Circulating Osteopontin Predicts Clinical and Radiological Response in First-Line Treatment of Advanced Non-Small Cell Lung Cancer.

PURPOSE: Pembrolizumab-based regimens are conditioned by the expression of PD-L1, but durable response rate is limited by innate and acquired resistance mechanisms. Here, we focus on osteopontin (OPN), an upfront biomarker of senescence, which closely associated with natural history of non-small cell lung cancer (NSCLC). METHODS: Seventy-nine patients eligible to pembrolizumab regimens-alone or in combination with chemotherapy-as first-line treatment of advanced NSCLC were enrolled. Predictive value of OPN toward iRECIST progression disease (PD) was set as first outcome. Secondary ones included performance status (ECOG) at baseline, early (first and best) responses, and overall survival (OS). RESULTS: High Serum OPN characterized patients with worse ECOG-PS (p = 0.015) at baseline and subjects experienced PD/death at first (OR 1.17 [1.02 to 1.35]; p = 0.030) and best responses (0.04 [0.00 to 0.81]; p = 0.035). OPN was associated with time-to-progression (B -2.74 [-4.46 to -1.01]) and time-to death (-0.13 [-0.20 to -0.05]). Cox regression models unveil a predictive value for iRECIST-PD (HR 1.01 [1.00 to 1.02]; p = -0.005), RECIST-PD (HR 1.01 [1.00 to 1.02]; p = 0.017), and OS (HR 1.02 [1.01 to 1.03]; p = 0.001). These models were internally validated through bootstrap resampling and characterized by relevant discrimination ability at ROC curve analyses. CONCLUSION: Baseline levels of serum OPN is closely associated with performance status and short/long term outcomes in patients with advanced NSCLC, which are candidate to pembrolizumab-based regimens. As upfront biomarker of senescence, OPN may pave the way for future studies focusing on senescence patterns in NSCLC.

METTL3/16-mediated m6A modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells.

Macrophages are the major components of tumour microenvironment, which play critical roles in tumour development. N6-methyladenosine (m6A) also contributes to tumour progression. However, the potential roles of m6A in modulating macrophages in hepatocellular carcinoma (HCC) are poorly understood. Here, we identified ZNNT1 as an HCC-related m6A modification target, which was upregulated and associated with poor prognosis of HCC. METTL3 and METTL16-mediated m6A modification contributed to ZNNT1 upregulation through stabilizing ZNNT1 transcript. ZNNT1 exerted oncogenic roles in HCC. Furthermore, ZNNT1 recruited and induced M2 polarization of macrophages via up-regulating osteopontin (OPN) expression and secretion. M2 Macrophages-recruited by ZNNT1-overexpressed HCC cells secreted S100A9, which further upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling. Thus, this study demonstrates that m6A modification activated the ZNNT1/OPN/S100A9 positive feedback loop, which promoted macrophages recruitment and M2 polarization, and enhanced malignant features of HCC cells. m6A modification-triggered ZNNT1/OPN/S100A9 feedback loop represents potential therapeutic target for HCC.

Osteopontin modulates microglial activation states and attenuates inflammatory responses after subarachnoid hemorrhage in rats.

AIMS: Osteopontin (OPN) has demonstrated neuroprotective effects in various stroke models. Its role in neuroinflammation after brain injury remains to be elucidated. This study aims to clarify the effect of OPN on neuroinflammation, particularly on the functional states of microglia after subarachnoid hemorrhage (SAH). METHODS: 77 rats were randomly divided into the following groups: Sham, SAH 24 h, SAH + rOPN, SAH + Vehicle (PBS), SAH + OPN siRNA, and SAH + Scr siRNA, SAH + rOPN+Fib-14 and SAH + rOPN+DMSO. Modified Garcia and beam balance tests were used to evaluate neurobehavioral outcomes. Semi-quantitative immunofluorescence staining was performed to measure expression of myeloperoxidase (MPO) and microglia activation state markers CD16, CD206 after SAH and recombinant OPN treatment. The quantification of microglia activation and functional markers CD16, CD206, TNF-α and IL-10 were further evaluated using Western-blotting. RESULTS: Nasal administration of rOPN improved neurological dysfunction, attenuated neutrophil infiltration, and decreased expression of phenotypic and functional markers of pro-inflammatory microglia CD16 and TNF-α. It also promoted an anti-inflammatory microglial state, as evidenced by increased expression of CD206 and IL-10. Furthermore, after blocking the phosphorylation of FAK signaling, the effects of rOPN on microglial activation states were partially reversed. The downstream pathways of STAT3 and NF-κB also exhibited consistent changes, suggesting the involvement of the STAT3 and NF-κB pathways in OPN's modulation of microglial activation via integrin-FAK signaling. CONCLUSION: OPN attenuates inflammatory responses after SAH by promoting an anti-inflammatory microglial state, potentially mediated through the integrin-FAK-STAT3 and NF-κB signaling pathways.

Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the induction of chemo-resistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma (HCC). METHODS: We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively, to identify key molecules that might induce resistance to TKIs. We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms. The associations of plasma secreted phosphoprotein 1 (SPP1) expression levels before sorafenib/lenvatinib treatment with progression-free survival (PFS) and overall survival (OS) of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis. RESULTS: Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance. SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo. CAF-derived SPP1 activated rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) through the integrin-protein kinase C-alpha (PKCα) signaling pathway and promoted epithelial-to-mesenchymal transition (EMT). A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS (P = 0.026) and OS (P = 0.047) in patients with advanced HCC after TKI treatment. CONCLUSIONS: CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion. Its inhibition represents a promising therapeutic strategy against TKI resistance in HCC. Moreover, plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

Osteopontin phosphopeptide mitigates calcium oxalate stone formation in a Drosophila melanogaster model.

Kidney stone disease affects nearly one in ten individuals and places a significant economic strain on global healthcare systems. Despite the high frequency of stones within the population, effective preventative strategies are lacking and disease prevalence continues to rise. Osteopontin (OPN) is a urinary protein that can inhibit the formation of renal calculi in vitro. However, the efficacy of OPN in vivo has yet to be determined. Using an established Drosophila melanogaster model of calcium oxalate urolithiasis, we demonstrated that a 16-residue synthetic OPN phosphopeptide effectively reduced stone burden in vivo. Oral supplementation with this peptide altered crystal morphology of calcium oxalate monohydrate (COM) in a similar manner to previous in vitro studies, and the presence of the OPN phosphopeptide during COM formation and adhesion significantly reduced crystal attachment to mammalian kidney cells. Altogether, this study is the first to show that an OPN phosphopeptide can directly mitigate calcium oxalate urolithiasis formation in vivo by modulating crystal morphology. These findings suggest that OPN supplementation is a promising therapeutic approach and may be clinically useful in the management of urolithiasis in humans.

The combination of olive oil and Lepidium sativum improves the deleterious effects resulting from dexamethasone-induced osteoporosis in rats.

INTRODUCTION: Osteoporosis is characterized by deterioration of bone microarchitecture and reduced bone mass and can increase the risk of fracture. To reduce this risk, the aim of this study was to compare the combination effects of olive oil and Lepidium sativum compared to the conventional drug therapy alendronate. METHODS: Osteoporosed-induced rat model was established by administration of dexamethasone in female adult albino rats. The serum level of Ca2+, P3+, and osteocalcin was assessed. In addition, histopathological changes and immunohistochemical expression of osteopontin within bone specimens were performed. RESULTS: Our results showed that a combination of olive oil and Lepidium sativum had a beneficial therapeutic effect in the treatment of osteoporosis as compared to alendronate therapy. This was demonstrated by increase of serum Ca2+, P3+, and osteocalcin levels in treated compared to control groups. Intriguingly, the highest effect was noticed in rats that received a combination of olive oil and Lepidium sativum compared to the individual treatment. This was reflected by an increase in the cortical bone thickness and a decrease in immunohistochemical expression of osteopontin compared to individual treated groups. CONCLUSION: We concluded that the administration of a combination of olive oil and Lepidium sativum improves bone mineral health and intensity and reduces the risk of osteoporosis in a rat model.

Osteopontin Exacerbates High-Fat Diet-Induced Metabolic Disorders in a Microbiome-Dependent Manner.

