UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
Macrophage regulatory mechanisms in atherosclerosis
The interplay of lipids and inflammation
Neele, A.E.
Publication date
2018
Document Version
Other version
License
Other
Link to publication
Citation for published version (APA):
Neele, A. E. (2018). Macrophage regulatory mechanisms in atherosclerosis: The interplay of
lipids and inflammation.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 199PDF page: 199PDF page: 199PDF page: 199
Chapter 10
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 200PDF page: 200PDF page: 200PDF page: 200
200 201
Atherosclerosis is a multifactorial disease driven by lipids and inflammation (1). Monocytes, macrophages and foam cells are the central players in atherosclerosis and contribute to all stages of lesion formation (2, 3). The overall aim of this thesis was to identify novel regulators in monocytes and macrophages to combat atherosclerosis
(chapter 1).
Epigenetic pathways have been identified to play a key role in monocyte-to-macrophage differentiation and activation (chapter 2) (4-6). We hypothesized that
interference with these pathways in macrophages can improve atherosclerosis outcome (chapter 3) (7). In this thesis we validated the role of the repressive
H3K27me3 methyltransferase enhancer of the zeste homolog 2 (Ezh2) and lysine demethylase 6b (Kdm6b) in macrophage foam cells and atherosclerotic lesion development in mice. In humans, we studied DNA methylation in monocyte-to-macrophage differentiation and subsequent activation. Moreover, we studied the link between low-density lipoprotein (LDL) and monocyte activation in patients with familial hypercholesterolemia (FH).
The main findings of the studies in this thesis are:
- Inhibition of epigenetic enzymes, especially Hdacs, by pharmacological inhibitors can improve atherogenic macrophage functions.
- Foam cells induce a pro-fibrotic transcriptome signature, which is partly regulated by Kdm6b.
- Myeloid Kdm6b deficiency results in advanced atherosclerosis, with lesions that contain more collagen and necrosis.
- Myeloid Ezh2 deficiency limits atherosclerosis, due to impaired neutrophil migration and a partly reduced inflammatory response of foam cells.
- DNA methylation changes occur mostly during monocyte-to-macrophage differentiation, rather than upon activation of macrophages.
- Both gain and loss of DNA methylation occurs during monocyte-to-macrophage differentiation at single CpGs or small regions at enhancer sites.
- Elevated levels of LDL-cholesterol associate with a pro-atherogenic profile of circulating monocytes, which is reversed upon lipid lowering.
Targeting histone modifying enzymes in macrophages to combat atherosclerosis
We hypothesized that skewing of macrophages to cells with anti-atherogenic functions would benefit atherosclerosis outcome. Histone modifications and its corresponding enzymes are important regulators of macrophage function and might therefore be a good target for atherosclerosis intervention (chapter 2 and 3). In chapter 4 we
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 201PDF page: 201PDF page: 201PDF page: 201
200 201
Atherosclerosis is a multifactorial disease driven by lipids and inflammation (1). Monocytes, macrophages and foam cells are the central players in atherosclerosis and contribute to all stages of lesion formation (2, 3). The overall aim of this thesis was to identify novel regulators in monocytes and macrophages to combat atherosclerosis
(chapter 1).
Epigenetic pathways have been identified to play a key role in monocyte-to-macrophage differentiation and activation (chapter 2) (4-6). We hypothesized that
interference with these pathways in macrophages can improve atherosclerosis outcome (chapter 3) (7). In this thesis we validated the role of the repressive
H3K27me3 methyltransferase enhancer of the zeste homolog 2 (Ezh2) and lysine demethylase 6b (Kdm6b) in macrophage foam cells and atherosclerotic lesion development in mice. In humans, we studied DNA methylation in monocyte-to-macrophage differentiation and subsequent activation. Moreover, we studied the link between low-density lipoprotein (LDL) and monocyte activation in patients with familial hypercholesterolemia (FH).
The main findings of the studies in this thesis are:
- Inhibition of epigenetic enzymes, especially Hdacs, by pharmacological inhibitors can improve atherogenic macrophage functions.
- Foam cells induce a pro-fibrotic transcriptome signature, which is partly regulated by Kdm6b.
- Myeloid Kdm6b deficiency results in advanced atherosclerosis, with lesions that contain more collagen and necrosis.
- Myeloid Ezh2 deficiency limits atherosclerosis, due to impaired neutrophil migration and a partly reduced inflammatory response of foam cells.
- DNA methylation changes occur mostly during monocyte-to-macrophage differentiation, rather than upon activation of macrophages.
- Both gain and loss of DNA methylation occurs during monocyte-to-macrophage differentiation at single CpGs or small regions at enhancer sites.
- Elevated levels of LDL-cholesterol associate with a pro-atherogenic profile of circulating monocytes, which is reversed upon lipid lowering.
Targeting histone modifying enzymes in macrophages to combat atherosclerosis
We hypothesized that skewing of macrophages to cells with anti-atherogenic functions would benefit atherosclerosis outcome. Histone modifications and its corresponding enzymes are important regulators of macrophage function and might therefore be a good target for atherosclerosis intervention (chapter 2 and 3). In chapter 4 we
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 202PDF page: 202PDF page: 202PDF page: 202
202
performed an in vitro screening assay with inhibitors for different classes of histone modifying enzymes (HMEs) and demonstrated that broad range inhibition of histone deacetylases (HDACs) and histone methyl transferases (HMTs) had both pro- and anti-atherogenic effects in macrophages. It was already shown that broad range inhibition of Hdacs by trichostatin A (TSA) enhances atherosclerosis as a result of enhanced lipid uptake in mice (8). Since histone modifications are regulated by a wide variety of HMEs, a better approach would be to target specific HMEs. We demonstrated that inhibition of Hdac3, switched macrophages to cells with anti-atherogenic properties (9). Hdac3-deleted mouse macrophages show an enhanced IL-4 response and a diminished LPS response, making Hdac3 a favorable target for atherosclerosis management (10, 11). Within our group, we showed that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and induces a stable plaque phenotype, confirming that Hdac3 is one of the epigenetic enzymes that can be regarded as a promising target for atherosclerosis therapy (12).
Next, we validated the role of novel HMEs in macrophages as targets for atherosclerosis. Based on the in vitro screening assay and literature we studied the enzymes involved in H3K27 methylation. H3K27me3 is a repressive histone mark catalyzed by the polycomb repressor complex 2 (PRC2) with Ezh2 containing the catalytic subunit of the complex (13). The H3K27 demethylases Kdm6b, Utx and Uty remove repressive histone marks (14). Both LPS and IL-4 induce Kdm6b expression (15, 16) and knockdown studies show that Kdm6b is involved in both the inflammatory and anti-inflammatory response of macrophages, and is not necessary associated with a specific macrophage activation state (17-19). On top of regulating inflammatory responses, in chapter 5 we showed that kdm6b also controls the expression of
pro-fibrotic genes in foam cells, describing an additional regulatory function for Kdm6b (20). Since foam cell formation, inflammatory responses and anti-inflammatory responses all occur in atherosclerosis, we studied the involvement of myeloid Kdm6b in atherosclerosis. In chapter 6 we observed that atherosclerotic lesions of myeloid
Kdm6b-deleted transplanted mice were more advanced as indicated by enhanced collagen and necrosis area. Whether this is beneficial or undesirable cannot be concluded based on these results. Increased cap thickness suggests that this is favorable but enhanced necrosis area on the other hand is associated with plaque instability (21). The pharmacological Kdm6b/Utx inhibitor GSK-J4 seemed promising in human macrophages by reducing inflammatory responses (22), but our study demonstrates that myeloid kdm6b-deficiency results in pro- and anti-atherogenic properties, making kdm6b not the best target for treatment of atherosclerosis.
