These findings may have broad applications in understanding and treatment of CNS disorders involving BBB disruption.Disruption of the blood-brain barrier (BBB) causes or contributes to neuronal dysfunction and several central nervous system (CNS) disorders. Wnt/β-catenin signaling is essential for maintaining the integrity of the adult BBB in physiological and pathological conditions, including stroke. However, how the impairment of the endothelial Wnt/β-catenin signaling results in BBB breakdown remains unclear. Furthermore, the individual contributions of different BBB permeability-inducing mechanisms, including intercellular junction damage, endothelial transcytosis, and fenestration, remains unexplored. Here, we induced β-catenin endothelial-specific conditional knockout (ECKO) in adult mice and determined its impact on BBB permeability and the underlying mechanism. β-catenin ECKO reduced the levels of active β-catenin and the mRNA levels of Wnt target genes in mice, indicating downregulation of endothelial Wnt/β-catenin signaling. β-catenin ECKO mice displayed severe and widespread leakage of plasma IgG and albumin into the cerebral cortex, which was absent in wild-type controls. Mechanistically, both the paracellular and transcellular transport routes were disrupted in β-catenin ECKO mice. First, β-catenin ECKO reduced the tight junction protein levels and disrupted the intercellular junction ultrastructure in the brain endothelium. Second, β-catenin ECKO substantially increased the number of endothelial vesicles and caveolae-mediated transcytosis through downregulating Mfsd2a and upregulating caveolin-1 expression. Interestingly, fenestration and upregulated expression of the fenestration marker Plvap were not observed in β-catenin ECKO mice. Overall, our study reveals that endothelial Wnt/β-catenin signaling maintains adult BBB integrity  The central nervous system (CNS) is separated from the blood circulation by a special interface called the blood-brain barrier (BBB). The BBB maintains the CNS microenvironment homeostasis by restraining the entry of neurotoxic substances and pathogens. The BBB also allows the selective influx of nutrients and hormones, and the efflux of metabolic wastes, to facilitate proper neuronal functions . BBB disBlood-brain barrier integrity is collectively determined by a multicellular microstructure consisting of endothelial cells (ECs), pericytes, astrocytes, neurons, and the extracellular matrix connecting them; all these components collectively form the neurovascular unit (NVU) . MicroglThe endothelial Wnt/β-catenin signaling pathway plays an essential role in BBB formation and maintenance in health, as well as in diseases such as ischemic stroke, glioblastoma, medulloblastoma, and multiple sclerosis . In miceHere, we first generated an adult mouse model with endothelial-specific deletion of β-catenin, and then comprehensively examined the cellular, molecular, and ultrastructural alterations of the BBB upon impairment of endothelial Wnt/β-catenin signaling. Our results demonstrated that suppressed endothelial β-catenin signaling increased paracellular and transcellular permeabilities by damaging the intercellular junctions, and by substantially increasing the caveolae-mediated transcytosis. However, fenestration was not observed in brain ECs. Our findings provide mechanistic insights into the regulation of BBB integrity by Wnt/β-catenin signaling and have broad applications in understanding and treatment of CNS disorders involving BBB disruption.The antibodies, ligands, and chemicals used in this study are listed in 2 and set at 37°C. After no more than three passages, cells at 80–90% confluency were seeded in a 12-well plate. The cells were serum-starved for 12 h and treated with different concentrations of Wnt3a and LiCl for 24 h.Mouse brain microvascular endothelial cells (bEnd.3) were purchased from the ATCC cell bank  and grown in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% serum and antibiotics, in a humidified incubator supplied with 5% COEthanol and sunflower seed oil were used to dissolve tamoxifen  by sonication (2 mg/ml) for immediate use or stored at –20°C. Tamoxifen was administered orally to all mice .The animals were bred at the specific pathogen free animal care houses. The research protocols and the use of animals were approved by the ethical committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.Ctnnb1 (β-catenin) flox mice and Cdh5-CreER mice were generated as previously described  β-catenin knockout mice loxp/loxp; Cdh5-CreER+Ctnnb1 (β-cat ECKO) and loxp/loxp; Cdh5-CreER–Ctnnb1 control mice (β-cat WT). Male 8–10-week-old mice were used and sacrificed at 2–3 days after the last dose of tamoxifen was administered.The escribed . Ctnnb1l® reagent  following manufacturer’s protocol. Complementary DNA (cDNA) was generated by performing real-time quantitative PCR (RT-qPCR) using 200 ng RNA and iScript Reverse Transcription Supermix . To perform RT-qPCR cDNA (0.5 μg), specific primers and SYBR Green qPCR Master Mix were used. A StepOnePlus Real-Time PCR System  was used for RT-qPCR. Relative mRNA levels were quantified using Ct values and 2–ΔΔCt method after normalizing their expression to that of β-actin. The sequence-specific PCR primers used in this study are listed in Total RNA extraction from whole mouse brains as well as bEnd.3 cells treated with Wnt3a and/or LiCl by using TRIzolRadio-immunoprecipitation assay buffer  containing protease and phosphatase inhibitor was used to lyse the whole cell lysate and mouse brain tissue. The protein concentrations were quantified using the standard BCA  . Equal protein amount from each sample was separated by performing SDS-PAGE and using 8–10% resolving gels. The resolved proteins were then transferred onto polyvinylidene difluoride membranes (PVDF). Non-fat milk solution (5%) was used to block the membranes for 1 h at room temperature (RT). The membrane was then incubated with primary antibodies  β-catenin, Claudin-5, Occludin, ZO-1 (Zona occludens 1), Mfsd2a, Plvap, Caveolin-1, IgG, and Albumin using immunofluorescence staining (IF). 10% ketamine and xylazine were used as anesthetic agens for the mice, and ice-cold phosphate-buffered saline (PBS) was used for cardiac perfusion. Brain tissues were collected and fixed in 4% paraformaldehyde (PFA) for 1 h at RT. Brain tissues were first dehydrated in 15% sucrose at 4°C overnight, followed by 3–6 h incubation in 30% sucrose at 4°C. Brain tissues were then embedded in optimal cutting temperature (OCT) compound for 10–15 min at RT, and stored at –20 or –80°C. To stain PFA fixation-sensitive antibodies , fresh brain tissues were embedded in OCT and placed in a –80°C freezer utill being used.Frozen brain sections of 10 μm thickness were incubated with blocking buffer [10% normal goat serum  + 0.2% Triton X-100] for 1 h at RT. The sections were then incubated with primary antibodies  immediately after sacrifice without cardiac perfusion and fixed in 2.5% glutaraldehyde/0.1 M PBS (pH 7.2) at 4°C overnight. Graded ethanol was used for tissue dehydration, and epoxy resin was used to embed the dehydrated brain tissues. Uranyl acetate and lead citrate were then used to stain the ultrathin sections (80 nm) collected on copper grids. JEM-1400Plus TEM  was used to analyze the ultrastructure of brain microvessels. To quantify vesicles and calculate the percentage of damaged adherens junctions and tight junctions per mouse, at least five microvessels were examined in the cerebral cortex of each mouse.t-tests. Statistical significance was set at P values < 0.05.All results were obtained from at least three independent experiments. GraphPad Prism 6.01 was used to analyze the data. Data are presented as the mean ± SD. Variations between the two groups were analyzed by using two-tailed unpaired loxp/loxp; Cdh5-CreER–Ctnnb1 mice were bred with loxp/loxp; Cdh5-CreER+Ctnnb1 mice to generate loxp/loxp; Cdh5-CreER–Ctnnb1 mice (termed as β-cat WT or WT) and loxp/loxp; Cdh5-CreER+Ctnnb1 mice (termed as β-cat ECKO or ECKO) mice  mice . Oral adKO) mice .Axin2, Lef1, Nkd1, Apcdd1, Spock2, and Notum. All these Wnt target genes were downregulated in the brain tissues of β-cat ECKO mice compared with WT mice  and Caveolin-1 . In line with previous reports, Mfsd2a expression was precisely observed in the endothelium of brain microvessels and the protein colocalized with CD31 in WT mice . HoweverNext, we used TEM to further assess the impact of Wnt/β-catenin signaling on endothelial transcytosis and fenestration. Our TEM analysis showed a substantial increase in transcytotic vesicles in the brain capillary ECs of β-catenin ECKO mice as compared to that of the WT controls . The vesin vitro study, we determined whether modulation of Wnt/β-catenin signaling by exogenous treatments in mouse brain endothelial cells bEnd.3 regulated the expression of transcytosis-related genes. This was assessed by treatment with the natural ligand of canonical Wnt/β-catenin signaling activator, Wnt3a, which binds to frizzled and LRP5/6 receptors  are overexpressed in brain ECs and regulate the paracellular permeability across the BBB . Genetic-catenin . In our via suppressing Mfsd2a. –/–Mfsd2a mice show leaky BBB and a dramatic increase in endothelial vesicular transcytosis (Caveolae-mediated transcytosis plays a major role in the regulation of BBB permeability under physiological and pathological conditions . Mfsd2a scytosis ; howeverscytosis . The impin vivo study, Mfsd2a genetic knockout increased in caveolae-mediated transcytosis (Transcytosis comprises both receptor/clathrin-mediated transcytosis and caveolae-mediated transcytosis . Clathriscytosis . ConsistThe fenestration marker gene, Plvap, is not expressed in brain ECs under normal conditions. along with absence of the fenestrations in the brain ECs with BBB function. Previous studies have reported that Plvap expression is regulated by Wnt/β-catenin signaling in both brain and retinal ECs , and forIn summary, our study provides the first evidence that β-catenin-dependent Wnt signaling maintains the integrity of the adult BBB by regulating both the intercellular junction-mediated paracellular transport route and the endothelial transcytosis-mediated transcellular transport route . We alsoThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.JC conceived and supervised the study, analyzed the results, and revised the manuscript. BH and CF performed most of the experiments and drafted the manuscript. XH, ZF, and YY participated in tissue collection, processing, and analysis. YW commented on the manuscript. All authors approved the final version of 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, the workability deteriorated drastically for BN/epoxy composites above a specific nanofiller concentration (2.0–3.0 wt%). BN prevents crack growth by drawing and bridging. SiO2 enhances performance by deflecting the crack direction and forming voids. Additionally, the dimension and content of nanofiller also influence glass transition temperature and storage modulus significantly.The mechanical properties of epoxy resin can be enhanced by adding nanofillers into its matrix. This study researches and compares the impacts of adding nanofillers with different dimensions, including two-dimensional boron nitride and zero-dimensional silica, on the mechanical and toughness properties of epoxy resin. At low fractions (0–2.0 wt%), 2DBN/epoxy composites have a higher Young’s modulus, fracture toughness and critical strain energy release rate compared to SiO In various areas, including the aerospace and aircraft industries, epoxy adhesives have been widely used ,2,3,4, e2) has been widely used for composites with enhanced performance [2. Roopesh Kumar Sinha et al. [2 also displays compatibility with the matrix’s polymer chains and high interfacial chemical reactivity with the fiber’s sizing [Silica nanoparticle  boron nitride (BN) and zero-dimensional (0D) silicon