/materials, setting, methods Human endometrial biopsies were enzymatically digested to isolate endometrial glands, embedded in matrigel and cultivated in a spinning bioreactor to achieve organoid formation. Thereafter EO were treated as following: 1) E2: (day 0–28); 2) E2 + P4: E2 (day 0–28), P4 (day 14–28); 3) E2 + P4 + dibutyryl cyclic Adenosine monophosphate (dbcAMP): E2 (day 0–28) + P4 (day 14–28) + dbcAMP (day 14–28) or 4) control group. Main results and the role of chance A total of 10 markers were selected for functional analysis and 3 showed a pattern of interest. The target gene expression included Forkhead box O1 (FOXO1), involved in regulating endometrium receptivity in human; progesterone associated endometrium protein (PAEP), a major protein in glandular endometrium secretions; and secreted phosphoprotein 1 (SPP1), an extracellular cytokine that increases in endometrium during the receptive phase. Gene expression of FOXO1, PAEP and SPP1 showed a significant increase in the organoids exposed to complete hormonal cycle, compared to controls. PAEP and SPP1 gene expression was lower up to day 21 but upregulated to 15-fold relative to non-treated group by day 28. This pattern recapitulates a similar response to the physiological conditions in vivo in the human endometrium. Although gene expression patterns followed similar trends, individual biopsies had slight variations in gene expression along 28-day hormone treatment. The 28 day-hormone treatment can stimulate EO for recapitulating histological and functional aspects. The dynamics resembled a timely correlation with gene expression changes accounting for some specific markers mostly involved in receptivity, and thus reinforcing the potential application of EO for further research purposes. Limitations, reasons for caution In vitro organoid culture conditions may have some influence over responsiveness and gene expression that requires further optimization to reproduce uterine environment in vitro and for other conditions related to infertility in a completely defined culture system. Wider implications of the findings Our findings enable the opportunity to expand experimental research over endometrial disorders affecting different stages of the menstrual cycle and also to improve research related to embryo-endometrium interactions. Trial registration number This research has been funded by grants from The Swedish Cancer Society, The Swedish Childhood Cancer Foundation, The Cancer Research Funds of Radiumhemmet, The Swedish Cancer Association BRO, the Stockholm County Council (CIMED) and Karolinska Institutet.

Osteopontin is a prognostic circulating biomarker in patients with neuroendocrine neoplasms

PurposeOsteopontin (OPN), also called secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and which has been shown to be involved in tumorigenesis and metastasis in many malignancies. Its role in neuroendocrine neoplasms (NEN) remains to be established. The aim of the study was to analyze plasma concentrations of OPN in patients with NEN and to explore its diagnostic and prognostic value as a clinical biomarker.MethodsOPN plasma concentrations were measured in a total of 38 patients with histologically proven NEN at three different time points during the course of disease and therapy (at the start of the study, after 3 and 12 months, respectively) as well as in healthy controls. Clinical and imaging data as well as concentrations of Chromogranin A (CgA) and Neuron Specific Enolase (NSE) were assessed.ResultsOPN levels were significantly higher in patients with NEN compared to healthy controls. High-grade tumors (grade 3) showed the highest OPN levels. OPN levels were neither different between male and female patients nor between different primary tumor sites. OPN correlated significantly with corresponding NSE levels, while there was no correlation with Chromogranin A. High OPN levels above a cutoff value of 200 ng/ml at initial analysis predicted a worsened prognosis with significantly shorter progression-free survival of patients with NEN, which also held true within the subgroup of well-differentiated G1/G2 tumors.ConclusionOur data indicate that high baseline OPN levels in patients with NEN are predictive of an adverse outcome with shorter progression-free survival, even within the group of well differentiated G1/G2 tumors. Therefore, OPN may be used as a surrogate prognostic biomarker in patients with NEN.

Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†.

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

SPP1 as a risk factor for patients with acute on chronic liver failure undergoing liver transplantation

BackgroundAcute on chronic liver failure (ACLF) is characterized by systemic inflammation and significant mortality, calling for accurate assessment due to the diverse prognosis of liver transplantation (LT). Methods8 patients with ACLF and 4 normal controls (NC) underwent peripheral blood mononuclear cells (PBMCs) transcriptomics, whereas 9 patients with ACLF and 3 NC had hepatic CD45+ T cells transcriptomics. Thecandidateindicatorfoundinthetranscriptomicswas confirmedbya retrospective cohort (n = 137) and one prospective cohort (n = 68). ResultsTranscriptomics revealed significant differentially expression genes (DEGs) and bioprocesses related to the PBMCs and hepatic CD45+ T cells. Secreted phosphoprotein 1 (SPP1) was identified as a potential indicator for ACLF patients receiving LT, which was supported by evidence from the cross-sectional cohorts. As the condition of ACLF got worse, so did SPP1 levels, which were associated with liver failure and coagulation failure. SPP1 levels prior to LT were considerably greater in non-survivors of ACLF within 90 days than that in survivors. In the derivation cohort and validation cohort, ACLF patients with elevated SPP1 levels had significantly shorter cumulative survival durations than those with low SPP1 levels, P = 0.02 and P < 0.001, respectively. The SPP1-MELD and SPP1-chronic liver failure consortium (CLIF-C) ACLF scores had comparatively larger areas under the receiver operating characteristic curves (AUCs) than MELD (P = 0.0388) and CLIF-C ACLF (P = 0.045). ConclusionsThe circulating SPP1 showed promise as a predictor for ACLF patients receiving LT, which demonstrated the need for tracking the clinical outcome of LT.

