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Histone Lysine Methylation
Modification and Its Role in
Vascular Calcification
Ye-Chi Cao
1
, Su-Kang Shan
1
, Bei Guo
1
, Chang-Chun Li
1
, Fu-Xing-Zi Li
1
,
Ming-Hui Zheng
1
, Qiu-Shuang Xu
1
, Yi Wang
1
, Li-Min Lei
1
, Ke-Xin Tang
1
,
Wen-Lu Ou-Yang
1
, Jia-Yue Duan
1
, Yun-Yun Wu
1
, Muhammad Hasnain Ehsan Ullah
1
,
Zhi-Ang Zhou
2
, Feng Xu
1
, Xiao Lin
3
, Feng Wu
4
, Xiao-Bo Liao
2
and Ling-Qing Yuan
1
*
1
National Clinical Research Center for Metabolic Diseases, Department of Metabolism and Endocrinology, The Second
Xiangya Hospital, Central South University, Changsha, China,
2
Department of Cardiovascular Surgery, The Second Xiangya
Hospital, Central South University, Changsha, China,
3
Department of Radiology, The Second Xiangya Hospital, Central
South University, Changsha, China,
4
Department of Pathology, The Second Xiangya Hospital, Central South University,
Changsha, China
Histone methylation is an epigenetic change mediated by histone methyltransferase, and
has been connected to the beginning and progression of several diseases. The most
common ailments that affect the elderly are cardiovascular and cerebrovascular disorders.
They are the leading causes of death, and their incidence is linked to vascular calcification
(VC). The key mechanism of VC is the transformation of vascular smooth muscle cells
(VSMCs) into osteoblast-like phenotypes, which is a highly adjustable process involving a
variety of complex pathophysiological processes, such as metabolic abnormalities,
apoptosis, oxidative stress and signalling pathways. Many researchers have
investigated the mechanism of VC and related targets for the prevention and treatment
of cardiovascular and cerebrovascular diseases. Their findings revealed that histone lysine
methylation modification may play a key role in the various stages of VC. As a result, a
thorough examination of the role and mechanism of lysine methylation modification in
physiological and pathological states is critical, not only for identifying specific molecular
markers of VC and new therapeutic targets, but also for directing the development of new
related drugs. Finally, we provide this review to discover the association between histone
methylation modification and VC, as well as diverse approaches with which to investigate
the pathophysiology of VC and prospective treatment possibilities.
Keywords: epigene tics modification, histone lysine methyl ation, histone lysine methyltransfe rases (HKMTs),
vascular calcification, signalling pathways
1 INTRODUCTION
Vascular calcification (VC) is a pathological condition prevalent in persons with atherosclerosis,
hypertension, chronic kidney disease and diabetic vascular disease. It presents a substantial risk
factor of cardiovascular disease (CVD) occurrence and death, as well as an independent predictor of
CVD occurrence (1). According to the location of calcified plaque formation and development,
Frontiers in Endocrinology | www.frontiersin.org June 2022 | Volume 13 | Article 8637081
Edited by:
Valentina Perissi,
Boston University, United States
Reviewed by:
Xiaoqiang Tang,
Sichuan University, China
Tao Yang,
Van Andel Institute, United States
*Correspondence:
Ling-Qing Yuan
allenylq@csu.edu.cn
Specialty section:
This article was submitted to
Cellular Endocrinology,
a section of the journal
Frontiers in Endocrinology
Received: 27 January 2022
Accepted: 06 May 2022
Published: 16 June 2022
Citation:
Cao Y-C, Shan S-K, Guo B, Li C-C,
Li F-X-Z, Zheng M-H, Xu Q-S,
Wang Y-L, Lei L-M, Tang K-X,
Ou-Yang W, Duan J-Y, Wu Y-Y,
Ullah MHE, Zhou Z-A, Xu F, Lin X,
Wu F, Liao X-B and Yuan L-Q (2022)
Histone Lysine Methylation
Modification and Its Role
in Vascular Calcification.
Front. Endocrinol. 13:863708.
doi: 10.3389/fendo.2022.863708
REVIEW
published: 16 June 2022
doi: 10.3389/fendo.2022.863708
VCis classified as either intimal calcification or medial
calcification (2). Intimal calcification is evident in
atherosclerotic plaques (3), where vascular cells undergo
metamorphosis and functional changes that encourage the
creation of calcium and phosphorus crystals in the lipid
necrosis nuclei of atheromatous plaques. Medial calcification is
seen in distal arteries (4), which can cause vascular compliance to
decrease and pulse pressure to rise, resulting in cardiac
insufficiency. Both forms of calcification can occur individually
or simultaneously in patients with CKD. VC is primarily caused
by phenotypic changes of vascular smooth muscle cells
(VSMCs), a highly changeable process analogous to bone
development. VC is caused by a variety of factors, including
oxidative stress, inflammatory response, autophagy (5), vesicle
production, vascular injury, high calcium and phosphorus levels,
and a lack of calcification inhibitory factors, all of which
contribute to mineral deposition in the extracellular matrix
and to VC (6,7). Various studies have been carried out to
understand the intricate molecular pathways that govern gene
expression and protein function, in order to slow down the
process of VC and identify appropriate treatments for
cardiovascular disease.
Post-translational regulations are covalent modifications that
occur after protein synthesis and are important targets for the
regulation of signalling pathways. Histone lysine methylation
modifications are one of these, mediated by histone lysine
methyltransferases (HKMTs), which may operate on both
histones and non-histones and play important roles in many
biological processes through heterochromatin formation,
transcriptional control, and so forth. A range of pathological
conditions can be caused by abnormalities in lysine methylation
modification, and studies have revealed that lysine methylation
modification is intimately linked to VC formation
and development.
This review summarizes well-known mechanisms of VC,
followed by recent updates regarding lysine methylation–
associated VC pathogenesis and related pathways. Further
studies are needed to uncover the complicated interactions
during lysine methylation modification and to achieve a
breakthrough in therapy for VC-associated diseases. These
complex and cross-talking mechanisms closely support the
postulate that VC is affected by histone lysine methylation.
Understanding specific VC pathologies associated with
HKMTs and exploring the potential application of epigenetics
to treatment would be of profound significance.
2 MECHANISMS OF VC
VSMCs exist in and can interconvert between two phenotypic
states, namely the contractile (differentiation) and synthetic
(de-differentiation) states (8). The change from a contractile to
a synthetic state, which is a pivotal phase at the onset of severe
vascular proliferative diseases, results in increased VSMC
proliferation and migration, extracellular matrix secretion and
synthesis, and the formation of neointimal membranes. Bone-
related genes, such as bone morphogenetic proteins (BMPs),
runt-related transcription factor 2 (Runx2) (8), and osteocalcin
can have increased expression followed by transition. Abnormal
calcium and phosphorus metabolism, inflammation and
oxidative stress, pro- and anti-calcification factor imbalance,
and autophagy, all of which interact to influence the formation
and progression of VC, have been identified as the etiology of
VC. In short, multiple factors that contribute to alterations of
body homeostasis have been shown to be strongly associated
with the onset and progression of VC.
