Extracellular Matrix Proteins Osteopontin Research Articles

Intrahepatic osteopontin signaling by CREBZF defines a checkpoint for steatosis-to-NASH progression.

NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.

Endothelial c-Myc knockout enhances diet-induced liver inflammation and fibrosis.

Endothelial cells play an essential role in inflammation through synthesis and secretion of chemoattractant cytokines and expression of adhesion molecules required for inflammatory cell attachment and infiltration. The mechanisms by which endothelial cells control the pro-inflammatory response depend on the type of inflammatory stimuli, endothelial cell origin, and tissue involved. In the present study, we investigated the role of the transcription factor c-Myc in inflammation using a conditional knockout mouse model in which Myc is specifically deleted in the endothelium. At a systemic level, circulating monocytes, the chemokine CCL7, and the extracellular-matrix protein osteopontin were significantly increased in endothelial c-Myc knockout (EC-Myc KO) mice, whereas the cytokine TNFSF11 was downregulated. Using an experimental model of steatohepatitis, we investigated the involvement of endothelial c-Myc in diet-induced inflammation. EC-Myc KO animals displayed enhanced pro-inflammatory response, characterized by increased expression of pro-inflammatory cytokines and leukocyte infiltration, and worsened liver fibrosis. Transcriptome analysis identified enhanced expression of genes associated with inflammation, fibrosis, and hepatocellular carcinoma in EC-Myc KO mice relative to control (CT) animals after short-exposure to high-fat diet. Analysis of a single-cell RNA-sequencing dataset of human cirrhotic livers indicated downregulation of MYC in endothelial cells relative to healthy controls. In summary, our results suggest a protective role of endothelial c-Myc in diet-induced liver inflammation and fibrosis. Targeting c-Myc and its downstream pathways in the endothelium may constitute a potential strategy for the treatment of inflammatory disease.

PCL and PCL/bioactive glass biomaterials as carriers for biologically active polyphenolic compounds: Comprehensive physicochemical and biological evaluation

In this work, polymeric and bioactive glass (BG)-modified composite films were successfully loaded with polyphenols (PPh) extracted from sage. It was hypothesized that PPh, alone and in combination with BGs particles, would affect physicochemical and biological properties of the films. Furthermore, sol-gel-derived BG particles would serve as an agent for control the release of the polyphenolic compounds, and other important properties related to the presence of PPh. The results showed that polyphenolic compounds significantly modified numerous material properties and also acted as biologically active substances. On the one hand, PPh can be considered as plasticizers for PCL, on the other hand, they can act as coupling agent in composite materials, improving their mechanical performance. The presence of PPh in materials improved their hydrophilicity and apatite-forming ability, and also provided antioxidant activity. What is important is that the aforementioned properties and kinetics of PPh release can be modulated by the use of various concentrations of PPh, and by the modification of PCL matrix with sol-gel-derived BG particles, capable of binding PPh. The films containing the lowest concentration of PPh exhibited cytocompatibility, significantly increased alkaline phosphatase activity and the expression of bone extracellular matrix proteins (osteocalcin and osteopontin) in human normal osteoblasts, while they reduced intracellular reactive oxygen species production in macrophages. Furthermore, materials loaded with PPh showed antibiofilm properties against Gram positive and Gram negative bacteria. The results suggest that obtained materials represent potential multifunctional biomaterials for bone tissue engineering with a wide range of tunable properties.

Methylsulfonylmethane increases osteogenesis and regulates the mineralization of the matrix by transglutaminase 2 in SHED cells.

Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM.

