Finally, we underline the potential function of microRNAs, epigenetic regulatory RNA factors reported to participate in several pathological responses in heart tissue and in the Wnt signaling network, as important modulators of Wnt/β-catenin signaling transduction in AC.Wnt/β-catenin signaling pathway plays essential roles in heart development as well as cardiac tissue homoeostasis in adults. Abnormal regulation of this signaling pathway is linked to a variety of cardiac disease conditions, including hypertrophy, fibrosis, arrhythmias, and infarction. Recent studies on genetically modified cellular and animal models document a crucial role of Wnt/β-catenin signaling in the molecular pathogenesis of arrhythmogenic cardiomyopathy (AC), an inherited disease of intercalated discs, typically characterized by ventricular arrhythmias and progressive substitution of the myocardium with fibrofatty tissue. In this review, we summarize the conflicting published data regarding the Wnt/β-catenin signaling contribution to AC pathogenesis and we report the identification of a new potential therapeutic molecule that prevents myocyte injury and cardiac dysfunction due to desmosome mutations Elucidation of the precise regulatory mechanism of Wnt/β-catenin signaling in AC molecular pathogenesis could provide fundamental insights for new mechanism-based therapeutic strategy to delay the onset or progression of this cardiac disease. The Wnt pathway is a highly conserved signaling pathway throughout evolution. It controls numerous stages of embryo development and adult tissue homeostasis through atiming- and location-specific activation , 2. The In this review, we aim to introduce the Wnt signaling pathway and to discuss its involvement in AC, together with the development of more selective therapeutic approaches based on Wnt targeting therapies.Drosophila melanogaster segment polarity gene Wingless and its vertebrate orthologous, Int-1, a proto-oncogene discovered in 1984 in mouse [The Wnt pathway is coupled to a highly conserved family of secreted protein growth factors (cysteine-rich glycoproteins) known as Wnt proteins, that have been identified in animals from Hydra to Human , 16, 17.in mouse , 18, 19.in mouse –22. Althin mouse .At the cell membrane, Wnt ligands bind the Frizzled receptors (Fzd) as well as low-density Lipoprotein Receptor-related Protein-5 or -6 (LRP5/6) co-receptors. Fzd receptor family includes eleven members in Humans, which show seven transmembrane-spanning regions, an extracellular N-terminus Wnt-binding domain, and an intercellular C-terminal tail, crucial in the signal transduction , 24, 25.2+ concentration and activating Ca2+-sensitive proteins, is unclear, but it seems to be also involved in the controlling of cell fate and cell migration [Different Wnt ligands activate different intracellular signal transduction pathways, including the canonical β-catenin-dependent Wnt pathway, and the non-canonical β-catenin-independent Wnt/Planar cell polarity and Wnt-calcium/protein kinase C pathways , 26–28. igration .In this review, we focus on the canonical Wnt pathway.The β-catenin protein is the main signal transducer in the canonical Wnt pathway Figure . β-catenIn the canonical Wnt pathway, the absence of Wnt ligands determines the rapid phosphorylation of the cytosolic β-catenin protein by a cytoplasmic destruction complex. This complex consists of the scaffolding protein axin, adenomatous polyposis coli (APC) tumour suppressor protein, glycogen synthase kinase 3β (GSK3β), directly responsible for β-catenin phosphorylation at key amino-terminal Ser33, Ser37 and Thr41 residues, and casein kinase 1 (CK1), which phosphorylates β-catenin at the amino acid Ser45 , 33. TheWhen Wnt ligands bind to Fzd receptor and LRP5/6 co-receptors, the Wnt signaling is activated. The binding of Wnt recruits the cytoplasmic protein dishevelled (Dvl) to the cell membrane, thus sequestering the rate-limiting components axin and GSK3β from the degradation complex and determining the destruction of the complex , 37. TheHeart development and morphogenesis are meticulously controlled by the relationship of several signaling pathways that cooperate to provide cell fate specification. The rigorous expression of Wnt growth factors, together with several families of secreted factors including TGFbs (Transforming Growth Factor-Betas), FGFs (Fibroblast Growth Factors), BMPs (bone morphogenetic proteins), Notch and Hippo proteins, is essentially responsible for heart processes, including myocardial specification and proliferation, cardiac septation, chamber and cardiac valve formation, as well as endothelial and vascular smooth muscle cell proliferation , 38–40.Knocking out β-catenin results in mouse embryonic defects at gastrulation, not allowing the investigation of a possible Wnt function during cardiogenesis . AdditioA delicate balance between canonical and noncanonical Wnt pathways has been implicated in cardiac precursor differentiation, as well as in epicardium formation and differentiation. The canonical Wnt signaling appears to retain the cardiac precursors in a proliferative and precursor state , whereasIn vitro treatment of neonatal and adult rat cardiomyocytes with BIO, a pharmacological agent inhibiting GSK3, results in increased β-catenin activity and promotes cell proliferation and mitosis. Thus, the activation of canonical Wnt signaling participates in controlling the proliferative capability of the differentiated cardiomyocytes by promoting maintenance of stem cell properties [The Wnt/β-catenin signaling also controls cardiomyocyte proliferation . In vitroperties . In embroperties .Wnt/β-catenin reporter mice unveiled the spatial and temporal distribution of the endogenous status of β-catenin activation in several tissues. Transgenic animals exhibit an evident reporter gene activity in the cardiac region, where it persists up to E10.5, whereas the Wnt/β-catenin pathway appears not active in the adult heart . TherefoThe adult mammalian heart includes interacting populations of cells, predominantly myocytes, fibroblasts, smooth muscle cells, endothelial and epicardial cells, all arranged in a precise three-dimensional network. Under pathological conditions, they can interact with each other directly or indirectly, through the expression of growth factors and cytokines, to affect a physiological cardiac response.wild-type littermates [In terminally differentiated cardiomyocytes, the canonical Wnt signaling is normally quiescent , but it termates , 60. On termates , 61.Many studies have emphasized an important role for the canonical Wnt signaling in cardiac fibrogenesis , 62, 63.Moreover, Wnt signaling has been suggested to inhibit the apoptotic process involved in the transition from hypertrophy to heart failure. Indeed, in heart from patients who have developed heart failure, Akt activation and consequent GSK3β inhibition seem to protect cardiac cells from apoptosis .Wnt1, a major element of the early Wnt/β-catenin signaling, is a specific and potent inducer of connexin-43 expression in cardiomyocytes. This effect results in enhanced accumulation of connexin-43 protein and formation of functional gap junction channels, supporting the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart .In conclusion, the Wnt/β-catenin signaling is a very complex system involved in a variety of cardiac pathologies with quite varied effects, according with the cell system and timing of intervention. For this reason, it can offer possibilities for new insights into disease pathogenesis and identification of new therapeutic targets.Arrhythmogenic cardiomyopathy (AC) is a rare, inherited cardiac disease characterized by ventricular arrhythmias, right ventricular dysfunction and sudden cardiac death , 71. It The early “concealed” phase is characterized by propensity toward ventricular tachyarrhythmia in the setting of well-preserved structural morphology and ventricular function. As the disease progresses, however, myocyte loss, inflammation, and accumulation of fibrofatty scar tissue become evident . The typA scoring system to establish the diagnosis of AC has been developed on the basis of the fulfilment of major and minor criteria encompassing structural, histological, electrocardiographic, arrhythmic and genetic features of the disease , 78.area composita (this latter found only in cardiac muscle) [via their extracellular domains, while their cytoplasmic domains interact with the armadillo proteins. These latter interact with desmoplakin, which in turn binds desmin intermediate filaments, thereby creating a continuous link between the cytoskeletons of adjacent cardiomyocytes. The area composita comprises some of the desmosomal proteins described above as well as proteins normally found in adherens junctions. Amongst these, N-cadherin interacts with the adjacent cell via its extracellular domain and, on the cytoplasmic side, is indirectly linked to α-catenin via β-catenin or plakoglobin. In turn, α-catenin binds the F-actin cytoskeleton through vinculin [AC is an inherited cardiomyopathy characterized by incomplete penetrance and variable expressivity and it is usually transmitted as an autosomal dominant trait, even though recessive forms have been reported . Since 1 muscle)   from affected patients, revealed the involvement of the canonical Wnt signaling in the molecular pathogenesis of AC Table .The progressive replacement of the cardiomyocytes by adipocytes and fibrosis is the pathological hallmark of AC, which contributes to both cardiac dysfunction and arrhythmias. In this regard, in addition to the inactivation of junctional mechanical functions, which is supposed to lead to myocyte death under physical stress, the alteration of canonical Wnt/β-catenin signaling could act as a major transcriptional switch regulator of myogenesis towards adipogenesis .+/− mouse model for AC [Several studies from Marian's group demonstrated the suppression of Wnt/β-catenin/Tcf/Lef pathway in AC due to nuclear translocation of plakoglobin , 91, 92.l for AC  -Cre deleter mice in order to obtain a novel mouse model with traceable cardiac fibroadipocyte progenitors (FAPs) lacking of Dsp. These mice showed increased fibroadipogenesis in the heart and mild cardiac dysfunction, if compared with control mice. FAPs isolated from the same mouse hearts showed enhanced differentiation to adipocytes through a Wnt-dependent mechanism .Finally, Wnt pathway perturbation, due to reduced AKT and GSK3β activation, has been recently associated to exercise-induced acceleration of AC phenotype in mice overexpressing a mutant form of desmoplakin and exposed to a daily running . The ideOn the other hand, conflicting evidences have been reported about the canonical Wnt signaling involvement in AC pathogenesis. In a conditional mouse model with cardiac-restricted JUP deletion showing cardiac alterations and dysfunctions similar to those of AC patients, the remodeling of the IDs causes an increased β-catenin stabilization associated with activated AKT and consequent inhibition of GSK3β. In these animals, a Wnt/β-catenin signaling activation was suggested to contribute to the AC cardiac phenotype  Figure . On the in vitro studies examined the effect of point mutations in the PKP2 gene using iPSC-derived cardiomyocytes (iPS-CMs) from patient affected with AC [Up to now, several  with AC –104. In  with AC .Altogether, these findings suggest a central role of the Wnt/β-catenin signaling in the disease pathogenesis and appear to expand the cellular spectrum of AC, commonly recognized as a disease of cardiac myocytes, towards nonmyocyte cells in the heart. However, the direct causal relationship between mutant junctional proteins and perturbed Wnt pathway remains