Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Dexamethasone induces trabecular meshwork cell myofibroblast transdifferentiation through ARHGEF26.

Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-ß pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Pregnane X receptor inhibits the transdifferentiation of hepatic stellate cells by down-regulating periostin expression.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that plays a key role in drug metabolism. Recently, PXR was found to attenuate the development of liver cancer by suppressing epithelial-mesenchymal transition (EMT) in liver cancer cells in a mouse model of two-stage chemical carcinogenesis. To elucidate the role of PXR in the EMT of liver cancer cells, we focused on its role in hepatic stellate cells (HSCs), which are components of the tumor microenvironment in hepatocellular carcinoma (HCC). Human HSC-derived LX-2 cells stably expressed destabilization domain (DD)-fused human PXR (hPXR-LX2 cells). Human HCC-derived HepG2 cells were transfected with the EMT marker VIM promoter-regulated reporter plasmid and co-cultured with hPXR-LX2 cells or treated with hPXR-LX2-derived conditioned medium (CM). Co-culture or CM treatment increased reporter activity in HepG2 cells. This induction was attenuated upon PXR activation in hPXR-LX2 cells by treatment with the DD-stabilizing chemical Shield-1 and the human PXR ligand rifampicin. PXR activation in hPXR-LX2 cells exhibited inhibition of TGF-ß1-induced transdifferentiation, supported by observations of morphological changes and protein or mRNA levels of the transdifferentiation markers COL1A1 and FN1. PXR activation in hPXR-LX2 cells also attenuated the mRNA levels of the key transdifferentiation factor, POSTN. Treatment of hPXR-LX2 cells with recombinant POSTN restored the PXR-mediated suppression of transdifferentiation. Reporter assays with the POSTN promoter showed that PXR inhibited the NF-κB-mediated transcription of POSTN. Consequently, PXR activation in HSCs is expected to inhibit transdifferentiation by down-regulating POSTN expression, thereby suppressing EMT of liver cancer cells.

Platelet Membrane-Coated Curcumin-PLGA Nanoparticles Promote Astrocyte-Neuron Transdifferentiation for Intracerebral Hemorrhage Treatment.

Intracerebral hemorrhage (ICH) is a hemorrhagic disease with high mortality and disability rates. Curcumin is a promising drug for ICH treatment due to its multiple biological activities, but its application is limited by its poor watersolubility and instability. Herein, platelet membrane-coated curcumin polylactic-co-glycolic acid (PLGA) nanoparticles (PCNPs) are prepared to achieve significantly improved solubility, stability, and sustained release of curcumin. Fourier transform infrared spectra and X-ray diffraction assays indicate good encapsulation of curcumin within nanoparticles. Moreover, it is revealed for the first time that curcumin-loaded nanoparticles can not only suppress hemin-induced astrocyte proliferation but also induce astrocytes into neuron-like cells in vitro. PCNPs are used to treat rat ICH by tail vein injection, using in situ administration as control. The results show that PCNPs are more effective than curcumin-PLGA nanoparticles in concentrating on hemorrhagic lesions, inhibiting inflammation, suppressing astrogliosis, promoting neurogenesis, and improving motor functions. The treatment efficacy of intravenously administered PCNPs is comparable to that of in situ administration, indicating a good targeting effect of PCNPs on the hemorrhage site. This study provides a potent treatment for hemorrhagic injuries and a promising solution for efficient delivery of water-insoluble drugs using composite materials of macromolecules and cell membranes.

Decellularized skin pretreatment by monophosphoryl lipid A and lactobacillus casei supernatant accelerate skin recellularization.

BACKGROUND: Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). METHODS AND RESULTS: The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. CONCLUSION: Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.

In situ reprogramming of cardiac fibroblasts into cardiomyocytes in mouse heart with chemicals.

