Osteopontin Secretion Research Articles

Functional, patient-derived 3D tri-culture models of the uterine wall in a microfluidic array.

Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues? We present a three-dimensional (3D) physiologically relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue. As a highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial-myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist. We employed microfluidic technology to characterize multiple miniaturized models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were assessed. Endometrial (n = 9) and myometrial biopsies (n = 4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5 × 5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterized by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both insulin-like growth factor binding protein 1 (IGFBP-1) and osteopontin secretion in response to hormone stimulation. When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of the protocol, epithelial cells localized to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate (cAMP) increased secretion of IGFBP-1, which indicates stromal decidualization, and enhanced epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes. Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may have influenced the myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having a hysterectomy, thus may not include the deeper basalis region, which may limit the physiological mimicry of the final models. Our novel approach to modelling the uterine wall in 3D captures all of the main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis. C.B. was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant. C.J.H. was supported by a Wellbeing of Women project grant (RG2137), SRI/Bayer and Wellcome Trust IFFS3. D.K.H. was supported by a Wellbeing of Women project grant (RG2137) and MRC clinical research training fellowship (MR/V007238/1). M.Z. is Director and Co-Founder of ScreenIn3D Limited. The other authors declare no conflict of interest. N/A.

Endothelial metabolic control of insulin sensitivity through resident macrophages.

Endothelial cells (ECs) not only form passive blood conduits but actively contribute to nutrient transport and organ homeostasis. The role of ECs in glucose homeostasis is, however, poorly understood. Here, we show that, in skeletal muscle, endothelial glucose transporter 1 (Glut1/Slc2a1) controls glucose uptake via vascular metabolic control of muscle-resident macrophages without affecting transendothelial glucose transport. Lowering endothelial Glut1 via genetic depletion (Glut1ΔEC) or upon a short-term high-fat diet increased angiocrine osteopontin (OPN/Spp1) secretion. This promoted resident muscle macrophage activation and proliferation, which impaired muscle insulin sensitivity. Consequently, co-deleting Spp1 from ECs prevented macrophage accumulation and improved insulin sensitivity in Glut1ΔEC mice. Mechanistically, Glut1-dependent endothelial glucose metabolic rewiring increased OPN in a serine metabolism-dependent fashion. Our data illustrate how the glycolytic endothelium creates a microenvironment that controls resident muscle macrophage phenotype and function and directly links resident muscle macrophages to the maintenance of muscle glucose homeostasis.

Oxygen tension regulating hydrogels for vascularization and osteogenesis via sequential activation of HIF-1α and ERK1/2 signaling pathways in bone regeneration

Angiogenesis plays a crucial role in bone regeneration. Hypoxia is a driving force of angiogenesis at the initial stage of tissue repair. The hypoxic microenvironment could activate the hypoxia-inducible factor (HIF)-1α signaling pathway in cells, thereby enhancing the proliferation, migration and pro-angiogenic functions of stem cells. However, long-term chronic hypoxia could inhibit osteogenic differentiation and even lead to apoptosis. Therefore, shutdown of the HIF-1α signaling pathway and providing oxygen at later stage probably facilitate osteogenic differentiation and bone regeneration. Herein, an oxygen tension regulating hydrogel that sequentially activate and deactivate the HIF-1α signaling pathway were prepared in this study. Its effect and mechanism on stem cell differentiation were investigated both in vitro and in vivo. We proposed a gelatin-based hydrogel capable of sequentially delivering a hypoxic inducer (copper ions) and oxygen generator (calcium peroxide). The copper ions released from the hydrogels significantly enhanced cell viability and VEGF secretion of BMSCs via upregulating HIF-1α expression and facilitating its translocation into the nucleus. Additionally, calcium peroxide promoted alkaline phosphatase activity, osteopontin secretion, and calcium deposition through the activation of ERK1/2. Both Cu2+ and calcium peroxide demonstrated osteogenic promotion individually, while their synergistic effect within the hydrogels led to a superior osteogenic effect by potentially activating the HIF-1α and ERK1/2 signaling pathways.

Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression.

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

Denervation alters the secretome of myofibers and thereby affects muscle stem cell lineage progression and functionality

Skeletal muscle function crucially depends on innervation while repair of skeletal muscle relies on resident muscle stem cells (MuSCs). However, it is poorly understood how innervation affects MuSC properties and thereby regeneration of skeletal muscle. Here, we report that loss of innervation causes precocious activation of MuSCs concomitant with the expression of markers of myogenic differentiation. This aberrant activation of MuSCs after loss of innervation is accompanied by profound alterations on the mRNA and protein level. Combination of muscle injury with loss of innervation results in impaired regeneration of skeletal muscle including shifts in myogenic populations concomitant with delayed maturation of regenerating myofibers. We further demonstrate that loss of innervation leads to alterations in myofibers and their secretome, which then affect MuSC behavior. In particular, we identify an increased secretion of Osteopontin and transforming growth factor beta 1 (Tgfb1) by myofibers isolated from mice which had undergone sciatic nerve transection. The altered secretome results in the upregulation of early activating transcription factors, such as Junb, and their target genes in MuSCs. However, the combination of different secreted factors from myofibers after loss of innervation is required to cause the alterations observed in MuSCs after loss of innervation. These data demonstrate that loss of innervation first affects myofibers causing alterations in their secretome which then affect MuSCs underscoring the importance of proper innervation for MuSC functionality and regeneration of skeletal muscle.

MULTILINEAGE DIFFERENTIATION OF C2C12 CELLS AND MONOCYTES IN VITRO WITHIN A 6-BROMOINDIRUBIN-3’-OXIME-INCORPORATED CHITOSAN-BASED SCAFFOLD FOR ENHANCED BONE REGENERATION

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2.Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining.Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation.Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S.

Universal cell membrane camouflaged nano-prodrugs with right-side-out orientation adapting for positive pathological vascular remodeling in atherosclerosis.

A right-side-out orientated self-assembly of cell membrane-camouflaged nanotherapeutics is crucial for ensuring their biological functionality inherited from the source cells. In this study, a universal and spontaneous right-side-out coupling-driven ROS-responsive nanotherapeutic approach, based on the intrinsic affinity between phosphatidylserine (PS) on the inner leaflet and PS-targeted peptide modified nanoparticles, has been developed to target foam cells in atherosclerotic plaques. Considering the increased osteopontin (OPN) secretion from foam cells in plaques, a bioengineered cell membrane (OEM) with an overexpression of integrin α9β1 is integrated with ROS-cleavable prodrugs, OEM-coated ETBNPs (OEM-ETBNPs), to enhance targeted drug delivery and on-demand drug release in the local lesion of atherosclerosis. Both in vitro and in vivo experimental results confirm that OEM-ETBNPs are able to inhibit cellular lipid uptake and simultaneously promote intracellular lipid efflux, regulating the positive cellular phenotypic conversion. This finding offers a versatile platform for the biomedical applications of universal cell membrane camouflaging biomimetic nanotechnology.

Immunomodulatory differences between mesenchymal stem cells from different oral tissues

Mesenchymal stem cells (MSCs) have recently been identified as having potentially therapeutic immunomodulatory properties. MSCs isolated from different oral tissues have similar morphology and immunophenotypes, however, direct comparisons of their gene expression and immunomodulatory properties have not been conducted. We isolated alveolar bone-derived MSCs (aBMSCs), dental pulp stem cells (DPSCs) and gingiva-derived MSCs (GMSCs) from the same patients and compared their immunophenotypes and transcriptomes. Additionally, we compared their production of soluble immunomodulatory cytokines as well as their immunoregulatory properties in coculture with THP-1 human monocytic cells. RNA sequencing revealed distinct gene expression in DPSCs while aBMSCs and GMSCs had less differentially expressed genes. DPSCs also had significantly less secretion of osteopontin compared to aBMSCs and GMSCs. Finally, DPSCs did not exhibit an immunosuppresive effect on THP-1 cells to the same degree as aBMSCs and GMSCs. These findings demonstrate that MSCs from different oral tissues have distinct transcriptomes and immunoregulatory properties.