poorly understood.The intricate involvement of Wnt signaling in so many biological processes and disease conditions made this pathway an obvious but yet difficult target for therapeutic intervention. Numerous fields of study have investigated both natural and synthetic compounds able to induce or inhibit Wnt at multiple stages within the pathway. However, despite the evidence of successful methods to rapidly screen thousands of compounds to identify Wnt pathway modulators, unfortunately these molecules cannot be translated yet into the clinical practice .Na and IK1 exhibited by mutant fish myocytes. Interestingly, SB216763 is annotated as an inhibitor of GSK3β thereby increasing canonical Wnt signaling [To discover potential chemical modifiers in AC, a zebrafish model with cardiac-specific expression of a truncated form of plakoglobin and manifesting a fully penetrant cardiomyopathy, has been generated and used to screen a library of bioactive compounds for disease modifiers  Table 22. SB2167ignaling . Additioignaling .in vivo in two murine models of AC at baseline and in response to exercise  targeting life-threatening ventricular arrhythmias and congestive heart once they occur.Tissue development, physiology, and functions are strictly regulated by multiple factors. Among them, microRNAs (miRNAs) have been recently described as 22–25 nucleotides long noncoding RNAs which regulate gene expression at post-transcriptional level. This is achieved through imperfect base-pairing with complementary sequences in the 3′UTR of their target mRNA, leading to translational repression or transcript degradation . A singlIncreasing evidence indicates that miRNAs participate in signaling networks, such as the canonical Wnt/β-catenin signaling, and in promoting or inhibiting the progression of many human diseases, including cardiomyopathies. In the last years, several studies described the correlation between miRNA expression level and the regulation of the Wnt/β-catenin signaling, mostly during cardiac differentiation. Overexpression of miR-499 in rat bone marrow-derived mesenchymal stem cells is sufficient to induce cardiac differentiation, as indicated by increased expression of cardiac specific markers Nkx2.5, GATA4, MEF2C, and cTnl, through Wnt/β-catenin signaling pathway . In partAmong the most expressed miRNAs in the heart, miR-1 is known to promote cardiomyocyte differentiation. Lu and colleagues showed that this miRNA suppresses the canonical Wnt/β-catenin signaling in human iPSCs by targeting the receptor Fzd7 and antagonizing the ligand Wnt3A. As a consequence, cardiomyocyte differentiation is promoted and endothelial cell commitment from multipotent cardiovascular progenitors is suppressed .Moreover, several papers reported miR-19b as a negative regulator of the Wnt/β-catenin signaling. Overexpression of miR-19b has been shown to cause aberrant heart tube and chamber development and to regulate the contractile function of the heart in zebrafish embryos by inhibiting the Wnt signaling pathway directly targeting Ctnnb1β . FurtherLess frequently, miRNAs have been described to modulate Wnt/β-catenin signaling also during cardiac diseases. Gain and loss of function studies show that miR-145 overexpression leads to an increase in Wnt/β-catenin signaling activity and the downregulation of Dab2, a TGFβ receptor adaptor protein required for cardiac protein expression in mesenchymal stem cells, in response to TGFβ1 . Interesvice versa, the activation of the canonical Wnt signaling does not affect miR-184 levels in the in vitro model. The authors show the relationship between the downregulation of miR-184 and perturbations in E2F1 pathway, suggesting a novel mechanism concurring to AC development [Interestingly, the expression profile of miRNAs recently performed by qRT-PCR in 24 heart samples from AC patients revealed that miR-21-5p and miR-135b, which are correlated with Wnt and Hippo pathway in cancer, are significantly associated with both the myocardium adiposis and fibrosis, and might to be useful as therapeutic targets . On the elopment .Taken together, all these data underline the crucial function of miRNAs in the regulation of theWnt/β-catenin signaling and are suggestive of a potential role of these small RNAs in the pathogenesis and the phenotypic variability of cardiac diseases such as AC. Further efforts will be needed to find out whether miRNAs represent the link between the perturbations at the IDs and the alteration of the Wnt/β-catenin signaling activity observed in AC.vice versa, needs to be further investigated to contribute in the understanding not only of AC pathogenesis, but also of other, more common forms of heart disease, as well as to provide the basis to develop mechanism-based therapies to treat or prevent diseases at high risk of sudden death.Increasing evidence from experimental studies defines the canonical Wnt/β-catenin signaling as a critical player in the regulation of cardiac function and dysfunctions. To date, the function of this pathway in cardiac hypertrophy, fibrosis and arrhythmias have been well established, however further investigations are required to unravel its role in other pathological conditions. Controversial results have been reported about the involvement of Wnt/β-catenin pathway in the development of AC, an inherited cardiomyopathy determined by mutations mostly in genes encoding for ID proteins and typically characterised by the fibrofatty replacement of the myocardium. Current investigations in zebrafish and mammalian models of AC mainly indicate that suppression of the canonical Wnt/β-catenin signaling promotes cardiac remodelling in affected hearts. The dual function of β-catenin and plakoglobin both in cadherin-mediated adhesion and in gene transcription determines a highly regulated and complex protein network seemingly able to integrate disease signals from the membrane with cellular programs of target gene transcription. However, there are still some questions to be answered, particularly, which signal molecule(s) can play a dominant role in Wnt/β-catenin downregulation upon molecular destabilization of ID junctional structures. A more comprehensive understanding of the dynamics of this signaling in AC cardiomyocytes is therefore required, also for the design of safe and effective drug molecules. Recently, a small molecule activating the Wnt pathway has been experimented and it appears to be remarkably effective in preventing or reversing selected features of the molecular AC pathology. Based on these promising results, Wnt signaling components could represent interesting therapeutic targets for novel treatment strategy in AC, hopefully in the near future. Furthermore, a deeper investigation on the cross-talk between Wnt and other cellular pathways, such as Hippo or TGFβ, may explain other molecular mechanisms leading to AC and assist the design of efficient combinatorial therapeutic protocols. In addition, the relationship between miRNAs-mediated regulation of Wnt signaling, and


In addition, previous investigations in our group demonstrated the presence of a Wnt/β-catenin/COX-2 axis in dorsal root ganglions (DRGs). Therefore, here in this paper, we tested whether there is an association of such axis with NMDA-induced RGC loss. Rat retinal damage models generated by intravitreal injection of NMDA were used to measure the expression levels of β-catenin, COX-2, and VEGF in retinas, and the neuron numbers of the retinal GCL of rats were counted. Then, pharmacological tools  were introduced to evaluate the detailed roles of Wnt/β-catenin, COX-2, and VEGF in retinopathy of rats. Results demonstrated that all three factors in sequence are positively regulated neuronal loss induced by NMDA. These observations indicated that the Wnt pathway/COX-2/VEGF axis plays a pathogenic role in retinopathy and represented novel therapeutic targets.NMDA, a molecule that is capable of producing the loss of retinal ganglia cells (RGCs), has been widely studied; however, the detailed mechanism is not yet clarified. Previously, Wnt/ This excess intracellular Ca2+ causes predominant neuronal excitotoxicity mechanisms and is thought to be an underlying mechanism of glaucoma-induced neuronal cell death [Retinal ganglion cell death is a characteristic of many ophthalmological diseases, such as glaucoma, proliferative diabetic retinopathy, and retinal vein occlusion , but thell death .β-Catenin is an essential downstream effector in the canonical Wnt/β-catenin signaling pathway. In the absence of Wnt ligands, β-catenin is phosphorylated by a protein complex containing glycogen synthase kinase-3β (GSK-3β) and is constantly degraded to prevent its accumulation [β-catenin is translocated from the cytoplasm to the nucleus, where it interacts with members of the T-cell factor for DNA binding and regulates the expression of target genes, including inflammatory factors, such as COX-2 [Recent evidence indicates that the canonical Wnt pathway was activated with the NMDA receptor activation , 8. β-Camulation . Upon exas COX-2 . Retinalas COX-2 –14. Incrβ-catenin accumulation on the loss of neurons in the damaged retinas of model animals. The Wnt/β-catenin pathway in the rat retina was further mimicked by treatment with TWS119 (a GSK-3β inhibitor), which caused abnormal β-catenin accumulation [β-catenin pathway), NS-398 (a COX-2 inhibitor), and bevacizumab  can modulate β-catenin, COX-2, and VEGF expression levels and the neuronal loss in the retinas of the rats.In the present study, we used NMDA-treated rats to examine the possible role of mulation . We subsExperimental male Sprague-Dawley rats (weighing 180–200 g) were provided by Hubei Center for Disease Control and Prevention (China). The animals were acclimatized to the laboratories for one week prior to manipulation and were group housed  in a controlled environment (temperature 20 ± 2°C and humidity 70%) under a 12-hour light-dark cycle. Water and food were supplied ad libitum. All experiments followed the WHO Guidance of Humane Care and Use of Laboratory Animals. The protocols were approved by the Committee on the Ethics of Animal Experiments of the South-Central University for Nationalities, China (permit number: 2013-SCUEC-AEC-006). Every effort was made to minimize the number of animals used and their suffering.μl, 40 nM) was intravitreally injected with a 33 gauge needle connected to a microsyringe after dilation of the rat pupil with tropicamide  in both eyes of each animal. MK801 , a NMDA receptor antagonist; NS-398 , a COX-2 selective inhibitor; DKK-1 , a Wnt/β-catenin signaling inhibitor; and bevacizumab , an anti-VEGF antibody were intravitreally injected with the 80 nmol NMDA in some rats. Besides, TWS119  was intravitreally injected after dilation of the rat pupil with tropicamide. IVB (0.25–25 mg/ml) was intravitreally injected with 1 μΜ TWS119 at the same rate. NS398  was intravitreally injected with 1 μΜ TWS119 in the other rats [n = 4) were injected with phosphate-buffered saline (PBS). The tip of the needle was inserted through the dorsal limbus of the eye [μl. Animals with lens damage or vitreal haemorrhage were excluded from the study. The rats were sacrificed on the seventh day after treatments. The eyes were enucleated, formalin-fixed for 24 h, and paraffin-embedded for sectioning.The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital . NMDA  of the central retina (1-2 mm from the optic disc) was counted in individual sections. A total of six sections from every five serial sections were examined for each retina. The morphological characteristics of adjacent tissue sections were assessed using a Nikon Eclipse Ti fluorescence microscope .The slides made from the eyes of the rats were stained with hematoxylin and eosin (H&E). The number of neurons in the RGC layer within an area . Samples were treated according to a simple immunohistochemical staining method using a Histofine rat MAX-PO (MULTI) kit  . Brieflyβ-catenin expression was performed with an anti-β-catenin polyclonal antibody . The tissue sections were then incubated for 1 h at room temperature with Alexa Fluor 546 goat anti-rabbit IgG , washed three times with PBS, and visualized under a Nikon Eclipse Ti fluorescence microscope [The immunofluorescence analysis of croscope .