203
Because atherosclerotic lesions of Kdm6b-deleted transplanted mice were more advanced we hypothesized that inhibition of the H3K27 methyltransferase Ezh2 is a better approach, since Ezh2 has opposite effects on H3K27 methylation compared to Kdm6b. In chapter 7 we demonstrated that lesion size of myeloid Ezh2-deleted
transplanted mice were smaller, contained less neutrophils and had a partly reduced inflammatory response of the foam cells. This suggests that compared to Kdm6b, Ezh2 has more potency as novel target for treatment of atherosclerosis. The beneficial effects on atherosclerosis are probably not all due to macrophage specific effects, since also neutrophils were deleted for Ezh2. These neutrophils showed reduced migration in vitro and were less present in atherosclerotic lesions.
Ezh2 inhibition as novel drug target in atherosclerosis
Recent years, several Ezh2 inhibitors have been developed as cancer therapeutics, but their impact on macrophages remains unknown (23). The use of these inhibitors in macrophages can help to validate the potency of Ezh2 as novel drug target in atherosclerosis. 3-deazaneplanocin A (DZNep) was the first established inhibitor and is most commonly used, yet this compound appeared to be non-specific for Ezh2 as it targets lysine methyltransferase activity in general (24). Therefore, several selective S-adenosylmethionine (SAM)-competitive Ezh2 inhibitors with high specificity have been developed, which act on the catalytic site of Ezh2 and affect tumor development (25-28). To make these drugs more valuable for clinical use, orally bioavailable inhibitors were synthesized (29-31). Phase 1/2 clinical trials are currently being performed with one of these compounds (EPZ-6438) in patients with advanced solid tumors or B cell lymphomas. Ezh2 is also described to have non-enzymatic functions (discussed in next paragraph), therefore inhibitors that block Ezh2 activity may not block all of its functions. A small molecule inhibitor that does not target the catalytic domain of Ezh2 but blocks the Ezh2-Eed interaction and thereby disrupts the PRC2 complex is now available (32). The use of these inhibitors should thus be tested on macrophages. Macrophage-specific targeting of compounds in atherosclerosis
We suggest that the use of Ezh2 inhibitors should be tested in experimental settings of atherosclerosis, however we cannot estimate the outcome on atherosclerosis when inhibiting Ezh2 in non-myeloid cells. Previously we have shown that Hdac3 deletion in macrophages is beneficial in a mouse model of atherosclerosis (12), but blocking Hdac3 in endothelial cells increases plaque development (33). Therefore, cell-specific targeting of compounds to macrophages is of great interest. Statin-loaded HDL-nanoparticles accumulate in atherosclerotic lesions of mice, where it reduced macrophages numbers and inflammation (34). Loading of HDL-nanoparticles with
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 203PDF page: 203PDF page: 203PDF page: 203
202
performed an in vitro screening assay with inhibitors for different classes of histone modifying enzymes (HMEs) and demonstrated that broad range inhibition of histone deacetylases (HDACs) and histone methyl transferases (HMTs) had both pro- and anti-atherogenic effects in macrophages. It was already shown that broad range inhibition of Hdacs by trichostatin A (TSA) enhances atherosclerosis as a result of enhanced lipid uptake in mice (8). Since histone modifications are regulated by a wide variety of HMEs, a better approach would be to target specific HMEs. We demonstrated that inhibition of Hdac3, switched macrophages to cells with anti-atherogenic properties (9). Hdac3-deleted mouse macrophages show an enhanced IL-4 response and a diminished LPS response, making Hdac3 a favorable target for atherosclerosis management (10, 11). Within our group, we showed that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and induces a stable plaque phenotype, confirming that Hdac3 is one of the epigenetic enzymes that can be regarded as a promising target for atherosclerosis therapy (12).
Next, we validated the role of novel HMEs in macrophages as targets for atherosclerosis. Based on the in vitro screening assay and literature we studied the enzymes involved in H3K27 methylation. H3K27me3 is a repressive histone mark catalyzed by the polycomb repressor complex 2 (PRC2) with Ezh2 containing the catalytic subunit of the complex (13). The H3K27 demethylases Kdm6b, Utx and Uty remove repressive histone marks (14). Both LPS and IL-4 induce Kdm6b expression (15, 16) and knockdown studies show that Kdm6b is involved in both the inflammatory and anti-inflammatory response of macrophages, and is not necessary associated with a specific macrophage activation state (17-19). On top of regulating inflammatory responses, in chapter 5 we showed that kdm6b also controls the expression of
pro-fibrotic genes in foam cells, describing an additional regulatory function for Kdm6b (20). Since foam cell formation, inflammatory responses and anti-inflammatory responses all occur in atherosclerosis, we studied the involvement of myeloid Kdm6b in atherosclerosis. In chapter 6 we observed that atherosclerotic lesions of myeloid
Kdm6b-deleted transplanted mice were more advanced as indicated by enhanced collagen and necrosis area. Whether this is beneficial or undesirable cannot be concluded based on these results. Increased cap thickness suggests that this is favorable but enhanced necrosis area on the other hand is associated with plaque instability (21). The pharmacological Kdm6b/Utx inhibitor GSK-J4 seemed promising in human macrophages by reducing inflammatory responses (22), but our study demonstrates that myeloid kdm6b-deficiency results in pro- and anti-atherogenic properties, making kdm6b not the best target for treatment of atherosclerosis.
203
Because atherosclerotic lesions of Kdm6b-deleted transplanted mice were more advanced we hypothesized that inhibition of the H3K27 methyltransferase Ezh2 is a better approach, since Ezh2 has opposite effects on H3K27 methylation compared to Kdm6b. In chapter 7 we demonstrated that lesion size of myeloid Ezh2-deleted
transplanted mice were smaller, contained less neutrophils and had a partly reduced inflammatory response of the foam cells. This suggests that compared to Kdm6b, Ezh2 has more potency as novel target for treatment of atherosclerosis. The beneficial effects on atherosclerosis are probably not all due to macrophage specific effects, since also neutrophils were deleted for Ezh2. These neutrophils showed reduced migration in vitro and were less present in atherosclerotic lesions.
