Publications by authors named "Bart Westendorp"

23 Publications

  • Page 1 of 1

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage.

Cell Rep 2020 Dec;33(9):108449

Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. Electronic address:

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/C via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/C to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.
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http://dx.doi.org/10.1016/j.celrep.2020.108449DOI Listing
December 2020

PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.

EMBO Rep 2020 12 23;21(12):e50893. Epub 2020 Nov 23.

Institute of Developmental Immunology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients.
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http://dx.doi.org/10.15252/embr.202050893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726793PMC
December 2020

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice.

Hepatology 2021 Jan;73(1):303-317

Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

Background And Aims: Up-regulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking.

Approach And Results: Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine-induced liver tumors.

Conclusion: Collectively, our findings demonstrate that atypical E2Fs can override cell-cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.
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http://dx.doi.org/10.1002/hep.31259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898887PMC
January 2021

E2F-Family Members Engage the PIDDosome to Limit Hepatocyte Ploidy in Liver Development and Regeneration.

Dev Cell 2020 02 23;52(3):335-349.e7. Epub 2020 Jan 23.

Division of Developmental Immunology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria; Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, 1090 Vienna, Austria; CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria. Electronic address:

E2F transcription factors control the cytokinesis machinery and thereby ploidy in hepatocytes. If or how these proteins limit proliferation of polyploid cells with extra centrosomes remains unknown. Here, we show that the PIDDosome, a signaling platform essential for caspase-2-activation, limits hepatocyte ploidy and is instructed by the E2F network to control p53 in the developing as well as regenerating liver. Casp2 and Pidd1 act as direct transcriptional targets of E2F1 and its antagonists, E2F7 and E2F8, that together co-regulate PIDDosome expression during juvenile liver growth and regeneration. Of note, whereas hepatocyte aneuploidy correlates with the basal ploidy state, the degree of aneuploidy itself is not limited by PIDDosome-dependent p53 activation. Finally, we provide evidence that the same signaling network is engaged to control ploidy in the human liver after resection. Our study defines the PIDDosome as a primary target to manipulate hepatocyte ploidy and proliferation rates in the regenerating liver.
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http://dx.doi.org/10.1016/j.devcel.2019.12.016DOI Listing
February 2020

Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression.

EMBO J 2019 10 2;38(20):e101430. Epub 2019 Sep 2.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S-phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/C during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCF during G2. Cyclin F binds via its cyclin domain to a conserved C-terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCF remains stable during G2. Furthermore, SCF can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCF nor the CY motifs in the C-terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical-E2F-dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F-dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression.
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http://dx.doi.org/10.15252/embj.2018101430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792010PMC
October 2019

Chk1 and 14-3-3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest.

EMBO J 2018 03 23;37(5). Epub 2018 Jan 23.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes providing them with the ability to induce a permanent S-phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post-translational mechanisms. Here, we show that atypical E2Fs can be directly phosphorylated by checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that 14-3-3 protein isoforms interact with both E2Fs in a Chk1-dependent manner. Strikingly, Chk1 phosphorylation and 14-3-3-binding did not relocate or degrade atypical E2Fs, but instead, 14-3-3 is recruited to E2F7/8 target gene promoters to possibly interfere with transcription. We observed that high levels of 14-3-3 strongly correlate with upregulated transcription of atypical E2F target genes in human cancer. Thus, we reveal that Chk1 and 14-3-3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to DNA-damaging therapeutic reagents.
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http://dx.doi.org/10.15252/embj.201797877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830916PMC
March 2018

DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells.

Stem Cells Dev 2018 01 3;27(2):133-146. Epub 2018 Jan 3.

1 Department of Clinical Sciences of Companion Animals, Utrecht University , Utrecht, the Netherlands .

Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage, HPC proliferation is triggered by external activation mechanisms from their niche. Although several important proproliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high-throughput kinome small interfering RNA (siRNA) screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing, the percentage of EdU- and phosphorylated histone H3 (pH3)-positive cells was increased, and total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F, and multivulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared with wild-type organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S-phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.
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http://dx.doi.org/10.1089/scd.2017.0139DOI Listing
January 2018

Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma.

J Clin Invest 2017 Mar 30;127(3):830-842. Epub 2017 Jan 30.

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.
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http://dx.doi.org/10.1172/JCI87583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330731PMC
March 2017

E2f8 mediates tumor suppression in postnatal liver development.

