Publications by authors named "Nils Bomer"

22 Publications

  • Page 1 of 1

Selenoprotein DIO2 Is a Regulator of Mitochondrial Function, Morphology and UPRmt in Human Cardiomyocytes.

Int J Mol Sci 2021 Nov 2;22(21). Epub 2021 Nov 2.

Department of Cardiology, University Medical Centre Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.

Members of the fetal-gene-program may act as regulatory components to impede deleterious events occurring with cardiac remodeling, and constitute potential novel therapeutic heart failure (HF) targets. Mitochondrial energy derangements occur both during early fetal development and in patients with HF. Here we aim to elucidate the role of DIO2, a member of the fetal-gene-program, in pluripotent stem cell (PSC)-derived human cardiomyocytes and on mitochondrial dynamics and energetics, specifically. RNA sequencing and pathway enrichment analysis was performed on mouse cardiac tissue at different time points during development, adult age, and ischemia-induced HF. To determine the function of DIO2 in cardiomyocytes, a stable human hPSC-line with a DIO2 knockdown was made using a short harpin sequence. Firstly, we showed the selenoprotein, type II deiodinase (DIO2): the enzyme responsible for the tissue-specific conversion of inactive (T4) into active thyroid hormone (T3), to be a member of the fetal-gene-program. Secondly, silencing DIO2 resulted in an increased reactive oxygen species, impaired activation of the mitochondrial unfolded protein response, severely impaired mitochondrial respiration and reduced cellular viability. Microscopical 3D reconstruction of the mitochondrial network displayed substantial mitochondrial fragmentation. Summarizing, we identified DIO2 to be a member of the fetal-gene-program and as a key regulator of mitochondrial performance in human cardiomyocytes. Our results suggest a key position of human DIO2 as a regulator of mitochondrial function in human cardiomyocytes.
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http://dx.doi.org/10.3390/ijms222111906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8584701PMC
November 2021

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy.

Nat Commun 2021 08 30;12(1):5180. Epub 2021 Aug 30.

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

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca handling is a key feature of HF pathophysiology. Restoring the Ca regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.
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http://dx.doi.org/10.1038/s41467-021-25439-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405807PMC
August 2021

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

Sci Transl Med 2021 07;13(603)

Regenerative Biomaterials and Therapeutics Group, Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.
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http://dx.doi.org/10.1126/scitranslmed.abd1817DOI Listing
July 2021

ATPase Inhibitory Factor-1 Disrupts Mitochondrial Ca Handling and Promotes Pathological Cardiac Hypertrophy through CaMKIIδ.

Int J Mol Sci 2021 Apr 23;22(9). Epub 2021 Apr 23.

Department of Cardiology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.

ATPase inhibitory factor-1 (IF1) preserves cellular ATP under conditions of respiratory collapse, yet the function of IF1 under normal respiring conditions is unresolved. We tested the hypothesis that IF1 promotes mitochondrial dysfunction and pathological cardiomyocyte hypertrophy in the context of heart failure (HF). Methods and results: Cardiac expression of IF1 was increased in mice and in humans with HF, downstream of neurohumoral signaling pathways and in patterns that resembled the fetal-like gene program. Adenoviral expression of wild-type IF1 in primary cardiomyocytes resulted in pathological hypertrophy and metabolic remodeling as evidenced by enhanced mitochondrial oxidative stress, reduced mitochondrial respiratory capacity, and the augmentation of extramitochondrial glycolysis. Similar perturbations were observed with an IF1 mutant incapable of binding to ATP synthase (E55A mutation), an indication that these effects occurred independent of binding to ATP synthase. Instead, IF1 promoted mitochondrial fragmentation and compromised mitochondrial Ca handling, which resulted in sarcoplasmic reticulum Ca overloading. The effects of IF1 on Ca handling were associated with the cytosolic activation of calcium-calmodulin kinase II (CaMKII) and inhibition of CaMKII or co-expression of catalytically dead CaMKIIδC was sufficient to prevent IF1 induced pathological hypertrophy. Conclusions: IF1 represents a novel member of the fetal-like gene program that contributes to mitochondrial dysfunction and pathological cardiac remodeling in HF. Furthermore, we present evidence for a novel, ATP-synthase-independent, role for IF1 in mitochondrial Ca handling and mitochondrial-to-nuclear crosstalk involving CaMKII.
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http://dx.doi.org/10.3390/ijms22094427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122940PMC
April 2021

Selenium, Selenoproteins, and Heart Failure: Current Knowledge and Future Perspective.

