The Role of Osteopontin (OPN) in Regulating Microglia Phagocytosis in Nervous System Diseases.

Phagocytosis is the process by which certain cells or organelles internalise foreign substances by engulfing them and then digesting or disposing of them. Microglia are the main resident phagocytic cells in the brain. It is generally believed that microglia/macrophages play a role in guiding the brain's repair and functional recovery processes. However, the resident and invading immune cells of the central nervous system can also exacerbate tissue damage by stimulating inflammation and engulfing viable neurons. The functional consequences of microglial phagocytosis remain largely unexplored. Overall, phagocytosis is considered a beneficial phenomenon in acute brain injury because it eliminates dead cells and induces an anti-inflammatory response. Osteopontin (OPN) is a phosphorylated glycoprotein induced by injury in various tissues, including brain tissue. In acute brain injuries such as hemorrhagic stroke and ischemic stroke, OPN is generally believed to have anti-inflammatory effects. OPN can promote the reconstruction of the blood-brain barrier and up-regulate the scavenger receptor CD36. But in chronic diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), OPN can cause microglia to engulf neurons and worsen disease progression. We explored the role of OPN in promoting microglial phagocytosis in nervous system disorders.

Milk osteopontin promotes brain development by up-regulating osteopontin in the brain in early life.

Osteopontin (OPN) is a pleiotropic protein and is abundantly present in milk. Its functions include immune modulation and cellular proliferation and differentiation. OPN is highly expressed in the brain. We investigated the effects of milk-derived OPN on brain development of mouse pups. Wild-type (WT) dams producing OPN+ milk and OPN knockout (KO) dams producing OPN- milk nursed WT pups (OPN+/+), yielding 2 pup treatment groups, OPN+ OPN+/+ and OPN- OPN+/+, for comparison. Preliminary studies supported use of this model by showing high concentrations of OPN in milk of WT dams and no OPN in milk of OPN KO dams, and production of similar amounts of milk by WT and KO dams. The ability of ingested milk OPN to enter the brain was revealed by appearance of orally gavaged [125I]-labeled and antibody-probed milk OPN in brains of pups. Brain OPN mRNA levels were similar in both nursed groups, but the brain OPN protein level was significantly lower in the OPN- OPN+/+ group at postnatal days 6 and 8. Behavior tests showed impaired memory and learning ability in OPN- OPN+/+ pups. In addition, our study revealed increased expression of myelination-related proteins and elevated proliferation and differentiation of NG-2 glia into oligodendrocytes in the brain of OPN+ OPN+/+ pups, accompanied by increased activation of ERK-1/2 and PI3K/Akt signaling. We concluded that milk OPN can play an important role in brain development and behavior in infancy by promoting myelination.-Jiang, R., Prell, C., Lönnerdal, B. Milk osteopontin promotes brain development by up-regulating osteopontin in the brain in early life.

Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic-to-pulmonary shunt.

Recent studies indicated that osteopontin (OPN) was involved in the genesis and progression of pulmonary arterial hypertension (PAH); however, its role in congenital heart disease-associated PAH (CHD/PAH) remains unknown. Our results showed that OPN was increased in lungs and plasma of patients with Eisenmenger syndrome; moreover, OPN and αVß3-integrin expression levels were augmented in rat lungs exposed to systemic-to-pulmonary shunt. Cell culture assay demonstrated that distal pulmonary arterial smooth muscle cells (PASMCs) from rat lungs suffering from volume and pressure overload exhibited enhanced proliferation compared with those from healthy rats. Mechanical stretch (20% at 1 Hz) increased OPN expression and activated ERK1/2 and protein kinase B (Akt) signal pathway in distal PASMCs from healthy rats. Interestingly, OPN enhanced the proliferation and migration of PASMCs while blocking αVß3-integrin with neutralizing antibody LM609 or Arg-Gly-Asp peptidomimetic antagonist cyclo(Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (XJ735), rectified the proliferative and migratory effects of OPN, which were partially mediated via ERK1/2 and Akt signaling pathways. Furthermore, surgical correction of systemic-to-pulmonary shunt, particularly XJ735 supplementation after surgical correction of systemic-to-pulmonary shunt, significantly alleviated the pulmonary hypertensive status in terms of pulmonary hemodynamic indices, pulmonary vasculopathy, and right ventricular hypertrophy. In summary, OPN alteration in lungs exposed to systemic-to-pulmonary shunt exerts a deteriorative role in pulmonary vascular remodeling through modulating the proliferation and migration of PASMCs, at least in part, via ανß3-ERK1/2 and ανß3-Akt signaling pathways. Antagonizing OPN receptor ανß3-integrin accelerated the regression of pulmonary vasculopathy after surgical correction of systemic-to-pulmonary shunt, indicating a potential therapeutic strategy for patients with CHD/PAH.-Meng, L., Liu, X., Teng, X., Gu, H., Yuan, W., Meng, J., Li, J., Zheng, Z., Wei, Y., Hu, S. Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic-to-pulmonary shunt.

