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.
Osteopontin (OPN) is a multifunctional molecule highly expressed in chronic inflammatory and autoimmune diseases, and it is specifically localized in and around inflammatory cells. OPN is a secreted ...adhesive molecule, and it is thought to aid in the recruitment of monocytes-macrophages and to regulate cytokine production in macrophages, dendritic cells, and T-cells. OPN has been classified as T-helper 1 cytokine and thus believed to exacerbate inflammation in several chronic inflammatory diseases, including atherosclerosis. Besides proinflammatory functions, physiologically OPN is a potent inhibitor of mineralization, it prevents ectopic calcium deposits and is a potent inducible inhibitor of vascular calcification. Clinically, OPN plasma levels have been found associated with various inflammatory diseases, including cardiovascular burden. It is thus imperative to dissect the OPN proinflammatory and anticalcific functions. OPN recruitment functions of inflammatory cells are thought to be mediated through its adhesive domains, especially the arginine-glycine-aspartate (RGD) sequence that interacts with several integrin heterodimers. However, the integrin receptors and intracellular pathways mediating OPN effects on immune cells are not well established. Furthermore, several studies show that OPN is cleaved by at least 2 classes of proteases: thrombin and matrix-metalloproteases (MMPs). Most importantly, at least in vitro, fragments generated by cleavage not only maintain OPN adhesive functions but also expose new active domains that may impart new activities. The role for OPN proteolytic fragments in vivo is almost completely unexplored. We believe that further knowledge of the effects of OPN fragments on cell responses might help in designing therapeutics targeting inflammatory and cardiovascular diseases.
Osteocalcin (OC) and osteopontin (OPN) are major non‐collagenous proteins (NCPs) involved in bone matrix organization and deposition. In spite of this, it is currently unknown whether OC and OPN ...alter bone morphology and consequently affect bone fracture resistance. The goal of this study is to establish the role of OC and OPN in the determination of cortical bone size, shape, and mechanical properties. Our results show that Oc–/– and Opn–/– mice were no different from each other or wild type (WT) with respect to bone morphology (P > 0.1). Bones from mice lacking both NCPs (Oc–/–Opn–/–) were shorter, with thicker cortices and larger cortical areas, compared with the WT, Oc–/–, and Opn–/– groups (P < 0.05), suggesting a synergistic role for NCPs in the determination of bone morphology. Maximum bending load was significantly different among the groups (P = 0.024), while tissue mineral density and measures of stiffness and strength were not different (P > 0.1). We conclude that the removal of both OC and OPN from bone matrix induces morphological adaptation at the structural level to maintain bone strength.
Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a ...crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvβ5 (ITGαvβ5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.
•Bovine lactoferrin (LF) and osteopontin (OPN) interact with each other to form a complex.•LF has the capacity to bind iron, whereas OPN binds calcium.•The apo-LF&holo-OPN complex (AH) exhibits the ...strongest pro-proliferative effect of the four LF-OPN complexes.•AH is resistant to in vitro gastrointestinal digestion, and co-localizes with both LF and OPN receptors.•AH stimulates proliferation of HIECs 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.
Human milk contains several bioactive proteins, including lactoferrin (LF) and osteopontin (OPN). These two proteins have been shown to form a complex, which shows increased bioactivities. Bovine LF ...and OPN can also form such a complex. We assessed bioactivities of the bovine LF-OPN complex (at molar ratios of LF:OPN = 3:1, 5:1, or 8:1) in a formula protein matrix, including LF, OPN, bovine whey protein hydrolysate, and α-lactalbumin. Our results show that the bovine LF-OPN complex together with formula proteins is resistant to
digestion, stimulates intestinal cell proliferation (by 15-50%) and differentiation (by 30-50%), increases antibacterial activity (by 25-50%), and enhances intestinal immunity. The 3:1 ratio of LF to OPN exhibits the most potent effects, as compared with the other two ratios. In conclusion, adding bovine LF and OPN to infant formulas may result in increased stability of the two components and enhanced bioactivities, possibly improving outcomes in formula-fed infants.
Osteopontin (OPN) also known by its official gene designation secreted phosphoprotein‐1 (SPP1) is a fascinating, multifunctional protein expressed in a number of cell types that functions not only in ...intercellular communication, but also in the extracellular matrix (ECM). OPN/SPP1 possesses cytokine, chemokine, and signal transduction functions by virtue of modular structural motifs that provide interaction surfaces for integrins and CD44‐variant receptors. In humans, there are three experimentally verified splice variants of OPN/SPP1 and CD44’s ten exons are also alternatively spiced in a cell/tissue‐specific manner, although very little is known about how this is regulated in the central nervous system (CNS). Post‐translational modifications of phosphorylation, glycosylation, and localized cleavage by specific proteases in the cells and tissues where OPN/SPP1 functions, provides additional layers of specificity. However, the former make elucidating the exact molecular mechanisms of OPN/SPP1 function more complex. Flexibility in OPN/SPP1 structure and its engagement with integrins having the ability to transmit signals in inside‐out and outside‐in direction, is likely why OPN/SPP1 can serve as an early detector of inflammation and ongoing tissue damage in response to cancer, stroke, traumatic brain injury, pathogenic infection, and neurodegeneration, processes that impair tissue homeostasis. This review will focus on what is currently known about OPN/SPP1 function in the brain.
