Osteopontin Protein Levels Research Articles

Osteopontin: A potential biomarker for heart failure and reverse remodeling after left ventricular assist device support

Left ventricular assist device (LVAD) support in end-stage heart failure (HF) leads to recovery of the patient's condition, size reduction of cardiomyocytes, and also volume reduction and change in the composition of the extracellular matrix (ECM). Myocardial expression of ECM osteopontin (OPN) protein increases with the severity of HF. We analyzed whether OPN messenger RNA expression in heart tissue and/or OPN protein in plasma are associated with reverse remodeling during LVAD support. Plasma and heart tissue specimens of 22 end-stage HF patients before and after LVAD implantation and subsequent heart transplantation (HTx) were used to determine the concentrations of OPN protein (EIA) and OPN messenger RNA (mRNA) by quantitative polymerase chain reaction. Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to locate OPN protein and mRNA. The high OPN protein levels in plasma of HF patients did not differ significantly before and after LVAD support in ischemic heart disease (IHD) (pre-LVAD 167 ± 32 ng/ml; post-LVAD 165 ± 28 ng/ml) and in dilated cardiomyopathy (DCM) (pre-LVAD 99 ± 12 ng/ml; post-LVAD (142 ± 6 ng/ml). The OPN plasma levels after HTx decreased to control levels (IHD, 48 ± 6; DCM, 40 ± 5; control, 31 ± 3 ng/ml). In contrast, expression of OPN mRNA in heart biopsy specimens decreased significantly after LVAD support (the relative quantity decreased > 90% in IHD and 50% in DCM). ISH and IHC revealed that OPN was present in cardiomyocytes and in the ECM. Levels of OPN mRNA in the myocardium of HF patients showed a significant decrease after LVAD support but OPN protein expression did not. LVAD support only induced a decrease of OPN plasma levels in individual patients, whereas OPN plasma levels reduced significantly in all patients after HTx.

Plasma concentrations of osteopontin, but not thrombin-cleaved osteopontin, are associated with the presence and severity of nephropathy and coronary artery disease in patients with type 2 diabetes mellitus

BackgroundThe aim of the present cross-sectional study was to assess possible associations between osteopontin (OPN), and thrombin-cleaved (N-half) OPN, and nephropathy and coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM).MethodsPlasma levels of OPN, N-half OPN, and high-sensitivity C-reactive protein (hsCRP) were determined in 301 diabetic patients with (n = 226) or without (n = 75) angiographically documented CAD (luminal diameter narrowing >50%), as well as in 75 non-diabetic controls with normal angiography. The estimated glomerular filtration rate (eGFR) was calculated in all patients.ResultsPlasma levels of OPN and hsCRP were significantly higher in patients with T2DM compared with controls. In addition, there was a higher occurrence of moderate renal insufficiency and lower eGFR in patients with T2DM (all P < 0.01). T2DM patients in whom OPN levels were greater than the median value had higher serum creatinine levels, a greater prevalence of mild or moderate renal insufficiency, a higher incidence of CAD, and lower eGFR (all P < 0.05) than T2DM patients in whom OPN levels were the same as or lower than the median value. However, there were no differences in these parameters when patients were stratified according to plasma N-half OPN levels. Furthermore, there was a significant correlation between OPN, but not N-half OPN, and the severity of nephropathy and CAD in diabetes. After adjustment for potential confounders and treatments, multiple linear regression analysis demonstrated an independent association between OPN, but not N-half OPN, and eGFR. Multivariate logistic regression revealed that higher OPN levels conferred a fourfold greater risk of renal insufficiency and CAD in patients with T2DM.ConclusionsThe results of the present study demonstrate that there is an independent association between plasma levels of OPN, but not N-half OPN, and the presence and severity of nephropathy and CAD in diabetes.

