Endothelial cell transcriptional regulation in vascular disease
Sol, Marloes
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10.33612/diss.136419916
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2020
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Chapter 1
General Introduction
GENERAL INTRODUCTION
The blood vascular system in the human body consists of the heart and blood vessels. Blood vessels are important as the conduits for the transportation of blood and the regulation of exchange of substances in the perfused tissues. Blood vessels can be divided in five different types that differ in diameter, morphology and function: arteries, arterioles, capillaries, venules, and veins. The arteries are responsible for transportation of blood from the heart into organs and tissues. The arteries enter organs and tissues and branch into smaller arterioles, which distribute blood into the capillaries. Capillaries enable the exchange of oxygen, nutrients and catabolites across the endothelium. Arterioles provide resistance to blood flow and consequently regulate blood pressure and tissue perfusion [1]. Subsequently, the blood is collected in post-capillary venules and then flows into veins that are essential for the vascular reserve capacity and take care of the transportation of blood back to the heart.
THE ENDOTHELIUM
The endothelium constitutes the inner monolayer of cells that lines all blood vessels and covers a surface area of approximately 1000 m2 in an average 70-kg human
[2]. The endothelium is a dynamic tissue consisting of heterogenic populations of endothelial cells that differ in morphology and function, but together maintain circulatory homeostasis [3]. The endothelium is pivotal in the regulation of multiple processes, including the exchange of oxygen and nutrients, vascular tone, vascular permeability, inflammation, angiogenesis, and haemostasis. Via molecular cross-talk, the endothelium also influences the function of cells in the underlying tissues. An example of molecular cross-talk by endothelial cells is the regulation of vascular tone via smooth muscle cell relaxation by the production of endothelial nitric oxide (NO) [4-6]. Proper functioning of endothelial cells is essential to maintain vascular health. Dysfunction of the endothelium is a key initiating step in the pathophysiology of several cardiovascular and renal diseases, including glomerulosclerosis and atherosclerosis [3, 7-12]. In glomerulosclerosis and atherosclerosis, endothelial homeostasis is disturbed leading to endothelial cell dysfunction.
ENDOTHELIAL CELL FUNCTION IN GLOMERULOSCLEROSIS
Blood filtration and urine production take place in the functional units of the kidney, i.e. the nephrons. Each nephron consists of a glomerulus, where the blood filtration takes place, and a tubule, a segmented compartment responsible for reabsorption of water, small molecules and ions, and the excretion of toxins. The glomerulus is a tuft of microcapillaries enclosed by the Bowman’s capsule lined with parietal epithelial cells.
In the glomeruli, the glomerular filtration barrier (GFB) is responsible for the size- and charge-selective filtration of molecules from the blood. The GFB is composed of non-diaphragmed fenestrated glomerular endothelial cells (GEnCs), which are covered by the endothelial glycocalyx at the luminal side, the glomerular basement membrane (GBM), and podocytes with interdigitating foot processes wrapped around the exterior of glomerular capillaries (fig.1). Intercallating mesangial cells are present between the capillaries and are in direct contact with the glomerular endothelium.
Figure 1. Schematic representation of the glomerular filtration barrier. The glomerular filtration barrier
(GFB) is composed of fenestrated glomerular endothelial cells (GEnCs), which are covered by the endothelial glycocalyx, the glomerular basement membrane (GBM), and podocytes with interdigitating foot processes. The GFB filters water and small molecules out of the blood into the urinary space. Mesangial cells are in direct contact with the glomerular endothelium.
Glomerulosclerosis is the scarring of the glomeruli (fig. 2). Glomerulosclerosis is an end-stage chronic pathologic condition that is often observed in kidney diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN). Glomerulosclerosis is a common pathway leading to the development of renal failure and end-stage renal disease (ESRD), irrespective of the underlying primary renal disease. Glomerulosclerosis is characterized by damage to podocytes and dysfunction of glomerular endothelial cells [10, 13-15], which culminate into proteinuria (the loss of blood-derived proteins into the urine). Furthermore, glomerulosclerosis is characterized by activation of inflammatory pathways in the glomerulus [16, 17], mesangial cell proliferation, and the accumulation of extracellular matrix in the glomerulus.
Glomerulosclerosis ultimately leads to obliteration of glomerular capillaries, due to the intracapillary accumulation of proteinaceous material (hyalinosis), extracellular matrix, and occasionally inflammatory cells [18, 19].