dioxide (SiO2) on the mechanical properties and toughness of epoxy resin. Studying of the different strengthening effects and mechanisms of SiO2 and BN on epoxy resin can expand the idea of epoxy resin modification and make it more convenient for these composites to be applied in practical production. Moreover, it is important to determine unambiguously how silica and BN nanofillers influence the fracture behaviors of epoxy. This article clarifies and understands the complicated enhancement between the different dimension fillers on toughening epoxy. In this article, specimens of pure epoxy and nanocomposites are reinforced with 2D and 0D inorganic nanomaterials. This paper studied and compared the impact of adding SiO2 and BN to the epoxy-based adhesive on its lap shear strength, toughness and dynamic mechanical behaviors in the range of loading 0–3 wt%. The mechanism of action of different dimensional materials in epoxy resin was revealed.At present, some scholars have studied the improvement of the properties of epoxy resin by nanofillers. These studies show that different kinds and different forms of nanofillers have different effects on the properties of epoxy resin. However, it is not common to compare SiO2 is shown in Nano silica (Nano-poxF400) as a colloidal solution (40 wt%) has been dispersed in epoxy resin by Hanse Chemie AG, Geesthacht, Germany. The diameter is 20–23 nanometers, and the shape is spherical. Nano hexagonal crystal BN  was purchased as a solid from Shanghai Chaowei Nanotechnology Co., Ltd., Shanghai, China. Epoxy resin, diglycidyl ether of bisphenol A  with an epoxide equivalent weight of 182–196 g/equiv, and the viscosity is 11,000~14,000 mPa.s, was supplied by Ciba-Geigy, Australia. Hardener Jeffamine D230 (denoted J230) was kindly provided by Huntsman, China. The basic physical properties of BN and SiO2) composite adhesive (ii) epoxy/BN nanosheets composite adhesive. The fabrication of the composite adhesive was carried out as follows. Each epoxy resin was mechanically mixed with a pre-weighed amount of BN nanoparticle to produce a composite adhesive with a fraction range of 1.0–3.0 wt%. Ultrasonic oscillation was a simple and effective method to uniformly disperse nanomaterials in epoxy resin. The epoxy resin mixed with the nanofiller is ultrasonically shaken to make the nanofiller uniformly dispersed. Magnetic stirring at 80 °C effectively removed acetone and obtained a resin matrix uniformly dispersed with nanofillers. A calculated quantity of J230 (weight ratio of DGEBA/J230 = 3.3:1) was added to the mixture. Each mixture was carefully degassed in a vacuum oven for 10 min to remove bubbles and the mixture was poured into molds. A two-stage curing process was then carried out: 80 °C for 2 h and then 120 °C for 10 h. Both sides of the samples were polished with sandpaper until all visible marks had disappeared. The samples were then thermally treated at 120 °C for 120 min to reduce the defects caused by polishing. The preparation process of epoxy/BN composites is shown in 2 composite was similar to that of the epoxy/BN composite. In this study, two epoxy composites were prepared: (i) epoxy/silica nanoparticle (SiOThe lap shear strength (LSS) test measures the joint strength by dividing the failure load by adhesive bond area. According to ASTM D5573–99, the structure of the single lap bonded specimen is shown in ΙC) of epoxy nanomaterials. The dimensions of the CT samples were 30 mm × 30 mm × (5–6) mm. Each component was tested at least three times and the average was recorded. Scanning electron microscopy (SEM) was carried out to observe the fracture surfaces (crack tip and propagation zone) of compact tension (CT) specimens using SEM, ZEISS Sigma 300 . A thin layer of platinum was used to coat the fractured surface and then examined at an accelerating voltage of 10 kV.Dynamic mechanical analysis (DMA) of nanocomposites was performed using the DMA 2980, TA Instruments (USA). Single cantilever clamped rectangle samples had tests conducted at a frequency of 1 Hz. The temperature range was 20−150 °C, and the heating rate was 5 °C/min. The dimensions of the samples were 35 mm × 10 mm × 3.5 mm. Dumbbell samples were used for static tensile tests at a speed of 0.5 mm/min. ISO13586 standard indicated that compact tension (CT) can test fracture toughness (K2 and BN nanoparticles are plotted in ΙC), critical strain energy release rate (GΙC), Young’s modulus and tensile strength. KΙC, which measures the absorbed energy to propagate sharp crack, and GΙC are primary ways to evaluate the toughness of an epoxy nanocomposite. With the changes to the two nanofiller components, ΙC) and critical strain energy release rate (GΙC) of epoxy composites. Both KΙC and GΙC of epoxy nanocomposites are obviously enhanced by adding BN and SiO2 up to 2.0 wt% at different increments. For instance, at 2.0 wt% filler, the KΙC and GΙC of epoxy/SiO2 composites are improved by 42% and 83%, respectively, while there was a 101% and 210% increase in the case of BN. The increase in attributes of nanocomposites will slowly decrease once the BN is increased to 2.0 wt%; at 3.0 wt%, KΙC and GΙC the benefits begin to decrease but are still higher than that of neat epoxy. In contrast, the performance growth of epoxy/SiO2 is stable and sustainable. At 3.0 wt% nano-0DSiO2, KIC and GΙC of nanocomposites are enhanced by 90% and 195%.Mechanical properties of epoxy resin after incorporating SiO2 composites. With the addition of BN and SiO2, Young’s modulus will continue and slowly increase, respectively. Nevertheless, the 2D materials BN had a better effect on the improvement of Young’s moduli of the epoxy compound. The reason is that BN can absorb more energy when the epoxy compound fractures as shown in 2 exhibited a special self-lubrication phenomenon, as a result of which the workability did not deteriorate [2 nanocomposites steadily increase. This conforms with the previous studies [eriorate ,14. With studies ,29,30.2 for resin composites when the content of the filler is beyond 2.0 wt%. The enhancement value of SiO2 exceeds BN in all behaviors tested including KΙC, GΙC and Young’s modulus. Among them, the tensile strength decreases slowly due to the addition of BN. The details of the properties of epoxy composites are given in ΙC, GΙC, Young’s modulus and tensile strength of epoxy/SiO2 are 1.135 MPa.m0.5, 517.11 J/m2, 2.27 GPa and 69.18 MPa compared to 1.020 MPa.m0.5, 380.53 J/m2, 2.49 GPa and 31.94 MPa in epoxy/BN composites, respectively.(i)2/epoxy due to the structure of SiO2 nanomaterials.In some sections of epoxy/BN nanocomposites, interlayer van der Waals forces lead to the accumulation and irregular dispersion of BN nanosheets, causing a reduction in the monotonous load transfer from resin to nanomaterials. This process occurs less frequently in the SiO(ii)Compared with zero-dimensional silica, BN with two-dimensional structure can form better mechanical interlocking with epoxy resin and improve the mechanical proper-ties of the composite.The reasons for the results are as follows. At low content (0–2 wt%), there is enough space for nanomaterials to be dispersed. They strengthen the resin matrix and improve stress transfer and load distribution between the epoxy matrix and nanofillers. While at a high fraction, because of the 2D structure of BN, the phenomenon of aggregation and clustering will appear earlier. These aggregated nanofillers formed defects, caused stress concentration and accelerated crack propagation. The addition of 2D and 0D nanomaterials will have different effects on the properties of epoxy composites, the reason is as follows:2 composite adhesives to 2D dimensional epoxy/BN composite adhesives. It is clear that the two nanomaterials enhance the LSS of the epoxy resin at all fractions (<3.0 wt%), which is attributed to the intrinsic outstanding strength of nanomaterials; nanomaterials generally reinforce polymeric materials. For example, at 3.0 wt%, SiO2 increased the epoxy’s LSS by 29% and BN increased the epoxy’s LSS by 30%. The addition of nanofillers can improve the interface performance of resin and aluminum, and thus nanofillers can enhance single lap shear strength [strength ,32.2 nanocomposite. The reason is that compared with spherical SiO2, sheet BN with two-dimensional structure can form better mechanical interlocking with epoxy resin, resulting in higher lap shear strength of the sample. Specifically, epoxy/BN nanocomposite showed 10%, 6%, and 5% increases in LSS at fraction levels of 1, 2, and 3 wt%, respectively, when compared to the epoxy/SiO2 nanocomposite. With the addition of BN and SiO2, the LSS steadily increases, but the growth curve of the epoxy/BN composites gradually flattens as the amount of nanofiller increases. The reason for this is that the 2D nanofillers became entangled, and the workability deteriorates as the content of nanofiller is increased.Moreover, these results indicate that the epoxy/BN nanocomposite had a higher LSS than the epoxy/SiO2 and BN to epoxy adhesives enhances the adhesive fracture toughness. For example, the adhesive toughness of epoxy/3wt%2DBN reached 121 J/m2 while it was 115 J/m2 for an epoxy/SiO2 composite adhesive. It confirms the results obtained for bulk composites in Specimens under loading I, double cantilever beam (DCB) can have their delamination toughness calculated . The adh2 with the increase of nanofiller content. The main reason is the high concentration of BN in the epoxy resin which probably led to its agglomeration.g) and damping factor based on DMA. These properties are tested by studying the behavior of composites under different frequencies, stresses and temperatures [Generally, we determined loss modulus, storage modulus, glass transition temperature  to 92 °C when BN is added, and to 102 °C in the case of using SiO2. When more nanofiller (>1 wt%) is added to epoxy, Tg steadily declines.The damping ratio (tan δ) is driven by the ratio of loss and storage modulus. It gives the equilibrium between the elastic and viscous phase. The peak value of tan δ determines the value of Tg . The tanThe reason for the improved storage modulus is that the evenly dispersed nanofillers are entangled with epoxy resin, which improves the resistance of the composite material to mechanical deformation. Simultaneously, the filler increases the crosslink density, restricts the fluidity of the epoxy resin chain, leading to increased stiffness. As the temperature increases (exceeding the glass transition temperature), the composite transforms into a viscoelastic, the fluidity increases, and the storage modulus decreases.The mechanical properties shown at macroscopic level can be examined at microscopic level. Here, we researched the fracture toughening mechanism of nanocomposites by studying the rough fracture surface of CT specimens using SEM analysis. The SEM micrographs show the dispersion and toughening of nanofillers.2 and epoxy/BN nanocomposites with different magnifications at 1.0 wt%. The image shows that SiO2 has good dispersibility, and the red mark in 2 has a positive effect on the fracture of epoxy resin. 