SPP1 expression indicates outcome of immunotherapy plus tyrosine kinase inhibition in advanced renal cell carcinoma.

Clinical guidelines have recently advised combination therapy involving immunotherapy (IO) and tyrosine kinase inhibitors (TKI) as the first-line therapy approach for advanced renal cell carcinoma (RCC). Nevertheless, there is currently no available biomarker that can effectively distinguish the progression-free survival (PFS). RNA-sequencing and immunohistochemistry were conducted on our cohort of metastatic RCC patients, namely ZS-MRCC, who received combination therapy consisting of IO and TKI. We further applied RNA-sequencing, immunohistochemistry, and flow cytometry to examine the immune cell infiltration and functionality inside the tumor microenvironment of high-risk localized RCC samples. SPP1 expression was significantly higher in non-responders to IO-TKI therapy. Elevated levels of SPP1 were associated with poor PFS in both the ZS-MRCC cohort (HR = 2.73, p = .018) and validated in the JAVELIN Renal 101 cohort (HR = 1.61, p = .004). By multivariate Cox analysis, SPP1 was identified as a significant independent prognosticator. Furthermore, there existed a negative correlation between elevated levels of SPP1 and the presence of GZMB+CD8+ T cells (Spearman's ρ= -0.48, p < .001). Conversely, SPP1 expression is associated with T cell exhaustion markers. A significant increase in the abundance of Tregs was observed in tumors with high levels of SPP1. Additionally, a machine-learning-based model was constructed to predict the benefit of IO-TKI treatment. High SPP1 is associated with therapeutic resistance and unfavorable PFS in IO-TKI therapy. SPP1 expression have also been observed to be indicative of malfunction and exhaustion in T cells. Increased SPP1 expression has the potential to serve as a potential biomarker for treatment selection of metastatic RCC.

Selected osteointegration markers in different timeframes after dental implantation: findings and prognostic value.

The study aimed to determine the osteointegration markers after dental implantation and evaluate their predictive value. The study was performed on 60 practically healthy persons who needed teeth rehabilitation using dental implants. The conical-shaped implants (CI) and hexagonal implants (HI) were used. The content of Osteopontin (OPN), Osteocalcin (OC), Alkaline Phosphatase (ALP), Osteoprotegerin (OPG), and nitric oxide (NO) was determined in patients' gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF), collected 1, 3, and 6 months after implantation. During the 3-6 months of observation level of OPN increased in patients with CIs (<50 years > 50 years) and HIs (<50 years) (CI: <50 years F = 36.457, p < 0.001; >50 years F = 30.104, p < 0.001; HI < 50 years F = 2.246, p < 0.001), ALP increased in patients with CIs (<50 years: F = 19.58, p < 0.001; >50 years: F = 12.01; p = 0.001) and HIs (<50 years) (F = 18.51, p < 0.001), OC increased in patients <50 years (CI: F = 33.72, p < 0.001; HI: F = 55.57, p < 0.001), but in patients >50 years - on the 3 days month (CI: F = 18.82, p < 0.001; HI: F = 26.26, p < 0.001), but sharply decreased at the end of sixth month. OPG increased during 1-3 months of the observation in patients <50 years (CI: F = 4.63, p = 0.037; HI: F = 2.8927, p = 0.046), but at the end of the sixth month returned to the initial level; NO content in PISF increased in patients with CI (>50 years) during 1-6 months of the observation (F = 27.657, p < 0.001). During the post-implantation period, age-related differences in osteointegration were observed. Patients <50 years old had relatively high levels of OPN, ALP, OC, and OPG in PISF, resulting in less alveolar bone destruction around dental implants and more intensive osteointegration. These indicators may be used as biological markers for monitoring implant healing. The process of osseointegration was more intense in CIs due to their comparatively high mechanical loading.

Osteopontin drives neuroinflammation and cell loss in MAPT-N279K frontotemporal dementia patient neurons.

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.

Microglial- neuronal crosstalk in chronic viral infection through mTOR, SPP1/OPN and inflammasome pathway signaling.

