University of Groningen Endothelial plasticity in fibrosis and epigenetics as a therapeutic target Hulshoff, Melanie

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University of Groningen

Endothelial plasticity in fibrosis and epigenetics as a therapeutic target

Hulshoff, Melanie

DOI:

10.33612/diss.146265795

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Publication date: 2020

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Hulshoff, M. (2020). Endothelial plasticity in fibrosis and epigenetics as a therapeutic target. University of Groningen. https://doi.org/10.33612/diss.146265795

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ABSTRACT

In this thesis, we aim to (1) uncover novel (epi)genetic regulatory mechanisms that affect endothelial plasticity as an underlying cause of organ fibrosis, and (2) to therapeutically target (epi)genetic regulatory mechanisms to combat organ fibrosis. We identified relevant epigenetic changes during endothelial-to-mesenchymal transition (EndMT), a specific form of endothelial plasticity, in different disease contexts and investigated the impact of different EndMT triggers (such as hypoxia and high glucose) and unraveled the epigenetic changes they induce. Moreover, we have investigated their relevance to the induction and progression of EndMT. Also, we performed stage-specific mapping of EndMT to identify genetic regulatory mechanisms that drive the active stage of transition. For the second aim, we demonstrate that Serelaxin, a recombinant form of the human Relaxin-2 hormone, alleviates cardiac fibrosis in part through the inhibition of EndMT. We also discussed the mechanisms involved in the reciprocal relationship between chronic kidney disease and cardiac fibrosis and identified microRNAs as potential profibrotic kidney-heart messengers. Moreover, we identified relevant possibilities and in vivo applications of CRISPR/Cas derivatives. This gene modulating tool is applied to target epigenetic modifications which alleviate both renal and cardiac fibrosis. In conclusion, in this thesis we have shown that in fibroproliferative diseases, endothelial plasticity is highly regulated by epigenetic modifications (part I). These epigenetic modifications may offer new therapeutic approaches. Indeed, we demonstrate that epigenetic modifications can be targeted in vivo, which results in alleviated organ fibrosis (part II).

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ABSTRACT, INTRODUCTION AND AIMS

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1 INTRODUCTION AND AIMS

Organ fibrosis is characterized by the progressive deposition of extracellular matrix which replaces functional tissue and results in structural alterations and eventually loss of function, contributing to organ dysfunction (e.g. heart failure). The excessive extracellular matrix is produced by different cell types, such as activated fibroblasts, so-called myofibroblasts, and myofibroblast-like cells derived from endothelial cells via a process called endothelial-to-mesenchymal transition (EndMT) [1-4]. As of yet, no specific anti-fibrotic therapy is available.

Endothelial cells line the inner side of blood (or lymphatic) vessels and are in direct contact with the blood. Endothelial cells form the barrier between the blood and the surrounding tissue, and have several important functions such as regulating vascular permeability and the trafficking of leukocytes to the surrounding tissue [5,6]. The dynamic environment (including both biochemical stimuli and mechanical forces) requires endothelial cells to be highly adaptive [7]. This endothelial plasticity can be advantageous, but adverse plasticity can also contribute to disease.

EndMT is a specific form of endothelial plasticity, and was originally discovered during cardiac valve development, but is now increasingly recognized for its contribution to pathologies such as cardiovascular disease [8-10], organ fibrosis [3,4,11] and cancer [12,13]. EndMT is characterized by the loss of endothelial markers and the upregulation of EndMT transcription factors as well as mesenchymal markers. EndMT-derived mesenchymal cells acquire a highly migratory and invasive phenotype which is accompanied by morphological changes from a cobble-stone endothelial morphology into a spindle-shaped myofibroblast-like morphology. EndMT is triggered by a variety of external stimuli such as the lack of oxygen (hypoxia), high glucose concentrations or pro-inflammatory cytokines [14-16]. These environmental factors trigger the activation of signaling pathways which induce EndMT such as the canonical Transforming Growth Factor Beta (TGF-β) pathway or the non-canonical pathways such as Notch [17]. However, the underlying molecular mechanisms that drive EndMT remain largely unknown.

