Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_3608_Библиотеки_им_академика_М_И_Перельмана
.pdf
30
Even though the closely homologous KLF4 and KLF5 TFs share
similar developmental and tissue pattern expression, they exert
different, often opposing, effects on gene regulation and proliferation/differentiation.
33
Whereas KLF4 is associated with growth arrest,
KLF5 exerts pro-proliferative effects, particularly in vascular remodeling in response to injury. KLF5 expression, abundant in fetal VSMCs
CH
but down-regulated in the adult (reviewed in
3
vascular injury by activation of immediate early response genes by
Ang II and ROS.
41
KLF5 in turn mediates re-expression of SMemb/
39
), is induced after
NMHC-B, a marker for the dedifferentiated phenotype, and activates
other critical injury response genes involved in remodeling, such as
PDGF-A/B, Egr-1, plasminogen activator inhibitor 1 (PAI-1), inducible
nitric oxide synthase (iNOS), and VEGFR, implicating KLF5 as a key
regulator for VSMC response to injury.
39
In additional injury responses,
KLF5 increases cyclin D1 expression and inhibits the cyclin kinase
inhibitor p21, thus leading to vascular remodeling by increased cell
proliferation.
42
Similar to KLF4 regulation, KLF5 expression and activ-
ity are regulated at multiple levels, including upstream Ras/MAPK,
PKC, and TGF-β signaling pathways; downstream interactions with
TFs, including retinoic acid receptor α (RARα), NF-κB, and peroxi-
some proliferator–activated receptor gamma (PPARγ); as well as
posttranslational modifications that can positively or negatively
regulate KLF activity.
39
In addition, KLF5 activity is regulated in the
nucleus by chromatin remodeling factors such as SET, a histone
chaperone that inhibits the DNA binding activity of KLF5,
(a coactivator/acetylase that coactivates KLF5 transcription), and
HDAC1, which inhibits KLF5 binding to DNA.
Two additional KLFs have been identified in VSMCs: KLF13 and
32
KLF15.
After vascular injury, KLF13 is induced and activates the
32
promoter for the VSMC differentiation marker SM22α, while KLF15
expression is down-regulated, implicating KLF15 as a negative regulator of VSMC proliferation and a counterbalance to the growthpromoting effects of KLF5 in vascular injury response.
Posttranscriptional Regulation of Vascular Smooth
Muscle Cell Diversity: Noncoding microRNAs
Upstream signaling and downstream transcriptional pathways
in VSMCs are intertwined with a multitude of micro ribonucleic
acid (miRNAs) that act as “rheostats” and “switches” in regulating
43
p300
protein activity in development, function, and disease.44 miRNAs
are small, noncoding RNAs (20-25 nucleotides in length) that associate with a miRNA-induced silencing complex (miRISC) of regulatory proteins, including Argonaute family proteins, Argonaute
interacting proteins of the GW182 family, eukaryotic initiation
factors (eIFs), polyA-binding complexes, decapping enzymes/
activators, and deadenylases, to induce posttranscriptional silencing of their target genes.
45
These multiple components are assembled
and interact in a multistep process with components of the translational machinery to inhibit translation initiation, mark mRNAs
for degradation through deadenylation, and sequester targets into
cytoplasmic P bodies.
44
Multiple mechanistic models for miRNAinduced gene silencing have been proposed that provide insights
into the molecular mechanisms of translational inhibition, deadenylation, and mRNA decay, but questions remain concerning the
kinetics and ordering of these translational events and whether
they are coupled or independent.
45
A recent unifying model for
miRNA-regulated gene repression is an attempt to reconcile the
often conflicting existing data. It proposes that recruitment of
Argonaute and associated GW182 proteins to miRNA induces
binding to the mRNA 5′m
7
Gcap, thus blocking translation initiation, potentially by mRNA deadenylation. Subsequent to miRNAmediated deadenylation, mRNA is degraded through recruitment
of decapping proteins.
46
In this model, inhibition of translation
initiation is linked to subsequent rapid mRNA decay in a coupled
process. Because miRNAs—which in general are negative regulators
of gene expression—may be almost as important as transcription factors in controlling gene expression in the pathogenesis
of human diseases,
noncoding RNAs are important in evaluating their potential use
as therapeutic targets.
Cardiovascular-specific, highly conserved miRNAs miR-143
and miR-145, the most abundant miRNA in the vascular wall,
47
insights into the functions of this class of
45
48
are
key players in programming VSMC fate from multipotent progenitors in embryonic development and in reprogramming VSMCs
during phenotypic modulation in the adult
44,49
(Fig. 3-5). miR-143
and miR-145 have distinct sequences but are clustered together
and transcribed as a bicistronic unit. Upstream in the genomic
sequence of miR-143/145 is a conserved SRF-binding CArG box
site, indicating control by SRF and myocardin.
