Veerle Kremer

Amsterdam University Medical Centers

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Reinier A. Boon

Amsterdam University Medical Centers

Diewertje I. Bink

Amsterdam University Medical Centers

Laura Stanicek

Science for Life Laboratory

A. Yaël Nossent

Leiden University Medical Center

Stefanie Dimmeler

Goethe University Frankfurt

Eva van Ingen

Centre for Human Drug Research

Robert Szulcek

German Center for Lung Research

Jan Voorberg

Sanquin

Elisabetta Michielon

Amsterdam University Medical Centers

Petr Symersky

Amsterdam University Medical Centers

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Amsterdam University Medical Centers

Vrije Universiteit Amsterdam

University of Amsterdam

Amsterdam UMC Location VUmc

Amsterdam Neuroscience

Leiden University Medical Center

Academic Medical Center

University Medical Center

German Centre for Cardiovascular Research

University Medical Center Utrecht

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C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2′O-ribose methylation

Human Molecular Genetics (2021)

Eva van Ingen Daphne A. L. van den Homberg M. Leontien van der Bent Hailiang Mei Nikolina Papac-Miličević

We have previously shown that C/D box small nucleolar RNAs (snoRNAs) transcribed from the DLK1-DIO3 locus on human chromosome 14 (14q32) are associated with cardiovascular disease. DLK1-DIO3 snoRNAs are 'orphan snoRNAs' that have no known targets. We aimed to identify RNA targets and elucidate the mechanism-of-action of human SNORD113-6 (AF357425 in mice). As AF357425-knockout cells were non-viable, we induced overexpression or inhibition of AF357425 in primary murine fibroblasts and performed RNA-Seq. We identified several pre-mRNAs with conserved AF357425/SNORD113-6 D'-seed binding sites in the last exon/3' untranslated region (3'UTR), which directed pre-mRNA processing and splice-variant-specific protein expression. We also pulled down the snoRNA-associated methyltransferase fibrillarin from AF357425-High versus AF357425-Low fibroblast lysates, followed by RNA isolation, ribosomal RNA depletion and RNA-Seq. Identifying mostly mRNAs, we subjected these to PANTHER pathway analysis and observed enrichment for genes in the integrin pathway. We confirmed 2'O-ribose methylation in six integrin pathway mRNAs (MAP2K1, ITGB3, ITGA7, PARVB, NTN4 and FLNB). Methylation and mRNA expressions were decreased while mRNA degradation was increased under AF357425/SNORD113-6 inhibition in both murine and human primary fibroblasts, but effects on protein expression were more ambiguous. Integrin signalling is crucial for cell-cell and cell-matrix interactions, and correspondingly, we observed altered human primary arterial fibroblast function upon SNORD113-6 inhibition.

Small nucleolar RNA

Fibrillarin

10.1093/hmg/ddab304

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Citations (17)

The long non-coding RNA MEG8 induces an endothelial barrier through regulation of microRNA-370 and -494 processing

Journal of Cell Science (2022)

Veerle Kremer Laura Stanicek Eva van Ingen Diewertje I. Bink Sarah Hilderink

ABSTRACT The 14q32 locus is an imprinted region in the human genome which contains multiple non-coding RNAs. We investigated the role of the long non-coding RNA maternally expressed gene 8 (MEG8) in endothelial function and its underlying mechanism. A 5-fold increase in MEG8 was observed with increased passage number in human umbilical vein endothelial cells (HUVECs), suggesting MEG8 is induced during aging. MEG8 knockdown resulted in a 1.8-fold increase in senescence, suggesting MEG8 might be protective during aging. The endothelial barrier was also impaired after MEG8 silencing. MEG8 knockdown resulted in reduced expression of microRNA (miRNA)-370 and -494 but not -127, -487b and -410. Overexpression of miRNA-370 or -494 partially rescued the MEG8-silencing-induced barrier loss. Mechanistically, MEG8 regulates expression of miRNA-370 and -494 at the mature miRNA level through interaction with the RNA-binding proteins cold-inducible RNA-binding protein (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multi-enzyme complex subunit β (HADHB). Mature miRNA-370 and miRNA-494 were found to interact with CIRBP, whereas precursor miRNA-370 and miRNA-494 were found to interact with HADHB. Individual CIRBP and HADHB silencing resulted in downregulation of miRNA-370 and induction of miRNA-494. These results suggest MEG8 interacts with CIRBP and HADHB and contributes to miRNA processing at the post-transcriptional level.