Effects of osteopontin inhibition on radiosensitivityof MDA-MB-231 breast cancer cells

BackgroundOsteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients. In patients with head and neck cancer, high OPN plasma levels have been associated with poor prognosis following radiotherapy. Since little is known about the relationship between OPN expression and radiosensitivity, we investigated the cellular and radiation induced effects of OPN siRNA in human MDA-MB-231 breast cancer cells.MethodsMDA-MB-231 cells were transfected with OPN-specific siRNAs and irradiated after 24 h. To verify the OPN knockdown, we measured the OPN mRNA and protein levels using qRT-PCR and Western blot analysis. Furthermore, the functional effects of OPN siRNAs were studied by assays to assess clonogenic survival, migration and induction of apoptosis.ResultsTreatment of MDA-MB-231 cells with OPN siRNAs resulted in an 80% decrease in the OPN mRNA level and in a decrease in extracellular OPN protein level. Transfection reduced clonogenic survival to 42% (p = 0.008), decreased the migration rate to 60% (p = 0.15) and increased apoptosis from 0.3% to 1.7% (p = 0.04). Combination of OPN siRNA and irradiation at 2 Gy resulted in a further reduction of clonogenic survival to 27% (p < 0.001), decreased the migration rate to 40% (p = 0.03) and increased apoptosis to 4% (p < 0.005). Furthermore, OPN knockdown caused a weak radiosensitization with an enhancement factor of 1.5 at 6 Gy (p = 0.09) and a dose modifying factor (DMF10) of 1.1.ConclusionOur results suggest that an OPN knockdown improves radiobiological effects in MDA-MB-231 cells. Therefore, OPN seems to be an attractive target to improve the effectiveness of radiotherapy.

Plasma decorin predicts the presence of esophageal squamous cell carcinoma

Using a microarray technique, we found decorin to be underexpressed, but osteopontin (OPN) to be overexpressed, in esophageal squamous cell carcinoma (ESCC). This study aims to examine whether plasma decorin and OPN plus personal substances use (tobacco, alcohol and areca) can serve as suitable clinical markers to predict the presence of ESCC. In total, 570 archived plasma specimens (275 patients and 295 controls) were collected from 2 medical centers in Taiwan between 2000 and 2008. Decorin and OPN protein levels were measured by ELISA. Means and standard deviation of plasma decorin were 5.6 + or - 3.6 ng/ml in case patients, which were significantly lower than those in controls (7.8 + or - 3.1, p < 0.0001). Plasma OPN levels in case patients were not significantly different from controls (p = 0.33). When compared to subjects with the lowest quartile of plasma decorin, those with the highest quartile one had a significantly lower risk to have ESCC (Adjusted OR = 0.03, p < 0.001). Receiver operator characteristic (ROC) analysis was performed for the combination of plasma decorin and 3 substances use (smoke, alcohol and areca) for the patients compared with the controls. The area under the curve was 88.6% and the optimal cut-point of ROC curve (any 3 factors) had 73.5% sensitivity and 90.2% specificity with approximately 82% of corrected classification. Plasma decorin, but not OPN, is a potential clinical marker for the detection of ESCC. When plasma decorin plus the use of the 3 substances are combined, this factor cluster could be used to detect the presence of ESCC.

Osteopontin is up-regulated and associated with neutrophil and macrophage infiltration in glioblastoma

Osteopontin (OPN) is a glycophosphoprotein with multiple intracellular and extracellular functions. In vitro, OPN enhances migration of mouse neutrophils and macrophages. In cancer, extracellular OPN facilitates migration of cancer cells via its RGD sequence. The present study was designed to investigate whether osteopontin is responsible for neutrophil and macrophage infiltration in human cancer and in particular in glioblastoma. We found that in vitro mouse neutrophil migration was RGD-dependent. In silico, we found that the OPN gene was one of the 5% most highly expressed genes in 20 out of 35 cancer microarray data sets in comparison with normal tissue in at least 30% of cancer patients. In some types of cancer, such as ovarian cancer, lung cancer and melanoma, the OPN gene was one of those with the highest expression levels in at least 90% of cancer patients. In glioblastoma, the most invasive type of brain tumours/glioma, but not in lower grades of glioma it was one of the 5% highest expressed genes in 90% of patients. In situ, we found increased protein levels of OPN in human glioblastoma versus normal human brain confirming in silico results. OPN protein expression was co-localized with neutrophils and macrophages. In conclusion, OPN in tumours not only induces migration of cancer cells but also of leucocytes.