Integrated bioinformatics analysis for exploring hub genes and related mechanisms affecting the progression of gastric cancer

ABSTRACT Objective Gastric cancer (GC) is a high-risk tumor disease worldwide. The goal of the current study was to explore new diagnostic and prognostic indicators for gastric cancer. Methods Database GSE19826 and GSE103236 were gained from the Gene Expression Omnibus (GEO) to screen for differentially expressed genes (DEGs), which were then grouped together as co-DEGs. GO and KEGG pathway analysis were used to investigate the function of these genes. The protein-protein interaction (PPI) network of DEGs was constructed by STRING. Results GSE19826 selected 493 DEGs in GC and gastric normal tissues, including 139 up-regulated genes and 354 down-regulated genes. A total of 478 DEGs were selected by GSE103236, including 276 up-regulated genes and 202 downregulated genes. 32 co-DEGs were overlapped from two databasesand involved in digestion, regulation of response to wounding, wound healing, potassium ion imports across plasma membrane, regulation of wound healing, anatomical structure homeostasis, and tissue homeostasis. KEGG analysis revealed that co-DEGs were mainly involved in ECM-receptor interaction, tight junction, protein digestion and absorption, gastric acid secretion and cell adhesion molecules. Twelve hub genes were screened by Cytoscape, including cholecystokinin B receptor (CCKBR), Collagen type I alpha 1 (COL1A1), COL1A2, COL2A1, COL6A3, COL11A1, matrix metallopeptidase 1 (MMP1), MMP3, MMP7, MMP10, tissue inhibitor of matrix metalloprotease 1 (TIMP1) and secreted phosphoprotein 1 (SPP1). Conclusions Twelve key genes affecting the progression of gastric cancer were obtained by bioinformatics, which may be potential biomarkers for the diagnosis and prognosis of GC.

Vitamin C protects retinal ganglion cells via SPP1 in glaucoma and after optic nerve damage.

Glaucoma is a common neurodegenerative disorder characterized by retinal ganglion cell death, astrocyte reactivity in the optic nerve, and vision loss. Currently, lowering the intraocular pressure (IOP) is the first-line treatment, but adjuvant neuroprotective approaches would be welcome. Vitamin C possesses neuroprotective activities that are thought to be related to its properties as a co-factor of enzymes and its antioxidant effects. Here, we show that vitamin C promotes a neuroprotective phenotype and increases gene expression related to neurotropic factors, phagocytosis, and mitochondrial ATP production. This effect is dependent on the up-regulation of secreted phosphoprotein 1 (SPP1) in reactive astrocytes via the transcription factor E2F1. SPP1+ astrocytes in turn promote retinal ganglion cell survival in a mouse model of glaucoma. In addition, oral administration of vitamin C lowers the IOP in mice. This study identifies an additional neuroprotective pathway for vitamin C and suggests a potential therapeutic role of vitamin C in neurodegenerative diseases such as glaucoma.

Vitamin D deficiency triggers intrinsic apoptosis by impairing SPP1-dependent antiapoptotic signaling in chronic hematogenous osteomyelitis

Chronic hematogenous osteomyelitis (CHOM) is a common bone disease characterized by the development of sequestra after bacterial infection. Emerging evidence has shown that vitamin D (VD) deficiency raises the risk of osteomyelitis, but the underlying mechanisms remain obscure. Here, we establish a CHOM model in VD diet-deficient mice by intravenous inoculation of Staphylococcus aureus. Whole-genome microarray analyses using osteoblast cells isolated from sequestra reveal significant downregulation of SPP1 (secreted phosphoprotein 1). Molecular basis investigations show that VD sufficiency activates the VDR/RXR (VD receptor/retinoid X receptor) heterodimer to recruit NCOA1 (nuclear receptor coactivator 1) and transactivate SPP1 in healthy osteoblast cells. Secreted SPP1 binds to the cell surface molecule CD40 to activate serine/threonine-protein kinase Akt1, which then phosphorylates forkhead box O3a (FOXO3a), blocking FOXO3a-mediated transcription. By contrast, VD deficiency impairs the NCOA1-VDR/RXR-mediated overexpression of SPP1, leading to the inactivation of Akt1 and the accumulation of FOXO3a. FOXO3a then upregulates the expression of the apoptotic genes BAX (Bcl2-associated X-protein), BID (BH3 interacting death domain), and BIM (Bcl2-interacting mediator of cell death), to induce apoptosis. Administration of the NCOA1 inhibitor gossypol to the CHOM mice also promotes the occurrence of sequestra. VD supplementation can reactivate the SPP1-dependent antiapoptotic signaling and improve the outcomes of CHOM. Collectively, our data reveal that VD deficiency promotes bone destruction in CHOM by the removal of SPP1-dependent antiapoptotic signaling.