μm, parallel to the retinal GCL) of the central retina (1-2 mm from the optic disc) in individual sections were observed. A total of six sections from every five serial sections were examined for each retina. The data are presented as the means ± SEM. All statistical analyses were performed using GraphPad Prism 5.0 software package. Comparisons between the mean variables of two groups were made by one-way ANOVA of Bonferroni. †P > 0.05, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control ‡P > 0.05, #P < 0.05, ##P < 0.01, and ###P < 0.001 versus model.In the immunohistochemical analysis, the retinal layers within an area  of rats treated with NMDA. The number of neurons in the retinal GCL of the NMDA-treated rats was decreased significantly (49 ± 4% of control) in comparison to that of normal rats . These data suggest that β-catenin accumulation might participate in the NMDA-induced loss of retinal neurons. Then, the effect of MK801 on the expression of β-catenin in the rats' retinas was investigated. After the treatment with different doses of MK801  in the presence of NMDA, the expression of β-catenin in the GCL was decreased significantly .Then, we investigated whether the Wnt/β-catenin, we detected the expression of COX-2 in retinopathy caused by NMDA. COX-2 expression of the rats' retinas examined by immunohistochemical assays was shown in μM) plus NMDA, the overexpressions of COX-2 were reversed  plus NMDA, the VEGF expression was lower in the GCL  than that of the NMDA-treated rats. These results showed that VEGF overexpression could be attenuated by blocking NMDA receptors.The VEGF expression level was examined by immunohistochemical assays and quantified by a multispectral image analysis. As shown in β-catenin, we treated some other rats with intravitreal injections of NMDA plus DKK-1, which is a widely used and effective inhibitor of Wnt/β-catenin signal pathway. Immunofluorescent images showed that DKK-1 can decrease the expression of β-catenin in retina tissue  to induce abnormal β-catenin accumulation which mimicked the activation of the canonical Wnt/β-catenin signaling pathway [β-catenin induced by TWS119 leads to neuronal loss.To further characterized the role of Wnt pathway, or more specifically a tissue . The neua tissue , indicat pathway . The neuβ-catenin was overexpressed in the retinal GCL of NMDA-treated rats (278 ± 9% of the control) and TWS119-treated rats (372 ± 13% of the control). Meanwhile, DKK-1  plus NMDA attenuated the overexpression of β-catenin . Meanwhile, with high expression of β-catenin (372 ± 13% of control) caused by TWS119, COX-2 was increased (169 ± 14% of control), and VEGF was increased (277 ± 9% of control) as well.The immunofluorescence analyses showed that β-catenin and COX-2, we treated some rats with an intravitreal injection of NMDA plus NS-398, an inhibitor of COX-2. The representative image of the rats treated with 10 μΜ NS-398 plus NMDA was shown in To further assess the relationship between In addition, the neuron numbers in the retinal GCL of the rats treated with NS-398  plus TWS119 elevated  compared with those in the TWS119 group (52 ± 3% of control). This indicated that the inhibition of COX-2 could block the TWS119-induced loss of neurons, which may contribute to retinal protection .β-catenin with NS398  plus NMDA, and we found that the expression of COX-2 in NMDA plus NS-398  was lower compared with the NMDA group  was unchanged in TWS119 plus NS-398 compared with TWS119 group  nor COX-2  in the retinas of rats treated with NMDA (278 ± 9% and 220 ± 18%). These findings demonstrate that the inhibition of VEGF did not modulate the β-catenin and COX-2 overexpression induced by NMDA, although the inhibition of VEGF helped attenuate the loss of neurons.Bevacizumab (IVB), an anti-VEGF antibody that could inhibit VEGF, was used to further characterize the retinopathy caused by NMDA. As shown in Figures β-catenin expression levels in the retinal GCL of TWS119-treated rats . The expression of COX-2 in the retinas of TWS119-treated rats was also unchanged after treatment with IVB  could decrease the NMDA-induced neuron loss. The activation of the Wnt/β-catenin signaling pathway was previously found in injured retinal tissues in vitro [β-catenin signaling pathway could alleviate diabetic retinopathies [β-catenin pathway in NMDA-induced retinal damage in rats, we examined retinal β-catenin levels in the NMDA-damaged retinas of rats. The immunofluorescence analyses demonstrated abnormal accumulation of β-catenin in the retinal GCL of the NMDA-treated rats in comparison to that of normal rats. COX-2 is one of the target genes and a modulating factor of the Wnt/β-catenin pathways, we checked the COX-2 expression and found that COX-2 increased after NMDA treatment. In addition, VEGF was increased after NMDA treatment. Notably, the inhibition of the NMDA receptor by MK801 could decrease the expression levels of β-catenin, COX-2, and VEGF (β-catenin, COX-2, and VEGF.Excessive activation of NMDA receptors  neurons –21. Our  neurons , 23. In in vitro . In addiopathies , 25. To and VEGF . These fβ-catenin, which is the upstream of COX-2. Similarly, DKK-1 also reduced the ischemia-induced retinal neovascularization, the VEGF expression. These results indicate that blockade of the Wnt pathway is sufficient to ameliorate the damage caused by NMDA. Further, we mimicked the activation of the β-catenin pathway using TWS119. By inhibiting GSK-3β activation, TWS119 leads to the accumulation of β-catenin in the cytoplasm, which subsequently translocates to the nucleus [β-catenin signaling pathway could induce neuronal loss. Further, activation of the β-catenin signaling pathway, without NMDA treatment, was sufficient to induce COX-2, VEGF expression, and neuronal loss.Since Wnt pathway is known to be activated under retinopathy conditions , we furt nucleus . Our immβ-catenin pathway in damaged retinas. We used specific inhibitors to examine the interaction between these various molecules.In the present study, the expression levels of both COX-2 and VEGF increased after the administration of either NMDA or TWS119. Therefore, COX-2 and VEGF might be regulated by the activation of the β-catenin overexpression that shows that COX-2 maybe upstream of β-catenin, which was confirmed by Li T that COX-2 can promote hepatocarcinogenesis through activation of β-catenin signaling pathway [β-catenin. This is in accordance with our previous work [β-catenin and COX-2.NS-398, an inhibitor of COX-2, could attenuate the loss of retinal ganglion cells. Interestingly, inhibiting COX-2 by NS398 could decrease the NMDA-induced  pathway . Similarous work  that theβ-catenin and maybe upstream of VEGF. We are continuing our investigations to further elucidate how COX-2 signaling regulates β-catenin and VEGF. We found that NS398 was administered with TWS119 which could protect retinal ganglion cell from losing. However, NS398 could not reverse the TWS119-induced β-catenin overexpression but could attenuate VEGF overexpression caused by TWS119 (These findings have shown that COX-2 is upstream of y TWS119 . This fuβ-catenin overexpression (Figures β-catenin and COX-2 expression suggesting that VEGF is downstream of COX-2, which was proved in gastric cancer [IVB is an anti-VEGF agent used to treat diabetic macular edema. In this study, we found that treatment with IVB could not reverse the NMDA- or TWS119-induced COX-2 and  Figures . But IVBc cancer .β-catenin/COX-2/VEGF signaling pathway is an important pathogenic mechanism underlying the NMDA-induced retinal damage in animal models. Thus, the β-catenin/COX-2/VEGF pathway might represent a new target for pharmaceutical intervention for retinal diseases.In summary, the present study provides the first evidence showing that activation of the


According to embodied cognition accounts, viewing others’ facial emotion can elicit the respective emotion representation in observers which entails simulations of sensory, motor, and contextual experiences. In line with that, published research found viewing others’ facial emotion to elicit automatic matched facial muscle activation, which was further found to facilitate emotion recognition. Perhaps making congruent facial muscle activity explicit produces an even greater recognition advantage. If there is conflicting sensory information, i.e., incongruent facial muscle activity, this might impede recognition. The effects of actively manipulating facial muscle activity on facial emotion recognition from videos were investigated across three experimental conditions: (a) explicit imitation of viewed facial emotional expressions (stimulus-congruent condition), (b) pen-holding with the lips (stimulus-incongruent condition), and (c) passive viewing (control condition). It was hypothesised that (1) experimental condition (a) and (b) result in greater facial muscle activity than (c), (2) experimental condition (a) increases emotion recognition accuracy from others’ faces compared to (c), (3) experimental condition (b) lowers recognition accuracy for expressions with a salient facial feature in the lower, but not the upper face area, compared to (c). Participants  underwent a facial emotion recognition experiment (ADFES-BIV) while electromyography (EMG) was recorded from five facial muscle sites. The experimental conditions’ order was counter-balanced. Pen-holding caused stimulus-incongruent facial muscle activity for expressions with facial feature saliency in the lower face region, which reduced recognition of lower face region emotions. Explicit imitation caused stimulus-congruent facial muscle activity without modulating recognition. Methodological implications are discussed. Embodied cognition accounts postulate that there are interrelations between bodily actions  and cognitions. When we acquire memory, we store all information of the specific situation  together in a representation of the situation also containing embodiments . When weSupport for the idea that stimulus-congruent facial muscle activation facilitates facial emotion recognition comes from a study investigating the effects of actively manipulating facial movements in observers on facial emotion recognition in others. Another explanation for diminished recognition accuracy when participants’ facial movements are actively manipulated  is that active manipulations themselves induce muscle feedback. Considering what is known from the literature on embodied cognition, it should be noted that such facial muscle feedback itself can have an effect on social processes such as facial emotion recognition. When mouth movement is actively manipulated, the activation in the observer’s face does not align with the activation in the observed expression, instead of being stimulus-congruent as during facial mimicry. This conflicting facial muscle activation could be causing interference during the decoding of the expression leading to decreased recognition accuracy. However, if automatic stimulus-congruent facial muscle feedback in observers  facilitates facial emotion recognition, it is plausible that more intense and deliberate muscle activation could facilitate decoding of the observed facial expression of emotion