Here you can find twelve selected publications, illustrating the excellent scientific work of each research theme within the RIMLS institute.

RIMLS highlights 2017

Dorien Kiers et al. Thromb Haemost

The effect of antiplatelet drugs on the inflammatory response during human endotoxemia

Platelet aggregation inhibitors are the pharmacological cornerstone in the primary and secondary prevention of cardiovascular events. In addition, it is increasingly recognized that platelets have potent immunomodulatory properties. The use of acetylsalicylic acid (ASA) is associated with improved outcome in patients with sepsis, and P2Y12 inhibitors (ticagrelor and clopidogrel) have been suggested to also have immunomodulatory effects.
Therefore, we evaluated the effects of clinically relevant combinations of antiplatelet therapy on the immune response in experimental endotoxemia in humans in vivo. 40 healthy subjects were randomized to seven days of placebo, placebo with ASA, ticagrelor and ASA, or clopidogrel and ASA treatment. Systemic inflammation was elicited at day seven by intravenous administration of Escherichia coli endotoxin.
ASA treatment profoundly augmented the plasma concentration of pro-inflammatory cytokines, but did not affect anti-inflammatory cytokines. Addition of the P2Y12 antagonist to ASA did not affect any of the circulating cytokines, except for an attenuation of the ASA-induced increase in TNFα by ticagrelor. Systemic inflammation increased plasma adenosine, without differences between groups, and although P2Y12 inhibition impaired platelet reactivity, there was no correlation with cytokine responses.

Kiers, D., van der Heijden, W.A., van Ede, L., Gerretsen, J., de Mast, Q., van der Ven, A.J., El Messaoudi, S., Rongen, G.A., Gomes, M., Kox, M., Pickkers, P., & Riksen, N.P. A randomised trial on the effect of anti-platelet therapy on the systemic inflammatory response in human endotoxaemia. Thromb Haemost. 117:1798-1807, 2017.

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Iris Nagtegaal et al. J Clin Oncol

Tumors of the digestive tract

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Iris Nagtegaal et al. J Clin Oncol

Tumor deposits in colorectal cancer: improving the value of modern staging

Colorectal cancer (CRC) treatment is largely determined by tumor stage and lymph node metastases (LNM) play an essential role. Deposits of malignant cells (tumor deposits (TD)) can be found in the fat surrounding the colon and rectum, without evidence of residual lymph node tissue, but their role in staging is debated. We investigated whether TD might add significant new information to staging. We performed a systematic literature search on the role of TD in CRC. Moreover, data from four large cohorts were analyzed for the relevance of TD, LNM and vascular invasion on the pattern of metastases and outcomes. Of the 10106 included patients with CRC from literature, 22% presented with TD. TD are invariably associated with poor outcome. In the four large cohorts the effects of TD were stronger than both LNM and vascular invasion. The presence of TD and LNM more than doubles the risk of liver metastases in comparison to LNM alone. We have shown that TDs are not equal to LNM or vascular invasion with respect to biology and outcome. By only considering TDs in the absence of LNM, valuable prognostic information is lost. Therefore, TNM staging in CRC should be changed.

Nagtegaal, I.D., Knijn, N., Hugen, N., Marshall, H.C., Sugihara, K., Tot, T., Ueno, H., & Quirke, P. Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis. J Clin Oncol. 35:1119-1127, 2017.

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Jasmijn van Kampen et al. Cancer Res

Reversal of Epithelial-to-Mesenchymal Transition of Tumor Cells by microRNA-520f

Despite continuous efforts to develop new therapeutic strategies for cancer, the overall survival of patients with metastatic disease is poor. Reversing epithelial-to-mesenchymal transition (EMT), a process that occurs in the majority of cancers, is widely regarded as an approach to combat cancer progression and therapeutic resistance. In this study, we screened a microRNA expression library to identify miRNAs that are able to activate transcriptional activity of the E-cadherin-encoding CDH1 gene as a surrogate for EMT reversal. MiR-520f expression was sufficient to restore endogenous levels of the cell adhesion molecule E-cadherin in cancer cell lines showing strong or intermediate mesenchymal phenotypes. In addition, miR-520f inhibited the invasive behavior of multiple cancer cell models and also inhibited the formation of lung metastasis in a mouse model. The inhibition of tumor cell invasion by miR-520f was accomplished by the direct knockdown of its target genes ADAM9, TGF-β receptor 2 (TGFBR2), and the EMT-inducing transcription factor-encoding genes SNAI2, ZEB1 and ZEB2. Our study has shown for the first time that miR-520f exerts anti-invasive and anti-metastatic effects in vitro and in vivo. Recent, unpublished data have revealed that miR-520f can also make cancer cells more susceptible to the chemotherapeutic drug cisplatin. Together these data show that this miRNA has a great therapeutic potential.