The expression profile of integrin receptors and osteopontin in thyroid malignancies varies depending on the tumor progression rate and presence of BRAF V600E mutation

Thyroid cancer (TC) is one of the most common malignancy of the human endocrine system. BRAF V600E mutation is the most frequent genetic alteration of papillary carcinoma, the most frequent TC, which effects RAS-RAF-MEK intracellular signaling pathway. These alterations in RAS-RAF-MEK pathway lead to changes in expression levels of cell membrane integrin receptors and their ligand - extracellular matrix protein osteopontin, which in turn increases the metastatic potential of tumor cells. Thus, integrins and their ligand osteopontin can be considered as potential biomarkers of tumor progression and aggressive tumor phenotypes.The aim of the study was to evaluate the expression levels of integrin receptors ITGA2, ITGA3, ITGAV, ITGA6, ITGA9, ITGB1, ITGB3 and their ligands OPNa, OPNb in the thyroid cancer with different BRAF V600E mutation status. MethodsThyroid tumor samples of 70 patients obtained during surgical treatment were analyzed. Expression levels of the investigated genes were evaluated by real time RT-PCR. Fluorescent immunohistochemistry (IHC) was used to confirm the PCR results and to estimate the amount of protein levels. For IHC frozen sections were used. BRAF V600E mutation was determined using allele-specific amplification. Nonparametric criteria (Kruskal Wallis, Wilcoxon and Mann-Whitney tests) were used to evaluate group differences. P values of less than 0.05 were considered as statistically significant. ResultsA higher gene expression level of ITGA2 (1.9-fold, p = 0.037), ITGA3 (21.1-fold, p = 0.041) and ITGA5 (2.08-fold, p = 0.048) was observed in papillary thyroid cancer (PTC) tissue in comparison with median expression level in control samples (conventionally normal tissue of thyroid gland). These changes were confirmed by IHC (significant changes for α2 integrin). ITGAV expression level was statistically significantly higher in follicular thyroid cancer (FTC) (2.0-fold, p = 0.040). Next, high gene expression levels in tissue samples of lymph node metastases were observed for ITGA5 (2.92-fold, p = 0.015), OPNb (4.36-fold, p = 0.037). For genes ITGA3 (37.48-fold, p = 0.017790), ITGA6 (18.76-fold, p = 0.028921) and ITGA9 (12.52-fold, p = 0.026710) higher expression level was detected in T3-4 tumors (TNM) compared to tumors classified as T1-2. Presence of BRAF V600E mutation was identified in 20 samples of PTC of 40 (50%). A significant increase of the expression level only of ITGA3 (3.1-fold, p = 0. 0422) was observed in BRAF V600E positive samples. Further, changes in expression levels of integrins and osteopontin were assessed in benign and malignant neoplasms. In PTC samples higher expression of ITGA2 (2.8-fold, p = 0.005), ITGA6 (2.11, p = 0.03) and ITGB1 (2.32-fold, p = 0.02) was detected. In FTC expression level of ITGA6 (2.67, p = 0.007) was higher than in benign thyroid nodules. ConclusionIdentified changes in expression levels of the studied genes indicate that they could play an important role in tumor progression, and their expression could be affected by the product of mutant BRAF gene. Integrins and their ligand osteopontin might be considered as potential markers in determining prognosis and treatment of TC.

Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line.