even further . This assumption is supported by the results of a study by A study considering these factors was conducted by Published research has included either an explicit imitation condition or a condition where participants held a pen in their mouth, alongside a condition without any facial movement manipulation. Much of the previous research testing the effects of facial muscle manipulations on emotion recognition ability has used either static images or morphed image sequences, which are limited in ecological validity compared to other types of stimuli. Many previous studies have only used a limited number of basic emotion categories, along only two or three muscle sites in the face to measure muscle activity, which limits the measures about emotion processing and activity in the face. The present study is the first report the authors are aware of to include all three experimental conditions in one experiment to assess how facial emotion recognition is affected by explicit facial muscle activation: (1) an Explicit Imitation condition where participants were told to exactly imitate the expressions they saw while they viewed video stimuli of others displaying various emotional expressions, (2) a Pen-Holding condition where participants held a pen tightly with the lips of their mouth while they watched the videos, and (3) a Passive Viewing control condition where participants just passively viewed the videos. The present study also increased the number of emotion categories included  and measured EMG from five different muscle sites .The aim of the present study was to induce explicit facial muscle activation and to investigate the effects of actively manipulating facial muscle activity to being stimulus-congruent and stimulus-incongruent on subsequent facial emotion recognition accuracy based on more ecologically valid stimuli. There were three hypotheses: (1) Enhanced facial muscle activity throughout the face was expected to result from the Explicit Imitation condition, and in the muscles of the lower face region from the Pen-Holding condition, compared to the Passive Viewing control condition. (2) It was hypothesised that enhanced congruency of facial muscle activity between the stimuli and observers (Explicit Imitation condition) would facilitate recognition of emotion compared to the Passive Viewing control condition. (3) It was further hypothesised that the Pen-Holding condition would induce stimulus-incongruent facial muscle activity in observers’ mouth region, resulting in poorer recognition of facial emotional expressions with salient facial features in the lower face region compared to the Passive Viewing control condition.SD = 3.6) were recruited through Campus advertising at the University of Bath and represented both Humanities and Science Departments (54 from Humanities and 32 from Sciences). Technical equipment failure resulted in the loss of data for two participants, resulting in a final sample of 84 participants . Based on a power analyses using G∗Power (t-tests), a sample size of 84 retrieves 0.78 power with an alpha level of 5% and a small effect size of dz = 0.3. The majority of participants in the final sample were undergraduate students (n = 82), with one participant enrolled in a Master’s Programme and another in a Ph.D. Programme. Two participants reported about a diagnosis of Major Depression and one participant reported about a diagnosis of an Anxiety Disorder. These participants reported to be on medication and not to experience any symptoms of their mental disorders at the time of participation. Thus, these participants were included in the analyses1. All participants had normal or corrected-to-normal vision. Ethical approval for the current study was granted by the Psychology Ethics Committee at the University of Bath.A total of 86 university students  and EMG110C units for each of the five facial muscle sites  were used for recording of the EMG data. Pairs of shielded surface silver–silver chloride (Ag–AgCl) electrodes (EL254S) filled with conductive gel  and with a contact area of 4mm diameter were used. The EMG signal was amplified by 2000 and online bandpass filtering of 10 Hz and 500 Hz was applied. Grounding was achieved through the VIN- of the TSD203 (GSR), the data of which is not reported in this paper. The sampling rate was 1000 Hz throughout the experiment.Participants were tested in a quiet testing laboratory at the University of Bath, and written consent was obtained prior to study participation. Participants were seated approximately 60 cm from the PC monitor. Before EMG electrode attachment, participants’ faces were cleaned with alcohol swabs. The 10 face EMG electrodes were then placed in pairs over the respective muscle sites on the left side of the face, which was done according to the guidelines by There were 90 trials within each of the three different conditions in the facial emotion recognition experiment: (1) Explicit Imitation, (2) Pen-Holding, and (3) Passive Viewing control condition. During the Explicit Imitation condition, participants were instructed to exactly imitate the facial expressions they observed in the videos  as soon as they perceived them. For the Pen-Holding condition, participants were told to hold a pen tightly with their lips, with one end of the pen sticking straight out of their mouth, with pressure applied by the lips (but not the teeth). This manipulation aimed to actively induce facial muscle activity, which also would be incongruent with the emotional expressions included in the study with facial feature saliency in the lower part of the face. The experimenter demonstrated to each participant how the pen was to be held in the mouth, and only after the experimenter was satisfied with the pen-holding technique, the experiment was started. The instruction for the Passive Viewing control condition was to simply watch the videos. Each trial started with a blank screen presented for 500 ms, which was followed by a fixation cross for 500 ms appearing in the centre of the screen. Immediately after the disappearance of the cross the stimulus appeared, followed by a blank screen for 500 ms before the answer screen appeared. The methods used for the facial emotion recognition task were the same as those reported in a previous study using the stimulus set and task . The ansFigure 1 displays the raw data of two selected participants across the whole experiment. Visually comparing the activity in the Passive Viewing control condition from Figures 1A,B clearly shows that the participant from Figure 1B explicitly imitated in the Passive Viewing control condition. The EMG activity of this participant was, for many trials, as intense in the Passive Viewing control condition as in the Explicit Imitation condition, whereas it should have been similar to the corrugator and frontalis channel during the pen-holding. Eighteen participants were subsequently identified to have shown explicit imitation in conditions other than the Explicit Imitation condition. Looking through the raw EMG data, a further two participants were identified who did not show constant elevated EMG activity in the muscles of the lower part of the face in the Pen-Holding condition, consistent with tightly holding a pen in their mouth . Another participant misunderstood the instructions and did the Explicit Imitation condition twice, so no data on the Passive Viewing control condition exists for this participant. Consequently, the EMG data of these participants for the experimental conditions where the instructions were not fully complied with were excluded from EMG data analyses. The same approach was taken for the accuracy of response data. In addition, there were errors for recording EMG from certain muscles for some participants, which meant the EMG data for some participants was not complete. Again, these participants were still included, but the EMG data of the muscles where problems occurred were excluded from the EMG data analyses. The resulting sample sizes per muscle in each analysis are reported in the respective results section. Participants were not fully excluded from analyses in order to retain enough power for the analyses.Several participants (who did not undergo the Explicit Imitation condition as last condition) verbally self-reported after the testing session that they were unable to stop themselves from imitating the observed facial expressions in subsequent conditions. Thus, the raw EMG data of all participants was visually inspected at trial level to identify participants whose EMG activity pattern suggested explicit imitation in other experimental conditions. Imitative activity on a trial basis during the Passive Viewing control condition was clearly visible in the raw data. The Autonomic Nervous System Laboratory 2.6 ANSLAB;  was usedWhen participants complete a task consisting of many trials or repeatedly do conditions of a new task, this produces learning effects and the participant’s performance will improve over time. t-tests to test for a significant increase in accuracy of response over the course of the experiment. The alpha-level of 5% was Bonferroni-corrected to account for multiple comparisons; the resulting p-values were compared to a p-value of 0.025 (p = 0.05/2) for significance determination. The within-task improvements analysis was conducted on a sample of N = 83 . Cohen’s d is presented as effect size measure. If the results show significant increase in accuracy of response from the first to the second and to the third Block, then this has important implications for the analyses. That is, between-subject analyses with only the first experimental condition each participant completed will be necessary instead of the planned within-subject analyses.To test for within-task improvements, the individual consecutive trials of the facial emotion recognition task were split into three equal ‘blocks,’ and accuracy of response was calculated for each block in order of their presentation for each participant . Then, difference scores were calculated between the accuracy of response from the first and second block and between the second and third block. The two resulting difference scores were tested for a significant change using one-sample t-tests showed that there was a significant increase in accuracy of response for participants from the first to the second Block , and from the second to the third Block . Since accuracy scores increased significantly over the course of the experiment, between-subject analyses needed to be conducted to test the hypotheses of the current study. . Due to the right-skewed nature of the EMG data, gamma distribution and log link function were specified in the conducted analyses. Pairwise comparisons were used to follow up significant main effects of Experimental Condition. Due to the necessary data eliminations described in Section “EMG Data Preparation,” the sample sizes for the EMG data per experimental condition varied. The resulting zygomaticus muscle showed a significant main effect of Experimental Condition ; see Figure 2. Pairwise comparisons showed that the EMG activity in the zygomaticus was significantly higher in the Explicit Imitation condition  than in the Passive Viewing control condition , but was not significantly different from the Pen-Holding condition . The EMG activity in the zygomaticus during the Pen-Holding condition was significantly higher than during the Passive Viewing control condition (p < 0.001).Generalised linear model results for the EMG activity in the depressor muscle showed a significant main effect of Experimental Condition ; see Figure 2. Pairwise comparisons showed that the EMG activity in the depressor was significantly lower in the Explicit Imitation