Curr Heart Fail Rep 2021 06 9;18(3):122-131. Epub 2021 Apr 9.

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

Purpose Of Review: (Mal-)nutrition of micronutrients, like selenium, has great impact on the human heart and improper micronutrient intake was observed in 30-50% of patients with heart failure. Low selenium levels have been reported in Europe and Asia and thought to be causal for Keshan disease. Selenium is an essential micronutrient that is needed for enzymatic activity of the 25 so-called selenoproteins, which have a broad range of activities. In this review, we aim to summarize the current evidence about selenium in heart failure and to provide insights about the potential mechanisms that can be modulated by selenoproteins.

Recent Findings: Suboptimal selenium levels (<100 μg/L) are prevalent in more than 70% of patients with heart failure and were associated with lower exercise capacity, lower quality of life, and worse prognosis. Small clinical trials assessing selenium supplementation in patients with HF showed improvement of clinical symptoms (NYHA class), left ventricular ejection fraction, and lipid profile, while governmental interventional programs in endemic areas have significantly decreased the incidence of Keshan disease. In addition, several selenoproteins are found impaired in suboptimal selenium conditions, potentially aggravating underlying mechanisms like oxidative stress, inflammation, and thyroid hormone insufficiency. While the current evidence is not sufficient to advocate selenium supplementation in patients with heart failure, there is a clear need for high level evidence to show whether treatment with selenium has a place in the contemporary treatment of patients with HF to improve meaningful clinical endpoints. Graphical summary summarizing the potential beneficial effects of the various selenoproteins, locally in cardiac tissues and systemically in the rest of the body. In short, several selenoproteins contribute in protecting the integrity of the mitochondria. By doing so, they contribute indirectly to reducing the oxidative stress as well as improving the functionality of immune cells, which are in particular vulnerable to oxidative stress. Several other selenoproteins are directly involved in antioxidative pathways, next to excreting anti-inflammatory effects. Similarly, some selenoproteins are located in the endoplasmic reticulum, playing roles in protein folding. With exception of the protection of the mitochondria and the reduction of oxidative stress, other effects are not yet investigated in cardiac tissues. The systemic effects of selenoproteins might not be limited to these mechanisms, but also may include modulation of endothelial function, protection skeletal muscles, in addition to thyroid metabolism.

Abbreviations: DIO, iodothyronine deiodinase; GPx, glutathione peroxidase; MsrB2, methionine-R-sulfoxide reductase B2; SELENOK, selenoprotein K; SELENON, selenoprotein N; SELENOP, selenoprotein P; SELENOS, selenoprotein S; SELENOT, selenoprotein T; TXNRD, thioredoxin reductase.
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http://dx.doi.org/10.1007/s11897-021-00511-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163712PMC
June 2021

Human iPSC-Derived Cardiomyocytes of Peripartum Patients With Cardiomyopathy Reveal Aberrant Regulation of Lipid Metabolism.

Circulation 2020 12 7;142(23):2288-2291. Epub 2020 Dec 7.

Department of Cardiology (M.F.H., N.B., K.F.A.G., P.v.d.M.), University Medical Center Groningen, University of Groningen, The Netherlands.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846285PMC
December 2020

Mass-spectrometric identification of carbamylated proteins present in the joints of rheumatoid arthritis patients and controls.

Clin Exp Rheumatol 2021 May-Jun;39(3):570-577. Epub 2020 Sep 1.

Department of Rheumatology, Leiden University Medical Center, and Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.

Objectives: Antibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in the sera of rheumatoid arthritis (RA) patients. These autoantibodies associate with increased risk of RA development and with severity of joint destruction. It is not known which proteins in the RA joint are recognised by anti-CarP antibodies. Therefore, we investigated the presence and identity of carbamylated proteins in the human (inflamed) joint.

Methods: We obtained synovium, cartilage and synovial fluid from RA joints. Cartilage and synovium were obtained from controls. Samples were processed and used for immunohistochemistry or mass-spectrometric analysis to investigate the presence of carbamylated proteins. Anti-CarP antibody reactivity towards identified carbamylated proteins was tested by ELISA.