2.1 Abnormal Metabolism of Calcium
and Phosphorus
Abnormal mineral homeostasis caused by elevated calcium and
phosphorus concentrations can mediate VC. High phosphate in
serum leads to VSMC calcification in vitro (9) and to coronary
artery calcification in human (10), suggesting that phosphate
plays a critical role in the pathophysiology of VC, and the degree
of calcification is dose-dependent on phosphorus concentration.
Meanwhile, it is worth noting that under conditions of normal
phosphorus concentration, the degree of VC in VSMCs can be
upregulated when the calcium concentration increases (11), and
high levels of calcium can lead to the formation and development
of hydroxyapatite crystals in the VSMCs. High phosphate
upregulates Pit1 to raise intracellular levels of inorganic
phosphate, followed by downregulation of calcification
inhibitors, release of extracellular vesicles, remodelling of the
extracellular matrix and apoptosis of VSMCs (9). Eventually,
various signalling pathways trigger phosphate-induced osteo-/
chondrogenic transdifferentiation of VSMCs, leading to VC, and
the main feature of the osteogenic signalling pathway in VC is
the upregulation of Runt-related transcription factor 2 (Runx 2).
In addition, previous studies showed that high extracellular
phosphate levels induced apoptosis and necrosis of VSMCs,
and so apoptotic bodies released from VSMCs could serve as a
nidus for calcium phosphate deposition (12). Briefly, the
disturbance in calcium and phosphorus metabolism has a
relatively direct role in the progression of VC, and attention to
the physiopathological processes associated with calcium and
phosphorus metabolism may provide direction for the treatment
and prevention of VC.
2.2 Inflammation and Oxidative Stress
The inflammatory response pathway is an important venue
associated with pathogenesis of VSMCs calcification (13), and
the progression of VC is aided by many inflammatory cells and
factors. Interleukin-1 beta (IL-1b) stimulates VSMC calcification
in vitro (14). Interleukin-6 (IL-6)/soluble interleukin-6 receptor
(sIL-6R) complexes induces VSMC transformation into an
osteoblast phenotype (15). IL-11 plays an important role in
VSMC phenotype switching and vascular inflammation (16).
When macrophages are exposed to calcium or phosphate
nanocrystals, they release inducible nitric oxide synthase
(iNOS) and tumour necrosis factor (TNF), implying that
inflammatory immune cells are recruited to the calcification
site (17). According to previous work, macrophages from
Cao et al. Histone Lysine Methylation Modification
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various subsets can undergo polarity drift, meaning that
macrophages from different subsets can turn into one another.
For example, under specific conditions, M1 and M2 may bio-
transform into each other. Macrophages can promote VC
through diverse mechanisms, such as the release of reactive
oxygen species, pro-inflammatory cytokines and matrix
vesicles (MVs) (18). This process is regulated by cartilage
oligomeric matrix protein (COMP), which can polarize
macrophages into M1 phenotypes (i.e., they tend to be
osteogenic phenotypes), and inhibits macrophages’
differentiation into M2 and osteoclast-like cells (19). M1 can
directly release oncostatin M (OSM) to promote the
differentiation of VSMCs into osteoblastic phenotypes through
the JAK3-STAT3 pathway (20), whereas M2 can secrete anti-
inflammatory factors, as well as phagocytize necrotic fragments
and apoptotic cells, to prevent the formation of calcified
nucleation sites (21). However, the persistent state of chronic
inflammation caused by M1 may also impair the normal
development and transformation of VSMCs into osteoblasts.
The macrophages can produce BMP2 as well as Runx2, and this
ability can be maintained in aortic plaques (18). This process
reveals a novel therapy avenue in which controlling the M1-to-
M2 transition and reducing inflammation may help to reduce
VC. Researchers found that inhibition of Runx2 regulation
mediates the anti-calcification effect by inhibiting the
inflammation-associated NF-kBpathway(22), suggesting a
strong link between inflammation and calcification in the
pathogenesis of VC. Thus, these discoveries indicate a strong
causal relationship between inflammation and VC, and the
research of VC should be accompanied by attention to
inflammatory indicators, suggesting that certain inflammatory
indicators can be used as tools to evaluate VC.
2.3 VC Activator
2.3.1 FGF23
FGF23 is produced by osteocytes, which have been found to be
one of the most powerful phosphomodulators (23)andan
important inducer of VC. It acts in collaboration with the
transmembrane protein Klotho as a cofactor, primarily
regulating the metabolic balance of blood phosphorus and
vitamin D in the body (24). Klotho has been identified as an
anti-aging gene (25) and Klotho protein has anti-aging and
cardiovascular protective effects. Lim et al. discovered that
human VSMCs express Klotho protein (26). They also
discovered that Klotho expression was significantly reduced
and Runx2 was increased in vascular tissues, and that Klotho
protein inhibited the transdifferentiation of VSMCs into
osteoblasts by inhibiting phosphorus uptake by cells. Klotho
deficiency can promote the osteogenic phenotype of VSMCs by
regulating phosphorus uptake in VSMCs, through the induction
of the sodium-phosphorus cotransporter Pit1/2 (27). Numerous
experiments have shown that Klotho deficiency is an important
causal factor in VC. Elevated levels of Klotho may regulate
phosphorus and calcium homeostasis in vivo, either directly in
the kidney and vascular cells or indirectly (28). The effect of
Klotho on calcification may be associated with the classic Wnt/-
catenin pathway (29,30), and a study involving stem cells
demonstrated Klotho may act as a Wnt antagonist and
immunoprecipitates with a number of Wnt isoforms, including
Wnt1, Wnt3, Wnt4 and Wnt5a (31). Hum et al. found that
upregulation of Klotho alleviated VC (32); for example,
intermedinl-53, a member of the adrenomedullin family, has
been shown to reduce the degree of VC by activating the cyclic
adenosine monophosphate/protein kinase A (cAMP/PKA)
signalling pathway (33). However, Lindberg (34)couldnot
detect mRNA expression of FGF23 or its coreceptor, Klotho, in
human or mouse VSMCs, nor normal or calcified mouse aorta.
There still exists controversy as to whether VSMCs express
Klotho, and whether Klotho is involved in the development
and progression of VC. Therefore, insights into FGF23 may
provide a new indication for the development of VC, which may
provide a direction for treatment and prevention, while a
complete understanding of this pathway and other roles in VC
remain to be confirmed.