2 delays entanglement due to its 0D shape. The main reason is that the nanomaterials with this structure exhibited the self-lubrication phenomenon [enomenon .2. SiO2 is a rigid nanomaterial. When the crack encounters SiO2 during the propagation process, it will be forced to deflect. In addition, when the resin matrix undergoes plastic deformation, the rigid SiO2 does not participate in the deformation. Therefore, SiO2 can form hollows in the resin matrix to enhance the toughness of the composite material.The continuous rough surface indicates the energy being absorbed by the crack propagating to a fracture . However2 delays entanglement and agglomeration by its self-lubricating ability and its properties are vitally enhanced.In short, with a low content, BN can absorb more energy by its 2D shape to further enhance the mechanical capabilities of the nanocomposite. At high content, SiO2 can improve the mechanical behaviors of the nanocomposites. In this paper, the mechanical properties and dynamic thermomechanical analysis of 2D and 0D inorganic-nanofiller/epoxy nanocomposites were researched. These results revealed that BN and SiO2 have different enhancement mechanisms due to different nanostructures. They improved the capabilities of epoxy nanocomposites at different increments. For example, at 2.0 wt%, the KΙC, GΙC, Young’s modulus and tensile strength of epoxy/SiO2 was 0.85 MPa.m0.5, 320 J/m2, 2.05 GPa and 63 MPa compared to 1.20 MPa.m0.5, 544 J/m2, 2.41 GPa and 41 MPa in epoxy/BN composites, respectively. At 3 wt%, KΙC and GIC of epoxy/BN started to decrease but were still higher than that of neat epoxy. However, BN and SiO2 reinforced composite, the maximum glass transition temperatures are 92.51 °C and 102.06 °C, respectively.Additionally, as the content of the two nanofillers changes, the glass transition temperature and storage modulus of the composite change. Among them, the changing trend of the storage modulus is similar to that of Young’s modulus, that is, it increases steadily with the increase of filler content. The reason is that the addition of nanofillers enhances the stiffness of the composite. The glass transition temperature reaches the maximum when the filler mass ratio is 1 wt%. Corresponding to BN and SiO2 strengthen the resin matrix through their respective structures and methods. At a high fraction, agglomeration of nanofillers began to occur. Due to the self-lubricating phenomenon of SiO2, the agglomeration phenomenon will be delayed. Epoxy composites with nanofillers withstand higher loads. It better transmits loads as a matrix for fiber composites or fiber metal laminates.SEM images of the rough fracture surface revealed the role of nanomaterials in the process of crack growth and specimen fracture. At a low fraction, BN and SiO2 is less likely to agglomerate at low fractions. This is be-cause of the larger van der Waals forces between the layers of the 2D sheet BN, which makes it easier to stick together.Compared with BN, SiO


However, these often apply viral vectors, feeder cells, or antibiotics to generate hiPSC and MNs, limiting their translational potential. In this study, a virus-, feeder-, and antibiotic-free method was used for reprogramming hiPSC, which were maintained in culture medium produced under clinical good manufacturing practice. Differentiation into MNs was performed with standardized, chemically defined, and antibiotic-free culture media. The identity of hiPSC, neuronal progenitors, and mature MNs was continuously verified by the detection of specific markers at the genetic and protein level via qRT-PCR, flow cytometry, Western Blot, and immunofluorescence. MNX1- and ChAT-positive motoneuronal progenitor cells were formed after neural induction via dual-SMAD inhibition and expansion. For maturation, an approach aiming to directly mature these progenitors was compared to an approach that included an additional differentiation step for further specification. Although both approaches generated mature MNs expressing characteristic postmitotic markers, the direct maturation approach appeared to be more efficient. These results provide new insights into the suitability of two standardized differentiation approaches for generating mature MNs, which might pave the way for future clinical applications.Motor neurons (MNs) derived from human-induced pluripotent stem cells (hiPSC) hold great potential for the treatment of various motor neurodegenerative diseases as transplantations with a low-risk of rejection are made possible. There are many hiPSC differentiation protocols that pursue to imitate the multistep process of motor neurogenesis  Human-induced pluripotent stem cells (hiPSC) are of great importance for regenerative medicine, as a wide variety of cell types can be generated, avoiding the controversially discussed use of embryonic stem cells (ESC). Transplantations with a low-risk of immunological rejection are made possible with autologous hiPSC-derived cells. Furthermore, reprogramming of cells from diseased individuals to hiPSC allows the analysis of pathomechanisms, e.g., of Alzheimer's disease, to be investigated and sets the course for specific drug discovery. With patient-derived hiPSC, it may soon be possible to predict the patient's individual response to drug-specific therapy .Currently, there is no appropriate therapy available for amyotrophic lateral sclerosis (ALS), a neuromuscular disease in which degeneration of motor neurons (MNs) occurs. Replacement of those degraded MNs would be desirable for the treatment of ALS but also other diseases involving degenerated, dysfunctional, or missing MNs. hiPSC are a promising tool for motor neuron (MN) generation, which exhibit the same MN differential potential in comparison to ESC. For clinical application, the generation of human mature and functional MNs needs to underly ensures quality and reproducibility with minimum use of xenogeneic material . Thus, tin vitro. Bianchi et al. , 18p < 0A scheme of 28 days NPC differentiation by direct maturation, approach A, and with a preceding differentiation step, approach B, is shown in p < 0.05).To further characterize the neurons derived from 28 days differentiation via approaches A and B, the expression of various genes characteristic of motor neurogenesis , neuronaIn approach A, some genes were selectively downregulated, including Sex Determining Region Y Box 2 , neurofilament protein Nestin , transcription factor Neurogenin 1 , and Hes Family BHLH Transcription Factor 1 , which are associated with a neural progenitor fate , 24. GenIn comparison, some genes involved in neurotransmission including Brain Derived Neurotrophic Factor , Cholinergic Receptor, Muscarinic 2 , Gamma-Aminobutyric Acid Type A Receptor Subunit Alpha 5 , Potassium Voltage-Gated Channel Subfamily H Member 2 , and doublecortin , a gene involved in neuronal migration, were selectively downregulated in approach B. Other genes associated with neurotransmission such as 5-Hydroxytryptamine Receptor 2A , Potassium Inwardly Rectifying Channel Subfamily J, Member 3 , and Glutamate Ionotropic Receptor Kainate Type Subunit 1  were found selectively upregulated. Besides, an increased expression of genes encoding, e.g., Fibroblast Growth Factor 1 , POU Class 4 Homeobox 1 , Paired Box Gene 3 , and S100 Calcium Binding Protein B  was observed.Concerning both approaches A and B, especially genes coding for motoneuronal proteins such as Motor Neuron And Pancreas Homeobox 1  and Insulin gene enhancer protein Islet-1  as well as genes coding for Glutamate Ionotropic Receptor AMPA Type Subunit 2 , Bone Morphogenetic Protein 2 , Potassium Voltage-Gated Channel Subfamily H Member 1 , and Adrenoceptor Alpha 1A  were upregulated.Early differentiation marker Paired Box 6  was downregulated in both approaches as were genes associated with synaptic transmission such as Tyrosine Hydroxylase , Choline O-Acetyltransferase , Solute Carrier Family 18 Member A3 , Solute Carrier Family 17 Member 6 , and GDNF Family Receptor Alpha 2 . Furthermore, genes involved in neuronal differentiation and migration like Achaete-Scute Family, BHLH Transcription Factor 1 , Dopamine Receptor D2 , and C-X-C Motif Chemokine Ligand 1  as well as genes regulating Notch signalling such as Hes Related Family BHLH Transcription Factor With YRPW Motif Like  and WNT signalling e.g., SHH  were less expressed in both approaches.p = 0.01), MNX1 , and GFRA2  were significantly differentially expressed in cells of approach A.When comparing gene expression of cells differentiated with approach A for 28 days with cells differentiated with approach B for 28 days, only S100B    and the  < 0.05) . Nuclear < 0.05) , thus coNestin expression was detected in NPC and cells of both approaches after 28 days of differentiation by immunofluorescence . In flowp < 0.05) (p < 0.05) in cells of approach A compared to cells of approach B. These results suggest that motoneuronal precursor cells are also present in the early neuronal differentiation stage of NPC and indicate that SHH- and RA-signalling pathways have been activated during dual-SMAD inhibition.To verify the generation of motoneuronal populations during 28-day differentiation of both approaches, the expression of MNX1, an early marker protein of postmitotic spinal MNs , was inv < 0.05) . FurtherTo validate both differentiation approaches in terms of their efficiency to generate mature MNs, cells differentiated by approaches A and B for 28 days were tested for postmitotic motoneuronal markers ChAT and Islet-1 (ISL1) in comparison to NPC. By immunofluorescence and flow cytometry, all approaches were positively stained for ChAT , an enzyp < 0.05) in comparison to NPC, confirming the suitability of both approaches to form mature MNs  Peripherin, and Neurofilament Heavy Polypeptide Smi-32) was examined by immunofluorescence. Syn I belongs to the phosphoprotein family of synapsins, which play a key role in neurite outgrowth, synapse formation, and synaptic transmission . Positiv was examThis study gives insight into the potential of two differentiation approaches A and B using standardized, chemically defined, and ready-to-use culture media to generate mature MNs from foodprint- and integration-free derived hiPSC. Both differentiation approaches avoid the use of antibiotics or feeder cells, thus the derived MNs may have the potential to be implemented in the therapy of motor neurodegenerative diseases. Since hiPSC differentiation into MNs is a multiple-step process including the formation of NPC, the identification and characterization of these generated NPC is essential.NPC were differentiated successfully under serum-free conditions from hiPSC, expressing early neuron differentiation markers such as Nestin, Sox2, Pax6, FABP7, and Tubb3 assessed by qRT-PCR, Western Blot, immunocytochemistry, and flow cytometry . Nestin,Maturation of MNs was achieved via both differentiation approaches A and B. Downregulation of Pax6 and Nestin is described in the course of MN maturation , 40 and Concerning the expression profile, both approaches differ in aspects of neurotrophic proteins . These are involved in the regulation of neuronal survival, axon outgrowth, dendritic pruning, and synaptic plasticity , 44 and Characteristic proteins essential for the identification of postmitotic and functional MNs are MNX1, ISL1, and ChAT . MNX1 (aGenes coding for neuronal ion channels being upregulated in approaches A and B can be considered for the functional classification of mature neurons . KCNH1, in vivo.To clarify if the generated MNs possess functionality, not only investigations on electrophysiological activity using, e.g., patch-clamp or multielectrode-array system need to be performed but also coculture experiments with skeletal muscle cells are required as the proper synaptic and signalling context is provided by neuromuscular junctions . ConsideThis study demonstrated that MNX1- and ChAT-positive MN precursors can be differentiated from integration-, feeder-, serum-, and antibiotic-free generated hiPSC by dual-SMAD inhibition and subsequent expansion. The derived MN precursor cells form mature and putative functional MNs after application of chemically defined, standardized, and antibiotic-free culture media. The quality of hiPSC, MN precursors, and mature MNs was continuously confirmed in terms of their morphology, protein, and gene expression, ensuring a robust and reproducible protocol. Comparison of differentiation approaches A and B revealed a significant difference in MNX1 gene expression. Thus, the approach A appears to be a more efficient differentiation method that might set the course for clinical translation if the entire process of hiPSC generation and differentiation, including all culture media and matrices used, complies with cGMP requirements.