The gut microbiome is involved in metabolic disorders. Osteopontin (OPN), as a key cytokine, contributes to various inflammation-related diseases. The underlying role of OPN in the microbiome remains poorly understood. Here, we investigated whether OPN could modulate metabolic disorders by affecting gut microbiota. In our present study, we found that the expression of OPN was elevated in individuals with obesity compared to that observed in healthy controls. There was a positive correlation between plasma OPN levels and body mass index (BMI) in humans. Moreover, OPN significantly exacerbated lipid accumulation and metabolic disorders in high-fat diet (HFD)-fed mice. Importantly, OPN significantly aggravated HFD-induced gut dysbiosis with a key signature profile. Fecal microbiota transplantation also supported the role of OPN in HFD-induced metabolic disorders in a microbiota-dependent manner. Moreover, the microbiome shift of OPN-deficient mice would be compensated to resemble those of wild-type mice by feeding with either OPN-containing milk or recombinant OPN protein in vivo. Furthermore, metagenomic analysis showed that OPN induced a higher abundance of Dorea and a lower abundance of Lactobacillus, which were positively and negatively correlated with body weight, respectively. Indeed, the abundance of Dorea was significantly decreased after Lactobacillus administration, suggesting that OPN may regulate the intestinal abundance of Dorea by reducing the colonization of Lactobacillus. We further confirmed that OPN decreased the adhesion of Lactobacillus to intestinal epithelial cells through the Notch signaling pathway. This study suggested that OPN could exacerbate HFD-induced metabolic dysfunctions through the OPN-induced alteration of the gut microbiome. Therefore, OPN could be a potential therapeutic target for metabolic syndrome. IMPORTANCE Gut microbiota are involved in metabolic disorders. However, microbiome-based therapeutic interventions are not always effective, which might be due to interference of the host factors. Here, we identified a strong positive correlation between OPN levels and BMI in humans. Next, we confirmed that OPN could aggravate high-fat diet-induced metabolic disorders in mice. Importantly, we found that fecal microbiota transplantation from OPN-deficient mice significantly alleviated metabolic disorders in WT mice. OPN directly induces the remodeling of the gut microbiota both in vitro and in vivo. These findings indicate that OPN could contribute to metabolic disorders by inducing an alteration of gut microbiota. OPN regulated the relative abundance of Lactobacillus by decreasing the adhesion of Lactobacillus to intestinal epithelial cells through the Notch signaling pathway. These data identify OPN as a potential pharmaceutical target for weight control and for the treatment of metabolic disorders.

Immune Regulatory Effect of Osteopontin Gene Therapy in a Murine Model of Multidrug Resistant Pulmonary Tuberculosis.

Tuberculosis (TB) has been for many years a major public health problem since treatment is long and sometimes ineffective favoring the increase of multidrug-resistant mycobacteria (MDR-TB). Gene therapy is a novel and effective tool to regulate immune responses. In this study we evaluated the therapeutic effect of an adenoviral vector codifying osteopontin (AdOPN), a molecule known for their roles to favor Th1 and Th17 type-cytokine expression which are crucial in TB containment. A single dose of AdOPN administration in BALB/c mice suffering late progressive pulmonary MDR-TB produced significant lower bacterial load and pneumonia, due to higher expression of IFN-γ, IL-12, and IL-17 in coexistence with increase of granulomas in number and size, resulting in higher survival, in contrast with mice treated with the control adenovirus that codify the green fluorescent protein (AdGFP). Combined therapy of AdOPN with a regimen of second line antibiotics produced a better control of bacterial load in lung during the first days of treatment, suggesting that AdOPN can shorten chemotherapy. Taken together, gene therapy with AdOPN leads to higher immune responses against TB infection, resulting in a new potential treatment against pulmonary TB that can co-adjuvant chemotherapy.

A Decreased Absolute Number of Treg Cells in Patients with Active Rheumatoid Arthritis is Associated with Elevated Serum Osteopontin Levels with Disease Progression.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic and refractory autoimmune disease characterized by synovial inflammation with unknown aetiology. Immune system dysfunction mediated by CD4+ T lymphocytes, which is regulated by the cytokine osteopontin (OPN), plays an important role in the pathogenesis of RA. METHODS: In this study, the levels of peripheral CD4+ T subsets and serum OPN in patients with active RA were measured and analysed to determine the possible pathogenesis of RA and to provide potential therapeutic targets. RESULTS: Serum OPN levels in both patients with active RA and patients with refractory RA were higher than those in healthy controls (HCs). Compared with HCs, the absolute numbers of Th2 cells increased in patients with active RA, while the absolute counts of Th1 and Treg cells decreased. There was no significant difference in CD4+ T subset levels between new-onset and refractory patients. As the condition persisted or deteriorated, a gradual increase in the levels of OPN and gradual declines in the absolute counts of Th1 and Treg cells were observed in patients with active RA. The fewest Th1 and Treg cells and the highest OPN levels were observed in patients with high disease activity. The serum OPN level was only significantly negatively correlated with the absolute counts of Treg cells in the CD4+ T lymphocyte subsets. CONCLUSIONS: Fewer Treg cells with the increase in disease activity may be related to the increased OPN concentration, which may provide new ideas and directions for the targeted immunoregulatory treatment of RA.

EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma.

BACKGROUND: Despite the remarkable breakthroughs achieved in the management of metastatic melanoma using immunotherapy and targeted therapies, long-term clinical efficacy is often compromised due to dose-limiting toxicity and innate or acquired resistance. Therefore, it is of vital importance to further explore the molecular mechanisms underlying melanoma progression and identify new targeted therapeutic approaches. METHODS: The function of eukaryotic elongation factor-2 kinase (EEF2K) in melanoma were investigated in vitro and in vivo. RNA-seq and chromatin immunoprecipitation (ChIP) assay were undertaken to explore the mechanisms. The antitumor effect of bromodomain and extra terminal domain (BET) inhibitors combined with cytarabine were assessed in melanoma both in vitro and in vivo. RESULTS: EEF2K silencing markedly attenuated the malignant phenotypes of melanoma cells, including proliferation, migration, invasion and metastasis. In contrast, EEF2K overexpression promoted melanoma cell proliferation, migration and invasion. Mechanistically, we demonstrated that EEF2K upregulates the phosphorylation of STAT3 (p-STAT3) at Tyr705, which binds to the promoter region of SPP1 and enhances its transcription, thus facilitating melanoma progression. Transfection-induced re-expression of SPP1 partly negated the inhibitory effect of EEF2K silencing on melanoma, whereas inhibition of SPP1 or STAT3 significantly abolished the efficacy of EEF2K on melanoma cells. Intriguingly, EEF2K silencing combined with BET inhibitor treatment further inhibited cell proliferation and promoted apoptosis in melanoma. We further screened the US FDA-approved antitumour drug library and identified cytarabine as a potential clinically applicable EEF2K inhibitor that could synergise with BET inhibitors in melanoma treatment. CONCLUSION: EEF2K/p-STAT3/SPP1 may be a novel oncogenic pathway in melanoma progression, which could be a target for novel combination therapy for melanoma.

SPP1 in infliximab resistant ulcerative colitis and associated colorectal cancer: an analysis of differentially expressed genes.

OBJECTIVE: Infliximab, a tumour necrosis factor-α (TNFα) antagonist, has advanced the management of ulcerative colitis. Although efficacious, considerable percentage of patients are resistant to treatment. Accumulative inflammatory burden in long-term ulcerative colitis patients refractory to therapy increases the risk of developing colorectal cancer (CRC). Our study investigated anti-TNFα-naïve patients with active ulcerative colitis to identify gene biomarkers whose dysregulated expression correlated with resistance to infliximab (IFX) treatment and poor prognosis in CRC. METHODS: Differentially expressed genes (DEGs) from two studies (GSE73661 and GSE14580) with colonic mucosal samples were retrieved. Noninflammatory bowel disease controls were compared with those with active ulcerative colitis that either responded or were resistant to IFX before treatment. DEGs from ulcerative colitis samples resistant to IFX were used to construct a protein-protein interaction network, and clustering gene modules were identified. Module DEGs that overlapped with ulcerative colitis samples responsive to IFX were analysed, based on topological closeness and radiality. Hub genes were obtained, and their correlation with CRC progression was evaluated. Their expression in CRC tissues and their tumour microenvironment immune status was estimated. RESULTS: Three clusters composed of 582 DEGs from ulcerative colitis samples resistant to IFX were retrieved. Comparative analysis identified 305 overlapping DEGs with ulcerative colitis samples responsive to IFX. Topological analysis revealed a hub gene - SPP1 - whose overexpression in CRC tissues and patients correlated with increased infiltration of immune signatures and poor prognosis. CONCLUSION: SPP1 may serve as potential gene biomarker and predictor of resistance to IFX therapy in ulcerative colitis and CRC development.