Osteopontin in Vascular Disease.

Inflammatory cytokines are necessary for an acute response to injury and the progressive healing process. However, when this acute response does not resolve and becomes chronic, the same proteins that once promoted healing then contribute to chronic inflammatory pathologies, such as atherosclerosis. OPN (Osteopontin) is a secreted matricellular cytokine that signals through integrin and CD44 receptors, is highly upregulated in acute and chronic inflammatory settings, and has been implicated in physiological and pathophysiologic processes. Evidence from the literature suggests that OPN may fit within the Goldilocks paradigm with respect to cardiovascular disease, where acute increases are protective, attenuate vascular calcification, and promote postischemic neovascularization. In contrast, chronic increases in OPN are clinically associated with an increased risk for a major adverse cardiovascular event, and OPN expression is a strong predictor of cardiovascular disease independent of traditional risk factors. With the recent finding that humans express multiple OPN isoforms as the result of alternative splicing and that these isoforms have distinct biologic functions, future studies are required to determine what OPN isoform(s) are expressed in the setting of vascular disease and what role each of these isoforms plays in vascular disease progression. This review aims to discuss our current understanding of the role(s) of OPN in vascular disease pathologies using evidence from in vitro, animal, and clinical studies. Where possible, we discuss what is known about OPN isoform expression and our understanding of OPN isoform contributions to cardiovascular disease pathologies.

Effect of ligand-receptor interaction of osteopontin-CD44 on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes in vitro. / 骨桥蛋白-CD44é ä½“å—ä½“ååº”å¯¹äººè†éª¨å ³èŠ‚ç‚Žè½¯éª¨ç»†èƒžé€æ˜Žè´¨é ¸è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To investigate the effect of ligand-receptor interaction of osteopontin (OPN)-CD44 on the expression of hyaluronic acid (HA) in the cultured human knee osteoarthritis (OA) chondrocytes via interfering the reaction between OPN and CD44 ligand-receptor. METHODS: The OA chondrocytes and normal chondrocytes were obtained from knee joint cartilage tissues in the patients with knee OA and malignant tumor respectively. The normal chondrocytes and OA chondrocytes were detected and analyzed, and then the intervention analysis of OA chondrocytes was carried out. The OA chondrocytes were divided into 4 groups: a negative control group, which was cultured with complete medium without any molecular intervention reagent; an OPN intervention group, which was cultured with recombinant human OPN (rhOPN) for 24 hours; a CD44 blocking group, which were pretreated with CD44 receptor specific antagonist for 1 hour to block the binding of OPN-CD44, and then treated with rhOPN for 23 hours; a CD44 homotype group, which was pretreated with CD44 for 1 hour and then treated with rhOPN for 23 hours. In addition, the study for OPN-CD44 axis was also divided into 4 groups: an OA-negative control group (OA-NC group), a si-OPN intervention group, a rhOPN intervention group, and a rhOPN + CD44 antibody (Ab) group. Western blotting, real-time PCR, and enzyme linked immunosorbent assay (ELISA) were used to detect the protein and mRNA expression levels of OPN, CD44, hyaluronate synthase (HAS), and HA, respectively. RESULTS: The protein expression levels of OPN, CD44, and HAS1 and the secretion levels of HA in the OA chondrocytes were higher than those in the normal chondrocytes. Compared with the OPN intervention group, the expression levels of HAS1, HAS2, HAS3 and HA in the CD44 blocking group were lower than those in OPN intervention group (all P<0.05); but there was no significant difference in the expression levels of HAS1, HAS2, HAS3 and HA between the CD44 homotype group and the OPN intervention group (all P>0.05). The results of OPN-CD44 axis study showed that: compared with the OA-NC group, the expression of CD44 in the rhOPN intervention group was slightly lower, but the protein and mRNA levels of HAS1 were significantly increased (all P<0.05); compared with the OA-NC group, the expression of CD44 was up-regulated, but the protein and mRNA level of HAS1 were significantly inhibited in the si-OPN intervention group (all P<0.05); compared with the OA-NC group, the protein and mRNA levels of HAS1 in the rhOPN+CD44 Ab group were also significantly inhibited (all P<0.05). CONCLUSIONS: The OPN in OA chondrocytes can promote the expression of HAS1, and the OPN can stimulate the secretion of HAS and induce HA expression by reacting with CD44 ligand receptor. They constitute the axis of OPN/CD44/HAS1, which plays an important role in regulating the expression of HA in chondrocytes.