HIV-infection of microglia and macrophages (MMs) induces neuronal injury and chronic release of inflammatory stimuli through direct and indirect molecular pathways. A large percentage of people with HIV-associated neurologic and psychiatric co-morbidities have high levels of circulating inflammatory molecules. Microglia, given their susceptibility to HIV infection and long-lived nature, are reservoirs for persistent infection. MMs and neurons possess the molecular machinery to detect pathogen nucleic acids and proteins to activate innate immune signals. Full activation of inflammasome assembly and expression of IL-1ß requires a priming event and a second signal. Many studies have demonstrated that HIV infection alone can activate inflammasome activity. Interestingly, secreted phosphoprotein-1 (SPP1/OPN) expression is highly upregulated in the CNS of people infected with HIV and neurologic dysfunction. Interestingly, all evidence thus far suggests a protective function of SPP1 signaling through mammalian target of rapamycin (mTORC1/2) pathway function to counter HIV-neuronal injury. Moreover, HIV-infected mice knocked down for SPP1 show by neuroimaging, increased neuroinflammation compared to controls. This suggests that SPP1 uses unique regulatory mechanisms to control the level of inflammatory signaling. In this mini review, we discuss the known and yet-to-be discovered biological links between SPP1-mediated stimulation of mTOR and inflammasome activity. Additional new mechanistic insights from studies in relevant experimental models will provide a greater understanding of crosstalk between microglia and neurons in the regulation of CNS homeostasis.

OPN silencing reduces hypoxic pulmonary hypertension via PI3K-AKT-induced protective autophagy.

Hypoxic pulmonary hypertension (HPH) is a pulmonary vascular disease primarily characterized by progressive pulmonary vascular remodeling in a hypoxic environment, posing a significant clinical challenge. Leveraging data from the Gene Expression Omnibus (GEO) and human autophagy-specific databases, osteopontin (OPN) emerged as a differentially expressed gene, upregulated in cardiovascular diseases such as pulmonary arterial hypertension (PAH). Despite this association, the precise mechanism by which OPN regulates autophagy in HPH remains unclear, prompting the focus of this study. Through biosignature analysis, we observed significant alterations in the PI3K-AKT signaling pathway in PAH-associated autophagy. Subsequently, we utilized an animal model of OPNfl/fl-TAGLN-Cre mice and PASMCs with OPN shRNA to validate these findings. Our results revealed right ventricular hypertrophy and elevated mean pulmonary arterial pressure (mPAP) in hypoxic pulmonary hypertension model mice. Notably, these effects were attenuated in conditionally deleted OPN-knockout mice or OPN-silenced hypoxic PASMCs. Furthermore, hypoxic PASMCs with OPN shRNA exhibited increased autophagy compared to those in hypoxia alone. Consistent findings from in vivo and in vitro experiments indicated that OPN inhibition during hypoxia reduced PI3K expression while increasing LC3B and Beclin1 expression. Similarly, PASMCs exposed to hypoxia and PI3K inhibitors had higher expression levels of LC3B and Beclin1 and suppressed AKT expression. Based on these findings, our study suggests that OPNfl/fl-TAGLN-Cre effectively alleviates HPH, potentially through OPN-mediated inhibition of autophagy, thereby promoting PASMCs proliferation via the PI3K-AKT signaling pathway. Consequently, OPN emerges as a novel therapeutic target for HPH.

Tumor-associated macrophage subtypes on cancer immunity along with prognostic analysis and SPP1-mediated interactions between tumor cells and macrophages.

Tumor-associated macrophages (TAM) subtypes have been shown to impact cancer prognosis and resistance to immunotherapy. However, there is still a lack of systematic investigation into their molecular characteristics and clinical relevance in different cancer types. Single-cell RNA sequencing data from three different tumor types were used to cluster and type macrophages. Functional analysis and communication of TAM subpopulations were performed by Gene Ontology-Biological Process and CellChat respectively. Differential expression of characteristic genes in subpopulations was calculated using zscore as well as edgeR and Wilcoxon rank sum tests, and subsequently gene enrichment analysis of characteristic genes and anti-PD-1 resistance was performed by the REACTOME database. We revealed the heterogeneity of TAM, and identified eleven subtypes and their impact on prognosis. These subtypes expressed different molecular functions respectively, such as being involved in T cell activation, apoptosis and differentiation, or regulating viral bioprocesses or responses to viruses. The SPP1 pathway was identified as a critical mediator of communication between TAM subpopulations, as well as between TAM and epithelial cells. Macrophages with high expression of SPP1 resulted in poorer survival. By in vitro study, we showed SPP1 mediated the interactions between TAM clusters and between TAM and tumor cells. SPP1 promoted the tumor-promoting ability of TAM, and increased PDL1 expression and stemness of tumor cells. Inhibition of SPP1 attenuated N-cadherin and ß-catenin expression and the activation of AKT and STAT3 pathway in tumor cells. Additionally, we found that several subpopulations could decrease the sensitivity of anti-PD-1 therapy in melanoma. SPP1 signal was a critical pathway of communication between macrophage subtypes. Some specific macrophage subtypes were associated with immunotherapy resistance and prognosis in some cancer types.

