A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing.

Diabetic wounds exhibit delayed and incomplete healing, usually due to vascular and nerve damage. Dysregulation of cellular Ca2+ homeostasis has recently been shown to be closely related to insulin resistance and type 2 diabetes mellitus. However, the involvement of this dysregulation in diabetic wound complications remains unknown. In this study, we found calcium dysregulation in patients with diabetic ulcers via tissue protein profiling. High glucose and glucometabolic toxicant stimulation considerably impaired the function of TRPC6, a pore subunit of transient receptor potential channels mediating Ca2+ influx, and mitochondria, which regulate calcium cycling and metabolism. Furthermore, we found that mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) could play a dual role in restoring the function of TRPC6 and mitochondria by delivering transcription factor SP2 and deubiquitinating enzyme USP9, respectively. MSC-sEVs could transfer SP2 that activated TRPC6 expression by binding to its specific promoter regions (-1519 to -1725 bp), thus recovering Ca2+ influx and downstream pathways. MSC-sEVs also promoted mitophagy to restore mitochondrial function by transporting USP9 that stabilized the expression of Parkin, a major player in mitophagy, thereby guaranteeing Ca2+ efflux and avoidance of Ca2+ overload. Targeting the regulation of calcium homeostasis provides a perspective for understanding diabetic wound healing, and the corresponding design of MSC-sEVs could be a potential therapeutic strategy.

Cancer-associated fibroblasts: from basic science to anticancer therapy.

Cancer-associated fibroblasts (CAFs), as a central component of the tumor microenvironment in primary and metastatic tumors, profoundly influence the behavior of cancer cells and are involved in cancer progression through extensive interactions with cancer cells and other stromal cells. Furthermore, the innate versatility and plasticity of CAFs allow their education by cancer cells, resulting in dynamic alterations in stromal fibroblast populations in a context-dependent manner, which highlights the importance of precise assessment of CAF phenotypical and functional heterogeneity. In this review, we summarize the proposed origins and heterogeneity of CAFs as well as the molecular mechanisms regulating the diversity of CAF subpopulations. We also discuss current strategies to selectively target tumor-promoting CAFs, providing insights and perspectives for future research and clinical studies involving stromal targeting.

Lactiplantibacillus plantarum NKK20 Alleviates High-Fat-Diet-Induced Nonalcoholic Fatty Liver Disease in Mice through Regulating Bile Acid Anabolism.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic disease in modern society. It is characterized by an accumulation of lipids in the liver and an excessive inflammatory response. Clinical trials have provided evidence that probiotics may prevent the onset and relapse of NAFLD. The aim of this study was to explore the effect of Lactiplantibacillus plantarum NKK20 strain (NKK20) on high-fat-diet-induced NAFLD in an ICR murine model and propose the underlying mechanism whereby NKK20 protects against NAFLD. The results showed that the administration of NKK20 ameliorated hepatocyte fatty degeneration, reduced total cholesterol and triglyceride concentrations, and alleviated inflammatory reactions in NAFLD mice. In addition, the 16S rRNA sequencing results indicated that NKK20 could decrease the abundance of Pseudomonas and Turicibacter and increase the abundance of Akkermansia in NAFLD mice. LC-MS/MS analysis showed that NKK20 could significantly increase the concentration of short-chain fatty acids (SCFAs) in the colon contents of mice. The obtained non-targeted metabolomics results revealed a significant difference between the metabolites in the colon contents of the NKK20 administration group and those in the high-fat diet group, in which a total of 11 different metabolites that were significantly affected by NKK20 were observed, and these metabolites were mainly involved in bile acid anabolism. UPLC-MS technical analysis revealed that NKK20 could change the concentrations of six conjugated and free bile acids in mouse liver. After being treated with NKK20, the concentrations of cholic acid, glycinocholic acid, and glycinodeoxycholic acid in livers of the NAFLD mice were significantly decreased, while the concentration of aminodeoxycholic acid was significantly increased. Thus, our findings indicate that NKK20 can regulate bile acid anabolism and promote the production of SCFA, which can inhibit inflammation and liver damage and thus prevent the development of NAFLD.