Matricellular proteins in cardiac adaptation and disease.

The term matricellular proteins describes a family of structurally unrelated extracellular macromolecules that, unlike structural matrix proteins, do not play a primary role in tissue architecture, but are induced following injury and modulate cell-cell and cell-matrix interactions. When released to the matrix, matricellular proteins associate with growth factors, cytokines, and other bioactive effectors and bind to cell surface receptors transducing signaling cascades. Matricellular proteins are upregulated in the injured and remodeling heart and play an important role in regulation of inflammatory, reparative, fibrotic and angiogenic pathways. Thrombospondin (TSP)-1, -2, and -4 as well as tenascin-C and -X secreted protein acidic and rich in cysteine (SPARC), osteopontin, periostin, and members of the CCN family (including CCN1 and CCN2/connective tissue growth factor) are involved in a variety of cardiac pathophysiological conditions, including myocardial infarction, cardiac hypertrophy and fibrosis, aging-associated myocardial remodeling, myocarditis, diabetic cardiomyopathy, and valvular disease. This review discusses the properties and characteristics of the matricellular proteins and presents our current knowledge on their role in cardiac adaptation and disease. Understanding the role of matricellular proteins in myocardial pathophysiology and identification of the functional domains responsible for their actions may lead to design of peptides with therapeutic potential for patients with heart disease.

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.

Acute ketamine administration corrects abnormal inflammatory bone markers in major depressive disorder.

Patients with major depressive disorder (MDD) have clinically relevant, significant decreases in bone mineral density (BMD). We sought to determine if predictive markers of bone inflammation-the osteoprotegerin (OPG)-RANK-RANKL system or osteopontin (OPN)-play a role in the bone abnormalities associated with MDD and, if so, whether ketamine treatment corrected the abnormalities. The OPG-RANK-RANKL system plays the principal role in determining the balance between bone resorption and bone formation. RANKL is the osteoclast differentiating factor and diminishes BMD. OPG is a decoy receptor for RANKL, thereby increasing BMD. OPN is the bone glue that acts as a scaffold between bone tissues matrix composition to bind them together and is an important component of bone strength and fracture resistance. Twenty-eight medication-free inpatients with treatment-resistant MDD and 16 healthy controls (HCs) participated in the study. Peripheral bone marker levels and their responses to IV ketamine infusion in MDD patients and HCs were measured at four time points: at baseline, and post-infusion at 230 min, Day 1, and Day 3. Patients with MDD had significant decreases in baseline OPG/RANKL ratio and in plasma OPN levels. Ketamine significantly increased both the OPG/RANKL ratio and plasma OPN levels, and significantly decreased RANKL levels. Bone marker levels in HCs remained unaltered. We conclude that the OPG-RANK-RANKL system and the OPN system play important roles in the serious bone abnormalities associated with MDD. These data suggest that, in addition to its antidepressant effects, ketamine also has a salutary effect on a major medical complication of depressive illness.

The role of osteopontin in kidney diseases.