49,50
These miRNAs
cooperatively feed back to modulate the actions of SRF by
VSMC
proliferation
KLF4
Elk-1
SRF SRF
CArG
miR-143/145
KLF4/5
MTRF
SRF SRF
CArG
VSMC
differentiation
Myocardin
SRF SRF
CArG
Actin cytoskeletal
remodeling
FIGURE 35 Model for regulation of vascular smooth
muscle cell (VSMC) phenotypes by cardiovascular-specific
micro ribonucleic acids (microRNAs) miR-143 and miR-145.
These miRNAs act as signaling nodes to modulate serum response
factor (SRF)-dependent transcription by regulating coactivators
and co-repressors to control VSMC proliferation or differentiation.
miR-145 represses proliferation by repressing KLF4 and promotes
differentiation by stimulating myocardin; miR-143 represses
proliferation by repressing Elk-1. miR-143/145 also controls actin/
cytoskeletal remodeling by repressing KLF4/5 and regulators
of actin dynamics, including MTRF/SRF activity. (Adapted from
Liu N, Olson EN: MicroRNA regulatory networks in cardiovascular
development. Dev Cell 18:510–525, 2010; Cordes KR, Sheehy NT, White
MP, et al: MiR-145 and miR-143 regulate smooth muscle cell fate and
plasticity. Nature 460:705–710, 2009; and Xin M, Small EM, Sutherland
LB, et al. MicroRNAs miR-143 and miR-145 modulate cytoskeletal
dynamics and responsiveness of smooth muscle cells to injury. Genes
Dev 23:2166–2178, 2009.)
targeting a network of transcription factors/coactivators/core-
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
pressors. This network includes miR-145-induced repression of
KLF4, a positive regulator of proliferation and myocardin repressor; miR-143-induced repression of Elk-1, a myocardin competitor and positive regulator of proliferation; and, contrary to the
usual inhibitory role of miRNA, miR-145-induced stimulation
of myocardin, a positive regulator of differentiation. Thus, miR145 is necessary and sufficient for VSMC differentiation, and the
miR-143/miR-145 cluster acts as an integrated signaling node
to promote differentiation while concurrently repressing proliferation.
49
Although mice with genetic deletions for miR-143/145
show no obvious abnormalities in early development, VSMCs
in the adult exhibit both structural and phenotypic differences
in injury- or stress-induced vascular remodeling. Ultrastructural
analysis of arteries from miR-143/145 knockout mice shows
reduced numbers of medial VSMCs with a contractile appearance, and an increase in synthetic VSMCs.
that miR-143 and miR-145 modulate cytoskeletal structure, actin
dynamics, and modulation to a dedifferentiated phenotype
51
These results suggest
50
(see
Fig. 3-5). Importantly, miR-143/145 knockout mice with increased
synthetic VSMCs develop spontaneous neointimal lesions in the
femoral artery in the absence of hyperlipidemia and inflammation, supporting a key role for phenotypically altered VSMCs in
the pathogenesis of lesion formation.
While miR-143 and miR-145 play keys roles in the contractile
phenotype of VSMCs and the response to injury,
51
52
miR-221 and
miR-222 are modulators of VSMC proliferation, although largely
by affecting growth-related signaling pathways rather than by
controlling VSMC phenotype. miR-221 and miR-222, encoded by a
gene cluster on the X chromosome, are up-regulated in VSMCs in
neointimal lesions and in proliferating cultured VSMCs stimulated
by PDGF-BB.
53
Studies show that two CDKIs, p27
KIP1
and p57
KIP2
have miR-221 and miR-222 binding sites and are gene targets
for miR-221 and miR-222 in the rat carotid artery
miR-221 and miR-222 are pro-proliferative because they repress
two CDKIs, p27
induction, inhibits VSMC differentiation via c-kit-induced inhibition of myocardin.
KIP1
and p57
54
KIP2
. Furthermore, PDGF, via miR-221
in vivo.53 Thus,
Posttranslational Regulation of Vascular Smooth
Muscle Cell Diversity: Epigenetics
The “epigenetic landscape” controls gene expression by chemical
modifications that mark regions of chromosomes either by methylation of promoter CpG sequences in the DNA itself, or by covalent
modification of histone proteins that package DNA by posttranslational addition of methyl, acetyl, phosphoryl, ubiquityl, or sumoyl
groups, leading to expression/repression of transcription (reviewed
55
in
). In VSMCs, multiple levels of epigenetic controls exist for gene
expression leading to differentiation or dedifferentiation programs
in healthy cells and for dysregulated gene expression in vascular
disease. These epigenetic changes in VSMCs involve both DNA and
histone methylation as well as histone acetylation/deacetylation.
Methylation of histones, catalyzed by histone methyltransferases
(HMTs), results in a tight, stable epigenetic mark between methylated histones and chromatin that can be passed to daughter cells,
thus providing “epigenetic memory” that defines cell lineage and
identity by controlling SRF access to VSMC-specific marker genes.
