Circulating Osteopontin Predicts Clinical and Radiological Response in First-Line Treatment of Advanced Non-Small Cell Lung Cancer.

PURPOSE: Pembrolizumab-based regimens are conditioned by the expression of PD-L1, but durable response rate is limited by innate and acquired resistance mechanisms. Here, we focus on osteopontin (OPN), an upfront biomarker of senescence, which closely associated with natural history of non-small cell lung cancer (NSCLC). METHODS: Seventy-nine patients eligible to pembrolizumab regimens-alone or in combination with chemotherapy-as first-line treatment of advanced NSCLC were enrolled. Predictive value of OPN toward iRECIST progression disease (PD) was set as first outcome. Secondary ones included performance status (ECOG) at baseline, early (first and best) responses, and overall survival (OS). RESULTS: High Serum OPN characterized patients with worse ECOG-PS (p = 0.015) at baseline and subjects experienced PD/death at first (OR 1.17 [1.02 to 1.35]; p = 0.030) and best responses (0.04 [0.00 to 0.81]; p = 0.035). OPN was associated with time-to-progression (B -2.74 [-4.46 to -1.01]) and time-to death (-0.13 [-0.20 to -0.05]). Cox regression models unveil a predictive value for iRECIST-PD (HR 1.01 [1.00 to 1.02]; p = -0.005), RECIST-PD (HR 1.01 [1.00 to 1.02]; p = 0.017), and OS (HR 1.02 [1.01 to 1.03]; p = 0.001). These models were internally validated through bootstrap resampling and characterized by relevant discrimination ability at ROC curve analyses. CONCLUSION: Baseline levels of serum OPN is closely associated with performance status and short/long term outcomes in patients with advanced NSCLC, which are candidate to pembrolizumab-based regimens. As upfront biomarker of senescence, OPN may pave the way for future studies focusing on senescence patterns in NSCLC.

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.

Osteopontin modulates microglial activation states and attenuates inflammatory responses after subarachnoid hemorrhage in rats.

AIMS: Osteopontin (OPN) has demonstrated neuroprotective effects in various stroke models. Its role in neuroinflammation after brain injury remains to be elucidated. This study aims to clarify the effect of OPN on neuroinflammation, particularly on the functional states of microglia after subarachnoid hemorrhage (SAH). METHODS: 77 rats were randomly divided into the following groups: Sham, SAH 24 h, SAH + rOPN, SAH + Vehicle (PBS), SAH + OPN siRNA, and SAH + Scr siRNA, SAH + rOPN+Fib-14 and SAH + rOPN+DMSO. Modified Garcia and beam balance tests were used to evaluate neurobehavioral outcomes. Semi-quantitative immunofluorescence staining was performed to measure expression of myeloperoxidase (MPO) and microglia activation state markers CD16, CD206 after SAH and recombinant OPN treatment. The quantification of microglia activation and functional markers CD16, CD206, TNF-α and IL-10 were further evaluated using Western-blotting. RESULTS: Nasal administration of rOPN improved neurological dysfunction, attenuated neutrophil infiltration, and decreased expression of phenotypic and functional markers of pro-inflammatory microglia CD16 and TNF-α. It also promoted an anti-inflammatory microglial state, as evidenced by increased expression of CD206 and IL-10. Furthermore, after blocking the phosphorylation of FAK signaling, the effects of rOPN on microglial activation states were partially reversed. The downstream pathways of STAT3 and NF-κB also exhibited consistent changes, suggesting the involvement of the STAT3 and NF-κB pathways in OPN's modulation of microglial activation via integrin-FAK signaling. CONCLUSION: OPN attenuates inflammatory responses after SAH by promoting an anti-inflammatory microglial state, potentially mediated through the integrin-FAK-STAT3 and NF-κB signaling pathways.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The combination of olive oil and Lepidium sativum improves the deleterious effects resulting from dexamethasone-induced osteoporosis in rats.