In-silico Validation of the Proposed Treatment Strategy of Periodontitis.

OBJECTIVE: The present study aims to assess a proposed treatment approach or therapy for periodontitis by using the in-silico technique. The proposed treatment strategy offers a singular vehicular system consisting of minocycline (antibiotic), celecoxib (selective COX-II inhibitor), doxycycline hyclate (matrix metalloproteinase inhibitor), and hydroxyapatite (osteogenic agent). MATERIAL AND METHODS: Molecular docking studies of drugs were performed using Maestro version 9.4 software Schrödinger, and 3-Dimensional Crystallographic X-ray protein structures of targeted proteins were downloaded from RCSB protein data bank in .pdb file format. These agents were docked, and their affinities towards the receptors/protein/enzyme were calculated. Furthermore, their affinities were compared with the standard drug. RESULTS: The study suggests that minocycline and metronidazole possess equal affinity towards the RGPB and Inlj protein of P.gingivalis. Celecoxib, a well-known inhibitor of the COX-II enzyme, showed very high affinity. Selective inhibitor of MMP-8 possessed higher affinity than doxycycline, whereas CMT-3 showed equal affinity as doxycycline for MMP-13. Similarly, hydroxyapatite and simvastatin also showed a comparatively similar affinity for osteopontin receptor. CONCLUSION: Based upon molecular docking results, it can be concluded that the proposed treatment strategy would be a suitable approach for periodontitis and all the selected therapeutic agents have potential similar to the standard drugs, thereby constituting a reliable system for periodontitis.

Liver osteopontin is required to prevent the progression of age-related nonalcoholic fatty liver disease.

Osteopontin (OPN), a senescence-associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age-dependent hepatosteatosis. Thus, we investigated the role of OPN in the age-related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild-type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN-deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN-KO mice liver were associated with the decrease of 78 kDa glucose-regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53-OPN axis is required to inhibit the onset of age-related hepatosteatosis.

Acute intranasal osteopontin treatment in male rats following TBI increases the number of activated microglia but does not alter lesion characteristics.

Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood-brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.

Effects of oral paricalcitol therapy on arterial stiffness and osteopontin in hypertensive patients with chronic kidney disease and secondary hyperparathyroidism.

BACKGROUND: Arterial stiffness is linked to the progression of atherosclerosis, while activation of vitamin D receptor exerts favorable cardiovascular effects in patients with renal insufficiency. In this study, we investigated the effects of oral treatment with paricalcitol, a potent vitamin D receptor activator, on arterial stiffness and osteopontin, a marker of atherosclerosis, in hypertensive patients with chronic kidney disease (CKD) and secondary hyperparathyroidism. METHODS: We followed up 29 treated hypertensive patients (mean age: 74.1 years, 19 men, office blood pressure = 132/85 mmHg) with CKD stages 3-5 (mean glomerular filtration rate [GFR] = 19.4 ml/min/1.73 m2) who were on therapy with oral paricalcitol for 1 year. The control group consisted of 10 age-, sex-, and GFR-matched hypertensive patients with secondary hyperparathyroidism. RESULTS: After 1 year of treatment with paricalcitol compared to baseline, there was no statistical difference in levels of GFR, office blood pressure, and osteopontin (p = NS for all), while carotid-femoral PWV was reduced from 11.8 ± 2.6 m/s to 11.2 ± 2.4 m/s (p < 0.05). The control group exhibited no significant changes in carotid-femoral PWV (p = NS). CONCLUSIONS: Treatment with oral paricalcitol in hypertensive subjects suffering from CKD stages 3-5 and secondary hyperparathyroidism is accompanied by amelioration of arterial stiffness as reflected by the reduction of carotid-femoral PWV.

Osteopontin isoforms differentially promote arteriogenesis in response to ischemia via macrophage accumulation and survival.

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

Recombinant Osteopontin Improves Neurological Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3ß Pathway Following Intracerebral Hemorrhage.

