Periostin Expression Research Articles

P11.12 Periostin is expressed by pericytes and is crucial for angiogenesis in glioma

Abstract BACKGROUND The expression of the matricellular protein periostin has recently been associated with glioma progression and angiogenesis. The aim of the present study was to identify the cellular source of periostin expression in human gliomas and to study the role of periostin in an in vitro model for angiogenesis. MATERIAL AND METHODS The expression of periostin in various types and grades of brain tumors was investigated at the genetics level by RT-PCR and at the protein level by immunohistochemistry. In addition, the expression of periostin in glioma samples was studied by double labeling and in situ RNA techniques. Subsequently, by silencing periostin in a 3D in vitro angiogenesis model, its effects on angiogenesis were studied. RESULTS Periostin RNA and protein expression was found to be elevated in pilocytic astrocytoma and glioblastoma, but not in grade II/III astrocytomas. The expression of periostin co-localized with PDGFRβ + cells, but not with Olig2+ / Sox2+ glioma stem cells. Silencing of periostin in pericytes resulted in the attenuation of the number and the length of the vessels, and in decrease in the number of vessel junctions formed. CONCLUSION The results prove that pericytes are the main source of periostin in human gliomas. We demonstrate that periostin plays an essential role in the growth and branching of blood vessels. Therefore, periostin and / or its molecular interactions should be explored as a novel target for developing a new anti-angiogenic therapy for glioma.

Periostin expression and characters of human adipose tissue-derived mesenchymal stromal cells were aberrantly affected by in vitro cultivation.

Human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) have been under focus in regenerative medicine since their discovery as a suitable source of MSCs. AD-MSCs are heterogeneous cells and exhibit variations in population doubling time, morphology and proliferative capacity. This study investigated if human AD-MSCs are developing, during in vitro long-term cultivation, in an unwanted or aberrant way. This study monitored AD-MSCs during their in vitro culture till the tenth passage investigating proliferation kinetics, DNA index and surface markers expression. Also, periostin gene expression was examined. The proliferation capacity and colony forming unit were decreased after passage 6 and the population doubling time was increased. Flow cytometric analysis revealed that newly cultivated population strongly expressed MSCs markers, furthermore, reduction of CD105 expression appeared in passage 5 onwards, the later was associated with significant increase in expression of CD34 (a hematopoietic cell marker). Also, reduction of CD73 and CD90 expression was observed from passage 8. Furthermore, during the first six passages, periostin expression was significantly unchanged, with significant upregulation in late passages. Long-term cultivation of human AD-MSCs changed their characters in an aberrant way and the first four passages might be the most appropriate passages for therapy. More investigation and understanding of these variations are needed to help in standardizing the expansion of MSCs-based therapies.

Periostin regulated by let-7/miR-98 family mediates the apoptosis and epithelial-mesenchymal transition of colon cancer

Objective: To observe the expressions of periostin (Postn) in colon cancer tissues and cells, and to investigate its biological effect and mechanism in colon cancer cells. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of Postn, let-7a and miR-98 in 20 pairs of colon cancer tissues and adjacent normal tissues, colon cancer cell lines including SW480, HT-29, HCT-116 and human normal colon epithelial cell NCM460. Small interfering RNAs (siRNAs) of Postn, pcDNA3.1-Postn plasmids, let-7a mimic and its negative control let-7a mimic-NC, miR-98 mimic and its negative control miR-98 mimic-NC were transfected into HCT-116 cells. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. Luciferase reporter gene assay was used to determine the targeting relationship between miRNAs and Postn. Results: Compared with adjacent normal tissues, Postn expression was up-regulated (P<0.05) while let-7a/miR-98 expression was down-regulated (P<0.05) in colon cancer tissues. Compared with NCM460 cells, Postn expression was up-regulated (P<0.05) while let-7a/miR-98 expression was down-regulated (P<0.05) in SW480, HT-29 and HCT-116 cells. In colon cancer tissues, the expression of Postn was negatively correlated with the expressions of let-7a and miR-98 (r=-0.69, P<0.001; r=-0.80, P<0.001). Inhibition of Postn in vitro reduced the viability of HCT-116 cells [(53.73±7.63)%, P<0.05], increased the apoptotic rate [(22.88±3.40)%, P<0.05], enhanced the expression of epithelial-mesenchymal transition (EMT) marker E-cadherin (2.44±0.39, P<0.05), while down-regulated the expressions of N-cadherin and Vimentin (0.44±0.07 and 0.38±0.06, P<0.05). Overexpression of Postn in vitro enhanced the cell viability of HCT-116 cells [(134.41±8.82) %, P<0.05], decreased the expression of E-cadherin (0.55±0.09, P<0.05), increased the expressions of N-cadherin and Vimentin (2.93±0.42 and 2.24±0.34, P<0.05), but had no effect on the apoptotic rate (P>0.05). Overexpression of let-7a or miR-98 partially reversed the biological effects of Postn overexpression in colon cancer cells, which implicated that Postn was a target gene of let-7a/miR-98. Conclusions: Postn is a cancer-promoting molecule of colon cancer, and inhibition of Postn expression can increase the apoptotic rate of colon cancer cells and repress EMT. Postn expression and function is regulated by let-7a/miR-98.