Results: Immunohistochemistry showed extensive staining of RA and control synovial tissue. Whole proteome analyses of the joint tissues revealed a large number of carbamylated peptidyllysine residues. We identified many carbamylated proteins in cartilage and were also able to detect carbamylation in synovial tissue and synovial fluid. Carbamylation was not exclusive to the RA joint and was also present in the joints of controls. Anti-CarP antibodies in the sera of RA patients were able to recognise the identified carbamylated proteins.

Conclusions: We conclude that numerous carbamylated proteins are present in the RA joint. These carbamylated proteins can be recognised by anti-CarP antibodies, substantiating the notion that anti-CarP antibodies may play a role in the pathogenesis of RA.
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May 2021

A Clinical Tool to Predict Low Serum Selenium in Patients with Worsening Heart Failure.

Nutrients 2020 Aug 21;12(9). Epub 2020 Aug 21.

Department of Cardiology, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, The Netherlands.

Selenium is an essential micronutrient, and a low selenium concentration (<100 µg/L) is associated with a poorer quality of life and exercise capacity, and an impaired prognosis in patients with worsening heart failure. Measuring selenium concentrations routinely is laborious and costly, and although its clinical utility is yet to be proven, an easy implemented model to predict selenium status is desirable. A stepwise multivariable logistic regression analysis was performed using routinely measured clinical factors. Low selenium was independently predicted by: older age, lower serum albumin, higher N-terminal pro-B-type natriuretic peptide levels, worse kidney function, and the presence of orthopnea and iron deficiency. A 10-points risk-model was developed, and a score of ≥6 points identified >80% of patients with low selenium (sensitivity of 44%, specificity of 80%). Given that selenium and iron overlap in their physiological roles, we evaluated the shared determinants and prognostic associates. Both deficiencies shared similar clinical characteristics, including the model risk factors and, in addition, a low protein intake and high levels of C-reactive protein. Low selenium was associated with a similar or worse prognosis compared to iron deficiency. In conclusion, although it is difficult to exclude low selenium based on clinical characteristics alone, we provide a prediction tool which identifies heart failure patients at higher risk of having a low selenium status.
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http://dx.doi.org/10.3390/nu12092541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551091PMC
August 2020

Selenium and outcome in heart failure.

Eur J Heart Fail 2020 08 6;22(8):1415-1423. Epub 2019 Dec 6.

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

Aims: Severe deficiency of the essential trace element selenium can cause myocardial dysfunction although the mechanism at cellular level is uncertain. Whether, in clinical practice, moderate selenium deficiency is associated with worse symptoms and outcome in patients with heart failure is unknown.

Methods And Results: BIOSTAT-CHF is a multinational, prospective, observational cohort study that enrolled patients with worsening heart failure. Serum concentrations of selenium were measured by inductively coupled plasma mass spectrometry. Primary endpoint was a composite of all-cause mortality and hospitalization for heart failure; secondary endpoint was all-cause mortality. To investigate potential mechanisms by which selenium deficiency might affect prognosis, human cardiomyocytes were cultured in absence of selenium, and mitochondrial function and oxidative stress were assessed. Serum selenium concentration (deficiency) was <70 μg/L in 485 (20.4%) patients, who were older, more often women, had worse New York Heart Association class, more severe signs and symptoms of heart failure and poorer exercise capacity (6-min walking test) and quality of life (Kansas City Cardiomyopathy Questionnaire). Selenium deficiency was associated with higher rates of the primary endpoint [hazard ratio (HR) 1.23; 95% confidence interval (CI) 1.06-1.42] and all-cause mortality (HR 1.52; 95% CI 1.26-1.86). In cultured human cardiomyocytes, selenium deprivation impaired mitochondrial function and oxidative phosphorylation, and increased intracellular reactive oxygen species levels.

Conclusions: Selenium deficiency in heart failure patients is independently associated with impaired exercise tolerance and a 50% higher mortality rate, and impaired mitochondrial function in vitro, in human cardiomyocytes. Clinical trials are needed to investigate the effect of selenium supplements in patients with heart failure, especially if they have low plasma concentrations of selenium.
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http://dx.doi.org/10.1002/ejhf.1644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540257PMC
August 2020

The role of cathepsin D in the pathophysiology of heart failure and its potentially beneficial properties: a translational approach.