Extracellular vesicles: emerging roles, biomarkers and therapeutic strategies in fibrotic diseases.

Extracellular vesicles (EVs), a cluster of cell-secreted lipid bilayer nanoscale particles, universally exist in body fluids, as well as cell and tissue culture supernatants. Over the past years, increasing attention have been paid to the important role of EVs as effective intercellular communicators in fibrotic diseases. Notably, EV cargos, including proteins, lipids, nucleic acids, and metabolites, are reported to be disease-specific and can even contribute to fibrosis pathology. Thus, EVs are considered as effective biomarkers for disease diagnosis and prognosis. Emerging evidence shows that EVs derived from stem/progenitor cells have great prospects for cell-free therapy in various preclinical models of fibrotic diseases and engineered EVs can improve the targeting and effectiveness of their treatment. In this review, we will focus on the biological functions and mechanisms of EVs in the fibrotic diseases, as well as their potential as novel biomarkers and therapeutic strategies.

HucMSC-derived exosomes delivered BECN1 induces ferroptosis of hepatic stellate cells via regulating the xCT/GPX4 axis.

Activated hepatic stellate cells (HSCs) are significant in liver fibrosis. Our past investigations have shown that human umbilical cord mesenchymal stem cells (hucMSCs) and their secreted exosomes (MSC-ex) could alleviate liver fibrosis via restraining HSCs activation. However, the mechanisms underlying the efficacy were not clear. Ferroptosis is a regulatory cell death caused by excessive lipid peroxidation, and it plays a vital role in the occurrence and development of liver fibrosis. In the present study, we aimed to study the proferroptosis effect and mechanism of MSC-ex in HSCs. MSC-ex were collected and purified from human umbilical cord MSCs. Proferroptosis effect of MSC-ex was examined in HSCs line LX-2 and CCl4 induced liver fibrosis in mice. Gene knockdown or overexpression approaches were used to investigate the biofactors in MSC-ex-mediated ferroptosis regulation. Results: MSC-ex could trigger HSCs ferroptosis by promoting ferroptosis-like cell death, ROS formation, mitochondrial dysfunction, Fe2+ release, and lipid peroxidation in human HSCs line LX-2. Glutathione peroxidase 4 (GPX4) is a crucial regulator of ferroptosis. We found that intravenous injection of MSC-ex significantly decreased glutathione peroxidase 4 (GPX4) expression in activated HSCs and collagen deposition in experimental mouse fibrotic livers. Mechanistically, MSC-ex derived BECN1 promoted HSCs ferroptosis by suppressing xCT-driven GPX4 expression. In addition, ferritinophagy and necroptosis might also play a role in MSC-ex-promoted LX-2 cell death. Knockdown of BECN1 in MSC diminished proferroptosis and anti-fibrosis effects of MSC-ex in LX-2 and fibrotic livers. MSC-ex may promote xCT/GPX4 mediated HSCs ferroptosis through the delivery of BECN1 and highlights BECN1 as a potential biofactor for alleviating liver fibrosis.

Gut Microbiota Targeted Approach in the Management of Chronic Liver Diseases.

The liver is directly connected to the intestines through the portal vein, which enables the gut microbiota and gut-derived products to influence liver health. There is accumulating evidence of decreased gut flora diversity and alcohol sensitivity in patients with various chronic liver diseases, including non-alcoholic/alcoholic liver disease, chronic hepatitis virus infection, primary sclerosing cholangitis and liver cirrhosis. Increased intestinal mucosal permeability and decline in barrier function were also found in these patients. Followed by bacteria translocation and endotoxin uptake, these will lead to systemic inflammation. Specific microbiota and microbiota-derived metabolites are altered in various chronic liver diseases studies, but the complex interaction between the gut microbiota and liver is missing. This review article discussed the bidirectional relationship between the gut and the liver, and explained the mechanisms of how the gut microbiota ecosystem alteration affects the pathogenesis of chronic liver diseases. We presented gut-microbiota targeted interventions that could be the new promising method to manage chronic liver diseases.