INTRODUCTION: Osteoporosis is characterized by deterioration of bone microarchitecture and reduced bone mass and can increase the risk of fracture. To reduce this risk, the aim of this study was to compare the combination effects of olive oil and Lepidium sativum compared to the conventional drug therapy alendronate. METHODS: Osteoporosed-induced rat model was established by administration of dexamethasone in female adult albino rats. The serum level of Ca2+, P3+, and osteocalcin was assessed. In addition, histopathological changes and immunohistochemical expression of osteopontin within bone specimens were performed. RESULTS: Our results showed that a combination of olive oil and Lepidium sativum had a beneficial therapeutic effect in the treatment of osteoporosis as compared to alendronate therapy. This was demonstrated by increase of serum Ca2+, P3+, and osteocalcin levels in treated compared to control groups. Intriguingly, the highest effect was noticed in rats that received a combination of olive oil and Lepidium sativum compared to the individual treatment. This was reflected by an increase in the cortical bone thickness and a decrease in immunohistochemical expression of osteopontin compared to individual treated groups. CONCLUSION: We concluded that the administration of a combination of olive oil and Lepidium sativum improves bone mineral health and intensity and reduces the risk of osteoporosis in a rat model.

Immune Regulatory Effect of Osteopontin Gene Therapy in a Murine Model of Multidrug Resistant Pulmonary Tuberculosis.

Tuberculosis (TB) has been for many years a major public health problem since treatment is long and sometimes ineffective favoring the increase of multidrug-resistant mycobacteria (MDR-TB). Gene therapy is a novel and effective tool to regulate immune responses. In this study we evaluated the therapeutic effect of an adenoviral vector codifying osteopontin (AdOPN), a molecule known for their roles to favor Th1 and Th17 type-cytokine expression which are crucial in TB containment. A single dose of AdOPN administration in BALB/c mice suffering late progressive pulmonary MDR-TB produced significant lower bacterial load and pneumonia, due to higher expression of IFN-γ, IL-12, and IL-17 in coexistence with increase of granulomas in number and size, resulting in higher survival, in contrast with mice treated with the control adenovirus that codify the green fluorescent protein (AdGFP). Combined therapy of AdOPN with a regimen of second line antibiotics produced a better control of bacterial load in lung during the first days of treatment, suggesting that AdOPN can shorten chemotherapy. Taken together, gene therapy with AdOPN leads to higher immune responses against TB infection, resulting in a new potential treatment against pulmonary TB that can co-adjuvant chemotherapy.

EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma.

BACKGROUND: Despite the remarkable breakthroughs achieved in the management of metastatic melanoma using immunotherapy and targeted therapies, long-term clinical efficacy is often compromised due to dose-limiting toxicity and innate or acquired resistance. Therefore, it is of vital importance to further explore the molecular mechanisms underlying melanoma progression and identify new targeted therapeutic approaches. METHODS: The function of eukaryotic elongation factor-2 kinase (EEF2K) in melanoma were investigated in vitro and in vivo. RNA-seq and chromatin immunoprecipitation (ChIP) assay were undertaken to explore the mechanisms. The antitumor effect of bromodomain and extra terminal domain (BET) inhibitors combined with cytarabine were assessed in melanoma both in vitro and in vivo. RESULTS: EEF2K silencing markedly attenuated the malignant phenotypes of melanoma cells, including proliferation, migration, invasion and metastasis. In contrast, EEF2K overexpression promoted melanoma cell proliferation, migration and invasion. Mechanistically, we demonstrated that EEF2K upregulates the phosphorylation of STAT3 (p-STAT3) at Tyr705, which binds to the promoter region of SPP1 and enhances its transcription, thus facilitating melanoma progression. Transfection-induced re-expression of SPP1 partly negated the inhibitory effect of EEF2K silencing on melanoma, whereas inhibition of SPP1 or STAT3 significantly abolished the efficacy of EEF2K on melanoma cells. Intriguingly, EEF2K silencing combined with BET inhibitor treatment further inhibited cell proliferation and promoted apoptosis in melanoma. We further screened the US FDA-approved antitumour drug library and identified cytarabine as a potential clinically applicable EEF2K inhibitor that could synergise with BET inhibitors in melanoma treatment. CONCLUSION: EEF2K/p-STAT3/SPP1 may be a novel oncogenic pathway in melanoma progression, which could be a target for novel combination therapy for melanoma.

