The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells.

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

Polymeric nanofibrous nerve conduits coupled with laminin for peripheral nerve regeneration.

Artificial nerve guidance conduits (NGCs) are being investigated as an alternative to autografts, since autografts are limited in supply. A polycaprolactone (PCL)-based spiral NGC with crosslinked laminin on aligned nanofibers was evaluated in vivo post a successful in vitro assessment. PC-12 cell assays confirmed that the aligned nanofibers functionalized with laminin were able to guide and enhance neurite outgrowth. In the rodent model, the data demonstrated that axons were able to regenerate across the critical nerve gap, when laminin was present. Without laminin, the spiral NGC with aligned nanofibers group resulted in a random cluster of extracellular matrix tissue following injuries. The reversed autograft group performed best, showing the most substantial improvement based on nerve histological assessment and gastrocnemius muscle measurement. Nevertheless, the recovery time was too short to obtain meaningful data for the motor functional assessments. A full motor recovery may take up to years. An interesting observation was noted in the crosslinked laminin group. Numerous new blood capillary-like structures were found around the regenerated nerve. Owing to recent studies, we hypothesized that new blood vessel formation could be one of the key factors to increase the successful rate of nerve regeneration in the current study. Overall, these findings indicated that the incorporation of laminin into polymeric nerve conduits is a promising strategy for enhancing peripheral nerve regeneration. However, the best combination of contact-guidance cues, haptotactic cues, and chemotactic cues have yet to be realized. The natural sequence of nerve regeneration should be studied more in-depth before modulating any strategies.

Polymeric ionically conductive composite matrices and electrical stimulation strategies for nerve regeneration: In vitro characterization.

Stem cell strategies and the use of electrical stimulation (ES) represent promising new frontiers for peripheral nerve regeneration. Composite matrices were fabricated by coating electrospun polycaprolactone/cellulose acetate micro-nanofibers with chitosan and ionically conductive (IC) polymers including, sulfonated polyaniline, and lignin sulfonate. These composite matrices were characterized for surface morphology, coating uniformity, ionic conductivity, and mechanical strength to explore as scaffold materials for nerve regeneration in conjunction with ES. Composite matrices measured conductivity in the range of 0.0049-0.0068 mS/m due to the uniform coating of sulfonated polymers on the micro-nanofibers. Thin films (2D) and composite fiber matrices (3D) of IC polymers seeded with human mesenchymal stem cells (hMSCs) were electrically stimulated at 0.5 V, 20 Hz for 1 h daily for 14 days to study the changes in cell viability, morphology, and expression of the neuronal-like phenotype. In vitro ES lead to changes in hMSCs' fibroblast morphology into elongated neurite-like structures with cell bodies for ES-treated and positive control growth factor-treated groups. Immunofluorescent staining revealed the presence of neuronal markers including ß3-tubulin, microtubule-associated protein 2, and nestin in response to ES. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1792-1805, 2019.

Nanomaterials/Nanocomposites for Osteochondral Tissue.

For many years, the avascular nature of cartilage tissue has posed a clinical challenge for replacement, repair, and reconstruction of damaged cartilage within the human body. Injuries to cartilage and osteochondral tissues can be due to osteoarthritis, sports, aggressive cancers, and repetitive stresses and inflammation on wearing tissue. Due to its limited capacity for regeneration or repair, there is a need for suitable material systems which can recapitulate the function of the native osteochondral tissue physically, mechanically, histologically, and biologically. Tissue engineering (TE) approaches take advantage of principles of biomedical engineering, clinical medicine, and cell biology to formulate, functionalize, and apply biomaterial scaffolds to aid in the regeneration and repair of tissues. Nanomaterial science has introduced new methods for improving and fortifying TE scaffolds, and lies on the forefront of cutting-edge TE strategies. These nanomaterials enable unique properties directly correlated to their sub-micron dimensionality including structural and cellular advantages. Examples include electrospun nanofibers and emulsion nanoparticles which provide nanoscale features for biomaterials, more closely replicating the 3D extracellular matrix, providing better cell adhesion, integration, interaction, and signaling. This chapter aims to provide a detailed overview of osteochondral regeneration and repair using TE strategies with a focus on nanomaterials and nanocomposites.

TNIP1 reduction sensitizes keratinocytes to post-receptor signalling following exposure to TLR agonists.