Acetylation is controlled by HATs, which promote gene transcription by destabilizing chromatin structure to an “open,” transcriptionally active conformation, and HDACs, which promote chromatin
condensation to a “closed,” transcriptionally silent conformation
with restricted access to DNA. Histone acetylation/deacetylation
thus serves to regulate transcription in a rapid and “on/off” manner
in response to dynamic environmental changes and links the cell's
genome with new extrinsic signals.
55
In VSMCs, SRF binding to CArG boxes in the promoters of
SMC marker genes to promote the VSMC differentiated phenotype depends upon alterations of chromatin structure, including
histone methylation and acetylation. In a model for epigenetic
55
regulation of VSMC phenotype,
56
SRF binding to CArG boxes in
VSMC marker gene promoters is blocked by conditions such as
PDGF-BB exposure or vascular injury. Such conditions promote
KLF4-induced myocardin suppression as well as KLF4-induced
recruitment of HDACs, resulting in “closed” deacetylated chromatin and transcriptional repression of VSMC marker genes. Histone
methylation, in contrast, is not affected by PDGF-BB and may serve
as a permanent “memory” for VSMC identity during repression
of SRF-dependent transcription and can, once repressive signals
are terminated, reactivate the differentiation program by recruiting myocardin/SRF complexes or HATs to VSMC marker genes for
reexpression. In the absence of KLF4 activation, SRF/myocardin
can bind to HAT-induced acetylated “open” chromatin at CArG
boxes for transcriptional activation of VSMC marker genes, thus
promoting VSMC differentiation. In addition, myocardin induces
acetylation of histones in the vicinity of SRF-binding promoters in
VSMC marker genes by association with p300, a ubiquitous transcriptional coactivator with its own intrinsic HAT activity, leading
to synergistic activation of VSMC marker gene expression. This promyogenic program is antagonized and repressed by myocardin
binding to class II HDACs, which strongly inhibits expression of
marker genes αSMA, SM22α, SMMLCK and SMMHC. These opposing
actions of HATs and HDACs on SRF/myocardin function to activate or repress, respectively, VSMC differentiation and serve to
regulate transcription in a rapid and reversible manner in response
to dynamic changes in the environment.
Often, transcription mediators play roles in both classic signal
transduction pathways and epigenetic programming.
55
57
Smad pro-
teins, for example, transmit TGF-β signals from the membrane to the
nucleus to mediate gene transcription and VSMC differentiation.
The balance between Smad-induced recruitment of corepressors
or coactivators to TGF-β-responsive genes is associated with activa-
,
tion of HDAC or HAT (p300), which then alters histone acetylation.
Transforming growth factor β induces histone hyperacetylation at the
VSMC marker gene SM22 promoter through recruitment of HATs,
Smad3, SRF, and myocardin, demonstrating a role for HATs and
HDACs in TGF-β activation of VSMC differentiation.
58
A proposed example of metabolic memory stored in the histone
code of VSMCs is found in the dysregulation of histone H3 methylation, an epigenetic mark usually associated with transcriptional
repression in type 2 diabetes.
59
In VSMCs derived from type 2 diabetic db/db mice, levels of H3K9me3 (H3 lysine-9 trimethylation),
as well as its HMT, are both reduced at the promoters of inflammatory
genes. This loss of repressive histone marks, leading to increased
inflammatory gene expression, is sustained in VSMCs from db/db
mice cultured
in vitro, suggesting persistence of metabolic memory. These results suggest that dysregulation in the histone code
in VSMCs is a potential mechanism for increased and sustained
inflammatory response in diabetic patients who continue to
exhibit “metabolic memory” and vascular complications after glucose normalization.
60
Influence of Cell-Cell and Cell-Matrix
Interactions
Many differential VSMC functions are influenced by cell-cell and
cell-matrix adhesion receptors that are altered during phenotypic
modulation and during response to injury or disease. Cell-cell
adhesion receptors include cadherins and gap junction connexins; cell-matrix interactions are dependent upon combinations of
integrins, syndecans, and α-dystroglycan.
Cell-Cell Adhesion Molecules: Cadherins
and Gap Junction Connexins
After investment of VSMCs to the EC layer of nascent vessels, vascular stabilization, also known as maturation,
the sphingosine 1-phosphate (S1P) receptor S1P1, a GPCR on
ECs. S1P1 activates the cell-cell adhesion molecule N-cadherin
11
61
is regulated by
31
CH
3
VASCULAR SMOOTH MUSCLE
32
in ECs and induces formation of direct N-cadherin-based junctions between ECs and VSMCs required for vessel stabilization.
