Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate renal fibrosis in diabetic nephropathy by targeting Hedgehog/SMO signaling.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease globally. Currently, there are no effective drugs for the treatment of DN. Although several studies have reported the therapeutic potential of mesenchymal stem cells, the underlying mechanisms remain largely unknown. Here, we report that both human umbilical cord MSCs (UC-MSCs) and UC-MSC-derived exosomes (UC-MSC-exo) attenuate kidney damage, and inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis in streptozotocin-induced DN rats. Strikingly, the Hedgehog receptor, smoothened (SMO), was significantly upregulated in the kidney tissues of DN patients and rats, and positively correlated with EMT and renal fibrosis. UC-MSC and UC-MSC-exo treatment resulted in decrease of SMO expression. In vitro co-culture experiments revealed that UC-MSC-exo reduced EMT of tubular epithelial cells through inhibiting Hedgehog/SMO pathway. Collectively, UC-MSCs inhibit EMT and renal fibrosis by delivering exosomes and targeting Hedgehog/SMO signaling, suggesting that UC-MSCs and their exosomes are novel anti-fibrotic therapeutics for treating DN.

A highly sensitive and specific Homo1-based real-time qPCR method for quantification of human umbilical cord mesenchymal stem cells in rats.

BACKGROUND: Owing to the characteristics of easier access in vitro, low immunogenicity, and high plasticity, human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are considered as a promising cell-based drugs for clinical application. No internationally recognized technology exists to evaluate the pharmacokinetics and distribution of cell-based drugs in vivo. METHODS: We determined the human-specific gene sequence, Homo1, from differential fragments Homo sapiens mitochondrion and Rattus norvegicus mitochondrion. The expression of Homo1 was utilized to determine the distribution of UC-MSCs in the normal and diabetic nephropathy (DN) rats. RESULTS: We observed a significant correlation between the number of UC-MSCs and the expression level of Homo1. Following intravenous transplantation, the blood levels of UC-MSCs peaked at 30 min. A large amount of intravenously injected MSCs were trapped in the lungs, but the number of them decreased rapidly after 24 h. Additionally, the distribution of UC-MSCs in the kidneys of DN rats was significantly higher than that of normal rats. CONCLUSIONS: In this study, we establish a highly sensitive and specific Homo1-based real-time quantitative PCR method to quantify the distribution of human UC-MSCs in rats. The method provides guidelines for the safety research of cells in preclinical stages.

Experimental study of the bond behavior of 400MPa grade hot-rolled ribbed steel bars in steel fibre reinforced concrete.

Present studies show that steel fibres can improve the bond of steel bar in steel fibre reinforced concrete (SFRC) with a correlation to the fibre factor and the fibre distribution uniformity. As a foundation of high-flowability SFRC working together with 400 MPa grade hot-rolled ribbed (HRB400) steel bar in reinforced structures, the bond between them was evaluated through a series of pull-out testing on 48 specimens with a central arranged steel bar. The bond behaviours of steel bar were estimated with a constant bond length of 5d (d is the diameter of steel bar) embedded in high-flowability SFRC, the main research parameters included the ingot mill steel fibres with a fibre volume fraction varied from 0.8 to 2.0%, the strength grade C40 and C50 of SFRC or referenced conventional concrete, and the diameter of steel bars varied from 14 to 20 mm. Results showed that the high-flowability SFRC compacted with a slight vibration is beneficial to improve the bond failure pattern since steel fibres effectively eliminate the crack appeared on the SFRC blocks during the pulling out of steel bar, leading to all specimens failed with the steel bar pull out of SFRC blocks. The bond strength was dominant by the SFRC strength, and obviously strengthened with the increase of fibre volume fraction, while the peak-slip was slightly influenced by the diameter of steel bar. By conducting analyses of test data, equations for calculating the bond strength and the peak-slip are proposed accounting for the effect of steel fibres. Then the predicting method for the anchorage length is suggested linking with different design codes for concrete structures. Compared with test results of this study, a little shorter anchorage length of steel bar in SFRC is obtained from the specification of Chinese code JGJ/T46, which should be noticed to ensure a rational anchorage of ribbed steel bar in SFRC with ingot mill steel fibres.

Loss of macrophage TSC1 exacerbates sterile inflammatory liver injury through inhibiting the AKT/MST1/NRF2 signaling pathway.