Proteomic analysis of synovial fluid identifies periostin as a biomarker for anterior cruciate ligament injury

Emerging evidence suggests that injury to the anterior cruciate ligament (ACL) typically initiates biological changes that contribute to the development of osteoarthritis (OA). The molecular biomarkers or mediators of these biological events remain unknown. The goal of this exploratory study was to identify novel synovial fluid biomarkers associated with early biological changes following ACL injury distinct from findings in end-stage OA. Synovial fluid was aspirated from patients with acute (≤30 days) and subacute (31-90 days) ACL tears and from patients with advanced OA and probed via tandem mass spectrometry for biomarkers to distinguish OA from ACL injury. Periostin (POSTN) was identified as a potential candidate. Further analyses of POSTN were performed in synovial fluid, OA cartilage, torn ACL remnants, and cultured cells and media by Western blot, PCR, immunostaining and ELISA. Synovial fluid analysis revealed that POSTN exhibited higher expression in subacute ACL injury than OA. POSTN expression was relatively low in cartilage/chondrocytes suggesting it is also produced by other intra-articular tissues. Conversely, high and time-dependent expression of POSTN in ACL tear remnants and isolated cells was consistent with the synovial fluid results. Elevated POSTN may provide a synovial fluid biomarker of subacute ACL injury setting separate from OA. Increased expression of POSTN in ACL suggests that the injured ACL may play a pivotal role in POSTN production, which is sensitive to time from injury. Previous studies have shown potential catabolic effects of POSTN, raising the possibility that POSTN contributes to the initiation of joint degeneration and may offer a window of opportunity to intervene in the early stages of post-traumatic OA.

Patterns of systemic and local inflammation in patients with asthma hospitalised with influenza.

Patients with asthma are at risk of hospitalisation with influenza, but the reasons for this predisposition are unknown. A prospective observational study of adults with PCR-confirmed influenza in 11 UK hospitals, measuring nasal, nasopharyngeal and systemic immune mediators and whole-blood gene expression. Of 133 admissions, 40 (30%) had previous asthma; these were more often female (70% versus 38.7%, OR 3.69, 95% CI 1.67-8.18; p=0.0012), required less mechanical ventilation (15% versus 37.6%, Chi-squared 6.78; p=0.0338) and had shorter hospital stays (mean 8.3 versus 15.3 days, p=0.0333) than those without. In patients without asthma, severe outcomes were more frequent in those given corticosteroids (OR 2.63, 95% CI 1.02-6.96; p=0.0466) or presenting >4 days after disease onset (OR 5.49, 95% CI 2.28-14.03; p=0.0002). Influenza vaccination in at-risk groups (including asthma) were lower than intended by national policy and the early use of antiviral medications were less than optimal. Mucosal immune responses were equivalent between groups. Those with asthma had higher serum interferon (IFN)-α, but lower serum tumour necrosis factor, interleukin (IL)-5, IL-6, CXCL8, CXCL9, IL-10, IL-17 and CCL2 levels (all p<0.05); both groups had similar serum IL-13, total IgE, periostin and blood eosinophil gene expression levels. Asthma diagnosis was unrelated to viral load, IFN-α, IFN-γ, IL-5 or IL-13 levels. Asthma is common in those hospitalised with influenza, but may not represent classical type 2-driven disease. Those admitted with influenza tend to be female with mild serum inflammatory responses, increased serum IFN-α levels and good clinical outcomes.