condition  than in the Pen-Holding condition  and significantly higher in the Explicit Imitation condition than in the Passive Viewing control condition . The EMG activity in the depressor during the Pen-Holding condition was significantly higher than during the Passive Viewing control condition (p < 0.001).Generalised linear model results for the EMG activity in the levator showed a significant main effect of Experimental Condition ; see Figure 2. Pairwise comparisons showed that the EMG activity in the levator was significantly lower in the Explicit Imitation condition  than in the Pen-Holding condition  and significantly higher in the Explicit Imitation condition than in the Passive Viewing control condition . The EMG activity in the levator during the Pen-Holding condition was significantly higher than during the Passive Viewing control condition (p < 0.001).Generalised linear model results for the EMG activity in the corrugator showed a significant main effect of Experimental Condition ; see Figure 2. Pairwise comparisons showed that the EMG activity in the corrugator was significantly higher in the Explicit Imitation condition  than in the Passive Viewing control condition  and the Pen-Holding condition . The EMG activity in the corrugator during the Pen-Holding condition was not significantly different than during the Passive Viewing control condition (p = 0.466).Generalised linear model results for the EMG activity in the frontalis showed a significant main effect of Experimental Condition ; see Figure 2. Pairwise comparisons showed that the EMG activity in the frontalis was significantly higher in the Explicit Imitation condition  than in the Pen-Holding condition  and the Passive Viewing control condition . The EMG activity in the frontalis during the Pen-Holding condition was not significantly different from the Passive Viewing control condition (p = 0.125).Generalised linear model results for the EMG activity in the Since it was hypothesised that the pen-holding would affect recognition of emotional expressions with facial feature saliency in the lower part of the face but not the upper part of the face, respective variables for the recognition scores were created. The ‘lower face saliency’ variable included accuracy scores for disgust, happiness, embarrassment, contempt, and pride. The ‘upper face saliency’ variable included accuracy scores for anger, fear, sadness, and surprise. This categorisation was based on the location of the facial features that are characteristic for each expression (and the number thereof) in the face stimulus set used; a table listing all facial features per emotion category is printed in n = 28 for the Passive Viewing control condition  and the Explicit Imitation condition . The sample size was 26  for the Pen-Holding condition. Three comparisons were conducted using independent samples t-tests to test the hypotheses of the current study. The accuracy scores of the variable ‘all emotion categories’ from the Explicit Imitation condition were compared to the accuracy scores from the Passive Viewing control condition to test whether enhanced stimulus-congruent facial muscle activation facilitated recognition. To test whether stimulus-incongruent facial muscle activation impeded recognition, the accuracy scores of the variables ‘lower face saliency’ and ‘upper face saliency’ from the Pen-Holding were compared to the Passive Viewing control condition. The alpha-level of 5% was Bonferroni-corrected to account for multiple comparisons. The resulting p-values were compared to a p-value of 0.017 (p = 0.05/3) for significance determination. Cohen’s d is presented as effect size measure.Only the first experimental condition a participant underwent was included in the between-subject analyses, as the first condition naturally could not have been influenced by former instructions. This between-subject approach decreased the sample size to t-test comparing accuracy of response across ‘all emotion categories’ included in the task from the Explicit Imitation condition  to the Passive Viewing control condition  showed no significant difference between the two experimental conditions ; see Figure 3A.The independent samples t-tests showed that the accuracy rates were significantly higher in the Passive Viewing control condition  than in the Pen-Holding condition  = 2.53, p = 0.014) with a medium to large effect size (Cohen’s d = 0.688); see Figure 3B.Comparing the accuracy rates of the ‘lower face saliency’ emotion category using independent samples t-tests showed that the accuracy rates from the Passive Viewing control condition  were not significantly different from the Pen-Holding condition ; see Figure 3C.Comparing the accuracy rates of the ‘upper face saliency’ emotion category using independent samples The current study investigated the effects of active facial muscle manipulations in observers on their ability to recognise emotions from others’ faces. Results showed that facial muscle manipulations effectively changed observers’ facial muscle activity. Holding a pen in the mouth increased the activity of facial muscles in the lower face region compared to a control condition with no facial movement manipulation, while explicit imitation of observed facial emotion produced enhanced facial muscle activity across the face compared to the control condition. In line with the facial muscle manipulation, holding a pen in the mouth was found to produce lower accuracy for recognising facial displays of emotion when the most salient facial feature was in the lower face region compared to passively viewing emotional expressions. In contrast, explicitly imitating the emotional expression seen in others did not result in greater recognition of these emotional expressions compared to passive viewing of the videos. The current findings provide support for embodied cognition accounts, but only when the experimental condition involved stimulus-incongruent facial muscle activity while observing emotional expressions in others, and not when the condition involved stimulus-congruent facial muscle activity. The methodological implications for investigations like the current research with a within-subject study design are discussed.Based on embodied cognition accounts, it was hypothesised that explicit facial muscle activity that is congruent with the observed facial expression would increase recognition rates compared to passive viewing. While explicitly imitating the perceived facial expressions of emotion by others in videos resulted in higher facial muscle activity compared to when they passively viewed the facial expressions, results showed the explicit imitation of others emotions had no facilitating effect on facial emotion recognition. These results are in line with those by As expected, holding a pen in the mouth caused increased EMG activity in the muscles of the lower face region, especially the depressor muscle. Further in line with the predictions, accuracy scores were significantly lower in the Pen-Holding condition compared to the Passive Viewing control condition when recognising emotional expressions with feature saliency in the lower face region. Effects for mouth movement manipulations on recognition of emotions with saliency in the lower face region are in line with previous studies. For example, disgust and happiness recognition are impaired when mouth movements are manipulated with a pen compared to passive viewing without facial movement manipulation . For botAn alternative interpretation is that facial muscle activations, as achieved through pen-holding, induce facial muscle feedback that is incongruent with the muscle activation underlying the observed facial expression. Since embodiments also include the typical facial expressions of emotions, it was proposed that facial muscle feedback in an observer that is in conflict with the perceived visual information might hamper recognition . StimuluThe Explicit Imitation condition and the Pen-Holding condition required additional action from the participants as opposed to the Passive Viewing condition. It could be argued that the results from the current study are based on the additional cognitive load the experimental conditions imposed rather than specific effects of the manipulations. However, The current study manipulated the muscles of the lower face region, but not the muscles of the upper face region. Future research should systematically test the effects of stimulus-incongruent muscle activity across the entire face on facial emotion recognition. Researchers have attempted to fix facial muscles in the upper face region by instructing participants to perform certain facial movements e.g., . It is lResults from the within-subject analyses of the current study showed that the accuracy rates from the Pen-Holding condition were comparable to the Passive Viewing control condition, against the expectation for the emotions with saliency in the lower face region. This finding can, however, be explained by a combination of two occurrences. The first occurrence was the necessary data eliminations, which lead to uneven numbers of participants for the six versions of the experiment. More participants underwent the Passive Viewing control condition first in the experiment sequence than last, while the number of participants per order in the Pen-Holding condition was similar. The second occurrence was the increase in recognition accuracy over the course of the experiment producing higher recognition rates in the last experimental condition a participant underwent. Combining these two occurrences resulted in lower mean accuracy scores for the Passive Viewing control condition, making the mean similar to the mean from the Pen-Holding condition instead of higher. The small albeit non-significant increase in facial emotion recognition when explicitly imitating observed facial expressions compared to the Passive Viewing control condition from the current study can also be explained by the combination of necessary data eliminations and an increase in recognition accuracy over the course of the experiment, as most participants included in the analyses underwent the Explicit Imitation condition last in the experiment. Consequently, theoretical interpretation of the findings from the within-subject analyses of the current study is problematic.The advantage of a within-subject design is usually that the found effects are the result of the experimental manipulations and not due to potential differences between samples as can be the case in between-subject designs, thereby reducing the error variance. However, the instruction to explicitly imitate the observed facial expressions turned out to have a lasting effect on more than a few participants in the current study. Those participants showed a similar pattern of facial muscle activation in the Pen-Holding condition and Passive Viewing control condition as during the Explicit Imitation condition when the Explicit Imitation condition preceded these conditions. This occurrence indicates that explicit imitation was carried out in the other experimental conditions as well (and led to data loss in the current study). This occurrence is very important to consider for researchers who are intending to conduct research similar to the current study. To avoid data eliminations and potential resulting data confounding effects (see next paragraph), it is advisable to apply a between-subject design. Nonetheless, the instruction to explicitly imitate facial emotional expressions having such a long-lasting effect constitutes an interesting finding in itself. The question why some people automatically keep imitating expressions against the task instructions should gain further attention in future