Tuberous sclerosis complex 1 (TSC1) plays important roles in regulating innate immunity. However, the precise role of TSC1 in macrophages in the regulation of oxidative stress response and hepatic inflammation in liver ischemia/reperfusion injury (I/R) remains unknown. In a mouse model of liver I/R injury, deletion of myeloid-specific TSC1 inhibited AKT and MST1 phosphorylation, and decreased NRF2 accumulation, whereas activated TLR4/NF-κB pathway, leading to increased hepatic inflammation. Adoptive transfer of AKT- or MST1-overexpressing macrophages, or Keap1 disruption in myeloid-specific TSC1-knockout mice promoted NRF2 activation but reduced TLR4 activity and mitigated I/R-induced liver inflammation. Mechanistically, TSC1 in macrophages promoted AKT and MST1 phosphorylation, and protected NRF2 from Keap1-mediated ubiquitination. Furthermore, overexpression AKT or MST1 in TSC1-knockout macrophages upregulated NRF2 expression, downregulated TLR4/NF-κB, resulting in reduced inflammatory factors, ROS and inflammatory cytokine-mediated hepatocyte apoptosis. Strikingly, TSC1 induction in NRF2-deficient macrophages failed to reverse the TLR4/NF-κB activity and production of pro-inflammatory factors. Conclusions: Macrophage TSC1 promoted the activation of the AKT/MST1 signaling pathway, increased NRF2 levels via reducing Keap1-mediated ubiquitination, and modulated oxidative stress-driven inflammatory responses in liver I/R injury. Our findings underscore the critical role of macrophage TSC1 as a novel regulator of innate immunity and imply the therapeutic potential for the treatment of sterile liver inflammation in transplant recipients. Schematic illustration of macrophage TSC1-mediated AKT/MST1/NRF2 signaling pathway in I/R-triggered liver inflammation. Macrophage TSC1 can be activated in I/R-stressed livers. TSC1 activation promotes phosphorylation of AKT and MST1, which in turn increases NRF2 expression and inhibits ROS production and TLR4/NF-κB activation, resulting in reduced hepatocellular apoptosis in I/R-triggered liver injury.

Fluoride-Free Synthesis of 2D Titanium Carbide (MXenes) Assisted by scCO2 -Based Ternary Solution.

2D MXene-Ti3 C2 Tx holds great promise in various electronic applications, especially for electromagnetic interference (EMI) shielding devices and supercapacitors. Ti3 C2 Tx synthesis typically involves the use of hazardous fluorine-containing chemicals that can result in the formation of inert fluoride functional groups on the surface of Ti3 C2 Tx , severely degrading its properties and posing a threat to the performance of electron transfer among electrical devices. Herein, a supercritical carbon dioxide-based ternary solution (scCO2 /DMSO/HCl) to produce fluoride-free Ti3 C2 Tx in mild conditions (via 0.5 m HCl, 20 MPa, 32 °C) is reported. The fluorine-free Ti3 C2 Tx films electrode presents an excellent gravimetric capacitance of 320 F g-1 at 2 mV s-1 in 1 m H2 SO4 . Besides, it is demonstrated that fluorine-free Ti3 C2 Tx films exhibit outstanding EMI shielding efficiency of 53.12 dB at 2.5 µm thickness. The findings offer a mild and practical approach to producing fluoride-free Ti3 C2 Tx and open opportunities for exploring MXenes' potential applications in various fields.

NIR-II light triggered burst-release cascade nanoreactor for precise cancer chemotherapy.

The current strategy of co-delivering copper ions and disulfiram (DSF) to generate cytotoxic CuET faces limitations in achieving rapid and substantial CuET production, specifically in tumor lesions. To overcome this challenge, we introduce a novel burst-release cascade reactor composed of phase change materials (PCMs) encapsulating ultrasmall Cu2-xSe nanoparticles (NPs) and DSF (DSF/Cu2-xSe@PCM). Once triggered by second near-infrared (NIR-II) light irradiation, the reactor swiftly releases Cu2-xSe NPs and DSF, enabling catalytic reactions that lead to the rapid and massive production of Cu2-xSe-ET complexes, thereby achieving in situ chemotherapy. The mechanism of the burst reaction is due to the unique properties of ultrasmall Cu2-xSe NPs, including their small size, multiple defects, and high surface activity. These characteristics allow DSF to be directly reduced and chelated on the surface defect sites of Cu2-xSe, forming Cu2-xSe-ET complexes without the need for copper ion release. Additionally, Cu2-xSe-ET has demonstrated a similar (to CuET) anti-tumor activity through increased autophagy, but with even greater potency due to its unique two-dimensional-like structure. The light-triggered cascade of interlocking reactions, coupled with in situ explosive generation of tumor-suppressive substances mediated by the size and valence of Cu2-xSe, presents a promising approach for the development of innovative nanoplatforms in the field of precise tumor chemotherapy.