Cxxc5−/− mice were found to be resistant to NASH pathogenesis with metabolic improvements. KY19334, a small molecule that activates the Wnt/β-catenin pathway via interference of the CXXC5-Dvl interaction, reversed the overall pathogenic features of NASH as Cxxc5−/− mice. The improvement in NASH by KY19334 is attributed to its regenerative effects through restorative activation of the suppressed Wnt/β-catenin signaling. Overall, the pronounced metabolic improvements with the stimulation of liver regeneration by interfering with the CXXC5-Dvl interaction provide a therapeutic approach for NASH.Non-alcoholic steatohepatitis (NASH) is a chronic liver disease that results from multiple metabolic disorders. Considering the complexity of the pathogenesis, the identification of a factor mediating the multiple pathogenic phenotypes of NASH will be important for treatment. In this study, we found that CXXC5, a negative feedback regulator of the Wnt/β-catenin pathway, was overexpressed with suppression of Wnt/β-catenin signaling and its target genes involved in hepatic metabolism in obese-NASH patients.  Cxxc5 knocked out were resistant to the worst effects of NASH. The researchers developed a treatment using a small molecule called KY19334 that interferes the function of CXXC5 via inhibition of its binding with Disheveled. This treatment suppressed inflammation, steatosis, oxidative stress and cell death, reversed liver fibrosis and enhanced liver regeneration.A small molecule treatment that restores the suppression of important signaling pathways could be effective against obesity-related liver disease. Non-alcoholic steatohepatitis (NASH) is a severe and prevalent condition in obese and diabetic patients. No specific NASH medications exist, but studies have shown that the Wnt/β-catenin signaling pathway is crucial in activating key genes that fight the disease. Kang-Yell Choi at Yonsei University in Seoul, South Korea, and co-workers investigated the role of CXXC5, which suppresses the Wnt/β-catenin pathway. They showed that mice with  Insulin resistance plays a critical role in the pathological process of NAFLD by increasing the free fatty acid (FFA) flux to the liver, resulting in hepatic steatosis and lipotoxicity3. Non-alcoholic steatohepatitis (NASH) is the most severe form of NAFLD and is often caused by hepatic steatosis, chronic inflammation, and liver injury-associated fibrosis2. Although NASH is a severe and prevalent liver disease, a clinically applicable medication for its treatment is not available5. Due to the complexity of the developmental process and the multiple pathological phenotypes, including insulin resistance, steatosis, inflammation, and fibrosis7, the development of drugs that cure one or several disease phenotypes of NASH has limited treatment efficacy10. Therefore, understanding the overall pathogenic process of NASH and identifying a factor that drives the multiple pathological phenotypes are attractive for the development of effective treatments for NASH patients.Non-alcoholic fatty liver disease (NAFLD) is prevalent among obese and diabetic patientsGLP-1), transcription factor-7-like-2 (TCF7L2), peroxisome proliferator-activated receptor delta (PPARδ), and WNT1-inducible signaling pathway protein 1 (WISP1)10. Wnt/β-catenin signaling is suppressed in the liver tissue of NASH patients, and the expression of its target genes involved in multiple pathological phenotypes of NASH is subsequently suppressed. For example, GLP-1, a direct transcriptional target of Wnt/β-catenin signaling, and its agonizts are suppressed by increased body weight in obese diabetic patients11. GLP-1 agonizts, such as liraglutide, exenatide, and semaglutide, are known to reduce hepatic steatosis in NAFLD patients by directly affecting lipid metabolism12. The inhibitor of dipeptidyl-peptidase 4 (DPP4), a GLP-1 destabilizer that is transcriptionally induced by Wnt/β-catenin signaling, prevents multiple NASH phenotypes14. In addition, activation of the Wnt/β-catenin pathway stimulates the neogenic replacement of damaged tissues such as the liver15, and overexpression of β-catenin after hepatotoxic liver injury enhances its regeneration via activation of its target genes17. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), one of the major Wnt/β-catenin signaling target genes involved in the activation of adult stem cells, plays a role in the self-renewal of tissues such as the small intestine, colon18, stomach19, hair follicles20, and liver21.The Wnt/β-catenin signaling pathway plays key roles in a wide range of metabolic disorders, such as inflammation, hepatic steatosis, gluconeogenesis, energy metabolism, and insulin resistance, which may be attributed to its target genes that play critical roles in metabolism, including glucagon-like peptide-1 (23.CXXC5-type zinc finger protein 5 (CXXC5) has been identified as a negative feedback regulator of the Wnt/β-catenin signaling pathway that functions through a direct interaction with the disheveled (Dvl) proteinGLP1, TCF7L2, WISP1, and fos-related antigen 1 (FOSL1) in the liver tissues of NASH patients with metabolic disorders. Multiple pathological phenotypes of NASH, including inflammation, steatosis, oxidative stress responses, and fibrosis, were not significantly observed in Cxxc5−/− mice. CXXC5 is specifically induced in the cytosol of cells in the adipose and liver tissues of NASH patients. CXXC5, especially cytosolic CXXC5, which binds Dvl, as a target for the development of drugs to treat NASH was confirmed by the similar improvement of NASH phenotypes in Cxxc5−/− mice and with oral application of KY19334, a small molecule interfering with CXXC5-Dvl protein‒protein interaction (PPI)24. The improvement in the multiple pathological phenotypes of Cxxc5−/− mice and with KY19334 treatment could be achieved by restoration of Wnt/β-catenin signaling followed by the activation of multiple target genes involved in NASH pathogenesis and liver tissue regeneration. Overall, a small molecule approach abolishing the cytosolic function of CXXC5 via interference of CXXC5-Dvl PPI is a potential treatment for improving NASH.In this study, we found that CXXC5 was specifically induced with suppressing Wnt/β-catenin pathway target genes such as Cxxc5−/− mice has been previously described23. Cxxc5 heterozygous mice were intercrossed for four generations to obtain littermate wild-type and Cxxc5−/− mice and maintained on a C57BL/6 background. Cxxc5+/+ and Cxxc5−/− mice exhibited NASH following HFD feeding containing CCl4 (Sigma‒Aldrich) or gold thioglucose . For the CCl4-induced NASH mouse model, 6-week-old male mice  were fed a 60% kcal HFD  for 12 weeks and administered 0.05 ml/kg CCl4 twice a week, and for the final 4 weeks, they were daily administered KY19334 (25 mg/kg), selonsertib , ocaliva  or vehicle25. For the GTG-induced NASH mouse model, 5-week-old male mice were intraperitoneally injected with GTG, followed by NCD for 1 week. Then, the mice were fed a HFD  or NCD for the next 11 weeks. The HFD-fed mice were orally administered KY19334 (25 mg/kg), selonsertib (25 mg/kg), ocaliva (25 mg/kg) or vehicle25 for the final 4 weeks. For the glucose tolerance test (GTT) or insulin tolerance test (ITT), mice were injected with D-glucose  after overnight fasting or human insulin  after 4 h of fasting. Tail blood was drawn at 0-, 15-, 30-, 60-, 120-, and 180-min intervals, and blood glucose levels were measured using a One Touch Ultra glucometer (LifeScan). All animals were housed on a 12-h light/12-h dark cycle, with free access to food and water. All protocols were reviewed and approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (09-013).The generation of g to obtain the supernatant. The supernatant was used to measure the metabolic parameters. ELISA kits were used to assess serum insulin , serum FFAs , and serum adiponectin . The homeostasis model assessment of insulin resistance (HOMA-IR) was used to calculate insulin resistance. Serum parameters, including total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides (TGs), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) concentrations, were measured using a blood chemistry analyzer (FUJI DRI-CHEM 4000i). Calibration was performed using the quality control card supplied with the FUJI DRI-CHEM slides whenever slides from a new lot were used.The total blood of the mice was collected by cardiac puncture after fasting. The blood was allowed to clot for 30 min and then centrifuged for 10 min at 1000 × Cytokines and chemokines in mouse serum were assessed using the Mouse XL Cytokine Array Kit , arrowing simultaneous detection of the expression levels of 111 different liver damage-related proteins. The array was