The impact of hydroxyapatite crystal structures and protein interactions on bone's mechanical properties.

Hydroxyapatite (HAP) constitutes the primary mineral component of bones, and its crystal structure, along with the surface interaction with proteins, significantly influences the outstanding mechanical properties of bone. This study focuses on natural hydroxyapatite, constructing a surface model with calcium vacancy defects. Employing a representative model of aspartic acid residues, we delve into the adsorption mechanism on the crystal surface and scrutinize the adsorption forms of amino acid residues on HAP and calcium-deficient hydroxyapatite (CDHA) surfaces. The research also explores the impact of different environments on adsorption energy. Furthermore, a simplified sandwich structure of crystal-polypeptide-crystal is presented, analyzing the distribution of amino acid residue adsorption sites on the crystal surface of the polypeptide fragment. This investigation aims to elucidate how the stick-slip mechanism of polypeptide molecules on the crystal surface influences the mechanical properties of the system. By uncovering the interface mechanical behavior between HAP and osteopontin peptides, this article offers valuable theoretical insights for the construction and biomimetic design of biocomposites.

Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

Osteopontin Rejuvenates Senescent Adipose-Derived Stem Cells and Restores their Bone Tissue Regenerative Function.

The regenerative function of stem cells is compromised when the proportion of senescent stem cells increases with ageing advance. Therefore, combating stem cell senescence is of great importance for stem cell-based tissue engineering in the elderly, but remains largely unexplored. Osteopontin (OPN), a glycosylated phosphoprotein, is one of the key extracellular matrix molecules in bone tissue. OPN activates various signalling pathways and modulates cellular activities, including cell senescence. However, the role of OPN in stem cell senescence remains largely unknown. This study aims to investigate if OPN modulates cell senescence and bone regenerative function in human adipose-derived mesenchymal stem cells (ASCs), and to determine the underlying mechanisms. We first developed a senescent ASC model using serial passaging until passage 10 (P10), in which senescent cells were characterised by reduced proliferation and osteogenic differentiation capacity compared to P4 ASCs. The conditioned medium from P10 ASCs exhibited a diminished trophic effect on human osteoblasts (HOBs), compared to that from P4 ASCs. P10 ASCs on OPN-coated surface showed rejuvenated phenotype and enhanced osteogenic differentiation. The conditioned medium from P10 ASCs on OPN-coating improved trophic effects on HOBs. OPN regulated the morphology of senescent ASCs, transforming them from a more rounded and flattened cell shape to an elongated shape with a smaller area. These findings demonstrated the effects of OPN in restoring senescent ASCs functions, possibly through a mechanism that involves the modulation of cell morphology, indicating that OPN might hold a great potential for rejuvenating senescent stem cells and could potentially open a new venue for regenerating bone tissue in age-related diseases.

SOCS3 regulates pathological retinal angiogenesis through modulating SPP1 expression in microglia and macrophages.

Pathological ocular angiogenesis has long been associated with myeloid cell activation. However, the precise cellular and molecular mechanisms governing the intricate crosstalk between the immune system and vascular changes during ocular neovascularization formation remain elusive. In this study, we demonstrated that the absence of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells led to a substantial accumulation of microglia and macrophage subsets during the neovascularization process. Our single-cell RNA sequencing data analysis revealed a remarkable increase in the expression of the secreted phosphoprotein 1 (Spp1) gene within these microglia and macrophages, identifying subsets of Spp1-expressing microglia and macrophages during neovascularization formation in angiogenesis mouse models. Notably, the number of Spp1-expressing microglia and macrophages exhibited further elevation during neovascularization in mice lacking myeloid SOCS3. Moreover, our investigation unveiled the Spp1 gene as a direct transcriptional target gene of signal transducer and activator of transcription 3. Importantly, pharmaceutical activation of SOCS3 or blocking of SPP1 resulted in a significant reduction in pathological neovascularization. In conclusion, our study highlights the pivotal role of the SOCS3/STAT3/SPP1 axis in the regulation of pathological retinal angiogenesis.