Targeting Microglia/Macrophages Notch1 Protects Neurons from Pyroptosis in Ischemic Stroke.

BACKGROUND AND AIMS: The immune-inflammatory cascade and pyroptosis play an important role in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). The maintenance of immune homeostasis is inextricably linked to the Notch signaling pathway, but whether myeloid Notch1 affects microglia polarization as well as neuronal pyroptosis in CIRI is not fully understood. This study was designed to clarify the role of myeloid Notch1 in CIRI, providing new therapeutic strategies for ischemic stroke. METHODS AND RESULTS: Myeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to middle cerebral artery occlusion (MCAO). After 3 days of CIRI, we evaluated the neurological deficit score and cerebral infarction volume. Immunofluorescence staining was used to detect the expression of Notch1 and microglial subtype markers. Cerebral infiltrating macrophages were detected by flow cytometry. RT-qPCR was used to detect pro-inflammatory cytokines. Western blot was used to detect the expression of pyroptosis related proteins. The Notch1-siRNA transfected BV2 cells were co-cultured with HT22 cells to investigate the potential mechanisms by which microglial Notch1 affects neuronal pyroptosis induced by anoxia/reoxygenation in vitro. We found that Notch1 was activated in cerebral microglia/macrophages after CIRI. Myeloid Notch1 deficiency decreased the cerebral infarct volume (24.17 ± 3.29 vs. 36.17 ± 2.27, p < 0.001), neurological function scores (2.33 ± 0.47 vs. 3.17 ± 0.37, p < 0.001) and the infiltration of peripheral monocytes/macrophages (3.26 ± 0.53 vs. 5.67 ± 0.57, p < 0.01). Strikingly, myeloid-specific Notch1 knockout alleviated pyroptosis. Compared with microglia M1, increased microglia M2 were detected in the ischemic penumbra. In parallel in vitro co-culture experiments, we found that Notch1 knockdown in microglial BV2 cells inhibited anoxia/reoxygenation-induced JAK2/STAT3 activation and pyroptosis in hippocampal neuron HT22 cells. CONCLUSIONS: Our findings elucidate the underlying mechanism of the myeloid Notch1 signaling pathway in regulating neuronal pyroptosis in CIRI, suggesting that targeting myeloid-specific Notch1 is an effective strategy for the treatment of ischemic stroke.

An Orthogonal Test Study on the Preparation of Self-Compacting Underwater Non-Dispersible Concrete.

To ensure a limited washout loss rate and the self-compaction of underwater concrete, the mix proportion design of underwater non-dispersible concrete is a key technology that has not been completely mastered. In view of this aspect, an orthogonal test study was carried out in this paper on the workability, washout resistance, and compressive strength of underwater non-dispersible concrete. Six factors with five levels were considered, which included the water/binder ratio, the sand ratio, the maximum particle size of the coarse aggregate, the content of the dispersion resistance agent, the content of superplasticizer, and the dosage of fly ash. Using a range and variance analysis, the sensitivity and significance of these factors were analyzed on the slump and slump-flow, the flow time, the washout loss rate, the pH value, and the compressive strength at the curing ages of 7 days and 28 days. The results indicated that the water/binder ratio and the content of the dispersion resistance agent were strong in terms of their sensitivity and significance on the workability and washout resistance, and the water/binder ratio and the dosage of fly ash were strong in terms of their sensitivity and significance on the compressive strength. With the joint fitness of the test results, formulas for predicting the slump-flow, washout loss rate, and compressive strength of underwater non-dispersible concrete were proposed considering the main impact factors.

Serum­derived exosomal hsa­let­7b­5p as a biomarker for predicting the severity of coronary stenosis in patients with coronary heart disease and hyperglycemia.

