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1.
J Hepatol ; 75(1): 34-45, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33571553

RESUMO

BACKGROUND & AIMS: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). METHODS: Serum Mg2+ 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 Mg2+ 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 Mg2+ 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 Mg2+ 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.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hepatócitos/metabolismo , Magnésio , Hepatopatia Gordurosa não Alcoólica , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Descoberta de Drogas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Magnésio/sangue , Magnésio/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia
2.
Br J Haematol ; 194(1): 200-210, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33942901

RESUMO

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 ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). 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 ß-thalassaemia and related disorders.


Assuntos
Deferiprona/uso terapêutico , Eritropoese/efeitos dos fármacos , Hepcidinas/biossíntese , Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/prevenção & controle , Proteínas de Membrana/antagonistas & inibidores , RNA Interferente Pequeno/uso terapêutico , Talassemia beta/tratamento farmacológico , Acetilgalactosamina/administração & dosagem , Animais , Deferiprona/administração & dosagem , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Perfilação da Expressão Gênica , Hepcidinas/genética , Humanos , Ferro/sangue , Quelantes de Ferro/administração & dosagem , Sobrecarga de Ferro/etiologia , Fígado/metabolismo , Magnésio/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Espécies Reativas de Oxigênio , Serina Endopeptidases/genética , Baço/metabolismo , Baço/ultraestrutura , Zinco/metabolismo , Talassemia beta/complicações , Talassemia beta/metabolismo , Talassemia beta/fisiopatologia
3.
Apoptosis ; 24(11-12): 934-945, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31576482

RESUMO

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, siRNABid 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 (vehicleHEPES) and siRNALuci 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 siRNABid 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.


Assuntos
Acetaminofen/toxicidade , Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/metabolismo , Falência Hepática Aguda/induzido quimicamente , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Família 2 do Citocromo P450/metabolismo , Glutationa/metabolismo , Proteína HMGB1/metabolismo , Hepatócitos/patologia , Inflamação/complicações , Inflamação/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/enzimologia , Falência Hepática Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína X Associada a bcl-2/metabolismo
4.
Atherosclerosis ; 349: 240-247, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35400495

RESUMO

BACKGROUND AND AIMS: The LPA gene encodes apolipoprotein (a), a key component of Lp(a), a potent risk factor for cardiovascular disease with no specific pharmacotherapy. Here we describe the pharmacological data for SLN360, a GalNAc-conjugated siRNA targeting LPA, designed to address this unmet medical need. METHODS: SLN360 was tested in vitro for LPA knockdown in primary hepatocytes. Healthy cynomolgus monkeys received single or multiple subcutaneous doses of the SLN360 sequence ranging from 0.1 to 9.0 mg/kg to determine the pharmacokinetic and pharmacodynamic effects. Liver mRNA and serum biomarker analyses were performed. RESULTS: In vitro, the SLN360 sequence potently reduces LPA mRNA in primary cynomolgus and human hepatocytes, while no effect was observed on the expression of APOB or PLG. In vivo, SLN360 exposure peaks 2 h after subcutaneous injection with near full elimination by 24 h. Specific LPA mRNA reduction (up to 91% 2 weeks after dosing) was observed with only the 3 mg/kg group showing appreciable return to baseline (40%). No consistent dose- or time-dependent effect on the expression of APOB, PLG or a panel of sensitive markers of liver lipid accumulation was observed. Potent (up to 95%) and long lasting (≥9 weeks) serum Lp(a) reduction was observed, peaking in all active groups at day 21. The minimally effective dose was determined to be 0.3 mg/kg with an ED50 of 0.6 mg/kg. CONCLUSIONS: SLN360 induces a sustained reduction in serum Lp(a) levels in cynomolgus monkeys following subcutaneous dosing. SLN360 has potential to address the unmet need of Lp(a) reduction in cardiovascular diseases.


Assuntos
Doenças Cardiovasculares , Hiperlipidemias , Apolipoproteínas A , Apolipoproteínas B , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Humanos , Lipoproteína(a) , RNA Mensageiro , RNA Interferente Pequeno/genética
5.
Nucleic Acids Res ; 31(11): 2705-16, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12771196

RESUMO

Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.