Ezh2 inhibition as novel drug target in atherosclerosis
Recent years, several Ezh2 inhibitors have been developed as cancer therapeutics, but their impact on macrophages remains unknown (23). The use of these inhibitors in macrophages can help to validate the potency of Ezh2 as novel drug target in atherosclerosis. 3-deazaneplanocin A (DZNep) was the first established inhibitor and is most commonly used, yet this compound appeared to be non-specific for Ezh2 as it targets lysine methyltransferase activity in general (24). Therefore, several selective S-adenosylmethionine (SAM)-competitive Ezh2 inhibitors with high specificity have been developed, which act on the catalytic site of Ezh2 and affect tumor development (25-28). To make these drugs more valuable for clinical use, orally bioavailable inhibitors were synthesized (29-31). Phase 1/2 clinical trials are currently being performed with one of these compounds (EPZ-6438) in patients with advanced solid tumors or B cell lymphomas. Ezh2 is also described to have non-enzymatic functions (discussed in next paragraph), therefore inhibitors that block Ezh2 activity may not block all of its functions. A small molecule inhibitor that does not target the catalytic domain of Ezh2 but blocks the Ezh2-Eed interaction and thereby disrupts the PRC2 complex is now available (32). The use of these inhibitors should thus be tested on macrophages. Macrophage-specific targeting of compounds in atherosclerosis
We suggest that the use of Ezh2 inhibitors should be tested in experimental settings of atherosclerosis, however we cannot estimate the outcome on atherosclerosis when inhibiting Ezh2 in non-myeloid cells. Previously we have shown that Hdac3 deletion in macrophages is beneficial in a mouse model of atherosclerosis (12), but blocking Hdac3 in endothelial cells increases plaque development (33). Therefore, cell-specific targeting of compounds to macrophages is of great interest. Statin-loaded HDL-nanoparticles accumulate in atherosclerotic lesions of mice, where it reduced macrophages numbers and inflammation (34). Loading of HDL-nanoparticles with
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 204PDF page: 204PDF page: 204PDF page: 204
204
inhibitors against HMEs might thus serve as a novel approach to specifically deliver compounds. Another approach is to develop drugs with an esterase-sensitive chemical motif (ESM). Carboxylestrease-1 is predominantly expressed in human monocytes and macrophages and drugs with this ESM motif can be hydrolyzed from inactive to active drugs. Hdac inhibitors with this ESM motif are anti-inflammatory and are another novel macrophage-specific drug targeting tool for the future (35). Upstream regulators of Ezh2 and functions beyond epigenetics
The upstream regulators of Ezh2 in monocytes and macrophages are not yet identified. In cancer, many regulators, including transcription factors (TFs) and non-coding RNAs (ncRNAs mainly microRNAs) control Ezh2 expression (36). We showed that LPS downregulated Ezh2 expression and upregulated Kdm6b expression in macrophages, suggesting that TLR signaling regulates the expression of these HMEs. Besides the induction, Ezh2 can bind to other proteins and ncRNAs, resulting in its recruitment to specific loci, thereby regulating gene expression (36). Ezh2 activity and stability is also regulated by post-translational modifications, like phosphorylation (37-41). Besides the direct epigenetic function of Ezh2 and other HMEs, also histone methylation- and demethylation-independent functions of these enzymes exist. In various cancers, Ezh2 acts as a transcriptional activator rather than a direct repressor, an effect which is independent of the PRC2 complex (42). Furthermore, Ezh2 can also methylate non-histones, like STAT3, thereby regulating its recruitment to the DNA (43, 44). In addition, methylation of non-histones can also result in degradation of methylated proteins via ubiquitination (45). It should thus be noted that Ezh2 is not only a H3K27 methyltransferase and transcriptional repressor, since it can methylate non-histones and have PRC2-independent effects as a co-activator. Therefore, future studies should shed a light on the direct and indirect effects of HMEs in immunological settings. Ideally, the performed RNA-sequencing analysis should be overlaid with ChIP analysis for H3K27me3 and its enzymes, to gain more insight in direct and indirect effects of these enzymes.
Regulation of macrophage cell-identity by DNA methylation
Next to histone modifications, DNA methylation is another important epigenetic regulator. In chapter 8 we revealed that DNA methylation changes mainly occur
during monocyte-to-macrophage differentiation and less during macrophage activation. While previous studies showed a role for DNA methylation in monocyte-to-macrophage differentiation in differentially methylated regions (DMRs) (46), we identified that DNA methylation changes occur at single CpGs or very small regions that contribute to monocyte-to-macrophage differentiation. In addition, it was
205
previously reported that mainly loss of methylation occurs during differentiation, while we also identified substantial gain of methylation (46, 47). We found that DNA methylation changes occur at positions that are enriched for enhancers and identified that gain of methylation occurs at positions that contain motifs, which can be bound by the lineage determining transcription factors (LDTFs) CEBP and ETS, while loss of methylation is enriched for motifs that bind AP1 factors. During monocyte-to-macrophage differentiation many epigenetic changes occur, of which enhancers are central regulatory regions necessary for macrophage identity (48). Gene transcription is regulated by multiple enhancers and their activity is cell-type specific (49). The selection and activity of enhancers is regulated by both LDTFs and stimuli-dependent TFs (SDTFs). The LDTFs are cell type-specific and regulate cellular identity, while SDTF are turned on based on environmental triggers thereby activating enhancers (50, 51). While DNA methylation in general is associated with gene repression, evidence is now showing that DNA methylation can influence TF binding, both positively and negatively, depending on the methylated motif (52). Binding of the TFs bLHL, bZIP and ETS is inhibited by CpG methylation, while transcription factors such as homeodomain, POU and NFAT preferred to bind methylated CpGs (52). This suggests that in our study, binding sites for LDTFs are switched off during monocyte-to-macrophage differentiation (ETS motifs), while positions that bind SDTFs are turned on (bZIP motifs; AP1 factors). The question remains whether differential methylation is a cause or consequence of TF-binding. DNA methylation is catalyzed by methylcytosine dioxygenases (DNMTs) and removed by ten-eleven translocation proteins (TETs), but recent studies suggest that also non-enzymatic TFs regulate DNA methylation (53). DNA methylation can thus influence TF binding, but TF-binding can also influence DNA methylation.
LDL and the inflammatory response of monocytes
Elevated LDL levels are associated with increased risk for atherosclerosis and lipid lowering by statins effectively lowers the risk for cardiovascular disease (54). Because atherosclerosis is driven by both lipids and inflammation, we studied the link between the two. We hypothesized that LDL has inflammatory effects on circulating monocytes, which can be reversed upon lipid lowering. Whether the cardiovascular benefit of statins is due to their direct lipid lowering actions or their anti-inflammatory pleiotropic effects is under debate (55). We therefore used PCSK9 monoclonal antibodies which selectively lower LDL and in contrast to statins do not lower the inflammation marker c-reactive protein (CRP) (56, 57).
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 205PDF page: 205PDF page: 205PDF page: 205
204
inhibitors against HMEs might thus serve as a novel approach to specifically deliver compounds. Another approach is to develop drugs with an esterase-sensitive chemical motif (ESM). Carboxylestrease-1 is predominantly expressed in human monocytes and macrophages and drugs with this ESM motif can be hydrolyzed from inactive to active drugs. Hdac inhibitors with this ESM motif are anti-inflammatory and are another novel macrophage-specific drug targeting tool for the future (35). Upstream regulators of Ezh2 and functions beyond epigenetics
The upstream regulators of Ezh2 in monocytes and macrophages are not yet identified. In cancer, many regulators, including transcription factors (TFs) and non-coding RNAs (ncRNAs mainly microRNAs) control Ezh2 expression (36). We showed that LPS downregulated Ezh2 expression and upregulated Kdm6b expression in macrophages, suggesting that TLR signaling regulates the expression of these HMEs. Besides the induction, Ezh2 can bind to other proteins and ncRNAs, resulting in its recruitment to specific loci, thereby regulating gene expression (36). Ezh2 activity and stability is also regulated by post-translational modifications, like phosphorylation (37-41). Besides the direct epigenetic function of Ezh2 and other HMEs, also histone methylation- and demethylation-independent functions of these enzymes exist. In various cancers, Ezh2 acts as a transcriptional activator rather than a direct repressor, an effect which is independent of the PRC2 complex (42). Furthermore, Ezh2 can also methylate non-histones, like STAT3, thereby regulating its recruitment to the DNA (43, 44). In addition, methylation of non-histones can also result in degradation of methylated proteins via ubiquitination (45). It should thus be noted that Ezh2 is not only a H3K27 methyltransferase and transcriptional repressor, since it can methylate non-histones and have PRC2-independent effects as a co-activator. Therefore, future studies should shed a light on the direct and indirect effects of HMEs in immunological settings. Ideally, the performed RNA-sequencing analysis should be overlaid with ChIP analysis for H3K27me3 and its enzymes, to gain more insight in direct and indirect effects of these enzymes.