Targeting osteopontin alleviates endometriosis and inflammation by inhibiting the RhoA/ROS axis and achieves non-invasive in vitro detection via menstrual blood.

STUDY QUESTION: How does osteopontin (OPN) in endometriosis ectopic stromal cells (EESCs) participate in the pathogenesis of endometriosis and achieve non-invasive detection in vitro? SUMMARY ANSWER: Targeted OPN regulates endometriosis's necroptosis and inflammatory state by inhibiting the RhoA/reactive oxygen species (ROS) axis, thereby alleviating endometriosis and enabling non-invasive detection of menstrual blood in vitro. WHAT IS KNOWN ALREADY: Endometriosis is a chronic inflammatory disease. Recent studies have shown that OPN plays an important role in disease progression by regulating cell death and inflammation. STUDY DESIGN, SIZE, DURATION: The study included 20 patients diagnosed with endometriosis (confirmed by laparoscopy and histology) and 10 controls without endometriosis. Endometriotic stromal cells were isolated from endometrial samples, while menstrual blood endometrial cells (MESCs) were isolated from menstrual blood. These cells were then cultured in vitro and utilized in subsequent experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: OPN expression in EESCs was assessed using inflammatory factor sequencing, immunohistochemical staining (IHC), quantitative real-time PCR (qRT-PCR) analysis, and Western blotting (WB). The biological behavior of OPN and its effects on inflammatory factors were examined using EdU, wound-healing, Transwell, and ELISA assays. Necroptosis in EESCs and its impact on inflammatory factors were detected through qRT-PCR, WB, and Calcein-AM/PI fluorescence assays. The examination of mitochondrial stress in EESCs involved the use of the Mitochondrial Membrane Potential (ΔΨm) Assay, ROS detection, and Calcein-AM Loading/cobalt chloride Quenching. qRT-PCR, WB, and other experiments were conducted to verify the regulation of necroptosis and inflammatory factor levels in EESCs by OPN through the RhoA/ROS axis. Knockdown of OPN and its inhibitory effect on endometriosis lesion size were confirmed using AAV9 virus, IHC, qRT-PCR, WB, and other experiments. Additionally, OPN expression in MESCs was detected using transcriptome sequencing, RT-PCR, WB, and other experiments. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro assays demonstrated a significant upregulation of OPN in EESCs, and the knockdown of OPN effectively inhibited necroptosis and the release of inflammatory factors. OPN inhibited necroptosis and inflammatory factor release by mediating RhoA-dependent ROS production and blocking mixed lineage kinase domain-like protein phosphorylation at the cell membrane. In vivo, targeting of OPN can inhibit the growth of endometriosis lesions. Clinically, OPN was also significantly upregulated in the menstrual blood of patients with endometriosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to limitations in obtaining surgical specimens, our study primarily involved collecting endometriosis tissues from women during the proliferative and secretory phases of the menstrual cycle. We observed a significant overexpression of OPN in the samples used for our investigation. However, the expression of OPN in endometriosis tissues during the intermenstrual phase remains unknown. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the pivotal role of the OPN/RhoA/ROS axis in the regulation of necroptosis and the release of inflammatory factors. OPN knockdown exerts a therapeutic effect in vivo, and the high expression detection of OPN in menstrual blood in vitro. In summary, targeting OPN provides possibilities for the treatment and detection of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071626), the Zhejiang Province Public Welfare Technology Application Research Project (LGF21H040010), and the Clinical Research project of the Second Affiliated Hospital of Wenzhou Medical University (1010293). The authors have no conflicts of interest.

Osteopontin promotes tumor growth and metastasis and GPX4-mediated anti-lipid peroxidation in triple-negative breast cancer by activating the PI3k/Akt/mTOR pathway.