Cardiomyocytes are terminal differentiated cells and have limited ability to proliferate or regenerate. Condition like myocardial infarction causes massive death of cardiomyocytes and is the leading cause of death. Previous studies have demonstrated that cardiac fibroblasts can be induced to transdifferentiate into cardiomyocytes in vitro and in vivo by forced expression of cardiac transcription factors and microRNAs. Our previous study have demonstrated that full chemical cocktails could also induce fibroblast to cardiomyocyte transdifferentiation both in vitro and in vivo. With the development of tissue clearing techniques, it is possible to visualize the reprogramming at the whole-organ level. In this study, we investigated the effect of the chemical cocktail CRFVPTM in inducing in situ fibroblast to cardiomyocyte transdifferentiation with two strains of genetic tracing mice, and the reprogramming was observed at whole-heart level with CUBIC tissue clearing technique and 3D imaging. In addition, single-cell RNA sequencing (scRNA-seq) confirmed the generation of cardiomyocytes from cardiac fibroblasts which carries the tracing marker. Our study confirms the use of small molecule cocktails in inducing in situ fibroblast to cardiomyocyte reprogramming at the whole-heart level and proof-of-conceptly providing a new source of naturally incorporated cardiomyocytes to help heart regeneration.

Glucagon blockade restores functional ß-cell mass in type 1 diabetic mice and enhances function of human islets.

We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted ß-cell survival and enhanced the recovery of insulin+ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of ß-cell apoptosis which allows for robust assessment of ß-cell regeneration following caspase-8-induced diabetes, Ab-4 drove a 6.7-fold increase in ß-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-ß-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin+ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion.

Inhibition of TRPC6 suppressed TGFß-induced fibroblast-myofibroblast transdifferentiation in renal interstitial NRK-49F cells.

Renal fibrosis is a global health concern with limited curative treatment. Canonical transient receptor potential channel 6 (TRPC6), a nonselective cation channel, has been shown to regulate the renal fibrosis in murine models. However, the molecular mechanism is unclear. Fibroblast-myofibroblast transdifferentiation is one of the critical steps in the progression of renal fibrosis. In the present study, we demonstrate that transforming growth factor (TGF)-ß1 exposure significantly increases the TRPC6 expression in renal interstitial fibroblast NRK-49F cells. Pharmacological inhibition of TRPC6 and knockdown of Trpc6 by siRNA alleviate TGF-ß1-increased expression levels of α-smooth muscle actin (α-SMA) and collagen I, two key markers of myofibroblasts. Although direct activation of TRPC6 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) does not affect the expression of α-SMA and collagen I, OAG potentiates TGF-ß1-induced fibroblast-myofibroblast transdifferentiation. Further study demonstrates that TGF-ß1 exposure increases the phosphorylation level of p38 and Yes-associated protein (YAP) translocation into the nuclei. Inhibition of p38 and YAP decreases TGF-ß1-enhanced TRPC6 and α-SMA expression. In conclusion, we demonstrate that TRPC6 is a key regulator of TGF-ß1-induced fibroblast-myofibroblast transdifferentiation and provides the mechanism of how TGF-ß1 exposure regulates TRPC6 expression in NRK-49F fibroblasts.

A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development.

An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the ß-cell lineage, where it plays a central role in ß-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic ß-cell differentiation and increase ß-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFß signaling led to α-cell to ß-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances ß-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and ß-cell development.

Cholesterol-Induced Phenotypic Modulation of Smooth Muscle Cells to Macrophage/Fibroblast-like Cells Is Driven by an Unfolded Protein Response.

OBJECTIVE: Vascular smooth muscle cells (SMCs) dedifferentiate and initiate expression of macrophage markers with cholesterol exposure. This phenotypic switching is dependent on the transcription factor Klf4 (Krüppel-like factor 4). We investigated the molecular pathway by which cholesterol induces SMC phenotypic switching. Approach and Results: With exposure to free cholesterol, SMCs decrease expression of contractile markers, activate Klf4, and upregulate a subset of macrophage and fibroblast markers characteristic of modulated SMCs that appear with atherosclerotic plaque formation. These phenotypic changes are associated with activation of all 3 pathways of the endoplasmic reticulum unfolded protein response (UPR), Perk (protein kinase RNA-like endoplasmic reticulum kinase), Ire (inositol-requiring enzyme) 1α, and Atf (activating transcription factor) 6. Blocking the movement of cholesterol from the plasma membrane to the endoplasmic reticulum prevents free cholesterol-induced UPR, Klf4 activation, and upregulation of the majority of macrophage and fibroblast markers. Cholesterol-induced phenotypic switching is also prevented by global UPR inhibition or specific inhibition of Perk signaling. Exposure to chemical UPR inducers, tunicamycin and thapsigargin, is sufficient to induce these same phenotypic transitions. Finally, analysis of published single-cell RNA sequencing data during atherosclerotic plaque formation in hyperlipidemic mice provides preliminary in vivo evidence of a role of UPR activation in modulated SMCs. CONCLUSIONS: Our data demonstrate that UPR is necessary and sufficient to drive phenotypic switching of SMCs to cells that resemble modulated SMCs found in atherosclerotic plaques. Preventing a UPR in hyperlipidemic mice diminishes atherosclerotic burden, and our data suggest that preventing SMC transition to dedifferentiated cells expressing macrophage and fibroblast markers contributes to this decreased plaque burden.

