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Background and Aims: Alcohol-associated liver disease (ALD) is a leading cause of liver-related mortality worldwide, with limited treatment options beyond abstinence and liver transplantation. Chronic alcohol consumption has been linked to magnesium (Mg 2+ ) deficiency, which can influence liver disease progression. The mechanisms underlying Mg 2+ homeostasis dysregulation in ALD remain elusive. This study aimed to investigate the role of the Mg 2+ transporter Cyclin M4 (CNNM4) in ALD by analyzing its expression patterns in patients with ALD and preclinical animal models., Approach and Results: In this study, CNNM4 is upregulated in the liver of both patients with ALD and animal models. CNNM4 overexpression triggers Mg 2+ homeostasis dysregulation, linked to ALD progression. We propose a novel therapeutic approach for ALD treatment using N -acetylgalactosamine silencing RNA technology to specifically modulate Cnnm4 expression in the liver, improving mitochondrial function and alleviating endoplasmic reticulum stress. Notably, silencing Cnnm4 restores protein isoaspartyl methyltransferase (PCMT1) activity, essential for repairing ethanol-induced protein damage. Enhancing mitochondrial activity through Cnnm4-dependent mechanisms increases S -adenosylmethionine levels, crucial for PCMT1 function, highlighting the interconnected roles of mitochondrial health and protein homeostasis in ALD treatment., Conclusions: These findings shed light on the dysregulation of Mg 2+ homeostasis in ALD, providing a promising therapeutic approach targeting CNNM4. N -acetylgalactosamine si Cnnm4 therapy boosts the repair processes of ethanol-damaged proteins through the upregulation of PCMT1 activity., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

SLN124, an N-acetylgalactosamine conjugated 19-mer short interfering RNA, is being developed to treat iron-loading anemias (including beta-thalassemia and myelodysplastic syndromes) and myeloproliferative neoplasms (polycythemia vera). Through hepatic targeting and silencing of the TMPRSS6 gene, SLN124 increases endogenous hepcidin synthesis. This is the first clinical report of an siRNA targeting a component of iron homeostasis. This first-in-human, phase 1 study assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending doses of SLN124 (1.0, 3.0, and 4.5 mg/kg) in healthy volunteers. Twenty-four participants were randomized in three sequential cohorts of eight subjects, each to receive a single dose of either SLN124 or placebo (6:2 randomization), administered subcutaneously. There were no serious or severe adverse events, or discontinuations due to adverse events, and most treatment-emergent adverse events were mild, including transient mild injection site reactions, resolving without intervention. SLN124 was rapidly absorbed, with a median t max of 4-5 h across all treatment groups, and largely eliminated from plasma by 48 h. Plasma concentrations increased in a greater than dose proportional fashion between treatment groups. In all SLN124 groups, a dose-related effect was observed across iron metabolism markers, and across erythroid markers, SLN124 resulted in increased plasma hepcidin levels, peaking around Day 29, and consequent dose-related sustained reductions in plasma iron and transferrin saturation with decreased reticulocyte production, MCHC, and MCV. Results suggest duration of action lasting up to 56 days after a single SLN124 dose, on hepcidin and hematological parameters of iron metabolism (serum iron and TSAT)., (© 2023 Silence Therapeutics plc. American Journal of Hematology published by Wiley Periodicals LLC.)

Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury., (© 2022. The Author(s).)

Background & Aims: Perturbations of intracellular magnesium (Mg 2+ ) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg 2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH)., Methods: Serum Mg 2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine., Results: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg 2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg 2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs., Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg 2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH., Lay Summary: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein., Competing Interests: Conflicts of interest The authors declare no conflicts of interest related to this submitted work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. All rights reserved.)