Cell level inflammatory signalling is a combination of initiation at cell membrane receptors and modulation by cytoplasmic regulatory proteins. For keratinocytes, the predominant cell type in the epidermis, this would include toll-like receptors (TLR) and cytoplasmic proteins that propagate or dampen post-receptor signalling. We previously reported that increased levels of tumor necrosis factor α induced protein 3-interacting protein 1 (TNIP1) in HaCaT keratinocytes leads to decreased expression of stress response and inflammation-associated genes. This finding suggested decreased TNIP1 levels, as seen in some cutaneous disease states, may produce the opposite effect, sensitizing cells to triggers of inflammatory signalling including those sensed by TLR. In this study of TNIP1-deficient HaCaT keratinocytes we examined intracellular signalling consequences especially those expected to produce gene expression changes downstream of TLR3 or TLR2/6 activation by Poly (I:C) or FSL-1, agonists modeling skin relevant pathogens. We found TNIP1-deficient keratinocytes are hyper-sensitive to TLR activation compared to control cells with a normal complement of TNIP1 and receiving the same agonist stimulation. TNIP1-deficient keratinocytes have increased levels of activated (phosphorylated) cytoplasmic mediators such as JNK and p38 and greater nuclear translocation of NF-κB and phospho-p38 when exposed to TLR ligands. This is consistent with significantly increased expression of several inflammatory cytokines and chemokines, such as IL-6 and IL-8. These results describe how decreased TNIP1 levels promote a hyper-sensitive state in HaCaT keratinocytes evidenced by increased activation of signalling molecules downstream of TLR agonists and increased expression of pro-inflammatory mediators. TNIP1 keratinocyte deficiency as reported for some skin diseases may predispose these cells to excessive inflammatory signalling upon exposure to viral or bacterial ligands for TLR.

Nuclear Receptors as Therapeutic Targets in Liver Disease: Are We There Yet?

Nuclear receptors (NR) are ligand-modulated transcription factors that play diverse roles in cell differentiation, development, proliferation, and metabolism and are associated with numerous liver pathologies such as cancer, steatosis, inflammation, fibrosis, cholestasis, and xenobiotic/drug-induced liver injury. The network of target proteins associated with NRs is extremely complex, comprising coregulators, small noncoding microRNAs, and long noncoding RNAs. The importance of NRs as targets of liver disease is exemplified by the number of NR ligands that are currently used in the clinics or in clinical trials with promising results. Understanding the regulation by NR during pathophysiological conditions, and identifying ligands for orphan NR, points to a potential therapeutic approach for patients with liver diseases. An overview of complex NR metabolic networks and their pharmacological implications in liver disease is presented here.

Oxidative stress-responsive transcription factor NRF2 is not indispensable for the human hepatic Flavin-containing monooxygenase-3 (FMO3) gene expression in HepG2 cells.

The flavin-containing monooxygenases (FMOs) are important for the oxidation of a variety of endogenous compounds and xenobiotics. The hepatic expression of FMO3 is highly variable and until recently, it was thought to be uninducible. In this study, human FMO3 gene regulation by the oxidative stress transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2) was examined. Constitutive FMO3 gene expression is repressed in HepG2 cells, thus this cell can be a good model for FMO3 gene regulation studies. Over-expression of NRF2 in HepG2 cells increased NRF2 target gene expression, heme oxygenase-1 (HMOX1) and NAD(P)H: quinone oxidoreductase-1 (NQO1), but did not alter FMO3 gene expression. Co-transfection studies with NRF2 or its cytosolic regulatory protein, Kelch-like ECH-associated protein 1 (KEAP1), expression vectors, along with FMO3 promoter luciferase reporter constructs of various lengths (5kb or 6kb), did not change FMO3 reporter gene activity significantly. Furthermore, treatment with tert-butyl hydroperoxide (tBHP) and tert-butyl hydroquinone (tBHQ) did not alter FMO3 reporter construct activity. In summary, in vitro results suggest that the transcriptional regulation of FMO3 might not involve the NRF2-KEAP1 regulatory pathway.

Antioxidant fractions of Khaya grandifoliola C.DC. and Entada africana Guill. et Perr. induce nuclear translocation of Nrf2 in HC-04 cells.