performed according to the manufacturer’s instructions. Blots were developed with enhanced chemiluminescence using a LAS-3000 luminescent imaging analyzer (Fujifilm).The dissected tissues were fixed in 4% neutral paraformaldehyde (PFA) and embedded in paraffin. The paraffin sections were cut to a thickness of 4 μm and subjected to H&E staining. Adipocyte cell size was measured in 20 randomly chosen microscopic areas from three independent animals using a Nikon bright-field optical microscope (Nikon TE-2000U). The average adipocyte size was determined using ImageJ software.2O2  for 30 min. Before incubating the sections with mouse primary antibody, mouse IgG was blocked using a M.O.M Mouse IgG blocking kit (Vector Laboratories). Sections were incubated with the following primary antibodies overnight at 4 °C: anti-β-catenin , anti-CXXC5 , anti-LGR5 , anti-CD133 , anti-HNF4a , and anti-Ki67 . The slides were washed with PBS, incubated with Alexa Fluor 488-  or Alexa Fluor 555-conjugated IgG secondary antibody  at 20 °C for 1 h, and counterstained with DAPI . The images were captured using an LSM700 META confocal microscope (Carl Zeiss) after excitation with 405-, 488-, or 543-nm lasers. To block endogenous peroxidase activity before peroxidase IHC analysis, tissues were incubated with 0.345% H2O2  for 30 min. Before incubating the sections with mouse primary antibody, mouse IgG was blocked using a M.O.M Mouse IgG blocking kit (Vector Laboratories). Sections were incubated with primary antibodies overnight at 4 °C at the following dilutions: anti-PPARγ , anti-F4/80 , anti-CD11b , anti-α-SMA , anti-collagen I , anti-desmin , and anti-UCP1 . Sections were then incubated with biotinylated anti-rabbit  secondary antibodies for 1 h at 20 °C. The samples were stained with 3,3-diaminobenzidine  for 3–7 min and counterstained with Mayer’s hematoxylin (Muto). All incubations were conducted in a humid chamber. Signals were analyzed using a bright-field microscope (Nikon TE-2000U).Paraffin sections of human liver tissue microarray (Xenotech) and mouse liver and scWAT were deparaffinized and rehydrated. For antigen retrieval, slides were autoclaved in 10 mM sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity, tissues were incubated with 0.345% Hg for 4 min. Stromal vascular fraction pellets were resuspended in 0.5 ml of RBC lysis buffer (BioLegend) for 5 min on ice, centrifuged for 5 min at 300 × g, and resuspended in PBS. Cells were incubated with the following fluorescently labeled antibodies for 30 min: anti-F4/80-FITC  or anti-CD11b-PE .Immediately after sacrifice, the liver tissues were excised and washed with PBS to remove blood. Tissues were minced and digested with 1 mg/mL collagenase type IV (Sigma‒Aldrich) in PBS containing 150 U/mL DNase I for 30 min at 37 °C. The digested liver was suspended in 50 mL of DMEM, and the dissociated cells were filtered through a 100-μm nylon strainer and collected by centrifugation at 300 × 26. Red cells were lysed with buffer  and resuspended in Williams E medium (Gibco) with 10% FCS. Following centrifugation at 300 × g for 5 min at 4 °C, the supernatant was collected, washed twice, and resuspended in PBS with 2% FCS for FACS staining. LPCs were stained with anti-LGR5  at 4 °C for 30 min and then incubated with Alexa 555-conjugated IgG secondary antibody  at 4 °C for 10 min. The stained cells were analyzed by flow cytometry (BD Bioscience).Isolation of LPCs was performed as previously describedParaffin-embedded liver tissue sections were deparaffinized and rehydrated. Tissues were stained with Weigert’s solution for 8 min and subsequently with picrosirius red for 1 h.Cryo-liver tissue sections were counterstained with DAPI (Sigma-Aldrich) and washed with PBS. The sections were then incubated with dihydroethidium  for 30 min at 37 °C in the dark and washed with PBS. The samples were mounted in 100% glycerol, and reactive oxygen species (ROS) levels in the liver were observed using an LSM700 META confocal microscope (Carl Zeiss) after excitation with a 405-nm laser.Cryo-liver tissue sections were fixed in PFA for 15 min and washed with 70% isopropanol . The lipid droplets were stained with ORO solution (Sigma-Aldrich) at 20 °C overnight. The samples were counterstained with Mayer’s hematoxylin. Images of ORO staining were visualized using a bright-field microscope.Liver tissues were incubated on ice in 100 μL of saline solution . Tissue suspensions were assayed for TG content using a triglyceride assay kit .GAPDH. The primer sequences are listed in Supplementary Table Total RNA was extracted from ground tissue powder using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen) and 2 μg of total RNA. The synthesized cDNA was diluted to a concentration of 100 ng/μL, and quantitative PCR analyses were performed in the Rotor-gene Q real-time PCR cycler (Qiagen) using SYBR green reagent (Qiagen) under the following conditions: 95 °C for 10 min followed by 40 cycles at 95 °C for 5 s and 60 °C for 15 s. Relative amounts of mRNA were quantified using the comparative Ct method (∆∆Ct). All mRNA values were normalized to those of www.broadinstitute.org/msigdb). Cross-species comparison of transcriptomic dysregulation was performed in the space of molecular pathway gene sets from the HALLMARK and KEGG databases, and statistically significant dysregulation was defined as a false discovery rate (FDR) < 0.01 in either of the two human liver tissue transcriptome datasets: normal (n = 12) vs. NASH (n = 13) subjects (GSE48452).Molecular pathway dysregulation in human visceral adipose tissues was determined by gene set enrichment analysis, surveying the molecular pathway gene set in the Molecular Signature Database (MsigDB) .Information about statistical details and the methods is indicated in the figure legends, tests, or methods. Data are presented as the mean ± SD. Statistical analyses were performed using an unpaired two-tailed Student’s TCF7L2, GLP1, AXIN2, FOSL1, and WISP1, were lower in liver tissues from NASH patients - or HFD plus gold thioglucose (HFD + GTG)-induced NASH model mice  assay and the expression of genes involved in apoptosis Cxxc5 knockout or CXXC5-Dvl PPI inhibition. This therapeutic approach is highly effective due to the restoration of the suppressed Wnt/β-catenin signaling target genes involved in liver regeneration and cellular metabolism. The pathological significance of controlling CXXC5 function was rationalized by the overexpression of CXXC5 following suppression of Wnt/β-catenin signaling and subsequent induction of its target genes in the liver tissue of NASH patients.In the present study, we suggest a new approach for improving NASH phenotypes by restoring the suppressed regenerative Wnt/β-catenin signaling by inhibiting CXXC5 function through either GLP1, PPARδ, TCF7L2, AXIN, and WISP1, and indirect effectors, such as PPARγ and C/EBPα, which are suppressed by WISP1, are involved in metabolic diseases and have been used as targets for the development of drugs34. For example, a GLP1 receptor agonist has been used for the development of clinically available drugs for diabetes and has also been tested as a therapeutic agent for NASH36. Induction of PPARδ expression, a direct transcriptional target of Wnt/β-catenin signaling37, by oral administration of KY19334 implies that systemic energy expenditure was increased by activation of this pathway. Furthermore, ectopic expression of UCP1 in scWAT from KY19334-treated mice was linked to protecting against diet-induced obesity by increasing fatty acid oxidation of scWAT.Various direct Wnt/β-catenin signaling target genes, including 4, administration of KY19334 increased energy metabolism, accompanied by the enhancement of adipose tissue browning. The role of CXXC5 in the regulation of Wnt/β-catenin signaling was shown by an increase in nuclear β-catenin with the accumulation of cytosolic Cxxc5 in adipocytes of NASH mice. Compared to the ASK-1 inhibitor selonsertib38 and the FXR agonist ocaliva39, KY19334 showed higher effectiveness in improving the pathological features of the liver in HFD + CCl4-induced NASH mouse models when administered at identical concentrations. Steatosis, which was weakly improved by selonsertib, was mostly inhibited by administration of the same amount of KY19334, as shown by various histochemical and biological analyses. Inflammation and ROS-mediated stress and apoptosis were also significantly improved by KY19334 but not by selonsertib or ocaliva.The hypothesis that the CXXC5-Dvl PPI is a target for the development of drugs for the treatment of NASH was supported by the improvement in metabolic dysfunctions and pathological features of NASH with the restoration of the suppressed Wnt/β-catenin