PURPOSE: Triple-negative breast cancer (TNBC) features high aggressiveness, metastasis rate, drug resistance as well as poor prognosis. Osteopontin (OPN) is a key protein in the process of osteogenesis and has emerged as a new tumor marker in recent years. METHODS: Cell viability was tested with the CCK-8 kit. Transwell and wound healing were adopted to test cell invasive and migratory abilities. Tumor sphere formation was detected by tumor sphere formation assay. Human umbilical vein endothelial cell (HUVEC) tube formation assay was used to measure the angiogenesis of tumor cells. Western blot was applied for the estimation of the expression of cancer stem cell markers, angiogenesis-, signaling pathway-related proteins as well as OPN. Bioinformatics tools predicted OPN expression in breast cancer tissues. The levels of oxidative stress-related markers were assessed with ELISA. Following the overexpression of OPN in MD-MB-436 cells and the addition of the PI3K/AKT/mTOR pathway inhibitor LY294002, the aforementioned functional experiments were implemented again to investigate the mechanism. Finally, in vivo experiments of tumor-bearing mice were performed for further verification. RESULTS: The proliferative, invasive, migratory and tumor sphere formation capabilities as well as angiogenesis of TNBC cells were conspicuously increased in contrast to non-TNBC cell lines. OPN expression in TNBC tissues and cells was dramatically enhanced. OPN upregulation significantly elevated cell proliferative, invasive and migratory capabilities as well as tumor sphere formation and angiogenesis. The mechanism might be achieved by activating PI3K/AKT/mTOR signaling to regulate glutathione peroxidase 4 (GPX4)-mediated anti-lipid peroxidation. CONCLUSION: OPN promoted tumor sphere formation and angiogenesis in TNBC by activating the PI3K/AKT/mTOR pathway to regulate GPX4-mediated anti-lipid peroxidation levels.

Increased Elastase and Matrix Metalloproteinase Levels in the Pulmonary Arteries of Infants With Congenital Diaphragmatic Hernia.

BACKGROUND: Pulmonary vascular disease (PVD) complicated with pulmonary hypertension (PH) is a leading cause of mortality in congenital diaphragmatic hernia (CDH). Unfortunately, CDH patients are often resistant to PH therapy. Using the nitrogen CDH rat model, we previously demonstrated that CDH-associated PVD involves an induction of elastase and matrix metalloproteinase (MMP) activities, increased osteopontin and epidermal growth factor (EGF) levels, and enhanced smooth muscle cell (SMC) proliferation. Here, we aimed to determine whether the levels of the key members of this proteinase-induced pathway are also elevated in the pulmonary arteries (PAs) of CDH patients. METHODS: Neutrophil elastase (NE), matrix metalloproteinase-2 (MMP-2), epidermal growth factor (EGF), tenascin-C, and osteopontin levels were assessed by immunohistochemistry in the PAs from the lungs of 11 CDH patients and 5 normal age-matched controls. Markers of proliferation (proliferating cell nuclear antigen (PCNA)) and apoptosis (cleaved (active) caspase-3) were also used. RESULTS: While expressed by both control and CDH lungs, the levels of NE, MMP-2, EGF, as well as tenascin-C and osteopontin were significantly increased in the PAs from CDH patients. The percentage of PCNA-positive PA SMCs were also enhanced, while those positive for caspase-3 were slightly decreased. CONCLUSIONS: These results suggest that increased elastase and MMPs, together with elevated tenascin-C and osteopontin levels in an EGF-rich environment may contribute to the PVD in CDH infants. The next step of this study is to expand our analysis to a larger cohort, and determine the potential of targeting this pathway for the treatment of CDH-associated PVD and PH. TYPE OF STUDY: Therapeutic. LEVEL OF EVIDENCE: LEVEL III.

Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

The immune escape stage in cancer immunoediting is a pivotal feature, transitioning immune-controlled tumor dormancy to progression, and augmenting invasion and metastasis. Tumors employ diverse mechanisms for immune escape, with generating immunosuppressive cells from skewed hematopoiesis being a crucial mechanism. This led us to suggest that tumor cells with immune escape properties produce factors that induce dysregulations in hematopoiesis. In support of this suggestion, this study found that mice bearing advanced-stage tumors exhibited dysregulated hematopoiesis characterized by the development of splenomegaly, anemia, extramedullary hematopoiesis, production of immunosuppressive mediators, and expanded medullary myelopoiesis. Further ex vivo studies exhibited that conditioned medium derived from EL4lu2 cells could mediate the expansion of myeloid derived suppressor cells (MDSCs) in bone marrow cell cultures. The protein array profiling results revealed the presence of elevated levels of osteopontin (OPN), prostaglandin E2 (PGE2) and interleukin 17 (IL-17) in the culture medium derived from EL4luc2 cells. Accordingly, substantial levels of these factors were also detected in the sera of mice bearing EL4luc2 tumors. Among these factors, only PGE2 alone could increase the number of MDSCs in the BM cell cultures. This effect of PGE2 was significantly potentiated by the presence of OPN but not IL-17. Finally, in vitro treatment of EL4luc2 cells with pioglitazone, a modulator of OPN and cyclooxygenase 2 (COX-2) resulted in a significant reduction in cell proliferation in EL4luc2 cells. Our findings highlight the significant role played by tumor cell-derived OPN and PGE2 in fostering the expansion of medullary MDSCs and in promoting tumor cell proliferation. Furthermore, these intertwined cancer processes could be key targets for pioglitazone intervention.