research of this type. Example research questions to address could be: Are these individuals more likely to experience emotion contagion? Do those individuals possess higher empathy?Further noteworthy is the continuous increase in accuracy of response over the course of the experiment in the current study, independent of the instructions given to participants for the various experimental conditions. The resulting methodological implication is the importance to balance the order of presentation of the experimental conditions when using a within-subject design (as done with the current study) or to apply a between-subject design. The latter option is recommendable when it is likely that unequal amounts of participants will be excluded per order of experimental condition. Nonetheless, that accuracy rates do increase even without the explicit feedback about the correctness of the response is interesting. It indicates some sort of underlying learning processes and it is possible that focussing attention on decoding of facial emotion also outside the laboratory in everyday social interactions might lead to improvements in facial emotion recognition, which could be particularly relevant for clinical populations with impairments in facial emotion recognition.Taken together, the current study showed that explicit stimulus-incongruent facial muscle activations in observers hamper recognition compared to passively viewing expressions. It was further demonstrated that explicit stimulus-congruent facial muscle activation does not lead to a facial emotion recognition advantage compared to passively viewing expressions. This latter finding is peculiar since awareness was added to the stimulus-congruent facial muscle activations and the facial muscle activation was explicit . Nonetheless, the results from the current study imply that stimulus-congruent facial muscle activations in observers have no facilitating effect on facial emotion recognition and that only stimulus-incongruent facial muscle activations hamper recognition. Given that observing facial emotion might elicit an emotion representation, incongruency between an observed emotion and the facial activity in the observer’s face might disrupt the encoding process due to the embodiment of facial emotional expressions, in line with embodied cognition accounts of emotion.This study was carried out in accordance with the recommendations of the University of Bath Psychology Ethics Committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University of Bath Psychology Ethics Committee.TW conceptualised, designed, and carried out the study. MB, CA, and MoP supervised TW. MiP and TW prepared the data for analyses. MoP provided the means for data preparation. TW analysed the data and interpreted the results. TW wrote the manuscript. All authors edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We found that cementoblasts with constitutively active β-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation. Importantly, cementoblasts transduced with retrovirus expressing constitutively active β-catenin exhibited upregulation of Osx expression through direct binding to the promoter region of Osx. Osx regulates Lef1 expression and consequently could regulate T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding activity in Wnt/β-catenin signaling. However, the loss of Tcf/Lef binding activity by Osx ablation was not rescued by transduction of retrovirus expressing constitutively active β-catenin or ectopic Lef1 overexpression. These results suggest that the Tcf/Lef binding activity of Wnt/β-catenin signaling is Osx-dependent during cementogenesis. Moreover, Osx differentially regulates the expression of various Tcf family members, suggesting that Osx regulates cementogenesis by utilizing various Tcf/Lef-dependent mechanisms. This is the first report to show that downstream Osx signaling through Tcf/Lefs is critical for cementogenesis.Although accumulating evidence indicates that both β-catenin and osterix (Osx) are essential for bone and tooth development, few studies have investigated the interaction of these two key proteins in the context of cementogenesis. In this study, we used transgenic mice with constitutively active  Although cementum has many similarities to bone, particularly in its biochemical composition and biomechanical properties, unlike bone, cementum is resistant to remodeling in its physiological state and is hard to regenerate.Cementum is a thin mineralized tissue covering the tooth root surface. Since cementum helps anchor the tooth to the surrounding alveolar bone and also provides a mechanical barrier to root resorption, this tissue is important for maintaining periodontal homeostasis and for resisting the continuous occlusal force4. Although its essential roles in tooth morphogenesis have been well studied, little is known about the involvement of Wnt/β-catenin signaling in cellular differentiation and dental hard tissue formation. The transcriptional activity of β-catenin is tightly controlled by targeting a protein complex consisting of Adenomatous polyposis coli (Apc), the scaffolding protein Axin, casein kinase 1 (CK-1), and glycogen synthase kinase 3β (Gsk-3β) for its proteasomal degradation6. After stabilization and nuclear accumulation, β-catenin engages T-cell factor/lymphoid enhancer factor (Tcf/Lef) transcription factors to activate the transcriptional program in the nucleus7. The Tcf family includes Lef1, Tcf1, Tcf3, and Tcf4; Tcf/Lefs are intensively studied as nuclear effectors of Wnt/β-catenin signaling9. These effectors cooperate with other factors to regulate Wnt-independent transcription as well as to mediate or suppress Wnt signaling11. Moreover, there is increasing evidence of functional diversity and non-redundant activities among Tcf family members12. Although Tcf/Lefs are context-dependent regulators of Wnt/β-catenin, their functional implications on cementogenesis and Osx-mediated regulation are largely unknown.Wnt/β-catenin signaling plays multiple roles in various stages of tooth morphogenesisOC-Cre:Catnblox/+ (OC-Catnb) transgenic mice13, in which stabilization of β-catenin is constitutively induced. This stabilization is achieved by the elimination of the entire exon 3 sequence, which encodes Gsk-3β phosphorylation targets in osteocalcin (OC)-expressing dental mesenchyme14. The excess cementum containing cementocyte-like cells within its matrix was deposited over the cervical and apical regions of the molar root surface. Histological studies of this excess cementum supported the proposal that stabilized β-catenin induces cementoblast differentiation and boosts matrix secretion; however, the precise molecular mechanisms of these processes remain unknown.We previously reported excessive cementum formation in 15. We recently showed that Osx is required for overall tooth root formation by regulating odontoblast differentiation, maturation, and root elongation in a site-specific manner16. In addition, a genetic study reported that Osx has a vital function in cementogenesis, particularly in cementoblast differentiation. This conclusion was supported by the finding that cementogenesis is impaired following conditional disruption of Osx in the dental mesenchyme and is restored by overexpression of Osx17. Studies of transgenic animals with stabilized β-catenin and transgenic animals overexpressing Osx found similar anabolic responses in cementum formation in both animal lines17, implying a linked molecular signaling cascade mediated by β-catenin and Osx.Osterix (Osx), a zinc finger-containing transcription factor, was initially identified as a key regulator of osteoblast differentiation during bone formationin vivo and in vitro approaches, we demonstrated a reciprocal interaction between β-catenin and Osx in cementogenesis and uncovered the underlying molecular mechanisms. Specifically, we showed that Wnt/β-catenin signaling regulates Osx expression for cementoblast differentiation and cementum matrix secretion; Osx, in turn, regulates Wnt/β-catenin activity by controlling Tcf/Lef expression. This is the first demonstration that the relationship between β-catenin and Osx is critical for cementum formation during postnatal tooth development. These results provide important insight into the mechanisms of cementum formation in tooth development and suggest future strategies for promoting cementum regeneration in periodontal disease.In this study, using OC-Cre:Catnblox/+ (OC-Catnb) mice13. Since Osx has been reported to be a key regulator in cellular cementum formation17, we hypothesized that the stabilized β-catenin might utilize Osx to drive excessive cementum formation in cementogenesis. To test this hypothesis, we generated and analyzed OC-Cre:Catnblox/+:Osxfl/fl (OC-Catnb:Osx) mice, which correspond to OC-Catnb mice with inactivation of Osx. Thus, stabilization of β-catenin is constitutively induced in the same OC-expressing cells. To analyze the role of each gene individually, we compared the double mutants with their wild type (WT) counterparts and with their corresponding single gene mutants, i.e., OC-Catnb and OC-Cre:Osxfl/fl (OC-Osx). The molars of OC-Catnb mice at postnatal week 6 (P6W) had a thicker cellular cementum layer on the overall root surface, including the cervical region with malformed dentin. Histological images of the cementum layer are shown in Fig. OC-Catnb mice exhibited much thicker apical cellular cementum compared to those of WT mice [Fig. OC-Osx mice had thin interradicular dentin and a smaller apical cementum layer, with no remarkable change of acellular cementum at P6W [Fig. OC-Catnb:Osx mice exhibited dramatically restored morphology. Specifically, they had thinner acellular cementum, without the excessive irregular cellular cementum observed in OC-Catnb mice and without any root dentin malformation, at the same age. The mean cervical cementum thickness was slightly thicker in molars from OC-Catnb:Osx mice than in molars from OC-Osx mice  of tooth development in OC-Catnb:Osx mice , and dentin matrix protein 1 (Dmp1) in fully differentiated  OCCM-30 cells  was increased up to 15-fold by transduction of β-Cat S33Y; this effect occurred in a concentration-dependent manner -qPCR analysis using OCCM-30 cells. As shown in Fig. Osx was observed in OCCM-30 cells transduced with retrovirus expressing β-Cat S33Y compared with control cells. However, no association with the regulatory regions of Runx2  were screened. Putative binding sites for Tcf/Lefs (CTTTGGG) in the −2036/+66 mouse Osx promoter were exhibited with consensus sites for Runx2 (TCCCAC/AACCACA), a well-known upstream regulator of Osx, and a preferred Osx binding site (CCACCC) for self-driving expression25 in Fig. Osx for cellular cementum formation.