Exosomal microRNAs (miRNAs/miRs) are potential biomarkers for the diagnosis and treatment of cardiovascular disease, and hyperglycemia serves an important role in the development of atherosclerosis. The present study aimed to investigate the expression profile of serum­derived exosomal miRNAs in coronary heart disease (CHD) with hyperglycemia, and to identify effective biomarkers for predicting coronary artery lesions. Serum samples were collected from eight patients with CHD and hyperglycemia and eight patients with CHD and normoglycemia, exosomes were isolated and differentially expressed miRNAs (DEMIs) were filtered using a human miRNA microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using standard enrichment computational methods for the target genes of DEMIs. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the values of the selected DEMIs in predicting the severity of coronary stenosis. A total of 10 DEMIs, including four upregulated miRNAs (hsa­let­7b­5p, hsa­miR­4313, hsa­miR­4665­3p and hsa­miR­940) and six downregulated miRNAs (hsa­miR­4459, hsa­miR­4687­3p, hsa­miR­6087, hsa­miR­6089, hsa­miR­6740­5p and hsa­miR­6800­5p), were screened in patients with CHD and hyperglycemia. GO analysis showed that the 'cellular process', 'single­organism process' and 'biological regulation' were significantly enriched. KEGG pathway analysis revealed that the 'mTOR signaling pathway', 'FoxO signaling pathway' and 'neurotrophin signaling pathway' were significantly enriched. Among these DEMIs, only hsa­let­7b­5p expression was positively correlated with both hemoglobin A1C levels and Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery score. ROC curves showed that hsa­let­7b­5p could serve as an effective biomarker for differentiating the severity of coronary stenosis. In conclusion, the present study demonstrated that serum­derived exosomal hsa­let­7b­5p is upregulated in patients with CHD and hyperglycemia, and may serve as a noninvasive biomarker for the severity of coronary stenosis.

Human Serum Albumin Based Nanodrug Delivery Systems: Recent Advances and Future Perspective.

With the success of several clinical trials of products based on human serum albumin (HSA) and the rapid development of nanotechnology, HSA-based nanodrug delivery systems (HBNDSs) have received extensive attention in the field of nanomedicine. However, there is still a lack of comprehensive reviews exploring the broader scope of HBNDSs in biomedical applications beyond cancer therapy. To address this gap, this review takes a systematic approach. Firstly, it focuses on the crystal structure and the potential binding sites of HSA. Additionally, it provides a comprehensive summary of recent progresses in the field of HBNDSs for various biomedical applications over the past five years, categorized according to the type of therapeutic drugs loaded onto HSA. These categories include small-molecule drugs, inorganic materials and bioactive ingredients. Finally, the review summarizes the characteristics and current application status of HBNDSs in drug delivery, and also discusses the challenges that need to be addressed for the clinical transformation of HSA formulations and offers future perspectives in this field.

Nature-inspired nanocarriers for improving drug therapy of atherosclerosis.

Atherosclerosis (AS) has emerged as one of the prevalent arterial vascular diseases characterized by plaque and inflammation, primarily causing disability and mortality globally. Drug therapy remains the main treatment for AS. However, a series of obstacles hinder effective drug delivery. Nature, from natural micro-/nano-structural biological particles like natural cells and extracellular vesicles to the distinctions between the normal and pathological microenvironment, offers compelling solutions for efficient drug delivery. Nature-inspired nanocarriers of synthetic stimulus-responsive materials and natural components, such as lipids, proteins and membrane structures, have emerged as promising candidates for fulfilling drug delivery needs. These nanocarriers offer several advantages, including prolonged blood circulation, targeted plaque delivery, targeted specific cells delivery and controlled drug release at the action site. In this review, we discuss the nature-inspired nanocarriers which leverage the natural properties of cells or the microenvironment to improve atherosclerotic drug therapy. Finally, we provide an overview of the challenges and opportunities of applying these innovative nature-inspired nanocarriers.

Vibronic and Cationic Features of 2-Fluorobenzonitrile and 3-Fluorobenzonitrile Studied by REMPI and MATI Spectroscopy and Franck-Condon Simulations.