METTL3/16-mediated m6A modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells.

Macrophages are the major components of tumour microenvironment, which play critical roles in tumour development. N6-methyladenosine (m6A) also contributes to tumour progression. However, the potential roles of m6A in modulating macrophages in hepatocellular carcinoma (HCC) are poorly understood. Here, we identified ZNNT1 as an HCC-related m6A modification target, which was upregulated and associated with poor prognosis of HCC. METTL3 and METTL16-mediated m6A modification contributed to ZNNT1 upregulation through stabilizing ZNNT1 transcript. ZNNT1 exerted oncogenic roles in HCC. Furthermore, ZNNT1 recruited and induced M2 polarization of macrophages via up-regulating osteopontin (OPN) expression and secretion. M2 Macrophages-recruited by ZNNT1-overexpressed HCC cells secreted S100A9, which further upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling. Thus, this study demonstrates that m6A modification activated the ZNNT1/OPN/S100A9 positive feedback loop, which promoted macrophages recruitment and M2 polarization, and enhanced malignant features of HCC cells. m6A modification-triggered ZNNT1/OPN/S100A9 feedback loop represents potential therapeutic target for HCC.

Targeted Macrophage CRISPR-Cas13 mRNA Editing in Immunotherapy for Tendon Injury.

CRISPR-Cas13 holds substantial promise for tissue repair through its RNA editing capabilities and swift catabolism. However, conventional delivery methods fall short in addressing the heightened inflammatory response orchestrated by macrophages during the acute stages of tendon injury. In this investigation, macrophage-targeting cationic polymers are systematically screened to facilitate the entry of Cas13 ribonucleic-protein complex (Cas13 RNP) into macrophages. Notably, SPP1 (OPN encoding)-producing macrophages are recognized as a profibrotic subtype that emerges during the inflammatory stage. By employing ROS-responsive release mechanisms tailored for macrophage-targeted Cas13 RNP editing systems, the overactivation of SPP1 is curbed in the face of an acute immune microenvironment. Upon encapsulating this composite membrane around the tendon injury site, the macrophage-targeted Cas13 RNP effectively curtails the emergence of injury-induced SPP1-producing macrophages in the acute phase, leading to diminished fibroblast activation and mitigated peritendinous adhesion. Consequently, this study furnishes a swift RNA editing strategy for macrophages in the inflammatory phase triggered by ROS in tendon injury, along with a pioneering macrophage-targeted carrier proficient in delivering Cas13 into macrophages efficiently.

Osteopontin Promotes Angiogenesis in the Spinal Cord and Exerts a Protective Role Against Motor Function Impairment and Neuropathic Pain After Spinal Cord Injury.

STUDY DESIGN: Basic science study using a hemisection spinal cord injury (SCI) model. OBJECTIVE: We sought to assess the effect of blocking osteopontin (OPN) upregulation on motor function recovery and pain behavior after SCI and to further investigate the possible downstream target of OPN in the injured spinal cord. SUMMARY OF BACKGROUND DATA: OPN is a noncollagenous extracellular matrix protein widely expressed across different tissues. Its expression substantially increases following SCI. A previous study suggested that this protein might contribute to locomotor function recovery after SCI. However, its neuroprotective potential was not fully explored, nor were the underlying mechanisms. MATERIALS AND METHODS: We constructed a SCI mouse model and analyzed the expression of OPN at different time points and the particular cell distribution in the injured spinal cord. Then, we blocked OPN upregulation with lentivirus-delivering siRNA targeting OPN specifically and examined its effect on motor function impairment and neuropathic pain after SCI. The underlying mechanisms were explored in the OPN-knockdown mice model and cultured vascular endothelial cells. RESULTS: The proteome study revealed that OPN was the most dramatically increased protein following SCI. OPN in the spinal cord was significantly increased three weeks after SCI. Suppressing OPN upregulation through siRNA exacerbated motor function impairment and neuropathic pain. In addition, SCI resulted in an increase in vascular endothelial growth factor (VEGF), AKT phosphorylation, and angiogenesis within the spinal cord, all of which were curbed by OPN reduction. Similarly, OPN knockdown suppressed VEGF expression, AKT phosphorylation, cell migration, invasion, and angiogenesis in cultured vascular endothelial cells. CONCLUSION: OPN demonstrates a protective influence against motor function impairment and neuropathic pain following SCI. This phenomenon may result from the proangiogenetic effect of OPN, possibly due to activation of the VEGF and/or AKT pathways.