To maintain VSMC quiescence within the vascular wall, cadherinmediated cell-cell adherens-type junctions between VSMCs inhibit
VSMC proliferation, possibly by inhibiting the transcriptional activity of β-catenin, a component of the Wnt signaling pathway, which
CH
interacts with the intracellular domain of cadherins.
3
β-catenin or stabilization of cadherin junctions in VSMCs may be
62
Inhibition of
useful in treating vascular disease or injury.
Another type of direct intercellular junction between cells
in the vasculature is the gap junction.
63
Gap junctions, formed
by connexin proteins between ECs and VSMCs and between
VSMCs, are intercellular channels that allow movement of metabolites, small signaling molecules, and ions between cells.
the four connexin proteins expressed in VSMCs (Cx37, Cx40, Cx43,
and Cx45), Cx45 is exclusively found in VSMCs, while Cx43 is the
most prominent and is essential for coordination of proliferation
and migration.
63
Homotypic gap junctions between VSMCs coordinate changes in membrane potential and intracellular Ca
heterotypic contacts between ECs and VSMCs at the myoendothelial junction control vascular tone by EC-mediated VSMC hyperpolarization. Notably, expression and/or activity of vascular
connexins are altered in vascular diseases such as hypertension,
atherosclerosis, or restenosis
64
and in diabetes.
63
Cell-Matrix Adhesion Molecules: Integrins
and Syndecans
INTEGRINS
Transmembrane integrin receptors are composed of combinations of α and β subunits, each combination with its own ligandbinding specificity and signaling properties. Integrins link the
ECM with the actin cytoskeleton within VSMCs. The β1 subunit
is the main β subunit in VSMCs
α integrin subunits expressed in VSMCs
11
α5.
Integrin α1β1 is involved in collagen remodeling after
injury, and integrin α5β1 binds to fibronectin (FN) and effects FN
polymerization.
Activation of different VSMC integrins results in expression of
differential phenotypic programs. Beta-1 expression contributes to
maintenance of the VSMC contractile phenotype, whereas integrins
α2β1 , α5β1 , α7β1, and αvβ3 participate in SMC migration indica-
tive of the synthetic phenotype.
injury is reduced by blocking αvβ3, but apoptosis in the injured vessel
is increased, potentially promoting plaque rupture. In addition, neointimal formation is prevented and the VSMC contractile phenotype is maintained by binding of α7β1 integrins to COMP (cartilage
oligomeric matrix protein), a macromolecular ECM protein.
SYNDECAN CORECEPTOR
Syndecans are members of a family of four transmembrane heparan sulfate proteoglycans (HSPGs) consisting of a core protein
covalently coupled with (GAGs).
ceptors with growth factor or adhesion receptors and function to
“tune” extracellular signal transfer across the cell surface to the
cytoskeleton and cytoplasmic mediators to effect activation of a
variety of intracellular signaling cascades. All four syndecans are
expressed in the artery, and VSMC syndecans bind to ECM proteins,
cell adhesion molecules, heparin-binding growth factors such
as fibroblast growth factor (FGF) and EGF, lipoproteins, lipoprotein lipases, and components of the blood coagulation cascade.
Syndecan-1 inhibits VSMC growth in response to PDGF-BB and
FGF2 after vascular injury.
thrombin-induced VSMC migration and proliferation by acting
both as a mediator for bFGF signaling and as a cofactor for fibroblast growth factor receptor 1 (FGFR-1), suggesting that syndecan-4
is an early response gene after injury, whereas syndecan-1 is active
during the proliferative and migratory phase.
in vivo and in vitro; the major
in vivo are α1 , α3, and
11
Neointimal formation after vessel
67,68
Syndecans function as core-
69
Syndecan-4 has been implicated in
68
63–65
2+
Of
, and
66
INSOLUBLE EXTRACELLULAR MATRIX COMPONENTS
61
One of the most important functions of VSMCs is to secrete, organize, and maintain an elaborate ECM architecture, an “extended
cytoskeleton” that varies according to the biomechanical stresses
of the differing vascular beds. Large elastic arteries (e.g., thoracic
aorta, carotid, renal arteries) are characterized by multiple concentric elastic lamellae that distribute cardiac-driven pulsatile stress
evenly throughout the vessel wall. Smaller muscular arteries that
experience less force (e.g., coronary, cerebral, mesenteric) contain only two elastic laminae. Elaboration of the ECM synthesized
and organized by VSMCs is considered to be a major part of their
“differentiated” phenotype
70
because ECM components influence
the same pathways regulated by growth/differentiation factors
(see
Fig. 3-3). Changes acquired by VSMCs during acquisition
of contractile properties are in turn maintained by the ECM in
“dynamic reciprocity” between the matrix and gene expression. In
addition to providing a structural elastic scaffold for the extensible vessels, the ECM regulates gene expression through binding of
matrix receptors on the cell surface and through acting as a reservoir for growth factors such as PDGF and FGF that regulate cell
function (reviewed in
71
).