signaling target genes following KY19334 treatment. In the NASH model induced by HFD combined with CCl4-induced NASH model. Similar improvements in NASH phenotypes were also observed in Cxxc5−/− mice, supporting the therapeutic effects of interference with Cxxc5-Dvl PPI. Moreover, the absence of pathological phenotypes in Cxxc5−/− mice following NASH induction by either HFD + CCl4 or HFD + GTG shows that CXXC5 is a key regulator in the pathogenesis of NASH. The critical role of CXXC5 as a mediator of the pathogenesis of NASH was further confirmed by the absence of CXXC5 induction and any apparent pathological phenotypes by administration of KY19334 when the mice were fed a NCD (Supplementary Fig. Finally, fibrosis was not observed in the liver tissue of mice treated with KY19334 compared to mice administered selonsertib or ocaliva, which showed relatively weak improvement, especially in the HFD + CClCxxc5 knockout or KY19334 treatment could be attributed to the enhancement of the regeneration system through activation of Wnt/β-catenin signaling and subsequent induction of genes involved in regeneration and metabolism, such as Glp-1, Pparδ, and Lgr541. Lgr5+ stem cells involved in liver regeneration accompanied by hepatocyte proliferation42 were observed in the liver tissue of NASH mice. The liver tissues of the Cxxc5−/− mice, as well as those of mice administered KY19334, showed an enhanced liver regeneration system in the HFD + CCl4-induced NASH model as monitored by the measurement of the stem cell markers including EpCam, Sox9, Cd44, Prom1, and Lgr5. The induction of these multiple stem cell markers may be attributed to the activation of direct Wnt/β-catenin signaling target genes, including Lgr526. The enhancement of liver regeneration by KY19334 in NASH mice was further confirmed by the increased numbers of both Hnf4α- and Ki67-positive cells. Overall, these results indicate that the improvement in the multiple pathological phenotypes of NASH by KY19334 is attributed to the enhancement of the regeneration system of the liver through the restorative activation of the suppressed Wnt/β-catenin pathway.The improvement in multiple NASH phenotypes, including steatosis, ballooning, inflammation, oxidative stress, and fibrosis, by 24. However, the inhibitory role of KY19334 on GSK3β alone may be limited due to attenuation of Wnt/β-catenin signaling activation by the increase in the negative feedback regulator CXXC5. Therefore, the inhibitory effect of the CXXC5-Dvl PPI is greater than that of GSK3β on the activation of Wnt/β-catenin signaling and the subsequent therapeutic effects of KY19334 on NASH.The enhancement of the regeneration system of the liver by KY19334 provides a further advantage in the efficient activation of the Wnt/β-catenin pathway by its effect on GSK3β inhibition as well as the blockade of CXXC5-Dvl PPITaken together, a small molecular approach, which is able to restore the overall NASH phenotype by metabolic improvement and regenerative tissue restoration, provides a potential therapy for curing chronic fibrotic diseases.Supplemental information


Prmt5-KO mice were generated to define the role of PRMT5 in hindlimb ischemia–induced angiogenesis. We found that these mice exhibited impaired recovery of blood perfusion and motor function of the lower limbs, an impairment that was accompanied by decreased vascular density and increased necrosis as compared with their WT littermates. Furthermore, both pharmacological and genetic inhibition of PRMT5 significantly attenuated EC proliferation, migration, tube formation, and aortic ring sprouting. Mechanistically, we showed that inhibition of PRMT5 markedly attenuated hypoxia-induced factor 1-α (HIF-1α) protein stability and vascular endothelial growth factor–induced (VEGF-induced) signaling pathways in ECs. Our results provide compelling evidence demonstrating a crucial role of PRMT5 in hypoxia-induced angiogenesis and suggest that inhibition of PRMT5 may provide novel therapeutic strategies for the treatment of abnormal angiogenesis-related diseases, such as cancer and diabetic retinopathy.Arginine methylation mediated by protein arginine methyltransferases (PRMTs) has been shown to be an important posttranslational mechanism involved in various biological processes. Herein, we sought to investigate whether PRMT5, a major type II enzyme, is involved in pathological angiogenesis and, if so, to elucidate the molecular mechanism involved. Our results show that PRMT5 expression is significantly upregulated in ischemic tissues and hypoxic endothelial cells (ECs). Endothelial-specific   Angiogenesis is the process of capillary formation from preexisting blood vessels, and it is important for normal development, tissue homeostasis, and various physiological and pathological processes , 2. UndeProtein arginine methyltransferases (PRMTs) have been characterized as critical regulators of cell homeostasis and are essentially involved in several biological processes, including RNA regulation, signal transduction, and chromatin regulation , 13. PRMIn this study, we sought to investigate the role of PRMT5 in hypoxia-induced angiogenesis and molecular mechanisms involved. We showed that genetic and pharmacological inhibition of PRMT5 markedly attenuated hindlimb ischemia–induced angiogenesis and VEGF-induced endothelial proliferation, migration, and tube formation, through decreasing HIF-1α protein stability and suppressing VEGF-induced signal transduction in vascular endothelial cells (ECs).PRMTs in human umbilical vein ECs (HUVECs), which express many important endothelial markers and signaling molecules and are widely used for in vitro studies of the vasculature and angiogenesis   , which h ECs (2) , while treatment . Twenty-Prmt5 has been shown to induce embryonic lethality  were given to both fl/fl/Cdh5-ERT/Cre+/–Prmt5 and fl/flPrmt5 mice. After recombination of LoxP, the exon 7 that encodes part of PRMT5 methyltransferase domain was removed in EC-Prmt5/ΔΔ mice  and murine aortic ECs (MAECs) from fl/flPrmt5 and EC-Prmt5/ΔΔ mice were isolated to detect the mRNA levels of Prmt5 using quantitative PCR (qPCR). As shown in Prmt5 in MLECs and MAECs isolated from EC-Prmt5/ΔΔ mice were significantly decreased compared with the fl/flPrmt5 group. In addition, Western blot demonstrated similar results as shown in Prmt5-depleted ECs. Immunofluorescent staining indicated that PRMT5 expression was virtually absent in mouse blood vessels as indicated by immunofluorescent staining of PRMT5 and CD31 in the blood vessels of GC muscles of fl/flPrmt5 and EC-Prmt5/ΔΔ mice  . Together, these results suggest that Prmt5 KO in ECs attenuates ischemia-induced angiogenesis and injury-induced muscle regeneration.To substantiate the function of PRMT5 in angiogenesis, a mouse model of hindlimb ischemia was established, and the angiogenesis was determined by measuring blood flow recovery in the hindlimb and the area of new capillaries in distal ischemic GC. g (LSCI) . The blorol mice . Similar surgery . Ligatio surgery . The GC ol group . Inadequol group , 31. To termates . Twenty-termates , was subese mice . FurtherPRMT5 also significantly attenuated the tube formation of ECs , suggesting that a posttranslational mechanism may be involved in regulating HIF-1α protein levels by PRMT5.HIF-1α and its downstream proangiogenic molecules such as VEGF-A play important roles in ischemia/hypoxia-induced angiogenesis . Thus, w protein , while tPRMT5-knockdown cells , which blocks translation and neo protein synthesis. In this regard, ECs were subjected to the treatment of either 10.0 μM EPZ015666 or vehicle for 2 days and then exposed to chemical hypoxia for 24 hours. Fresh complete ECM was then replaced with CHX supplement. The degradation rate of HIF-1α protein was then determined by Western blot. As shown in wn cells . Furtherwn cells , suggestwn cells , were down cells , as detePrmt5 KO were unable to produce embryos  (Tg(Cdh5-cre/ERT2)1Rha mice were obtained from Ralf H. Adams  (fl/flPrmt5 mice were bred with Tg(Cdh5-cre/ERT2)1Rha mice to create inducible EC–specific Prmt5-KO mice (fl/fl/Cdh5-ERT/Cre+/–Prmt5). Prmt5 gene deletion in ECs of fl/fl/Cdh5-ERT/Cre+/–Prmt5 mice was induced by i.p. injection of tamoxifen (MilliporeSigma) at a dose of 75 mg/kg body weight for 5 consecutive days, and mice were allowed to recover for 1 week before being used for experiments. All mice were genotyped by PCR. The genomic DNA isolated from mice tail biopsies was analyzed by PCR to detect Prmt5 WT allele (product size 247 bp), loxP-flanked allele (465 bp), and VE-Cadherin-Cre allele . Genotyping primers for Prmt5 allele were 5′-TGGAACTGCAGGCATATGCC-3′ (forward [F]) and 5′-TTCTGGCCTCCATGGGGGAA-3′ (reverse [R]), and the ERT allele were 5′-ATCCGAAAAGAAAACGTTGA-3′ (F) and 5′-ATCCAGGTTACGGATATAGT-3′ (R). All mice were on the C57BL/6J background and were maintained under specific pathogen–free conditions at 22°C with a 12-hour light/ 12-hour dark cycle. Age-matched male littermates (8–12 weeks old) were used for the experiments.da, USA) , and C57Germany) . Prmt5fl2 at 37°C. HUVEC from passage 2 to 6 were used in experiments. To induce a hypoxic environment in ECs, cells were exposed to 200 μM CoCl2 (MilliporeSigma) in HUVEC culture medium for 3–36 hours as a chemical hypoxia model.HUVECs were obtained from Thermo Fisher Scientific  and cultured in complete EC medium  supplemented with 5% FBS (ScienCell), 1% EC growth supplement , and 1% antibiotic solution  under 5% COg for 5 minutes at room temperature), cells were resuspended in DMEM and incubated with anti-CD31–coated  Dynal magnetic beads (Thermo Fisher Scientific) on an end-over-end rotator for 10 minutes at room temperature. The beads/cell pellets were washed 5 times using the magnetic separator (Thermo Fisher Scientific) and transferred to a 0.1% gelatin-coated (MilliporeSigma) 100 mm cell culture dish (Corning) with complete ECM medium (ScienCell) supplemented with 20% FBS, 1% ECGS, and 1% P/S. To improve MLEC purity, the primary cells that reached around 80% confluence were sorted a second time using anti-CD102–coated  magnetic beads.ECs from fresh lung tissue of mice were isolated as described previously . BrieflyUnder anesthesia, mice were dissected from the midline of the abdomen and slowly perfused with PBS containing 1000 U/mL heparin (MilliporeSigma). Under a dissecting microscope, the adipose tissue attached to the thoracic aorta was carefully removed. On the distant end, the aorta was ligated by sutures. On the proximal end, a small incision was made, and the PBS solution was carefully expelled with tweezers. TRIzol (Invitrogen) reagent was slowly injected into the aorta for about 20 seconds and was then slowly withdrawn. This procedure was repeated several times. Finally, TRIzol containing MAECs was stored at –80°C for further qPCR assay.PRMT1, 5′-CTTTGACTCCTACGCACACTT-3′ (F) and 5′-GTGCCGGTTATGAAACATGGA-3′ (R); human PRMT2, 5′-ACATTCCGGCAAACCATGTG-3′ (F) and 5′-GGATGACTTTATCCGTCAGGGA-3′ (R); human PRMT3, 5′-GTACCCTTCTCATACCCCAATGG-3′ (F) and 5′-GACGAGCAGGTTCTGACATCT-3′ (R); human PRMT4, 5′-TCGCCACACCCAACGATTT-3′ (F) and 5′-GTACTGCACGGCAGAAGACT-3′ (R); human PRMT5 5′-CTGTCTTCCATCCGCGTTTCA-3′ (F) and 5′-GCAGTAGGTCTGATCGTGTCTG-3′ (R); human PRMT6 5′-TACCGCCTGGGTATCCTTCG-3′ (F) and 5′-CCTGTTCCGGCAACTCTACA-3′ (R); human PRMT7 5′-GGTGTCACCAGCCGACTTTAC-3′ (F) and 5′-TGAGCTACTGACTTGCTTGCT-3′ (R); human PRMT8 5′-CCTGCTAAGCCCGTGCAAT-3′ (F) and 5′-TGGGCATAGGAGTCGAAGTAA-3′ (R); human PRMT9 5′-GCCTGTGAGTTATCCAAGACC-3′ (F) and 5′-ACAACTAGGGACACTCTTTCGG-3′ (R); human HIFIA, 5′-CACCTCTTTTGGCAAGCATCCTG-3′ (F) and 5′-TATGAGCCAGAAGAACTTTTAGGC-3′ (R); human VEGFA, 5′-GATGGCAGTAGCTGCGCTGATA-3′ (F) and 5′-TTGCCTTGCTGCTCTACCTCCA-3′ (R); human GAPDH, 5′-TGTGGGCATCAATGGATTTGG-3′ (F) and 5′-ACACCATGTATTCCGGGTCAAT-3′ (R); mouse Prmt5, 5′-CTGAATTGCGTCCCCGAAATA-3′ (F) and 5′-AGGTTCCTGAATGAACTCCCT-3′ (R); and mouse Gapdh, 5′-AGGTCGGTGTGAACGGATTTG-3′ (F) and 5′-GGGGTCGTTGATGGCAACA-3′ (R).Total RNAs were extracted from ECs by using miRNeasy Mini Kit (Qiagen) or the miRNeasy Serum/Plasma Kit (Qiagen). qPCR for messenger RNA levels was performed using the miRCURY LNATM Universal cDNA Synthesis and SYBR Green Master Mix Kit (Exqion) as described . The priWestern blot analysis was performed as described previously . Brieﬂy,Mice were anesthetized with isoflurane inhalation and were placed on a heating mat at 37°C. The hair in the surgical area was completely removed with depilation cream, and the left lower limb area of mice was disinfected with 70% ethanol. A skin incision about 1 cm from the knee to the inside of the thigh was made to expose the femoral artery, where the anatomical markers of the femoral nerve, artery, and venous neurovascular bundle were identified. The femoral nerve, artery, and vein neurovascular bundles were separated by pulling the connective tissue next to them. After the separation, 2 surgical knots were made to ligate the proximal femoral artery. Next, the femoral artery was separated from the femoral vein distally near the knee joint, and another surgical line was placed at its lower end to close the femoral artery by 2 surgical knots. The femoral artery was transected between the distal and proximal ends of the ligated femoral artery using ophthalmic scissors. The hindlimb incisions were sutured, and the mice were s.c. injected with 0.1 mg/kg buprenorphine to relieve pain.LSCI was used to monitor blood perfusion before and after surgery and on days 1, 3, 7, 14, 21, and 28 postoperatively in both groups of mice. The laser scanning instrument  was fixed about 11 cm above the mice, and the background threshold was adjusted. Two hindfoot regions were selected as the region of interest (ROI), and the perfusion images were obtained and quantitated using PeriCam PSI System (PERIMED).Tarlov functional scoring was used in this project to evaluate movement of hindlimbs of mice . The detSections were fixed with 4% formaldehyde and permeabilized with 0.2% Triton-X 100 at room temperature. Nonspecific binding was covered by incubating with 3% BSA at room temperature for 60 minutes and then incubated with rat anti-CD31  and rabbit anti-PRMT5  antibodies at 4°C overnight. Slides were washed with PBS 3 times for 5 minutes and incubated with Alexa Fluor–conjugated secondary antibodies  at room temperature for 1 hour. After staining with DAPI , slides were embedded with antifade mountant reagent (Invitrogen) and photographed under a Nikon fluorescence confocal microscope.Mice were euthanized and perfused with PBS containing 0.2% heparin, followed by 4% paraformaldehyde through the left ventricle. Whole hindlimbs were then dissected and fixed overnight. After decalcification by 10% formic acid, the hindlimbs were cut into the proximal, mid, and distal segments as described previously . The segThe Matrigel plug assay is widely used to assess the angiogenic potential or antiangiogenic activity of different compounds in vivo . BrieflyAn aortic ring assay is a simple and effective method to detect new vessels sprouting . BrieflyGene knockdown in ECs was performed by lentivirus transduction. pLKO-PRMT5 constructs (TRCN0000107086) and Non-Target shRNA control (SHC016) were from Sigma-Aldrich. Lentiviral pLKO constructs were transfected with packaging and envelope plasmids to HEK293T cells. Viral supernatant was harvested at 72 hours after transfection, filtered through a 0.45 μm filter, and then added to the cultured cells in the media supplemented with 10 μg/mL polybrene (Sigma-Aldrich). Twenty-four hours after infection, the virus-containing media were removed and replaced with fresh media. Knockdown efficiency was detected after infection of virus for 3 days.5 cells per milliliter. Mixed cell suspension  was seeded on the Matrigel in 96-well plates and incubated from 4 to 16 hours. The images were captured using the EVOS cell imaging system, and the vascular network was quantified using the ImageJ software (NIH) with the angiogenesis analyzer plug-in.Tube formation assay was performed as previously described . In totaThe wound healing assay is a simple and economical method to study cell migration . HUVECs ECs at a density of 70% were digested with trypsin and counted. A total of 1000 cells/well were added into 96-well plates and then incubated with different concentrations of PRMT5 inhibitor EPZ015666 or control vehicle. Six replicates were set at each concentration. In total, 10 μL of WST-8 and 1-methoxyphenazine methyl sulfate  were added to each well and incubated at 37°C for 2–3 hours. The absorbance was measured at 450 nm.2–) and nitrate (NO3–), the major oxidation product of NO, were determined using a standard calibration curve of potassium iodide detected by chemiluminescence NO detector .To detect endothelial NO production, cell culture supernatants were freshly collected and kept on ice until measurement. The levels of nitrite (NO450) using a plate reader (BioTek Instruments).VEGF-A levels in cell culture supernatants were detected using a human ELISA kit , according to the manufacturer’s instructions. Briefly, collected cell culture supernatants and standards were added in various dilutions to 96-well plates coated with capture antibody, and they were then incubated with biotinylated detection antibody. After incubation with streptavidin-peroxidase and TMB substrate, the protein contents in supernatants were determined by measuring the absorbance at 450 nm .


Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary disease of the heart muscle. Clinical challenges remain, however, in identifying patients with ARVC in the early or concealed stages with subtle clinical manifestations. Therefore, we wanted to identify potential targets by immunohistochemical (IHC) analysis in comparison with controls. Pathogenic mutations were identified in 11 of 37 autopsied patients with ARVC. As observed from IHC analysis of the RV, expression of αT-catenin and plakophilin-2 is significantly decreased in autopsied patients with ARVC as compared to controls, and the decreased expression is consistent in patients with and without pathogenic mutations. Furthermore, ARVC specimens demonstrated a reduced localization of αT-catenin, desmocollin-2, desmoglein-2, desmoplakin, and plakophilin-2 on intercalated discs. These findings have been validated by comparing RV specimens obtained via endomyocardial biopsy between patients with ARVC and those without. The pathogenic mutation was present in 3 of 5 clinical patients with ARVC. In HL-1 myocytes, siRNA was used to knockdown CTNNA3, and western blotting analysis demonstrated that the decline in αT-catenin expression was accompanied by a significant decline in the expression of plakophilin-2. The aforementioned effect was directed towards protein degradation rather than mRNA stability. Plakophilin-2 expression decreases concurrently with the decline in CTNNA3 expression. Therefore, the expression of αT-catenin and plakophilin-2 could be potential surrogates for the diagnosis of ARVC. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary disorder of the heart muscle. Pathologically, ARVC is characterized by progressive fibro-fatty replacement of the right ventricle (RV) ,2. A priBy immunohistochemical analysis of endomyocardial biopsy samples, it has previously been demonstrated that a decreased expression of plakoglobin in the ICDs and connexin 43 could be identified in ARVC , which cThe purpose of the present study was to explore the expression of αT-catenin and desmosomal-associated proteins in RV tissues collected from autopsied participants and clinical patients with ARVC using IHC staining. The transient knockdown of CTNNA3 using cultured cardiomyocytes was used to validate the abovementioned findings by western blotting.First, 48 RV specimens were collected from 36 autopsied patients with ARVC and 12 controls autopsied for non-cardiac causes from the Taiwan National Forensic Institute database. Secondly, clinical RV specimens were collected via endomyocardial biopsy from eight and four patients with and without definite ARVC, respectively. Those samples were then stained by IHC. The Institute of Forensic Medicine and Taipei Veterans General Hospital reviewed and approved this study.Among 36 autopsied patients with ARVC from the Taiwan National Forensic Institute registry, the diagnosis was confirmed based on gross and histopathological findings, including extensive fibro-fatty replacement of the RV ,23,24. AA clinical diagnosis of ARVC is made according to the 2010 revised Task Force Criteria . None ofGenomes of heart tissues or blood samples collected from autopsied participants with ARVC and controls were sequenced by targeted next-generation sequencing on the Illumina Hi-seq2000 platform . These genomes contained PKP2, DSC2, DSG2, DSP, JUP, DES, TMEM43, TGFβ3, RYR2, CTNNA3, PKP4, and PLN according to the ARVC genetic variants database. Based on the American College of Medical Genetics and Genomics guidelines, we filtered and evaluated each variant for pathogenicity and identified pathogenic, likely pathogenic, and variants of uncertain significance .2O2 methanol and then incubated with DakoCytomation Dual Endogenous Enzyme Block  for 10 min; Ultra V Block  for 10 min; antibody dilution buffer  for 10 min; and antibodies against αT-catenin, plakophilin-2 , desmin , desmocollin-2 , desmoglein-2, ryanodine receptor 2, transmembrane protein 43, transforming growth factor-beta 3, tenascin N , desmoplakin , and plakoglobin  overnight at 4 °C. In accordance with the manufacturer’s instructions, immunoreactivity was assessed using the BioGenex Super Sensitive Link-Label IHC Detection System . We randomly selected and evaluated ten regions of the RV tissues. ImageJ was used to quantify the density of immunoreactivity in the regions of each protein.A 4-μm-thick section of right ventricular tissue, embedded in paraffin and deparaffinized with Trilogy Solution  at 121 °C for 10 min, was obtained from autopsied patients or patients with endomyocardial biopsy. The sections were treated with 3% H2.The HL-1 cells were derived from mouse atrial cardiomyocytes and cultured on Corning CellBIND surface culture dishes  in Claycomb medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, penicillin/streptomycin/amphotericin B , 0.1 mM norepinephrine, and 2 mM L-glutamine (Sigma-Aldrich) at 37 °C in a humidified atmosphere containing 5% OWe placed the cells in 60-mm dishes and incubated them at 37 °C overnight (for drug treatment). MG101  was diluted to a working concentration of 5 µM in the medium. The cells were also treated with an equal volume of DMSO (Sigma-Aldrich) as a control. The cell lysate was harvested after 24 h of treatment and analyzed by gel electrophoresis and western blotting.ON-TARGETplus Non-targeting Pool siRNA was obtained from Dharmacon and used as the control in this study. The pool contains four small interfering RNAs (siRNAs). The target sequences were as follows: UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, and UGGUUUACAUGUUUUCCUA. On-TARGETplus Mouse CTNNA3 siRNA was used against CTNNA3 in HL-1 cells, and the target sequences were as follows: GGAUGAAGGCUCGGCUAA, ACGGAGUACAUGAGUAAUA, CCGCAAGAGUAGCUCACAU, and GGGAGAACUCAUCGUAUCA.5 HL-1 cells were plated in six-well plates and incubated at 37 °C overnight. Transfection complexes (containing Lipofectamine 3000 and 75 pmol siRNA) were added directly to the medium. Protein samples were collected 48 h after transfection.The siRNA transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s recommended protocol. In the RNA silencing assay, 6 × 105 HL-1 cells were plated in 60-mm plates and incubated overnight at 37 °C. The cells were transduced using shLuc, shCTNNA3, or shPKP2 lentivirus from RNAi core  with polybrene (Sigma), and then selected after treating with 2 μg/mL puromycin.Following the establishment of stable cell lines, 5 × 10Western blot analysis was used to quantify the expression levels of the selected proteins in HL-1 myocytes. Proteins were extracted from cells using a RIPA lysis buffer containing 25 mM tris hydrochloride (pH 7.6), 150 mM sodium chloride, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor . The samples were homogenized by sonication and the cell debris was removed by centrifugation at 13,000 rpm for 30 min at 4 °C. We measured the protein concentration using a BCA protein quantitative analysis kit .Aliquots of 30 μg of protein extracts were denatured by boiling at 100 °C for 10 min in 5× Laemmli buffer . Denatured proteins were separated into 8–12% sodium dodecyl sulfate-polyacrylamide resolving gel, and then transferred to pre-equilibrated polyvinylidene fluoride membranes (Perkin Elmer Corporation). The membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature and then incubated with antibodies against desmosomal-associated proteins, which include: αT-catenin, connexin 43 (Sigma-Aldrich), desmocollin-2, desmoglein-2, desmoplakin, plakophilin-2 (Abcam), and plakoglobin ; fibrosis markers: transforming growth factor-beta 3 (Abcam); and internal control: glyceraldehyde-3-phosphate dehydrogenase  overnight at 4 °C. Membranes were washed with TBST 3 times for 10 min and then incubated with HRP-conjugated secondary antibody (DakoCytomation Inc.) at room temperature for 1 h. The immunoreactive proteins were visualized with thechemiluminescent reagent ECL . We used VisionWorks 8.0 software to perform a densitometric analysis of the protein expression level.RNA was isolated from HL-1 cardiomyocytes using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The total RNA was reverse-transcribed for 30 min at 50 °C using the UltraScript 2.0 cDNA Synthesis Kit  and random hexamer primers. Moreover, the mouse CTNNA3 and PKP2 transcript levels were analyzed using a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific) with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and the following primer sequences: CTNNA3 forward, 5′-TGAGATTGAGATATGGGATG-3′, and reverse, 5′-CAGTGGTATGCTTTAGTGGT-3′; PKP2 forward, 5′-CAGGTGCTGAAGCAAACCAGAG-3′, and reverse, 5′-GACACTCTCTGTCAAGGTGAGC-3′; and β-actin forward, 5′-GGCTGTATTCCCCTCCATCG-3′, and reverse, 5′-CCAGTTGGTAACAATGCCATGT-3′. β-actin was used as the internal control.p-value < 0.05 was considered statistically significant.The categorical variables were expressed as numbers and proportions and were compared using either chi-square (χ2) test or Fisher’s exact test. The continuous variables were expressed as mean ± standard deviation. The differences in the quantitative results of IHC staining between the two groups were compared using the Wilcoxon signed-rank test (Mann–Whitney U test). The Statistical Package for the Social Sciences software 22.0  was used in all analyses. A p = 0.021, From 2012 to 2017, we collected RV tissue samples from 49 Taiwanese  autopsied at Taiwan National Forensic Institute. Among them, 37 were diagnosed with ARVC based on histopathological findings, and 12 controls died from noncardiac causes. Furthermore, no structural abnormalities could be identified in the controls. The ARVC participants were younger than the controls  of 37 autopsied ARVC participants. Among these patients, one (9.09%) and ten (90.91%) had multiple gene mutations (DSP and CTNNA3) and single-gene mutations, respectively. The IHC results are shown in As shown in Furthermore, we analyzed the expression of desmosomal proteins in the ICD structure. IHC staining of the ICD shows that more than half of the autopsied ARVC patients have lost localization of αT-catenin, desmocollin-2, desmoglein-2, desmoplakin, plakoglobin, and plakophilin-2 compared with the controls d. The afp = 0.028, Ten clinical specimens from 10 patients, including 5 with definite ARVC and 5 with structurally normal heart, who underwent endomyocardial biopsy were collected for validation. We assessed the expression pattern of ICD-associated proteins using a cell model. Using siRNA, CTNNA3 gene expression was transiently knocked down to investigate the function of αT-catenin in mouse cardiomyocyte HL-1 cells. Western blotting was performed 48 h after siRNA transfection to determine protein levels. Then, we examined whether other ICD-associated molecules were affected by the silencing of CTNNA3. Notably, silencing CTNNA3 decreased the expression of desmocollin-2, plakophilin-2, and connexin 43 (86.70% ± 3.60%, 75.08% ± 11.58%, and 57.73% ± 18.62%; In light of the fact that depletion of αT-catenin reduces plakophilin-2 expression, it would be useful to identify the regulatory mechanism involving αT-catenin and plakophilin-2. Firstly, the proteins were knocked down using a lentiviral vector-based shRNA system, and it was observed that the knockdown of CTNNA3 and PKP2 did not affect each other’s mRNA expression a, but prIn the present study, IHC staining of autopsied patients with ARVC showed significant reductions in the expression of αT-catenin and plakophilin-2. These findings may provide crucial information that could assist in the accurate diagnosis of ARVC. A reduction in the expression of αT-catenin and plakophilin-2 has consistently been observed in clinical patients with definite ARVC who have undergone endomyocardial biopsy. Furthermore, after gene knockdown, αT-catenin and plakophilin-2 affect each other’s protein stability through post-translational proteolytic processing.The CTNNA3 gene encoding αT-catenin in human cells has been mapped to chromosome 10q21.3, which belongs to the αT-catenin family . Among tThe role of plakoglobin staining in the diagnosis of ARVC has been reported previously with a sensitivity of 76–91% and a specificity of 57–84% ,32,33,34The current study had several limitations. First, ARVC exhibits a wide spectrum of clinical manifestations. Selection bias could confound the present results. Therefore, a large cohort should be included to validate the generalizability of the present findings. Second, the expression of αT-catenin decreased significantly in autopsied patients with ARVC, which was not consistent in clinical patients with ARVC. The abovementioned findings highlighted that the combination of αT-catenin and plakophilin-2 expression should be used to facilitate the clinical diagnosis, particularly during the early stage. Further investigation should be conducted to validate the role of αT-catenin and plakophilin-2 expression in a large cohort. Third, the clinical phenotype was not associated with the expression of αT-catenin and plakophilin-2 in the current study. Whether the expression of ICD-associated proteins is associated with specific clinical manifestations needs to be elucidated. Lastly, the disease entities of specimens obtained from clinical endomyocardial biopsy could be varied. Therefore, further studies are warranted to assess the applicability of using the expression of αT-catenin and plakophilin-2 to differentiate patients with non-ischemic cardiomyopathy rather than ARVC.The depletion of αT-catenin reduced the expression of desmocollin-2, plakophilin-2, and connexin 43, thereby mimicking the pathogenesis of ARVC. However, αT-catenin and plakophilin-2 affect each other’s protein stability through post-translational proteolytic processing. The reduction and dislocalization of αT-catenin and plakophilin-2 on the ICD can be observed in autopsied patients and clinical patients with ARVC irrespective of the identifiable gene mutations. Therefore, it provides a diagnostic value using IHC staining to detect αT-catenin and plakophilin-2 to facilitate the diagnosis of ARVC.