β-Catenin is an established transcriptional activatorner Fig. . The clenx2 Fig.  and Alpllpl Fig.  was deteOsx prevents this abnormal accumulation and precludes the normal formation of apical cellular cementum. To further define the role of Osx in β-catenin-driven cementum formation, we compared various parameters of cementogenesis – differentiation, mineralization, and extracellular matrix secretion – in a similar in vitro genetic situation. As shown in Supplementary Figure Osx expression was silenced using small hairpin RNA (shRNA) specific for Osx (shOsx), real-time qPCR analysis of OC, Bsp, Opn, and Dmp1 transcripts showed that the stabilized β-catenin could not upregulate these transcripts  and in their corresponding control shRNA counterparts (shNC) by real-time qPCR  in cementoblasts. Most interestingly, the transcript level of Lef1 was remarkably decreased by ablation of Osx in cementoblasts. Furthermore, we also observed corresponding changes of these genes with ectopic overexpression of mouse Osx. Interestingly, the transcript levels of Axin2 and Lef1 were slightly increased by overexpression of Osx, whereas the level of β-Cat was not changed compared to the level in control cells  gradually increased as differentiation proceeded. In contrast, control cells (shNC) only exhibited upregulation of active β-catenin with short-term differentiation. However, the protein level of Axin2 remained low and Lef1 and Tcf1 were undetectable in Osx-ablated OCCM-30 cells (shOsx). In contrast, control cells (shNC) exhibited higher overall levels of Axin2, Lef1 and Tcf1 detected during differentiation. Correspondingly, Tcf/Lef binding activity was almost undetectable in Osx-ablated OCCM-30 cells (shOsx) compared to control (shNC) cells, as demonstrated using FOPflash/TOPflash luciferase reporters . Positive BSP staining was observed in the cervical cementum line close to the cemento-enamel junction in both mouse lines. Osx and Lef1 were expressed in the cementoblasts, as well as in odontoblasts and some periodontal ligament (PDL) cells at the growing roots of WT molars. However, the cementoblasts in close proximity to the newly formed cementum lining of OC-Osx molars did not exhibit staining of Osx or Lef1 . These results strongly suggest that the accumulated β-catenin in Osx-ablated OCCM-30 cells was still non-phosphorylated and stable against ubiquitination but inactive for transcription.To address the role of Osx in the relationship between β-catenin and Osx in cementogenesis, we analyzed the expression of genes associated with Wnt/β-catenin signaling in OCCM-30 cells lacking PCR Fig. . Interesers Fig. . In addief1 Fig. . Taken t9. Because of the importance of Lef1 in Tcf/Lef binding activity21, we next examined whether Tcf/Lef binding activity could be recovered by Lef1 introduction into Osx-ablated OCCM-30 cells. LEF1 expression was achieved by transient transfection of mouse Lef1 into OCCM-30 cells expressing control or shOsx. After confirming the expression of each transfected gene on the protein level by Western blotting . However, as shown in Fig. Osx-ablated OCCM-30 cells (shOsx). Compared to the control gene (Gfp), the transcriptional levels of Bsp and Opn were significantly increased by Lef1 overexpression in Osx-ablated OCCM-30 cells (shOsx), whereas those of OC and Dmp1 were decreased by Lef1 overexpression. To examine whether Tcf/Lef binding activity could be recovered by Lef1 overexpression, we next analyzed FOPflash/TOPflash luciferase reporter activity in OCCM-30 cells expressing control or Osx-targeting (shOsx) shRNA. Unexpectedly, Tcf/Lef binding activities in control OCCM-30 cells (shNC) were greatly decreased by transfection of Lef1  cells. Interestingly, however, the transcript level of Tcf3, a repressor of Wnt target genes28, was increased by as much as 9.8-fold by ablation of Osx. In addition, the transcript level of Tcf4 was also increased 2.5-fold by ablation of Osx. The full-length β-catenin-binding form of Tcf4 is sometimes associated with target gene repression29. When Osx was overexpressed via transfection of Osx into OCCM-30 cells, all transcripts of Tcf members were slightly upregulated compared to the control gene (Gfp) when assessed at full-term differentiation  What is the mechanism by which cementoblasts constitutively expressing stabilized β-catenin form excessive cellular cementum, such as that observed in OC-Catnb mice13? (2) At which step of cementogenesis do cementoblasts constitutively expressing stabilized β-catenin require Osx for cellular cementum formation, as observed in OC-Catnb:Osx mutant mice? (3) What is the mechanism by which Osx regulates β-catenin activity during cementogenesis?To develop new tissue regeneration strategies, it is important to identify how developmental cues and regulatory factors are integrated to accommodate the requirements for biological control of cell differentiation and tissue formation. In this study, using both OC-Catnb mice. In addition, stabilized β-catenin induced Osx expression in OCCM-30 cementoblast-like cells in vitro. Our ChIP analysis indicated that Osx expression is likely induced by direct regulation of the Osx promoter. However, indirect pathways are also likely to be involved in Osx induction. In agreement with our hypothesis, β-catenin signaling has been shown to upregulate Osx expression in osseous cells using human pre-osteoblastic and bone marrow stromal cells34. Regarding the mechanism, it was suggested that β-catenin signaling upregulates Osx expression by transactivating the Osx promoter, mainly through increased c-Jun binding at a putative c-Jun binding site. In addition to Osx induction, canonical Wnt signaling has been shown to promote osteogenesis. Specifically, the Runx2 gene is a direct target of the canonical Wnt/β-catenin signaling pathway via Tcf123. Cementoblasts share many properties with osteoblasts; therefore, similar regulatory mechanisms may be involved in control of tissue formation by Runx2 and Osx2. However, based on the effects observed after blocking TGF-β18 and Wnt/β-catenin signaling, the regulation of Osx expression is likely more critical than that of Runx2 during cementogenesis. In support of this idea, the expression level of Osx was sensitively altered by the modulation of TGF-β or Wnt/β-catenin signaling, while the gross level of Runx2 was not altered.Osx plays pivotal roles in cementogenesis and is a central molecule controlling cementogenesis through numerous signaling pathways. Histological analysis revealed that Osx expression was increased in the excessive cellular cementum in molars from OC-Catnb:Osx transgenic mice, we next addressed whether cementoblasts constitutively expressing stabilized β-catenin require Osx for cementogenesis. We found that cementoblasts constitutively expressing stabilized β-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation and precludes the normal formation of apical cellular cementum. We performed protein expression profiling to determine the kinetics of this phenomenon in OCCM-30 cementoblast-like cells in vitro and found that Wnt/β-catenin signaling appears to regulate cell proliferation in the early stages. Later, i.e., after differentiation, Wnt/β-catenin signaling contributes to further matrix secretion during cementogenesis18. These in vitro experimental results clearly show that cementogenesis is regulated in an Osx-dependent manner in the presence of stabilized β-catenin, a finding consistent with the results using our transgenic mice in which Osx expression is controlled by a stage-specific Cre recombinase in OC-expressing cells. Taken together, these results strongly suggest that stabilized β-catenin induces cementoblast differentiation by triggering Osx expression and further regulates cementogenesis following matrix secretion and mineralization in an Osx-dependent manner. As exhibited in vitro data with Osx-silenced cementoblast-like cells (shOsx) in Fig. Osx ablation was only partially recovered by stimulation with constitutively active β-catenin supports the idea that Tcf/Lef binding activity is also largely Osx-dependent in cementoblasts.Using Osx ablation. The results strongly imply that extraordinarily low levels of Tcf1 and Lef1 in Osx-ablated cells are one of the major mechanisms for impaired β-catenin activity even with its higher protein level. The nuclear mediators most closely associated with Wnt/β-catenin action are the Tcf/Lefs, a high-mobility group of DNA-binding proteins with multiple domains for protein interaction and regulation9. Lef1 is one of four transcription factors in the Tcf family21. Since we found that the protein and mRNA expression levels of Lef1 were very low in Osx-ablated OCCM-30 cells and that Tcf/Lef binding activity was also markedly reduced, we examined whether the Tcf/Lef binding activity lost after Osx ablation could be recovered by ectopic overexpression of Lef1 in Osx-ablated OCCM-30 cells. Some matrix transcripts such as Bsp were remarkablely recovered by ectopic overexpression of Lef1 in Osx-ablated OCCM-30 cells. However, unexpectedly, overexpression of Lef1 could not rescue all defects, including the lost TCF/LEF binding activity. These findings reflect that, in cementogenesis, complicated pathways downstream of Osx acting through Lef1–such as crosstalk between Lef1 and other negative factors, or other factors regulated by Osx–affect Tcf/Lef binding activity, in addition to Lef1. To investigate the mechanisms by which Osx controls the effects of β-catenin on Tcf/Lef binding activity in cementogenesis, we extended our study to all Tcf family members in Osx-ablated cementoblasts. One of our most interesting and novel findings is that Osx differentially regulates Tcf/Lef family members in cementum formation. This finding supports the hypothesis that Osx regulates cementogenesis through Wnt/β-catenin signaling by controlling Tcf/Lef expression andutilizes different Tcf/Lefs to act by distinct mechanisms. This explanation would account for the ability of Wnt/β-catenin signaling to elicit distinct responses at different time points and in different tissues. This type of response pattern depends critically upon the major regulator of the target tissue, such as Osx in the case of cementum, to control target gene expression in a highly context-dependent manner36. For example, Osx-ablated cementoblasts have decreased levels of Lef1 and Tcf1 (activators of Wnt/β-catenin), but increased levels of some family member such as Tcf3, which appears to function primarily as a repressor of Wnt/β-catenin. This finding implies that Osx helps fine-tune the signal specificity and strength of Wnt/β-catenin signaling for cementogenesis through different Tcf/Lefs. However, the mechanism by which Osx regulates Tcf/Lefs in cementoblasts and the relevant roles of these proteins in downstream pathways of Osx in cementogenesis still need to be determined.To gain insight into the mechanism by which Osx regulates β-catenin activity during cementogenesis, we analyzed the nuclear translocation of β-catenin and the expression of Tcf/Lef genes with in vivo and in vitro approaches, our study provides compelling evidence to support a mechanism in which Wnt/β-catenin promotes cementum formation through Tcf/Lef-mediated activation of the master cementogenic transcription factor Osx. Osx, in turn, regulates β-catenin activity by controlling Tcf/Lef expression during cementum formation. This is the first demonstration of a reciprocal interaction between β-catenin and Osx and the first report to show that a pathway downstream of Osx that signals through Tcf/Lefs is critical for cementogenesis during tooth development. Our report will potentially stimulate interest in local modulation of β-catenin and Osx and could ultimately be applied to develop therapies aimed at improving periodontal structure, including cementum regeneration.In summary, using Catnblox(ex3)/lox(ex3) (Catnblox/lox), Osx-floxed allele (Osxfl/fl), and OC-Cre mice have been previously described38. OC-Cre:Catnblox/+ (OC-Catnb) and OC-Cre:Osxfl/fl (OC-Osx) mice were generated as described16. To generate OC-Cre:Catnblox/+:Osxfl/fl (OC-Catnb:Osx) mice, OC-Cre;Osxfl/+ (control) mice were crossed with Catnblox/lox:Osxfl/fl mice, and the offspring were genotyped by polymerase chain reaction (PCR) analysis using previously described primers38.All procedures were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. All experimental protocols and animal care methods were approved by the Animal Welfare Committee of Chonbuk National University. For immunostaining, sections were treated with 3% hydrogen peroxide and incubated with rabbit polyclonal antibodies against β-catenin , Osx , and bone sialoprotein . The Histostain Plus Rabbit