SPP1 in infliximab resistant ulcerative colitis and associated colorectal cancer: an analysis of differentially expressed genes.

OBJECTIVE: Infliximab, a tumour necrosis factor-α (TNFα) antagonist, has advanced the management of ulcerative colitis. Although efficacious, considerable percentage of patients are resistant to treatment. Accumulative inflammatory burden in long-term ulcerative colitis patients refractory to therapy increases the risk of developing colorectal cancer (CRC). Our study investigated anti-TNFα-naïve patients with active ulcerative colitis to identify gene biomarkers whose dysregulated expression correlated with resistance to infliximab (IFX) treatment and poor prognosis in CRC. METHODS: Differentially expressed genes (DEGs) from two studies (GSE73661 and GSE14580) with colonic mucosal samples were retrieved. Noninflammatory bowel disease controls were compared with those with active ulcerative colitis that either responded or were resistant to IFX before treatment. DEGs from ulcerative colitis samples resistant to IFX were used to construct a protein-protein interaction network, and clustering gene modules were identified. Module DEGs that overlapped with ulcerative colitis samples responsive to IFX were analysed, based on topological closeness and radiality. Hub genes were obtained, and their correlation with CRC progression was evaluated. Their expression in CRC tissues and their tumour microenvironment immune status was estimated. RESULTS: Three clusters composed of 582 DEGs from ulcerative colitis samples resistant to IFX were retrieved. Comparative analysis identified 305 overlapping DEGs with ulcerative colitis samples responsive to IFX. Topological analysis revealed a hub gene - SPP1 - whose overexpression in CRC tissues and patients correlated with increased infiltration of immune signatures and poor prognosis. CONCLUSION: SPP1 may serve as potential gene biomarker and predictor of resistance to IFX therapy in ulcerative colitis and CRC development.

Effects of oral paricalcitol therapy on arterial stiffness and osteopontin in hypertensive patients with chronic kidney disease and secondary hyperparathyroidism.

BACKGROUND: Arterial stiffness is linked to the progression of atherosclerosis, while activation of vitamin D receptor exerts favorable cardiovascular effects in patients with renal insufficiency. In this study, we investigated the effects of oral treatment with paricalcitol, a potent vitamin D receptor activator, on arterial stiffness and osteopontin, a marker of atherosclerosis, in hypertensive patients with chronic kidney disease (CKD) and secondary hyperparathyroidism. METHODS: We followed up 29 treated hypertensive patients (mean age: 74.1 years, 19 men, office blood pressure = 132/85 mmHg) with CKD stages 3-5 (mean glomerular filtration rate [GFR] = 19.4 ml/min/1.73 m2) who were on therapy with oral paricalcitol for 1 year. The control group consisted of 10 age-, sex-, and GFR-matched hypertensive patients with secondary hyperparathyroidism. RESULTS: After 1 year of treatment with paricalcitol compared to baseline, there was no statistical difference in levels of GFR, office blood pressure, and osteopontin (p = NS for all), while carotid-femoral PWV was reduced from 11.8 ± 2.6 m/s to 11.2 ± 2.4 m/s (p < 0.05). The control group exhibited no significant changes in carotid-femoral PWV (p = NS). CONCLUSIONS: Treatment with oral paricalcitol in hypertensive subjects suffering from CKD stages 3-5 and secondary hyperparathyroidism is accompanied by amelioration of arterial stiffness as reflected by the reduction of carotid-femoral PWV.