Figure 2. Healthy glomerulus and glomerulosclerosis. Light microscopy photomicrographs of glomeruli,
stained with hematoxylin-eosin. Nuclei are stained in blue. Cytoplasm, cell membranes and extracellular matrix are stained in pink. (A) In a healthy glomerulus, multiple capillary loops are observed (red arrows). (B) A glomerulus from a diabetic nephropathy patient presenting glomerulosclerosis, representing an increase in extracellular matrix in the glomerulus, by which capillary loops are obliterated. Scale bars represent 50µm. Courtesy of Dr. M. C. van den Heuvel.
GEnCs are the first cells of the GFB in contact with the blood. At the luminal side, GEnCs are covered by the endothelial glycocalyx, a gel-like layer consisting of proteoglycans with covalently bound glycosaminoglycan (GAG) side chains [20-22]. The endothelial glycocalyx is negatively charged and provides a barrier to proteins and hereby contributes to a large extent to the charge and size-selective filtration of blood by the GFB [20, 21, 23-25]. Furthermore, the endothelial glycocalyx is essential for vascular protection, attenuation of blood cell-vessel wall interactions, mechanotransduction as it serves as the primary sensor of shear stress, and signaling processes such as NO production. In glomerulosclerosis, the endothelial glycocalyx is degraded. Degradation of the glomerular endothelial glycocalyx has been shown to be important in the development of proteinuria and kidney failure [26, 27]. Different types of GAGs are located in the endothelial glycocalyx, including heparan sulphate (HS). HS plays an important role in regulating the specific permeability of the GFB [28, 29]. Heparanase (HPSE), the only mammalian enzyme that can cleave HS, is involved in the pathogenesis of several glomerular diseases by reducing HS in the endothelial glycocalyx and other compartments of the GFB, including the GBM
[20, 27, 30, 31]. Next to HPSE, matrix metalloproteases are known to be involved in the degradation of the endothelial glycocalyx as well [32, 33]. Other mechanisms involved in endothelial glycocalyx degradation remain largely unknown.
ENDOTHELIAL CELL FUNCTION IN ATHEROSCLEROSIS
Under physiological conditions, the arterial endothelium, together with some subendothelial connective tissue and an elastic membrane (i.e. the internal elastic lamina), form the tunica intima. Vascular smooth muscle cells, elastic lamellae and collagen comprise the tunica media. The outer layer of arteries is composed of fibroblasts and collagens, called the tunica externa (or tunica adventitia) (fig. 3).
Figure 3. Schematic representation of a normal artery. The endothelium lines the inner layer of the artery
and together with elastic tissue (the internal elastic lamina) and some subendothelial connective tissue forms the tunica intima. Smooth muscle cells are present in the middle layer, and together with elastin lamellae and collagens form the tunica media. Fibroblasts are found on the outer layer of the artery, and together with collagens form the tunica externa (or tunica adventitia).
Atherosclerosis is a chronic cardiovascular disease. Atherogenesis comprises a process in which a new intimal layer is formed (neo-intima) [34, 35]. Atherogenesis is characterized by accumulation of oxidized low-density lipoprotein cholesterol (ox-LDL) in the vessel wall [36], the infiltration of inflammatory cells [37], smooth muscle cell migration and proliferation in the neo-intima [38], and the accumulation of extracellular matrix proteins in the neo-intima. Together, these processes result in intimal thickening [39] that ultimately restricts blood flow through the artery (fig. 4).
Figure 4. Healthy (A) and atherosclerotic (B) coronary artery. Light microscopy photomicrographs of arteries,
stained with hematoxylin-eosin. Nuclei are stained in blue. Cytoplasm, cell membranes and extracellular matrix are stained in pink. (A) In a healthy artery, the vessel wall normal layers of tunica intima (I), media (II) and externa (III). (B) In atherosclerosis, a new intimal layer is formed (neo-intima, IV) and restricts the artery. Scale bars represent 50µm.