Extracellular matrix components are classified as fiber-forming
molecules (certain collagens and elastin), non-fiber-forming or
interfibrillar molecules (proteoglycans and glycoproteins), and
matricellular proteins (thrombospondin-1 and -2, secreted protein acidic and rich in cysteine [SPARC/osteonectin], tenascin-C,
and osteopontin) that modulate cell-matrix interactions and tissue
72
repair.
A list of ECM molecules and diseases resulting from ECM
alterations can be found in a recent review
72
(also see Chapter 4).
BASEMENT MEMBRANE
Vascular smooth muscle cells in the intact vessel are surrounded
by a basement membrane composed primarily of type IV collagen and laminin.11 Laminins are basement membrane modular
glycoproteins that interact with both cells and ECM to affect proliferation, migration, and differentiation.
70
Evidence from cultured
VSMCs suggests that laminin induces expression of contractile
proteins and moderates the proliferative response to mitogens
such as PDGF through a mechanism involving the laminin receptor α7β1, which links the basement membrane to the VSMC
contractile apparatus.
3
FIBRONECTIN
Fibronectin is present in developing tissues prior to collagen, and
there is evidence that FN has an organizing role in ECM assembly as a “master orchestrator” for matrix assembly, organization, and
73,74
stability.
Fibronectin binding to α5β1 induces integrin-bound
FN clustering, resulting in activation of actin polymerization, actinmyosin interactions, and signaling through kinase cascades. Thus,
FN modulates VSMCs toward the synthetic phenotype.
COLLAGENS
Differential phenotypic modulation of VSMCs in response to different forms of collagen or to different isotypes of collagen illustrates the importance of cues from the physical and chemical ECM
environment that regulate VSMC physiology in normal and disease
75
states.
Cells cultured on fibrillar vs. monomeric collagen type 1
exhibit very different gene expression profiles, responses to growth
factors such as PDGF-BB, and migration properties.
11
lagen type 1 promotes the contractile phenotype, whereas mono-
12
meric collagen type 1, found in the degraded matrix of vascular
lesions (“atherosclerotic matrix”), activates proliferation,
contractile gene expression, and promotes a VSMC inflammatory
phenotype with increased VCAM-1 expression.
75
Vascular smooth
muscle cells also exhibit different phenotypic profiles depending upon contact with different collagen isotypes: collagen type
IV, a component of the basement membrane surrounding VSMCs
12
Fibrillar col-
76
reduces
(“protective” matrix), promotes expression of contractile proteins
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
by regulating the SRF coactivator myocardin expression and mediating recruitment of SRF to CArG boxes in αSMA and SMMHC
promoters.
75
ELASTINS AND ELASTIN-ASSOCIATED PROTEINS
Elastic fibers are composed of tropoelastin, fibrillin-1, and
fibrillin-2 and are assembled and deposited in a tightly regulated,
hierarchical manner.
77,78
They provide not only unique elastomeric properties to the vessel wall but also influence phenotypes
of VSMCs, directly through adhesion and indirectly through TGFβ signaling,
77
to regulate migration, survival, and differentiation.78
Elastin maintains the quiescent, contractile phenotype of VSMCs
by specifically regulating actin polymerization and organization
via a signal transduction pathway involving Rho GTPases and
their effector proteins.
79
Mechanical injury or inflammation that
results in focal destruction of insoluble elastin into soluble elastinderived peptides induces VSMC dedifferentiation and migration.
Elastin-derived peptides can activate cyclins/cyclin-dependent
kinases, leading to cell cycle progression and proliferation found
in neointimal formation.
FIBRILLINS
Fibrillins are large cysteine-rich glycoproteins that serve dual roles:
(1) providing stability and elasticity to tissues and (2) sequestering
TGF-β and BMP complexes in the ECM to limit their bioavailability,
providing for spatial and temporal growth factor signaling during
remodeling or repair.
in Marfan syndrome, a heritable disease associated with disorganized elastic fibers in defective aorta and excess TGF-β signaling.
80,81
Mutations in the fibrillin-1 gene are found
78
FIBULINS
78
Fibulins are elastic fiber–associated proteins.
Vascular smooth
muscle cells from fibulin-5 null mice exhibit enhanced proliferation and migration, indicating an inhibitory role for fibulin-5 in
VSMC response to mitogenic stimuli.
82
Vascular smooth muscle
cell- specific deletion of the fibulin-4 gene results in large aneurysm
formation exclusively in the ascending aorta and down-regulation
of SMC-specific contractile proteins and transcription factors for
SMC differentiation. Thus fibulin-4 may serve a dual role in both
elastic fiber formation and SMC differentiation, and therefore may
protect the aortic wall against aneurysm formation
in vivo and
may also maintain an ECM environment for VSMC differentiation.