Osteopontin isoforms differentially promote arteriogenesis in response to ischemia via macrophage accumulation and survival.

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

Recombinant Osteopontin Improves Neurological Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3ß Pathway Following Intracerebral Hemorrhage.

BACKGROUND This study aimed to investigate the potential neuroprotective effect of recombinant osteopontin (r-OPN) on apoptotic changes via modulating phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK-3ß) signaling in a rat model of intracerebral hemorrhage (ICH). MATERIAL AND METHODS We subjected 10-12-week-old Sprague-Dawley male rats (n=120) to injection of autologous blood into the right basal ganglia to induce ICH or sham surgery. ICH animals received vehicle administration, r-OPN (4 µL/pup), or r-OPN combined with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (86 ng/pup) at 30 min after injury. Neurological scores and rotarod latencies were evaluated on days 1-5 post-ICH. Brain water content was evaluated on days 1-3 post-ICH. The number of apoptotic cells changes were evaluated by terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) and hematoxylin staining. Apoptosis-related proteins Bcl-2, Bax, and cleaved caspase-3 (CC3), and the phosphorylation levels of Akt and GSK-3b were assayed by Western blot. RESULTS Neurological deficits, rotarod latencies, and brain water content following ICH were reduced in the r-OPN group compared to the vehicle group. r-OPN also attenuated cell death in ICH. Furthermore, treatment with r-OPN significantly increased p-Akt expression and decreased p-GSK-3ß. These effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of CC3 at 24 h after ICH. Importantly, all the beneficial effects of r-OPN in ICH were abrogated by the PI3K inhibitor wortmannin. CONCLUSIONS r-OPN may provide a wide range of neuroprotection by suppressing apoptosis through the PI3K/Akt/GSK-3ß signaling pathway after ICH.

Role of vascular calcification inhibitors in preventing vascular dysfunction and mortality in hemodialysis patients.

Cardiovascular events make up the primary cause of death in hemodialysis patients, and the risk for cardiovascular mortality is significantly increased by vascular calcification, a condition observed frequently in this patient population. The mechanisms underlying the pathogenesis of vascular calcification are complex, and many factors facilitate or hinder the development of calcification. In this review, we first summarize the main factors contributing to the pathogenesis of vascular calcification in patients with end-stage renal disease. We then explore the role of calcification inhibitors in the calcification process, as well as their effect on vascular dysfunction and mortality in hemodialysis patients.

Osteopontin Augments M2 Microglia Response and Separates M1- and M2-Polarized Microglial Activation in Permanent Focal Cerebral Ischemia.

BACKGROUND: Focal cerebral ischemia induces distinct neuroinflammatory processes. We recently reported the extracellular phosphor-glyco-protein osteopontin (OPN) to directly affect primary microglia in vitro, promoting survival while shifting their inflammatory profile towards a more neutral phenotype. We here assessed the effects of OPN on microglia after stroke in vivo, with focus on infarct demarcation. METHODS: Animals underwent focal photothrombotic stroke and were injected intracerebroventricularly with 500 µg OPN or vehicle. Immunohistochemistry assessed neuronal damage and infarct volume, neovascularisation, glial scar formation, microglial activation, and M1 and M2 polarisation. RESULTS: After photothrombotic stroke, areas covered by M1 and M2 microglia substantially overlapped. OPN treatment reduced that overlap, with microglia appearing more spread out and additionally covering the infarct core. OPN additionally modulated the quantity of microglia subpopulations, reducing iNOS+ M1 cells while increasing M2 microglia, shifting the M1/M2 balance towards an M2 phenotype. Moreover, OPN polarized astrocytes towards the infarct. CONCLUSION: Microglial activation and M1 and M2 polarization have distinct but overlapping spatial patterns in permanent focal ischemia. Data suggest that OPN is involved in separating M1 and M2 subpopulations, as well as in shifting microglia polarization towards the M2 phenotype modulating beneficially inflammatory responses after focal infarction.