Vascular smooth muscle cells (VSMC) dedifferentiation from a contractile to a synthetic phenotype contributes to atherosclerosis. Atherosclerotic tissue has a chronic inflammatory component with high levels of tumor necrosis factor-α (TNF-α). VSMC of atheromatous plaques have increased autophagy, a mechanism responsible for protein and intracellular organelle degradation. The aim of this study was to evaluate whether TNF-α induces phenotype switching of VSMCs and whether this effect depends on autophagy. Rat aortic Vascular smooth A7r5 cell line was used as a model to examine the phenotype switching and autophagy. These cells were stimulated with TNF-α 100 ng/mL. Autophagy was determined by measuring LC3-II and p62 protein levels. Autophagy was inhibited using chloroquine and siRNA Beclin1. Cell dedifferentiation was evaluated by measuring the expression of contractile proteins α-SMA and SM22, extracellular matrix protein osteopontin and type I collagen levels. Cell proliferation was measured by [3H]-thymidine incorporation and MTT assay, and migration was evaluated by wound healing and transwell assays. Expression of IL-1β, IL-6 and IL-10 was assessed by ELISA. TNF-α induced autophagy as determined by increased LC3-II (1.91±0.21, p<0.001) and decreased p62 (0.86±0.02, p<0.05) when compared to control. Additionally, TNF-α decreased α-SMA (0.74±0.12, p<0.05) and SM22 (0.54±0.01, p<0.01) protein levels. Consequently, TNF-α induced migration (1.25±0.05, p<0.05), proliferation (2.33±0.24, p<0.05), and the secretion of IL-6 (258±53, p<0.01), type I collagen (3.09±0.85, p<0.01) and osteopontin (2.32±0.46, p<0.01). Inhibition of autophagy prevented all the TNF-α-induced phenotypic changes. TNF-α induces phenotype switching in A7r5 cell line by a mechanism that required autophagy. Therefore, autophagy may be a potential therapeutic target for the treatment of atherosclerosis.

Involvement of the accumbal osteopontin-interacting transmembrane protein 168 in methamphetamine-induced place preference and hyperlocomotion in mice

Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.

Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes metastasis-related properties via TGF\u03b22/T\u03b2R and CD44 in MDA-MB-231 breast cancer cells

BackgroundTartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness.MethodsMDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes.ResultsWe identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFβ) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFβ isoform 2 (TGFβ2), TGFβ receptor type 1 (TβR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFβ2/TβR, whereas proliferation beyond basal levels is regulated through CD44.ConclusionAltogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFβ2/TβR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.

Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer.

BackgroundExcess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC), promoting a microenvironment favorable for tumor growth.MethodsSerum samples obtained from 79 subjects [26 lean (LN) and 53 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC). Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN), chitinase-3-like protein 1 (YKL-40), tenascin C (TNC) and lipocalin-2 (LCN-2) were determined by ELISA.ResultsSignificant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01) and colon cancer (P<0.05). LCN-2 levels were affected by obesity (P<0.05), but no differences were detected regarding the presence or not of CC. A positive association (P<0.05) with different inflammatory markers was also detected.ConclusionsTo our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics.

Selective Expression of Osteopontin in ALS-resistant Motor Neurons is a Critical Determinant of Late Phase Neurodegeneration Mediated by Matrix Metalloproteinase-9.

Differential vulnerability among motor neuron (MN) subtypes is a fundamental feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) MNs are more vulnerable than fast fatigue-resistant (FR) or slow (S) MNs. The reason for this selective vulnerability remains enigmatic. We report here that the extracellular matrix (ECM) protein osteopontin (OPN) is selectively expressed by FR and S MNs and ALS-resistant motor pools, whereas matrix metalloproteinase-9 (MMP-9) is selectively expressed by FF MNs. OPN is secreted and accumulated as extracellular granules in ECM in three ALS mouse models and a human ALS patient. In SOD1G93A mice, OPN/MMP-9 double positivity marks remodeled FR and S MNs destined to compensate for lost FF MNs before ultimately dying. Genetic ablation of OPN in SOD1G93A mice delayed disease onset but then accelerated disease progression. OPN induced MMP-9 up-regulation via αvβ3 integrin in ChAT-expressing Neuro2a cells, and also induced CD44-mediated astrocyte migration and microglial phagocytosis in a non-cell-autonomous manner. Our results demonstrate that OPN expressed by FR/S MNs is involved in the second-wave neurodegeneration by up-regulating MMP-9 through αvβ3 integrin in the mouse model of ALS. The differences in OPN/MMP-9 expression profiles in MN subsets partially explain the selective MN vulnerability in ALS.

Osteoblast differentiation is enhanced by a nano-to-micro hybrid titanium surface created by Yb:YAG laser irradiation.

