Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Safdar, Huma; Cheung, Ka Lei; Vos, Hans L.; Gonzalez, Frank J.; Reitsma, Pieter H.; Inoue, Yusuke; Vlijmen, Bart J.M. van

doi:10.1371/journal.pone.0038104

Cited by 29 publications

(23 citation statements)

References 35 publications

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“…8,9 Mouse heads were fixed in 4% formaldehyde. All experimental procedures were approved by the Institutional Animal Welfare Committee.…”

Section: Methodsmentioning

confidence: 99%

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Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice

Heestermans¹,

Salloum-Asfar²,

Salvatori³

et al. 2016

Blood

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Key Points• Platelets, neutrophils, and coagulation factor XII are implicated as important players in experimental venous thrombosis pathophysiology.• We demonstrate that platelets, but not neutrophils, are critical in spontaneous venous thrombosis, whereas low factor XII aggravates thrombosis.Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNAmediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology. (Blood. 2016;127(21):2630-2637

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“…8,9 Mouse heads were fixed in 4% formaldehyde. All experimental procedures were approved by the Institutional Animal Welfare Committee.…”

Section: Methodsmentioning

confidence: 99%

“…8,9 siRNA-mediated hepatic silencing of Serpinc1 and Proc silencing were routinely confirmed. 6 Liver fibrin deposition was determined by immunoblotting using the monoclonal antibody 59D8.…”

Section: Liver and Blood Analysesmentioning

confidence: 96%

Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice

Heestermans¹,

Salloum-Asfar²,

Salvatori³

et al. 2016

Blood

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“…3 Selected siSerpinc1, siProc (sequences, see supplemental Methods), and control siNEG (Life Technologies) complexed with Invivofectamine (Life Technologies) were injected into the tail veins of female C57Black/6J mice (Charles River, Maastricht, the Netherlands) age 8-to 10-weeks, alone or in combination. Mice were euthanized at different time points and subjected to necropsy according to international pathology guidelines.…”

Section: Methodsmentioning

confidence: 99%

Acute and severe coagulopathy in adult mice following silencing of hepatic antithrombin and protein C production

Safdar¹,

Cheung²,

Salvatori³

et al. 2013

Blood

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Key Points• RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes, alone or in combination, in normal adult mice.• RNA interference of Serpinc1and Proc provides a novel, controlled mouse model for spontaneous venous thrombosis.Mice deficient in the anticoagulants antithrombin (Serpinc1) or protein C (Proc) display premature death due to thrombosis-related coagulopathy, thereby precluding their use in gene function studies and thrombosis models. We used RNA interference to silence Serpinc1 and/or Proc in normal adult mice. The severe coagulopathy that followed combined "knockdown" of these genes is reported. Two days after siRNA injection, thrombi (occlusive) were observed in vessels (large and medium-sized) in multiple tissues, and hemorrhages were prominent in the ocular, mandibular, and maxillary areas. Tissue fibrin deposition and reduction of plasma fibrinogen accompanied this phenotype. The coagulopathy was prevented by dabigatran etexilate treatment. Silencing of Serpinc1 alone yielded a comparable but milder phenotype with later onset. The phenotype was absent when Proc was targeted alone. We conclude that RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes in vivo and provides a novel, controlled mouse model for spontaneous venous thrombosis. (Blood. 2013;121(21):4413-4416)

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“…# 4404020, Ambion, Life Technologies Corporation, USA) and a siRNA tested in mouse primary hepatocytes to be effective to reduce HNF4α transcript and protein levels by 90% at a concentration of 3 nM were used as described previously [13]. The sequences of the two siHNF4α RNA-strands were, sense: 5 ´-AGA GGU CCA UGG UGU UUA AUU-3 ´ and antisense: 5 ´-UUA AAC ACC AUG GAC CUC UUG-3 ´ (siHNF4α, cat.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the F11, Klkb1, Cyp4v3 Gene Cluster in Livers of Metabolically Challenged Mice

et al. 2013

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Single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that harbors the coagulation factor XI (F11), prekallikrein (KLKB1), and a cytochrome P450 family member (CYP4V2) genes are associated with deep venous thrombosis (DVT). These SNPs exert their effect on DVT by modifying the circulating levels of FXI. However, SNPs associated with DVT were not necessarily all in F11, but also in KLKB1 and CYP4V2. Here, we searched for evidence for common regulatory elements within the 4q35.2 locus, outside the F11 gene, that might control FXI plasma levels and/or DVT risk. To this end, we investigated the regulation of the orthologous mouse gene cluster under several metabolic conditions that impact mouse hepatic F11 transcription. In livers of mice in which HNF4α, a key transcription factor controlling F11, was ablated, or reduced by siRNA, a strong decrease in hepatic F11 transcript levels was observed that correlated with Cyp4v3 (mouse orthologue of CYP4V2), but not by Klkb1 levels. Estrogens induced hepatic F11 and Cyp4v3, but not Klkb1 transcript levels, whereas thyroid hormone strongly induced hepatic F11 transcript levels, and reduced Cyp4v3, leaving Klkb1 levels unaffected. Mice fed a high-fat diet also had elevated F11 transcription, markedly paralleled by an induction of Klkb1 and Cyp4v3 expression. We conclude that within the mouse F11, Klkb1, Cyp4v3 gene cluster, F11 and Cyp4v3 frequently display striking parallel transcriptional responses suggesting the presence of shared regulatory elements.

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Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Cited by 29 publications

References 35 publications

Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice

Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice

Acute and severe coagulopathy in adult mice following silencing of hepatic antithrombin and protein C production

Regulation of the F11, Klkb1, Cyp4v3 Gene Cluster in Livers of Metabolically Challenged Mice

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