Regulation of macrophage cell-identity by DNA methylation
Next to histone modifications, DNA methylation is another important epigenetic regulator. In chapter 8 we revealed that DNA methylation changes mainly occur
during monocyte-to-macrophage differentiation and less during macrophage activation. While previous studies showed a role for DNA methylation in monocyte-to-macrophage differentiation in differentially methylated regions (DMRs) (46), we identified that DNA methylation changes occur at single CpGs or very small regions that contribute to monocyte-to-macrophage differentiation. In addition, it was
205
previously reported that mainly loss of methylation occurs during differentiation, while we also identified substantial gain of methylation (46, 47). We found that DNA methylation changes occur at positions that are enriched for enhancers and identified that gain of methylation occurs at positions that contain motifs, which can be bound by the lineage determining transcription factors (LDTFs) CEBP and ETS, while loss of methylation is enriched for motifs that bind AP1 factors. During monocyte-to-macrophage differentiation many epigenetic changes occur, of which enhancers are central regulatory regions necessary for macrophage identity (48). Gene transcription is regulated by multiple enhancers and their activity is cell-type specific (49). The selection and activity of enhancers is regulated by both LDTFs and stimuli-dependent TFs (SDTFs). The LDTFs are cell type-specific and regulate cellular identity, while SDTF are turned on based on environmental triggers thereby activating enhancers (50, 51). While DNA methylation in general is associated with gene repression, evidence is now showing that DNA methylation can influence TF binding, both positively and negatively, depending on the methylated motif (52). Binding of the TFs bLHL, bZIP and ETS is inhibited by CpG methylation, while transcription factors such as homeodomain, POU and NFAT preferred to bind methylated CpGs (52). This suggests that in our study, binding sites for LDTFs are switched off during monocyte-to-macrophage differentiation (ETS motifs), while positions that bind SDTFs are turned on (bZIP motifs; AP1 factors). The question remains whether differential methylation is a cause or consequence of TF-binding. DNA methylation is catalyzed by methylcytosine dioxygenases (DNMTs) and removed by ten-eleven translocation proteins (TETs), but recent studies suggest that also non-enzymatic TFs regulate DNA methylation (53). DNA methylation can thus influence TF binding, but TF-binding can also influence DNA methylation.
LDL and the inflammatory response of monocytes
Elevated LDL levels are associated with increased risk for atherosclerosis and lipid lowering by statins effectively lowers the risk for cardiovascular disease (54). Because atherosclerosis is driven by both lipids and inflammation, we studied the link between the two. We hypothesized that LDL has inflammatory effects on circulating monocytes, which can be reversed upon lipid lowering. Whether the cardiovascular benefit of statins is due to their direct lipid lowering actions or their anti-inflammatory pleiotropic effects is under debate (55). We therefore used PCSK9 monoclonal antibodies which selectively lower LDL and in contrast to statins do not lower the
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 206PDF page: 206PDF page: 206PDF page: 206
206
In chapter 9, we showed that in patients with familial hypercholesterolemia (FH), not using statins, elevated levels of circulating LDL induce pro-inflammatory and migratory changes in monocytes, which coincide with an increase in cytoplasmic lipid droplets compared to controls (58). This implies that there is a relation between intracellular lipid accumulation and inflammatory changes in monocytes. We showed that treatment with PCSK9 monoclonal antibodies in these FH patients reversed the migratory capacity, inflammatory response and intracellular lipid content, similar to patients using statins. Since PCSK9 antibody treatment selectively lowers LDL without decreasing CRP, these data imply that LDL lowering itself, rather than the pleiotropic effects, contributes to anti-inflammatory effects on monocytes.
Foamy monocytes
The idea that also monocytes encounter lipids in circulation, a concept called “foamy monocytes” was already described in mice (59, 60). We here show that this is also true in humans with hyperlipidemia and that lipid lowering reduces the amount of lipid droplets in monocytes. Questions that still need to be answered from a mechanistic point of view are: What type of lipids accumulate in these monocytes and how are these lipids taken up by the cell (61). Data from our study suggest that lipid uptake is not directly regulated via the LDLR, since the expression was virtually absent on circulating monocytes. Furthermore, PCSK9 antibodies enhance LDLR surface expression, but the monocytes of PCKS9 antibody treated-patients showed reduced accumulation of lipids. Homozygous FH-patients, characterized by the absence of functional LDLRs also show accumulation of lipids in monocytes and in addition, foamy monocytes were seen in Ldlr-deficient mice (59, 60, 62). We and others observed that scavenger receptors like CD36 are increased and thus might play a role in lipid uptake (59, 62). Furthermore, LDL can also be engulfed by macrophages via micropinocytosis, independent of receptor-mediated phagocytosis, a process which might also occur in monocytes (63). It is also possible that lipid synthesis is affected rather than the actual lipid uptake. We also observed a correlation between LDL and CCR2 surface expression, suggesting that LDL influences CCR2 expression. The exact mechanism via which LDL regulates CCR2 expression also needs to be addressed.
Trained immunity
In vitro, it has been shown that oxLDL priming of monocytes results in an enhanced cytokine response after re-stimulation with TLR ligands that was accompanied by epigenetic changes, a concept called trained immunity (64). These data support the idea that pro-atherogenic mediators can have long lasting effects on monocytes. In our study, we observed that LDL lowering by PCSK9 monoclonal antibodies reverse the
207
pro-atherogenic properties of monocytes, suggesting that the memory induced by lipids can be partly reduced. The question remains whether this is true for all inflammatory and atherosclerosis-related genes and whether epigenetic modifications are altered in our study. RNA-sequencing and ChIP-sequencing could help to identify the complete transcriptional profile and epigenetic landscape induced by lipids. It can also help to answer the question whether epigenetic changes are reversed upon lipid lowering.
Targeting inflammation
Besides these lipid-lowering actions, patients still have a strong residual risk for cardiovascular events (65). As hypothesized before, this might be due to some kind of epigenetic memory, but can also be due to the residual inflammatory risk. Recently it was shown that anti-inflammatory therapy targeting IL-1β in cardiovascular patients, significantly lowered recurrent cardiovascular events, independent of lipid lowering, highlighting the importance of inflammation in cardiovascular disease (66). Targeting both lipids and inflammation can thus improve atherosclerosis outcome.