Fibulin-2 and fibulin-5 double knockout mice have vessels that
exhibit disorganized internal elastic lamina and an inability to
remodel after carotid artery ligation-induced injury,
83
which was
not observed in single knockout mice for fibulin-2 or fibulin-5.
These data suggest that fibulins 2 and 5 function cooperatively to
form the internal elastic laminae and protect vessel integrity.
EMILINS
EMILINs (elastin microfibril interface-located proteins) act as an
extracellular negative regulator of TGF signaling.
mice (Emilin1
blood vessels, altered elastic fibers,
resistance, causing hypertension.
[−/−]
) exhibit inhibition of cell proliferation, smaller
85
and increased peripheral
84
These data indicate a role for
84
EMILIN null
EMILIN in elastogenesis, maintenance of VSMC morphology, and—
importantly—in blood pressure control.
GLYCOSAMINOGLYCANS, PROTEOGLYCANS,
AND MATRICELLULAR PROTEINS
Glycosaminoglycans in the vascular ECM, including heparin and
the related heparan sulfate, inhibit VSMC migration and proliferation. Heparin also induces expression of contractile markers for
maintenance of the differentiated phenotype.
GAG chains, the proteoglycans,
86
which include syndecan trans-
3
Proteins bearing
membrane HSPG and perlecan basement membrane HSPG,
interact with FN in matrix assembly.74 Different proteoglycans can
have opposing effects on VSMCs: the HSPG perlecan inhibits VSMC
proliferation and intimal thickening by sequestering FGF-2,
while versican, a chondroitin sulphate proteoglycan, promotes
VSMC proliferation.
as endothelin-1 and Ang II stimulate elongation of GAG chains on
the proteoglycan core proteins.
87
Vasoactive agents acting through GPCRs such
88
These elongated GAG chains
12,69
exhibit enhanced binding to low-density lipoprotein (LDL), providing a mechanism for atherogenic lipid retention in the vessel
wall. Finally, matricellular proteins (e.g., thrombospondins, tenascins, SPARC), are thought to be “antiadhesive proteins” with effects
on VSMC migration and adhesion.70 CCN (cysteine-rich protein, Cyr
61/CCN1) is a family of secreted matricellular proteins that mediate cellular responses to environmental stimuli through interaction with a variety of cell surface proteins and adhesion receptors
including Notch receptors and integrins.
lated in the VSMCs of injured arteries, stimulates VSMC proliferation
through CCN1/α6β1 integrin interactions.
89
CCN1, which is up-regu-
90
Knockdown of CCN1
in injury models suppresses neointimal hyperplasia. In contrast,
CCN3 protein inhibits VSMC proliferation in a TGF-β-independent
manner by increasing the CDKI p21, partly through Notch signaling, thus suppressing neointimal thickening.
91
These contrast-
ing roles for pro-proliferative CCN1/α6β1 integrin signaling and
antiproliferative CCN3/Notch signaling in VSMCs offer therapeutic
strategies for reducing neointimal hyperplasia.
91
MATRIX METALLOPROTEINASES AND TISSUE
INHIBITORS OF MATRIX METALLOPROTEINASES
Matrix metalloproteinases are zinc-containing enzymes that, along
with extracellular proteases in the plasminogen activation system, induce remodeling of VSMC cell-matrix and cell-cell interactions (reviewed in
92–94
) and release ECM-bound growth factors,
cytokines, and proteolyzed ECM fragments, or “matrikines,” with
cytokine-like properties into the ECM. Members of the MMP family found in vascular tissues (listed in Ref. 95) include interstitial collagenases, basement membrane gelatinases, stromelysins,
matrilysins, and membrane type (MT)-MMPs and metalloelastase
(see Chapter 4). In the vascular wall, production of pro-MMP-2,
MMP-14, and TIMP-1 and -2 is constitutive,
96
while other MMPs can
be induced by inflammatory cytokines (interleukin [IL]-1 and
-4 and tumor necrosis factor α [TNF-α]), hemodynamics, vessel
injury, and ROS.
93
In addition, MMPs can act synergistically with
growth factors such as PDGF and FGF-2.
Matrix metalloproteinase induced remodeling of basement
membrane components laminin, polymerized type IV collagen,
and HSPGs promotes a VSMC migratory phenotype. In addition,
MMP cleavage and shedding of non-matrix substrates—in particular, adherens junction cadherins—act to remove physical constraints on cell movement.
integrin signaling from the cell surface to focal adhesions, modulating cell cycle components cyclin D1 and p21/p27 CDKIs.
93
Furthermore, ECM remodeling enables
96
In vascular remodeling, MMP activities are tightly regulated at
several levels: transcriptional level, activation of pro-forms, interaction with specific ECM components, and inhibition by TIMPs.