Eur J Heart Fail 2020 11 3;22(11):2102-2111. Epub 2019 Dec 3.

Department of Cardiology, University of Groningen, Groningen, The Netherlands.

Aims: Cathepsin D is a ubiquitous lysosomal protease that is primarily secreted due to oxidative stress. The role of circulating cathepsin D in heart failure (HF) is unknown. The aim of this study is to determine the association between circulating cathepsin D levels and clinical outcomes in patients with HF and to investigate the biological settings that induce the release of cathepsin D in HF.

Methods And Results: Cathepsin D levels were studied in 2174 patients with HF from the BIOSTAT-CHF index study. Results were validated in 1700 HF patients from the BIOSTAT-CHF validation cohort. The primary combined outcome was all-cause mortality and/or HF hospitalizations. Human pluripotent stem cell-derived cardiomyocytes were subjected to hypoxic, pro-inflammatory signalling and stretch conditions. Additionally, cathepsin D expression was inhibited by targeted short hairpin RNAs (shRNA). Higher levels of cathepsin D were independently associated with diabetes mellitus, renal failure and higher levels of interleukin-6 and N-terminal pro-B-type natriuretic peptide (P < 0.001 for all). Cathepsin D levels were independently associated with the primary combined outcome [hazard ratio (HR) per standard deviation (SD): 1.12; 95% confidence interval (CI) 1.02-1.23], which was validated in an independent cohort (HR per SD: 1.23, 95% CI 1.09-1.40). In vitro experiments demonstrated that human stem cell-derived cardiomyocytes released cathepsin D and troponin T in response to mechanical stretch. ShRNA-mediated silencing of cathepsin D resulted in increased necrosis, abrogated autophagy, increased stress-induced metabolism, and increased release of troponin T from human stem cell-derived cardiomyocytes under stress.

Conclusions: Circulating cathepsin D levels are associated with HF severity and poorer outcome, and reduced levels of cathepsin D may have detrimental effects with therapeutic potential in HF.
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http://dx.doi.org/10.1002/ejhf.1674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754332PMC
November 2020

In peripartum cardiomyopathy plasminogen activator inhibitor-1 is a potential new biomarker with controversial roles.

Cardiovasc Res 2020 09;116(11):1875-1886

Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany.

Aims: Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease occurring in previously heart-healthy women. A common pathomechanism in PPCM involves the angiostatic 16 kDa-prolactin (16 kDa-PRL) fragment, which via NF-κB-mediated up-regulation of microRNA-(miR)-146a induces vascular damage and heart failure. We analyse whether the plasminogen activator inhibitor-1 (PAI-1) is involved in the pathophysiology of PPCM.

Methods And Results: In healthy age-matched postpartum women (PP-Ctrl, n = 53, left ventricular ejection fraction, LVEF > 55%), PAI-1 plasma levels were within the normal range (21 ± 10 ng/mL), but significantly elevated (64 ± 38 ng/mL, P < 0.01) in postpartum PPCM patients at baseline (BL, n = 64, mean LVEF: 23 ± 8%). At 6-month follow-up (n = 23), PAI-1 levels decreased (36 ± 14 ng/mL, P < 0.01 vs. BL) and LVEF (49 ± 11%) improved. Increased N-terminal pro-brain natriuretic peptide and Troponin T did not correlate with PAI-1. C-reactive protein, interleukin (IL)-6 and IL-1β did not differ between PPCM patients and PP-Ctrl. MiR-146a was 3.6-fold (P < 0.001) higher in BL-PPCM plasma compared with PP-Ctrl and correlated positively with PAI-1. In BL-PPCM serum, 16 kDa-PRL coprecipitated with PAI-1, which was associated with higher (P < 0.05) uPAR-mediated NF-κB activation in endothelial cells compared with PP-Ctrl serum. Cardiac biopsies and dermal fibroblasts from PPCM patients displayed higher PAI-1 mRNA levels (P < 0.05) than healthy controls. In PPCM mice (due to a cardiomyocyte-specific-knockout for STAT3, CKO), cardiac PAI-1 expression was higher than in postpartum wild-type controls, whereas a systemic PAI-1-knockout in CKO mice accelerated peripartum cardiac fibrosis, inflammation, heart failure, and mortality.