This work was supported by InteRNA Technologies BV and by the Dutch Technology Foundation STW (project number: 12439).

van Kampen, J.G.M., van Hooij, O., Jansen, C.F., Smit, F.P., van Noort, P.I., Schultz, I., Schaapveld, R.Q.J., Schalken, J.A., & Verhaegh, G.W. miRNA-520f Reverses Epithelial-to-Mesenchymal Transition by Targeting ADAM9 and TGFBR2. Cancer Res. 77:2008-2017, 2017.

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Joost te Riet et al. Sci Rep

Sweet but strong: how the glycocalyx strengthens pathogen binding

We have demonstrated that the pericellular layer of sugar chains (named glycocalyx) that surrounds every cell of our body can help strengthening host-pathogen interactions. By using an atomic force microscope (AFM) – a very sensitive technique to manipulate single cells – we were able to measure the binding strength between single human dendritic cells and single cells of the fungus Candida albicans. Although the primary players in these interactions are receptors on the dendritic cells that bind to specific molecules on the Candida cell wall, the glycocalyx of the immune cells now appears responsible for strengthening binding to the fungus by increasing cell stiffening at the contact site with the pathogen.
Our findings demonstrate for the first time the direct involvement of this sugar layer in strengthening cell-pathogen interactions and put forward a possible role for the glycocalyx as extracellular ”skeleton”. This exo-skeleton could possibly synergize with the intracellular actin cytoskeleton to reinforce cell-pathogen interactions, eventually facilitating pathogen uptake by the dendritic cell.
Glycocalyx defects are associated with pathologies such as cancer and diabetes and are known to affect the immune system. Unraveling the repertoire of molecular mechanisms involved in host-pathogen interactions may provide novel leads for treatments of infectious diseases.

te Riet, J., Joosten, B., Reinieren-Beeren, I., Figdor, C.G., & Cambi, A. N-glycan mediated adhesion strengthening during pathogen-receptor binding revealed by cell-cell force spectroscopy. Sci Rep. 7:6713, 2017.

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Louis van der Putten et al. Carcinogenesis

Molecular carcinogenesis of (pre)malignant endometrial lesions

Most endometrial carcinomas have endometrioid histology, and a favorable prognosis. Histological studies have proposed that these carcinomas arise from (benign) endometrial hyperplasia, which can have simple or complex architecture, and normal of atypical nuclei. However, endometrium surrounding endometrioid carcinomas sometimes is atrophic. We therefore studied the presence of mutations in benign and malignant endometrium.
KRAS, PIK3CA, AKT1, CTNNB1, BRAF, EGFR and NRAS mutations were determined by single molecule molecular inversion probes in endometrioid carcinomas next to atrophic or hyperplastic endometrium, and in benign endometrial hyperplasia.
As shown in Figure 1, the KRAS gene was most often mutated in carcinomas next to hyperplastic endometrium, whereas PIK3CA and CTNNB1 mutations were found in carcinomas next to atrophic endometrium. In benign hyperplasia, the presence of mutations appeared to be related to complex architecture, regardless of the presence of atypical nuclei.
Based on mutation analysis, carcinogenesis of endometrioid carcinomas next to atrophic endometrium appears to be different to that of those next to hyperplasia. Moreover, especially the presence of complex architecture in benign endometrial hyperplasia appears to be related to the presence of mutations. The value of mutation analysis in managing endometrial hyperplasia and carcinoma should therefore be studied.

Figure: Percentage of cases with a mutation in the studied genes, stratified by histological type. Several cases had multiple mutations.

van der Putten, L.J.M., van Hoof, R., Tops, B.B.J., Snijders, M.P.L.M., van den Berg-van Erp, S.H., van der Wurff, A.A.M., Bulten, J., Pijnenborg, J.M.A., & Massuger, L.F.A.G. Molecular profiles of benign and (pre)malignant endometrial lesions. Carcinogenesis 38:329-335, 2017.