Under physiological conditions, endothelial cells are in a quiescent state, and they have a vasodilatory function, and an anti-inflammatory, anti-thrombotic, and anti-proliferative phenotype [40]. Fluid shear stress (FSS), the frictional force induced by blood flow to which endothelium is continuously exposed, is a major determinant of vascular health [41]. Areas in arteries exposed to laminar fluid shear stress (i.e. unidirectional flow) are protected from atherosclerosis. Areas exposed to non-unidirectional (disturbed) or oscillatory flow are more prone to the development of atherosclerotic lesions [42, 43]. FSS is important for endothelial homeostasis via mechanotransduction within the endothelial cell. Laminar shear stress protects the endothelial cells via mechanotransduction and the subsequent initiation of essential signalling pathways, including the MAP2K5-MAPK7 signalling pathway, which results in the expression of the transcription factors Krüppel-like factor-2 and 4 (KLF2 and KLF4) [44, 45]. KLF2 and KLF4 drive the expression of downstream atheroprotective genes such as endothelial nitric oxide synthase (eNOS) [46]. Contrary, upon non-unidirectional or oscillatory shear stress mechanotransduction and signalling are lost. Subsequent endothelial homeostasis is disturbed, resulting in endothelial cell dysfunction. Endothelial dysfunction in these atherosusceptible regions is characterized by a reduced production of NO leading to impaired vasorelaxation and platelet adhesion [7], the expression of pro-inflammatory genes, increased adhesiveness for inflammatory cells that consequently migrate into the arterial wall, increased permeability to plasma particles such as ox-LDL, and a proliferative phenotype. Additionally, endothelial cells can obtain a fibro-proliferative phenotype via the process of endothelial-to-mesenchymal transition (EndMT) [42, 47].
EndMT is a process wherein endothelial cells lose their endothelial-specific phenotype, including the expression of endothelial cell-specific markers such as CD31 and VE-cadherin. Endothelial cells conversely acquire a mesenchymal phenotype, including the expression of mesenchymal markers such as alpha smooth muscle actin (αSMA), smooth muscle protein 22 alpha (SM22α) and calponin, and start to display contractile behaviour. Additionally, EndMT is associated with the increased production of extracellular matrix proteins [48, 49]. EndMT is important during embryonic cardiac valve formation [50], but in adulthood EndMT contributes to the development of various fibrotic diseases [51, 52]. In atherosclerosis, EndMT has been shown to play a pivotal role in the formation of neointimal lesions [53]. Notably, our lab previously established a pivotal role for FSS-responsive MAPK7 signalling in the inhibition of EndMT [54].
MOLECULAR MECHANISMS INVOLVED IN ENDOTHELIAL HOMEOSTASIS: REGULATION OF THE ENDOTHELIAL TRANSCRIPTOME
A quiescent endothelial phenotype is secured by the tight regulation of the endothelial transcriptome, i.e. the full array of RNA transcripts produced [55-57]. The transcriptome is regulated at multiple levels, such as activation of specific signal-transduction pathways, binding of transcription factors, chromatin packaging of the DNA, and post-transcriptional regulation via microRNAs [58]. Dysregulation of any of these aforementioned factors might drastically change the endothelial transcriptome and culminate in endothelial dysfunction and concurrently the pathogenesis of atherosclerosis and glomerulosclerosis [59]. In this thesis, we focussed on three players of regulation of the transcriptome, i.e. epigenetic processes including histone modifications, binding of transcription factors, and microRNAs, which are further detailed below.
Nuclear DNA is wrapped around an octamer of histones, consisting of two molecules of each of the four histone proteins (H2A, H2B, H3 and H4), and a linker histone (H1). The DNA and histones together form the nucleosome, the basic building block of chromatin. All nucleosomes together are called chromatin. Histone proteins contain flexible tails that protrude from the nucleosome and are prone to a variety of post-translational modifications, called histone modifications. The best characterized histone modifications involve methylation, acetylation, and phosphorylation. Histone modifications drive epigenetic processes and dictate the conformation of chromatin, and thereby the accessibility of the DNA for proteins, including transcription factors. Consequently, histone modifications modulate the initiation of gene transcription, but without altering the DNA sequence of the respective gene [60]. Histone modifications result either in accessible chromatin (“open” euchromatin) or not accessible chromatin (“condensed” or “closed” heterochromatin) for transcription-associated proteins in order to facilitate or prevent gene expression [61].