Modulation of MMP activity is evident in VSMC migration and
neointima formation after injury, plaque destabilization in atherosclerosis, aneurysm formation, hypertension, and coronary restenosis.
95
In atherosclerosis, MMPs have potential either to promote plaque
instability, as in advanced plaques of hypercholesterolemia models,
or to stabilize plaques by increasing VSMC migration/proliferation.
Up-regulation of MMPs in VSMCs may contribute to aneurysm
formation.
3
MECHANICAL EFFECTS
Data on VSMC phenotypic modulation by the mechanical environment indicate that continuous cyclic mechanical strain acting
directly on VSMCs increases collagen and fibronectin synthesis,
possibly by paracrine release of TGF-β1, resulting in increased ECM
remodeling indicative of a VSMC synthetic phenotype.
12
In contrast,
33
CH
3
VASCULAR SMOOTH MUSCLE
34
some studies have shown that mechanical strain can also stimulate
expression of contractile genes.
ways are induced following initiation of cyclic strain, mechanisms
for this induction are unclear. Activation of ion channels and tyrosine kinases, and paracrine release of soluble mediators such as
Ang II, PDGF , and IGF , are postulated to play a role.
CH
Mechanical signals play a role in stimulating cell cycle progres-
3
sion. Actin filament polymerization and organization induced
by integrin ligation generate intracellular mechanical tensional
forces that promote cell cycle progression.
or compliance of the ECM, can direct cellular functions through
integrin-dependent signaling pathways involving FAK, the canonical mediator of integrin signaling, Rho family GTPase Rac and
cyclin D1.
98
3
Although MAPK signaling path-
3
97
In addition, “stiffness,”
Phenotype-Specific Vascular Smooth
Muscle Cell Functions
Contraction
The primary function of differentiated VSMCs is to maintain vascular tone. This is an active process requiring significant energy
expenditure, especially in resistance arterioles. A number of hormones and peptides regulate VSMC contraction, including catecholamines, Ang II, and endothelin-1. Contractions can be phasic,
lasting only minutes, or tonic, depending on the stimulus.
In nearly all cases, stimulation of VSMC with contractile agents
results in activation of a specific GPCR (
res ponse is activation of PLC, which cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP
Fig. 3-6). The immediate
2
) to release
inositol 1,4,5-trisphosphate (IP
turn, binds to its receptor (a channel) on the sarcoplasmic reticulum (SR), creating an open conformation and translocating Ca
) and diacylglycerol (DAG). IP3, in
3
2+
to the cytoplasm. Simultaneously, receptor activation depolarizes
the plasma membrane by altering the activity of pumps such as
the sodium/potassium–adenosine triphosphate (Na
and channels that include Ca
channels.
99
Membrane depolarization leads to activation of voltage-dependent L-type Ca
sustained but less robust elevation of cytosolic calcium. Moreover,
2+
Ca
entry through these channels activates ryanodine receptors
on the SR, further increasing Ca
2+
-sensitive K+ channels and TRP
2+
channels, calcium influx, and a more
2+
release into the cytosol.
+/K+
-ATPase),
The increased cytoplasmic calcium binds to calmodulin
(CaM) at a ratio of four calcium ions to one CaM molecule.
Calmodulin then undergoes a conformational change, and binds
to and activates myosin light chain kinase (MLCK), the enzyme
responsible for phosphorylation of the 20-kD regulatory myosin
light chain (LC20) on serine 19. Activated LC20 facilitates actinmediated myosin adenosine triphosphate (ATPase) activity and
cyclic interaction of myosin and actin,
100
leading to contraction.
Contraction is maintained even when calcium drops, suggesting
that LC20 becomes sensitized to calcium, likely by inhibition of
myosin phosphatase (see later discussion).
101
Because the increase in intracellular calcium caused by
vasoconstrictors is largely responsible for activation of the
contractile apparatus, essential mechanisms exist to limit Ca2+
entry and clear Ca
cluster to release calcium sparks, which in turn stimulate Ca
activated large conductance K channels (BK channels) to cause
hyperpolarization and limit L-type calcium channel activity.
2+
from the cytosol. Ryanodine receptors
2+
102
-
Rho-GTP
Hormones Peptides
GEF
Rho-kinase
LC20
MLCP
P
2
Ca
CaM
MLCK
MLCK
Ca
CaM
GPCR
G
Y
PLC
2
Ca
CaM
ATP
LC20-P Actin
PIP
DAG
2
IP
3
FIGURE 36 Model for contraction
cascade in vascular smooth muscle
cell (VSMC). Binding of contractile
agonists to G protein-coupled receptors
(GPCRs) activates phospholipase C (PLC)
and subsequent PLC-mediated
hydrolysis of phosphatidylinositol 4,5bisphos phate (PIP2) to release inositol
1,4,5-trisphosphate (IP3) and diacylglycerol
(DAG), leading to increased mobilization
of Ca2+. Ca2+ combines with calmodulin
(CaM) and activates myosin light chain
ATP ADP
Actomyosin-P
Contraction
kinase (MLCK)-induced phosphorylation
of myosin light chain (MLC), which,
together with actin, initiates contraction.