Fate mapping of Spp1 expression reveals age-dependent plasticity of disease-associated microglia-like cells after brain injury.

Microglial reactivity to injury and disease is emerging as a heterogeneous, dynamic, and crucial determinant in neurological disorders. However, the plasticity and fate of disease-associated microglia (DAM) remain largely unknown. We established a lineage tracing system, leveraging the expression dynamics of secreted phosphoprotein 1（Spp1） to label and track DAM-like microglia during brain injury and recovery. Fate mapping of Spp1+ microglia during stroke in juvenile mice revealed an irreversible state of DAM-like microglia that were ultimately eliminated from the injured brain. By contrast, DAM-like microglia in the neonatal stroke models exhibited high plasticity, regaining a homeostatic signature and integrating into the microglial network after recovery. Furthermore, neonatal injury had a lasting impact on microglia, rendering them intrinsically sensitized to subsequent immune challenges. Therefore, our findings highlight the plasticity and innate immune memory of neonatal microglia, shedding light on the fate of DAM-like microglia in various neuropathological conditions.

Gastro-Intestinal Digested Bovine Milk Osteopontin Modulates Gut Barrier Biomarkers In Vitro.

SCOPE: Osteopontin (OPN) is a multifunctional protein naturally present in mammals' milk, associated with immune homeostasis and intestinal maturation. This study aims to investigate the protein digestion pattern and the cellular bioactivity of bovine milk OPN digesta in vitro. METHODS AND RESULTS: A modified INFOGEST static in vitro infant digestion protocol and a Caco-2/HT-29 co-culture cell model are employed to evaluate the digestion properties and the anti-inflammatory effects of OPN. OPN is resistant to gastric hydrolysis but degraded into large peptides during intestinal digestion. Its 10 kDa digesta permeate with predicted extensive bioactivities protects the co-culture cell model from the inflammation-induced dysfunction by dose-dependently recovering the expression of occludin, claudin-3, and ZO-1. Low dosage of OPN significantly decreases the production of IL-8 and IL-6, and downregulates the mRNA and protein expression of MyD88, NF-κB p65, and IκB-α, whereas a high dose evokes a mild pro-inflammatory response. Interestingly, anti-inflammatory effect of OPN digesta is stronger than lactoferrin and whey protein concentrate counterparts. CONCLUSION: The findings demonstrate that the bioactive peptides released from in vitro infant gastrointestinal digestion of bovine milk OPN alleviates intestinal epithelial cell inflammation by inhibiting NF-κB pathway activation and potentiates the barrier function of the intestinal epithelium.

Immunoregulation of bovine lactoferrin together with osteopontin promotes immune system development and maturation.

The immune system of infants is partly weak and immature, and supplementation of infant formula can be of vital importance to boost the development of the immune system. Lactoferrin (LF) and osteopontin (OPN) are essential proteins in human milk with immunoregulation function. An increasing number of studies indicate that proteins have interactions with each other in milk, and our previous study found that a ratio of LF : OPN at 1 : 5 (w/w, denoted as LOP) had a synergistic effect on intestinal barrier protection. It remains unknown whether LOP can also exert a stronger effect on immunoregulation. Hence, we used an in vitro model of LPS-induced macrophage inflammation and in vivo models of LPS-induced intestinal inflammation and early life development. We showed that LOP increased the secretion of the granulocyte-macrophage colony-stimulating factor (132%), stem cell factor (167%) and interleukin-3 (176%) in bone marrow cells, as well as thymosin (155%) and interleukin-10 (161%) in the thymus, more than LF or OPN alone during development, and inhibited changes in immune cells and cytokines during the LPS challenge. In addition, analysis of the components of digested proteins in vitro revealed that differentially expressed peptides may provide immunoregulation. Lastly, LOP increased the abundance of Rikenellaceae, Muribaculum, Faecalibaculum, and Elisenbergiella in the cecum content. These results imply that LOP is a potential immunomodifier for infants and offers a new theoretical basis for infant formula innovation.