J Clin Invest 2016 08 25;126(8):2955-69. Epub 2016 Jul 25.

E2F-mediated transcriptional repression of cell cycle-dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8's tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8's DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development.
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http://dx.doi.org/10.1172/JCI85506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966321PMC
August 2016

Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.

EMBO Rep 2016 Mar 5;17(3):414-27. Epub 2016 Feb 5.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.
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http://dx.doi.org/10.15252/embr.201540984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772983PMC
March 2016

Physiological significance of polyploidization in mammalian cells.

Trends Cell Biol 2013 Nov 10;23(11):556-66. Epub 2013 Jul 10.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands.

Programmed polyploidization occurs in all mammalian species during development and aging in selected tissues, but the biological properties of polyploid cells remain obscure. Spontaneous polyploidization arises during stress and has been observed in a variety of pathological conditions, such as cancer and degenerative diseases. A major challenge in the field is to test the predicted functions of polyploidization in vivo. However, recent genetic mouse models with diminished polyploidization phenotypes represent novel, powerful tools to unravel the biological function of polyploidization. Contrary to a longstanding hypothesis, polyploidization appears to not be required for differentiation and has no obvious impact on proliferation. Instead, polyploidization leads to increased cell size and genetic diversity, which could promote better adaptation to chronic injury or stress. We discuss here the consequences of reducing polyploidization in mice and review which stress responses and molecular signals trigger polyploidization during development and disease.
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http://dx.doi.org/10.1016/j.tcb.2013.06.002DOI Listing
November 2013

HIF proteins connect the RB-E2F factors to angiogenesis.

Transcription 2013 Mar-Apr;4(2):62-6. Epub 2013 Feb 14.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Recently, we showed that E2F7 and E2F8 (E2F7/8) are critical regulators of angiogenesis through transcriptional control of VEGFA in cooperation with HIF. (1) Here we investigate the existence of other novel putative angiogenic E2F7/8-HIF targets, and discuss the role of the RB-E2F pathway in regulating angiogenesis during embryonic and tumor development.
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http://dx.doi.org/10.4161/trns.23680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646055PMC
December 2013

E2F8 is essential for polyploidization in mammalian cells.

Nat Cell Biol 2012 Nov 14;14(11):1181-91. Epub 2012 Oct 14.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht, The Netherlands.

Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.
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http://dx.doi.org/10.1038/ncb2585DOI Listing
November 2012

E2F7 and E2F8 promote angiogenesis through transcriptional activation of VEGFA in cooperation with HIF1.

EMBO J 2012 Oct 17;31(19):3871-84. Epub 2012 Aug 17.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis.
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http://dx.doi.org/10.1038/emboj.2012.231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463843PMC
October 2012

The E2F6 repressor activates gene expression in myocardium resulting in dilated cardiomyopathy.

FASEB J 2012 Jun 7;26(6):2569-79. Epub 2012 Mar 7.

Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, ON K1H 8M5, Canada.

The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P<0.05) in left ventricular end-diastolic diameter and reduced ejection fraction (<40%, P<0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F-responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P<0.01) decrease in gap junction protein connexin-43, resulting in abnormal electrocardiogram including a 24% (P<0.05) increase in PR interval. Further, mir-206, which targets connexin-43, was up-regulated 10-fold (P<0.05) in Tg myocardium. The mitogen-activated protein kinase pathway, which regulates the levels of miR-206 and connexin-43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post-transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.
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http://dx.doi.org/10.1096/fj.11-203174DOI Listing
June 2012

Tail-anchored membrane protein SLMAP is a novel regulator of cardiac function at the sarcoplasmic reticulum.

Am J Physiol Heart Circ Physiol 2012 Mar 16;302(5):H1138-45. Epub 2011 Dec 16.

Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.
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http://dx.doi.org/10.1152/ajpheart.00872.2011DOI Listing
March 2012

E2F7 represses a network of oscillating cell cycle genes to control S-phase progression.