10.1242/jcs.259671

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The PNUTS-PP1 axis regulates endothelial aging and barrier function via SEMA3B suppression

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Noelia Lozano‐Vidal Laura Stanicek Diewertje I. Bink Veerle Kremer Alyson W. MacInnes

ABSTRACT Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTS fl/fl ), termed PNUTS EC-KO . Two weeks after PNUTS deletion, PNUTS EC-KO mice presented severe multiorgan failure and vascular leakage. We showed that the PNUTS binding motif for protein phosphatase 1 (PP1) is essential to maintain endothelial barrier function. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTS EC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a PP1-SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

Protein phosphatase 1

10.1101/2020.08.10.243170

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COVID19 Impairs Cardiac Function via Endothelial H19 and IL6 Signaling

Circulation Research (2023)

Rio P. Juni P Phelp Diewertje I. Bink Veerle Kremer Anke van Bergen

10.1161/circresaha.122.322166

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Neuro-oncological Ventral Antigen 2 Regulates Splicing of Vascular Endothelial Growth Factor Receptor 1 and Is Required for Endothelial Function

Reproductive Sciences (2022)

Veerle Kremer Jetta J. Oppelaar Theresa Gimbel Susanne Koziarek Wessel Ganzevoort

Abstract Pre-eclampsia (PE) affects 2–8% of pregnancies and is responsible for significant morbidity and mortality. The maternal clinical syndrome (defined by hypertension, proteinuria, and organ dysfunction) is the result of endothelial dysfunction. The endothelial response to increased levels of soluble FMS-like Tyrosine Kinase 1 (sFLT1) is thought to play a central role. sFLT1 is released from multiple tissues and binds VEGF with high affinity and antagonizes VEGF. Expression of soluble variants of sFLT1 is a result of alternative splicing; however, the mechanism is incompletely understood. We hypothesize that neuro-oncological ventral antigen 2 (NOVA2) contributes to this. NOVA2 was inhibited in human umbilical vein endothelial cells (HUVECs) and multiple cellular functions were assessed. NOVA2 and FLT1 expression in the placenta of PE, pregnancy-induced hypertension, and normotensive controls was measured by RT-qPCR. Loss of NOVA2 in HUVECs resulted in significantly increased levels of sFLT1 , but did not affect expression of membrane-bound FLT1 . NOVA2 protein was shown to directly interact with FLT1 mRNA. Loss of NOVA2 was also accompanied by impaired endothelial functions such as sprouting. We were able to restore sprouting capacity by exogenous VEGF. We did not observe statistically significant regulation of NOVA2 or sFLT1 in the placenta. However, we observed a negative correlation between sFLT1 and NOVA2 expression levels. In conclusion, NOVA2 was found to regulate FLT1 splicing in the endothelium. Loss of NOVA2 resulted in impaired endothelial function, at least partially dependent on VEGF. In PE patients, we observed a negative correlation between NOVA2 and sFLT1 .

Endothelial Dysfunction

10.1007/s43032-022-01044-4

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MEG8 regulates Tissue Factor Pathway Inhibitor 2 (TFPI2) expression in the endothelium

Scientific Reports (2022)

Veerle Kremer Diewertje I. Bink Laura Stanicek Eva van Ingen Theresa Gimbel

Abstract A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. The 14q32 locus contains many non-coding RNAs such as Maternally Expressed Gene 8 (MEG8). We observed an induction of this gene in ischemic heart disease. We investigated the role of MEG8 specifically in endothelial function as well as the underlying mechanism. We hypothesized that MEG8 plays an important role in cardiovascular disease via epigenetic regulation of gene expression. Experiments were performed in human umbilical vein endothelial cells (HUVECs). In vitro silencing of MEG8 resulted in impaired angiogenic sprouting. More specifically, total sprout length was reduced as was proliferation, while migration was unaffected. We performed RNA sequencing to assess changes in gene expression after loss of MEG8. The most profoundly regulated gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), was fivefold increased following MEG8 silencing. TFPI2 has previously been described as an inhibitor of angiogenesis. Mechanistically, MEG8 silencing resulted in a reduction of the inhibitory histone modification H3K27me3 at the TFPI2 promoter. Interestingly, additional silencing of TFPI2 partially restored angiogenic sprouting capacity but did not affect proliferation of MEG8 silenced cells. In conclusion, silencing of MEG8 impairs endothelial function, suggesting a potential beneficial role in maintaining cell viability. Our study highlights the MEG8/TFPI2 axis as potential therapeutic approach to improve angiogenesis following ischemia.