2.3.2 BMP
BMPs are powerful osteogenic differentiation activators, and
were discovered in calcified VSMCs. BMP-2 and BMP-4, in
particular, are intimately linked to VC among BMP family
members. BMP-2 may promote VC by activating muscle
segment homeobox2 (MSX2) and inhibiting matrix Gla protein
(MGP), and may also promote apoptosis of VSMCs (35). BMP-2
and MSX2 can activate the Wnt/b-catenin pathway, one of the
major osteo-inductive signalling pathways in VC, and then
induce VSMC calcification (36). Nuclear factor-kappa B ligand
(RANKL) promotes VC by inducing the release of BMP-2 from
human aortic endothelial cells, which, in turn, acts in a paracrine
manner on the adjacent human aortic smooth muscle cells to
increase osteoblastic activity (37). In vascular media, BMP2 was
observed to act through the type III sodium-dependent
phosphate cotransporter, Pit1, and downregulate microRNA-
30b and 30c (38), resulting in an increased expression of Runx2,
calcium deposition, and mineralization to accelerate medial or
intimal calcification. BMP‐2 signalling is involved in the
maintenance of the contractile phenotype and has been shown
to inhibit VSMCs proliferation and neointimal hyperplasia (39).
Serum BMP4 levels are higher in patients with chronic renal
disease and coronary artery disease, and they are independently
and positively linked with coronary artery calcification indices
(40). BMP-4 may participate in leptin-induced calcification of
VSMCs via ERK1/2/RANKL/BMP-4 and PI3K/Akt/RANKL/
BMP-4 signalling pathways (41,42). In addition, BMP4 can
promote foam cell production, inhibit lipid carrier expression
and lipid export, and contribute to atherosclerosis through the
BMPR1/2/Smad1/5/8 signalling pathway (43). These discoveries
systematically and clearly illustrate the role of BMP as an
activator in VC, which affects the onset and progression of
the disease.
In addition to the above two activators of calcification that are
more associated with histone lysine methylation modifications,
there exist many other risk factors that promote calcification,
including osteocalcin (OC) (44), alkaline phosphatase
Cao et al. Histone Lysine Methylation Modification
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(ALP) (45), osteopontin (OPN) (46), PTH (47) and Cathepsin K
(48). Together, these activators of calcification are involved in
key aspects of the VC signalling pathway and focusing on these
activators to find HKMTs which produce inhibitory effects is an
important therapeutic direction for the alleviation and treatment
of VC.
2.4 VC Inhibitors
As the external environment changes, the corresponding
decrease in calcification inhibitors and functional deficiencies
can exacerbate VC. There are natural inhibitors of calcification
including osteoprotegerin (OPG) (49), matrix Gla protein
(MGP) (50), fetuin-A (Fet-A) (51), BMP7 (35)and
pyrophosphate (PPi) (52). Paloian identified a novel protein
involved in the bone-vascular axis, osteosclerin (OPN), which is
mainly secreted by osteoblasts and chondrocytes. As a specific
and regulated negative regulator of the Wnt pathway,
osteosclerin plays a protective role in the development of VC
(53). These findings about VC inhibitors provide some directions
for the identification of new therapeutic targets, suggesting
analysis of the differential expression of these inhibitors may
provide novel outlooks on diagnosis and therapy.
2.5 Disturbed Autophagy Regulation
Autophagy is a cell protective mechanism occurring through
removal of mistake proteins, damaged organelles or unwanted
metabolites (54), which is vital to maintain normal VSMC
function (55–57). However, excessively activated autophagy
can induce autophagic death of VSMCs (58). For example,
hyperphosphatemia can induce calcification by promoting
osteogenic transformation of VSMCs through activating
abnormal autophagy (54,59). Cell death by apoptosis or
necrosis leads to the release of apoptotic bodies, or necrotic
debris, which may act as nucleation sites for calcium phosphate
deposition and further aggravate calcification. In contrast,
research showed that autophagy could counteract against ROS-
induced VC at high Pi concentrations in vitro (59). In addition,
emerging evidences indicates that autophagy also regulates
extracellular matrix homeostasis and mitigates VC process
(60). To summarise, whether autophagy is a protective or
harmful mechanism in VC pathology remains controversial.
The idea of modulating autophagy offers an attractive direction
to treat or prevent VC. For example, oestrogen-induced
autophagy inhibits the osteogenic differentiation of VSMCs
and arterial calcification via the ERapathway (57). Therefore,
further studies are required to investigate autophagy and their
significance in pathophysiology of VC.
3 HISTONE LYSINE METHYLATION
In eukaryotes the nucleosome is the main structural element of
chromatin, which consists of DNA and a core histone octamer
including two copies of H2A, H2B, H3 and H4, with histone H1
acting as a junction between the nucleosome and DNA (61).
Histone acetyltransferase, histone methyltransferase and histone
phosphotransferase can all produce post-translational
modifications, of which histone lysine methylation is one of
the most characterized post-translational modification. With
more clues uncovered, lysine methylation modification plays a
significant role in the progress of VC. Mechanistically speaking,
only lysine and arginine were thought to be the locations of
histone methyltransferase action, based on early investigations
on a vast number of protein sequences, and the N-terminal tails
of histone residues can be methylated once or repeatedly,
resulting in monomethylation, dimethylation or trimethylation.
HKMTs are a family of proteins that include the SET domain,
named after the first three genes that expressed it: Su(var)3-9,
enhancer of zeste [E(z)] and trithorax (trx) (62). Apart from
Dot1 enzyme (63), which methylates H3K79, all HKMTs have a
SET domain. Histone lysine methylation is directly linked to
chromatin concentration and gene silencing (64), and can play a
role in physiological and pathological states via a variety of
pathways. Thus, HKMTs can be summarized into two categories
based on their different functions on the substrate, one activating
and the other inhibiting. H3K4, K26, K36, K79 and H4K12
methylations are mostly engaged in gene activation, whereas
H3K9, K27, K56, H4K5, and K20 methylations are involved in
gene silencing (Figure 1).
4 HKMTS SUBSTRATES LYSINE FROM
HISTONE TO CONTROL VC
4.1 Transcriptional Activation-Associated
Histone Lysine Methylation Modifications
in VC
4.1.1 H3K4 Locus Methylation and its Potential Role
in Facilitating VC
H3K4 methylation is recognized as a marker of gene
transcriptional activation. H3K4 methyltransferases, also
known as MLL (mixed lineage leukaemia) family proteins,
include MLLl, MLL2, MLL3, MLLL4, SET1A, SET1B, SETD7,
SMYD2 and SMYD3. SMYD2 methylates histone H3K4 and
H3K36 with the SET-dependent manner. TGF -binduces
increased deposition of the extracellular matrix when the level
of H3K4me methylated by SMYD2 increases (65), and TGF-b1
or BMP2-stimulated valvular endothelial cells transform into
osteoblast-like cells by increasing ALP expression, eventually
resulting in VC (66). SMYD3 catalyzes dimethylation and
trimethylation of H3K4 to form H3K4me2 and H3K4me3 near
the promoter regions of target genes, which usually serve as
transcriptional activators involved in promoting cell growth (67).