Conclusion: In PPCM patients, circulating and cardiac PAI-1 expression are up-regulated. While circulating PAI-1 may add 16 kDa-PRL to induce vascular impairment via the uPAR/NF-κB/miR-146a pathway, experimental data suggest that cardiac PAI-1 expression seems to protect the PPCM heart from fibrosis. Thus, measuring circulating PAI-1 and miR-146a, together with an uPAR/NF-κB-activity assay could be developed into a specific diagnostic marker assay for PPCM, but unrestricted reduction of PAI-1 for therapy may not be advised.
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http://dx.doi.org/10.1093/cvr/cvz300DOI Listing
September 2020

Increased WISP1 expression in human osteoarthritic articular cartilage is epigenetically regulated and decreases cartilage matrix production.

Rheumatology (Oxford) 2019 06;58(6):1065-1074

Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.

Objectives: Previously, we have shown the involvement of Wnt-activated protein Wnt-1-induced signaling protein 1 (WISP1) in the development of OA in mice. Here, we aimed to characterize the relation between WISP1 expression and human OA and its regulatory epigenetic determinants.

Methods: Preserved and lesioned articular cartilage from end-stage OA patients and non-OA-diagnosed individuals was collected. WISP1 expression was determined using immunohistochemistry and damage was classified using Mankin scoring. RNA expression and DNA methylation were assessed in silico from genome-wide datasets (microarray analysis and RNA sequencing, and 450 k-methylationarrays, respectively). Effects of WISP1 were tested in pellet cultures of primary human chondrocytes.

Results: WISP1 expression in cartilage of OA patients was increased compared with non-OA-diagnosed controls and, within OA patients, WISP1 was even higher in lesioned compared with preserved regions, with expression strongly correlating with Mankin score. In early symptomatic OA patients with disease progression, higher synovial WISP1 expression was observed as compared with non-progressors. Notably, increased WISP1 expression was inversely correlated with methylation levels of a positional CpG-dinucleotide (cg10191240), with lesioned areas showing strong hypomethylation for this CpG as compared with preserved cartilage. Additionally, we observed that methylation levels were allele-dependent for an intronic single-nucleotide polymorphism nearby cg10191240. Finally, addition of recombinant WISP1 to pellets of primary chondrocytes strongly inhibited deposition of extracellular matrix as reflected by decreased pellet circumference, proteoglycan content and decreased expression of matrix components.

Conclusion: Increased WISP1 expression is found in lesioned human articular cartilage, and appears epigenetically regulated via DNA methylation. In vitro assays suggest that increased WISP1 is detrimental for cartilage integrity.
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http://dx.doi.org/10.1093/rheumatology/key426DOI Listing
June 2019

Concise Review: The Current State of Human In Vitro Cardiac Disease Modeling: A Focus on Gene Editing and Tissue Engineering.

Stem Cells Transl Med 2019 01 9;8(1):66-74. Epub 2018 Oct 9.

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

Until recently, in vivo and ex vivo experiments were the only means to determine factors and pathways involved in disease pathophysiology. After the generation of characterized human embryonic stem cell lines, human diseases could readily be studied in an extensively controllable setting. The introduction of human-induced pluripotent stem cells, a decade ago, allowed the investigation of hereditary diseases in vitro. In the field of cardiology, diseases linked to known genes have successfully been studied, revealing novel disease mechanisms. The direct effects of various mutations leading to hypertrophic cardiomyopathy, dilated cardiomyopathy, arrythmogenic cardiomyopathy, or left ventricular noncompaction cardiomyopathy are discovered as a result of in vitro disease modeling. Researchers are currently applying more advanced techniques to unravel more complex phenotypes, resulting in state-of-the-art models that better mimic in vivo physiology. The continued improvement of tissue engineering techniques and new insights into epigenetics resulted in more reliable and feasible platforms for disease modeling and the development of novel therapeutic strategies. The introduction of CRISPR-Cas9 gene editing granted the ability to model diseases in vitro independent of induced pluripotent stem cells. In addition to highlighting recent developments in the field of human in vitro cardiomyopathy modeling, this review also aims to emphasize limitations that remain to be addressed; including residual somatic epigenetic signatures induced pluripotent stem cells, and modeling diseases with unknown genetic causes. Stem Cells Translational Medicine 2019;8:66-74.
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http://dx.doi.org/10.1002/sctm.18-0052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312446PMC
January 2019

Annotating Transcriptional Effects of Genetic Variants in Disease-Relevant Tissue: Transcriptome-Wide Allelic Imbalance in Osteoarthritic Cartilage.