Osteopontin promotes dendritic cell maturation and function in response to HBV antigens.

PURPOSE: Dendritic cells (DCs) play critical roles in promoting innate and adaptive immunity in microbial infection. Functional impairment of DCs may mediate the suppression of viral-specific T-cell immune response in chronic hepatitis B (CHB) patients. Osteopontin (OPN) is involved in several liver diseases and infectious diseases. However, whether OPN affects DC function in hepatitis B virus (HBV) infection is unknown. METHODS: Twenty CHB patients and 20 healthy volunteers were recruited. OPN secreted by DCs was compared. Peripheral blood mononuclear cells cultured with OPN antibody were examined to study the costimulatory molecular expression and interleukin (IL)-12 production of DCs after HBV antigenic stimulation. OPN-deficient mice were used to investigate the influence of OPN on DC maturation and function after HBV antigenic stimulation in vitro and in vivo. Exogenous OPN was administrated to further verify the functioning of DCs from CHB patients upon HBV antigenic stimulation. RESULTS: We found that OPN production of DCs from CHB patients was significantly lower than those from healthy volunteers. The absence of OPN impaired IL-12 production and costimulatory molecular expression of DCs upon stimulation with HBV antigens. Defective DC function led to reduced activation of Th1 response to HBV antigens. In addition, OPN deficiency in DCs reduced the HBV antigen-induced inflammatory response in the liver of mice. Importantly, OPN administration significantly promoted the maturation of DCs from CHB patients in vitro. CONCLUSION: These findings suggested that OPN could improve the maturation and functioning of DCs in the immune response to HBV antigens, which might be useful to further improve the effect of DC vaccine.

Interactions between osteopontin and vascular endothelial growth factor: Implications for cancer.

For this comprehensive review, 257 publications with the keywords "osteopontin" or "OPN" and "vascular endothelial growth factor" or "VEGF" in PubMed were screened (time frame from year 1996 to year 2014). 37 articles were excluded because they were not focused on the interactions between these molecules, and papers relevant for transformation-related phenomena were selected. Osteopontin (OPN) and vascular endothelial growth factor (VEGF) are characterized by a convergence in function for regulating cell motility and angiogenesis, the response to hypoxia, and apoptosis. Often, they are co-expressed or one molecule induces the other, however, in some settings OPN-associated pathways and VEGF-associated pathways are distinct. Their relationships affect the pathogenesis in cancer, where they contribute to progression and angiogenesis and serve as markers for poor prognosis. The inhibition of OPN may reduce VEGF levels and suppress tumor progression. In vascular pathologies, these two cytokines mediate remodeling, but may also perpetuate inflammation and narrowing of the arteries. OPN and VEGF are elevated and contribute to vascularization in inflammatory diseases.

Effects of osteopontin on the expression of IL-6 and IL-8 inflammatory factors in human knee osteoarthritis chondrocytes.

OBJECTIVE: Osteoarthritis (OA) is a chronic musculoskeletal disease characterized by progressive destruction of articular cartilage, OA lead to chronic pain and functional restrictions in affected joints. The present study was to investigate the role of osteopontin (OPN) in the athogenesis of OA through studying the effect of OPN on expression of IL-6 and IL-8 inflammatory factors in human OA chondrocytes. PATIENTS AND METHODS: One-step type II collagenase digestive method was used to isolate OA chondrocytes from sectional cartilage specimens of 16 primary knee OA patients received total knee replacement surgery. Synchronized first-generation chondrocytes were then treated with OPN (100 ng/ml or 1 µg/mL). The changes in cell morphology of OA chondrocytes were analyzed before and after treated with OPN; and the expression levels of IL-6 and IL-8 were evaluated by real-time q-PCR. RESULTS: Chondrocytes were successfully isolated from human OA knee cartilage, and the viability of isolated chondrocytes was 92.11±3.13%. Adherent chondrocytes formed clusters of irregular polygonal shape with intercellular pseudopodia extension. After OPN treatment, cells became fusiform or irregularly shaped, and the number of intercellular pseudopodia decreased significantly. The mRNA expression of IL-6 increased to 1.83 times at 0.1 µg/ml of OPN and 3.1 times at the dose of 1 µg/ml; the expression of IL-8 increased to 1.57 and 3.27 times at the dose of 0.1 µg/ml and 1 µg/ml respectively. CONCLUSIONS: OPN could up-regulate expression of IL-6 and IL-8 cytokines in human OA chondrocytes, and the expression increased with the increasing concentration of OPN, which might be one of the potential mechanisms of OPN in the development of OA.