SMYD3 promotes vascular cellular senescence by binding to the
promoter region of p21 gene, implicated in cell cycle arrest and
cellular senescence via H3K4 methylation (68). Other research
also shows that SMYD3 can bound to promotors of PARP16
through increased H3K4me3 levels, to mediate vascular
senescence and ER stress existing in cell models (69). Research
on SMYD3 in VC has not yet been carried out, but there are
many common points of contact between vascular senescence
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and calcification in terms of their mechanisms, which can
provide a novel direction for the study of VC. Increasing
evidence suggests that SETD7 plays a critical role in a number
of physiological and pathological processes, such as metabolism,
immunity, vascular pathology and cancer (70). Hypoxia-
inducible factor 1a(HIF-1a) is a transcription factor
upregulated by hypoxia, and SETD7 can inhibit its
transcriptional activity by methylating H3K4 (71). HIF-1acan
upregulate Runx2 to induce VC (72) and plays a critical role in
Pi-induced VC (73). However, our understanding of the
relationship between H3K4 methylation and VC is
rudimentary and is based on the association of other vascular
pathological alterations and VC in previous studies. In
conclusion, H3K4 methylation contributes to an enhanced
interpretation of VSMCs physiology and pathology.
Current studies on MLL family methylation modifications are
mainly in the tumour area, with few reports investigating the VC
area. It is noteworthy that MLL1 facilitates the proliferation of
myoblastsbyepigeneticallyregulatingMyf5via mediating
H3K4me3 on its promoter (74). Also, SETD7 has been shown
to be associated with transcriptional activation of myogenic
differentiation genes, such as MYOD, MYOGENIN, MHC and
MCK, via H3K4 methylation (75), providing an insight into the
mechanisms of HKMTs in VSMC pathophysiology. These
discoveries further support the potential role of HKMTs acting
on H3K4 in VC progression.
4.1.2 H3K36 Locus Methylation and its Potential
Associated Role in VC
H3K36 plays an important role in transcriptional elongation, and
its methylation modifications may be seen in abundance in the
coding areas of transcriptionally active genes (76). H3K36me1 is
considered an intermediate modification without any significant
role, whereas H3K36me2 and H3K36me3 each play a major role
in histone modifications (77). Zhou et al. revealed that NSD2
could serve a new function in the etiology of pulmonary arterial
hypertension by elevating the H3K36me2 level, which regulates
trehalose metabolism and autophagy (78). According to a
genome-wide investigation, DNMT3A binding and activity co-
localize with H3K36me2 at non-coding areas of euchromatin,
and NSD1-mediated H3K36me2 is required for the recruitment
of DNMT3A and the maintenance of DNA methylation at
intergenic regions, showing the intrinsic interactions of histone
and DNA in epigenetic modifications (79). SMYD2 inhibits
macrophage activation by facilitating H3K36 dimethylation at
TNF and IL6 promoters, and increased SMYD2 expression
decreases the production of pro-inflammatory cytokines
including IL-6 and TNF. Then, as a result of enhanced TGF-
production and reduced IL-6 release, macrophages with elevated
SMYD2 expression promote regulatory T cell differentiation
(80). Although no direct links between VC and H3K36
methylation modifications have been discovered, several
studies in other diseases have shown that H3K36 methylation
modifications are involved in the regulation of VC risk factors,
suggesting that there may exist potential mechanisms for
researchers to investigate.
4.1.3 H3K79 Locus Methylation and its Potential
Associated Role in VC
H3K79me is a marker of activated chromosomes and is
significantly expressed in areas with high gene transcriptional
activity (81). Disruptor of telomeric silencing 1-like (DOT1L) is a
methyltransferase that acts on lysine 79 of histone H3 (H3K79).
In addition to regulating transcriptional activation of certain
genes, DOT1L is also involved in DNA repair, cell differentiation,
and cell cycle regulation (82,83). In the G1 phase, DOT1L-
deficient cells experience irreversible cell cycle arrest, resulting in
premature senescence (74). Increased levels of SIRT1 caused by
FIGURE 1 | Histones and the DNA wound around the histones together constitute the nucleosomes, which are the main structural elements of chromatin, mainly
composed of H2A, H2B, H3 and H4. Histone lysine methylation occurs mainly at H3 and H4, of which six sites are currently well studied. This figure summarizes the
role of lysine methylation activation or inhibition at these six sites and the frequently regulated enzymes for them. Created with BioRender.com.
Cao et al. Histone Lysine Methylation Modification
Frontiers in Endocrinology | www.frontiersin.org June 2022 | Volume 13 | Article 8637085
AMPK activation can lead to an increase in H3K79me3 via
DOT1L upregulation, and then to H3K79me3-induced up-
regulation of SIRT3 levels, enhancing mitochondrial biogenesis
function as well as delaying vascular senescence (84). Vascular
senescence and VC have many common contributors to the
mechanism, and this has given researchers new ideas as to
whether H3K79 methylation plays a similar role in VC. In a
nutshell, these results present the probably function of H3K79
associated enzymes in VC.
4.2 Transcriptional Repression-Associated
Histone Lysine Methylation Modifications
in VC
4.2.1 H3K9 Locus Methylation–Associated Protective
Mechanisms of VC
H3K9 methylation plays an important role in X-chromosome
silencing, heterochromatin formation, DNA methylation and
transcriptional regulation. Researchers have confirmed that
methylation of H3K9 was significantly reduced in
atherosclerotic plaques in SMCs and inflammatory cells (85),
which illustrates the important role of H3K9 methylation in the
activation of SMCs in atherosclerosis and macrophages. G9a,
SUV39H2, SETDB1 and SUV39H1 are the primary
methyltransferases at this locus. G9a is the main euchromatin
H3K9 methyltransferase, catalyzing monomethylation and
dimethylation of H3K9 (86) in euchromatic regions, and is
involved in trimethylation of H3K9 (87). The methylation of
H3K9 by G9a is mostly associated with gene silencing. G9a can
epigenetically silence Klotho expression by monomethylating
H3K9 on the Klotho promoter (88), whereas silencing G9a has
the reverse effect, apparently reversing the repressive effect of
Klotho expression (89). As a result, inhibiting G9a expression
can control the development of VC via FGF23/Klotho axis,
which may provide an important therapeutic strategy for VC
and deserves consideration. Vascular inflammation is an
important contributor to the development of VC, and
SUV39H1 exerts anti-inflammatory effects in the vascular
inflammatory response by inhibiting the transcription of
downstream target genes, such as NOS (90), and modulating
the NF-kB signalling pathway (91). Whether or not this role in
inhibiting the development of inflammation delays VC deserves
further investigation. Therefore, future studies are required to
explore these mechanisms in detail. As more studies are
conducted, targeted therapies aimed at H3K9 methylation may
provide clinicians with a new therapy to attenuate
cardiovascular diseases.
4.2.2 H3K27 Locus Methylation–Associated
Protective Mechanisms of VC
H3K27 is associated with the transcriptional repression of genes
and can result in the inactivation of X-chromosomes (92). The
results of several studies suggest that the overall level of
H3K27me3 modification is reduced in VSMCs containing
atherosclerotic plaques (85,93). EZH2 is regarded as a
transcriptional suppressor that targets genes to alter cell
biological behaviour, by generally silencing the expression of
thetargetgene(94). EZH2 acts on H3K27 and catalyzes
methylation to form H3K27me3, which affects chromatin
configuration and genome stability. In addition to generating
H3K27me3, EZH2 can also recruit DNA methyltransferase 1
(DNMT1) (95) in the promoter region of target genes to directly
silence their expression, and DNA damage can, to a certain
extent, promote VC (96). Several studies have confirmed that
EZH2 expression is abnormally elevated in cancer tissues and is
positively correlated with the degree of malignancy of cancer
(97). Apart from functioning in cancer, EZH2 was also involved
in several signalling pathway modulations, including playing a
negative regulatory role in the process of muscle cell
differentiation and the pathophysiologic processes of VC (98).