Histone modifications are regulated by enzymes that place or remove histone marks. For example, histone acetyltransferases and histone deacetyl transferases (HDAC) can respectively add and remove acetyl-modifications from histone tails. Acetylation of histones generally results in euchromatin formation and thus enhances gene expression. Histone methyltransferases and demethylases add and remove methyl groups on histone tail residues, respectively. Methylation of lysine residues of histone tails can either enhance or inhibit gene transcriptional activity, depending on the position of the modified residue within the histone tail [62]. Polycomb proteins form chromatin-modifying complexes and are important epigenetic regulators. Two main families of Polycomb complexes exist, called Polycomb Repressive Complex 1 (PRC1) and PRC2. PRC2 contains the catalytic subunit Enhancer of Zeste Homolog 2 (EZH2), a highly conserved methyltransferase. EZH2 methylates histone 3 on lysine 27 (H3K27me3). H3K27me3 associates with gene repression [63, 64]. EZH2 is previously identified as essential player in developmental processes, and has recently also been shown to play an important role in endothelial homeostasis. EZH2 is a modulator of a number of endothelial cell functions, such as endothelial-leukocyte interactions and angiogenesis [65, 66] (fig. 5).
Figure 5. Schematic representation of EZH2-mediated H3K27me3 and concomitant gene repression in endothelial cells. In loosely packed chromatin (euchromatin), genes that are essential for endothelial cells, are
accessible for transcription. Enhancer of Zeste Homolog 2 (EZH2) methylates lysine 27 on histone 3 (H3K27me3) which leads to densely packed chromatin (heterochromatin) thereby preventing transcription. Histone modifications are reversible and H3K27me3 can be removed by Jumonji demethylase 3 (JMJD3) or Ubiquitously Transcribed X Chromosome Tetratricopeptide Repeat Protein (UTX).
Next to histone modifications, epigenetic modifications also include DNA methylation. DNA methylation is in general associated with gene repression due to changes in the biophysical characteristics of the DNA [61, 67].
When the chromatin has an open structure (euchromatin), transcription factors can bind to a transcription factor binding site in the promoter region of a gene [68]. Once a transcription factor is bound to this specific site in the DNA, a co-activator complex is recruited and RNA polymerase II can start gene transcription (fig. 6) [69].
However, the presence of euchromatin does not necessarily imply that transcription occurs. Instead of the recruitment of co-activator complexes, also co-repressor complexes can be recruited to DNA-bound transcription factors [70]. Co-repressor complexes, at their turn, recruit HDACs, which remove acetyl groups from histone tails leading to a more compact structure of the chromatin and a reduced accessibility of the DNA, ultimately leading to gene repression [71].
Once a gene is transcribed into messenger RNA (mRNA), the mRNA transcript can be translated into the encoded protein on the ribosome complex (polysome) in the cytoplasm. However, translation can be inhibited by post-translational regulation. More specifically, a mRNA molecule can be degraded prior to translation, a process that is initiated by the binding of microRNAs (miRNAs) to the mRNA transcript. MiRNAs are small single-stranded non-coding RNAs with an average length of 22 nucleotides. MiRNAs mostly interact with the 3’ UTR of a target mRNA to induce translational repression by inhibition of ribosome binding. Multiple different mRNA transcripts can be targeted by one miRNA sequence, as long as the miRNA binding sequence matches with the 3’ UTR of the mRNAs [72-74] (fig. 6).
Figure 6. Three levels of regulation of the transcriptome: histone modifications, binding of transcription factors, and microRNAs. [1] The first level of regulation of the transcriptome is formed by histone modifications.
Histone modifications stably alter the confirmation of the chromatin, and thereby the accessibility of the DNA for transcription-associated proteins. Repressive histone modifications (marks) reduce the accessibility of the chromatin, whereas activating marks lead to an open chromatin structure (euchromatin). [2] In an open chromatin structure, transcription factors can bind the DNA and recruit co-activators and RNA polymerase II to initiate transcription of the DNA into mRNA. To inhibit transcription, transcription factors recruit co-repressors, which at their turn recruit histone deacetylases (HDACs), to remove activating acetyl groups on the histones leading to a closed chromatin structure (heterochromatin) and transcriptional repression (indicated by the lock). Binding of transcription factors leading to transcriptional activation or repression is a second level of regulation of the transcriptome. [3] In case mRNA is transcribed from the DNA, a third layer of regulation exists. The mRNA can be translated into protein in the ribosome, or degraded by microRNAs (miRNAs) to inhibit protein expression.
