The bovine Lactoferrin-Osteopontin complex increases proliferation of human intestinal epithelial cells by activating the PI3K/Akt signaling pathway.

Lactoferrin (LF) and osteopontin (OPN), multifunctional proteins involved in cell proliferation, can form a complex. LF binds iron, whereas OPN binds calcium. We investigated whether iron- and calcium-binding influences complex formation and the pro-proliferation property of the LF-OPN complex, and the mechanism behind this effect. LF-OPN complexes were prepared using bovine milk LF and OPN, and effects on proliferation of human intestinal epithelial cells (HIECs) were evaluated using a BrdU proliferation assay. Of the four complexes formed by apo- and holo-LF/OPN, the apo-Lf&holo-OPN complex (AH) exhibited the strongest pro-proliferative effect on HIECs, and we therefore focused on AH. AH was resistant to in vitro gastrointestinal digestion, co-localized with both LF and OPN receptors as revealed by confocal microscopy, and stimulated proliferation of HIECs by activating PI3K/Akt signaling. In conclusion, forming a LF-OPN complex may help both proteins to resist digestion and increase the capacity to promote intestinal development in infants.

Assessment of bioactivities of the human milk lactoferrin-osteopontin complex in vitro.

Lactoferrin (LF) and osteopontin (OPN) are both multi-functional whey proteins present at high levels in human milk. These two proteins have a high affinity for each other due to their opposite charges; LF is a basic glycoprotein while OPN is an acidic phosphorylated glycoprotein. LF and OPN were identified to bind to each other over a decade ago, but potential functions of their complex remain unexplored. In this work, we investigated the characteristics of the LF-OPN complex with a focus on its bioactivities. Our results reveal a stronger stability of the LF-OPN complex towards in vitro digestion and more effective binding and uptake by human intestinal cells (HIEC) than LF or OPN alone show. Moreover, the LF-OPN complex promotes proliferation and differentiation of intestinal cells significantly more than the individual proteins do and shows an effect on anti-bacterial function and immune-stimulatory activities intermediate between those of LF and OPN. Thus, by forming a complex in human milk, LF and OPN may protect each other against proteolysis and enhance their individual bioactivities.

Assessing the Feasibility of Neutralizing Osteopontin with Various Therapeutic Antibody Modalities.

Osteopontin is a secreted glycophosphoprotein that is highly implicated in many physiological and pathological processes such as biomineralization, cell-mediated immunity, inflammation, fibrosis, cell survival, tumorigenesis and metastasis. Antibodies against osteopontin have been actively pursued as potential therapeutics for various diseases by pharmaceutical companies and academic laboratories. Many studies have demonstrated the efficacy of osteopontin inhibition in a variety of preclinical models of diseases such as rheumatoid arthritis, cancer, nonalcoholic steatohepatitis, but clinical utility has not yet been demonstrated. To evaluate the feasibility of osteopontin neutralization with antibodies in a clinical setting, we measured its physiological turnover rate in humans, a sensitive parameter required for mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Results from a stable isotope-labelled amino acid pulse-chase study in healthy human subjects followed by mass spectrometry showed that osteopontin undergoes very rapid turnover. PK/PD modeling and simulation of different theoretical scenarios reveal that achieving sufficient target coverage using antibodies can be very challenging mostly due to osteopontin's fast turnover, as well as its relatively high plasma concentrations in human. Therapeutic antibodies against osteopontin would need to be engineered to have much extended PK than conventional antibodies, and be administered at high doses and with short dosing intervals.

MRI/optical dual-modality imaging of vulnerable atherosclerotic plaque with an osteopontin-targeted probe based on Fe3O4 nanoparticles.

Rupture of vulnerable atherosclerotic plaque is the major pathological cause of luminal thrombosis in acute coronary syndromes. Since foamy macrophages have been identified as a prominent component in vulnerable atherosclerotic lesions and osteopontin (OPN) is reported to be highly expressed in foamy macrophages, OPN could be a potential target for vulnerable atherosclerotic plaque imaging. The current study designed an OPN-specific MRI/optical dual-modality probe to detect vulnerable plaques. Fluorescence imaging revealed that 24 h after injection of the Cy5.5-OPN-DMSA-MNPs (COD-MNPs), the atherosclerotic plaques in carotid artery exhibited significant higher signals in high fat diet (HFD) fed mice in comparison to the group injected with Cy5.5-IgG-DMSA-MNPs (CID-MNPs) or normal diet fed group injected with COD-MNPs (1.87 ± 0.19 × 1010 vs. 0.74 ± 0.04 × 1010, 0.73 ± 0.03 × 1010 p/sec/cm2/sr, P < 0.05). Meanwhile, MRI displayed stronger T2 contrast enhancement 24 h post-injection at the area of atherosclerotic plaques in the carotid of HFD fed group injected with COD-MNPs than group injected with CID-MNPs or normal diet fed group injected with COD-MNPs (post/pre signal ratio: 0.64 ± 0.04 vs. 0.95 ± 0.02, 0.98 ± 0.01, P < 0.05). As a dual-modality molecular probe, the resulting COD-MNPs conjugates exhibit promising potentials for noninvasive detection of vulnerable atherosclerotic plaque in vivo.

