Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis

Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background

Emodin Prevents Intrahepatic Fat Accumulation, Inflammation and Redox Status Imbalance During Diet-Induced Hepatosteatosis in Rats

High-fat and/or high-carbohydrate diets may predispose to several metabolic disturbances including liver fatty infiltration (hepatosteatosis) or be associated with necro-inflammation and fibrosis (steatohepatitis). Several studies have emphasized the hepatoprotective effect of some natural agents. In this study, we investigated the potential therapeutic effects of the treatment with emodin, an anthraquinone derivative with anti-oxidant and anti-cancer abilities, in rats developing diet-induced hepatosteatosis and steatohepatitis. Sprague-Dawley rats were fed a standard diet (SD) for 15 weeks, or a high-fat/high-fructose diet (HFD/HF). After 5 weeks, emodin was added to the drinking water of some of the SD and HFD/HF rats. The experiment ended after an additional 10 weeks. Emodin-treated HFD/HF rats were protected from hepatosteatosis and metabolic derangements usually observed in HFD/HF animals. Furthermore, emodin exerted anti-inflammatory activity by inhibiting the HFD/HF-induced increase of tumor necrosis factor (TNF)-α. Emodin also affected the hepatocytes glutathione homeostasis and levels of the HFD/HF-induced increase of glutathionylated/phosphorylated phosphatase and tensin homolog (PTEN). In conclusion, we demonstrated that a natural agent such as emodin can prevent hepatosteatosis, preserving liver from pro-inflammatory and pro-oxidant damage caused by HFD/HF diet. These findings are promising, proposing emodin as a possible hindrance to progression of hepatosteatosis into steatohepatitis

Mutated FANCA Gene Role in the Modulation of Energy Metabolism and Mitochondrial Dynamics in Head and Neck Squamous Cell Carcinoma

Fanconi Anaemia (FA) is a rare recessive genetic disorder characterized by a defective DNA repair mechanism. Although aplastic anaemia is the principal clinical sign in FA, patients develop a head and neck squamous cell carcinoma (HNSCC) with a frequency 500-700 folds higher than the general population, which appears more aggressive, with survival of under two years. Since FA gene mutations are also associated with a defect in the aerobic metabolism and an increased oxidative stress accumulation, this work aims to evaluate the effect of FANCA mutation on the energy metabolism and the relative mitochondrial quality control pathways in an HNSCC cellular model. Energy metabolism and cellular antioxidant capacities were evaluated by oximetric, luminometric, and spectrophotometric assays. The dynamics of the mitochondrial network, the quality of mitophagy and autophagy, and DNA double-strand damage were analysed by Western blot analysis. Data show that the HNSCC cellular model carrying the FANCA gene mutation displays an altered electron transport between respiratory Complexes I and III that does not depend on the OxPhos protein expression. Moreover, FANCA HNSCC cells show an imbalance between fusion and fission processes and alterations in autophagy and mitophagy pathways. Together, all these alterations associated with the FANCA gene mutation cause cellular energy depletion and a metabolic switch to glycolysis, exacerbating the Warburg effect in HNSCC cells and increasing the growth rate. In addition, the altered DNA repair due to the FANCA mutation causes a higher accumulation of DNA damage in the HNSCC cellular model. In conclusion, changes in energy metabolism and mitochondrial dynamics could explain the strict correlation between HNSCC and FA genes, helping to identify new therapeutic targets

Mutated FANCA Gene Role in the Modulation of Energy Metabolism and Mitochondrial Dynamics in Head and Neck Squamous Cell Carcinoma

Fanconi Anaemia (FA) is a rare recessive genetic disorder characterized by a defective DNA repair mechanism. Although aplastic anaemia is the principal clinical sign in FA, patients develop a head and neck squamous cell carcinoma (HNSCC) with a frequency 500–700 folds higher than the general population, which appears more aggressive, with survival of under two years. Since FA gene mutations are also associated with a defect in the aerobic metabolism and an increased oxidative stress accumulation, this work aims to evaluate the effect of FANCA mutation on the energy metabolism and the relative mitochondrial quality control pathways in an HNSCC cellular model. Energy metabolism and cellular antioxidant capacities were evaluated by oximetric, luminometric, and spectrophotometric assays. The dynamics of the mitochondrial network, the quality of mitophagy and autophagy, and DNA double-strand damage were analysed by Western blot analysis. Data show that the HNSCC cellular model carrying the FANCA gene mutation displays an altered electron transport between respiratory Complexes I and III that does not depend on the OxPhos protein expression. Moreover, FANCA HNSCC cells show an imbalance between fusion and fission processes and alterations in autophagy and mitophagy pathways. Together, all these alterations associated with the FANCA gene mutation cause cellular energy depletion and a metabolic switch to glycolysis, exacerbating the Warburg effect in HNSCC cells and increasing the growth rate. In addition, the altered DNA repair due to the FANCA mutation causes a higher accumulation of DNA damage in the HNSCC cellular model. In conclusion, changes in energy metabolism and mitochondrial dynamics could explain the strict correlation between HNSCC and FA genes, helping to identify new therapeutic targets