Assuntos
Proteínas Proto-Oncogênicas , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Endorribonucleases/metabolismo , Células HeLa , Humanos , Metilação , Oligonucleotídeos Antissenso/genética , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genética , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt , Estabilidade de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Transfecção , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética
6.
Oncogene ; 22(43): 6748-63, 2003 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-14555988

RESUMO

Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-beta (TGF-beta) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-beta-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-beta-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiation-competent cells by using improved antisense molecules. We found that TGF-beta mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-beta-induced cell cycle arrest and to induce or suppress endogenous cell cycle regulators. Inhibition of Smad4 expression exhibited a partial effect, whereas inhibition of Smad2 expression had no effect. By gene expression profiling, we identified TGF-beta-dependent genes that are differentially regulated by Smad2 and Smad3 under regular growth conditions on a genome-wide scale. We show that Smad2, Smad3 and Smad4 contribute to the regulation of TGF-beta responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Transdução de Sinais , Transativadores/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Western Blotting , Ciclo Celular , Linhagem Celular , Análise Mutacional de DNA , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Immunoblotting , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteína Smad2 , Proteína Smad3 , Proteína Smad4 , Transfecção , Fator de Crescimento Transformador beta/metabolismo
7.
Res Dev Disabil ; 43-44: 123-35, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26183337

RESUMO

UNLABELLED: The MUSAD was developed as a diagnostic observational instrument in an interactional music framework. It is based on the ICD-10/DSM-5 criteria for autism spectrum disorder (ASD) and was designed to assess adults on a lower level of functioning, including individuals with severe language impairments. This study aimed to evaluate the psychometric properties of the newly developed instrument. METHODS: Calculations were based on a consecutive clinical sample of N=76 adults with intellectual and developmental disabilities (IDD) suspected of ASD. Objectivity, test-retest reliability, and construct validity were calculated and a confirmatory factor analysis was applied to verify a reduced and optimized test version. RESULTS: The structural model showed a good fit, while internal consistency of the subscales was excellent (ω>.92). Item difficulties ranged between .04≤pi≤.82 and item-total correlation from .21 to .85. Objectivity was assessed by comparing the scorings of two external raters based on a subsample of n=12; interrater agreement was .71 (ICC 2, 1). Reliability was calculated for four test repetitions: the average ICC (3, 1) was .69. Convergent ASD measures correlated significantly with the MUSAD, while the discriminant Modified Overt Aggression Scale (MOAS) showed no significant overlap. CONCLUSION: Confirmation of factorial structure and acceptable psychometric properties suggest that the MUSAD is a promising new instrument for diagnosing ASD in adults with IDD.


Assuntos
Transtorno Autístico/diagnóstico , Deficiências do Desenvolvimento/psicologia , Deficiência Intelectual/psicologia , Música , Adolescente , Adulto , Idoso , Transtorno Autístico/complicações , Transtorno Autístico/psicologia , Estudos de Coortes , Deficiências do Desenvolvimento/complicações , Análise Fatorial , Feminino , Humanos , Deficiência Intelectual/complicações , Masculino , Pessoa de Meia-Idade , Psicometria , Reprodutibilidade dos Testes , Adulto Jovem
9.
Clin Cancer Res ; 16(22): 5469-80, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21062934

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Lewis/prevenção & controle , Carcinoma Pulmonar de Lewis/secundário , Modelos Animais de Doenças , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Humanos , Injeções Intravenosas , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancer Res ; 68(23): 9788-98, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19047158

RESUMO

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.


Assuntos
Neoplasias Pancreáticas/terapia , Neoplasias da Próstata/terapia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Animais , Processos de Crescimento Celular/fisiologia , Progressão da Doença , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células HeLa , Humanos , Lipossomos/administração & dosagem , Metástase Linfática , Macaca fascicularis , Masculino , Camundongos , Camundongos SCID , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Interferência de RNA , Ratos , Transfecção/métodos
11.
EMBO J ; 23(16): 3303-13, 2004 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-15282551

RESUMO

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Membrana Basal/enzimologia , Membrana Basal/metabolismo , Membrana Basal/patologia , Catálise , Divisão Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/genética , Neoplasias da Próstata/genética , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
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