Arthritis Rheumatol 2019 04 23;71(4):561-570. Epub 2019 Feb 23.

Leiden University Medical Center, Leiden, The Netherlands.

Objective: Multiple single-nucleotide polymorphisms (SNPs) conferring susceptibility to osteoarthritis (OA) mark imbalanced expression of positional genes in articular cartilage, reflected by unequally expressed alleles among heterozygotes (allelic imbalance [AI]). We undertook this study to explore the articular cartilage transcriptome from OA patients for AI events to identify putative disease-driving genetic variation.

Methods: AI was assessed in 42 preserved and 5 lesioned OA cartilage samples (from the Research Arthritis and Articular Cartilage study) for which RNA sequencing data were available. The count fraction of the alternative alleles among the alternative and reference alleles together (φ) was determined for heterozygous individuals. A meta-analysis was performed to generate a meta-φ and P value for each SNP with a false discovery rate (FDR) correction for multiple comparisons. To further validate AI events, we explored them as a function of multiple additional OA features.

Results: We observed a total of 2,070 SNPs that consistently marked AI of 1,031 unique genes in articular cartilage. Of these genes, 49 were found to be significantly differentially expressed (fold change <0.5 or >2, FDR <0.05) between preserved and paired lesioned cartilage, and 18 had previously been reported to confer susceptibility to OA and/or related phenotypes. Moreover, we identified notable highly significant AI SNPs in the CRLF1, WWP2, and RPS3 genes that were related to multiple OA features.

Conclusion: We present a framework and resulting data set for researchers in the OA research field to probe for disease-relevant genetic variation that affects gene expression in pivotal disease-affected tissue. This likely includes putative novel compelling OA risk genes such as CRLF1, WWP2, and RPS3.
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http://dx.doi.org/10.1002/art.40748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593438PMC
April 2019

Modeling Human Cardiac Hypertrophy in Stem Cell-Derived Cardiomyocytes.

Stem Cell Reports 2018 03 15;10(3):794-807. Epub 2018 Feb 15.

Department of Cardiology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, PO Box 30.001, Groningen, the Netherlands. Electronic address:

Cardiac hypertrophy accompanies many forms of cardiovascular diseases. The mechanisms behind the development and regulation of cardiac hypertrophy in the human setting are poorly understood, which can be partially attributed to the lack of a human cardiomyocyte-based preclinical test system recapitulating features of diseased myocardium. The objective of our study is to determine whether human embryonic stem cell-derived cardiomyocytes (hESC-CMs) subjected to mechanical stretch can be used as an adequate in vitro model for studying molecular mechanisms of cardiac hypertrophy. We show that hESC-CMs subjected to cyclic stretch, which mimics mechanical overload, exhibit essential features of a hypertrophic state on structural, functional, and gene expression levels. The presented hESC-CM stretch approach provides insight into molecular mechanisms behind mechanotransduction and cardiac hypertrophy and lays groundwork for the development of pharmacological approaches as well as for discovering potential circulating biomarkers of cardiac dysfunction.
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http://dx.doi.org/10.1016/j.stemcr.2018.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918264PMC
March 2018

Aberrant Calreticulin Expression in Articular Cartilage of Dio2 Deficient Mice.

PLoS One 2016 10;11(5):e0154999. Epub 2016 May 10.

Department of Molecular Epidemiology, LUMC, Leiden, Netherlands.

Objective: To identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis.

Methods: Previously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition.

Results: Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > |1,5|) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.

Conclusion: We here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154999PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862667PMC
July 2017

Translating genomics into mechanisms of disease: Osteoarthritis.

Best Pract Res Clin Rheumatol 2015 12 4;29(6):683-91. Epub 2016 Mar 4.