BACKGROUND: Osteopontin (OPN) is a pleiotropic glycoprotein expressed in various cell types in animals and in humans, including bone, immune, smooth muscle, epithelial and endothelial cells. Moreover, OPN is found in kidneys (in the thick ascending limbs of the loop of Henle and in distal nephrons) and urine. The protein plays an important role in mineralization and bone resorption. In addition, OPN is involved in the regulation of immunity and inflammation, angiogenesis and apoptosis. It was demonstrated that OPN and some OPN gene polymorphic variants are associated with the pathogenesis and progression of multiple disorders, such as cancer, autoimmune, neurodegenerative and cardiovascular diseases. Moreover, recent studies suggested that OPN is associated with the pathogenesis of renal failure. METHODS: In this review, I briefly discussed the role of OPN and its gene polymorphisms in kidney physiology, as well as in various kidney diseases. FINDINGS AND CONCLUSION: Most studies reported that OPN expression is elevated in urolithiasis, and also in acute and chronic kidney diseases, and in renal allograft dysfunction. Moreover, it was demonstrated that polymorphic variants of the OPN gene may be associated with renal failure. However, some reports suggested that OPN is essential for tubulogenesis, and that it inhibits calcium oxalate crystal formation and retention, nitric oxide synthesis, cell apoptosis and promotes cell regeneration. Thus, further studies are required to fully understand the role of OPN in kidney physiology and pathology. Eventually, these studies may result in the identification of OPN as a valuable marker for renal dysfunction prognosis and treatment.

The role of osteopontin as a candidate biomarker of renal involvement in systemic lupus erythematosus.

OBJECTIVES: Kidney biopsy is the gold standard for the diagnosis of lupus nephritis (LN). Conventional biomarkers of disease activity or renal function, such as complement levels, anti-dsDNA, serum creatinine, urinary sediment and proteinuria, do not have a sensitive diagnostic and prognostic value, therefore new biomarkers are needed to help predict or monitor LN. Osteopontin (OPN) is a pro-inflammatory molecule detectable in serum and renal tissue. The aim of this study was to evaluate OPN as a biomarker of renal involvement in patients with systemic lupus erythematosus (SLE) and correlate its levels with disease activity and laboratory features. METHODS: OPN was measured in the serum and urine of SLE patients with active LN (n=14), LN in remission (n=20), SLE without kidney involvement (n=22) and age- and sex-matched healthy controls (HC, n=20). RESULTS: OPN levels were significantly higher in urine than in serum in both groups of patients and controls (p<0.001). Serum OPN levels were higher in the LN patients than in HC and in SLE patients without renal involvement (p<0.0001 and 0.0032, respectively), regardless of the phase of renal activity. SLE patients without renal involvement and controls showed similar serum levels. We detected a direct correlation between low complement levels and OPN serum levels in patients with LN (p=0.014; R=0.438). Moreover, a higher percentage of patients with LN, compared to SLE without LN and HC, showed abnormal serum OPN. CONCLUSIONS: Our data suggest that serum OPN could be considered a biomarker of renal involvement, without differentiating between active and remission LN.

Endogenous osteopontin induces myocardial CCL5 and MMP-2 activation that contributes to inflammation and cardiac remodeling in a mouse model of chronic Chagas heart disease.

Cardiac dysfunction with progressive inflammation and fibrosis is a hallmark of Chagas disease caused by persistent Trypanosoma cruzi infection. Osteopontin (OPN) is a pro-inflammatory cytokine that orchestrates mechanisms controlling cell recruitment and cardiac architecture. Our main goal was to study the role of endogenous OPN as a modulator of myocardial CCL5 chemokine and MMP-2 metalloproteinase, and its pathological impact in a murine model of Chagas heart disease. Wild-type (WT) and OPN-deficient (spp1 -/-) mice were parasite-infected (Brazil strain) for 100days. Both groups developed chronic myocarditis with similar parasite burden and survival rates. However, spp1 -/- infection showed lower heart-to-body ratio (P<0.01) as well as reduced inflammatory pathology (P<0.05), CCL5 expression (P<0.05), myocyte size (P<0.05) and fibrosis (P<0.01) in cardiac tissues. Intense OPN labeling was observed in inflammatory cells recruited to infected heart (P<0.05). Plasma concentration of MMP-2 was higher (P<0.05) in infected WT than in spp1 -/- mice. Coincidently, specific immunostaining revealed increased gelatinase expression (P<0.01) and activity (P<0.05) in the inflamed hearts from T. cruzi WT mice, but not in their spp1 -/- littermates. CCL5 and MMP-2 induction occurred preferentially (P<0.01) in WT heart-invading CD8+ T cells and was mediated via phospho-JNK MAPK signaling. Heart levels of OPN, CCL5 and MMP-2 correlated (P<0.01) with collagen accumulation in the infected WT group only. Endogenous OPN emerges as a key player in the pathogenesis of chronic Chagas heart disease, through the upregulation of myocardial CCL5/MMP-2 expression and activities resulting in pro-inflammatory and pro-hypertrophic events, cardiac remodeling and interstitial fibrosis.