Lesional macrophage activation
Shifting monocytes and macrophages to cells with anti-atherogenic properties as treatment for atherosclerosis also highlights the importance of understanding the process of macrophage activation in atherosclerotic lesions. Activation of macrophages and combinations of activation triggers results in a spectrum of macrophage activation states in humans (67). Furthermore, macrophages from different tissues have a specific transcriptional and epigenetic profile, dependent on the environment in mice (68). Although the concept of classical (M1) and alternatively (M2) activation can be used as a reductionist tool to study macrophage activation, it should be taken into account that these states are at the ends of the macrophage activation spectrum, and that wide variety of macrophage activation states are observed based on their origin and environmental triggers (69). It is thus of great interest to know what is the origin of monocytes and macrophages present in atherosclerotic lesions, what is their lifespan and which activation states are present. Although efforts have been made to answer some of these questions, by use of reporter mice, the question remains whether the same holds true in human atherosclerotic lesions. In mice, monocyte-derived cells can replenish tissue resident macrophages, based on environmental triggers. Whether monocyte-derived cells turn into tissue resident macrophages in atherosclerotic lesions remains to be answered. Novel technologies like single-cell RNA sequencing and Mass cytometry (CyTOF)
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 207PDF page: 207PDF page: 207PDF page: 207
206
In chapter 9, we showed that in patients with familial hypercholesterolemia (FH), not using statins, elevated levels of circulating LDL induce pro-inflammatory and migratory changes in monocytes, which coincide with an increase in cytoplasmic lipid droplets compared to controls (58). This implies that there is a relation between intracellular lipid accumulation and inflammatory changes in monocytes. We showed that treatment with PCSK9 monoclonal antibodies in these FH patients reversed the migratory capacity, inflammatory response and intracellular lipid content, similar to patients using statins. Since PCSK9 antibody treatment selectively lowers LDL without decreasing CRP, these data imply that LDL lowering itself, rather than the pleiotropic effects, contributes to anti-inflammatory effects on monocytes.
Foamy monocytes
The idea that also monocytes encounter lipids in circulation, a concept called “foamy monocytes” was already described in mice (59, 60). We here show that this is also true in humans with hyperlipidemia and that lipid lowering reduces the amount of lipid droplets in monocytes. Questions that still need to be answered from a mechanistic point of view are: What type of lipids accumulate in these monocytes and how are these lipids taken up by the cell (61). Data from our study suggest that lipid uptake is not directly regulated via the LDLR, since the expression was virtually absent on circulating monocytes. Furthermore, PCSK9 antibodies enhance LDLR surface expression, but the monocytes of PCKS9 antibody treated-patients showed reduced accumulation of lipids. Homozygous FH-patients, characterized by the absence of functional LDLRs also show accumulation of lipids in monocytes and in addition, foamy monocytes were seen in Ldlr-deficient mice (59, 60, 62). We and others observed that scavenger receptors like CD36 are increased and thus might play a role in lipid uptake (59, 62). Furthermore, LDL can also be engulfed by macrophages via micropinocytosis, independent of receptor-mediated phagocytosis, a process which might also occur in monocytes (63). It is also possible that lipid synthesis is affected rather than the actual lipid uptake. We also observed a correlation between LDL and CCR2 surface expression, suggesting that LDL influences CCR2 expression. The exact mechanism via which LDL regulates CCR2 expression also needs to be addressed.
Trained immunity
In vitro, it has been shown that oxLDL priming of monocytes results in an enhanced cytokine response after re-stimulation with TLR ligands that was accompanied by epigenetic changes, a concept called trained immunity (64). These data support the idea that pro-atherogenic mediators can have long lasting effects on monocytes. In our study, we observed that LDL lowering by PCSK9 monoclonal antibodies reverse the
207
pro-atherogenic properties of monocytes, suggesting that the memory induced by lipids can be partly reduced. The question remains whether this is true for all inflammatory and atherosclerosis-related genes and whether epigenetic modifications are altered in our study. RNA-sequencing and ChIP-sequencing could help to identify the complete transcriptional profile and epigenetic landscape induced by lipids. It can also help to answer the question whether epigenetic changes are reversed upon lipid lowering.
Targeting inflammation
Besides these lipid-lowering actions, patients still have a strong residual risk for cardiovascular events (65). As hypothesized before, this might be due to some kind of epigenetic memory, but can also be due to the residual inflammatory risk. Recently it was shown that anti-inflammatory therapy targeting IL-1β in cardiovascular patients, significantly lowered recurrent cardiovascular events, independent of lipid lowering, highlighting the importance of inflammation in cardiovascular disease (66). Targeting both lipids and inflammation can thus improve atherosclerosis outcome.
Lesional macrophage activation
Shifting monocytes and macrophages to cells with anti-atherogenic properties as treatment for atherosclerosis also highlights the importance of understanding the process of macrophage activation in atherosclerotic lesions. Activation of macrophages and combinations of activation triggers results in a spectrum of macrophage activation states in humans (67). Furthermore, macrophages from different tissues have a specific transcriptional and epigenetic profile, dependent on the environment in mice (68). Although the concept of classical (M1) and alternatively (M2) activation can be used as a reductionist tool to study macrophage activation, it should be taken into account that these states are at the ends of the macrophage activation spectrum, and that wide variety of macrophage activation states are observed based on their origin and environmental triggers (69). It is thus of great interest to know what is the origin of monocytes and macrophages present in atherosclerotic lesions, what is their lifespan and which activation states are present. Although efforts have been made to answer some of these questions, by use of reporter mice, the question remains whether the same holds true in human atherosclerotic lesions. In mice, monocyte-derived cells can replenish tissue resident macrophages, based on environmental triggers. Whether monocyte-derived cells turn into tissue resident macrophages in atherosclerotic lesions remains to be answered. Novel technologies like single-cell RNA sequencing and Mass cytometry (CyTOF)
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 208PDF page: 208PDF page: 208PDF page: 208
208
analysis could help to identify different transcriptional macrophage activation states present in atherosclerotic lesions for both mice and humans.
Summary
In conclusion, we identified the H3K27me3 methyltransferase Ezh2 as a novel target in myeloid cells to combat atherosclerosis. Inhibitors against Ezh2 should be tested in macrophages and experimental atherosclerosis, to validate the potency of Ezh2 inhibition as treatment for atherosclerosis. In addition to histone modifications, we highlight that also DNA methylation is an important regulator in human monocyte-to-macrophage differentiation. Finally, by use of PCSK9 monoclonal antibodies we showed that LDL lowering itself contributes to anti-inflammatory effects on monocytes.
References
1. Shapiro MD, Fazio S. From Lipids to Inflammation: New Approaches to Reducing Atherosclerotic Risk. Circulation research. 2016;118(4):732-49.
2. Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nature reviews Immunology. 2013;13(10):709-21.
3. Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011;145(3):341-55. 4. Hoeksema MA, de Winther MP. Epigenetic Regulation of Monocyte and Macrophage Function. Antioxidants & redox signaling. 2016;25(14):758-74.
5. Amit I, Winter DR, Jung S. The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis. Nature immunology. 2016;17(1):18-25.
6. Van den Bossche J, Neele AE, Hoeksema MA, de Winther MP. Macrophage polarization: the epigenetic point of view. Curr Opin Lipidol. 2014;25(5):367-73.
7. Neele AE, Van den Bossche J, Hoeksema MA, de Winther MP. Epigenetic pathways in macrophages emerge as novel targets in atherosclerosis. Eur J Pharmacol. 2015;763(Pt A):79-89.
8. Choi JH, Nam KH, Kim J, Baek MW, Park JE, Park HY, et al. Trichostatin A exacerbates atherosclerosis in low density lipoprotein receptor-deficient mice. Arteriosclerosis, thrombosis, and vascular biology. 2005;25(11):2404-9.
9. Van den Bossche J, Neele AE, Hoeksema MA, de Heij F, Boshuizen MC, van der Velden S, et al. Inhibiting epigenetic enzymes to improve atherogenic macrophage functions. Biochemical and biophysical research communications. 2014;455(3-4):396-402.
10. Mullican SE, Gaddis CA, Alenghat T, Nair MG, Giacomin PR, Everett LJ, et al. Histone deacetylase 3 is an epigenomic brake in macrophage alternative activation. Genes & development. 2011;25(23):2480-8.