Nucleic Acids Res 2012 Apr 17;40(8):3511-23. Epub 2011 Dec 17.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

E2F transcription factors are known to be important for timely activation of G(1)/S and G(2)/M genes required for cell cycle progression, but transcriptional mechanisms for deactivation of cell cycle-regulated genes are unknown. Here, we show that E2F7 is highly expressed during mid to late S-phase, occupies promoters of G(1)/S-regulated genes and represses their transcription. ChIP-seq analysis revealed that E2F7 binds preferentially to genomic sites containing the TTCCCGCC motif, which closely resembles the E2F consensus site. We identified 89 target genes that carry E2F7 binding sites close to the transcriptional start site and that are directly repressed by short-term induction of E2F7. Most of these target genes are known to be activated by E2Fs and are involved in DNA replication, metabolism and DNA repair. Importantly, induction of E2F7 during G(0)-G(1)/S resulted in S-phase arrest and DNA damage, whereas expression of E2F7 during G(2)/M failed to disturb cell cycle progression. These findings provide strong evidence that E2F7 directly controls the downswing of oscillating G(1)/S genes during S-phase progression.
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http://dx.doi.org/10.1093/nar/gkr1203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333892PMC
April 2012

Adverse renal effects of hydrochlorothiazide in rats with myocardial infarction treated with an ACE inhibitor.

Eur J Pharmacol 2009 Jan 3;602(2-3):373-9. Epub 2008 Dec 3.

Department of Experimental Cardiology, University Medical Center Groningen, Groningen, The Netherlands.

Diuretics, when added to angiotensin-converting enzyme inhibitors (ACE inhibitors) treatment, can augment the response to ACE inhibitors, but may have adverse effects on renal function, which negatively affect prognosis. While in heart failure rats combined therapy initially improved cardiac function and prognosis, this benefit was completely lost at later stages. We now studied renal effects of adding hydrochlorothiazide to ACE inhibitor after myocardial infarction in rats. Rats were randomized to ACE inhibitor quinapril monotherapy or quinapril with add-on hydrochlorothiazide. Survival was monitored for 14 months. Plasma creatinine, measured at 4 months, was increased by 40% in quinapril with add-on hydrochlorothiazide compared to quinapril. Although overall 14-months mortality was similar in quinapril with add-on hydrochlorothiazide and quinapril, stratification based on plasma creatinine showed increased mortality in the tertile with highest plasma creatinine (P=0.03, Log rank). With add-on hydrochlorotiazide, renal morphology displayed severe renal interstitial lesions; tubular dilatation and fibrosis. Interstitial myofibroblast transformation (alpha-smooth muscle actine staining) was increased at 8 and 14 months, and coincided with collagen deposition and interstitial inflammation (macrophage influx). In rats with quinapril monotherapy or untreated rats, renal structure was normal. Thus, adding hydrochlorotiazide to ACE inhibitor detrimentally affected not only renal function, but also renal structure in rats with myocardial infarction. Altered pharmacokinetics, resulting from a vicious circle of reduced renal function and increased circulating drug levels, may provide an explanation for the adverse renal effects and may exert unfavorable effects on long-term prognosis after myocardial infarction.
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http://dx.doi.org/10.1016/j.ejphar.2008.11.055DOI Listing
January 2009

Beneficial effects of add-on hydrochlorothiazide in rats with myocardial infarction optimally treated with quinapril.

Eur J Heart Fail 2005 Dec;7(7):1085-94

Department of Clinical Pharmacology, University of Groningen, The Netherlands.

Background: The antihypertensive and renoprotective effects of ACE inhibitor (ACEi) therapy are enhanced by inducing a negative sodium balance. Whether this strategy also improves outcome of chronic ACEi treatment after myocardial infarction (MI) is unknown. Therefore, we investigated whether hydrochlorothiazide (HCTZ) or dietary sodium restriction further improves survival in ACEi-treated rats with MI.

Methods: MI was induced by coronary ligation. After 2 weeks rats were randomised to quinapril (QUI), HCTZ added to quinapril (QUI+HCTZ), or low sodium diet added to quinapril (QUI+LS). Survival was monitored for 62 weeks, after which left ventricular (LV) pressures were measured and blood for neurohumoral characterisation was collected. A separate group of rats, subjected to the same procedure, was evaluated after 35 weeks.

Results: After 62 weeks, mortality was comparable in all groups. However, survival was improved by HCTZ until 35 weeks. This effect on survival was paralleled by decreased proteinuria and LV end-diastolic pressures in QUI+HCTZ rats at 35, but not 62 weeks. Plasma renin activity was significantly decreased in QUI+HCTZ rats at 35 weeks. Contrary to HCTZ, LS added to QUI caused no benefit.

Conclusions: Adding HCTZ, but not LS, to quinapril improved survival, neurohumoral status, and proteinuria during the early chronic phase of experimental post-MI LV dysfunction. Since no adverse effects were observed, HCTZ may safely be used to improve ACEi therapy.
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http://dx.doi.org/10.1016/j.ejheart.2004.12.002DOI Listing
December 2005

Hydrochlorothiazide increases plasma or tissue angiotensin-converting enzyme-inhibitor drug levels in rats with myocardial infarction: differential effects on lisinopril and zofenopril.