Abstract 521: Cardiac Fibroblasts are Activated During Postnatal Extracellular Matrix Remodeling

Objectives: During the postnatal period, P0 to P30 in the mouse, the myocardial extracellular matrix (ECM) transitions to a mature profile necessary for increased cardiac output. We hypothesize that, during the postnatal period, cardiac fibroblasts (CF) become activated, as indicated by Periostin (Postn) expression, and remodel the ECM, followed by quiescence in the mature heart. Our goal is to study CF activation and its role in postnatal heart development. Methods: CF activation was monitored by Postn MerCreMer(MCM) ;Rosa GFP lineage analysis and the resident CF were studied using a TCF21 MCM ;R26 GFP model. Tamoxifen induction of Cre MCM was initiated in the postnatal period and lineage-specific CF were quantified. Cardiac ECM maturation and CF proliferation, together with bone marrow-derived and endothelial cells, were assessed by Immunofluorescence (IF). TCF21 , Postn , Col1a1 , TnnI and CD31 transcripts were examined by RNAscope, and Fibronectin (FN) compartmentalization, an indicator of ECM maturation, also was examined. Results: FN, Postn, and Postn MCM R26 GFP lineage cells were more prominent at P0-7 than at P30. Postn -expressing cells also co-express Tcf21 and Col1a1 , but not TnnI (myocytes) or PECAM (endothelial cells), as determined by RNAscope. In agreement, Postn MCM R26 GFP CF do not express CD31, CD45, or the myofibroblast marker alphaSMA at P7. At P30, Postn, FN, and Col1a1 expression is reduced in CF, but TCF21 MCM ;R26 GFP is maintained, suggesting a transient period of CF activation followed by quiescence. Similarly, proliferation rates of the Postn MCM R26 GFP cells are higher than TCF21 MCM ;R26 GFP CF at P7, followed by decreased proliferation at P30. Conclusions: Activation and proliferation of CFs and ECM gene expression is increased in the week after birth relative to quiescent CFs at P30. Postn+ cells also peak at P7 but are not detected in appreciable numbers at P30, suggesting a critical role in ECM remodeling and myocardial maturation in the postnatal period. Studies are ongoing to determine the codependence of postnatal activated CF, ECM maturation and myocardial regeneration.

Expression and Significance of Periostin in Tissues and Serum in Oral Leukoplakia and Squamous Cell Carcinoma.

Objective: This study aims to investigate the expression changes of periostin (PN or OSF-2) in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), and analyze its significance in the development of OSCC. Study Design: The expression of periostin was detected from tissue specimens and serum obtained from normal mucosa, OLK and OSCC by immunohistochemistry, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction. Results: Periostin was significantly overexpressed in OLK and OSCC, when compared with normal controls (p < 0.05). Furthermore, the overexpression of periostin was positively correlated with TNM stage, depth of invasion, and lymph node metastasis (p < 0.05). Conclusion: The overexpression of periostin may be involved in the carcinogenesis process of OLK, which may be used as a marker for detecting OLK. In addition, periostin serum levels can be used as a potential indicator of invasion and a prognosis target for OSCC.

Prognostic significance of combining immunohistochemical markers for cancer-associated fibroblasts in lung adenocarcinoma tissue.

Cancer-associated fibroblasts (CAFs), activated fibroblasts in a cancer microenvironment, exert various effects upon carcinoma cells including lung adenocarcinoma cells. Various markers identifying CAFs have been proposed, but the correlations among these markers proposed and their clinicopathological significance have remained largely unknown. Therefore, in this study, we immunohistochemically evaluated the expression of alpha-smooth muscle actin (α-SMA), podoplanin, and periostin among these proposed markers in 92 cases of lung adenocarcinoma. These three markers were weakly correlated, but the relative abundance of α-SMA was significantly associated with high Ki-67 labelling index (LI), lymph node metastasis, and low 5-year overall survival (OS) rate of the patients. That of podoplanin was significantly associated with high pT and Ki-67 LI, distant metastasis, and low 5-year OS rate and that of periostin with high pT and Ki-67 LI. We then tentatively subclassified these cases into four groups according to high or low status of each of paired markers: α-SMA/podoplanin, α-SMA/periostin, and periostin/podoplanin. The α-SMA high/podoplanin high group was associated with the lowest survival rate (53.3%) among the four groups with significance. However, there were no significant differences in overall survival when the patients were classified according to the combinations of α-SMA/periostin or periostin/podoplanin. Results of our study firstly revealed the heterogeneity of CAFs in human lung adenocarcinoma tissue, and the analysis employing multiple markers of CAFs is generally required to study the clinical significance of CAFs in clinical materials.

