Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the ...rate-limiting step of de novo lipogenesis and regulates fatty acid β-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in patients with NASH; however, the effect of ACC inhibition on liver fibrosis has not been reported.
A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, diethylnitrosamine. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments.
Inhibition of ACC reduced the activation of TGF-β-stimulated HSCs, as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which inhibition of de novo lipogenesis blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require de novo lipogenesis for activation. Consistent with this direct antifibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat choline-deficient, high-fat diet model and in response to chronic diethylnitrosamine-induced liver injury (in the absence of hepatic lipid accumulation).
In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the profibrogenic activity of HSCs. Thus, small molecule inhibitors of ACC may lessen fibrosis by reducing lipotoxicity in hepatocytes and by preventing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH.
Hepatic fibrosis is the most important predictor of liver-related outcomes in patients with non-alcoholic steatohepatitis (NASH). Small molecule inhibitors of acetyl-CoA carboxylase (ACC) reduce hepatic fat content and markers of liver injury in patients with NASH. Herein, we report that inhibition of ACC and de novo lipogenesis also directly suppress the activation of hepatic stellate cells – the primary cell responsible for generating fibrotic scar in the liver – and thus fibrosis. These data provide further evidence for the use of ACC inhibitors to treat patients with NASH and advanced fibrosis.
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•ACC inhibition blocks TGF-β-induced hepatic stellate cell activation.•ACC inhibition blocks tension-mediated activation of primary rat and human hepatic stellate cells.•Inhibiting de novo lipogenesis targets hepatic stellate cells’ reliance on increased glycolysis and oxidative phosphorylation.•ACC inhibition in vivo significantly reduces fibrosis in 4 models of non-alcoholic steatohepatitis.
Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the ...phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.
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•Sequence-independent monoclonal antibodies against phosphohistidine were developed•pHis mAbs are isomer specific (1-pHis or 3-pHis) and do not bind pTyr•pHis mAbs can be used in immunoblotting, IF, and immunoaffinity purification•Immunofluorescence reveals roles for 1-pHis and 3-pHis in phagocytosis and mitosis
Sequence-independent monoclonal antibodies that specifically recognize histidine phosphorylation sites allow identification of pHis substrates and functional studies of this posttranslational modification, using a variety of immunological, proteomic, and biological assays.
The Scandinavian Caledonides comprise nappe stacks of far-travelled allochthons that record the opening and closure of the Iapetus Ocean, culminating with the collision of Baltica and Laurentia. The ...Seve Nappe Complex (SNC) of the Scandinavian Caledonides comprises relics of the outermost Baltoscandian passive margin that were subducted to mantle depths during ocean closure. Subduction of these rocks overprinted much of the Neoproterozoic record for Iapetus Ocean formation, and as a result, much of the work conducted in the SNC has focused solely on the Caledonian orogenic history.
In this study, we combined petrological and geochronological work to expand the knowledge about the Neoproterozoic metamorphic history of the Baltoscandian margin in the understudied Váivančohkka-Salmmečohkat region of the northern Scandinavian Caledonides. The work focused on rocks that belong to the upper gneiss unit, which constitutes part of SNC in the region. The unit comprises migmatitic paragneisses and garnet-mica schist containing metamafic bodies. U-Pb zircon and Th-U-total Pb monazite dating of the migmatitic paragneiss yielded consistent age of metamorphism in 602 ± 5 Ma and 599 ± 3 Ma, respectively. A similar U-Pb age of 604 ± 8 Ma was obtained for the zircon from the leucocratic vein transecting the amphibolite within the studied gneiss. Interestingly, no Caledonian ages were identified. Likewise, no evidence for high or ultra-high pressure conditions was found, neither in the gneisses/schists nor in the metamafic rocks. Petrographic observations and calculated metamorphic P-T conditions indicate that rocks belonging to upper gneiss unit underwent upper-amphibolite facies metamorphism in a melt stability field; 8.0–10.5 kbar at 750–790 °C.
Since the studied rocks underwent high-grade metamorphism in the Ediacaran and lack obvious evidence for Caledonian high-pressure metamorphism, the studied part of the SNC offers an extraordinary insight into the Late Neoproterozoic history of the hot, extended Baltoscandian margin.
•The Salmmečohkat Core Complex developed in hyperextended magma-rich Baltoscandian margin.•The upper gneiss unit of the SNC underwent high-grade metamorphism in the Ediacaran.•No evidence for Caledonian high pressure metamorphism.
Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild ...type mice, maximal expression of HO-1 in the skin was observed on the 2(nd) and 3(rd) days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.
The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as ...phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.
Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal-regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates ...hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.
The Seve Nappe Complex exposed in the Kittelfjäll area of the northern Scandinavian Caledonides comprises a volcano-sedimentary succession representing the Baltica passive margin, which was ...metamorphosed during the Iapetus Ocean closure. Garnet amphibolites, together with their host migmatitic paragneisses, record a potential (U)HP event followed by decompression-driven migmatization. The garnet amphibolites were originally thought to represent retrogressively altered granulites. The petrological and geochemical features of a studied garnet amphibolite allow for speculation about a peridotitic origin. Zirconium (Zr) content in rutile inclusions hosted in garnet in paragneisses points to near-peak temperatures between 738 °C and 780 °C, which is in agreement with the c. 774 °C obtained from the matrix rutile in the garnet amphibolite. The matrix rutile in multiple paragneiss samples records temperatures below 655 °C and 726 °C. Whereas the LA-ICP-MS U-Pb dating of zircon cores revealed the age spectrum from Paleoproterozoic to early Paleozoic, suggesting a detrital origin of zircon cores in paragneisses, the metamorphic zircon rims show an Early Ordovician cluster c. 475–469 Ma. Additionally, zircon cores and rims from the garnet amphibolite yielded an age of c. 473 Ma. The REE patterns of the Caledonian zircon rims from the paragneisses show overall low LREE concentrations, different from declining to rising trends in HREE (LuN/GdN = 0.49–38.76). Despite the textural differences, the cores and rims in zircon from the garnet amphibolite show similar REE patterns of low LREE and flat to rising HREE (LuN/GdN = 3.96–65.13). All zircon rims in both lithologies display a negative Eu anomaly. Hence, we interpret the reported ages as the growth of metamorphic zircon during migmatization, under granulite facies conditions related to exhumation from (U)HP conditions.
The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We ...have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.
The article presents methods of making the rock bolt support more yieldable, especially for a stratified roof. Alongside the increasing depth of exploitation of raw material deposits, rock bolt ...support units are more often designed taking into account more intensive deformations and displacements of underground excavations. In the article, a room and pillar method with mined roof bending and roof reinforcement with bolt patterns of 1 m × 1 m, 1.5 m × 1.5 m and 2 m × 2 m is presented. Moreover, the laboratory tests included 1.8 m long bolts, which were embedded segmentally on the lengths of 100 mm, 150 mm and 200 mm were tested. Based on the load–displacement characteristics, the deformation energy for flat and profiled dome bearing plates was calculated. Making the segmentally embedded resin rock bolt support yieldable enabled it to perform additional work. Furthermore, it was found that rock bolt support with a dome bearing plate took over 2.5 times more energy compared to a rock bolt support equipped with a flat bearing plate.
Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor ...tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson's disease. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.
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