Eur J Pharmacol 2005 Dec 28;527(1-3):141-9. Epub 2005 Nov 28.

Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Sodium depletion with diuretics augments the efficacy of angiotensin-converting enzyme-inhibitor therapy for hypertension and renal dysfunction, and possibly for left ventricular dysfunction after myocardial infarction. Underlying mechanisms may involve altered angiotensin-converting enzyme-inhibitor pharmacokinetics. We hypothesized that the diuretic hydrochlorothiazide causes increased steady-state levels of the angiotensin-converting enzyme-inhibitors lisinopril and zofenopril in rats with myocardial infarction. Rats were subjected to coronary ligation to induce myocardial infarction. After 1 week, rats were randomized to 50 mg/kg/day hydrochlorothiazide or control treatment for 3 weeks. The last week, rats received lisinopril or zofenopril in equipotentent dosages (3.3 and 10 mg/kg/day, respectively). Rats were sacrificed at Tmax after the last dose of angiotensin-converting enzyme-inhibitor, and tissues were collected for analysis of drug concentrations. Lisinopril concentrations in plasma were significantly increased by hydrochlorothiazide, at unchanged tissue concentrations. This increase could be fully explained by decreased renal function, as evidenced by increased plasma creatinine levels (lisinopril + hydrochlorothiazide 82+/-5 microM versus lisinopril 61+/-5 microM, P < 0.001). In contrast, zofenoprilat levels in kidney and non-infarcted left ventricle were markedly increased by hydrochlorothiazide, whereas plasma concentrations were unchanged. Although hydrochlorothiazide tended to increase plasma creatinine in zofenopril-treated rats as well, this increase was less pronounced (zofenopril + hydrochlorothiazide 61+/-3 microM versus zofenopril 54+/-2 microM, P = 0.15). Hydrochlorothiazide increases steady-state angiotensin-converting enzyme-inhibitor drug levels, most likely by affecting their renal clearance. Notably, the lipophilic angiotensin-converting enzyme-inhibitor zofenopril accumulated in tissue, whereas the hydrophilic lisinopril increased in plasma. Whether combining different angiotensin-converting enzyme-inhibitors with hydrochlorothiazide translates into distinct clinical profiles requires further study.
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http://dx.doi.org/10.1016/j.ejphar.2005.10.019DOI Listing
December 2005

Improvement of EDHF by chronic ACE inhibition declines rapidly after withdrawal in rats with myocardial infarction.

J Cardiovasc Pharmacol 2005 Dec;46(6):766-72

Department of Clinical Pharmacology, University Medical Center, Groningen, The Netherlands.

Heart failure after myocardial infarction (MI) is associated with endothelial dysfunction. There is conflicting evidence on the exact nature of this endothelial dysfunction and how endothelium-dependent vasodilation is affected by angiotensin-converting enzyme inhibitor (ACE-I) therapy. Furthermore, consequences of acute ACE-I withdrawal are largely unknown. Therefore, we studied the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the effects of ACE-I therapy and its withdrawal on endothelial function in MI rats. Rats were subjected to coronary ligation to induce MI and were assigned to quinapril or vehicle from 2 weeks to 8 months post-MI. In parallel, MI rats treated for 14 months with quinapril were subjected to treatment withdrawal for 0, 4, and 6 weeks. Acetylcholine (ACh)-induced relaxation and underlying endothelium-derived mediators were studied in isolated aortic rings. Long-term quinapril (8 months) resulted in markedly improved endothelium-dependent vasodilation in rats with myocardial infarction, which could be attributed to marked improvement in non-NO/prostanoid-mediated relaxation (ie, EDHF). After 14 months of follow-up, maximum vasodilation was still preserved by quinapril. Withdrawal after 14 months of treatment caused significantly impaired ACh-induced EDHF-mediated relaxation within 4 weeks. A marked reduction in EDHF-mediated relaxation caused this impairment. NO-mediated relaxation was unaffected. These findings highlight the importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction and the possibility of improving EDHF-mediated vasodilation with chronic ACE inhibitor therapy. In addition, withdrawal of chronic ACE inhibition after MI should be considered carefully, as profound endothelial dysfunction may develop rapidly.
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http://dx.doi.org/10.1097/01.fjc.0000187175.05780.e2DOI Listing
December 2005

Progressive left ventricular hypertrophy after withdrawal of long-term ACE inhibition following experimental myocardial infarction.