10.1038/s41598-022-04812-z

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Citations (13)

RNA-based therapeutics in cardiovascular disease

Current Opinion in Cardiology (2020)

Tan Phát Pham Veerle Kremer Reinier A. Boon

Cardiovascular disease is the leading cause of death globally, with the number of deaths rising every year. Much effort has gone into development of new treatment strategies. Many RNA species have important regulatory functions in disease initiation and progression, providing interesting new treatment options. This review focuses on different classes of RNA-based therapeutics and provides examples of current clinical and preclinical studies. Current challenges that prevent clinical translation and possibilities to overcome them will be discussed.Different RNA-based molecules have been developed, such as antisense oligos, microRNA mimics and small interfering RNAs. Modifications are used to prevent degradation and immune activation and improve affinity. Additionally, in order to improve delivery of the RNA molecules to the target tissues, viral or nonviral vectors can be used.RNA-based therapy has been shown to be a promising new treatment strategy for different disorders. However, several challenges, such as delivery problems and low efficacy remain. Future research will likely focus on effective delivery to target tissues in order to improve efficacy and avoid harmful side-effects.

10.1097/hco.0000000000000724

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Citations (12)

Tiotropium inhibits airway inflammation and goblet cell metaplasia in a model of relapsing asthma in mice

Stan de Kleijn Yolanda van Wijck Veerle Kremer Hermelijn H. Smits Michael Pieper

The anticholinergic Tiotropium has recently been shown to reduce exacerbations in patients with asthma. It is currently not completely understood how Tiotropium exerts its effect. In the current study, we designed a house dust mite (HDM) extract-induced rechallenge model of allergic airway inflammation in mice, to mimic the symptoms of asthma exacerbations. C57BL/6 mice were sensitized intranasally (i.n.) with 1 µg of HDM extract followed by a primary challenge regime (5 consecutive days 10 µg HDM extract i.n.) after one week. Five weeks after sensitization, a secondary challenge period was carried out. Mice were treated with Tiotropium (1, 10 and 100 µg/kg) i.n. prior to and during the secondary challenge period. Three days after the last challenge, bronchoalveolar lavage (BAL) fluid and lung single cell suspensions were analysed by flow cytometry analysis. In addition, lung histology was analysed via H&E and PAS staining. Mouse serum was collected to determine HDM specific IgE and IgG1 antibodies. Secondary challenge with HDM extract strongly induced allergic airway disease reflected by inflammatory cell infiltration (H&E and BAL) and goblet cell metaplasia (PAS staining). Tiotropium treatment reduced numbers of inflammatory cells in BAL, tissue inflammation and PAS positive cells in a dose-dependent manner. In contrast, serum levels of HDM specific IgE and IgG1 were unchanged. We showed that Tiotropium inhibits allergic airway inflammation and goblet cell metaplasia in a model of relapsing allergic airway disease. These data suggest that Tiotropium may prevent exacerbations by reducing inflammation and mucus production in the airways.

Goblet cell

Tiotropium bromide

Allergic Inflammation

Metaplasia

10.1183/13993003.congress-2015.pa1901

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Aging-regulated TUG1 is dispensable for endothelial cell function

PLoS ONE (2022)

Anna Theresa Gimbel Susanne Koziarek Kosta Theodorou Jana Felicitas Schulz Laura Stanicek

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

Ectopic expression

10.1371/journal.pone.0265160

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Citations (6)

Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner

Nature Communications (2021)

Monika M Gladka Arwa Kohela Bas Molenaar Daniëlle Versteeg Lieneke Kooijman

Abstract The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.

Contractility

10.1038/s41467-020-20361-3

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Citations (75)