The aim of this study was to analyze the capacity of a new modified laser surface to stimulate calvarial osteoblasts isolated from neonatal mouse bones to differentiate and form mineralized nodules. Titanium discs were subjectezd or not to laser irradiation according to specific parameters and characterized. Osteoblasts isolated from neonatal mouse calvaria were cultured over the discs, and the capacity of these cells to proliferate (MTT assay), form mineralized nodules (Alizarin red assay), and enhance alkaline phosphatase activity (ALPase activity) was analyzed. Real-time PCR was used for quantification of gene expression. Laser-irradiated titanium discs (L) presented a rough nano-to-micrometric oxidized surface contrasting with the smooth pattern on polished discs (P). The Ra on the micrometric level increased from 0.32 ± 0.01 μm on P surfaces to 10.57 ± 0.39 μm on L surfaces. When compared with P, L promoted changes in osteoblast morphology, increased mineralized nodule formation in osteoblasts cultured on the surfaces for 14 days, and enhanced ALPase activity at days 7 and 14. Transcription factors triggering osteoblast differentiation (Runx2 and Sp7) and genes encoding the bone extracellular matrix proteins collagen type-1 (Col1a1), osteopontin (Spp1), and osteocalcin (Bglap) were upregulated in cells on L surfaces compared with those on P surfaces at days 1-14. Laser treatment of titanium surfaces created a rough surface that stimulated osteoblast differentiation. Laser treatment of titanium generates a reproducible and efficient surface triggering osteoblast differentiation that can be of importance for osteointegration.

Osteopontin Gene Expression is Inversely Regulate 5-Fluorouracil Drug Resistance in Colon Cancer

Background: Resistance to chemotherapeutic drugs complicates the treatment of cancer patients. The extracellular matrix protein osteopontin (OPN) plays multiple roles in the proliferation and metastasis of cancer cells. We therefore attempted to determine whether OPN expression correlated with drug resistance. Methods: OPN expression in the HCT 116 colon cancer cell line was inhibited by an OPN-short interfering RNA (siRNA). We determined the cytotoxic effect (IC50) of 5-fluorouracil (5FU) on these cells. Patients with recurrence or colorectal cancer or harbored residual tumor cells were treated with S-1-based chemotherapy. The levels of OPN mRNA expression in the tumors were determined and compared with the patients’ responses to chemotherapy. Results: The IC50 of 5FU for HCT 116 cells transfected with the OPN-siRNA was 32.9 μM. In contrast, the IC50 values for cells transfected with a negative control siRNA, mock-transfected cells, or untreated cells were 6.58 μM, 7.08 μM, and 6.76 μM, respectively (P<0.001). The level of OPN mRNA expression in the S-1 non-responder group was significantly lower than that of the responder group (P = 0.0387). Survival analysis revealed no significant difference between the responder and non-responder groups (P = 0.8737). Conclusions: The level of OPN expression plays a role in the resistance of colorectal tumor cells to 5FU.

The effect of extracellular matrix proteins on the cellular response of HUVECS and HOBS after covalent immobilization onto titanium.

Biomimetic surface modifications are regarded as promising approach to stimulate cellular behavior at the interface of implant materials. Aim of the study was an evaluation of the cellular response of human umbilical cord cells (HUVECS) and human osteoblasts (HOBS) on titanium covalently coated with the extracellular matrix (ECM) proteins fibrinogen, collagen, laminin, and osteopontin. For the surface modification, titanium discs were first amino-functionalized by plasma polymerization of allylamine. The ECM protein conjugation was performed using the linker molecule α, ω-bis-N-hydroxysuccinimide polyethylene glycol (Di-NHS linker). For surface characterization, infrared spectroscopy and fluorescein isothiocyanate staining (FITC) were used to evaluate the presence and distribution of primary amines in the plasma polymer film. Real-time analyses of the respective protein conjugation processes were performed via surface plasmon resonance kinetic measurements. All ECM proteins were immobilized successfully. Furthermore, the biological functionality of the conjugated factors fibronectin and collagen could be proven as they led to a distinct stimulation of cell adhesion of HUVECS and HOBS when compared to the control group. The highest cell coverage of HUVECS was observed on fibronectin-modified surfaces with approximately 35% and on collagen with 33% after 24 h (PT: 9.4%). For laminin, no additional effect was observed, and for osteopontin, only a slight enhancement of cell adhesion was found. A similar, cell-stimulating tendency of fibronectin and collagen was seen as well after 3 and 7 days. Biomimetic surface modification via plasma polymerization is a powerful method for biomolecule conjugation with a high retention of biological functionality and offer promising clinical perspectives.