Epigenetics is a way of regulating gene expression independent of changes in the nucleotide sequence, and has been increasingly linked to EndMT [18,19]. Epigenetic regulation includes DNA promoter methylation, histone modifications

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and non-coding RNAs, which each individually or in concert influence gene transcription. Epigenetic regulation was originally identified as a determinant for cellular differentiation during development but is increasingly recognized for its association with cardiovascular disease, organ fibrosis and cancer [20-25]. Because EndMT (1) is originally a developmental process, (2) is a form of cellular plasticity by which extensive gene expression changes are necessary, and (3) contributes to diseases which are associated with epigenetic regulation, epigenetics is considered as a strong candidate regulator of EndMT [18].

The aims of this thesis are to (1) uncover novel (epi)genetic regulatory mechanisms that affect EndMT and (2) to therapeutically target (epi)genetic regulatory mechanisms to combat organ fibrosis.

OUTLINE OF THE THESIS

The first part of the thesis focuses on the epigenetic regulation of EndMT in the context of cardiovascular disease. Chapter 2 starts with an overview of the signaling pathways and modulators of EndMT including profibrotic cytokines (e.g. TGF-β), hypoxia (the lack of oxygen) and high glucose. The focus of Chapter 2 includes a summary of the epigenetic mechanisms (i.e. histone modifications, DNA methylation and non-coding RNAs) which influence EndMT in the context of chronic heart disease. Having identified the relevant epigenetic changes (Chapter

2), in the next two chapters we investigated the impact of different EndMT

triggers (i.e. hypoxia and high glucose) and unraveled the epigenetic changes they induce. In Chapter 3 we examine the possible involvement of DNA promoter methylation of the anti-fibrotic gene RASAL1 in hypoxia-induced EndMT. In

Chapter 4 we investigate the contribution of the non-coding RNA miR-132-3p and

the transcription factor KLF7 in glucose-induced EndMT and the related aortic stiffening in diabetes. Besides cardiovascular disease, EndMT contributes to other pathologies including chronic kidney disease. Chapter 5 describes the mechanisms by which non-coding RNAs facilitate or inhibit EndMT during development and diseases. In the last chapter of part I, Chapter 6, we performed stage-specific mapping of EndMT to identify regulatory mechanisms that drive the active stage of transition.

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The second part of the thesis focuses on the therapeutic targeting of epigenetic mechanisms to combat organ fibrosis. We explore in Chapter 7 the anti-fibrotic effect of Serelaxin, a recombinant form of the human Relaxin-2 hormone, in the heart. Serelaxin exerts vasodilating, inflammatory and anti-fibrotic effects and might therefore be of interest to target chronic heart failure.

Chapter 8 describes the mechanisms by which chronic kidney disease may cause

cardiac fibrosis. Several (potential) modulators are discussed including non-coding RNAs. Chapter 9 describes different genome editing tools, the variety of Cas proteins and most importantly the possibilities and in vivo applications of CRISPR/Cas derivatives to perform gene modulation. The CRISPR/Cas sytem was originally discovered in bacteria and is now used to cleave mammalian DNA or RNA. Deactivation of the cleavage capacity of the CRISPR/Cas system enables to fuse Cas to other effector domains thereby enabling distinct possibilities to use the CRISPR/Cas system. In Chapter 10 we use the deactivated CRISPR/Cas system to perform gene-specific reactivation of the anti-fibrotic genes (including RASAL1) which results in the amelioration of kidney fibrosis. After the successful establishment of this system to prevent experimental kidney fibrosis, we transfer this system to the heart to treat experimental cardiac fibrosis (Chapter 11).

The final part of the thesis includes a Research Summary and an Epilogue including discussion and future perspectives.

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PART

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(EPI)GENETIC REGULATION OF

ENDOTHELIAL PLASTICITY

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