BACKGROUND This study aimed to investigate the potential neuroprotective effect of recombinant osteopontin (r-OPN) on apoptotic changes via modulating phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK-3ß) signaling in a rat model of intracerebral hemorrhage (ICH). MATERIAL AND METHODS We subjected 10-12-week-old Sprague-Dawley male rats (n=120) to injection of autologous blood into the right basal ganglia to induce ICH or sham surgery. ICH animals received vehicle administration, r-OPN (4 µL/pup), or r-OPN combined with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (86 ng/pup) at 30 min after injury. Neurological scores and rotarod latencies were evaluated on days 1-5 post-ICH. Brain water content was evaluated on days 1-3 post-ICH. The number of apoptotic cells changes were evaluated by terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) and hematoxylin staining. Apoptosis-related proteins Bcl-2, Bax, and cleaved caspase-3 (CC3), and the phosphorylation levels of Akt and GSK-3b were assayed by Western blot. RESULTS Neurological deficits, rotarod latencies, and brain water content following ICH were reduced in the r-OPN group compared to the vehicle group. r-OPN also attenuated cell death in ICH. Furthermore, treatment with r-OPN significantly increased p-Akt expression and decreased p-GSK-3ß. These effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of CC3 at 24 h after ICH. Importantly, all the beneficial effects of r-OPN in ICH were abrogated by the PI3K inhibitor wortmannin. CONCLUSIONS r-OPN may provide a wide range of neuroprotection by suppressing apoptosis through the PI3K/Akt/GSK-3ß signaling pathway after ICH.

Role of vascular calcification inhibitors in preventing vascular dysfunction and mortality in hemodialysis patients.

Cardiovascular events make up the primary cause of death in hemodialysis patients, and the risk for cardiovascular mortality is significantly increased by vascular calcification, a condition observed frequently in this patient population. The mechanisms underlying the pathogenesis of vascular calcification are complex, and many factors facilitate or hinder the development of calcification. In this review, we first summarize the main factors contributing to the pathogenesis of vascular calcification in patients with end-stage renal disease. We then explore the role of calcification inhibitors in the calcification process, as well as their effect on vascular dysfunction and mortality in hemodialysis patients.

Osteopontin Augments M2 Microglia Response and Separates M1- and M2-Polarized Microglial Activation in Permanent Focal Cerebral Ischemia.

BACKGROUND: Focal cerebral ischemia induces distinct neuroinflammatory processes. We recently reported the extracellular phosphor-glyco-protein osteopontin (OPN) to directly affect primary microglia in vitro, promoting survival while shifting their inflammatory profile towards a more neutral phenotype. We here assessed the effects of OPN on microglia after stroke in vivo, with focus on infarct demarcation. METHODS: Animals underwent focal photothrombotic stroke and were injected intracerebroventricularly with 500 µg OPN or vehicle. Immunohistochemistry assessed neuronal damage and infarct volume, neovascularisation, glial scar formation, microglial activation, and M1 and M2 polarisation. RESULTS: After photothrombotic stroke, areas covered by M1 and M2 microglia substantially overlapped. OPN treatment reduced that overlap, with microglia appearing more spread out and additionally covering the infarct core. OPN additionally modulated the quantity of microglia subpopulations, reducing iNOS+ M1 cells while increasing M2 microglia, shifting the M1/M2 balance towards an M2 phenotype. Moreover, OPN polarized astrocytes towards the infarct. CONCLUSION: Microglial activation and M1 and M2 polarization have distinct but overlapping spatial patterns in permanent focal ischemia. Data suggest that OPN is involved in separating M1 and M2 subpopulations, as well as in shifting microglia polarization towards the M2 phenotype modulating beneficially inflammatory responses after focal infarction.

A Sensitized IGF1 Treatment Restores Corticospinal Axon-Dependent Functions.

A major hurdle for functional recovery after both spinal cord injury and cortical stroke is the limited regrowth of the axons in the corticospinal tract (CST) that originate in the motor cortex and innervate the spinal cord. Despite recent advances in engaging the intrinsic mechanisms that control CST regrowth, it remains to be tested whether such methods can promote functional recovery in translatable settings. Here we show that post-lesional AAV-assisted co-expression of two soluble proteins, namely insulin-like growth factor 1 (IGF1) and osteopontin (OPN), in cortical neurons leads to robust CST regrowth and the recovery of CST-dependent behavioral performance after both T10 lateral spinal hemisection and a unilateral cortical stroke. In these mice, a compound able to increase axon conduction, 4-aminopyridine-3-methanol, promotes further improvement in CST-dependent behavioral tasks. Thus, our results demonstrate a potentially translatable strategy for restoring cortical dependent function after injury in the adult.