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Menno ter Huurne et al. Cell Stem Cell

Cell cycle control and pluripotency

Two major hallmarks of cancer are aberrant cell cycle control and the inability of tumor cells to differentiate. Several studies have shown a mechanistic link between these two processes and it has been postulated that a short G1-phase during the cell cycle prevents differentiation of pluripotent cells. To study the relationship between the G1-phase and pluripotency we have used two different flavors of pluripotent mouse Embryonic Stem Cells (ESCs). Mouse ESCs classically cultured in serum-rich condition (serum ESCs) lack G1-phase control and rapidly progress through the cell cycle. Here, we show for the first time that ESCs grown in the presence of two small-molecule inhibitors that ensure maintenance of pluripotency (2i ESCs) have a significantly longer G1-phase and possess a functional G1 checkpoint. In addition, we have identified the signaling pathway that is responsible for abrogation of the G1 checkpoint in serum ESCs. Altogether, these results indicate that the length of G1-phase does not affect the maintenance of pluripotency. This finding provides a paradigm shift in how the cell cycle impacts on the pluripotent potential of cells and could have important implications for future research on cancer treatment.

ter Huurne, M., Chappell, J., Dalton, S., & Stunnenberg, H.G. Distinct Cell-Cycle Control in Two Different States of Mouse Pluripotency. Cell Stem Cell. 21:449-455, 2017.

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Richard Rodenburg et al. Hum Mutat

Functional confirmation of disease-causing mutations in mitochondrial disorders

Mitochondrial disorders are caused by defects in more than 300 different genes. Whole exome and genome sequencing make it possible to detect all genetic variants in patients by a single diagnostic test. However, a correct clinical interpretation of these genetic variants remains a challenge, especially in disorders with a poor genotype-phenotype correlation and a large number of candidate genes, such as mitochondrial disorders. Functional studies of genetic variations (“functional genomics”) can help to discriminate disease-causing mutations from harmless genetic variants. A recent example is a study of mitochondrial disease patients from five different families, with only partially overlapping clinical symptoms. All patients had genetic variants in the WARS2 gene, encoding mitochondrial tryptophanyl-tRNA synthase (mtTrpRS). This enzyme plays an essential role in the synthesis of proteins involved in mitochondrial energy metabolism. Functional studies of patient-derived cells confirmed that mtTrpRS  function was compromised. This study establishes mtTrpRS deficiency as a mitochondrial protein synthesis defect causing a clinically heterogeneous disease. It illustrates the power of the combination of functional and genetic tests in diagnosing patients with mitochondrial disorders. The knowledge about mitochondrial function in health and disease that is gained from investigations of novel gene defects, such as WARS2, is of great importance for research aimed at developing novel treatment approaches for mitochondrial disorders, for which there is no cure yet.

Wortmann, S.B., Timal, S., Venselaar, H., Wintjes, L.T., Kopajtich, R., Feichtinger, R.G., Onnekink, C., Mühlmeister, M., Brandt, U., Smeitink, J.A., Veltman, J.A., Sperl, W., Lefeber, D., Pruijn, G., Stojanovic, V., Freisinger, P., van Spronsen, F., Derks, T.G., Veenstra-Knol, H.E., Mayr, J.A., Rötig, A., Tarnopolsky, M., Prokisch, H., & Rodenburg, R.J. Biallelic variants in WARS2 encoding mitochondrial tryptophanyl-tRNA synthase in six individuals with mitochondrial encephalopathy. Hum Mutat. 38:1786-1795, 2017.