Transcriptomic analysis of patients with tetralogy of Fallot reveals the effect of chronic hypoxia on myocardial gene expression

In cyanotic patients undergoing repair of heart defects, chronic hypoxia is thought to lead to greater susceptibility to ischemia and reoxygenation injury. We sought to find an explanation to such a hypothesis by investigating the cardiac gene expression in patients with tetralogy of Fallot undergoing cardiac surgery. The myocardial gene profile was investigated in right ventricular biopsy specimens obtained from 20 patients with a diagnosis of cyanotic (n = 11) or acyanotic (n = 9) tetralogy of Fallot undergoing surgical repair. Oligonucleotide microarray analyses were performed on the samples, and the array results were validated with Western blotting and enzyme-linked immunosorbent assay. Data revealed 795 differentially expressed genes in cyanotic versus acyanotic hearts, with 198 upregulated and 597 downregulated. Growth/morphogenesis, remodeling, and apoptosis emerged as dominant functional themes for the upregulated genes and included the apoptotic gene TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), the remodeling factor OPN (osteopontin), and the mitochondrial function gene COX11 (cytochrome-c oxidase 11). In contrast, transcription, mitogen-activated protein kinase signaling, and contractile machinery were the dominant functional classes for the downregulated genes, which included the calcium-handling gene NCX1 (sodium-calcium exchanger). Protein levels of COX11, NCX1, OPN, and LYZ (lysozyme) in the myocardium followed the same pattern obtained by means of transcriptomics. The TRAIL level did not change in myocardium but increased in circulating blood of cyanotic patients, suggesting the myocardium as a possible source. Additionally, our data showed increased protein expression of apoptosis markers in cyanotic myocardium. Chronic hypoxia in cyanotic children with tetralogy of Fallot induced the expression of genes associated with apoptosis and remodeling and reduced the expression of genes associated with myocardium contractility and function.

Elevated tumor and serum levels of the hypoxia-associated protein osteopontin are associated with prognosis for soft tissue sarcoma patients

BackgroundOsteopontin (OPN) overexpression is correlated with a poor prognosis for tumor patients. However, only a few studies investigated the prognostic impact of expression of OPN in soft tissue sarcomas (STS) yet.MethodsThis study is based on tumor and serum samples from 93 adult STS patients. We investigated OPN protein levels in serum (n = 86) and tumor tissue (n = 80) by ELISA and OPN mRNA levels in tumor tissue (n = 68) by quantitative real-time PCR.ResultsNo correlation was found between OPN levels in serum and tumor tissue. Moreover, an elevated OPN protein level in the serum was significantly associated with clinical parameters such as higher stage (p = 0.004), higher grade (p = 0.003), subtype (p = 0.002) and larger tumor size (p = 0.03). OPN protein levels in the tumor tissue were associated with higher stage (p = 0.06), higher grade (p = 0.003), subtype (p = 0.07) and an increased rate of relapse (p = 0.02). In addition, using a Cox's proportional hazards regression model, we found that an elevated OPN protein level in the serum and tumor tissue extracts is a significant negative prognostic factor for patients with STS. The relative risks of tumor-related death were 2.2 (p < 0.05) and 3.7 (p = 0.01), respectively.ConclusionOur data suggest OPN protein in serum as well as in tumor tissue extracts is an important prognostic factor for soft tissue sarcoma patients.

Elevated Osteopontin Expression and Proliferative/Apoptotic Ratio in the Colorectal Adenoma–Dysplasia–Carcinoma Sequence

Colorectal cancer progression is characterized by altered epithelial proliferation and apoptosis and by changed expression of tumor development regulators. Our aims were to determine the proliferative/apoptotic epithelial cell ratio (PAR) in the adenoma-dysplasia-carcinoma sequence (ADCS), and to examine its association with osteopontin (OPN), a previously identified protein product related to cancer development. One mm diameter cores from 13 healthy colons, 13 adenomas and 13 colon carcinoma samples were included into a tissue microarray (TMA) block. TUNEL reaction and Ki-67 immunohistochemistry were applied to determine the PAR. The osteopontin protein was also immunodetected. Stained slides were semiquantitatively evaluated using digital microscope and statistically analyzed with logistic regression and Fisher's exact test. The PAR continuously increased along the ADCS. It was significantly (p < 0.001) higher in cancer epithelium (8.84 ± 7.01) than in adenomas (1.40 ± 0.78) and in normal controls (0.89 ± 0.21) (p < 0.001). Also, significant positive correlation was observed between elevated PAR and the expression of osteopontin. Cytoplasmic OPN expression was weak in healthy samples. In contrast, cytoplasmic immunoreaction was moderately intensive in adenomas, while in colon cancer strong, diffuse cytoplasmic immune staining was detected. Increasing PAR and OPN expression along ADCS may help monitoring colorectal cancer progression. The significantly elevated OPN protein levels we found during normal epithelium to carcinoma progression may contribute to the increased fibroblast-myofibroblast transition determining stem cell niche in colorectal cancer.