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in β-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β-thalassaemia mice (Hbb th3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of β-thalassaemia and related disorders., (© 2021 Silence Therapeutics. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Increased vascular permeability and leakage are hallmarks of several pathologies and determine disease progression and severity by facilitating inflammatory/metastatic cell infiltration. Using tissue-specific genetic ablation in endothelial cells, we have investigated in vivo the role of Tumor progression locus 2 (Tpl2), a mitogen-activated protein kinase kinase kinase (MAP3K) member with pleiotropic effects in inflammation and cancer. In response to proinflammatory stimuli, endothelial Tpl2 deletion alters tight junction claudin-5 protein expression through inhibition of JNK signaling and lysosomal degradation activation, resulting in reduced vascular permeability and immune cell infiltration. This results in significantly attenuated disease scores in experimental autoimmune encephalomyelitis and fewer tumor nodules in a hematogenic lung cancer metastasis model. Accordingly, pharmacologic inhibition of Tpl2 or small interfering RNA (siRNA)-mediated Tpl2 knockdown recapitulates our findings and reduces lung metastatic tumor invasions. These results establish an endothelial-specific role for Tpl2 and highlight the therapeutic potential of blocking the endothelial-specific Tpl2 pathway in chronic inflammatory and metastatic diseases., Competing Interests: Declaration of interests Ute Schaeper is employed by Silence Therapeutics GmbH. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Circulating levels of endothelial miR-150 are reduced in pulmonary arterial hypertension (PAH) and act as an independent predictor of patient survival, but links between endothelial miR-150 and vascular dysfunction are not well understood. We studied the effects of endothelial miR-150 supplementation and inhibition in PAH mice and cells from patients with idiopathic PAH. The role of selected mediators of miR-150 identified by RNA sequencing was evaluated in vitro and in vivo . Endothelium-targeted miR-150 delivery prevented the disease in Sugen/hypoxia mice, while endothelial knockdown of miR-150 had adverse effects. miR-150 target genes revealed significant associations with PAH pathways, including proliferation, inflammation, and phospholipid signaling, with PTEN-like mitochondrial phosphatase (PTPMT1) most markedly altered. PTPMT1 reduced inflammation and apoptosis and improved mitochondrial function in human pulmonary endothelial cells and blood-derived endothelial colony-forming cells from idiopathic PAH. Beneficial effects of miR-150 in vitro and in vivo were linked with PTPMT1-dependent biosynthesis of mitochondrial phospholipid cardiolipin and reduced expression of pro-apoptotic, pro-inflammatory, and pro-fibrotic genes, including c-MYB , NOTCH3 , transforming growth factor β ( TGF-β ), and Col1a1 . In conclusion, we are the first to show that miR-150 supplementation attenuates pulmonary endothelial damage induced by vascular stresses and may be considered as a potential therapeutic strategy in PAH., Competing Interests: This work involved the collaboration of B.W.-S. with Silence Therapeutics (London, UK), who provided DACC/miRNA preparations. The remaining authors declare no competing interests., (© 2020 The Author(s).)

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNA Bid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicle HEPES ) and siRNA Luci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNA Bid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

Rationale: Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood., Objective: To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension., Methods and Results: Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition., Conclusions: Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

There is an increasing prevalence of obesity and metabolic syndrome, which promote the development of non-alcoholic fatty liver disease (NAFLD), a disease that can evolve into cirrhosis and hepatocellular carcinoma. Repin1 loss was previously shown to have beneficial effects on lipid and glucose metabolism and obesity regulation. Herein, we characterized NAFLD in mice with hepatic deletion of Repin1 (LRep1-/-). For this purpose, liver disease was analysed in male LRep1-/- and wild-type mice treated with streptozotocin/high fat diet or a control diet over a period of 20 wks. Streptozotocin/high fat diet treated LRep1-/- mice showed a significant decrease in systemic and hepatic lipid accumulation, accompanied by diminished chronic inflammation and a subsequent reduction in liver injury. Remarkably, Repin1-deficient mice exhibited a lower tumour prevalence and tumour frequency, as well as a reduced liver weight/body weight index. A therapeutic approach using Repin1 siRNA in the early phase of NAFLD verified the observed beneficial effects of Repin1 deficiency. This study provides evidence that loss of Repin1 in the liver attenuates NAFLD progression, most likely by reducing fat accumulation and alleviating chronic tissue inflammation. Thus, modulating Repin1 expression may become a novel strategy and potential tool to inhibit NAFLD progression.