Osteopontin secreted from obese adipocytes enhances angiogenesis and promotes progression of pancreatic ductal adenocarcinoma in obesity.

Obesity is a risk factor and poor prognostic factor for pancreatic ductal adenocarcinoma (PDAC), but the underlying mechanisms remain unclear. PDAC cells and obese visceral adipocytes (O-Ad) derived from mice and humans were used to analyze interactions between the two cell types, and human microvascular endothelial cells were used for angiogenesis assay. A xenograft mouse model with subcutaneously injected PDAC cells was used for animal studies. The relationship between visceral fat and prognosis was analyzed using resected tissues from PDAC patients with and without obesity. Conditioned media (CM) from O-Ad significantly increased PDAC cell growth and migration and angiogenic capacity in both human and mice cells, and blocking osteopontin (OPN) in O-Ad canceled O-Ad-induced effects in both mouse and human cells. In addition, O-Ad directly increased the migratory and tube-forming capacities of endothelial cells, while blocking OPN canceled these effects. O-Ad increased AKT phosphorylation and VEGFA expression in both PDAC and endothelial cells, and OPN inhibition in O-Ad canceled those O-Ad-induced effects. In the xenograft model, PDAC tumor volume was significantly increased in obese mice compared with lean mice, whereas blocking OPN significantly inhibited obesity-accelerated tumor growth. OPN expression in adipose tissues adjacent to human PDAC tumor was significantly higher in obese patients than in non-obese patients. In PDAC patients with obesity, high OPN expression in adipose tissues was significantly associated with poor prognosis. Obese adipocytes trigger aggressive transformation in PDAC cells to induce PDAC progression and accelerate angiogenesis via OPN secretion.

Abstract 526: Activated Platelets And Osteopontin Contribute To Arterial Stiffness And Are Involved In Abdominal Aortic Aneurysm

Significance: Chronic inflammation is a consistent theme in the progression of cardiovascular disease including AAA. Chronic inflammation contributes to aortic wall weakening in AAA by disrupting the ECM and promoting aortic stiffness. Various cell types (fibroblast, macrophages, VSMCs, and circulating platelets) and inflammatory signaling cascades are implicated in aortic wall weakening. Osteopontin (OPN) is a well-known pro-inflammatory cytokine, which is secreted by pro-inflammatory cells and has been found upregulated in AAA patients. However, its mechanistical pathway in disease’ progression and which cells participate in OPN signaling are unclear. Interestingly, literature suggests that activated platelets can drive OPN secretion in proximal inflammatory cells. Therefore, the aim of this project was to determine whether a mechanistic link exists among the activated platelets and OPN production in cells associated with AAA that eventually increases local aortic stiffness and promotes AAA progression. Methods: Human platelets were isolated from donor blood and activated to obtain activated platelet supernatants (APS). M1 macrophages (M1), adventitial fibroblasts (Fib) and vascular smooth-muscle cells (VSMC) were exposed to APS.Full-length OPN and its fragments, generated by thrombin, plasmin and MMP-2 and -9 cleavage, were incubated with the same cell populations as mentioned above. Stiffness-related gene expression and MMP-2 and -9 activity was investigated utilizing qRT-PCR and gelatin zymography, respectively. Results: On exposure to APS, stiffness related gene expression as well as MMPs activity was significantly altered in the vascular cell types. Downstream effects of OPN on aortic stiffness presented changes in OPN treated cells compared to control. Conclusion: Our results indicate that activated platelets contribute to aortic stiffness and induction of vascular inflammation by modulating OPN expression, suggesting a potential role in AAA pathobiology.

The enhancer landscape predetermines the skeletal regeneration capacity of stromal cells.

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

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.