Osteopontin in Spontaneous Germinal Centers Inhibits Apoptotic Cell Engulfment and Promotes Anti-Nuclear Antibody Production in Lupus-Prone Mice.

Disposal of apoptotic cells is important for tissue homeostasis. Defects in this process in immune tissues may lead to breakdown of self-tolerance against intracellular molecules, including nuclear components. Development of diverse anti-nuclear Abs (ANAs) is a hallmark of lupus, which may arise, in part, due to impaired apoptotic cell clearance. In this work, we demonstrate that spontaneous germinal centers (GCs) in lupus-prone mice contain significantly elevated levels of unengulfed apoptotic cells, which are otherwise swiftly engulfed by tingible body macrophages. We indicate that osteopontin (OPN) secreted by CD153(+) senescence-associated T cells, which selectively accumulate in the GCs of lupus-prone mice, interferes with phagocytosis of apoptotic cells specifically captured via MFG-E8. OPN induced diffuse and prolonged Rac1 activation in phagocytes via integrin αvß3 and inhibited the dissolution of phagocytic actin cup, causing defective apoptotic cell engulfment. In wild-type B6 mice, administration of TLR7 ligand also caused spontaneous GC reactions with increasing unengulfed apoptotic cells and ANA production, whereas B6 mice deficient for Spp1 encoding OPN showed less apoptotic cells and developed significantly reduced ANAs in response to TLR7 ligand. Our results suggest that OPN secreted by follicular CD153(+) senescence-associated T cells in GCs promotes a continuous supply of intracellular autoantigens via apoptotic cells, thus playing a key role in the progression of the autoreactive GC reaction and leading to pathogenic autoantibody production in lupus-prone mice.

Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.

Myeloid-derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two-photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor-bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN-deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.

Osteopontin has a protective role in prostate tumor development in mice.

Osteopontin (OPN) is a protein, generally considered to play a pro-tumorigenic role, whereas several reports have demonstrated the anti-tumorigenic function of OPN during tumor development. These opposing anti- and pro-tumorigenic functions are not fully understood. Here, we report that host-derived OPN plays an anti-tumorigenic role in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model and a TRAMP tumor transplant model. Tumor suppression mediated by OPN in Rag2-/- mice suggests that OPN is dispensable in the adaptive immune response. We found that host-derived OPN enhanced infiltration of natural killer (NK) cells into TRAMP tumors. The requirement of OPN in NK cell migration towards TRAMP cells was confirmed by an ex vivo cell migration assay. In contrast to TRAMP cells, in vivo B16 tumor development was not inhibited by OPN, and B16 tumors did not show OPN-mediated cell recruitment. It is possible that low levels of chemokine expression by B16 cells do not allow OPN to enhance immune cell recruitment. In addition to demonstrating the anti-tumorigenic role of OPN in TRAMP tumor development, this study also suggests that the contribution of OPN to tumor development depends on the type of tumor as well as the source and isoform of OPN.

Mediation of the single-walled carbon nanotubes induced pulmonary fibrogenic response by osteopontin and TGF-ß1.