Although commensal flora is involved in the regulation of immunity, the interplay between cytokine signaling and microbiota in atherosclerosis remains unknown. We found that interleukin (IL)-23 and ...its downstream target IL-22 restricted atherosclerosis by repressing pro-atherogenic microbiota. Inactivation of IL-23-IL-22 signaling led to deterioration of the intestinal barrier, dysbiosis, and expansion of pathogenic bacteria with distinct biosynthetic and metabolic properties, causing systemic increase in pro-atherogenic metabolites such as lipopolysaccharide (LPS) and trimethylamine N-oxide (TMAO). Augmented disease in the absence of the IL-23-IL-22 pathway was mediated in part by pro-atherogenic osteopontin, controlled by microbial metabolites. Microbiota transfer from IL-23-deficient mice accelerated atherosclerosis, whereas microbial depletion or IL-22 supplementation reduced inflammation and ameliorated disease. Our work uncovers the IL-23-IL-22 signaling as a regulator of atherosclerosis that restrains expansion of pro-atherogenic microbiota and argues for informed use of cytokine blockers to avoid cardiovascular side effects driven by microbiota and inflammation.
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•IL-23 and IL-22 cytokines restrict atherosclerosis development•IL-23 and IL-22 control intestinal barrier and pro-atherogenic bacteria expansion•Bacterial metabolites activate myeloid cells and drive atherosclerosis
While anti-cytokine therapies show promising results in autoimmune diseases, its effects on cardiovascular disease development are not well understood. Fatkhullina and colleagues show that inactivation of IL-23-IL-22 signaling leads to deterioration of the intestinal barrier, expansion of pro-atherogenic bacteria, and production of metabolites promoting macrophage activation and atherosclerosis.
Aging induces cardiac structural and functional changes linked to the increased deposition of extracellular matrix proteins, including OPN (osteopontin), conducing to progressive interstitial ...fibrosis. Although OPN is involved in various pathological conditions, its role in myocardial aging remains unknown.
OPN deficient mice (OPN-/-) with their wild-type (WT) littermates were evaluated at 2 and 14 months of age in terms of cardiac structure, function, histology and key molecular markers. OPN expression was determined by reverse-transcription polymerase chain reaction, immunoblot and immunofluorescence. Luminex assays were performed to screen plasma samples for various cytokines/adipokines in addition to OPN. Similar explorations were conducted in aged WT mice after surgical removal of visceral adipose tissue (VAT) or treatment with a small-molecule OPN inhibitor agelastatin A. Primary WT fibroblasts were incubated with plasma from aged WT and OPN-/- mice, and evaluated for senescence (senescence-associated β-galactosidase and p16), as well as fibroblast activation markers (Acta2 and Fn1).
Plasma OPN levels increased in WT mice during aging, with VAT showing the strongest OPN induction contrasting with myocardium that did not express OPN. VAT removal in aged WT mice restored cardiac function and decreased myocardial fibrosis in addition to a substantial reduction of circulating OPN and transforming growth factor β levels. OPN deficiency provided a comparable protection against age-related cardiac fibrosis and dysfunction. Intriguingly, a strong induction of senescence in cardiac fibroblasts was observed in both VAT removal and OPN-/- mice. The addition of plasma from aged OPN-/- mice to cultures of primary cardiac fibroblasts induced senescence and reduced their activation (compared to aged WT plasma). Finally, Agelastatin A treatment of aged WT mice fully reversed age-related myocardial fibrosis and dysfunction.
During aging, VAT represents the main source of OPN and alters heart structure and function via its profibrotic secretome. As a proof-of-concept, interventions targeting OPN, such as VAT removal and OPN deficiency, rescued the heart and induced a selective modulation of fibroblast senescence. Our work uncovers OPN's role in the context of myocardial aging and proposes OPN as a potential new therapeutic target for a healthy cardiac aging.
Osteopontin (OPN) is a highly modified integrin-binding extracellular matrix glycophosphoprotein produced by cells of the immune system, epithelial tissue, smooth muscle cells, osteoblasts, and tumor ...cells. Extensive research has elucidated the pivotal role of OPN in cell signaling that controls inflammation, tumor progression, and metastasis. OPN interaction with the integrin receptors expressed on inflammatory cells through its arginine–glycine–aspartate (RGD) and non-RGD motifs promote migration and adhesion of cells. In the liver, it has been reported that hepatic Kupffer cells secrete OPN facilitating macrophage infiltration into necrotic areas following carbon tetrachloride liver toxicity. Recent work has highlighted the role of OPN in inflammatory liver diseases such as alcoholic and nonalcoholic liver disease and T-cell–mediated hepatitis. The role of OPN in hepatocellular carcinoma (HCC) has also generated significant interest, especially with regards to its role as a prognostic factor. OPN therefore appears to play an important role during liver inflammation and cancer. In this review we will present data to demonstrate the key role played by OPN in mediating hepatic inflammation (neutrophils, monocytes/macrophages, and lymphocytes) and its role in HCC. Greater understanding of the pathophysiologic role of OPN in hepatic inflammation and cancer may enable development of novel inflammation and cancer treatment strategies.
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