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Tania Crişan et al. PNAS

Monocyte exposure to soluble urate results in enhanced proinflammatory cytokine production

Cells originating from gout patients differ in their cytokine production capacity compared to control volunteers in a serum urate dependent manner. The elevation of urate levels is associated to several other diseases except gout: metabolic syndrome, atherosclerosis, hypertension, chronic kidney disease, aging, or cancer, making uric acid a link between the diseases of modern society associated with low-grade chronic inflammation. We extensively studied the involvement of soluble uric acid in modifying the behavior of myeloid cells in response to TLR ligands. We show that urate priming significantly, dose-dependently, shifts the transcription and protein levels of pro-inflammatory cytokines (such as IL-1β) while uniquely decreasing anti-inflammatory IL-1 receptor antagonist (IL-1Ra). We showed that pathways are modulated to explain at least in part how urate determines this behavior: AKT is upregulated, autophagy is downregulated, reactive oxygen species production is inhibited, and epigenetic modifications could be induced by uric acid. The finding that basal autophagy levels are diminished shows that autophagy could be a possible new source for intervention in gout. Moreover, the indications that urate can exert stable inflammatory effects non-restricted to MSU crystallization, are likely to impact on a broad range of associated inflammatory diseases in the future.

Figure: Schematic overview of effects induced by uric acid in monocytes. A. Uric acid is a purine catabolism product. B. In vitro pre-treatment of monocytes with increasing concentrations of soluble urate results in increasing IL-1β production but in a reduction in natural antagonist IL-1Ra. C. This effect is obtained through activation of AKT and PRAS40 phosphorylation which reduces autophagic activity as observed by fluorescence microscopy of LC3-GFP-overexpressing HeLa cells.

Crişan, T.O., Cleophas, M.C.P., Novakovic, B., Erler, K., van de Veerdonk, F.L., Stunnenberg, H.G., Netea, M.G., Dinarello, C.A., & Joosten, L.A.B. Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway. Proc Natl Acad Sci USA. 114(21):5485-5490, 2017.

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Tom Nijenhuis et al. JASN

Viagra® in kidney disease: erecting the injured podocyte

Worldwide millions suffer from kidney injury, which impacts quality-of-life and has a large economic burden due to costly treatments for kidney failure (dialysis and kidney transplantation).
Proper function of glomerular epithelial cells, called podocytes, is of crucial importance for the integrity of the glomerular filtration barrier (GFB). GFB injury induces proteinuria and loss of renal function. Gain-of-function mutations in the TRPC6 ion channel and enhanced TRPC6 expression in inherited and acquired glomerular disease, respectively, demonstrated the importance of enhanced Ca2+ influx into podocytes, activating downstream injurious effector mechanisms. Our previous work revealed a deleterious feedforward signaling loop in which TRPC6 activity stimulates its own expression. Therefore, we hypothesized there must also be pathways inhibiting ongoing TRPC6-mediated signaling, thereby protecting the podocyte from further harm.
We now demonstrated that a cGMP-PKG-PPARɣ signaling cascade downregulates TRPC6 expression, which  reduces TRPC6-mediated Ca2+ influx in the podocyte. In vivo, PPARɣ knockout increased TRPC6 expression and induced proteinuria, whereas PPARɣ agonism did the opposite. We made use of this pathway by repurposing the phosphodiesterase 5A inhibitor sildenafil (Viagra®) to increase cGMP and prevent TRPC6 overexpression, podocyte injury and proteinuria in animal models of primary and secondary glomerular injury, e.g. diabetic nephropathy. This identifies sildenafil as a novel treatment possibility.

Sonneveld, R., Hoenderop, J.G., Isidori, A.M., Henique, C., Dijkman, H.B., Berden, J.H., Tharaux, P.L., van der Vlag, J., & Nijenhuis, T. Sildenafil Prevents Podocyte Injury via PPAR-γ-Mediated TRPC6 Inhibition. J Am Soc Nephrol. 28:1491-1505, 2017.

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Eline Boon et al. Head Neck

Prostate cancer treatment in a subtype of salivary gland cancer – finding similarities across cancer types

Salivary gland cancers are a heterogeneous group of cancers; each subtype has different clinical characteristics and course of disease. Salivary duct carcinoma is one of the most aggressive subtypes; many patients develop distant metastasis, leading to a poor prognosis. Interestingly, salivary duct carcinoma shows similarities with prostate cancer. Both of these cancers express the well-known male hormone receptor, the androgen receptor. In prostate cancer, androgen receptor blockade is a known effective treatment. Therefore, we investigated the potential of androgen blockade in salivary duct carcinoma.
In this paper we present data from a national cohort of 35 patients with advanced androgen receptor positive salivary duct carcinoma treated with androgen deprivation therapy (ADT). We compared the outcomes of these patients to 43 patients with advanced salivary duct carcinoma which received best supportive care. We demonstrated that patients treated with ADT had 20% partial response and 30% stable disease, leading to a clinical benefit of 50%. Furthermore, the overall survival of treated patients was significantly better compared to the patients with best supportive care.
This research contributes to the expanding knowledge of using ADT as it is one of the largest series of salivary duct carcinoma patients treated with this type of hormonal therapy. Close collaboration with prostate cancer researchers may lead to valuable new insights in treating salivary duct carcinoma patients.