Primary (DAB) kit  and goat ImmunoCruz staining system  were used following the manufacturers’ instructions. Cervical cementum thickness was measured as the shortest vertical distance at a site 100 μm apical from the cemento–enamel junction in the mid-sagittal section of the mandibular first molar using the analySIS Pro imaging system . The five starting points of the proximal line for measurement were randomly selected within the apical part of each root. Experiments were performed three times with representative slides from each group, and statistical analysis was performed to evaluate the significance of differences between values (p < 0.01). The average apical cementum area was calculated using the analySIS Pro imaging system. For this calculation, three measurements from five representative slides in each group were used; the analyzed tissue was harvested from 6-week-old mice.39. To induce cell differentiation and mineralization, 95% confluent cells were cultured in osteogenic medium (OM), which consisted of medium supplemented with 5% fetal bovine serum, 50 μg/ml ascorbic acid , and 10 mM β-glycerophosphate (Sigma Aldrich), for up to 4 days.OCCM-30, a mouse cementoblast cell line, was kindly provided by Dr. Martha J. Somerman  and cultured as described previouslyOsx promoter plasmid −1269/+91 was kindly provided by Dr. Mark Nanes . The TOPflash β-catenin reporter construct containing the Tcf/Lef binding sites and the FOPflash control reporter containing mutated Tcf/Lef binding sites were gifts from Randall Moon (Addgene plasmids #12456 and #12457). Plasmids driving the expression of mouse β-catenin deltaGSK (β-Cat GSK) and S33Y were gifts from Tannishtha Reya and Shinya Yamanaka, respectively (Addgene plasmids #14717 and #13371). Four serine and threonine residues  of β-catenin in recognition sites for GSK-3β were changed to alanine in β-Cat GSK40. Flag-tagged mouse Osx (accession no. NM_130458) and Myc-tagged Lef1 (accession no. BC057543) constructs in the pCMV6 backbone were purchased from OriGene Technologies . A Gfp construct was also transfected as a control to analyze GFP protein production18. Transfection experiments were performed with LipofectamineTM LTX and PLUS Reagent  according to the manufacturer’s instructions. After 24 h, transfected cells were harvested for whole cell lysate preparation or cultured with OM for further differentiation. Retroviral particles harboring short hairpin RNA (shRNA) against mouse Osx (TG514041) or control shRNA (TR30013) (OriGene Technologies) were generated for stable cell lines with shRNA as described previously18. To establish stable cell lines by viral transductions, subconfluent OCCM-30 cells were transduced for 24 h with the viral particles expressing shRNA directed against mouse Osx or control shRNA in the presence of polybrene (4 μg/ml) (Sigma Aldrich). Forty-eight hours later, transduced OCCM-30 cells were selected using growth medium containing 10 µg/ml puromycin (Santa Cruz Biotechnology). For retroviral transduction of β-Cat GSK and S33Y into OCCM-30 cells, viral particles were generated by transfecting a 293T-based amphotropic retroviral packaging cell line, Phoenix, with the plasmids for 24 h using LipofectamineTM LTX and PLUS reagent (Invitrogen) according to the manufacturer’s instructions. Supernatants containing viral particles were collected between 48 after transfection, filtered through a 0.45-μm filter, and then used after 100-fold concentration using a Retro-X Concentrator  according to the manufacturer’s instructions. Finally, cells were transduced by adding 25 μl of concentrated viral supernatant into one well of a 12-well plate where cell density is at 80–90% for 24 h in the presence of polybrene. This amount of viral particles is estimated to infect target cells at a MOI (multiplicity of infection) of 50.The luciferase 18 (details are described in Supplementary Methods). The ChIP experiments used an anti-β-catenin rabbit polyclonal antibody  and normal rabbit IgG (Santa Cruz Biotechnology). PCR primers are listed in Supplementary Table Osx gene promoter sequence were identified using online PROMO software , as previously described42.We performed ChIP as previously describedData are presented as mean ± standard deviation (SD) of three or more separate experiments, as indicated. Normal data with equal variance were analyzed using Student’s t-test or one-way analysis of variance with Tukey’s procedure. Significance was assigned for p-values as indicated.Supplementary Information


To improve the compatibility between the organic and inorganic phases, a coupling agent was used to modify the diglycidyl ether of bisphenol A (DGEBA) epoxy. The sol-gel technique enables the silica to be successfully incorporated into the network of the hybrids, increasing the thermal stability and improving the mechanical properties of the prepared epoxy/silica nano-hybrids. An autocatalytic mechanism of the epoxy/SiO2 nanocomposites was observed. The low reaction rate of epoxy in the nanocomposites is caused by the steric hindrance in the network of hybrids that arises from the consuming of epoxide group in the network of hybrids by the silica. In the nanocomposites, the nano-scale silica particles had an average size of approximately 35 nm, and the particles were well dispersed in the epoxy matrix, according to the TEM images.The sol-gel technique was used to prepare epoxy/silica nano-hybrids. The thermal characteristics, curing kinetics and structure of epoxy/silica nano-hybrids were studied using differential scanning calorimetry (DSC),  Epoxies have many favorable properties such as high tensile strength and modulus, excellent chemical resistance, and high thermal stability. These characteristics make them ideal matrices for many applications, such as epoxy molding compounds (EMCs) , printedSol gel-derived products have various applications, such as thin films ,9, proteHydrolysis:Condensation:Polymerization is associated with the formation of a one-, two- or three-dimensional network of siloxane (Si–O–Si) bonds, accompanied by the formation of H–O–H and R–O–H species ,17,18,19Epoxy/silica hybrids that comprise nanoparticles that are dispersed throughout an epoxy matrix have been widely studied because they exhibit a desired combination of the flexibility of epoxy and the hardness of silica nanoparticles ,21. The The epoxy resin that is used in this work is the diglycidyl ether of bisphenol A (DGEBA) . The epoxy resin was cured with 2.4-methylhexanhydrophthalic anhydride (MHHPA) . The alkoxides, 4.3-isocyanatopropyltriethoxysilane (IPTES) and tetraethoxysilane (TEOS), were purchased from Shin-Etsu Inc. . The chemical structures of DGEBA, MHHPA, IPTES and TEOS are as follows.2 hybrids. A mixture of epoxy and IPTES, with an epoxy/IPTES weight ratio of 100/5, was stirred vigorously at 80 °C for 2 h to yield the silanized epoxy. HIO (Jg−1). To determine the residual heat of reaction, ΔHR (Jg−1), the cured samples were scanned at 10 °C/min from 40 °C to 200 °C. The sum of both the isothermal heat (ΔHIO) and the residual heat (ΔHR) of reaction was taken to represent the total heat of reaction (ΔHT). The isothermal conversion at time t was defined as αI(t) = ΔHI(t)/ΔHT.A differential scanning calorimeter  was used in the isothermal curing experiments to obtain data for analysis. The isothermal curing reaction was conducted at five temperatures ; it was considered to be complete when the isothermal DSC thermogram leveled off to the baseline. The total area under the exothermal curve, which was based on the extrapolated baseline at the end of the reaction, was used to calculate the isothermal heat of curing, Δ29Si NMR measurements were made at room temperature using a Bruker Avance 400 NMR spectrometer that was operated at 400 MHz. The chemically modified silica was examined using solid-state 29Si cross-polarization and magic angle spinning nuclear magnetic resonance spectroscopy. The morphology of epoxy/silica nanocomposites was analyzed by a transmission electron microscopy  with an accelerating voltage of 100 kV.Solid-state 29Si NMR. 29Si NMR spectra of the epoxy/silica nanocomposite, which exhibited major chemical shifts at −91 ppm, −102 ppm, and −110 ppm, which were attributed to silsesquioxane absorption by dihydroxy-substituted silica (Q2), monohydroxy-substituted silica (Q3) and nonhydroxy-substituted silica (Q4), respectively. These chemical shifts involved the Si–O–Si bonding of the silsesquioxane [3 and Q4 environment could be considered to include a highly condensed silica phase, the formation of the SiO2 network structure was complete in the prepared samples. The incomplete reaction of siloxane was not observed in the epoxy/silica nano-hybrids. Additionally, the amount of Q4 was observed to increase with TEOS content, indicating that a large amount of introduced TEOS may have promoted the formation of the silica network.The chemical structure of silica in the epoxy/silica nanocomposites was examined using quioxane ,25. Schek1 and k2 are the apparent rate constants; r is the rate of the reaction, and m and n are the kinetic exponents of the reactions. The constant k1 in Equation (1) can be calculated if the initial reaction rate at α = 0 can be estimated. Kinetic constants k1 and k2 are assumed to be of the Arrhenius form: aE is the activation energy; R is the gas constant, and T is the absolute temperature.A general equation for the autocatalytic curing reactions of many epoxy systems is as follows .(1)r=dαThe epoxy resin contained 10 phr (based on 100 parts of the epoxy) of silica and was cured at five isothermal temperatures of 100 °C, 110 °C, 120 °C, 130 °C, and 140 °C. Kinetic analysis was performed using the above kinetic models. E1 and ΔE2 for anhydride-cured DGEBA epoxy that were obtained in this study were 80 kJmol−1 and 60 kJmol−1, respectively. The autocatalytic kinetic model and the obtained rate constants were used to calculate the empirical curves of conversions as a function of time for the epoxy/silica hybrids at five isothermal curing temperatures. vs. conversion for the epoxy-silica hybrids with 10 phr silica, which were cured at five isothermal temperatures. The conversion rate above which the curing rate varied coincided with the formation of the gel point, at which the curing reaction of the gelled epoxy network began to slow down.2 network structure in the prepared samples was complete. The difference in polarity between the inorganic and organic materials may have formed the nanophase-separated structure, which is shown in the TEM microphotographs. The sizes of the nano-scale silica particles in the nanocomposites varied in the range of 15–60 nm, averaging 35 nm, and the particles were well dispersed in the epoxy matrix, according to the TEM images. An autocatalytic mechanism of the curing of 10 phr epoxy/silica hybrids at various isothermal temperatures was observed. Kinetic parameters for the epoxy/silica hybrids were obtained and the proposed kinetic model was found to describe accurately the curing behavior of the epoxy/silica hybrids up to the vitrification point. A kinetic modeling approach is used to demonstrate how thermosetting epoxy resins that have been modified with silica behave during curing. The curing kinetics were corrected for the morphological development of the heterogeneous epoxy/silica hybrids during the curing progress.A new class of epoxy/silica nano-hybrids was prepared using a sol-gel technique. To improve the compatibility between the organic and inorganic phases, a coupling agent was used to modify the diglycidyl ether of bisphenol A (DGEBA) epoxy. Siloxane bridges in the silica, which were highly crosslinked in the epoxy/silica hybrid materials, were observed. The formation of the SiO