Effects of first-line diabetes therapy with biguanides, sulphonylurea and thiazolidinediones on the differentiation, proliferation and apoptosis of islet cell populations.

AIMS: Metformin, rosiglitazone and sulfonylureas enhance either insulin action or secretion and thus have been used extensively as early stage anti-diabetic medication, independently of the aetiology of the disease. When administered to newly diagnosed diabetes patients, these drugs produce variable results. Here, we examined the effects of the three early stage oral hypoglycaemic agents in mice with diabetes induced by multiple low doses of streptozotocin, focusing specifically on the developmental biology of pancreatic islets. METHODS: Streptozotocin-treated diabetic mice expressing a fluorescent reporter specifically in pancreatic islet α-cells were administered the biguanide metformin (100 mg/kg), thiazolidinedione rosiglitazone (10 mg/kg), or sulfonylurea tolbutamide (20 mg/kg) for 10 days. We assessed the impact of the treatment on metabolic status of the animals as well as on the morphology, proliferative potential and transdifferentiation of pancreatic islet cells, using immunofluorescence. RESULTS: The effect of the therapy on the islet cells varied depending on the drug and included enhanced pancreatic islet ß-cell proliferation, in case of metformin and rosiglitazone; de-differentiation of α-cells and ß-cell apoptosis with tolbutamide; increased relative number of ß-cells and bi-hormonal insulin + glucagon + cells with metformin. These effects were accompanied by normalisation of food and fluid intake with only minor effects on glycaemia at the low doses of the agents employed. CONCLUSIONS: Our data suggest that metformin and rosiglitazone attenuate the depletion of the ß-cell pool in the streptozotocin-induced diabetes, whereas tolbutamide exacerbates the ß-cell apoptosis, but is likely to protect ß-cells from chronic hyperglycaemia by directly elevating insulin secretion.

Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoforms: Possible Implications for New Antifibrotic Therapies.

Cardiac fibrosis is characterized by accumulation and activation of fibroblasts and excessive production of extracellular matrix, which results in myocardial stiffening and eventually leads to heart failure. Although previous work suggests that protein kinase C (PKC) isoforms play a role in cardiac fibrosis and remodeling, the results are conflicting. Moreover, the potential of targeting PKC with pharmacological tools to inhibit pathologic fibrosis has not been fully evaluated. Here we investigated the effects of selected PKC agonists and inhibitors on cardiac fibroblast (CF) phenotype, proliferation, and gene expression using primary adult mouse CFs, which spontaneously transdifferentiate into myofibroblasts in culture. A 48-hour exposure to the potent PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 nM concentration reduced the intensity of α-smooth muscle actin staining by 56% and periostin mRNA levels by 60% compared with control. The decreases were inhibited with the pan-PKC inhibitor Gö6983 and the inhibitor of classical PKC isoforms Gö6976, suggesting that classical PKCs regulate CF transdifferentiation. PMA also induced a 33% decrease in 5-bromo-2'-deoxyuridine-positive CFs, which was inhibited with Gö6983 but not with Gö6976, indicating that novel PKC isoforms (nPKCs) regulate CF proliferation. Moreover, PMA downregulated the expression of collagen-encoding genes Col1a1 and Col3a1 nPKC-dependently, showing that PKC activation attenuates matrix synthesis in CFs. The partial PKC agonist isophthalate derivative bis(1-ethylpentyl) 5-(hydroxymethyl)isophthalate induced parallel changes in phenotype, cell cycle activity, and gene expression. In conclusion, our results reveal distinct PKC-dependent regulation of CF transdifferentiation and proliferation and suggest that PKC agonists exhibit potential as an antifibrotic treatment. SIGNIFICANCE STATEMENT: Cardiac fibrosis is a pathological process that contributes to the development of heart failure. The molecular mechanisms regulating fibrosis in the heart are, however, not fully understood, which hinders the development of new therapies. Here, we demonstrate that classical and novel protein kinase C (PKC) isoforms distinctly regulate cardiac fibroblast transdifferentiation and proliferation, the two central processes in fibrosis. Our results indicate that pharmacological PKC activation may be a promising strategy to inhibit myocardial fibrosis.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

