Within most matrix projects (see ‘Projects’), there are opportunities for life sciences-, medical- and applied sciences university students to perform internships, under the supervision of a PhD student or a postdoctoral fellow. Below is a list of general eligible topics; the specific research topics will be defined together with the supervisor. Please address contact persons listed below for further inquiries.
Together we’re strong: Formation of multinucleated giant cells
Contact: Prof Dr. R.A. Bank
After implantation of a biomaterial, inflammatory cells, such as neutrophils and macrophages, recognize the biomaterial as a foreign body and mount a reaction that aims at removing it. When macrophages do not succeed in destroying the biomaterial by phagocytosis and proteolytic enzymes, they fuse and form a multinucleated giant cell. The mechanisms resulting in the formation of such cells are not fully understood.
In this project, we will use in vitro models to investigate macrophage fusion mechanisms by using various cytokines to stimulate macrophage fusion, siRNA’s for blocking candidate fusion pathways, and cell tracking dyes to visualize macrophage fusion. Students will explore these models and will use molecular and cellular techniques relevant to our study question. (See also Multinucleated giant cell formation: Molecular mediators and signal transduction routes under ‘Projects’ for further information)
Epigenetic regulation of fibrosis-related genes
Contact: Rutger Gjaltema , MSc
The general focus of this project is epigenetic regulation of fibrosis-related genes. From literature it is known that posttranslational histone modifications and DNA methylation are important epigenetic regulators of gene expression. Several authors have showed a clear role for epigenetic modifications in myofibroblast differentiation, which is fundamental to fibrotic diseases. We aim to identify specific epigenetic modifications, and the enzymes responsible for these modifications in relation to myofibroblast differentiation in general and for a specific subsets of fibrosis-related genes in particular, and find novel ways to interfere in this process. In our experiments we use primary human skin fibroblasts stimulated with TGFb1 as an in vitro model system for fibrosis.
One of the genes we focus on is plod2, which codes for lysyl hydroxylase 2 (LH2). LH2 is upregulated in many fibrotic tissues and is involved in collagen cross-linking. Fundamental knowledge how this gene is regulated is of high value for making fibrosis reversible, as it has been shown that less cross-links enables collagenases to cleave the high amount of deposited collagen. We identified by chromatin immunoprecipitations that several histone modifications (acetylation and methylation) in the plod2 promoter are changed in a TGFb1 rich environment. siRNA knockdown or chemical inhibition of several histone modifying enzymes resulted in a sharp decrease of LH2 expression. Indicating that these modifications are a potential targets for interfering in TGFb1 induced LH2 expression. In order to reduce LH2 expression in the presence of TGFb1 for extended time periods, we developed Zinc-finger proteins (ZFPs) that can be used as a tool to target specific regions in the genome. To these ZFPs we fused various effector domains that can either introduce epigenetic modifications or attract other enzymes that can. This enables us to introduce targeted epigenetic modification at the plod2 promoter, resulting in reduced LH2 expression. These modifications should be stable enough (inherited by daughter cells) to keep LH2 expression silenced over a prolonged period during TGFb1. With this strategy we were able to significantly reduce LH2 expression by introducing DNA methylation or histone modifications in the plod2 gene. We are currently identifying their long-term silencing effects in our in vitro model system and in Dupuytren's patient-derived fibroblasts.
Adjacent to this project, we are deciphering the role of active DNA demethylation (5mC) in myofibroblast differentiation. Active DNA demethylation can occur via oxidation of 5mC through 5hmC, which can then be either directly deaminated to 5hmU or further oxidized to 5fC and 5caC. These oxidized and deaminated forms are thought to be replaced by a unmethylated cytosine by base-pair excision repair. Since myofibroblast differentiation is a process defined by strong changes in gene expression patterns, we hypothesized a fundamental role for active DNA demethylation of specific genes in this process. We are currently identifying the enzymes responsible for active DNA demethylation in myofibroblast differentiation, and the genomic distribution of these enzymes and their corresponding modifications in our in vitro model.
Interleukin-beta 1 in Fibrosis
contact: prof dr. R.A. Bank
Fibrosis is a defective repair processes often seen after chronic injury and inflammation in a large variety of organs and tissues, such as the kidney, heart, liver, lung and skin. The hallmark of fibrosis is an excessive accumulation of extracellular matrix (ECM), especially due to an imbalance between collagen synthesis and degradation. One of the key processes in fibrosis is the activation of fibroblasts into myofibroblasts, a process that seems to be dependent on the activation of the Hedgehog pathway.
Various cytokines play a role in the differentiation of fibroblasts into myofibroblasts. One of the major pro-fibrotic cytokines is transforming growth factor-beta 1 (TGFb1), as it induces the differentiation of fibroblasts into myofibroblasts. Myofibroblasts are characterized by the presence of cytoplasmic stress fibers and show an excessive production of collagen. TGFb1 is also involved in the disbalance regarding the expression of matrix metalloproteinases capable of degrading collagen versus their inhibitors.
Interleukin-1 beta (IL-1b), a classical pro-inflammatory cytokine, has been implicated as one of the dominant players in the development of fibrosis. It is expressed in the acute phase of inflammation, but is also elevated in the later stages of inflammation and tissue repair. Despite its assumed deleterious role in fibrosis, relatively little is known about the direct effect of IL-1b on fibroblasts. Although both TGFb1 and IL-1b are present during tissue repair processes, however not much is known whether these cytokines have co-stimulatory effects.