PURPOSE OF THE STUDY: A number of in vivo studies have shown that pulmonary exposure to carbon nanotubes (CNTs) may lead to an acute local inflammatory response, pulmonary fibrosis, and granulomatous lesions. Among the factors that play direct roles in initiation and progression of fibrotic processes are epithelial-mesenchymal transition and myofibroblasts recruitment/differentiation, both mediated by transforming growth factor-ß1 (TGF-ß1). Yet, other contributors to TGF-ß1 associated signaling, such as osteopontin (OPN) has not been fully investigated. MATERIALS AND METHODS: OPN-knockout female mice (OPN-KO) along with their wild-type (WT) counterparts were exposed to single-walled carbon nanotubes (SWCNT) (40 µg/mouse) via pharyngeal aspiration and fibrotic response was assessed 1, 7, and 28 days post-exposure. Simultaneously, RAW 264.7 and MLE-15 cells were treated with SWCNT (24 hours, 6 µg/cm2 to 48 µg/cm2) or bleomycin (0.1 µg/ml) in the presence of OPN-blocking antibody or isotype control, and TGF-ß1 was measured in supernatants. RESULTS AND CONCLUSIONS: Diminished lactate dehydrogenase activity at all time points, along with less pronounced neutrophil influx 24 h post-exposure, were measured in broncho-alveolar lavage (BAL) of OPN-KO mice compared to WT. Pro-inflammatory cytokine release (IL-6, TNF-α, MCP-1) was reduced. A significant two-fold increase of TGF-ß1 was found in BAL of WT mice at 7 days, while TGF-ß1 levels in OPN-KO animals remained unaltered. Histological examination revealed marked decrease in granuloma formation and less collagen deposition in the lungs of OPN-KO mice compared to WT. RAW 264.7 but not MLE-15 cells exposed to SWCNT and bleomycin had significantly less TGF-ß1 released in the presence of OPN-blocking antibody. We believe that OPN is important in initiating the cellular mechanisms that produce an overall pathological response to SWCNT and it may act upstream of TGF-ß1. Further investigation to understand the mechanistic details of such interactions is critical to predict outcomes of pulmonary exposure to CNT.

Genetic determinants and postorthodontic external apical root resorption in Czech children.

OBJECTIVE: Genes, involved in the modulation of inflammatory response and bone remodeling, play a role in the development of postorthodontic external apical root resorption (EARR). The aim of our study was to analyze possible associations between seven single nucleotide polymorphisms (SNPs) in interleukin-17A (IL-17), osteopontin (SPP1), purinoreceptor P2X7 (P2RX7), and tumor necrosis factor receptor superfamily member 11B (TNFRSF11B) genes and EARR in children after orthodontic treatment. SUBJECTS AND METHODS: This case-control study comprised 99 orthodontically treated patients (69 controls and 30 subjects with EARR). Genotype determinations of rs2275913, rs11730582, rs9138, rs208294, rs1718119, rs3102735, and rs2073618 were based on polymerase chain reaction using 5' nuclease TaqMan® assays. RESULTS: While no significant differences were observed in allele or genotype frequencies of all seven studied SNPs, specific haplotype of P2RX7 (rs208294 and rs1718119) modified the risk of EARR development (P < 0.05). In addition, the length of treatment with a fixed orthodontic appliance positively correlated with the presence of EARR (P < 0.05). CONCLUSIONS: Although the effect of individual SNPs studied on the EARR development was not confirmed in the Czech population, complex analysis suggested that variability in the P2RX7 gene and the length of orthodontic treatment may be important factors contributing to the etiopathogenesis of postorthodontic EARR.

Actinide(IV) Deposits on Bone: Potential Role of the Osteopontin-Thorium Complex.