Macrophage-lineage TRAP+ cells recruit periosteum-derived cells for periosteal osteogenesis and regeneration.

The periosteum, a thin tissue that covers almost the entire bone surface, accounts for more than 80% of human bone mass and is essential for bone regeneration. Its osteogenic and bone regenerative abilities are well studied, but much is unknown about the periosteum. In this study, we found that macrophage-lineage cells recruit periosteum-derived cells (PDCs) for cortical bone formation. Knockout of colony stimulating factor-1 eliminated macrophage-lineage cells and resulted in loss of PDCs with impaired periosteal bone formation. Moreover, macrophage-lineage TRAP+ cells induced transcriptional expression of periostin and recruitment of PDCs to the periosteal surface through secretion of platelet-derived growth factor-BB (PDGF-BB), where the recruited PDCs underwent osteoblast differentiation coupled with type H vessel formation. We also found that subsets of Nestin+ and LepR+ PDCs possess multipotent and self-renewal abilities and contribute to cortical bone formation. Nestin+ PDCs are found primarily during bone development, whereas LepR+ PDCs are essential for bone homeostasis in adult mice. Importantly, conditional knockout of Pdgfrβ (platelet-derived growth factor receptor beta) in LepR+ cells impaired periosteal bone formation and regeneration. These findings uncover the essential role of periosteal macrophage-lineage cells in regulating periosteum homeostasis and regeneration.

Sonic Hedgehog Ligand: A Role in Formation of a Mesenchymal Niche in Human Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterised by desmoplasia, thought to support progression and chemotherapeutic resistance. The Hedgehog pathway is known to play an important role in this cancer. While the upregulation of Sonic hedgehog (Shh) in the epithelium of PDAC is known, we investigated its expression in the tumour microenvironment in order to find new targets for new chemotherapeutical approaches. Immunohistochemistry was used for the investigation of Shh and Vimentin in primary human pancreatic tissues. Gene (qRT-PCR) and protein (immunofluorescence) expression of Shh, αSMA (a marker of the mesenchymal phenotype) and periostin (a marker of mesenchymal cells within a mixed population) were investigated in in vitro cell models. Shh expression was significantly upregulated in the stromal and epithelial compartments of poorly-differentiated PDAC samples, with a strong correlation with the amount of stroma present. Characterisation of stromal cells showed that there was expression of Shh ligand in a mixed population comprising αSMA+ myofibroblasts and αSMA− mesenchymal stem cells. Moreover, we demonstrated the interaction between these cell lines by showing a higher rate of mesenchymal cell proliferation and the upregulation of periostin. Therefore, targeting stromal Shh could affect the equilibrium of the tumour microenvironment and its contribution to tumour growth.

Periostin is induced by IL-4/IL-13 in dermal fibroblasts and promotes RhoA/ROCK pathway-mediated TGF-β1 secretion in abnormal scar formation

Excess scar formation can occur after skin injurふy and lead to abnormal scar formation, such as keloids and hypertrophic scars, which are characterised by substantial deposition of extracellular matrix in the dermis. Periostin, an extracellular matrix protein that plays a crucial role in skin development and maintaining homeostasis, is also involved in skin disorders such as systemic/limited scleroderma, wound closure, and abnormal scar formation. However, the mechanism of periostin involvement in abnormal scar formation is not yet fully understood. In this study, we investigated the mechanism by which periostin is involved in abnormal scar formation. Treatment of human dermal fibroblasts (HDFs) with IL-4 and IL-13, which are cytokines of Th2 type immune responses that are up-regulated in abnormal scars, dramatically elevated the levels of periostin mRNA and protein, and also promoted the secretion of periostin by HDFs. Transforming growth factor-β1 (TGF-β1) had the same effect on HDFs as IL-4 and IL-13. Stimulation of HDFs with periostin promoted RhoA/ROCK pathway-mediated TGF-β1 secretion from HDFs. Our results suggest that IL-4 and IL-13 induce periostin expression and secretion, and in turn, secreted periostin induces RhoA/ROCK pathway-mediated TGF-β1 secretion. Secreted TGF-β1 then induces further periostin production and secretion, thereby promoting abnormal scar formation.