Exosomes: Emerging Cell-Free Based Therapeutics in Dermatologic Diseases.

Exosomes are lipid bilayer vesicles released by multiple cell types. These bioactive vesicles are gradually becoming a leading star in intercellular communication involving in various pathological and physiological process. Exosomes convey specific and bioactive transporting cargos, including lipids, nucleic acids and proteins which can be reflective of their parent cells, rendering them attractive in cell-free therapeutics. Numerous findings have confirmed the crucial role of exosomes in restraining scars, burning, senescence and wound recovery. Moreover, the biology research of exosomes in cutting-edge studies are emerging, allowing for the development of particular guidelines and quality control methodology, which favor their possible application in the future. In this review, we discussed therapeutic potential of exosomes in different relevant mode of dermatologic diseases, as well as the various molecular mechanisms. Furthermore, given the advantages of favorable biocompatibility and transporting capacity, the bioengineering modification of exosomes is also involved.

Neutrophil Extracellular Traps: A New Player in Cancer Metastasis and Therapeutic Target.

Neutrophil Extracellular Traps (NETs) are neutrophil-derived extracellular scaffolds, which typically consist of fibrous decondensed chromatins decorated with histones and granule proteins. Initially discovered as a host defence mechanism of neutrophil against pathogens, they have also been implicated in the progression of sterile inflammation-associated diseases such as autoimmune disease, diabetes, and cancer. In this review, we highlight and discuss the more recent studies on the roles of NETs in cancer development, with a special focus on cancer metastasis. Moreover, we present the strategies for targeting NETs in pre-clinical models, but also the challenging questions that need to be answered in the field.

Isoflurane decreases substantia gelatinosa neuron excitability and synaptic transmission from periphery in the rat spinal dorsal horn.

Isoflurane is an inhaled anesthetic, though its actions at the cellular level remain controversial. By using acute spinal cord slices from adult rats and the whole-cell recording technique, we found that aqueous isoflurane at the minimum alveolar concentration decreased postsynaptic neural excitability and enhanced membrane conductance, while suppressing glutamate release from presynaptic afferent onto substantia gelatinosa (lamina II) neurons in the dorsal horn. The data demonstrate that isoflurane modulates synaptic transmission from peripheral to the spinal cord via both pre- and postsynaptic effects and these actions may underlie its spinal anesthesia.

MicroRNA-125b-5p Regulates Hepatocyte Proliferation During the Termination Phase of Liver Regeneration.

The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life-threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)-mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well-established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno-associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR-125b-5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV-mediated miR-125b-5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR-125b-5p in primary mouse and human hepatocytes and contributes to the pro-proliferative activity of miR-125b-5p by forkhead box G1 (FOXG1) and the cyclin-dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR-125b-5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.

Targeting extracellular vesicles-mediated hepatic inflammation as a therapeutic strategy in liver diseases.

Extracellular Vesicles (EVs) are nano- to micro-sized membranous vesicles that can be produced by normal and diseased cells. As carriers of biologically active molecules including proteins, lipids and nucleic acids, EVs mediate cell-to-cell communication and execute diverse functions by delivering their cargoes to specific cell types. Hepatic inflammation caused by virus infection, autoimmunity and malignancy is a common driver of progressive liver fibrosis and permanent liver damage. Emerging evidence has shown that EVs-mediated inflammation as critical player in the progression of liver diseases since they shuttle within the liver as well as between other tissues with inflammatory signals. Therefore, targeting inflammatory EVs could represent a potential therapeutic strategy in liver diseases. Moreover, EVs are emerging as a promising tool for intracellular delivery of therapeutic nucleic acid. In this review, we will discuss not only recent advances on the role of EVs in mediating hepatic inflammation and present strategies for targeted therapy on the context of liver diseases but also the challenging questions that need to be answered in the field.