Transient hepatic steatosis upon liver resection supposes functional relationships between lipid metabolism and liver regeneration. Repin1 has been suggested as candidate gene for obesity and dyslipidemia by regulating key genes of lipid metabolism and lipid storage. Herein, we characterized the regenerative potential of mice with a hepatic deletion of Repin1 (LRep1-/-) after partial hepatectomy (PH) in order to determine the functional significance of Repin1 in liver regeneration. Lipid dynamics and the regenerative response were analyzed at various time points after PH. Hepatic Repin1 deficiency causes a significantly decreased transient hepatic lipid accumulation. Defects in lipid uptake, as analyzed by decreased expression of the fatty acid transporter Cd36 and Fatp5, may contribute to attenuated and shifted lipid accumulation, accompanied by altered extent and chronological sequence of liver cell proliferation in LRep1-/- mice. In vitro steatosis experiments with primary hepatocytes also revealed attenuated lipid accumulation and occurrence of smaller lipid droplets in Repin1-deficient cells, while no direct effect on proliferation in HepG2 cells was observed. Based on these results, we propose that hepatocellular Repin1 might be of functional significance for early accumulation of lipids in hepatocytes after PH, facilitating efficient progression of liver regeneration.

Background & Aims: Liver fibrosis is the outcome of chronic liver injury. Transforming growth factor-β (TGF-β) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF-β type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC-specific endoglin deletion., Methods: Eng expression was measured in human and murine samples of liver injury. After generating GFAP Cre(+) Eng Δ HSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Eng flox/flox HSCs were infected with Cre-expressing virus to deplete Endoglin and fibrogenic responses were analysed., Results: Endoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L-Eng splice variant. Comparing GFAP C re(-) Eng f/f to GFAP C re(+) Eng Δ HSC mice in toxic liver injury, livers of GFAP C re(+) Eng Δ HSC mice showed 39.9% (P < .01) higher Hydroxyproline content compared to GFAP C re(-) Eng f/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P < .05) more Collagen deposition in livers of GFAP C re(+) Eng Δ HSC mice. Similar results were obtained in mice subjected to cholestatic injury., Conclusion: Endoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α-SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF-β signalling., (© 2017 The Authors Liver International Published by John Wiley & Sons Ltd.)

Development of portosystemic collateral vessels and gastroesophageal varices is responsible for the most serious clinical consequences of portal hypertension, but effective clinical therapies are limited. Here we developed and investigated the therapeutic potential of an innovative liposomally-formulated short-interfering RNA (siRNA) technology based on clinical stage components, capable to attenuate production of the endothelial kinase insert domain receptor (KDR), which controls portosystemic collateralization and contributes to disease progression and aggravation. These siRNAs were first validated in vitro, and then, their therapeutic potential on portosystemic collateralization and pathological angiogenesis was tested in vivo in mouse models of portal hypertension (portal vein-ligation). siRNA KDR -lipoplexes efficiently transported siRNA KDR to vascular endothelial cells in mesenteric microvenules and portal vein of portal hypertensive mice, where collaterogenesis and angiogenesis take place. This systemic treatment significantly downregulated pathological KDR overexpression, without causing complete KDR knockout, preserving homeostatic baseline KDR levels and thus limiting adverse effects. siRNA KDR -lipoplex-induced endothelial-specific KDR knockdown drastically reduced by 73% the portosystemic collateralization, and impaired the pathologic angiogenic potential of vascular endothelial cells at different levels (cell proliferation, sprouting and remodeling). Targeting endothelial KDR with therapeutic siRNA KDR -lipoplexes could be a promising and plausible treatment modality for attenuating the formation of portosystemic collaterals in a clinical setting.