11. Chen X, Barozzi I, Termanini A, Prosperini E, Recchiuti A, Dalli J, et al. Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(42):E2865-74.
12. Hoeksema MA, Gijbels MJ, Van den Bossche J, van der Velden S, Sijm A, Neele AE, et al. Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions. EMBO molecular medicine. 2014;6(9):1124-32.
13. Di Croce L, Helin K. Transcriptional regulation by Polycomb group proteins. Nature structural & molecular biology. 2013;20(10):1147-55.
14. Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rappsilber J, et al. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature. 2007;449(7163):731-4.
15. De Santa F, Totaro MG, Prosperini E, Notarbartolo S, Testa G, Natoli G. The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell. 2007;130(6):1083-94.
209 16. Ishii M, Wen H, Corsa CA, Liu T, Coelho AL, Allen RM, et al. Epigenetic regulation of the alternatively activated macrophage phenotype. Blood. 2009;114(15):3244-54.
17. De Santa F, Narang V, Yap ZH, Tusi BK, Burgold T, Austenaa L, et al. Jmjd3 contributes to the control of gene expression in LPS-activated macrophages. EMBO J. 2009;28(21):3341-52.
18. Satoh T, Takeuchi O, Vandenbon A, Yasuda K, Tanaka Y, Kumagai Y, et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nature immunology. 2010;11(10):936-44.
19. Yan Q, Sun L, Zhu Z, Wang L, Li S, Ye RD. Jmjd3-mediated epigenetic regulation of inflammatory cytokine gene expression in serum amyloid A-stimulated macrophages. Cell Signal. 2014;26(9):1783-91.
20. Neele AE, Prange KH, Hoeksema MA, van der Velden S, Lucas T, Dimmeler S, et al. Macrophage Kdm6b controls the pro-fibrotic transcriptome signature of foam cells. Epigenomics. 2017;9(4):383-91.
21. Halvorsen B, Otterdal K, Dahl TB, Skjelland M, Gullestad L, Oie E, et al. Atherosclerotic plaque stability--what determines the fate of a plaque? Progress in cardiovascular diseases. 2008;51(3):183-94.
22. Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature. 2012;488(7411):404-8. 23. Kim KH, Roberts CW. Targeting EZH2 in cancer. Nature medicine. 2016;22(2):128-34.
24. Miranda TB, Cortez CC, Yoo CB, Liang G, Abe M, Kelly TK, et al. DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Molecular cancer therapeutics. 2009;8(6):1579-88.
25. Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nature chemical biology. 2012;8(11):890-6. 26. Verma SK, Tian X, LaFrance LV, Duquenne C, Suarez DP, Newlander KA, et al. Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2. ACS medicinal chemistry letters. 2012;3(12):1091-6.
27. McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012;492(7427):108-12.
28. Qi W, Chan H, Teng L, Li L, Chuai S, Zhang R, et al. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(52):21360-5.
29. Konze KD, Ma A, Li F, Barsyte-Lovejoy D, Parton T, Macnevin CJ, et al. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS chemical biology. 2013;8(6):1324-34.
30. Knutson SK, Kawano S, Minoshima Y, Warholic NM, Huang KC, Xiao Y, et al. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Molecular cancer therapeutics. 2014;13(4):842-54.
31. Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(19):7922-7.
32. Kim W, Bird GH, Neff T, Guo G, Kerenyi MA, Walensky LD, et al. Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer. Nature chemical biology. 2013;9(10):643-50.
33. Zampetaki A, Zeng L, Margariti A, Xiao Q, Li H, Zhang Z, et al. Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow. Circulation. 2010;121(1):132-42.
34. Duivenvoorden R, Tang J, Cormode DP, Mieszawska AJ, Izquierdo-Garcia D, Ozcan C, et al. A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation. Nature communications. 2014;5:3065.
35. Needham LA, Davidson AH, Bawden LJ, Belfield A, Bone EA, Brotherton DH, et al. Drug targeting to monocytes and macrophages using esterase-sensitive chemical motifs. The Journal of pharmacology and experimental therapeutics. 2011;339(1):132-42.
36. Yamaguchi H, Hung MC. Regulation and Role of EZH2 in Cancer. Cancer research and treatment : official journal of Korean Cancer Association. 2014;46(3):209-22.
37. Cha TL, Zhou BP, Xia W, Wu Y, Yang CC, Chen CT, et al. Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3. Science. 2005;310(5746):306-10.
38. Kaneko S, Li G, Son J, Xu CF, Margueron R, Neubert TA, et al. Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA. Genes & development. 2010;24(23):2615-20. 39. Minnebo N, Gornemann J, O'Connell N, Van Dessel N, Derua R, Vermunt MW, et al. NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes. Nucleic acids research. 2013;41(2):842-54.
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 209PDF page: 209PDF page: 209PDF page: 209
208
analysis could help to identify different transcriptional macrophage activation states present in atherosclerotic lesions for both mice and humans.
Summary
In conclusion, we identified the H3K27me3 methyltransferase Ezh2 as a novel target in myeloid cells to combat atherosclerosis. Inhibitors against Ezh2 should be tested in macrophages and experimental atherosclerosis, to validate the potency of Ezh2 inhibition as treatment for atherosclerosis. In addition to histone modifications, we highlight that also DNA methylation is an important regulator in human monocyte-to-macrophage differentiation. Finally, by use of PCSK9 monoclonal antibodies we showed that LDL lowering itself contributes to anti-inflammatory effects on monocytes.
References
1. Shapiro MD, Fazio S. From Lipids to Inflammation: New Approaches to Reducing Atherosclerotic Risk. Circulation research. 2016;118(4):732-49.
2. Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nature reviews Immunology. 2013;13(10):709-21.
3. Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011;145(3):341-55. 4. Hoeksema MA, de Winther MP. Epigenetic Regulation of Monocyte and Macrophage Function. Antioxidants & redox signaling. 2016;25(14):758-74.
5. Amit I, Winter DR, Jung S. The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis. Nature immunology. 2016;17(1):18-25.
6. Van den Bossche J, Neele AE, Hoeksema MA, de Winther MP. Macrophage polarization: the epigenetic point of view. Curr Opin Lipidol. 2014;25(5):367-73.
7. Neele AE, Van den Bossche J, Hoeksema MA, de Winther MP. Epigenetic pathways in macrophages emerge as novel targets in atherosclerosis. Eur J Pharmacol. 2015;763(Pt A):79-89.
8. Choi JH, Nam KH, Kim J, Baek MW, Park JE, Park HY, et al. Trichostatin A exacerbates atherosclerosis in low density lipoprotein receptor-deficient mice. Arteriosclerosis, thrombosis, and vascular biology. 2005;25(11):2404-9.
9. Van den Bossche J, Neele AE, Hoeksema MA, de Heij F, Boshuizen MC, van der Velden S, et al. Inhibiting epigenetic enzymes to improve atherogenic macrophage functions. Biochemical and biophysical research communications. 2014;455(3-4):396-402.
10. Mullican SE, Gaddis CA, Alenghat T, Nair MG, Giacomin PR, Everett LJ, et al. Histone deacetylase 3 is an epigenomic brake in macrophage alternative activation. Genes & development. 2011;25(23):2480-8.
11. Chen X, Barozzi I, Termanini A, Prosperini E, Recchiuti A, Dalli J, et al. Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(42):E2865-74.