SCOPE AND OUTLINE OF THIS THESIS
Significant advancements have been made in the elucidation of the pathogenetic pathways underlying atherosclerosis and glomerulosclerosis. However, there are still no definite preventative or curative treatments available for these pathologies. To halt the development and progression of glomerulosclerosis and atherosclerosis, a deeper understanding of the pathogenetic mechanisms involved as well as the identification of novel therapeutic targets is needed. Dysfunction of endothelial cells is a critical initiating process in both the pathogenesis of glomerulosclerosis and atherosclerosis. Differential gene and protein expression patterns lie at the basis of endothelial dysfunction and the concomitant phenotypic changes of endothelial cells. Aberrant
epigenetic, transcriptional, and translational regulation of genes therefore underlies the development of endothelial dysfunction during pathogenesis. The overall aim of this thesis is to identify epigenetic, transcriptional and translational mechanisms involved in the modulation of the endothelial transcriptome underlying endothelial dysfunction in glomerulosclerosis and atherosclerosis.
Glomerular endothelial cell (GEnC) dysfunction is critical in the development of glomerulosclerosis and comprises multiple facets. In chapter 2 we postulate that GEnC dysfunction is a pivotal and early factor in the development of glomerulosclerosis and at the basis of the podocyte dysfunction and mesangial expansion, which is observed later during disease progression. We review the importance of the cross-talk between GEnCs and podocytes, and GEnCs and mesangial cells, and further describe that GEnC dysfunction comprises multiple facets, precedes podocyte damage, and is sufficient for the development of proteinuria and glomerulosclerosis in Focal Segmental Glomerulosclerosis (FSGS) and diabetic nephropathy (DN).
Endothelial cell function is hampered by aberrant changes to the endothelial transcriptome, some of which involve the epigenetic silencing of genes. The glomerular endothelial glycocalyx is reduced in glomerular sclerotic diseases, including DN. In
chapter 3, we investigated whether the expression of the histone methyltransferase
Enhancer of Zeste Homolog 2 (EZH2) and its concomitant histone modification H3K27me3 are changed in GEnCs, and associate with a reduced glomerular endothelial glycocalyx in experimental DN in mice. In vitro, we investigated whether, and if so, how EZH2 reduces the endothelial glycocalyx. Furthermore, in vitro and in vivo findings are translated to the clinical setting by studying renal biopsies obtained from DN patients. In chapter 3, it is shown that EZH2-mediated H3K27me3 is increased in human and experimental DN and associated with a compromised endothelial glycocalyx. In chapter
4, it is evaluated whether downregulation of (glomerular) endothelial EZH2, i.e. reduced
H3K27me3 activity, can be achieved in vivo, and if so whether this results in ameliorated DN. To decrease EZH2 and H3K27me3, an activated endothelial cell-specific siRNA-based targeting approach is used in which cationic liposomes, called SAINT-O-Somes (SOS), containing siRNA against EZH2 are administered to diabetic BTBRob/ob mice. The
delivery of the siRNA-encapsulating SOS to GEnCs, and the decrease of EZH2 and H3K27me3 by the encapsulated siRNA are assessed. Proteinuria, plasma creatinine, the endothelial glycocalyx, and renal histology are evaluated.
The heparan sulphate cleaving enzyme heparanase (HPSE) is involved in the pathogenesis of glomerular sclerotic diseases. Aberrant transcriptional regulation of the HPSE gene could be involved in an increased expression of HPSE, culminating in glomerular damage. In chapter 5, we investigated in GEnC and podocytes whether an increased expression of HPSE results from decreased transcriptional inhibition of the HPSE gene by the transcription factor peroxisome proliferator-activated receptor gamma (PPARɣ). It is investigated whether, and if so, how PPARɣ agonists reduce the expression of HPSE at the transcriptional level, and modulate glomerular disease development in adriamycin-induced glomerulopathy, an experimental model for FSGS. Unidirectional flow is a major determinant for endothelial health and activates Mitogen-Activated Protein Kinase 7 (MAPK7) signalling, which suppresses endothelial dysfunction and mesenchymal transition (i.e. EndMT). Conversely, loss of MAPK7 signalling leads to endothelial dysfunction and EndMT. Currently, it is unknown how MAPK7 activity is regulated in coronary artery disease. The histone methyltransferase EZH2, responsible for H3K27 methylation (H3K27me3), silences gene expression and plays a pivotal role in endothelial dysfunction. It was previously found that uniform flow reduces the expression of EZH2, whereas repression of EZH2 reciprocally activates MAPK7 signalling. In chapter 6, the reciprocal cross-talk between MAPK7 and EZH2 in endothelial cells and the process of EndMT is studied. To translate in vitro findings to the clinical setting, it is investigated whether the balance between MAPK7 and EZH2 is disturbed in human coronary artery stenosis.
In chapter 7, the findings of the studies presented in this thesis are summarized, the translational aspects are discussed and directions of future research are proposed.
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