LncRNA H19/miR-let-7 axis participates in the regulation of ox-LDL-induced endothelial cell injury via targeting periostin

Oxidized-low density lipoprotein (ox-LDL)-induced endothelial cell dysfunction is a crucial event in the pathogenesis of atherosclerosis (AS). Long noncoding RNAs (lncRNAs) have been shown to play important roles in this process. The purpose of this study was to investigate the biological effects of lncRNA H19 on the ox-LDL-induced endothelial cell injury and to explore the underlying molecular mechanisms. In the present study, the expression of H19 in the serum of patients with AS and in the ox-LDL-treated human umbilical vein endothelial cells (HUVECs) was significantly up-regulated. H19 knockdown by transfection with H19 siRNAs in ox-LDL-treated HUVECs remarkably promoted cell viability, suppressed the secretion of interleukin (IL)-1β, IL-6, and tumour necrosis factor (TNF)-α, decreased the expression of vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1) and E-selectin, reduced levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) levels, and reduced cell apoptosis. Moreover, H19 knockdown significantly down-regulated the ox-LDL-induced expression of periostin, but did not affect the expression of let-7a, which directly targets the 3′-UTR of periostin mRNA. In addition, periostin overexpression partly reversed the biological effects of H19 knockdown in ox-LDL-treated HUVECs, which were almost recapitulated by let-7 overexpression. In conclusion, these data suggest that H19 knockdown suppressed ox-LDL-induced inflammation, apoptosis and oxidative stress in HUVECs, which may be related to the down-regulation of periostin by interfering with let-7 bioavailability.

Oscillatory shear potentiates latent TGF-\u03b21 activation more than steady shear as demonstrated by a novel force generator

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor β1 ubiquitously in a latent form (LTGF-β1). While complex shear stresses can activate LTGF-β1, the mechanisms underlying LTGF-β1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-β1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-β1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-β1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-β1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-β1 activation. OSS-generated active TGF-β1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-β1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-β1 to drive vascular remodeling.

The transcriptional regulation of periostin by scleraxis in cardiac myofibroblasts

Extracellular matrix (ECM) remodeling resulting in cardiac fibrosis is a crucial factor in determining the severity of myocardial dysfunction during heart failure. In response to stress or damage, cardiac fibroblasts undergo phenotype conversion to myofibroblasts, the primary cell type responsible for ECM synthesis and remodeling in the heart. Periostin, a marker of the myofibroblast phenotype, is a matricellular secreted protein that contributes to cardiac remodeling. Periostin plays important roles in mediating cell–matrix signal transduction and in promoting pro‐fibrotic TGF‐β signaling via coupling with matrix associated lysyl oxidase; it is highly expressed in myofibroblasts, but is not expressed in fibroblasts. We have shown that the transcription factor scleraxis induces the phenotype conversion of cardiac fibroblasts to myofibroblasts by directly transactivating the expression of proteins highly up‐regulated in myofibroblasts, including collagen 1α2, fibronectin and α‐smooth muscle actin. Here we report that the treatment of primary adult rat cardiac myofibroblasts with angiotensin II significantly increases periostin gene expression in parallel with increased scleraxis expression. Over‐expression of scleraxis in myofibroblasts increased periostin expression nearly 19‐fold at the mRNA level. TGF‐b treatment induced periostin expression in cardiac myofibroblasts isolated from wild type mice, but failed to show a similar effect in cells isolated from scleraxis null mice. Using in silico analysis, we identified putative E‐box sites within the periostin gene promoter to which scleraxis may bind to potentially regulate promoter transactivation. Our data suggests that scleraxis is necessary and sufficient for TGF‐β mediated periostin expression. Our findings are consistent with a potential role for scleraxis in cardiac fibrosis, where periostin and fibrillar collagen gene expression are elevated in parallel with scleraxis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ALDH2 Activation Inhibited Cardiac Fibroblast-to-Myofibroblast Transformation Via the TGF-β1/Smad Signaling Pathway.