Fluorinated organic compounds have superior physicochemical properties than general organic compounds due to the strong C-F single bond; they are widely used in medicine, biology, pesticides, and materials science. In order to gain a deeper understanding of the physicochemical properties of fluorinated organic compounds, fluorinated aromatic compounds have been investigated by various spectroscopic techniques. 2-fluorobenzonitrile and 3-fluorobenzonitrile are important fine chemical intermediates and their excited state S1 and cationic ground state D0 vibrational features remain unknown. In this paper, we used two-color resonance two photon ionization (2-color REMPI) and mass analyzed threshold ionization (MATI) spectroscopy to study S1 and D0 state vibrational features of 2-fluorobenzonitrile and 3-fluorobenzonitrile. The precise excitation energy (band origin) and adiabatic ionization energy were determined to be 36,028 ± 2 cm-1 and 78,650 ± 5 cm-1 for 2-fluorobenzonitrile and 35,989 ± 2 cm-1 and 78,873 ± 5 cm-1 for 3-fluorobenzonitrile, respectively. The density functional theory (DFT) at the levels of RB3LYP/aug-cc-pvtz, TD-B3LYP/aug-cc-pvtz, and UB3LYP/aug-cc-pvtz were used to calculate the stable structures and vibrational frequencies for the ground state S0, excited state S1, and cationic ground state D0, respectively. Franck-Condon spectral simulations for transitions of S1 ← S0 and D0 ← S1 were performed based on the above DFT calculations. The theoretical and experimental results were in good agreement. The observed vibrational features in S1 and D0 states were assigned according to the simulated spectra and the comparison with structurally similar molecules. Several experimental findings and molecular features were discussed in detail.

Hyaluronic Acid-Based Nanocarriers for Anticancer Drug Delivery.

Hyaluronic acid (HA), a main component of the extracellular matrix, is widely utilized to deliver anticancer drugs due to its biocompatibility, biodegradability, non-toxicity, non-immunogenicity and numerous modification sites, such as carboxyl and hydroxyl groups. Moreover, HA serves as a natural ligand for tumor-targeted drug delivery systems, as it contains the endocytic HA receptor, CD44, which is overexpressed in many cancer cells. Therefore, HA-based nanocarriers have been developed to improve drug delivery efficiency and distinguish between healthy and cancerous tissues, resulting in reduced residual toxicity and off-target accumulation. This article comprehensively reviews the fabrication of anticancer drug nanocarriers based on HA in the context of prodrugs, organic carrier materials (micelles, liposomes, nanoparticles, microbubbles and hydrogels) and inorganic composite nanocarriers (gold nanoparticles, quantum dots, carbon nanotubes and silicon dioxide). Additionally, the progress achieved in the design and optimization of these nanocarriers and their effects on cancer therapy are discussed. Finally, the review provides a summary of the perspectives, the lessons learned so far and the outlook towards further developments in this field.

Experimental Study on the Performance of Steel-Fiber-Reinforced Concrete for Remote-Pumping Construction.

Remote-pumped concrete for infrastructure construction is a key innovation of the mechanized and intelligent construction technology. This has brought steel-fiber-reinforced concrete (SFRC) into undergoing various developments, from conventional flowability to high pumpability with low-carbon features. In this regard, an experimental study on the mixing proportion design and the pumpability and mechanical properties of SFRC was conducted for remote pumping. Using the absolute volume method based on the steel-fiber-aggregate skeleton packing test, the water dosage and the sand ratio were adjusted with an experimental study on reference concrete with the premise of varying the volume fraction of steel fiber from 0.4% to 1.2%. The test results of the pumpability of fresh SFRC indicated that the pressure bleeding rate and the static segregation rate were not the controlling indices due to the fact that they were far below the limits of the specifications, and the slump flowability fitted for remote-pumping construction was verified by a lab pumping test. Although the rheological properties of the SFRC charactered by the yield stress and the plastic viscosity increased with the volume fraction of steel fiber, those of mortar used as a lubricating layer during the pumping was almost constant. The cubic compressive strength of the SFRC had a tendency to increase with the volume fraction of steel fiber. The reinforcement effect of steel fiber on the splitting tensile strength of the SFRC was similar to the specifications, while its effect on the flexural strength was higher than the specifications due to the special feature of steel fibers distributed along the longitudinal direction of the beam specimens. The SFRC had excellent impact resistance with an increased volume fraction of steel fiber and presented acceptable water impermeability.

Indoor Robot Path Planning Using an Improved Whale Optimization Algorithm.

An improved whale optimization algorithm is proposed to solve the problems of the original algorithm in indoor robot path planning, which has slow convergence speed, poor path finding ability, low efficiency, and is easily prone to falling into the local shortest path problem. First, an improved logistic chaotic mapping is applied to enrich the initial population of whales and improve the global search capability of the algorithm. Second, a nonlinear convergence factor is introduced, and the equilibrium parameter A is changed to balance the global and local search capabilities of the algorithm and improve the search efficiency. Finally, the fused Corsi variance and weighting strategy perturbs the location of the whales to improve the path quality. The improved logical whale optimization algorithm (ILWOA) is compared with the WOA and four other improved whale optimization algorithms through eight test functions and three raster map environments for experiments. The results show that ILWOA has better convergence and merit-seeking ability in the test function. In the path planning experiments, the results are better than other algorithms when comparing three evaluation criteria, which verifies that the path quality, merit-seeking ability, and robustness of ILWOA in path planning are improved.