In case of a nuclear event, contamination (broad or limited) of the population or of specific workers might occur. In such a senario, the fate of actinide contaminants may be of first concern, in particular with regard to human target organs like the skeleton. To improve our understanding of the toxicological processes that might take place, a mechanistic approach is necessary. For instance, ∼50% of Pu(IV) is known from biokinetic data to accumulate in bone, but the underlining mechanisms are almost unknown. In this context, and to obtain a better description of the toxicological mechanisms associated with actinides(IV), we have undertaken the investigation, on a molecular scale, of the interaction of thorium(IV) with osteopontin (OPN) a hyperphosphorylated protein involved in bone turnover. Thorium is taken here as a simple model for actinide(IV) chemistry. In addition, we have selected a phosphorylated hexapeptide (His-pSer-Asp-Glu-pSer-Asp-Glu-Val) that is representative of the peptidic sequence involved in the bone interaction. For both the protein and the biomimetic peptide, we have determined the local environment of Th(IV) within the bioactinidic complex, combining isothermal titration calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy, theoretical calculations with density functional theory, and extended X-ray absorption fine structure spectroscopy at the Th LIII edge. The results demonstrate a predominance of interaction of metal with the phosphate groups and confirmed the previous physiological studies that have highlighted a high affinity of Th(IV) for the bone matrix. Data are further compared with those of the uranyl case, representing the actinyl(V) and actinyl(VI) species. Last, our approach shows the importance of developing simplified systems [Th(IV)-peptide] that can serve as models for more biologically relevant systems.

A preliminary in vivo study of the effects of OPN on rat liver regeneration induced by partial hepatectomy.

Osteopontin (OPN) is a member of Th1 cytokine secreted by activated lymphocytes and macrophages. However, it deserves to be studied whether OPN could promote cell activation or proliferation, and then facilitate hepatic self-repair during liver regeneration (LR). This study is designed to further reveal the effects of OPN on LR in vivo. Firstly, quantitative reverse transcription-PCR (qRT-PCR) and western blot (WB) were utilized to validate the expression profile of endogenous OPN in rat regenerating livers after partial hepatectomy (PH). Then OPN expression vector, two shRNA expression vectors and their respective test vectors were successfully constructed. Afterwards, test vectors were administrated into mouse livers via tail vein to find the more efficient shRNA. Furthermore, OPN expression vector and the more efficient shRNA expression vector were injected into rat regenerating livers, and then the changes in liver regeneration and hepatic microstructure were respectively detected by liver regeneration rate and HE staining, while the expressions of several marker genes were detected by qRT-PCR and WB. Endogenous OPN was strikingly up-regulated in both mRNA and protein level during LR, especially at 12 and 72 h after PH. The shRNA expression vector Opn(313) was found to be more efficient than Opn(887) in silencing the expression of Opn. Then OPN expression vector and Opn(313) were injected into rat remnant livers, and it showed that OPN overexpression aggravated hepatic necrosis and leukocytes infiltration, while OPN silencing inhibited liver regeneration rate and the expressions of PCNA and CCL2, but augmented that of BAX. In conclusion, OPN might enhance inflammation and cell proliferation, attenuate cell apoptosis, and ultimately facilitate liver regeneration at the termination stage of liver regeneration.

Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis.

AIMS: Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process. METHODS AND RESULTS: OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo. CONCLUSION: In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.

The expression of osteopontin in breast cancer tissue and its relationship with p21ras and CD44V6 expression.

OBJECTIVE: This study aimed to investigate the expression of osteopontin (OPN), p2lras, and CD44V6 in breast cancer tissues, and to analyze the relationships between their expression and a patient's clinicopathological characteristics and five-year survival rate. MATERIALS AND METHODS: Streptavidin-peroxidase immunohistochemistry was used to detect the expression of OPN, p2lras, and CD44V6 in tissue samples from 96 breast cancer patients, and the multivariate Cox proportional hazards model (mCOX-PHM) was used to analyze the factors that affect prognosis. RESULTS: Among the 96 breast cancer patients studied, positive staining for OPN, CD44V6, and p21ras was observed in 54.2%, 58.3%, and 43.8% of samples, respectively. The expression of OPN and CD44V6 were positively correlated (r = 0.58), and the expression of OPN and p21ras were also positively correlated (r = 0.25). Coexpression OPN, CD44V6, and p21ras was negatively correlated with a patient's five-year survival rate (p < 0.05). Kaplan-Meier analysis indicated that a patient without OPN, CD44V6, or p21ras expression had an improved survival (p < 0.05). Results from the mCOX-PHM analysis indicated that CD44V6 expression, the degree of tumor differentiation, and lymph node metastasis were all independent factors that indicate prognosis. The combined detection of OPN, CD44V6, and p21ras could contribute to a more accurate assessment of the biological behavior of breast cancers, and could help to indicate the prognosis of breast cancer patients.