Eur J Heart Fail 2006 Mar 5;8(2):122-30. Epub 2005 Aug 5.

Department of Clinical Pharmacology, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Background: Although discontinuation of chronic ACE inhibitor (ACEi) therapy after myocardial infarction (MI) is common in clinical practice, some clinical studies reported an increased incidence of ischemia-related events after withdrawal. To further address this issue, we assessed hemodynamic, neurohormonal and vascular consequences of withdrawing long-term ACEi treatment after experimental MI.

Methods: Rats were subjected to coronary ligation to induce MI, and received quinapril (15 mg/kg/day) from 2 weeks to 14 months post-MI. Subsequently, surviving rats were randomized to sacrifice at 0, 4, and 6 weeks after ACEi withdrawal. Rats were studied for signs of heart failure, hemodynamics and cardiac function, neurohormones, and vascular edothelial function.

Results: After discontinuation of ACEi treatment, plasma aldosterone levels increased between 0-4 weeks without further increment thereafter, suggesting persistent RAAS activation. Acetylcholine-induced aortic relaxation was impaired at 4 and 6 weeks, indicating rapid and sustained development of endothelial vasodilator dysfunction after withdrawal. Moreover, 24% of the rats developed heart failure signs (edema, dyspnea), and 3 rats died, all within 4 weeks after withdrawal. Significantly increased N-ANP levels and lung weights at 4, but not at 6 weeks suggest a transient volume overload. Finally, LV/body weight ratios significantly increased between 0-4 as well as 4-6 weeks, indicating progressive LV hypertrophy.

Conclusions: The observed alterations after withdrawing long-term post-MI quinapril treatment in the present study may account for an increased risk for ischemic events. Thus, our findings highlight the potentially harmful effects associated with abrupt discontinuation of long-term post-MI ACE inhibition, and imply careful clinical consideration in this matter.
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http://dx.doi.org/10.1016/j.ejheart.2005.04.014DOI Listing
March 2006

Dietary sodium restriction specifically potentiates left ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction.

J Renin Angiotensin Aldosterone Syst 2004 Mar;5(1):27-32

Department of Clinical Pharmacology, University of Groningen, Groningen, The Netherlands.

Introduction: In patients with myocardial infarction (MI)-induced heart failure, angiotensin-converting enzyme (ACE) inhibitors are proven effective therapy in inhibiting the progression towards overt heart failure. However, the prognosis in these patients is still very poor, and optimisation of therapy is warranted. The antihypertensive and renoprotective effects of ACE inhibitors (ACE-Is) can be substantially enhanced by dietary sodium restriction. In line with the latter, the aim of the present study was to explore whether dietary sodium restriction enhances the efficacy of ACE-I after MI.

Methods: Rats with MI-induced left ventricular (LV) dysfunction received ACE-I therapy with zofenopril (5.5 mg/kg/day orally), with or without dietary sodium restriction. ACE activity was measured in non-infarcted LV tissue, kidneys and plasma. Effects on cardiac hypertrophy were examined by means of organ weight/body weight ratios. After blood pressure (BP) measurements, functional consequences of therapy were evaluated as LV pressure development in isolated perfused hearts.

Results: Dietary sodium restriction alone had no effect on any of the measured parameters, whereas zofenopril alone significantly reduced plasma and kidney ACE activity, but not LV ACE activity, nor LV weight/body weight ratio. However, only when ACE-I therapy was combined with dietary sodium restriction was LV ACE activity significantly reduced. This effect was paralleled by inhibition of LV hypertrophy. BP was reduced after infarction, and further reduced by zofenopril, but not affected by dietary sodium. Neither treatment was associated with effects on the MI-induced reduction of LV function in vitro.

Conclusions: Effects of ACE inhibition with zofenopril can be potentiated by additional dietary sodium restriction. However, these effects were tissue-specific, since LV, but not kidney or plasma, ACE activity was affected by the additional dietary sodium restriction. Effects on LV ACE activity were paralleled by reduced LV hypertrophy. Since the measured parameters did not indicate any adverse side-effects, dietary sodium restriction may provide a safe strategy to improve ACE-I efficacy in patients with infarction-induced LV dysfunction.
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http://dx.doi.org/10.3317/jraas.2004.004DOI Listing
March 2004