Human Umbilical Cord Mesenchymal Stem Cells Inhibit Pyroptosis of Renal Tubular Epithelial Cells through miR-342-3p/Caspase1 Signaling Pathway in Diabetic Nephropathy.

Diabetic nephropathy (DN) is one of the microvascular complications of diabetes. Recent studies suggest that the pyroptosis of renal tubular epithelial cell plays a critical role in DN. Currently, effective therapeutic strategies to counteract and reverse the progression of DN are lacking. Mesenchymal stem cells (MSCs) represent an attractive therapeutic tool for tissue damage and inflammation owing to their unique immunomodulatory properties. However, the underlying mechanisms remain largely unknown. In the present study, we found that human umbilical cord MSCs (UC-MSCs) can effectively ameliorate kidney damage and reduce inflammation in DN rats. Importantly, UC-MSC treatment inhibits inflammasome-mediated pyroptosis in DN. Mechanistically, we performed RNA sequencing and identified that miR-342-3p was significantly downregulated in the kidneys of DN rats. Furthermore, we found that miR-342-3p was negatively correlated with renal injury and pyroptosis in DN rats. The expression of miR-342-3p was significantly increased after UC-MSC treatment. Moreover, miR-342-3p decreased the expression of Caspase1 by targeting its 3'-UTR, which was confirmed by double-luciferase assay. Using miRNA mimic transfection, we demonstrated that UC-MSC-derived miR-342-3p inhibited pyroptosis of renal tubular epithelial cells through targeting the NLRP3/Caspase1 pathway. These findings would provide a novel intervention strategy for the use of miRNA-modified cell therapy for kidney diseases.

Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice.

BACKGROUND & AIMS: Hepatic immune system disorder plays a critical role in the pathogenesis of acute liver injury. The intrinsic signaling mechanisms responsible for dampening excessive activation of liver macrophages are not completely understood. The Notch and Hippo-YAP signaling pathways have been implicated in immune homeostasis. In this study, we investigated the interactive cell signaling networks of Notch1/YAP pathway during acute liver injury. METHODS: Myeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to lipopolysaccharide/D-galactosamine toxicity. Some mice were injected via the tail vein with bone marrow-derived macrophages transfected with lentivirus-expressing YAP. Some mice were injected with YAP siRNA using an in vivo mannose-mediated delivery system. RESULTS: We found that the activated Notch1 and YAP signaling in liver macrophages were closely related to lipopolysaccharide/D-galactosamine-induced acute liver injury. Macrophage/neutrophil infiltration, proinflammatory mediators, and hepatocellular apoptosis were markedly ameliorated in Notch1M-KO mice. Importantly, myeloid Notch1 deficiency depressed YAP signaling and facilitated M2 macrophage polarization in the injured liver. Furthermore, YAP overexpression in Notch1M-KO livers exacerbated liver damage and shifted macrophage polarization toward the M1 phenotype. Mechanistically, macrophage Notch1 signaling could transcriptionally activate YAP gene expression. Reciprocally, YAP transcriptionally upregulated the Notch ligand Jagged1 gene expression and was essential for Notch1-mediated macrophage polarization. Finally, dual inhibition of Notch1 and YAP in macrophages further promoted M2 polarization and alleviated liver damage. CONCLUSIONS: Our findings underscore a novel molecular insight into the Notch1-YAP circuit for controlling macrophage polarization in acute liver injury, raising the possibility of targeting macrophage Notch1-YAP circuit as an effective strategy for liver inflammation-related diseases.

Development of a high-throughput micropatterned agarose scaffold for consistent and reproducible hPSC-derived liver organoids.