Biodegradable gelatin microspheres enhance the neuroprotective potency of osteopontin via quick and sustained release in the post-ischemic brain.

Gelatin microspheres (GMSs) are widely used as drug carriers owing to their excellent biocompatibilities and toxicologically safe degradation products. The drug release profile is easily tailored by controlling the cross-linking density and surface-to-volume ratio, i.e. size, of the GMS. In this study, we employed GMSs which are 25 µm in diameter and cross-linked with 0.03125% glutaraldehyde, to enable rapid initial and a subsequent sustained release. Therapeutic potency of human recombinant osteopontin (rhOPN) with or without encapsulation into GMSs was investigated after administrating them to rat stroke model (Sprague-Dawley; middle cerebral artery occlusion, MCAO). The administration of rhOPN/GMS (100 ng/100 µg) at 1h post-MCAO reduced the mean infarct volume by 81.8% of that of the untreated MCAO control and extended the therapeutic window at least to 12h post-MCAO, demonstrating a markedly enhanced therapeutic potency for the use of OPN in the post-ischemic brain. Scanning electron microscopy micrographs revealed that GMSs maintained the three-dimensional shape for more than 5 days in normal brain but were degraded rapidly in the post-ischemic brain, presumably due to high levels of gelatinase induction. After encapsulation with GMS, the duration of OPN release was markedly extended; from the period of 2 days to 5 days in normal brain, and from 2 days to 4 days in the post-ischemic brain; these encompass the critical period for recovery processes, such as vascularization, and controlling inflammation. Together, these results indicate that GMS-mediated drug delivery has huge potential when it was used in the hyperacute period in the post-ischemic brain.

Oxidative nanopatterning of titanium surfaces promotes production and extracellular accumulation of osteopontin.

The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.

Oxidative nanopatterning of titanium surfaces promotes production and extracellular accumulation of osteopontin

The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.

A interface osso-implante é caracterizada pela presença de uma camada de matriz extracellular (MEC) afibrilar rica em proteínas não-colágenas, incluindo osteopontina (OPN), cujas funções no tecido ósseo estão relacionadas à adesão celular e ao controle do processo de mineralização da MEC (crescimento de cristais). Aspectos físicos e químicos das superfícies de biomateriais podem afetar as interações célula-MEC-substrato. O objetivo do presente estudo foi demonstrar a capacidade de aspectos nanotopográficos de superfície de titânio (Ti) de controlar a deposição extracelular de OPN in vitro. Discos de Ti foram tratados quimicamente por solução de H2SO4/H2O2 durante 30 min [Nano(30') Ti] ou 4 h [Nano(4h) Ti]. Superfícies de Ti não tratadas foram usadas como controle. Células osteogênicas primárias derivadas de calvárias de ratos recém-nascidos foram plaqueadas sobre os discos de Ti e cultivadas em condições osteogênicas por até 7 dias. Microscopia eletrônica de varredura de alta resolução revelou que os discos de Ti tratados quimicamente exibiam superfície nanoporosa, com áreas de nanoporos maiores para Nano(4h) Ti. Apenas para esse grupo detectavam-se acúmulos extensos de OPN extracelular, os quais se distribuíam em áreas adjacentes a células OPN-positivas, com aspectos morfológicos típicos de células em migração. Em conclusão, a nanoestruturação química de superfície de Ti pode favorecer o aumento da deposição extracelular de OPN endógena por células osteogênicas in vitro, dependendo do tempo de condicionamento utilizado, o que deve ser considerado no desenvolvimento de estratégias para funcionalizar superfícies de implantes com moléculas com reconhecido efeito no processo de adesão celular.

Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice.

Osteopontin (OPN) is a matricellular cytokine present in most tissues and body fluids; it is known to modulate immune responses. In previous studies using the dextran sulfate sodium (DSS) acute colitis model, we found exacerbated tissue destruction and reduced repair in OPN-null ((-/-)) mice compared with wild-type (WT) controls. As OPN is normally present in milk, we hypothesized that administration of OPN may protect the intestines from the adverse effects of experimental colitis. A volume of 20 or 2 microg/ml bovine milk OPN, dissolved in drinking water, was given to mice 24 h before, and during administration of DSS. Clinical parameters of colitis and neutrophil functions were analyzed as previously reported. Orally administered OPN was absorbed and detected in the colon mucosa by immunohistochemistry. The 20 microg/ml OPN- and DSS-treated WT mice showed 37% less weight loss and reduced colon shortening and spleen enlargements than control mice (P<0.05). OPN administration also reduced the disease activity index, improved red blood cell counts, and reduced gut neutrophil activity compared with the DSS-treated WT mice that were not administered OPN (P<0.05). Immunohistochemical detection of F4/80-labelled cells (macrophages) was also less frequent. The level of transforming growth factor beta1 (TGF-beta1) was increased and the levels of pro-inflammatory mediators decreased in colon tissue samples of OPN-treated mice analyzed by ELISA. The reversal of experimental colitis parameters by exogenous OPN was not as robust in the OPN(-/-) mice. Administration of prokaryotic-expressed recombinant OPN and bovine serum albumin were ineffective. This study shows that administration of a physiological concentration of milk OPN in drinking water ameliorates the destructive host response in DSS-induced acute colitis.