Acute liver failure (ALF) is a life threatening disease for which only few treatment options exist. The molecular pathways of disease progression are not well defined, but the death receptor Fas (CD95/Apo-1) appears to play a pivotal role in hepatocyte cell death and the development of ALF. Here, we explored posttranscriptional gene silencing of Fas by RNAi to inhibit pathophysiological gene expression. For targeting Fas expression in mice, Fas siRNA was formulated with the liver-specific siRNA delivery system DBTC. Treatment of mice with DBTC/siRNA(Fas) reduced Fas expression in the liver, but not in the spleen, lung, kidney or heart. Furthermore, silencing of Fas receptor was effective in blocking or reducing several aspects of ALF when it was tested in mice exposed to galactosamine/lipopolysaccharide (G/L), a well-known model of ALF. The application of DBTC/siRNA(Fas) 48 h prior G/L exposure resulted in amelioration of hepatic perfusion, reduction of hepatocellular death and increase of survival rate. The administration of DBTC/siRNA(Fas) formulation further diminished the inflammatory response upon G/L challenge, as indicated by a marked decrease of TNFα mRNA expression. However, IL-6 plasma concentration remained unaffectedly by DBTC/siRNA(Fas) formulation. Since the specific silencing of hepatic Fas expression only partially protected from inflammation, but completely attenuated apoptotic and necrotic cell death as well as microcirculatory dysfunction, the development of therapeutic strategies with DBTC lipoplex formulations to treat ALF should be combined with anti-inflammatory strategies to reach maximal therapeutic efficacy.

Activation of hepatic stellate cells (HSCs) is a key event in pathogenesis of liver fibrosis and represents an orchestral interplay of inhibiting and activating transcription factors like forkhead box f1 (Foxf1), being described to stimulate pro-fibrogenic genes in HSCs. Here, we evaluated a lipidbased liver-specific delivery system (DBTC) suitable to transfer Foxf1 siRNA specifically to HSCs and examined its antifibrotic potential on primary HSCs and LX-2 cells as well as in a murine model of bile duct ligation (BDL)-induced secondary cholestasis. Foxf1 silencing reduced proliferation capacity and attenuated contractility of HSCs. Systemic administration of DBTC-lipoplexes in mice was sufficient to specifically silence genes expressed in different liver cell types. Using intravital and immunofluorescence microscopy we confirmed the specific delivery of Cy3-labeled DBTC to the liver, and particularly to HSCs. Repeated treatment with DBTC-lipoplexes resulted in siRNA-mediated silencing of Foxf1 early after BDL and finally attenuated progression of the fibrotic process. Decreased HSC activation in-effect ameliorated liver injury as shown by substantial reduction of necrotic area and deposition of extracellular matrix. Our findings suggest that Foxf1 may serve as a target gene to disrupt progression of liver fibrosis and DBTC might provide a potentially feasible and effective tool for HSC-specific delivery of therapeutic RNA.

Objective: Angiopoietin-2, a protein secreted by stimulated endothelium and an antagonist of the endothelium-stabilizing receptor Tie2, contributes to the pathophysiology of septic multiple organ dysfunction. We tested the therapeutic potential of a pulmonary-endothelium-specific RNA interference-based angiopoietin-2 targeting strategy in sepsis., Design: Laboratory and animal research., Settings: Research laboratories of the Medical School Hannover, Department of Nephrology and Hypertension, Hannover and Silence Therapeutics GmbH, Berlin., Subjects: C57Bl/6 mice., Interventions: Lung-endothelium-specific angiopoietin-2 small interfering RNA was administered both before and after sepsis induction (cecal ligation and puncture or lipopolysaccharides) intravenously., Measurements and Main Results: Angiopoietin-2 small interfering RNA was highly specific and reduced angiopoietin-2 expression in the septic murine lungs up to 73.8% (p = 0.01) and enhanced the phosphorylation of Tie2 both in control and septic animals. Angiopoietin-2 small interfering RNA reduced pulmonary interleukin-6 transcription, intercellular adhesion molecule expression, neutrophil infiltration, and vascular leakage. Manifestations of sepsis were also attenuated in distant organs, including the kidney, where renal function was improved without affecting local angiopoietin-2 production. Finally, angiopoietin-2 small interfering RNA ameliorated the severity of illness and improved survival in cecal ligation and puncture, both as a pretreatment and as a rescue intervention., Conclusion: The Tie2 antagonist angiopoietin-2 represents a promising target against sepsis-associated multiple organ dysfunction. A novel RNA interference therapeutic approach targeting gene expression in the pulmonary endothelium could be a clinically relevant pharmacological strategy to reduce injurious angiopoietin-2 synthesis.