12. Hoeksema MA, Gijbels MJ, Van den Bossche J, van der Velden S, Sijm A, Neele AE, et al. Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions. EMBO molecular medicine. 2014;6(9):1124-32.
13. Di Croce L, Helin K. Transcriptional regulation by Polycomb group proteins. Nature structural & molecular biology. 2013;20(10):1147-55.
14. Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rappsilber J, et al. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature. 2007;449(7163):731-4.
15. De Santa F, Totaro MG, Prosperini E, Notarbartolo S, Testa G, Natoli G. The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell. 2007;130(6):1083-94.
209 16. Ishii M, Wen H, Corsa CA, Liu T, Coelho AL, Allen RM, et al. Epigenetic regulation of the alternatively activated macrophage phenotype. Blood. 2009;114(15):3244-54.
17. De Santa F, Narang V, Yap ZH, Tusi BK, Burgold T, Austenaa L, et al. Jmjd3 contributes to the control of gene expression in LPS-activated macrophages. EMBO J. 2009;28(21):3341-52.
18. Satoh T, Takeuchi O, Vandenbon A, Yasuda K, Tanaka Y, Kumagai Y, et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nature immunology. 2010;11(10):936-44.
19. Yan Q, Sun L, Zhu Z, Wang L, Li S, Ye RD. Jmjd3-mediated epigenetic regulation of inflammatory cytokine gene expression in serum amyloid A-stimulated macrophages. Cell Signal. 2014;26(9):1783-91.
20. Neele AE, Prange KH, Hoeksema MA, van der Velden S, Lucas T, Dimmeler S, et al. Macrophage Kdm6b controls the pro-fibrotic transcriptome signature of foam cells. Epigenomics. 2017;9(4):383-91.
21. Halvorsen B, Otterdal K, Dahl TB, Skjelland M, Gullestad L, Oie E, et al. Atherosclerotic plaque stability--what determines the fate of a plaque? Progress in cardiovascular diseases. 2008;51(3):183-94.
22. Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature. 2012;488(7411):404-8. 23. Kim KH, Roberts CW. Targeting EZH2 in cancer. Nature medicine. 2016;22(2):128-34.
24. Miranda TB, Cortez CC, Yoo CB, Liang G, Abe M, Kelly TK, et al. DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Molecular cancer therapeutics. 2009;8(6):1579-88.
25. Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nature chemical biology. 2012;8(11):890-6. 26. Verma SK, Tian X, LaFrance LV, Duquenne C, Suarez DP, Newlander KA, et al. Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2. ACS medicinal chemistry letters. 2012;3(12):1091-6.
27. McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012;492(7427):108-12.
28. Qi W, Chan H, Teng L, Li L, Chuai S, Zhang R, et al. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(52):21360-5.
29. Konze KD, Ma A, Li F, Barsyte-Lovejoy D, Parton T, Macnevin CJ, et al. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS chemical biology. 2013;8(6):1324-34.
30. Knutson SK, Kawano S, Minoshima Y, Warholic NM, Huang KC, Xiao Y, et al. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Molecular cancer therapeutics. 2014;13(4):842-54.
31. Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(19):7922-7.
32. Kim W, Bird GH, Neff T, Guo G, Kerenyi MA, Walensky LD, et al. Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer. Nature chemical biology. 2013;9(10):643-50.
33. Zampetaki A, Zeng L, Margariti A, Xiao Q, Li H, Zhang Z, et al. Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow. Circulation. 2010;121(1):132-42.
34. Duivenvoorden R, Tang J, Cormode DP, Mieszawska AJ, Izquierdo-Garcia D, Ozcan C, et al. A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation. Nature communications. 2014;5:3065.
35. Needham LA, Davidson AH, Bawden LJ, Belfield A, Bone EA, Brotherton DH, et al. Drug targeting to monocytes and macrophages using esterase-sensitive chemical motifs. The Journal of pharmacology and experimental therapeutics. 2011;339(1):132-42.
36. Yamaguchi H, Hung MC. Regulation and Role of EZH2 in Cancer. Cancer research and treatment : official journal of Korean Cancer Association. 2014;46(3):209-22.
37. Cha TL, Zhou BP, Xia W, Wu Y, Yang CC, Chen CT, et al. Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3. Science. 2005;310(5746):306-10.
38. Kaneko S, Li G, Son J, Xu CF, Margueron R, Neubert TA, et al. Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA. Genes & development. 2010;24(23):2615-20. 39. Minnebo N, Gornemann J, O'Connell N, Van Dessel N, Derua R, Vermunt MW, et al. NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes. Nucleic acids research. 2013;41(2):842-54.
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 210PDF page: 210PDF page: 210PDF page: 210
210
40. Chen S, Bohrer LR, Rai AN, Pan Y, Gan L, Zhou X, et al. Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2. Nature cell biology. 2010;12(11):1108-14.
41. Wu SC, Zhang Y. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability. The Journal of biological chemistry. 2011;286(32):28511-9.
42. Kim KH, Roberts CWM. Targeting EZH2 in cancer. Nature medicine. 2016;22(2):128-34.
43. Hamamoto R, Saloura V, Nakamura Y. Critical roles of non-histone protein lysine methylation in human tumorigenesis. Nature reviews Cancer. 2015;15(2):110-24.
44. Kim E, Kim M, Woo DH, Shin Y, Shin J, Chang N, et al. Phosphorylation of EZH2 Activates STAT3 Signaling via STAT3 Methylation and Promotes Tumorigenicity of Glioblastoma Stem-like Cells. Cancer Cell. 2013;23(6):839-52.
45. Lee JM, Lee JS, Kim H, Kim K, Park H, Kim JY, et al. EZH2 generates a methyl degron that is recognized by the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex. Molecular cell. 2012;48(4):572-86.
46. Wallner S, Schroder C, Leitao E, Berulava T, Haak C, Beisser D, et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics & chromatin. 2016;9:33.
47. Vento-Tormo R, Company C, Rodriguez-Ubreva J, de la Rica L, Urquiza JM, Javierre BM, et al. IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation. Genome biology. 2016;17:4.
48. Hoeksema MA, de Winther MP. Epigenetic Regulation of Monocyte and Macrophage Function. Antioxidants & redox signaling. 2016.
49. Romanoski CE, Link VM, Heinz S, Glass CK. Exploiting genomics and natural genetic variation to decode macrophage enhancers. Trends in immunology. 2015;36(9):507-18.
50. Glass CK, Natoli G. Molecular control of activation and priming in macrophages. Nature immunology. 2016;17(1):26-33.
51. Gosselin D, Glass CK. Epigenomics of macrophages. Immunological reviews. 2014;262(1):96-112. 52. Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, et al. Impact of cytosine methylation on DNA binding specificities of human transcription factors. Science. 2017;356(6337).
53. Zhu H, Wang G, Qian J. Transcription factors as readers and effectors of DNA methylation. Nature reviews Genetics. 2016;17(9):551-65.
54. Ference BA, Ginsberg HN, Graham I, Ray KK, Packard CJ, Bruckert E, et al. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. European heart journal. 2017. 55. Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM, Jr., Kastelein JJ, et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. The New England journal of medicine. 2008;359(21):2195-207.
56. Robinson JG, Farnier M, Krempf M, Bergeron J, Luc G, Averna M, et al. Efficacy and safety of alirocumab in reducing lipids and cardiovascular events. The New England journal of medicine. 2015;372(16):1489-99. 57. Sahebkar A, Di Giosia P, Stamerra CA, Grassi D, Pedone C, Ferretti G, et al. Effect of monoclonal antibodies to PCSK9 on high-sensitivity C-reactive protein levels: a meta-analysis of 16 randomized controlled treatment arms. British journal of clinical pharmacology. 2016;81(6):1175-90.