Abstract 779: Osteopontin gene expression is associated with 5-fluorouracil drug resistance in colon cancer

Abstract Background: Resistance to chemotherapeutic drugs complicates the treatment of cancer patients. The extracellular matrix protein osteopontin (OPN) plays multiple roles in the proliferation and metastasis of cancer cells. We therefore attempted to determine whether OPN expression correlated with drug resistance. Methods: OPN expression in the HCT 116 colon cancer cell line was inhibited by an OPN-short interfering RNA (siRNA). We determined the cytotoxic effect (IC50) of 5-fluorouracil (5FU) on these cells. Patients with recurrence or colorectal cancer or harbored residual tumor cells were treated with S-1-based chemotherapy. The levels of OPN mRNA expression in the tumors were determined and compared with the patients’ responses to chemotherapy. Results: The IC50 of 5FU for HCT 116 cells transfected with the OPN-siRNA was 32.9 μM. In contrast, the IC50 values for cells transfected with a negative control siRNA, mock-transfected cells, or untreated cells were 6.58 μM, 7.08 μM, and 6.76 μM, respectively (P&amp;lt;0.001). The level of OPN mRNA expression in the S-1 non-responder group was significantly lower than that of the responder group (P = 0.0387). Survival analysis revealed no significant difference between the responder and non-responder groups (P = 0.8737). Conclusions: The level of OPN expression plays a role in the resistance of colorectal tumor cells to 5FU. Citation Format: Go Nakajima, Hidekazu Kuramochi, Masakazu Yamamoto, Kazuhiko Hayashi. Osteopontin gene expression is associated with 5-fluorouracil drug resistance in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2014-779

Osteopontin: A novel regulator at the cross roads of inflammation, obesity and diabetes.

Since its first description more than 20 years ago osteopontin has emerged as an active player in many physiological and pathological processes, including biomineralization, tissue remodeling and inflammation. As an extracellular matrix protein and proinflammatory cytokine osteopontin is thought to facilitate the recruitment of monocytes/macrophages and to mediate cytokine secretion in leukocytes. Modulation of immune cell response by osteopontin has been associated with various inflammatory diseases and may play a pivotal role in the development of adipose tissue inflammation and insulin resistance. Here we summarize recent findings on the role of osteopontin in metabolic disorders, particularly focusing on diabetes and obesity.

Influence of pressurized cyclic stretch and endothelial cell presence on multipotent stem cell osteogenic commitment