EZH2 plays a critical role in the differentiation of skeletal muscle
cells in myoblast and myosatellite cells through miR-101a, which
promotes skeletal muscle cell differentiation directly through
EZH2 (99), instead of the terminal stage of differentiation (100).
Reduced EZH2 expression increases expression of ATG5 and
ATG7 and activates the MEK–ERK1/2 signalling pathway, which
induces excessive autophagosome formation and then leads to
VSMC loss (101). EZH2 promotes triple methylation of H3K27
in the promoter region of ATP-binding cassette transporter A1
gene and represses its transcriptional expression, which is
associated with atherosclerosis pathology and ultimately leads
to the appearance of VC. Han et al. discovered that H3K27me3
catalyzed by EZH2 was abundant in the promoter region of the
Klotho gene, due to directly binding to the Klotho promoter, and
suppressed Klotho gene expression (102) which also leads to VC.
H3K37 methylation changes can act on macrophages to promote
the process of VC, in addition to influencing the phenotypic
alteration of VSMCs. Increased H3K27me3 levels on the
promoter of nuclear factor of activated T cells type c-1
(NFATc-1) causes epigenetic impairment of the NFATc-1
gene, resulting in macrophages in the vicinity of calcium
deposits being phenotypically deficient and unable to resorb
calcification (103). The methylation modification H3K27 has
been shown to be a risk promoter for VC, and considering
H3K27 as a target for inhibition of methylation modification or
demethylation of H3K27me3 will provide new ideas for the
alleviation and treatment of VC.
4.2.3 H4K20 Locus Methylation–Associated
Protective Mechanisms of VC
H4K20 methylation plays key roles in DNA replication, gene
damage repair, and silenced heterochromatin (104), which was
once thought to be a methyltransferase target for gene silencing.
However, recent research suggests that H4K20-associated
methylation may play a dual roleinepigeneticregulation
(105). H4K20me1 is linked to transcriptional activity and
controls chromatin condensation, whereas H4K20me3 is a
hallmark of suppressed heterochromatic areas and is linked
to transcription repression and transposon activity (106).
SETD8 is the only methyltransferase known to catalyze
the monomethylation of H4K20, and can govern gene
transcription, maintain genomic integrity and regulate cell-
cycle progression by monomethylating H4K20. The vast
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majority of di- and tri-methylation modifications are mediated
by the SUV4-20H1 and SUV4-20H2 enzymes (107). Besides
acting on lysine, SET8 also can methylate non-histone proteins
such as TWIST and p53 (105). On the one hand, SET8 binds
directly to and inactivates the target gene by monomethylating
H4K20, resulting in the inhibition of its downstream pathways in
the pathogenesis of some diseases (108); on the other hand,
SET8/H4K20me1 is enriched in the promoter and coding regions
of transcriptionally active genes, for example, mediating the
transcriptional activation of Wnt target gene (109), which can
regulate the expression of the target protein RUNX2 and, in turn,
may regulate the phenotypic transformation of VSMCs (110).
Furthermore, downregulation of SET8-mediated H4K20me1 is
involved in the increasing modulation of PTEN expression,
which mediates endothelial inflammation to generate VC
(111). Yang et al. discovered that SET8 also acts as a protective
epigenetic modifier on the TWIST promoters via its H4K20
monomethylation activity. When factors that contribute to the
inhibition of SET8 expression, such as high phosphorus, TWIST
target genes can be monomethylated to inhibit AKT expression
by negatively regulating their transcription, and then Caspase-3
expression can be promoted, which, in turn, promotes apoptosis
and increases the development of calcification in VSMCs
(112,113).
Although SET8 has a complex mechanism of action in H4K20
and non-histone catalysis, in terms of overall effect SET8 is
significantly downregulated in the calcification model (114). The
methylation modification of H4K20 is highly complex due to the
different effects at different levels of methylation; therefore,
researchers may focus on the specific role of SET8 in different
pathways and the effects of other H4K20 methyltransferases
on VC.
5 HKMTS SUBSTRATES LYSINE FROM
NON-HISTONE TO CONTROL VC
According to current research, HKMTs have a broad function in
identifying methylated lysines on non-histone proteins. The area
of non-histone methylation is still in its early stages, and the
majority of applications identified thus far are p53-related.
Furthermore, HKMTs catalyze a variety of non-histone
proteins involved in the VC pathological process.
5.1 HKMTs Methylate p53 Lysine and
Participate in VC
The p53 oncogene, in addition to being one of the most
important tumour suppressors in cells, has recently been
discovered to play a vital role in VC (115,116). The HKMTs
SET9, SMYD2, G9a and SET8 can methylate four of the six lysine
sites at the C-terminus of the p53 protein to modulate its
function (117). Consequently, p53 activity can be enhanced or
lowered after methylation modification, and the response of p53-
mediated transcription activation suppression depends on target
genes. SET9 mono- and dimethylates p53K372 to regulate the
expression of p53 target genes, and positively affects p53 stability
(118,119). SMYD2 was thought to be a transcription coactivator
as it can deposit methyl groups on histones H3K4 and H3K36,
both of which are epigenetic signatures of active transcription
(120). However, unlike its action on histones, SMYD2 generally
performs an inhibitory role in regulating non-histone proteins.
For example, SMYD2 directly monomethylates p53 at K370 to
inhibit its transactivation (121). In addition, SMYD2 was
reported to act as an endogenous antagonist of p53-dependent
cardiomyocyte apoptosis (122), which may have a connection to
VSMCs. G9a dimethylates p53 at lysine 373 to inhabit p53
activity (123), and G9a inhibitors that restore p53 activity may
act as therapeutic agents for treating specific diseases (124). SET8
inhibits apoptosis and cell cycle arrest, by monomethylating the
lysine 382 site of p53 to p53K382me1 and decreasing p53
transcription (125). By regulating the p53/Bcl-2/caspase
signalling pathway, SET8 can downregulate the expression of
anti-apoptotic protein Bcl-2 and upregulate the expression of
pro-apoptotic proteins Bax and Caspase3, thus participating in
the regulation of calcification and apoptosis in VSMCs when its
expression is reduced by various factors (126). Meanwhile, lysine
methylation impairs the function of the Numb phosphotyrosine-
binding (PTB) domain, detaches Numb from p53, and prevents
it from performing its pro-apoptotic role (127). The mTOR
pathway may affect VSMC senescence through upregulation of
p53/p21/p16 (128). Upregulation of p53/p21/p16 by the mTOR
pathway impacts VSMC senescence (52), and downregulation of
p53 expression by activation of the ROS/p53/p21 pathway has
also been discovered to slow down the process of vascular aging
(129). Using p53 knockout or p21 knockout mice, however, it
was discovered that the atherosclerotic lesion developed faster
than the wild type, even though DNA damage and VSMC death
were reduced compared to the control. This could be due to the
multiple functions of the p53 and p21 genes, implying that there
may be another unknown mechanism in the p53-associated VC.