Dept. Medical Statistics and Bioinformatics, Section Molecular Epidemiology, Leiden University Medical Centre, LUMC Post-zone S-05-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Electronic address:

Osteoarthritis (OA) is the most common age-related arthritic disorder and is characterized by aberrant extracellular matrix (ECM) content and surface disruptions that range from fibrillation, clefting and delamination, leading to articular surface erosion. Worldwide, over 20% of the population is affected with OA and 80% of these patients have limitations in movement, whereas 25% experience inhibition in major daily activities of life. OA is the most common disabling arthritic disease; nevertheless, no disease-modifying treatment is available except for the expensive total joint replacement surgery at end-stage disease. Lack of insight into the underlying pathophysiological mechanisms of OA has considerably contributed to the inability of the scientific community to develop disease-modifying drugs. To overcome this critical barrier, focus should be on translation of identified robust gene deviations towards the underlying biological mechanisms.
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http://dx.doi.org/10.1016/j.berh.2016.01.001DOI Listing
December 2015

Transcriptional associations of osteoarthritis-mediated loss of epigenetic control in articular cartilage.

Arthritis Rheumatol 2015 May;67(8):2108-16

Leiden University Medical Center, Leiden, The Netherlands, and The Netherlands Genomics Initiative-sponsored Netherlands Consortium for Healthy Aging, Rotterdam, The Netherlands.

Objective: To identify osteoarthritis (OA) progression-modulating pathways in articular cartilage and their respective regulatory epigenetic and genetic determinants in end-stage disease.

Methods: Transcriptional activity of CpG was assessed using gene expression data and DNA methylation data for preserved and lesional articular cartilage samples. Disease-responsive transcriptionally active CpG were identified by means of differential methylation between preserved and lesional cartilage. Transcriptionally relevant genetic determinants were addressed by means of single-nucleotide polymorphisms (SNPs) proximal to the OA-responsive transcriptionally active CpG. Statistical analyses were corrected for age, sex, joint, and technical covariates. A random effect was included to correct for possible correlations between paired samples.

Results: Of 9,838 transcribed genes in articular cartilage, 2,324 correlated with the methylation status of 3,748 transcriptionally active CpG; both negative (n = 1,741) and positive (n = 2,007) correlations were observed. Hypomethylation and hypermethylation (false discovery rate of <0.05, |Δβ| > 0.05) were observed for 62 and 25 transcriptionally active CpG, respectively, covering 70 unique genes. Enrichment for developmental and extracellular matrix maintenance pathways indicated possible reactivation of endochondral ossification. Finally, we observed 31 and 26 genes for which methylation and expression, respectively, were additionally affected by genetic variation.

Conclusion: We identified tissue-specific genes involved in OA disease progression, reflected by genetic and pathologic epigenetic regulation of transcription, primarily at genes involved in development. Therefore, transcriptionally active SNPs near these genes may serve as putative susceptibility alleles. Our results constitute an important step toward understanding the reported widespread epigenetic changes occurring in OA articular cartilage and toward subsequent development of treatments targeting disease-driving pathways.
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http://dx.doi.org/10.1002/art.39162DOI Listing
May 2015

The effect of forced exercise on knee joints in Dio2(-/-) mice: type II iodothyronine deiodinase-deficient mice are less prone to develop OA-like cartilage damage upon excessive mechanical stress.

Ann Rheum Dis 2016 Mar 30;75(3):571-7. Epub 2014 Dec 30.

Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Centre, KU Leuven, Leuven, Belgium Division of Rheumatology, University Hospitals Leuven, Leuven, Belgium.

Objective: To further explore deiodinase iodothyronine type 2 (DIO2) as a therapeutic target in osteoarthritis (OA) by studying the effects of forced mechanical loading on in vivo joint cartilage tissue homeostasis and the modulating effect herein of Dio2 deficiency.

Methods: Wild-type and C57BL/6-Dio2(-/-) -mice were subjected to a forced running regime for 1 h per day for 3 weeks. Severity of OA was assessed by histological scoring for cartilage damage and synovitis. Genome-wide gene expression was determined in knee cartilage by microarray analysis (Illumina MouseWG-6 v2). STRING-db analyses were applied to determine enrichment for specific pathways and to visualise protein-protein interactions.

Results: In total, 158 probes representing 147 unique genes showed significantly differential expression with a fold-change ≥1.5 upon forced exercise. Among these are genes known for their association with OA (eg, Mef2c, Egfr, Ctgf, Prg4 and Ctnnb1), supporting the use of forced running as an OA model in mice. Dio2-deficient mice showed significantly less cartilage damage and signs of synovitis. Gene expression response upon exercise between wild-type and knockout mice was significantly different for 29 genes.