The changes of osteopontin expression in exprerimental murine colitis

Objective To investigate the changes of osteopontin (OPN) expression in murine colitis induced by dextran sodium sulfate (DSS) and its role in inflammatory bowel disease (IBD).Methods Thirty-two male BALB/C mice were randomly assigned into 4 groups: control group (group A), DSS-induced murine colitis model group (group B), salazosulfapyridine treatment group (group C) and infliximab treatment group (group D). Plasma OPN concentration was measured by enzyme linked immunosorbent assay (ELISA). OPN mRNA level was detect by using reverse transcriptase polymerase chain reaction (RT-PCR) and protein expression in colonic tissues was analyzed by using Western blotting. The location expression of OPN in colonic tissues was determined by immunohistochemical staining. Results In group A, B,C and D, the plasma concentrations of OPN were (5.26±1.93) ng/ml, (10.21±2.37) ng/ml, (4.58±1.83) ng/ml and (4.82±1.83) ng/ml,respectively: the mRNA levels of OPN in colonic tissues were (0.36±0.16), (0.71±0.17),(0.32±0.07) and (0. 34±0. 08), respectively; the protein levels of OPN in colonic tissues were (0.44±0.10), (0.85±0.04), (0.61±0.11) and (0.58±0.17), respectively. The OPN-positive cell numbers in lamina propria mononuclear were (46.6±10.9), (155.5±43.8), (73.1±6.8) and (70.6±8.3), respectively. The expression of OPN in group B was significantly higher than that in group A (P<0.05). Compared with group B, the expressions of OPN in group C and D were significantly decreased (all P valus <0. 05). No significant difference was detected between group C and D. Conclusions The study shows that the expression of OPN significantly increased in DSS-induced murine colitis and decreased after treated with drugs. OPN-mediated immune response contributes to DSS-induced murine colitis. Key words: Colitis; Osteopontin; Dextran sodium sulfate

Inhibitory effects on proliferation and invasion of lung cancer cells by RNAi-mediated knockdown of osteopontin

Objective To develop DNA vector-based RNA interference (RNAi) technology and block the expression of osteopontin (OPN) in lung cancer cell line A549 ,and further examine its possible effect on invasion and proliferation of lung cancer cells. Methods One double-stranded DNA vectors pEN-TRTM/U6-INF (pINF-1) targeting the mRNA of human OPN, and the control vector pENTRTM/U6-CTR (pCTR) mismatching with mRNA of OPN were constructed, and then they were transfected into human A549 cells with high metastatic potentials. Western blotting was used to quantify the protein level of OPN. The malignant phenotypes of transfected A549 cells including proliferation and invasive activities were ana-lyzed. Results As compared with control group only transfected with Lipofectamine~(TM) 2000, OPN protein lev-el in plNF-1 group could be decreased by 85% 72 h after transfection,and no significant difference was found in the group transfected with pCTR. In vitro cellular growth rate was decreased by 56% (P<0.05) in the group transfected with pINF-1 ,and migrating number of A549 cells (15.2±2.8) transfected with pINF-1 was also significantly decreased (P<0.01 ) as compared with the control group (72.4±6.5). Conclusion Se-quence-specific shRNA (pINF-1) can significantly reduce the OPN expression in A549 cell line. The sup-pression of OPN expression in A549 cells transfected with pINF-1 can induce the decline of invasive and pro-liferative ability of A549. This suggests that OPN may be an alternative target for treatment in lung cancer. Key words: Lung carcinoma; Osteopontin; RNA interference; Metastasis

Influence of osteopontin short hairpin RNA on the proliferation and invasion of human renal cancer cells

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by Lipofectamine 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.