Pathological stimulus-triggered differentiation of cardiac fibroblasts plays a major role in the development of myocardial fibrosis. Aldehyde dehydrogenase 2 (ALDH2) was reported to exert a protective role in cardiovascular disease, and whether ALDH2 is involved in cardiac fibroblast differentiation remains unclear. In this study, we used transforming growth factor-β1 (TGF-β1) to induce the differentiation of human cardiac fibroblasts (HCFs) and adopted ALDH2 activator Alda-1 to verify the influence of ALDH2 on HCF differentiation. Results showed that ALDH2 activity was obviously impaired when treating HCFs with TGF-β1. Activation of ALDH2 with Alda-1 inhibited the transformation of HCFs into myofibroblasts, demonstrated by the decreased smooth muscle actin (α-actin) and periostin expression, reduced HCF-derived myofibroblast proliferation, collagen production, and contractility. Moreover, application of Smad2/3 inhibitor alleviated TGF-β1-induced HCF differentiation and improved ALDH2 activity, which was reversed by the application of ALDH2 inhibitor daidzin. Finally, Alda-1-induced HCF alterations alleviated neonatal rat cardiomyocyte hypertrophy, supported by the immunostaining of α-actin. To summarize, activation of ALDH2 enzymatic activity inhibited the differentiation of cardiac fibroblasts via the TGF-β1/Smad signaling pathway, which might be a promising strategy to relieve myocardial fibrosis of various causes.

Generation of a Fully Human scFv that binds Tumor-Specific Glycoforms

Tumor-specific glycosylation changes are an attractive target for the development of diagnostic and therapeutic applications. Periostin is a glycoprotein with high expression in many tumors of epithelial origin including ovarian cancer. Strategies to target the peptide portion of periostin as a diagnostic or therapeutic biomarker for cancer are limited due to increased expression of periostin in non-cancerous inflammatory conditions. Here, we have screened for antibody fragments that recognize the tumor-specific glycosylation present on glycoforms of periostin containing bisecting N-glycans in ovarian cancer using a yeast-display library of antibody fragments, while subtracting those that bind to the periostin protein with glycoforms found in non-malignant cell types. We generated a biotinylated form of a fully human scFv antibody (scFvC9) that targets the bisecting N-glycans expressed by cancer cells. Validation studies in vitro and in vivo using scFvC9 indicate this antibody can be useful for the development of diagnostic, imaging, and therapeutic applications for cancers that express the antigen.

New Noonan syndrome model mice with RIT1 mutation exhibit cardiac hypertrophy and susceptibility to β-adrenergic stimulation-induced cardiac fibrosis

BackgroundNoonan syndrome (NS) is a genetic disorder characterized by short stature, a distinctive facial appearance, and heart defects. We recently discovered a novel NS gene, RIT1, which is a member of the RAS subfamily of small GTPases. NS patients with RIT1 mutations have a high incidence of hypertrophic cardiomyopathy and edematous phenotype, but the specific role of RIT1 remains unclear.MethodsTo investigate how germline RIT1 mutations cause NS, we generated knock-in mice that carried a NS-associated Rit1 A57G mutation (Rit1A57G/+). We investigated the phenotypes of Rit1A57G/+ mice in fetal and adult stages as well as the effects of isoproterenol on cardiac function in Rit1A57G/+ mice.FindingsRit1A57G/+ embryos exhibited decreased viability, edema, subcutaneous hemorrhage and AKT activation. Surviving Rit1A57G/+ mice had a short stature, craniofacial abnormalities and splenomegaly. Cardiac hypertrophy and cardiac fibrosis with increased expression of S100A4, vimentin and periostin were observed in Rit1A57G/+ mice compared to Rit1+/+ mice. Upon isoproterenol stimulation, cardiac fibrosis was drastically increased in Rit1A57G/+ mice. Phosphorylated (at Thr308) AKT levels were also elevated in isoproterenol-treated Rit1A57G/+ hearts.InterpretationThe A57G mutation in Rit1 causes cardiac hypertrophy, fibrosis and other NS-associated features. Biochemical analysis indicates that the AKT signaling pathway might be related to downstream signaling in the RIT1 A57G mutant at a developmental stage and under β-adrenergic stimulation in the heart.FundThe Grants-in-Aid were provided by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science KAKENHI Grant.

Relaxin 2 carried by magnetically directed liposomes accelerates rat midpalatal suture expansion and subsequent new bone formation