Liver organoids represent emerging human-relevantin vitroliver models that have a wide range of biomedical applications in basic medical studies and preclinical drug discovery. However, the generation of liver organoids currently relies on the conventional Matrigel dome method, which lacks precise microenvironmental control over organoid growth and results in significant heterogeneity of the formed liver organoids. Here, we demonstrate a novel high-throughput culture method to generate uniform liver organoids from human pluripotent stem cell-derived foregut stem cells in micropatterned agarose scaffold. By using this approach, more than 8000 uniformly-sized liver organoids containing liver parenchyma cells, non-parenchymal cells, and a unique stem cell niche could be efficiently and reproducibly generated in a 48-well plate with a size coefficient of variation significance smaller than that in the Matrigel dome. Additionally, the liver organoids highly expressed liver-specific markers, including albumin (ALB), hepatocyte nuclear factor 4 alpha (HNF4α), and alpha-fetoprotein (AFP), and displayed liver functions, such as lipid accumulation, glycogen synthesis, ALB secretion, and urea synthesis. As a proof of concept, we evaluated the acute hepatotoxicity of acetaminophen (APAP) in these organoids and observed APAP-induced liver fibrosis. Overall, we expect that the liver organoids will facilitate wide biomedical applications in hepatotoxicity analysis and liver disease modeling.

High-throughput bioengineering of homogenous and functional human-induced pluripotent stem cells-derived liver organoids via micropatterning technique.

Human pluripotent stem cell-derived liver organoids are emerging as more human-relevant in vitro models for studying liver diseases and hepatotoxicity than traditional hepatocyte cultures and animal models. The generation of liver organoids is based on the Matrigel dome method. However, the organoids constructed by this method display significant heterogeneity in their morphology, size, and maturity. Additionally, the formed organoid is randomly encapsulated in the Matrigel dome, which is not convenient for in situ staining and imaging. Here, we demonstrate an approach to generate a novel type of liver organoids via micropatterning technique. This approach enables the reproducible and high-throughput formation of bioengineered fetal liver organoids with uniform morphology and deterministic size and location in a multiwell plate. The liver organoids constructed by this technique closely recapitulate some critical features of human liver development at the fetal stage, including fetal liver-specific gene and protein expression, glycogen storage, lipid accumulation, and protein secretion. Additionally, the organoids allow whole-mount in-situ staining and imaging. Overall, this new type of liver organoids is compatible with the pharmaceutical industry's widely-used preclinical drug discovery tools and will facilitate liver drug screening and hepatotoxic assessment.

Notch-activated mesenchymal stromal/stem cells enhance the protective effect against acetaminophen-induced acute liver injury by activating AMPK/SIRT1 pathway.

BACKGROUND: Notch signaling plays important roles in regulating innate immunity. However, little is known about the role of Notch in mesenchymal stromal/stem cell (MSC)-mediated immunomodulation during liver inflammatory response. METHODS: Notch activation in human umbilical cord-derived MSCs was performed by a tissue culture plate coated with Notch ligand, recombinant human Jagged1 (JAG1). Mice were given intravenous injection of Notch-activated MSCs after acetaminophen (APAP)-induced acute liver injury. Liver tissues were collected and analyzed by histology and immunohistochemistry. RESULTS: MSC administration reduced APAP-induced hepatocellular damage, as manifested by decreased serum ALT levels, intrahepatic macrophage/neutrophil infiltration, hepatocellular apoptosis and proinflammatory mediators. The anti-inflammatory activity and therapeutic effects of MSCs were greatly enhanced by Notch activation via its ligand JAG1. However, Notch2 disruption in MSCs markedly diminished the protective effect of MSCs against APAP-induced acute liver injury, even in the presence of JAG1 pretreatment. Strikingly, Notch-activated MSCs promoted AMP-activated protein kinase (AMPKα) phosphorylation, increased the sirtuins 1 (SIRT1) deacetylase expression, but downregulated spliced X-box-binding protein 1 (XBP1s) expression and consequently reduced NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation. Furthermore, SIRT1 disruption or XBP1s overexpression in macrophages exacerbated APAP-triggered liver inflammation and augmented NLRP3/caspase-1 activity in MSC-administrated mice. Mechanistic studies further demonstrated that JAG1-pretreated MSCs activated Notch2/COX2/PGE2 signaling, which in turn induced macrophage AMPK/SIRT1 activation, leading to XBP1s deacetylation and inhibition of NLRP3 activity. CONCLUSIONS: Activation of Notch2 is required for the ability of MSCs to reduce the severity of APAP-induced liver damage in mice. Our findings underscore a novel molecular insights into MSCs-mediated immunomodulation by activating Notch2/COX2/AMPK/SIRT1 pathway and thus provide a new strategy for the treatment of liver inflammatory diseases.