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells

Exploring Charged Polymeric Cyclodextrins for Biomedical Applications

Over the years, cyclodextrin uses have been widely reviewed and their proprieties provide a very attractive approach in different biomedical applications. Cyclodextrins, due to their characteristics, are used to transport drugs and have also been studied as molecular chaperones with potential application in protein misfolding diseases. In this study, we designed cyclodextrin polymers containing different contents of β- or γ-cyclodextrin, and a different number of guanidinium positive charges. This allowed exploration of the influence of the charge in delivering a drug and the effect in the protein anti-aggregant ability. The polymers inhibit Amiloid β peptide aggregation; such an ability is modulated by both the type of CyD cavity and the number of charges. We also explored the effect of the new polymers as drug carriers. We tested the Doxorubicin toxicity in different cell lines, A2780, A549, MDA-MB-231 in the presence of the polymers. Data show that the polymers based on γ-cyclodextrin modified the cytotoxicity of doxorubicin in the A2780 cell line

Terpyridine functionalized cyclodextrin nanoparticles: metal coordination for tuning anticancer activity

Multi-metal and multi-cavity systems based on the coordination properties of terpyridine functionalized cyclodextrin polymers were synthesized and characterized. Nanoparticles decorated with terpyridine derivatives via metal coordination showed high antiproliferative activity in tumor cells

Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging

Inflammaging is one of the evolutionarily conserved mechanisms underlying aging and is defined as the long-term consequence of the chronic stimulation of the innate immune system. As macrophages are intimately involved in initiating and regulating the inflammatory process, their dysregulation plays major roles in inflammaging. The paracrine factors, and in particular extracellular vesicles (EVs), released by mesenchymal stromal cells (MSCs) retain immunoregulatory effects on innate and adaptive immune responses. In this paper, we demonstrate that EVs derived from MSCs preconditioned with hypoxia inflammatory cytokines exerted an anti-inflammatory role in the context of inflammaging. In this study, macrophages isolated from aged mice presented elevated pro-inflammatory factor levels already in basal conditions compared to the young counterpart, and this pre-activation status increased when cells were challenged with IFN-γ. EVs were able to attenuate the age-associated inflammation, inducing a decrease in the expression of TNF-α, iNOS, and the NADase CD38. Moreover, we demonstrate that EVs counteracted the mitochondrial dysfunction that affected the macrophages, reducing lipid peroxidation and hindering the age-associated impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis. These results indicate that preconditioned MSC-derived EVs might be exploited as new anti-aging therapies in a variety of age-related diseases

Polypyridine ligands as potential metallo-\u3b2-lactamase inhibitors

Bacteria have developed multiple resistance mechanisms against the most used antibiotics. In particular, zinc-dependent metallo-\u3b2-lactamase producing bacteria are a growing threat, and therapeutic options are limited. Zinc chelators have recently been investigated as metallo-\u3b2-lactamase inhibitors, as they are often able to restore carbapenem susceptibility. We synthesized polypyridyl ligands, N,N\u201f-bis(2-pyridylmethyl)-ethylenediamine, N,N,N\u201f-tris(2-pyridylmethyl)-ethylenediamine, N,N\u201f-bis(2-pyridylmethyl)-ethylenediamine-N-acetic acid (N,N,N\u201f-tris(2-pyridylmethyl)-ethylenediamine-N\u201f-acetic acid, which can form zinc(II) complexes. We tested their ability to restore the antibiotic activity of meropenem against three clinical strains isolated from blood and metallo-\u3b2-lactamase producers (Klebsiella pneumoniae, Enterobacter cloacae, and Stenotrophomonas maltophilia). We functionalized N,N,N\u201f-tris(2-pyridylmethyl)-ethylenediamine with D-alanyl-D-alanyl-D-alanine methyl ester with the aim to increase bacterial uptake. We observed synergistic activity of four polypyridyl ligands with meropenem against all tested isolates, while the combination N,N\u201f-bis(2-pyridylmethyl)-ethylenediamine and meropenem was synergistic only against New Delhi and Verona integron-encoded metallo-\u3b2-lactamase-producing bacteria. All synergistic interactions restored the antimicrobial activity of meropenem, providing a significant decrease of minimal inhibitory concentration value (by 8- to 128-fold). We also studied toxicity of the ligands in two normal peripheral blood lymphocytes

A Multidrug Approach to Modulate the Mitochondrial Metabolism Impairment and Relative Oxidative Stress in Fanconi Anemia Complementation Group A

Fanconi Anemia (FA) is a rare recessive genetic disorder characterized by aplastic anemia due to a defective DNA repair system. In addition, dysfunctional energy metabolism, lipid droplets accumulation, and unbalanced oxidative stress are involved in FA pathogenesis. Thus, to modulate the altered metabolism, Fanc-A lymphoblast cell lines were treated with quercetin, a flavonoid compound, C75 (4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid), a fatty acid synthesis inhibitor, and rapamycin, an mTOR inhibitor, alone or in combination. As a control, isogenic FA cell lines corrected with the functional Fanc-A gene were used. Results showed that: (i) quercetin recovered the energy metabolism efficiency, reducing oxidative stress; (ii) C75 caused the lipid accumulation decrement and a slight oxidative stress reduction, without improving the energy metabolism; (iii) rapamycin reduced the aerobic metabolism and the oxidative stress, without increasing the energy status. In addition, all molecules reduce the accumulation of DNA double-strand breaks. Two-by-two combinations of the three drugs showed an additive effect compared with the action of the single molecule. Specifically, the quercetin/C75 combination appeared the most efficient in the mitochondrial and lipid metabolism improvement and in oxidative stress production reduction, while the quercetin/rapamycin combination seemed the most efficient in the DNA breaks decrement. Thus, data reported herein suggest that FA is a complex and multifactorial disease, and a multidrug strategy is necessary to correct the metabolic alterations