Applied mechanical stretch and blood vessel invasion are key stimuli to which progenitor cells are exposed in post-natal endochondral bone formation. Understanding the combined effects of cyclic stretch and endothelial cell (EC) presence on multipotent stem cell (MSC) osteogenesis therefore has the potential to lead to improved MSC-based bone regeneration strategies. Toward this goal, 10T1/2 mouse MSCs were encapsulated in tubular poly(ethylene glycol) diacrylate [PEGDA] hydrogels with moduli within the "osteogenic" range in order to induce osteogenesis. Half of the constructs were fabricated with a luminal EC layer. All of the EC(+) (EC(+)/dyn(+)) and half of the EC(-) constructs (EC(-)/dyn(+)) were subjected to pressurized cyclic stretch in the absence of osteogenic media supplements, with remaining EC(-) constructs (EC(-)/dyn(-)) serving as static controls. At day 10 of culture, expression of the bone extracellular matrix protein osteopontin was over 3.3- and 1.9-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. At day 22 of culture, osteopontin levels could not be statistically distinguished from day 0 in the EC(+)/dyn(+) constructs and were one-third less than day 0 in the EC(-)/dyn(+) constructs. In contrast, at day 22 levels of an osteogenic marker alkaline phosphatase (AP) were over 2.4- and 1.4-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. Furthermore, at day 22 matrix mineralization in both dynamic groups was increased over 2.5-fold and over 9-fold relative to the EC(-)/dyn(-) and day 0 groups, respectively. Cumulatively, these results suggest that pressurized cyclic stretch alone significantly increases the rate/degree of osteogenesis relative to static culture. However, EC presence combined with pressured cyclic stretch appears to further enhance the rate/degree of MSC osteogenesis and/or to support a distinct osteogenic "fingerprint" compared to that promoted by cyclic stretch alone.

Structural Analysis of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Intracellular Domain Reveals a Conserved Interaction Epitope

Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

C-terminal Modification of Osteopontin Inhibits Interaction with the αVβ3-Integrin

Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the α(V)β(3)-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly well conserved among mammalian species, which suggests an important functional role of this region. In this study, we show that modification of the extreme C terminus of OPN plays an important regulatory role for the interaction with the α(V)β(3)-integrin. It is demonstrated that highly phosphorylated OPN has a much reduced capability to promote cell adhesion via the α(V)β(3)-integrin compared with lesser phosphorylated forms. The cell attachment promoted by highly phosphorylated OPN could be greatly increased by both dephosphorylation and proteolytic removal of the C terminus. Using recombinantly expressed OPN containing a tag in the N or C terminus, it is shown that a modification in the C-terminal part significantly reduces the adhesion of cells to OPN via the α(V)β(3)-integrin, whereas modification of the N terminus does not influence the binding. The inhibited binding of the α(V)β(3)-integrin to OPN could be restored by proteolytic removal of the C terminus by thrombin and plasmin. These data illustrate a novel mechanism regulating the interaction of OPN and the α(V)β(3)-integrin by modification of the highly conserved C-terminal region of the protein.

Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds

AbstractFragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size (< 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by “grafting” them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses.

Poly(Ethylene Glycol) and Hydroxy Functionalized Alkane Phosphate Self-Assembled Monolayers Reduce Bacterial Adhesion and Support Osteoblast Proliferation

Presently there is interest today in designing improved titanium surfaces capable of high bioactivity in order to promote strong anchorage of the bone surrounding implants while at the same time discouraging bioadhesion. Poly(ethylene glycol)-modified (PEG) alkane phosphate and OH-terminated alkane phosphates have been demonstrated to be spontaneously adsorbed onto titanium oxide surfaces and produce surfaces with different protein resistance in relation to the PEG surface density. This study aims to evaluate caries-associated Streptococcus mutans (S. mutans) adhesion and osteoblast proliferation while varying the PEG surface density of titanium surfaces. Bacterial adhesion was quantified by fluorescence microscopy and SAOS-2 human osteoblast proliferation was evaluated up to 7 days of culture in vitro. Metabolic activity of osteoblasts was measured by MTT test and the secretion of extracellular matrix proteins (osteopontin, osteocalcin and type I collagen) in culture medium was determined by immunoenzymatic assays. As the PEG surface density increased, the bacterial adhesion considerably decreased when compared to uncoated titanium surfaces. The monomolecular coatings proved to be capable of supporting osteoblast proliferation with the greatest levels of metabolic activity at the highest PEG surface concentrations. These results are extremely promising for potential clinical application in implant uses where both reduction of bacteria adhesion and stimulation of bone formation are highly desirable.