Albumin-Coated Polycaprolactone (PCL)-Decellularized Extracellular Matrix (dECM) Scaffold for Bone Regeneration.

With the emphasis on collagen and hydroxyapatite, the main structural components of bone tissue, synthetic grafts fall short of matching the clinical efficacy of autologous bone grafts. Excluded non-collagenous protein (NCPs) and carbohydrates also participate in critical cell signaling cascades and guide mineral deposition during intermediate stages of bone healing. By mimicking the native fracture repair process, polymeric scaffolds that incorporate calcium-binding moieties present in fibrocartilage can potentially enhance their bioactivity, mineralization, and bone growth. Likewise, coating polymeric fibers with serum albumin is an additional strategy that can impart collagen-like biofunctionality and further increase mineral deposition on the fibrous surface. Here, a combination of electrospun polycaprolactone (PCL) fibers with chondrocyte-derived decellularized extracellular matrix (dECM) and albumin coating were investigated as a fibrocartilage-mimetic scaffold that can serve as a woven bone precursor for bone regeneration. PCL fibrous constructs coated with dECM and albumin are shown to synergistically increase calcium concentration and calcium phosphate (CaP) deposition in a simulated body fluid biomineralization assay. Albumin/dECM coating increased osteoblast proliferation and mineral deposition in culture. In contrast, CaP coating transformed osteoblast bone lining morphology into cuboidal phenotype and arrested their proliferation. Cell sheets of osteoblasts cultured on dECM/albumin/CaP-coated constructs exhibited an increase in calcium deposition and secretion of collagen, osteopontin, osteocalcin, and bone morphogenetic protein. These results highlight the potential of biomolecular coatings to enhance bone-mimetic properties of synthetic nanofibrous scaffolds, stimulate critical protein and mineral deposition, and augment the bone's capacity to heal. Thus, mimicking the intermediate stages of bone regeneration by incorporating calcium-binding moieties may prove to be a useful strategy for improving the clinical outcomes of synthetic bone grafts.

Cathepsin L was involved in vascular aging by mediating phenotypic transformation of vascular cells

Vascular media and adventitia-induced remodeling plays an important role in vascular aging. However, the mechanism remains unclear. This study aims to investigate the mechanisms underlying vascular aging. Transcriptome analysis revealed that the expression of cathepsin L (CTSL) significantly decreased in arteries of old mice (24 months old) compared with that in arteries of young mice (4 months old), which was confirmed by immunohistochemistry and Western blot. The expression of CTSL in adventitia fibroblasts (AFs) and vascular smooth muscle cells (VSMCs) of aged mice was lower than that of young mice. Compared with wild-type control mice, CTSL knockout (CTSL - /−) mice had increased collagen deposition (fibrosis) and decreased telomerase activity and LC3Ⅱ/ LC3Ⅰratio. The expression of mammalian target of rapamycin (mTOR) and osteopontin (OPN) increased in aortas of CTSL−/−mice compared with that in aortas of wild-type control mice. In vitro, lentivirus-mediated CTSL knockdown induced VSMCs senescence and AFs transformed into myofibroblasts (MFs). Rapamycin, a mTOR inhibitor, inhibited CTSL deficiency induced VSMCs senescence, osteopontin (OPN) secretion and AFs migration. In conclusion, the decreased level of CTSL with age may participate in vascular aging by promoting the phenotypic transformation of vascular cells.

Induction of a cytokine storm involves suppression of the Osteopontin-dependent TH1 response.