Figure: Cox regression survival curves for all androgen deprivation therapy (ADT) treated versus best supportive care (BSC) patients. ADT treated patients had significantly better overall survival compared to patients receiving BSC (p = 0.024; HR 0.53; 95% CI 0.30 – 0.92).

Boon, E., van Boxtel, W., Buter, J., Baatenburg-de Jong, R.J., van Es, R.J.J., Bel, M., Fiets, E., Oosting, S.F., Slingerland, M., Hoeben, A., Tesselaar, M.E.T., Jonker, M.A., & Flucke, U.E. Nationwide Network and Registry of Histopathology and Cytopathology (PALGA) Group, van der Graaf WTA, van Herpen CML. Androgen deprivation therapy for androgen receptor-positive advanced salivary duct carcinoma: A nationwide case series of 35 patients in The Netherlands. Head Neck. Epub 2017.

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Jos Smits et al. Sci Rep

Immortalized N/TERT keratinocytes: a powerful tool for skin research

The strong societal urge to reduce the use of experimental animal has led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. Our publication compares two immortalized keratinocyte cell lines (N/TERT-1, N/TERT-2G) to primary human keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of three dimensional (3D) normal and inflammatory human epidermal equivalents (HEEs). Epidermal barrier permeability, morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are very useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, skin disease pathogenesis and therapeutics. In contrast to the most widely used aneuploid keratinocyte HaCaT cell line, N/TERT cells are diploid which enables us to use these cells for genome editing strategies. Currently, our group is editing the genome of the N/TERT keratinocytes using CRISPR-Cas9 to eventually generate genome-edited 3D human skin equivalents for studying gene function in an organ-like environment.

Smits, J.P.H., Niehues, H., Rikken, G., van Vlijmen-Willems, I.M.J.J., van de Zande, G.W.H.J.F., Zeeuwen, P.L.J.M., Schalkwijk, J., & van den Bogaard, E.H. Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models. Sci Rep. 7:11838, 2017.

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Mani Diba et al. Adv Mater

Highly elastic and self-healing composite colloidal gels for regenerative medicine 

Colloidal gels are three-dimensional interconnected networks of colloidal particles, which are formed within a liquid phase (Figure 1). These materials are increasingly used for applications in regenerative medicine in view of their favorable biological and rheological properties. It was shown recently that colloidal gels can be rendered self-healing when the interactions between the particles are non-covalent and reversible. Purely organic colloidal gels are, however, mechanically weak. Organic colloidal gels can be reinforced by stiff inorganic nanoparticles to overcome this mechanical weakness, but this strategy was shown to result into a strongly reduced self-healing capacity. In addition, composite colloidal gels which can resist substantial compressive and tensile loads have not been reported yet. Consequently, the self-healing ability of colloidal gels was only confirmed using rheological tests so far. In this study, novel composite colloidal gels were developed by electrostatic self-assembly of silica and gelatin nanoparticles into 3D porous networks, which combined a very strong capacity for self-healing with high mechanical robustness in terms of tensile and compressive strength. These unique properties were not reported previously for colloidal gel systems and were obtained by a delicate design and thorough mechanistic studies of the composition and structure of these composite gels. The results obtained in this study provide new, critical insight into the structural and mechanical properties of composite colloidal gels and, more importantly, open up a new horizon for applications of self-healing colloidal gel systems in regenerative medicine.

Figure: Schematic illustration of the porous structure of composite colloidal gels. Soft gelatin and stiff silica nanoparticles are shown in red and green, respectively.

Diba, M., Wang, H., Kodger, T.E., Parsa, S., & Leeuwenburgh, S.C. Highly Elastic and Self-Healing Composite Colloidal Gels. Adv Mater. 29, 2017.

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