Using human lung and dermal fibroblasts, we have shown that IL-1b alone has no obvious pro-fibrotic effects on fibroblasts. However, IL-1b is able to inhibit the TGFb1-induced myofibroblast formation as well as collagen synthesis. In addition, we found that lung and dermal fibroblasts do not always behave identical towards IL-1b. In general, our findings explain that IL-1b is able to suppress the pro-fibrotic features of TGFb1, and thus shows potential anti-fibrotic properties.
Currently, we are studying the IL-1b mediated signaling cascades (such as Nuclear factor kappa B and c-Jun N-terminal kinase pathway) that are possibly involved to inhibit the TGFb1-induced pro-fibrotic responses. We hypothesized that activation/blockade of specific signaling pathway could answer the underlying mechanism. We anticipate our findings would be a key step to develop the future anti-fibrotic therapy.
Hippo signalling in renal fibrosis
Contact: Bram Piersma, MSc
Renal fibrosis is the final common pathway towards ends stage renal disease (ESRD) and renal failure, and is characterised by excessive extra cellular matrix (ECM) production and the destruction of renal architecture. Myofibroblasts are the main ECM producing cells, and the phenotyping and targeting of these cells is a major focus in the field of fibrosis. Elucidating the mechanisms of ECM production and remodelling by myofibroblasts is of paramount importance in order to develop potent anti-fibrotic therapies.
The Hippo pathway has first been described in Drosophila melanogaster and regulates tissue growth. Many components were identified as a results of mutations in the fruit fly that led to tissue overgrowth. The pathway is conserved in vertebrates, including mammals, and since the discovery, has been implicated in various forms of cancers, in special in the liver. Next to the regulation of tissue growth, the Hippo pathway has been implicated in other processes like cell-fate differentiation, mitosis, and apoptosis. Although the Hippo pathway has mainly been studied in cancer pathology, a small body of evidence suggests that it is also implicated in the regulation of fibrogenic responses. This is to be expected, for in fibrosis exaggerated fibroblast proliferation, and aberrant apoptosis of myofibroblasts are key, and may be regulated by the Hippo pathway. In renal myofibroblasts, however, nothing is known about the Hippo pathway.
The aim of this project is to elucidate the role of the Hippo pathway in the functioning of myofibroblasts in fibrosis, with emphasis on the kidney. In our lab we have developed a culturing system using polyacrylamide gels with varying stiffness which allows us to mimic the stiffness of different tissues in the human body. Fibrotic tissue is stiffer than healthy interstitial tissue, and therefore this culturing system can aid us in the understanding of myofibroblast behaviour in vivo..
Macrophage phenotyping during the Foreign Body reaction
Contact: Prof dr. R.A. Bank
Biomaterials play a pivotal role within the field of tissue engineering. In this field an attempt is made to repair, restore or augment damaged or diseased tissue, usually through the use of biomaterials. In these approaches biomaterials act as a temporary scaffold, which provides structural support, delivers (stem)cells, and may release biologically active mediators. Introduction of biomaterials into the body elicits an inflammatory reaction called the foreign body reaction (FBR) (reviewed in [1-3]). Initially, the inflammatory reaction is very similar to a wound healing response. However, over time, the response changes from an acute inflammation in response to the damage caused by the implantation or injection procedure to a chronic inflammation in response to the presence of the biomaterial. The course and nature of the FBR that follows introduction into the body depends largely on the characteristics of the introduced biomaterial. In some cases the inflammatory response may be mild, in some cases it may be very severe, but in all cases the inflammatory response has a major impact on the ability of the biomaterial to function properly. Despite decades of research on the FBR, many processes that occur during the FBR and the mechanisms that are involved are poorly understood at best. By investigating the fundamental processes that occur during the FBR and by challenging the current dogmas, we aim to expand the fundamental knowledge on this topic. Doing so will improve our general understanding of the FBR and may provide clues about how biomaterials and the host's response may be improved.
Historically, the macrophage has been thought to be the most important immune cell during the foreign body reaction. They are supposed to be responsible for the generation and orchestration of the pro-inflammatory microenvironment, intra- and extracellular degradation and phagocytosis of the biomaterial and possibly for the instruction of the fibroblasts surrounding the biomaterial. We are currently investigating this dogma by studying the foreign body reaction in "MaFIA" mice [4-5], which enable conditional depletion of macrophages. Our preliminary data suggests that the macrophage is indeed of critical importance for the generation of the inflammatory microenvironment, inflammatory ingrowth into the biomaterial and its degradation. Capsule formation and the expression of genes encoding extracellular matrix proteins and fibroblast-activating growth factors were increased in the absence of macrophages, indicating that macrophages are not required for capsule formation or the instruction of fibroblasts.
Despite their apparent importance in the foreign body reaction, surprisingly little is known about the origin, phenotype and activity of macrophages in the (different phases of the) foreign body reaction. Furthermore, the exact role and importance of fibroblasts and their crosstalk with macrophages has never been addressed in the context of a biomaterial. These themes will be investigated in this project.
|16 December 2014 4.13 p.m.