Coordination of Necessary and Permissive Signals by PTEN Inhibition for CNS Axon Regeneration.

In the nearly 10 years since PTEN was identified as a prominent intrinsic inhibitor of CNS axon regeneration, the PTEN negatively regulated PI3K-AKT-mTOR pathway has been intensively explored in diverse models of axon injury and diseases and its mechanism for axon regeneration is becoming clearer. It is therefore timely to summarize current knowledge and discuss future directions of translational regenerative research for neural injury and neurodegenerative diseases. Using mouse optic nerve crush as an in vivo retinal ganglion cell axon injury model, we have conducted an extensive molecular dissection of the PI3K-AKT pathway to illuminate the cross-regulating mechanisms in axon regeneration. AKT is the nodal point that coordinates both positive and negative signals to regulate adult CNS axon regeneration through two parallel pathways, activating mTORC1 and inhibiting GSK3ßß. Activation of mTORC1 or its effector S6K1 alone can only slightly promote axon regeneration, whereas blocking mTORC1 significantly prevent axon regeneration, suggesting the necessary role of mTORC1 in axon regeneration. However, mTORC1/S6K1-mediated feedback inhibition prevents potent AKT activation, which suggests a key permissive signal from an unidentified AKT-independent pathway is required for stimulating the neuron-intrinsic growth machinery. Future studies into this complex neuron-intrinsic balancing mechanism involving necessary and permissive signals for axon regeneration is likely to lead eventually to safe and effective regenerative strategies for CNS repair.

miRNA-122 Protects Mice and Human Hepatocytes from Acetaminophen Toxicity by Regulating Cytochrome P450 Family 1 Subfamily A Member 2 and Family 2 Subfamily E Member 1 Expression.

Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF.

MicroRNA-125b-5p mimic inhibits acute liver failure.

The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF.

Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.

Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

MicroRNA-199a-5p inhibition enhances the liver repopulation ability of human embryonic stem cell-derived hepatic cells.

BACKGROUND & AIMS: Current hepatic differentiation protocols for human embryonic stem cells (ESCs) require substantial improvements. MicroRNAs (miRNAs) have been reported to regulate hepatocyte cell fate during liver development, but their utility to improve hepatocyte differentiation from ESCs remains to be investigated. Therefore, our aim was to identify and to analyse hepatogenic miRNAs for their potential to improve hepatocyte differentiation from ESCs. METHODS: By miRNA profiling and in vitro screening, we identified miR-199a-5p among several potential hepatogenic miRNAs. Transplantation studies of miR-199a-5p-inhibited hepatocyte-like cells (HLCs) in the liver of immunodeficient fumarylacetoacetate hydrolase knockout mice (Fah(-/-)/Rag2(-/-)/Il2rg(-/-)) were performed to assess their in vivo liver repopulation potential. For target determination, western blot and luciferase reporter assay were carried out. RESULTS: miRNA profiling revealed 20 conserved candidate hepatogenic miRNAs. By miRNA screening, only miR-199a-5p inhibition in HLCs was found to be able to enhance the in vitro hepatic differentiation of mouse as well as human ESCs. miR-199a-5p inhibition in human ESCs-derived HLCs enhanced their engraftment and repopulation capacity in the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Furthermore, we identified SMARCA4 and MST1 as novel targets of miR-199a-5p that may contribute to the improved hepatocyte generation and in vivo liver repopulation. CONCLUSIONS: Our findings demonstrate that miR-199a-5p inhibition in ES-derived HLCs leads to improved hepatocyte differentiation. Upon transplantation, HLCs were able to engraft and repopulate the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Thus, our findings suggest that miRNA modulation may serve as a promising approach to generate more mature HLCs from stem cell sources for the treatment of liver diseases.