Cytokine release syndromes represent a severe turn in certain disease states, which may be caused by several infections, including those with the virus SARS-CoV-2. This inefficient, even harmful, immune response has been associated with a broad release of chemokines. Although a cellular (type I) immune reaction is efficacious against viral infections, we noted a type I deficit in the cytokine patterns produced by cytokine storms of all reported etiologies. Agents including lipopolysaccharide (LPS, bacterial), anti-CD3 (antibody) and a version of the prominent SARS-CoV-2 viral surface molecule, Spike Glycoprotein, were individually sufficient to induce IL-6 and multiple chemokines in mice. They failed to upregulate the TH1 inducer cytokine Osteopontin, and the pathophysiologic triggers actually suppressed the PMA-induced Osteopontin secretion from monocytic cells. Osteopontin administration partially reversed the chemokine elevation, more effectively so in a mouse strain with TH1 bias. Corroboration was obtained from the inverse correlation in the levels of IL-6 and Osteopontin in plasma samples from acute COVID-19 patients. We hypothesize that the inhibition of Osteopontin by SARS-CoV-2 Spike Glycoprotein or LPS represents an immune evasion mechanism employed by the pathogens of origin. The ensuing dysfunctional inflammatory response promotes a vicious cycle of amplification, resulting in a cytokine storm.

Aryl hydrocarbon receptor activation-mediated vascular toxicity of ambient fine particulate matter: contribution of polycyclic aromatic hydrocarbons and osteopontin as a biomarker

BackgroundExposure to ambient fine particulate matter (PM2.5) is associated with vascular diseases. Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 are highly hazardous; however, the contribution of PM2.5-bound PAHs to PM2.5-associated vascular diseases remains unclear. The ToxCast high-throughput in vitro screening database indicates that some PM2.5-bound PAHs activate the aryl hydrocarbon receptor (AhR). The present study investigated whether the AhR pathway is involved in the mechanism of PM2.5-induced vascular toxicity, identified the PAH in PM2.5 that was the major contributor of AhR activation, and identified a biomarker for vascular toxicity of PM2.5-bound PAHs.ResultsTreatment of vascular smooth muscle cells (VMSCs) with an AhR antagonist inhibited the PM2.5-induced increase in the cell migration ability; NF-κB activity; and expression of cytochrome P450 1A1 (CYP1A1), 1B1 (CYP1B1), interleukin-6 (IL-6), and osteopontin (OPN). Most PM2.5-bound PAHs were extracted into the organic fraction, which drastically enhanced VSMC migration and increased mRNA levels of CYP1A1, CYP1B1, IL-6, and OPN. However, the inorganic fraction of PM2.5 moderately enhanced VSMC migration and only increased IL-6 mRNA levels. PM2.5 increased IL-6 secretion through NF-κB activation; however, PM2.5 and its organic extract increased OPN secretion in a CYP1B1-dependent manner. Inhibiting CYP1B1 activity and silencing OPN expression prevented the increase in VSMC migration ability caused by PM2.5 and its organic extract. The AhR activation potencies of seven PM2.5-bound PAHs, reported in the ToxCast database, were strongly correlated with their capabilities of enhancing the migration ability of VSMCs. Benzo(k)fluoranthene (BkF) contributed the most to the AhR agonistic activity of ambient PM2.5-bound PAHs. The association between PM2.5-induced vascular toxicity, AhR activity, and OPN secretion was further verified in mice; PM2.5-induced intimal hyperplasia in pulmonary small arteries and OPN secretion were alleviated in mice with low AhR affinity. Finally, urinary concentrations of 1-hydroxypyrene, a major PAH metabolite, were positively correlated with plasma OPN levels in healthy humans.ConclusionsThe present study offers in vitro, animal, and human evidences supporting the importance of AhR activation for PM2.5-induced vascular toxicities and that BkF was the major contributor of AhR activation. OPN is an AhR-dependent biomarker of PM2.5-induced vascular toxicity. The AhR activation potency may be applied in the risk assessment of vascular toxicity in PAH mixtures.

MyD88 in hepatic stellate cells promotes the development of alcoholic fatty liver via the AKT pathway.

Myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in the Toll-like receptors (TLRs) signalling pathway, is expressed in various liver cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs). And yet, the functional role of MyD88 in HSCs is poorly elucidated in alcoholic fatty liver (AFL). Here, to study the functional role of MyD88 in HSCs and the molecular mechanism related to the development of AFL, chronic-binge ethanol mouse models were established in mice with specific MyD88 knockout in quiescent (MyD88GFAP-KO) and activated HSCs (MyD88SMA-KO), respectively. Our results clearly showed an elevated expression of MyD88 in liver tissues of ethanol treated mouse model which harbours the wild type. Intriguingly, ethanol treatment profoundly inhibited inflammation in both MyD88GFAP-KO and MyD88SMA-KO mice, but the suppression of lipogenesis was only observed in MyD88GFAP-KO mice. Molecularly, our study indicated that MyD88 induced osteopontin (OPN) secretion in HSCs, which consequently resulted in activation of AKT signalling pathway and accumulation of fat in hepatocytes. Additionally, our data also suggested that OPN promoted inflammation by activating p-STAT1. Thus, targeting MyD88 may be a potentially represent a promising strategy for the prevention and treatment of AFL. KEY MESSAGES: The expression of MyD88 in HSCs was significantly increased in ethanol-induced liver tissues of wild-type mice. MyD88 deficiency in quiescent HSCs inhibited inflammation and lipogenesis under the ethanol feeding condition. MyD88 deficiency in activated HSCs only inhibited inflammation under the ethanol feeding condition. MyD88 promoted the OPN secretion of HSCs, which further activated the AKT signalling pathway of hepatocytes and upregulated lipogenic gene expression to promote fat accumulation. OPN also promotes inflammation by activating p-STAT1.

Human Gingival Fibroblast and Osteoblast Behavior on Groove-Milled Zirconia Implant Surfaces.

Two type of cells representing periodontal hard tissues (osteoblasts) and soft tissues (fibroblasts) were evaluated in response to microgroove-milled zirconia surfaces. A total of 90 zirconia discs were randomly assigned to four width-standardized milling microgroove-textured groups and a control group without grooves (UT). The sandblast and acid-etch protocol were applied to all samples. Both cell lines were cultured on zirconia discs from 1 day up to 14 days. Cell morphology and adhesion were evaluated after 1 day of culturing. Cell viability and proliferation of the cells were measured. Alkaline phosphatase activity, collagen I, osteopontin, interleukin 1β and interleukin 8 secretions were assessed at predefined times. The results obtained were presented in the form of bar graphs as means and standard deviations. Multi comparisons between groups were evaluated using two-away ANOVA or Mann–Whitney tests, and a p-value < 0.05 was established. Group comparisons with regard to cell viability, proliferation and secretion of collagen I, interleukin-1β and interleukin 8 revealed no statistically significant differences. The alkaline phosphatase activity and osteopontin secretion were significantly higher in the group with a large groove compared to the small one and the control group. Nevertheless, the viability of gingival and bone cells did not appear to be affected by the milled microgroove texture compared to the conventional sandblasted and acid-etched texture, but they seem to influence osteoblasts’ cellular differentiation.

Osteopontin in post-subarachnoid hemorrhage pathologies.

Rupture of intracranial aneurysms causes subarachnoid hemorrhage (SAH), of which the treatment remains the most difficult among cerebrovascular disorders even in this modern medical era. Following successful surgical ablation of ruptured intracranial aneurysms, other conditions may be encountered including delayed cerebral ischemia and chronic hydrocephalus, in addition to early brain injury. Osteopontin (OPN) is one of matricellular proteins that have cytokine-like effect on various cells and act as secretory extracellular matrix proteins between cells. The complexity of OPN functions is attributed to its several isoforms, cleavage sites and functional changes determined by its differing isoforms following various cleavages or other post-translational modifications. Notably, OPN functions beneficially or harmfully in accordance with the context of OPN upregulation. In the field of aneurysmal SAH, OPN has exerted neuroprotective effects against early brain injury and delayed cerebral ischemia by suppressing apoptosis of neurons, disruption of blood-brain barrier, and/or cerebrovascular constriction, while excessive and prolonged secretion of OPN can be harmful through the occurrence of chronic hydrocephalus requiring shunt surgery. This is a review article that is focused on OPN's potential roles in post-SAH pathologies.