Researchers have identified HKMTs as p53-modifying enzymes
and have suggested analyzing how methylation may help
maintain normal physiological function to regulate different
p53 functions, in search of a dynamic equilibrium that can
both inhibit apoptotic pathways and reduce VC. Further, the
influence of p53 is likely an important area of future research, as
their methylation state could affect different substrates to
modulate the dysregulation of multiple pathological states and
restore them to a relatively normal state.
5.2 Heat Shock Proteins Are Associated
With VC and Can Be Methylated by HKMTs
Several post-translational modifications are also present in HSPs,
including methylation modifications. The current research found
that the dimethylation of HSP70 lysine at position 561 was
catalyzed by SETD1A (112), while HSP90AB1 lysine at
position 531 and lysine at position 574 were dimethylated by
SMYD2 (130). Similarly, SMYD2 methylates Hsp90 in muscle to
maintain titin stability and muscular function (131). Yao et al.
illustrated that HSP70 mediated the procalcific effect on
calcifying vascular cells by binding to MGP and enhancing
BMP activity, showing a potential connection between cellular
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stress, inflammation, and BMP signalling (132). Together, these
studies demonstrated that HKMTs can methylate HSPs and
HSPs serve a role in the VC progress-related pathways, so
what requires further confirmation is the specific mechanism
of HSPs in VC and whether HKMTs play an HSP-associated role
in VC.
5.3 p65 Is Involved in VC and Plays a Dual
Role via Different HKMTs
P65 is one of the most important components of the
transcription factor nuclear factor-kB (NF-kB), which regulates
the expression of a wide variety of genes (133). NF-kB -p65
activated by phosphorylation, in response to various factors,
promotes transcription of target genes (134). NSD1 was
discovered to catalyze the methylation of p65 K218/K221me,
enhancing the transcriptional activity of the NF-kB pathway
(135). Increased circulating levels of other inflammatory factors,
such as TNF, IL-6 and NF-kB, can promote calcification in
VSMCs (136), and interleukin 1b(IL-1b) induces osteogenic
transformation of VSMCs through the activation of the NF-kB/
p53/p21 pathway, ultimately leading to VC (137). Six methylated
K sites, K37, 218, 221, 310, 314, and 315, have been identified on
the p65 subunit of NF-kB(138). Yang and colleagues reported
that p65 is monomethylated by SET9 on K314 and 315, resulting
in inhibition of NF-kB action by inducing the proteasome-
mediated degradation of the p65 promoter (139). However,
methylation of SET9 at the p65K37 site exhibited the opposite
effect, and could activate the NF-kB pathway (140). SETD6
monomethylates p65 on K310, leading to the induction of a
repressed state of NF-kB target genes through the binding of
G9a-like protein (141). Taken together, the NF-kB pathway is
critical in VC and in the development of many diseases. Various
HKMTs act on p65 in this pathway to inhibit or promote
pathogenesis, and it is worthwhile to investigate how to reduce
p65-related methylation activation, and, thus, control the NF-kB
pathway to reduce the occurrence of VC.
5.4 HKMTs Catalyze ERaLysine and
Control Its Downstream Function
Studies have shown that different HKMTs operate at different
locations on the oestrogen receptor alpha (ERa) and thus show
different effects in physiopathological processes. SETD7 catalyzes
the monomethylation of ERalysine position 302
(ERaK302me1) to stabilize the ERaprotein, which is
necessary for the efficient recruitment of ER to its target genes
and the activation of an oestrogen-driven transcriptional
response (142). SMYD2 catalyzes ERK266me1 and prevents
ER binding to chromatin, inhibiting ER target gene activation
(143). G9a methylates ERaat K235, attracting the PHF20/MOF
complex to deposit histone acetylation and boosting gene
activation (144). Oestrogen inhibits VC by modulating the
receptor activator of nuclear factor-kappa B (RANK) and
RANKL signalling pathways (145). Runx2, a major osteogenic
transcription factor expressed in calcified atherosclerotic
plaques, can be inhibited by estradiol in osteoblasts (132).
Meanwhile, McRobb et al. illustrated an opposite effect, that
oestrogen can promote calcification in advanced atherosclerotic
lesions by promoting the differentiation of VSMCs to osteoblast-
like cells, and this process could be augmented by inhibition of
ERaor ERbactivity (146). These findings provide some
directions for the identification of novel ERamethylation
targets associated with VC in the later stage, and for the
development of novel therapies. Researchers may further
investigate how ER methylation can play a role in reducing
VC, based on numerous studies that have demonstrated the
preventive effects of oestrogen in cardiovascular disease.
5.5 Methylated MAPK Participates in
Various Diseases
The mitogen-activated protein kinase (MAPK) route, the P13K
system, and the cyclic adenosine phosphate (CAMP) pathway
are all implicated in controlling the phenotypic transition of
VSMCs. RAS/RAF/MEK/ERK1/2 is a typical MAPK signal
transduction pathway (147) and MAPK/ERK has a verified
relationship with vascular dysfunction (148). SMYD3
methylates MAP3K2 at lysine 260, increasing MAPK signalling
(149), and SMYD3 overexpression is associated with poor
prognosis in a variety of diseases (150). Overall, we believe that
theimpactofMAPKlysinemethylationshouldnotbe
overlooked in the VC research. Exploration of MAPK
methylation modifications could explain the related pathway in
VC and become a new target with which to overcome VC in
the future.
6 HKMTS CATALYZE LYSINE
SUBSTRATES TO PARTICIPATE IN THE
MECHANISM OF VASCULAR
CALCIFICATION
As a classic saying goes, all roads lead to Rome. During
pathologic processes, many signal pathways work
independently to achieve the same goal. HKMTs have been
demonstrated to play an important role in each of these
pathways, either by inhibiting or boosting gene expression, or
by targeting critical components of a signalling system
implicated in physiopathological activity. The majority of
current research regarding the role of HKMTs in pathway
signalling–mediated disease development is concentrated on
tumours, and evidence has shown that HKMTs and
pathological pathways associated with VC have sophisticated
cross-talk. HKMTs are involved in various pathological
alterations such as vascular inflammation, atherosclerosis and
VC, and play seemingly minor epigenetic modifying roles
throughout the pathway. Because HKMTs are post-
translational modifying enzymes, they can affect gene
expression at almost every segment of the pathway. When all
pathways leading to VC are linked together, each location of the
pathway may behave as a possible target for HKMTs to act, either
activating or inhibiting the VC process. At the macroscopic level,
independent and distinct HKMTs may burst into a mighty flame
from little sparks (Figure 2).