Spatiotemporal Characterization of Human Early Intervertebral Disc Formation at Single-Cell Resolution.

The intervertebral disc (IVD) acts as a fibrocartilaginous joint to anchor adjacent vertebrae. Although several studies have demonstrated the cellular heterogeneity of adult mature IVDs, a single-cell transcriptomic atlas mapping early IVD formation is still lacking. Here, the authors generate a spatiotemporal and single cell-based transcriptomic atlas of human IVD formation at the embryonic stage and a comparative mouse transcript landscape. They identifytwo novel human notochord (NC)/nucleus pulposus (NP) clusters, SRY-box transcription factor 10 (SOX10)+ and cathepsin K (CTSK)+ , that are distributed in the early and late stages of IVD formation and they are validated by lineage tracing experiments in mice. Matrisome NC/NP clusters, T-box transcription factor T(TBXT)+ and CTSK+ , are responsible for the extracellular matrix homeostasis. The IVD atlas suggests that a subcluster of the vertebral chondrocyte subcluster might give rise to an inner annulus fibrosus of chondrogenic origin, while the fibroblastic outer annulus fibrosus preferentially expresseds transgelin and fibromodulin . Through analyzing intercellular crosstalk, the authors further find that notochordal secreted phosphoprotein 1 (SPP1) is a novel cue in the IVD microenvironment, and it is associated with IVD development and degeneration. In conclusion, the single-cell transcriptomic atlas will be leveraged to develop preventative and regenerative strategies for IVD degeneration.

Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib.

Cancer-associated fibroblasts (CAFs) play an important role in the induction of chemo-resistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma (HCC). We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively, to identify key molecules that might induce resistance to TKIs. We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms. The associations of plasma secreted phosphoprotein 1 (SPP1) expression levels before sorafenib/lenvatinib treatment with progression-free survival (PFS) and overall survival (OS) of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis. Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance. SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo. CAF-derived SPP1 activated rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) through the integrin-protein kinase C-alpha (PKCα) signaling pathway and promoted epithelial-to-mesenchymal transition (EMT). A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS (P = 0.026) and OS (P = 0.047) in patients with advanced HCC after TKI treatment. CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion. Its inhibition represents a promising therapeutic strategy against TKI resistance in HCC. Moreover, plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

Abstract TP225: Cell-type-based Pathway Analysis In Experimental Subarachnoid Hemorrhage

Introduction: Recently, increasing evidence points to a pivotal role of neuroinflammation in the pathogenesis of Subarachnoid hemorrhage (SAH). However, the pattern of cell interaction after SAH is not clear. Methods: Endovascular perforation (EVP) murine SAH model was established to reproduce experimental SAH. Post-SAH CD11b+ single-cell suspension was harvested at day 3 and sequenced using 10X single-cell RNA-sequencing platform. Then, the detailed single-cell information of post-SAH cells was analyzed with bioinformatics. The same pathways and related ligand-receptors in microglia and other cells were compared. Results: More than 51 pathways were found in the crosstalk of brain cells after SAH, these cells including: microglia (microglia worked in 25 pathways), CNS-associated macrophage, astrocyte, neuron, monocyte and macrophage and Oligodendrocyte. In the microglia subsets’ interaction, 15 pathways upregulated, of which four pathways were specific to microglia subsets, including amyloid-β precursor protein (APP), Tumor necrosis factor (TNF), growth differentiation factor (GDF) and oncostatin M (OSM) signaling pathways. We precisely distinguished the ligand-receptor pattern in which microglia differ with other cells in 7 pathways, including chemokine ligands (CCL), colony-stimulating factor (CSF), GALECTIN, growth arrest-specific (GAS), intercellular adhesion molecule (ICAM), junction adhesion molecule (JAM) and secreted phosphoprotein 1 (SPP1) signaling pathways. Gene ontology (GO) results showed that compared with non-microglia cells, post-SAH microglia with upregulated function like leukocyte, glia cell activation and migration, while have downregulated functions of protein-lipid complex remodeling. Conclusions: Collectively, we first systematically report the interaction patterns of seven brain cells after SAH. Further the cell type based ligand-receptor analysis of microglia and other cells in the same pathway was comparatively conducted. These results revealed the cellular mechanisms for neuroinflammation after SAH, and the ligand-receptor results provided a theoretical basis and opportunities for accurate monitoring and modulation of SAH in the future.