Relaxin (RLN) is an insulin-like peptide hormone that enables softening and lengthening of the pubic symphysis and uterine cervix. Here, we analyzed the effects of RLN2 on the expansion of rat midpalatal suture (MPS) using a magnetically directed liposome-based drug delivery system. Thirty-six male rats were divided into three groups: control (MPS was not expanded), lipo (expanded for 1 week with vehicle liposomes encapsulating ferric oxide and Cy5.5), and RLN-lipo (expanded for 1 week with the liposomes coated with RLN2). Rats were sacrificed after 1 week of expansion or after 2 weeks of retention. To accumulate RLN2-liposomes, a magnetic sheet was fixed to the palatal mucosa of the MPS. In vivo imaging showed magnetically controlled accumulation of liposomes in the MPS for 72 h. Immunohistochemistry revealed RLN2 expression in the MPS after expansion and relaxin receptor (RXFP) 2 expression at the osteogenic front (OF) in the RLN-lipo group; all groups expressed RXFP1 in the MPS. MPS expansion and bone formation were significantly accelerated at the OF in RLN-lipo group compared with the other groups. In the RLN-lipo group, significantly accelerated serrate bone deposition and elevated periostin (POSTN), iNOS, and MMP-1 levels were observed in the MPS. Sclerostin (SOST) expression was significantly reduced in newly formed bone in the RLN-lipo group. Our data revealed that RLN2 enhanced suture expansion via MMP-1 and iNOS secretion in the sutural fibroblasts and new bone formation via POSTN expression in osteoblasts at the OF. These properties may be useful for developing a new less-invasive orthopedic treatment aiming at sutural modification of cranio- and maxillofacial deformity patients.

Osteitis in Chronic Rhinosinusitis.

Osteitis is recognized as a common factor in recalcitrant chronic rhinosinusitis (CRS). There is evidence for the association of osteitis with revision surgeries and CRS severity, in terms of higher Lund-Mackay scores. This is a narrative review on the osteitis in CRS patients. Evidence to date is inconclusive with regard to the etiology and pathogenesis of this bony thickening. Histopathology of osteitis in primary CRS is likely a process of neo-osteogenesis and bone remodeling. For better understanding, various associating factors have been studied including an inflammatory pattern of rhinosinusitis. Recent studies have associated osteitis with nasal polyps and tissue eosinophilia with the increase in periostin expression and P-glycoprotein mucosal expression. There is no association of osteitis to symptoms or quality of life. Osteitis is an outcome of neo-osteogenesis rather than inflammatory processes in CRS patients without a prior history of surgery. While CT has become a staple in osteitis assessment, the standards for grading osteitic severity remain in an experimental stage. There is no association between the presence or severity of osteitis at the time of surgery and clinical outcomes at 1year after surgery. This review provides a comprehensive overview of the pathogenesis, epidemiology, and correlation with clinical and biological factors of osteitis in CRS patients.

A humanized bone microenvironment uncovers HIF2 alpha as a latent marker for osteosarcoma

The quest for predictive tumor markers for osteosarcoma (OS) has not well progressed over the last two decades due to a lack of preclinical models. The aim of this study was to investigate if microenvironmental modifications in an original humanized in vivo model alter the expression of OS tumor markers. Human bone micro-chips and bone marrow, harvested during hip arthroplasty, were implanted at the flanks of NOD/scid mice. We administered recombinant human bone morphogenetic protein 7 (rhBMP-7) in human bone micro-chips/bone marrow group I in order to modulate bone matrix and bone marrow humanization. Ten weeks post-implantation, human Luc-SAOS-2 OS cells were injected into the humanized tissue-engineered bone organs (hTEBOs). Tumors were harvested 5 weeks post-implantation to determine the expression of the previously described OS markers ezrin, periostin, VEGF, HIF1α and HIF2α. Representation of these proteins was analyzed in two different OS patient cohorts. Ezrin was downregulated in OS in hTEBOs with rhBMP-7, whereas HIF2α was significantly upregulated in comparison to hTEBOs without rhBMP-7. The expression of periostin, VEGF and HIF1α did not differ significantly between both groups. HIF2α was consistently present in OS patients and dependent on tumor site and clinical stage. OS patients post-chemotherapy had suppressed levels of HIF2α. In conclusion, we demonstrated the overall expression of OS-related factors in a preclinical model, which is based on a humanized bone organ. Our preclinical research results and analysis of two comprehensive patient cohorts imply that HIF2α is a potential prognostic marker and/or therapeutic target. Statement of SignificanceThis study demonstrates the clinical relevance of the humanized organ bone microenvironment in osteosarcoma research and validates the expression of tumor markers, especially HIF2α. The convergence of clinically proven bone engineering concepts for the development of humanized mice models is a new starting point for investigations of OS-related marker expression. The validation and first data set in such a model let one conclude that further clinical studies on the role of HIF2α as a prognostic marker and its potential as therapeutic target is a condition sine qua non.