The O­glycosylation mutant osteopontin alters lung cancer cell growth and migration in vitro and in vivo.

Osteopontin (OPN) is an acidic, glycosylated and phosphorylated protein that plays an essential role in determining the aggressiveness and oncogenic potential of several types of cancer, including lung cancer. The OPN function is highly dependent on post-translational modification (PTM) and regulation of the processes that involve OPN can be mediated through glycosylation. However, the connection between OPN function and its O-glycosylation in lung cancer cells has yet to be investigated. In the present study, this issue was addressed by studying the effects of wild-type (WT) OPN and a triple mutant (TM) of OPN, which was mutated at three O-glycosylation sites in lung cancer cells. It was shown that OPN WT rather than OPN TM induced the OPN­mediated signaling pathway. The OPN WT expression enhanced cap-dependent protein translation, NF-κB activity and glucose uptake, whereas a reduction was observed in cells treated with OPN TM. The results clearly demonstrated that unlike OPN WT, OPN TM did not increase lung cancer cell growth and migration both in vitro and in a xenograft mouse model. Thus, results of the present study suggested that targeting OPN by introducing OPN TM may be a good strategy for treating lung cancer.

Nasal administration of recombinant osteopontin attenuates early brain injury after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Neuronal apoptosis is a key pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Given that recombinant osteopontin (rOPN), a promising neuroprotectant, cannot pass through the blood-brain barrier, we aimed to examine whether nasal administration of rOPN prevents neuronal apoptosis after experimental SAH. METHODS: Male Sprague-Dawley rats (n=144) were subjected to the endovascular perforation SAH model. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 and 72 hours after SAH induction. The expressions of cleaved caspase-3, phosphorylated focal adhesion kinase (FAK), and phosphorylated Akt were examined using Western blot analysis. Neuronal cell death was demonstrated with terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling. We also administered FAK inhibitor 14 and phosphatidylinositol 3-kinase inhibitor, Wortmannin, prior to rOPN to establish its neuroprotective mechanism. ELISA was used to measure rOPN delivery into the cerebrospinal fluid. RESULTS: Cerebrospinal fluid level of rOPN increased after its nasal administration. This was associated with improved neurological scores and reduced brain edema at 24 hours after SAH. rOPN increased phosphorylated FAK and phosphorylated Akt expressions and decreased caspase-3 cleavage, resulting in attenuation of neuronal cell death within the cerebral cortex. These effects were abolished by FAK inhibitor 14 and Wortmannin. CONCLUSIONS: Nasal administration of rOPN decreased neuronal cell death and brain edema and improved the neurological status in SAH rats, possibly through FAK-phosphatidylinositol 3-kinase-Akt-induced inhibition of capase-3 cleavage.

Milk osteopontin, a nutritional approach to prevent alcohol-induced liver injury.