Pharmacological modulation of RORα controls fat browning, adaptive thermogenesis, and body weight in mice.

Beiging is an attractive therapeutic strategy to fight against obesity and its side metabolic complications. The loss of function of the nuclear transcription factor RORα has been related to a lean phenotype with higher thermogenesis in sg/sg mice lacking this protein. Here we show that pharmacological modulation of RORα activity exerts reciprocal and cell-autonomous effect on UCP1 expression ex vivo, in cellulo, and in vivo. The RORα inverse-agonist SR3335 upregulated UCP1 expression in brown and subcutaneous white adipose tissue (scWAT) explants of wild-type (WT) mice, whereas the RORα agonist SR1078 had the opposite effect. We confirmed the reciprocal action of these synthetic RORα ligands on gene expression, mitochondrial mass, and uncoupled oxygen consumption rate in cultured murine and human adipocytes. Time course analysis revealed stepwise variation in gene expression, first of TLE3, an inhibitor of the thermogenic program, followed by a reciprocal effect on PRDM16 and UCP1. Finally, RORα ligands were shown to be useful tools to modulate in vivo UCP1 expression in scWAT with associated changes in this fat depot mass. SR3335 and SR1078 provoked the opposite effects on the WT mice body weight, but without any effect on sg/sg mice. This slimming effect of SR3335 was related to an increased adaptive thermogenesis of the mice, as assessed by the rectal temperature of cold-stressed mice and induction of UCP1 in scWAT, as well as by indirect calorimetry in presence or not of a ß3-adrenoceptor agonist. These data confirmed that RORα ligands could be useful tools to modulate thermogenesis and energy homeostasis.NEW & NOTEWORTHY The regulation of adipose tissue browning was not fully deciphered and required further studies explaining how the regulation of this process may be of interest for tackling obesity and related metabolic disorders. Our data confirmed the involvement of the transcription factor RORα in the regulation of nonshivering thermogenesis, and importantly, revealed the possibility to in vivo modulate its activity by synthetic ligands with beneficial consequences on fat mass and body weight of the mice.

L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes.

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Transdifferentiation of goat ear fibroblasts into lactating mammary epithelial cells induced by small molecule compounds.

Mammary epithelial cells are the only cells in the mammary glands that are capable of lactation and they are ideal for studying cellular and molecular biology mechanisms during growth, development and lactation of the mammary glands. The limiting factors in most of the currently available mammary epithelial cells are low cell viability, transgenerational efficiency and lactation function that renders them unsuitable for subsequent studies on mammary gland's cellular and lactation mechanisms and utilizing them as bioreactors. Hence, new methods are required to obtain mammary epithelial cells with high transgenerational efficiency and lactation function. In this study, transdifferentiation of goat ear fibroblasts (GEFs) into goat mammary epithelial cells (CiMECs) was induced in only eight days by five small molecule compounds, including 500 µg/mL VPA, 10 µM Tranylcypromine, 10 µM Forskolin, 1 µM TTNPB, 10 µM RepSox. Morphological observation, marker genes comparison, specific antigen expression and comparison of gene expression levels by transcriptome sequencing between the two types of cells that led to the primary deduction that CiMECs have similar biological properties to goat mammary epithelial cells (GMECs) and comparatively more lactation capacity. Therefore, we establish a novel reprogramming route to convert fibroblasts into CiMECs under fully chemically conditions. This study is expected to provide an in vitro platform for understanding cellular mechanisms such as mammary epithelial cells' fate determination and developmental differentiation, and also to find a new way to obtain a large number of functional mammary epithelial cells in vitro.