Conclusions: Mice subjected to a running regime have significant increased cartilage damage and synovitis scores. Lack of Dio2 protected against cartilage damage in this model and was reflected in a specific gene expression profile, and either mark a favourable effect in the Dio2 knockout (eg, Gnas) or an unfavourable effect in wild-type cartilage homeostasis (eg, Hmbg2 and Calr). These data further support DIO2 activity as a therapeutic target in OA.
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http://dx.doi.org/10.1136/annrheumdis-2014-206608DOI Listing
March 2016

Genes involved in the osteoarthritis process identified through genome wide expression analysis in articular cartilage; the RAAK study.

PLoS One 2014 23;9(7):e103056. Epub 2014 Jul 23.

Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands; The Netherlands Genomics Initiative, sponsored by the NCHA, Leiden-Rotterdam, The Netherlands.

Objective: Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.

Methods: Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.

Results: Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. TNFRSF11B and FRZB). Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.

Conclusion: We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103056PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108379PMC
November 2015

Severe osteoarthritis of the hand associates with common variants within the ALDH1A2 gene and with rare variants at 1p31.

Nat Genet 2014 May 13;46(5):498-502. Epub 2014 Apr 13.

1] Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands. [2] Department of Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands.

Osteoarthritis is the most common form of arthritis and is a major cause of pain and disability in the elderly. To search for sequence variants that confer risk of osteoarthritis of the hand, we carried out a genome-wide association study (GWAS) in subjects with severe hand osteoarthritis, using variants identified through the whole-genome sequencing of 2,230 Icelanders. We found two significantly associated loci in the Icelandic discovery set: at 15q22 (frequency of 50.7%, odds ratio (OR) = 1.51, P = 3.99 × 10(-10)) in the ALDH1A2 gene and at 1p31 (frequency of 0.02%, OR = 50.6, P = 9.8 × 10(-10)). Among the carriers of the variant at 1p31 is a family with several members in whom the risk allele segregates with osteoarthritis. The variants within the ALDH1A2 gene were confirmed in replication sets from The Netherlands and the UK, yielding an overall association of OR = 1.46 and P = 1.1 × 10(-11) (rs3204689).
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http://dx.doi.org/10.1038/ng.2957DOI Listing
May 2014

Underlying molecular mechanisms of DIO2 susceptibility in symptomatic osteoarthritis.

Ann Rheum Dis 2015 Aug 2;74(8):1571-9. Epub 2014 Apr 2.

Department of Molecular Epidemiology, LUMC, Leiden, The Netherlands Genomics Initiative, sponsored by the NCHA, Leiden, The Netherlands.

Objectives: To investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches.

Methods: Epigenetically regulated expression of DIO2 was explored by assessing methylation of positional CpG-dinucleotides and the respective DIO2 expression in OA-affected and macroscopically preserved articular cartilage from end-stage OA patients. In a human in vitro chondrogenesis model, we measured the effects when thyroid signalling during culturing was either enhanced (excess T3 or lentiviral induced DIO2 overexpression) or decreased (iopanoic acid).

Results: OA-related changes in methylation at a specific CpG dinucleotide upstream of DIO2 caused significant upregulation of its expression (β=4.96; p=0.0016). This effect was enhanced and appeared driven specifically by DIO2 rs225014 risk allele carriers (β=5.58, p=0.0006). During in vitro chondrogenesis, DIO2 overexpression resulted in a significant reduced capacity of chondrocytes to deposit extracellular matrix (ECM) components, concurrent with significant induction of ECM degrading enzymes (ADAMTS5, MMP13) and markers of mineralisation (ALPL, COL1A1). Given their concurrent and significant upregulation of expression, this process is likely mediated via HIF-2α/RUNX2 signalling. In contrast, we showed that inhibiting deiodinases during in vitro chondrogenesis contributed to prolonged cartilage homeostasis as reflected by significant increased deposition of ECM components and attenuated upregulation of matrix degrading enzymes.

Conclusions: Our findings show how genetic variation at DIO2 could confer risk to OA and raised the possibility that counteracting thyroid signalling may be a novel therapeutic approach.
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http://dx.doi.org/10.1136/annrheumdis-2013-204739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516000PMC
August 2015
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