Alcohol consumption is a leading cause of liver disease worldwide; thus, there is an urgent need to develop novel therapeutic interventions. Key events for the onset and progression of alcoholic liver disease result in part from the gut-to-liver interaction. Osteopontin is a cytokine present at high concentration in human milk, umbilical cord, and infants' plasma with beneficial potential. We hypothesized that dietary administration of milk osteopontin could prevent alcohol-induced liver injury perhaps by maintaining gut integrity and averting hepatic inflammation and steatosis. Wild-type mice were fed either the control or the ethanol Lieber-DeCarli diets alone or in combination with milk osteopontin for 3 wk, and parameters of gut and liver damage were measured. Milk osteopontin protected the stomach and the gut by increasing gland height, crypt cell plus enterocyte proliferation, and mucin content in addition to lowering macrophages, plasmacytes, lymphocytes, and neutrophils in the mucosa and submucosa in alcohol-fed mice. Milk osteopontin targeted the gut-liver axis, preserving the expression of tight-junction proteins in alcohol-fed mice thus maintaining intestinal integrity and permeability. There was protection from liver injury since transaminases, the activity scores, triglyceride levels, neutrophil infiltration, 3-nitrotyrosine residues, lipid peroxidation end products, translocation of gram-negative bacteria, lipopolysaccharide levels, and tumor necrosis factor-α were lower in cotreated than in ethanol-fed mice. Furthermore, milk osteopontin diminished ethanol-mediated liver injury in OPN knockout mice. Milk osteopontin could be a simple effective nutritional therapeutic strategy to prevent alcohol hepatotoxicity due, among others, to gut protective, anti-inflammatory, and anti-steatotic actions.

Expressão da família de proteínas SIBLING nos tecidos regenerados em defeitos de furca em câes / The SIBLING family of proteins expression in regenerative tissues in furcation defects in dogs

O presente estudo teve como objetivo caracterizar a expressão da família SIBLING (Small Integrin-Binding Ligand, N-linked Glycoproteins) após tratamento regenerativo de furca com enxerto de tecido reparativo de alvéolos dentários. Para isto, os 2os e 3os pré-molares superiores foram extraídos em quatro cães s.r.d. Cinco dias após as extrações, defeitos padronizados de furca classe II foram criados nos 2os, 3os e 4os pré-molares inferiores, bilateralmente. Estes defeitos foram tratados imediatamente com raspagem, alisamento e polimento corono-radicular (RAPCR) e retalho deslocado coronariamente (RDC) (Grupo Controle) ou com RAPCR + RDC + enxerto de tecido reparativo de alvéolos dentários (Grupo Teste) em um experimento de boca-dividida. Após um período de 6 semanas de reparação, os animais foram sacrificados e foi realizada análise imuno-histoquímica para avaliar a localização dos membros da família de proteínas SIBLING, composta pelas seguintes proteínas nãocolágenas da matriz extracelular: osteopontina (OPN), sialoproteína óssea (BSP), proteína da matriz dentinária 1 (DMP1), sialofosfoproteína da dentina (DSPP) e fosfoglicoproteína da matriz extracelular (MEPE). Não foram encontradas diferenças na expressão da família SIBLING entre os grupos teste e controle. Todas as proteínas foram expressas no novo osso, novo cemento e novo ligamento periodontal, em ambos os grupos. Os osteoclastos demonstraram imunolocalização intracelular intensa somente para a OPN.

The present study aimed in characterizing the expression of the SIBLING (Small Integrin- Binding Ligand, N-linked Glycoproteins) family in a regenerative treatment of furcation defects with a reparative tissue graft obtained from extraction sockets. The second and third upper premolars were extracted in four mixed breed dogs. Five days later, standardized class II furcation defects were created in the second, third and fourth mandibular premolars, bilaterally. The defects were immediately treated with either debridement and root planning (DRP) combined with a coronally positioned flap (CPF) (Control Group), or with DRP+CPF + a reparative tissue graft derived from the second and third premolar extraction sockets (Experimental Group) in a split-mouth design. After 6 weeks period of healing, the animals were sacrificed and immunohistochemistry was carried out to assess the localization of members of the SIBLING family of noncollagenous extracellular matrix proteins, namely osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). No differences in the SIBLING family of proteins expression were noted between the control and experimental group. All proteins were expressed in new bone, new cementum and new periodontal ligament in both groups...