Osteopontin silencing by small interfering RNA induces apoptosis and suppresses invasion in human renal carcinoma Caki-1 cells

Antiapoptosis and invasion are the causes for the failure of conventional radiotherapy and chemotherapy in human renal carcinomas. Osteopontin (OPN), a ligand for vß3 integrin and CD44 receptors, is a phosphorylated glycoprotein with diverse functions including tumorigenesis and tumor cell metastasis. Recently, OPN has been detected in human renal carcinomas and assessed as a potential prognostic marker of renal carcinomas. However, the function and mechanism of OPN in renal carcinomas remain unknown. In this study, we used OPN siRNA to silence the expression of OPN in renal carcinoma Caki-1 cells. Silent effect showed that sequence-specific siRNA targeting OPN suppressed OPN mRNA expression by 81% and OPN protein level by 91% in vitro. The apoptosis ability was significantly increased while the invasion ability was decreased in Caki-1 cells transfected with OPN siRNA. Western blot demonstrated that the effects of OPN silence were significantly accompanied by the activation of mitochondria-related apoptosis pathway involving cytochrome c, Apaf-1, cleaved caspase-3 and Bcl-2/Bax, and the downregulation of invasion-related proteins of MMP-2 and uPA expression. These results suggest that the downregulation of OPN expression can induce apoptosis increase and invasion suppression in renal carcinoma Caki-1 cells through mitochondria-related apoptosis pathway and MMP-2 and uPA-related invasion proteins, respectively.

Expression and regulation of osteopontin in type 1 diabetes

Osteopontin (OPN) is a secreted acidic phosphoprotein that is involved in many inflammatory and immune-modulating disorders. We previously demonstrated that OPN is a novel islet protein and a pro survival factor that may serve as an intrinsic feedback regulator of nitric oxide signaling in β-cells. Here, we investigated the endogenous expression of pancreatic OPN in non obese diabetic (NOD) mice and explored its regulation in the islets and b-cells. High levels of pancreatic OPN mRNA and protein were seen in the prediabetic NOD mice pancreata. The temporal pattern of OPN expression inversely correlated with progression of insulitis and β-cell destruction. Immunostaining of pancreatic serial sections showed co localization of OPN with most of the islet hormones. Next we investigated the regulation of OPN in the islets and β-cells. Naturally occurring early upregulation of OPN transcription was seen after exposure of native normoglycemic NOD islets and β-cells to a high-dose combination of IL-1β, TNF-α and IFN-γ. To distinguish between the effect of cytokines and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with cytokines or glucose. Cytokines induced upregulation of OPN promoter activity within one hour, while glucose induced a dose-dependent upregulation of OPN promoter activity after 24 hrs. Long-term exposures to cytokines or glucose reduced OPN expression and promoter activity. Our data provide the first observations into the presence of a positive intrinsic mechanism that regulates pancreatic OPN expression. Based upon previous studies that support a protective role of OPN in the islets, our data suggest that exhaustion of this local OPN system is implicated in the associated loss of endogenous islet protection and progression of the destructive insulitis and diabetes severity in the NOD mouse model.

Effects of Corticosteroids on Osteopontin Expression in a Murine Model of Allergic Asthma

Background: Osteopontin (OPN) contributes to the development of T helper 1 (Th1)-mediated immunity and Th1-associated diseases. However, the role of OPN in bronchial asthma is unclear. Corticosteroids reduce airway inflammation, as reflected by the low eosinophil and T-cell counts, and the low level of cytokine expression. We investigated OPN production and the inhibitory effects of corticosteroids on OPN production in a murine model of allergic asthma. Methods: BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Some mice received daily injections of dexamethasone (DEX) or phosphate-buffered saline for 1 week. All OVA-challenged mice were exposed to aerosolized 1% OVA for 30 min an hour after these injections. After the OVA challenge, the mice were killed, and bronchoalveolar lavage (BAL) fluid and lung tissue were examined. Results: The levels of OPN protein in BAL fluid and OPN mRNA in lung tissue increased after OVA challenge. Most OPN-expressing cells were CD11c+ cells and some were T cells. DEX decreased the levels of OPN protein in the BAL fluid, and those of OPN mRNA and OPN protein in lung tissue. Conclusions: OPN may play an important role in allergic bronchial asthma. Corticosteroids inhibit OPN production in mice with allergic asthma. The beneficial effect of corticosteroids in bronchial asthma is partly due to their inhibitory effects on OPN production.

IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice

Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

Influence of osteopontin short hairpin RNA on the proliferation and activity of rat vascular smooth muscle cells

To investigate the influence of osteopontin (OPN) short hairpin RNA (shRNA) on the proliferation and activity of rat vascular smooth muscle cells (VSMCs), the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 (PG1), pGenesil-1/OPNshRNA2 (PG2) and pGenesil-1/OPNshRNAHK (PGH) were transfected into the cultured rat VSMC by Lipofectamine 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and III collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% (P<0.01) by PG1, 73% and 52% (P<0.01) by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type I and III collagen. It is concluded that recombinant plasmid can be successfully transfected into VSMCs by Lipofectamine 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type I and III collagen is inhibited after knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention (PCI) by RNA interference (RNAi) technology.

The effects of natalizumab on inflammatory mediators in multiple sclerosis: prospects for treatment‐sensitive biomarkers

Natalizumab affects systemic cytokine expressions and clinical course in relapsing-remitting multiple sclerosis (RRMS). We analyzed levels of inflammatory cytokines in cerebrospinal fluid (CSF) cells and peripheral blood mononuclear cells (PBMCs), levels of matrix metalloproteinase (MMP)-9 and osteopontin (OPN) in CSF, and clinical outcome measures in 22 natalizumab-treated RRMS patients. mRNA levels of cytokines in cells were detected with real-time RT-PCR. Protein levels of OPN and MMP-9 were measured by ELISA. Natalizumab reduced CSF cell counts (P < 0.0001). Tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) mRNAs were significantly increased in PBMCs. In contrast, expressions of IFN-gamma and interleukin (IL)-23 were decreased but IL-10 increased in the CSF cells. OPN and MMP-9 were reduced in the CSF. Patients being in remission at baseline showed the same deviations of mediators as those in relapse after natalizumab treatment. The open label clinical outcome measures were either stable or improved during therapy. Natalizumab attenuates pro-inflammatory mediators intrathecally and the reduced pro-inflammatory milieu may allow increased production of the anti-inflammatory mediator IL-10. The increased systemic cytokines may impede the improvement of certain clinical measures like fatigue. The affected mediators seem to be sensitive to an immune-modifying treatment which could be used as biomarkers for this therapy.

Senescent Stromal-Derived Osteopontin Promotes Preneoplastic Cell Growth

Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression.

Prognostic Significance of Plasma Osteopontin Levels in Patients Undergoing Percutaneous Coronary Intervention

Osteopontin (OPN) mRNA is highly expressed in atherosclerotic plaques and plasma OPN levels are high in patients with coronary artery disease and in those with restenosis. OPN-overexpressing transgenic mice show markedly increased neointimal formation after arterial injury. The prognostic significance of the preprocedural plasma OPN level was investigated in 130 patients undergoing percutaneous coronary intervention (PCI). Patients were followed up for restenosis and major adverse cardiovascular events (MACE). At 7+/-3 months after PCI, angiography was performed again in 91 patients, of whom 40 had restenosis. Between patients with and without restenosis, OPN (492+/-200 vs 482+/-224 ng/ml) and C-reactive protein (CRP: 0.78 vs 0.70 mg/L) levels did not differ. During a 3-year follow-up, MACE occurred in 21 patients, who had higher OPN (586+/-230 vs 438+/-195 ng/ml) and CRP (1.30 vs 0.70 mg/L) levels than those without MACE (P<0.005). Both OPN and CRP levels were independent predictors for MACE. Hazard ratios for MACE were 1.3 (95% confidence interval (CI) 1.1-1.5) for a 100 ng/ml increase in OPN and 3.6 (95%CI 1.4-9.3) for CRP >1.0 mg/L. In patients undergoing PCI, the preprocedural OPN and CRP levels are independent predictors for further cardiovascular events, but not for restenosis.

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45 kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45 kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal–fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.