Cardiometabolic Changes in Sirtuin1-Heterozygous Mice on High-Fat Diet and Melatonin Supplementation.

A hypercaloric fatty diet predisposes an individual to metabolic syndrome and cardiovascular complications. Sirtuin1 (SIRT1) belongs to the class III histone deacetylase family and sustains anabolism, mitochondrial biogenesis, and fat distribution. Epididymal white adipose tissue (eWAT) is involved in inflammation, whilst interscapular brown adipose tissue (iBAT) drives metabolism in obese rodents. Melatonin, a pineal indoleamine, acting as a SIRT1 modulator, may alleviate cardiometabolic damage. In the present study, we morphologically characterized the heart, eWAT, and iBAT in male heterozygous SIRT1+/- mice (HET mice) on a high-fat diet (60%E lard) versus a standard rodent diet (8.5% E fat) and drinking melatonin (10 mg/kg) for 16 weeks. Wild-type (WT) male C57Bl6/J mice were similarly fed for comparison. Cardiomyocyte fibrosis and endoplasmic reticulum (ER) stress response worsened in HET mice on a high-fat diet vs. other groups. Lipid peroxidation, ER, and mitochondrial stress were assessed by 4 hydroxy-2-nonenal (4HNE), glucose-regulated protein78 (GRP78), CCAA/enhancer-binding protein homologous protein (CHOP), heat shock protein 60 (HSP60), and mitofusin2 immunostainings. Ultrastructural analysis indicated the prevalence of atypical inter-myofibrillar mitochondria with short, misaligned cristae in HET mice on a lard diet despite melatonin supplementation. Abnormal eWAT adipocytes, crown-like inflammatory structures, tumor necrosis factor alpha (TNFα), and iBAT whitening characterized HET mice on a hypercaloric fatty diet and were maintained after melatonin supply. All these data suggest that melatonin's mechanism of action is strictly linked to full SIRT1 expression, which is required for the exhibition of effective antioxidant and anti-inflammatory properties.

Multiplexing Biosensor for the Detection of Extracellular Vesicles as Biomarkers of Tissue Damage and Recovery after Ischemic Stroke.

The inflammatory, reparative and regenerative mechanisms activated in ischemic stroke patients immediately after the event cooperate in the response to injury, in the restoration of functions and in brain remodeling even weeks after the event and can be sustained by the rehabilitation treatment. Nonetheless, patients' response to treatments is difficult to predict because of the lack of specific measurable markers of recovery, which could be complementary to clinical scales in the evaluation of patients. Considering that Extracellular Vesicles (EVs) are carriers of multiple molecules involved in the response to stroke injury, in the present study, we have identified a panel of EV-associated molecules that (i) confirm the crucial involvement of EVs in the processes that follow ischemic stroke, (ii) could possibly profile ischemic stroke patients at the beginning of the rehabilitation program, (iii) could be used in predicting patients' response to treatment. By means of a multiplexing Surface Plasmon Resonance imaging biosensor, subacute ischemic stroke patients were proven to have increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) and translocator protein (TSPO) on the surface of small EVs in blood. Besides, microglia EVs and endothelial EVs were shown to be significantly involved in the intercellular communications that occur more than 10 days after ischemic stroke, thus being potential tools for the profiling of patients in the subacute phase after ischemic stroke and in the prediction of their recovery.

Immunostaining patterns reveal potential morphogenetic role of Toll-like receptors 4 and 7 in the development of mouse respiratory system, liver and pancreas.

Toll-like receptors (TLRs) are the mammalian ortholog of Drosophila melanogaster protein Toll, originally identified for its involvement in embryonic development. In mammals, TLRs are mainly known for their ability to recognize pathogen- or damage-associated molecular patterns and, consequently, to initiate the immune response. However, it is becoming clear that TLRs can play a role also in mammal embryo development. We have previously described TLR4 and TLR7 expression in developing mouse peripheral nervous system and gastrointestinal tract. In the present study, we extended the investigation of TLR4 and TLR7 to the respiratory system and to the two main accessory organs of the digestive system, the liver and pancreas. TLR4 and TLR7 immunostaining was performed on mouse conceptuses collected at different stages, from E12 to E18. TLR4 and TLR7 immunoreactivity was evident in the embryo pancreas and liver at E12, while, in the respiratory apparatus, appeared at E14 and E17, respectively. Although further studies are required to elucidate the specific role of these TLRs in embryo development, the differential spatiotemporal TLR4 and TLR7 appearance may suggest that TLR expression in developing embryos is highly regulated for a possible their direct involvement in the formation of the organs and in the acquisition of immune-related features in preparation for the birth.

The Educational Program of Macrophages toward a Hyperprogressive Disease-Related Phenotype Is Orchestrated by Tumor-Derived Extracellular Vesicles.