Purpose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node metastasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the pharmacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four different preclinical mouse models., Experimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes was tested in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous metastasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules (repeated low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. Primary tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistochemistry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to provide an insight into molecular changes on depletion of PKN3, in support of efficacy results., Results: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formation of spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well as in mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an increase in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change endothelial function, resulting in reduced colonization and micrometastasis formation., Conclusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation, which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy., (©2010 AACR.)

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density.

Gab1 is a multiadaptor protein that has been shown to be required for multiple processes in embryonic development and oncogenic transformation. Gab1 functions by amplifying signal transduction downstream of various receptor tyrosine kinases through recruitment of multiple signaling effectors, including phosphatidylinositol 3-kinase and Shp2. Until now, the functional significance of individual interactions in vivo was not known. Here we have generated knockin mice that carry point mutations in either the P13K or Shp2 binding sites of Gab1. We show that different effector interactions with Gab1 play distinct biological roles downstream of Gab1 during the development of different organs. Recruitment of phosphatidylinositol 3-kinase by Gab1 is essential for EGF receptor-mediated embryonic eyelid closure and keratinocyte migration, and the Gab1-Shp2 interaction is crucial for Met receptor-directed placental development and muscle progenitor cell migration to the limbs. Furthermore, we investigate the dual association of Gab1 with the Met receptor. By analyzing knockin mice with mutations in the Grb2 or Met binding site of Gab1, we show that the requirements for Gab1 recruitment to Met varies in different biological contexts. Either the direct or the indirect interaction of Gab1 with Met is sufficient for Met-dependent muscle precursor cell migration, whereas both modes of interaction are required and neither is sufficient for placenta development, liver growth, and palatal shelf closure. These data demonstrate that Gab1 induces different biological responses through the recruitment of distinct effectors and that different modes of recruitment for Gab1 are required in different organs.

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the EpoR.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

The docking protein Gab1 binds phosphorylated c-Met receptor tyrosine kinase directly and mediates signals of c-Met in cell culture. Gab1 is phosphorylated by c-Met and by other receptor and nonreceptor tyrosine kinases. Here, we report the functional analysis of Gab1 by targeted mutagenesis in the mouse, and compare the phenotypes of the Gab1 and c-Met mutations. Gab1 is essential for several steps in development: migration of myogenic precursor cells into the limb anlage is impaired in Gab1-/- embryos. As a consequence, extensor muscle groups of the forelimbs are virtually absent, and the flexor muscles reach less far. Fewer hindlimb muscles exist, which are smaller and disorganized. Muscles in the diaphragm, which also originate from migratory precursors, are missing. Moreover, Gab1-/- embryos die in a broad time window between E13.5 and E18.5, and display reduced liver size and placental defects. The labyrinth layer, but not the spongiotrophoblast layer, of the placenta is severely reduced, resulting in impaired communication between maternal and fetal circulation. Thus, extensive similarities between the phenotypes of c-Met and HGF/SF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1-/-:c-Met+/- embryos. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin receptors, which require IRS1 and IRS2 as specific docking proteins.

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

Adenovirus E1B 19 kDa protein protects against cell death induced by viral infection and certain external stimuli. The Bcl-2 protein can functionally substitute for the E1B 19 kDa protein. To identify cellular targets for the 19 kDa protein, we used the two-hybrid screen in yeast. We have isolated cDNAs for three different proteins, designated Nip1, Nip2, and Nip3, that interact with the 19 kDa protein. Mutational analysis indicates that these proteins do not associate with 19 kDa mutants defective in suppression of cell death, suggesting a correlation between interaction of these proteins and suppression of cell death. These proteins also associate with discrete sequence motifs in the Bcl-2 protein that are homologous to motifs of the 19 kDa protein. Our results suggest that two diverse proteins, the E1B 19 kDa and the Bcl-2 proteins, promote cell survival through interaction with a common set of cellular proteins.