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6.1 Wnt/b-Catenin Pathway
The Wnt/b-catenin signalling pathway is a cell/receptor context–
dependent route to activate the nuclear functions of b-catenin
and can activate the expression of target genes, and this pathway
is involved in virtually all physiological or pathological
mechanisms in a variety of organisms. In the classical Wnt
signalling pathway, when the Wnt ligand binds to the FZD-
LRP5-LRP6 co-receptor, it promotes the phosphorylation of
glycogen synthase kinase 3b(GSK3b), thereby b-catenin can
be prevented from being degraded. In addition, b-catenin
accumulates stably in the cytoplasm before it enters the
nucleus to bind to T-cell factor/Lymphoid enhancer-binding
factor (Tcf/Lef), triggering target gene transcription (151).
Multiple studies have suggested that the Wnt signalling
pathway is involved in VSMC calcification (152,153), and Hao
et al. identified elevated expressions of b-catenin and Wnt-5a in
VC (154). Meanwhile, abnormalities in Wnt signalling are
associated with aberrant epigenetic modification mechanisms
(155). The histone H3K27 methyltransferase EZH2 is abundant
in Wnt promoters, according to genome-wide profiling studies
(156). H3K27me3 is an important modification involved in Wnt/
b-catenin pathways, acting as a marker of gene repression (157).
EZH2-regulated H3K27me3 plays a negative role on the b-
catenin promoter during the odontogenic differentiation of
hDPCs (158), and Lu et al. indicated that activation of EZH2
can inhibit the expression of Wnt and BMP targets and that
PRC2 dysfunction can elevate Wnt signalling, shown by genomic
occupancy and transcriptomic analyses. Together, reducing
EZH2 activity and H3K27me3 levels may induce
demyelinating diseases (159). Researchers have recently begun
to investigate the existence and significance of histone lysine
methylation modifications in the Wnt/b⁃catenin pathway in
investigations into the mechanisms of VC. Advanced glycation
end products (AGEs) can activate the Wnt/b⁃catenin signalling
pathway by binding to the receptor for advanced glycation end
products (RAGE) on the cell membrane (160), and it has been
shown that AGEs significantly stimulate the expression of
osteopontin (OPN), osteocalcin (OC) and Runx2 mRNA in rat
aortic smooth muscle cells (161), during which b-catenin and
OPG gene expressions are upregulated, leading to the
differentiation of VSMCs into osteoblasts and ultimately to the
development of VC (162). RAGE induces the conversion of
VSMCs to osteoblasts through activation of signalling pathways
such as ERK (163), NF-kB(164)andWnt(160); thus,
participating in VC. AGEs decrease EZH2 expression in
podocytes and, consequently, reduces H3K27me3, causing an
upregulated expression of pathological factors and contributing
to podocyte injury in diabetic kidney disease (165). EZH2 is
required in Wilm’stumour1(WT1)-mediatedb-catenin
inactivation via repression of secreted frizzled-related protein 1
(SFRP-1), which is a Wnt antagonist, and EZH2-mediated
silencing of SFRP-1 is due to increased H3K27me3 at its
promoter area(s) (166). H3K27 methyltransferase EZH2
represses Wnt genes directly to facilitate adipogenesis, and
deletion of EZH2 eliminates H3K27me3 on WNT promoters
and de-represses Wnt expression, leading to activation of Wnt/
beta-catenin signalling (167). The mechanism of SET8 activation
of the Wnt signalling pathway is yet to be revealed, despite
numerous studies (104). However, after activation of the Wnt
signalling pathway, SET8 has been found to act as a
transcriptional activation cofactor for H4K20me1 modification
of histones, regulating chromosomal conformation and thus
recruiting more transcription factors to accumulate and initiate
downstream gene transcription (105). Through these findings, it
is evident that the Wnt/b-catenin pathway plays an essential role
in vascular calcification, as well as that HKMTs regulate VC
FIGURE 2 | Complex network of relevant pathways involving mutual interactions in vascular calcification via HKMTs. The entire process of vascular calcification is
the consequence of a complex integrated effect which is determined by multiple pathways. HKMTs, as a post-translational modifying enzyme that can act on both
histone lysine and catalyze lysine from non-histone, have been shown to perform a sophisticated function in inhibiting or promoting multiple pathways of VC
progression. This figure summarizes the pathways constituted by the mechanisms involved in vascular calcification by HKMTs and their role in the process of
vascular calcification. Created with BioRender.com.
Cao et al. Histone Lysine Methylation Modification
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progression by methylating critical factors in the pathway.
Considering that HKMTs have a bidirectional effect on the
regulation of the pathway, we couldn’t help wondering which
modality of regulation is more beneficial in reducing the
occurrence of vascular calcification?
6.2 NF-kB Pathway
In normally unstimulated conditions, nuclear factor kappa B
(NF-kB) in the cytoplasm is inactivated and binds to inhibitor of
kappa B proteins (IkBs), forming a trimeric complex. In the
presence of external stimuli, TNF receptors on the cell
membrane surface bind cytokines and multimerize, and then
interact with TRADD molecules in the cytoplasm. IkB then
dissociates from the p50/p65/IkB heterotrimer and is degraded;
thus, NF-kB is released from repression and enters the nucleus,
where it binds to specific sequences on intranuclear DNA to
initiate or enhance the transcription of related genes (168). NF-
kB signalling has been implicated in osteogenic differentiation of
VSMCs in response to diverse stimuli such as high glucose (169),
high phosphate (9,170) and oxidative stress (171), as well as pro-
inflammatory cytokines. TNF–NF-kB signalling can significantly
increase ALP activity and TNF-activated NF-kBpromotes
inflammation-accelerated VC (172). In addition to enhancing
ALP activity, NF-kB activation also upregulates BMP-2 and
Runx2 expression, thereby interfering with the anti-
calcification pathway in VSMCs (172). Barroso et al. revealed
that EZH2 suppression promoted the expression of
inflammatory cytokines by the reduction of H3K27me3 in
promoters of related genes, and activated the NF-kB pathway
in the vasculature (173). For example, the decrease of H3K27me3
at the IL-1bpromoter can increase IL-1bexpression, which acts
on IL-1bR to activate the NF-kB pathway (174). EZH2 deficiency
enhances tumour necrosis factor receptor-associated factor 2
(TRAF2) expression, thereby enhancing TNF-a-induced NF-kB
signalling (175). SMYD2-mediated TRAF2 methylation
continuously activates the NF-kB signalling axis by sustaining
its own stability (176). In addition to the amplified inflammatory
program caused by inhibition of EZH2, inhibition of G9a
expression can also enhance the NF-kB pathway by reducing
H3K9me2 expression, inducing an inflammatory response in
VSMCs (177). Therefore, increasing H3K9me2 or H3K27me3
levels of NF-kB pathway–associated sites, by regulating HKMTs’
activity or inhibiting relative demethylases, may provide a novel
target strategy for VC.