58. Bernelot Moens SJ, Neele AE, Kroon J, van der Valk FM, Van den Bossche J, Hoeksema MA, et al. PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia. European heart journal. 2017;38(20):1584-93.
59. Xu L, Dai Perrard X, Perrard JL, Yang D, Xiao X, Teng BB, et al. Foamy monocytes form early and contribute to nascent atherosclerosis in mice with hypercholesterolemia. Arteriosclerosis, thrombosis, and vascular biology. 2015;35(8):1787-97.
60. Jackson WD, Weinrich TW, Woollard KJ. Very-low and low-density lipoproteins induce neutral lipid accumulation and impair migration in monocyte subsets. Scientific reports. 2016;6:20038.
61. Wu H, Ballantyne CM. Dyslipidaemia: PCSK9 inhibitors and foamy monocytes in familial hypercholesterolaemia. Nature reviews Cardiology. 2017;14(7):385-6.
62. Mosig S, Rennert K, Buttner P, Krause S, Lutjohann D, Soufi M, et al. Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism. BMC medical genomics. 2008;1:60.
63. Kruth HS, Jones NL, Huang W, Zhao B, Ishii I, Chang J, et al. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. The Journal of biological chemistry. 2005;280(3):2352-60.
64. Bekkering S, Quintin J, Joosten LA, van der Meer JW, Netea MG, Riksen NP. Oxidized low-density lipoprotein induces long-term proinflammatory cytokine production and foam cell formation via epigenetic reprogramming of monocytes. Arteriosclerosis, thrombosis, and vascular biology. 2014;34(8):1731-8.
211 65. Ridker PM. How Common Is Residual Inflammatory Risk? Circulation research. 2017;120(4):617-9. 66. Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, et al. Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. The New England journal of medicine. 2017.
67. Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity. 2014;40(2):274-88.
68. Lavin Y, Winter D, Blecher-Gonen R, David E, Keren-Shaul H, Merad M, et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell. 2014;159(6):1312-26. 69. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014;41(1):14-20.
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
Processed on: 1-12-2017 PDF page: 211PDF page: 211PDF page: 211PDF page: 211
210
40. Chen S, Bohrer LR, Rai AN, Pan Y, Gan L, Zhou X, et al. Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2. Nature cell biology. 2010;12(11):1108-14.
41. Wu SC, Zhang Y. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability. The Journal of biological chemistry. 2011;286(32):28511-9.
42. Kim KH, Roberts CWM. Targeting EZH2 in cancer. Nature medicine. 2016;22(2):128-34.
43. Hamamoto R, Saloura V, Nakamura Y. Critical roles of non-histone protein lysine methylation in human tumorigenesis. Nature reviews Cancer. 2015;15(2):110-24.
44. Kim E, Kim M, Woo DH, Shin Y, Shin J, Chang N, et al. Phosphorylation of EZH2 Activates STAT3 Signaling via STAT3 Methylation and Promotes Tumorigenicity of Glioblastoma Stem-like Cells. Cancer Cell. 2013;23(6):839-52.
45. Lee JM, Lee JS, Kim H, Kim K, Park H, Kim JY, et al. EZH2 generates a methyl degron that is recognized by the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex. Molecular cell. 2012;48(4):572-86.
46. Wallner S, Schroder C, Leitao E, Berulava T, Haak C, Beisser D, et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics & chromatin. 2016;9:33.
47. Vento-Tormo R, Company C, Rodriguez-Ubreva J, de la Rica L, Urquiza JM, Javierre BM, et al. IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation. Genome biology. 2016;17:4.
48. Hoeksema MA, de Winther MP. Epigenetic Regulation of Monocyte and Macrophage Function. Antioxidants & redox signaling. 2016.
49. Romanoski CE, Link VM, Heinz S, Glass CK. Exploiting genomics and natural genetic variation to decode macrophage enhancers. Trends in immunology. 2015;36(9):507-18.
50. Glass CK, Natoli G. Molecular control of activation and priming in macrophages. Nature immunology. 2016;17(1):26-33.
51. Gosselin D, Glass CK. Epigenomics of macrophages. Immunological reviews. 2014;262(1):96-112. 52. Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, et al. Impact of cytosine methylation on DNA binding specificities of human transcription factors. Science. 2017;356(6337).
53. Zhu H, Wang G, Qian J. Transcription factors as readers and effectors of DNA methylation. Nature reviews Genetics. 2016;17(9):551-65.
54. Ference BA, Ginsberg HN, Graham I, Ray KK, Packard CJ, Bruckert E, et al. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. European heart journal. 2017. 55. Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM, Jr., Kastelein JJ, et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. The New England journal of medicine. 2008;359(21):2195-207.
56. Robinson JG, Farnier M, Krempf M, Bergeron J, Luc G, Averna M, et al. Efficacy and safety of alirocumab in reducing lipids and cardiovascular events. The New England journal of medicine. 2015;372(16):1489-99. 57. Sahebkar A, Di Giosia P, Stamerra CA, Grassi D, Pedone C, Ferretti G, et al. Effect of monoclonal antibodies to PCSK9 on high-sensitivity C-reactive protein levels: a meta-analysis of 16 randomized controlled treatment arms. British journal of clinical pharmacology. 2016;81(6):1175-90.
58. Bernelot Moens SJ, Neele AE, Kroon J, van der Valk FM, Van den Bossche J, Hoeksema MA, et al. PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia. European heart journal. 2017;38(20):1584-93.
59. Xu L, Dai Perrard X, Perrard JL, Yang D, Xiao X, Teng BB, et al. Foamy monocytes form early and contribute to nascent atherosclerosis in mice with hypercholesterolemia. Arteriosclerosis, thrombosis, and vascular biology. 2015;35(8):1787-97.
60. Jackson WD, Weinrich TW, Woollard KJ. Very-low and low-density lipoproteins induce neutral lipid accumulation and impair migration in monocyte subsets. Scientific reports. 2016;6:20038.
61. Wu H, Ballantyne CM. Dyslipidaemia: PCSK9 inhibitors and foamy monocytes in familial hypercholesterolaemia. Nature reviews Cardiology. 2017;14(7):385-6.
62. Mosig S, Rennert K, Buttner P, Krause S, Lutjohann D, Soufi M, et al. Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism. BMC medical genomics. 2008;1:60.
63. Kruth HS, Jones NL, Huang W, Zhao B, Ishii I, Chang J, et al. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. The Journal of biological chemistry. 2005;280(3):2352-60.
64. Bekkering S, Quintin J, Joosten LA, van der Meer JW, Netea MG, Riksen NP. Oxidized low-density lipoprotein induces long-term proinflammatory cytokine production and foam cell formation via epigenetic reprogramming of monocytes. Arteriosclerosis, thrombosis, and vascular biology. 2014;34(8):1731-8.
211 65. Ridker PM. How Common Is Residual Inflammatory Risk? Circulation research. 2017;120(4):617-9. 66. Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, et al. Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. The New England journal of medicine. 2017.
67. Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity. 2014;40(2):274-88.
68. Lavin Y, Winter D, Blecher-Gonen R, David E, Keren-Shaul H, Merad M, et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell. 2014;159(6):1312-26. 69. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014;41(1):14-20.