Epigallocatechin-3-gallate increases autophagic activity attenuating TGF-ß1-induced transformation of human Tenon's fibroblasts.

We previously found that epigallocatechin-3-gallate (EGCG) could inhibit the myofibroblast transformation of human Tenon's fibroblasts, however, the underlying mechanism remained unclear. We therefore investigated whether the autophagic regulation involved in the anti-fibrotic function of EGCG. The fibroblasts were subjected to transforming growth factor beta-1 (TGF-ß1) induction followed by EGCG treatments. The autophagic flux was examined by transmission electron microscopy and autophagic flux analysis. The levels of autophagy-related proteins (LC3ß and p62) and alpha-smooth muscle actin (α-SMA) were measured by Western blot and immunofluorescence. Results showed that TGF-ß1 partially inhibited the autophagic function of Tenon's fibroblasts. But this inhibition effect was rescued by LY2157299, a TGF-ßR1 selective inhibitor. Compared with the cells treated with TGF-ß1 alone, EGCG treatments increased the amount of autophagosomes and autolysosomes, evaluated the ratio of LC3-II to LC3-I and decreased p62 level. Our results indicated that EGCG could recover the activity of autophagy in the TGF-ß1-treated cells. Moreover, treatments with EGCG significantly decreased the α-SMA expression. Taken together, these findings revealed that autophagic regulation involved in the action of EGCG against TGF-ß1-induced transformation of Tenon's fibroblasts. Through increasing intracellular autophagy, EGCG could be a potential anti-fibrotic reagent for preventing subconjunctival fibrosis after glaucoma filtration surgery.

Up-regulation of FUT8 inhibits TGF-ß1-induced activation of hepatic stellate cells during liver fibrogenesis.

Liver fibrosis is a continuous wound healing response caused by chronic liver injury, and the activation of hepatic stellate cells (HSCs) is considered as the main event for it. Core fucosylation catalyzed by FUT8 refers to adding the fucosyl moiety to the innermost GlcNAc residue of N-linked oligosaccharides and is involved in many biological processes such as cell differentiation, migration, and signaling transduction. Aberrant core fucosylation is associated with a variety of diseases including cardiovascular disease, tumors and neuroinflammation, but much less is understood in liver fibrosis. Herein, we reported FUT8 mRNA level was increased in patients with liver fibrosis from GEO database and positively correlated with fibrosis progression. FUT8 expression and the core fucosylation were also elevated in TAA-induced mouse liver fibrosis model, and were mainly distributed in the fibrous septum of mouse liver. TGF-ß1, as the most pro-fibrogenic cytokine, could promote the expression of FUT8 and total core fucosylation levels in HSCs in vitro. However, up-regulation of FUT8 in turn inhibited TGF-ß1-induced trans-differentiation, migration and pro-fibrogenic signaling pathways in HSCs. In conclusion, our results suggest that the up-regulation of FUT8 inhibits TGF-ß1-induced HSC activation in a negative feedback loop, and provide potential new therapeutic strategy for liver fibrosis by targeting FUT8.

Direct Reprogramming of Somatic Cells to Neurons: Pros and Cons of Chemical Approach.

Translating successful preclinical research in neurodegenerative diseases into clinical practice has been difficult. The preclinical disease models used for testing new drugs not always appear predictive of the effects of the agents in the human disease state. Human induced pluripotent stem cells, obtained by reprogramming of adult somatic cells, represent a powerful system to study the molecular mechanisms of the disease onset and pathogenesis. However, these cells require a long time to differentiate into functional neural cells and the resetting of epigenetic information during reprogramming, might miss the information imparted by age. On the contrary, the direct conversion of somatic cells to neuronal cells is much faster and more efficient, it is safer for cell therapy and allows to preserve the signatures of donors' age. Direct reprogramming can be induced by lineage-specific transcription factors or chemical cocktails and represents a powerful tool for modeling neurological diseases and for regenerative medicine. In this Commentary we present and discuss strength and weakness of several strategies for the direct cellular reprogramming from somatic cells to generate human brain cells which maintain age-related features. In particular, we describe and discuss chemical strategy for cellular reprogramming as it represents a valuable tool for many applications such as aged brain modeling, drug screening and personalized medicine.

Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFß1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFß1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.