6.3 BMP Pathway
BMP signalling exists mainly in the form of specific binding of
ligands to serine/threonine kinase receptors on the cell
membrane, forming a ligand-receptor binary complex. The
type I receptors phosphorylate Smad proteins (Smad1, Smad5,
and Smad8), prompting Smad molecules to detach from the cell
membrane and enter the nucleus after binding Smad4 molecules
(common-Smad, Co-Smad) in the cytoplasm. In the nucleus,
the Smad multiplex acts on specific target genes with the
participation of other DNA-binding proteins, regulating the
transcription of the target genes (178). Researchers have
confirmed since decades ago that BMP signalling was involved
in VC (179) and that the BMP signalling pathway cross‐talks
with the Wnt signalling pathway (180). SMYD2 was shown to
have a strong association with the BMP pathway by methylating
BMPR2, which, in turn, facilitates Smad1/5 phosphorylation,
nuclear entry, and interaction with Smad4 and, consequently,
BMP target gene expression (181). EZH2 deletion increases the
BMP-dependent Smad1/5 phosphorylation by decreasing
H3K27me3 near transcriptional start sites (182). Suv39h2
interacts with Smad5, and can silence the myogenic promoters
by methylation of histone H3K9 which induces the expression of
osteoblast-specific genes (183). Wang et al. described the reduced
level of H3K9me3 and H3K27me3 and their occupancy at
promoters of Bmp2 and Bmp4 without affecting the expression
of HKMTs, implying that histone demethylases may be
responsible for the reduction in methylation (184). In
summary, according to the most recent findings, HKMTs act
at several levels of the BMP signalling pathway, and BMP is
regarded as a major contributor in VC promotion. Researchers
should investigate other undiscovered mechanisms of the BMP
pathway in VC advancement, as well as ways to control VC
progression by controlling HKMTs’activation or inhabitation.
6.4 PI3K/AKT Pathway
PI3K/AKT signalling plays a critical role in cellular physiology
(185) and it is confirmed that calcification of VSMCs could be
significantly reduced by applying LY294002, a specific inhibitor
of the PI3K/AKT pathway, indicating that activation of the PI3K/
AKT pathway could promote VC (186). One of the pathways of
AGEs that lead to VC includes activation of the PI3K/AKT
signalling pathway (187). Researchers have also identified that
AKT can increase the stability and transcriptional activity of
Runx2 protein to regulate osteoblast differentiation (188). In
addition, PI3K/AKT and ERK signalling pathways can elevate
BMP-4 and ALP expression through activation of RANKL, and
this process can be reversed after pretreatment with LY294002
(42). The mechanism of interaction between the PI3K/AKT
pathway and histone lysine methylation has also been widely
studied. EZH2 can act as a substrate for AKT, and the activation
of AKT signalling pathway phosphorylates EZH2, which
decreases the affinity between EZH2 and histone, leading to
the loss of EZH2 methylation histone function and the decrease
of H3K27me3 level, promoting the development of disease (189).
These discoveries demonstrate that the interaction of the PI3K/
AKT pathway with altered methylation catalyzed by HKMTs has
critical properties for VC progression. In conclusion, enhanced
understanding of the role of HKMTs in the PI3K/AKT pathway
affecting VC helps to enhance the physiological and
pathological interpretation.
7 HKMTS REGULATOR AND THE CLINICAL
APPLICATION PROSPECTS IN VC
Given that HKMTs-mediated histone or non-histone lysine
methylation plays an important role in various pathways of VC
development, we can investigate how targeting HKMTs or
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demethylases through small molecules can affect enzyme
function, which may be an efficient therapy for VC. Since the
discovery of histone lysine specific demethylase 1 (LSD1) (190),
the thought that histone methylation is an irreversible process
has been overturned, and researchers have begun to recognize
that the histone methylation process is dynamically active, so
how to use this dynamism to regulate disease onset and
progression deserves the attention of researchers. Based on
the previous studies, we expect mis-regulated genes can be re-
expressed after treatment with methyltransferase inhibitors or
activators in vascular calcification. A variety of researches have
shownthatinhibitorsofHKMTscanbeusedincancer
treatment, for example, Tazemetostat, an EHZ2 inhibitor, for
which researchers have started clinical trials to prove its efficacy
in tumors such as lymphoma (191). However, few studies have
investigated the treatment of VC by targeting methylated lysine
associated with HKMTs, and the epigenetic regulatory
medicines that researchers have identified that may be
applied to control VC include inhibitors of histone
deacetylase and histone acetyltransferases (192,193). To date,
these epigenetic mechanism-related small molecule inhibitors
or activators have not been applied to clinical trials for the
treatment of VC and related cardiovascular diseases. Therefore,
researchers can focus on molecules which can regulate
HKMTsanddevelopnewepi-drugstargetingHKMTsto
maintain the physiological homeostasis of the organism. It is
expected to find an effective way to target VC through histone
methylation modifications.
In addition, another application of epigenetic modifications
in VC can be the determination of disease progression and
prognosis by specific biomarkers. The current research
indicates that methylated lysine sites such as H3K4me3 can be
identified as epigenetic marks associated with Myh7 gene
expression (194). And EZH2 has been proven to be a
promising therapeutic and prognostic biomarker for tumors
(195). In summary, investigators have confirmed in studies on
other diseases that HKMTs and methylated substrates can be
used as biomarkers for diagnosis and prognosis of diseases.
Although there are no research results so far to suggest that
HKMTs can be used as biomarkers for VC, it also gives
researchers ideas to identify new markers that can contribute
to the diagnosis and prognosis of VC.
8 CONCLUSION
In summary, histone lysine methylation modification is an
important component of epigenetics, and their abnormal
expression and function are receiving increasing attention with
regard to the pathogenesis of VC. The study of histone lysine
methylation in relation to VC has also made initial progress,
showing that histone lysine methylation is associated with
transcriptional activation or repression in different conditions
in VC, and inhibiting or activating the expression of HKMTs can
affect the progression. However, the exact mechanism of histone
methylation modification is not well understood to date.
Therefore, although abnormal histone lysine methylation has
been verified to be associated with VC, further research is needed
to elucidate the relationship between the two phenomena at the
molecular level. An in-depth study of the pathogenesis of VC in
combination with the characteristics of HKMTs will help to gain
a deeper understanding of the “bridge”between histone lysine
methylation modification and VC. The development of effective
prevention and treatment tools based on this mechanism will be
of great significance in controlling the development of VC and
reducing mortality in the elderly, especially in patients with
concomitant cardiovascular disease.
AUTHOR CONTRIBUTIONS
L-QY wrote the manuscript and approved the final version of the
manuscript. Y-CC contributed to study conduct, data analysis,
and manuscript writing. S-KS, BG, C-CL, F-X-ZL, M-HZ, Q-SX,
YW, L-ML, K-XT, W-LO-Y, J-YD, Y-YW, MU, Z-AZ, FX, X-BL,
FW and XL contributed to data analysis. All authors reviewed the
manuscript. All authors contributed to the article and approved
the submitted version.
FUNDING
This work was supported by funding from the National Natural
Science Foundation of China (Nos. 81770881 and 82070910).
Key R & D plan of Hunan Province (2020SK2078). Natural
Science Foundation of Hunan Province (2021JJ30036).
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