The in vitro antioxidant properties, cytoprotective activity, and ability to induce nuclear translocation of nuclear factor E2-related factor-2 (Nrf-2) of five solvent fractions of the methylene chloride/methanol (1:1 v/v) extract of Khaya grandifoliola (Meliaceae) and Entada africana (Fabaceae) were evaluated. Five antioxidant endpoints were used in the antioxidant activity investigation. The total phenolic content of the fractions was assessed as to the Folin-Ciocalteu method and the profile of interesting fractions analyzed by high-performance liquid chromatography (HPLC). The cytoprotective activity of fractions was determined by H2O2-induced oxidative stress in HC-04 cells by measuring lactate dehydrogenase (LDH) leakage into culture medium. HC-04 cells were used to investigate the ability to induce nuclear translocation of Nrf2. For both plants, the methylene chloride/methanol (90/10; v/v) fraction (F10), methylene chloride/methanol (75/25; v/v) (F25), and the methanolic fraction (F100) were found to have the highest total polyphenol content and exhibited high antioxidant activity strongly correlated with total polyphenol content. The cytoprotective activity of fraction F25 from both plants was comparable to that of quercetin (3.40 ± 0.05 µg/mL), inhibiting LDH leakage with a low half inhibition concentration (IC50) of 4.05 ± 0.03 and 3.8 ± 0.02 µg/mL for K. grandifoliola and E. africana, respectively. Lastly, fraction F25 of K. grandifoliola significantly (P < 0.05) induced nuclear Nrf2 translocation by sixfold, whereas that from E. africana and quercetin was only twofold. The results indicate for the first time that fraction F25 of the studied plants is more antioxidant and cytoprotective and induces nuclear translocation of Nrf2 in a human hepatocyte cell line.

Interactions Between Nuclear Receptor SHP and FOXA1 Maintain Oscillatory Homocysteine Homeostasis in Mice.

BACKGROUND & AIMS: Hyperhomocysteinemia is often associated with liver and metabolic diseases. We studied nuclear receptors that mediate oscillatory control of homocysteine homeostasis in mice. METHODS: We studied mice with disruptions in Nr0b2 (called small heterodimer partner [SHP]-null mice), betaine-homocysteine S-methyltransferase (Bhmt), or both genes (BHMT-null/SHP-null mice), along with mice with wild-type copies of these genes (controls). Hyperhomocysteinemia was induced by feeding mice alcohol (National Institute on Alcohol Abuse and Alcoholism binge model) or chow diets along with water containing 0.18% DL-homocysteine. Some mice were placed on diets containing cholic acid (1%) or cholestyramine (2%) or high-fat diets (60%). Serum and livers were collected during a 24-hour light-dark cycle and analyzed by RNA-seq, metabolomic, and quantitative polymerase chain reaction, immunoblot, and chromatin immunoprecipitation assays. RESULTS: SHP-null mice had altered timing in expression of genes that regulate homocysteine metabolism compared with control mice. Oscillatory production of S-adenosylmethionine, betaine, choline, phosphocholine, glyceophosphocholine, cystathionine, cysteine, hydrogen sulfide, glutathione disulfide, and glutathione, differed between SHP-null mice and control mice. SHP inhibited transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1. Expression of Bhmt and cystathionine γ-lyase was decreased when mice were fed cholic acid but increased when they were placed on diets containing cholestyramine or high-fat content. Diets containing ethanol or homocysteine induced hyperhomocysteinemia and glucose intolerance in control, but not SHP-null, mice. In BHMT-null and BHMT-null/SHP-null mice fed a control liquid, lipid vacuoles were observed in livers. Ethanol feeding induced accumulation of macrovesicular lipid vacuoles to the greatest extent in BHMT-null and BHMT-null/SHP-null mice. CONCLUSIONS: Disruption of Shp in mice alters timing of expression of genes that regulate homocysteine metabolism and the liver responses to ethanol and homocysteine. SHP inhibits the transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1.

Is nuclear factor erythroid 2-related factor 2 responsible for sex differences in susceptibility to acetaminophen-induced hepatotoxicity in mice?

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that positively regulates the expression and activity of cytoprotective genes during periods of oxidative stress. It has previously been shown that some Nrf2 genes are more highly expressed in livers of female than male mice. This could explain previously reported sex-related differences in susceptibility to acetaminophen (APAP) hepatotoxicity in mice, where females show greater resistance to APAP hepatotoxicity. Here, we examined, for the first time, differences in mRNA and protein expression for Nrf2 and a battery of Nrf2-dependent genes in naïve wild-type (WT) and overnight-fasted WT and Nrf2-null male and female mice following APAP treatment. Alanine aminotransferase (ALT) activity was measured as an indicator of hepatotoxicity. Hepatic mRNA and protein levels were measured by quantitative polymerase chain reaction and western blotting, respectively. Contrary to expectations, basal Nrf2 mRNA and protein expression were significantly lower in livers of naïve female than male mice. Although mRNA and/or protein expression of quinone oxidoreductase 1 and multidrug resistance-associated protein 4 was more pronounced in livers of female than male mice under some of the conditions examined, no higher global expression of Nrf2-dependent genes was detected in female mice. Furthermore, ALT activity was significantly elevated in overnight-fasted WT and Nrf2-null male mice following APAP treatment, but no increases in ALT were observed in either genotype of female mice. These results indicate that factors other than Nrf2 are responsible for the lower susceptibility of female mice to APAP hepatotoxicity.