Hyperprogressive disease (HPD), an aggressive acceleration of tumor growth, was observed in a group of cancer patients treated with anti-PD1/PDL1 antibodies. The presence of a peculiar macrophage subset in the tumor microenvironment is reported to be a sort of "immunological prerequisite" for HPD development. These macrophages possess a unique phenotype that it is not clear how they acquire. We hypothesized that certain malignant cells may promote the induction of an "HPD-related" phenotype in macrophages. Bone-marrow-derived macrophages were exposed to the conditioned medium of five non-small cell lung cancer cell lines. Macrophage phenotype was analyzed by microarray gene expression profile and real-time PCR. We found that human NSCLC cell lines, reported as undergoing HPD-like tumor growth in immunodeficient mice, polarized macrophages towards a peculiar pro-inflammatory phenotype sharing both M1 and M2 features. Lipid-based factors contained in cancer cell-conditioned medium induced the over-expression of several pro-inflammatory cytokines and the activation of innate immune receptor signaling pathways. We also determined that tumor-derived Extracellular Vesicles represent the main components involved in the observed macrophage re-education program. The present study might represent the starting point for the future development of diagnostic tools to identify potential hyperprogressors.

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study.

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.

Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia.

BACKGROUND: HDL (high-density lipoprotein) and its major protein component, apoA-I (apolipoprotein A-I), play a unique role in cholesterol homeostasis and immunity. ApoA-I deficiency in hyperlipidemic, atheroprone mice was shown to drive cholesterol accumulation and inflammatory cell activation/proliferation. The present study was aimed at investigating the impact of apoA-I deficiency on lipid deposition and local/systemic inflammation in normolipidemic conditions. METHODS: ApoE deficient mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I, and C57Bl/6J control mice were fed normal laboratory diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric, and whole transcriptome analyses. RESULTS: DKO were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to control mice. Only DKO showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4+ T effector memory cells was detected in blood, spleen, and skin-draining lymph nodes of DKO. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO versus apoE deficient. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and halt atherosclerosis development. CONCLUSIONS: HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation.

Mechanical Cues, E-Cadherin Expression and Cell "Sociality" Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior.

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior.

Simvastatin Prevents Liver Microthrombosis and Sepsis Induced Coagulopathy in a Rat Model of Endotoxemia.

Background: Endotoxemia causes endothelial dysfunction and microthrombosis, which are pathogenic mechanisms of coagulopathy and organ failure during sepsis. Simvastatin has potential anti-thrombotic effects on liver endothelial cells. We investigated the hemostatic changes induced by lipopolysaccharide (LPS) and explored the protective effects of simvastatin against liver vascular microthrombosis. Methods and results: We compared male Wistar rats exposed to LPS (5 mg/kg one i.p. dose) or saline in two experimental protocols­placebo (vehicle) and simvastatin (25 mg/kg die, orally, for 3 days before LPS). Morphological studies were performed by light- and electron-microscopy analyses to show intravascular fibrin deposition, vascular endothelial structure and liver damage. Peripheral- and organ-hemostatic profiles were analyzed using whole blood viscoelastometry by ROTEM, liver biopsy and western-blot/immunohistochemistry of thrombomodulin (TM), as well as immunohistochemistry of the von Willebrand factor (VWF). LPS-induced fibrin deposition and liver vascular microthrombosis were combined with a loss of sinusoidal endothelial TM expression and VWF-release. These changes were associated with parenchymal eosinophilia and necrosis. ROTEM analyses displayed hypo-coagulability in the peripheral blood that correlated with the degree of intrahepatic fibrin deposition (p < 0.05). Simvastatin prevented LPS-induced fibrin deposition by preserving TM expression in sinusoidal cells and completely reverted the peripheral hypo-coagulability caused by endotoxemia. These changes were associated with a significant reduction of liver cell necrosis without any effect on eosinophilia. Conclusions: Simvastatin preserves the antithrombotic properties of sinusoidal endothelial cells disrupted by LPS, deserving pharmacological properties to contrast sepsis-associated coagulopathy and hepatic failure elicited by endotoxemia

Macrophages Impair TLR9 Agonist Antitumor Activity through Interacting with the Anti-PD-1 Antibody Fc Domain.

BACKGROUND: A combination of TLR9 agonists and an anti-PD-1 antibody has been reported to be effective in immunocompetent mice but the role of innate immunity has not yet been completely elucidated. Therefore, we investigated the contribution of the innate immune system to this combinatorial immunotherapeutic regimens using an immunodeficient mouse model in which the effector functions of innate immunity can clearly emerge without any interference from T lymphocytes. METHODS: Athymic mice xenografted with IGROV-1 human ovarian cells, reported to be sensitive to TLR9 agonist therapy, were treated with cytosine-guanine (CpG)-oligodeoxynucleotides (ODNs), an anti-PD-1 antibody or their combination. RESULTS: We found that PD-1 blockade dampened CpG-ODN antitumor activity. In vitro studies indicated that the interaction between the anti-PD-1 antibody fragment crystallizable (Fc) domain and macrophage Fc receptors caused these immune cells to acquire an immunoregulatory phenotype, contributing to a decrease in the efficacy of CpG-ODNs. Accordingly, in vivo macrophage depletion abrogated the detrimental effect exerted by the anti-PD-1 antibody. CONCLUSION: Our data suggest that if TLR signaling is active in macrophages, coadministration of an anti-PD-1 antibody can reprogram these immune cells towards a polarization state able to negatively affect the immune response and eventually promote tumor growth.