In addition, guanine nucleotide exchange
factor (GEF) activation of Rho leads to Rho
kinase stimulation and inhibition of myosin
light chain phosphatase (MLCP), resulting
in enhancement of contraction. ADP,
adenosine diphosphate; ATP, adenosine
triphosphate; GTP, guanosine triphosphate.
(Adapted from Griendling K, Harrison D,
Alexander R: Biology of the vessel wall. In
Fuster V, Walsh R, O'Rourke R, Poole-Wilson P,
editors. Hurst's the heart. 12th ed. New York,
2008, McGraw-Hill, pp 135–154.)
Additionally, the sarcoplasmic reticulum Ca2+-ATPase (SERCA)
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
mediates Ca
extrusion from the cell because the newly taken-up SR Ca
released in a directed manner towards the plasma membrane,
where a plasma membrane Ca
cell. Importantly, SERCA is inhibited by CaM kinase II–mediated
phosphorylation.
Recently, ROS and reactive nitrogen species (RNS) have emer-
ged as effective modulators of contractile signaling.
2+
reuptake into the SR and serves to maximize Ca2+
2+
-ATPase extrudes Ca2+ from the
103
104
Specifically,
2+
is
high levels of ROS oxidize SERCA, thereby inhibiting its activity.
Hydrogen peroxide applied externally increases IP
mediated release of Ca
2+
into the cytosol, and activation of NADPH
oxidases by contractile agonists sensitizes the IP
Ryanodine receptors are also redox-sensitive. S-nitrosylation acti-
receptor-
3
receptor to IP3.
3
vates them, and exposure to endogenous levels of ROS and RNS
can protect these receptors from inhibition by CaM at high concentrations of calcium. Both hydrogen peroxide and super oxide
can stimulate Ca2+ entry via L-type or T-type calcium channels
(including TRP channels), but S-nitrosylation by nitric oxide (NO)
is inhibitory. Thus, in general, ROS and RNS inhibit Ca
activate Ca
lular Ca
2+
entry and release, resulting in an increase in intracel-
2+
concentration.
2+
pumps and
Myosin light chain phosphatase (MLCP) is also a vital regulator
of vascular contraction. It is a multimeric enzyme composed of
a regulatory myosin-binding subunit (MYPT1), a catalytic subunit
(PP1c), and a 20-kD protein (M20). The activity of MLCP is largely
regulated by Rho kinase-mediated phosphorylation of MYPT1 on
Thr695, either directly or via Rho-kinase activation of ZIP kinase.
101
Myosin light chain phosphatase activity can also be inhibited by
CPI-17 (PKC-potentiated PP1 inhibitory protein of 17 kD), which
when phosphorylated by PKC, acts as a pseudosubstrate, binds to
PP1c, and competes with LC20 for phosphorylation. Inhibition of
MLCP activity enhances contraction, as mentioned, by inducing
2+
Ca
sensitization of the contractile apparatus.
Rho kinase has thus emerged as an important part of the contraction cascade.
106
In addition to its role in enhancing contraction,
105
such as in response to Ang II, it is a major regulator of relaxation.
Its activator, the small-molecular-weight GTPase RhoA, is a target of
NO, which by activating protein kinase G (PKG), inactivates Rho,
thus indirectly inhibiting Rho kinase, increasing MLCP activity, and
inhibiting contraction.
It is noteworthy that paracrine factors such as NO secreted by
neighboring ECs represent the major mechanism of vasorelaxation. Shear stress forces and hormones such as acetylcholine
or bradykinin stimulate ECs to secrete NO, which in turn initiates
VSMC relaxation.
3,107,108
Nitric oxide induces relaxation of smooth
muscle potentially via a number of pathways, the most important
of which depend on its ability to release cyclic guanosine monophosphate (cGMP). It can directly (via S-nitrosylation of cysteine
residues) or indirectly (through PKG) activate BK channels,
thus causing membrane hyperpolarization and reducing influx
through L-type Ca
2+
channels. In addition, PKG phosphorylates IP3
receptor-associated PKG-I substrate (IRAG), which inhibits Ca
release from IP
via S-glutathionylation of SERCA and decreases the Ca
receptors. Nitric oxide also increases Ca2+ uptake
3
2+
109
2+
sensitivity of contractile proteins. This pathway is perturbed in diabetic
animal models, in which high levels of ROS derived from NADPH
oxidase 4 irreversibly oxidize SERCA, rendering it insensitive to
110
NO.
In addition to regulating Ca2+ levels, NO-mediated activation of PKG can phosphorylate PP1c and/or MYPT1 to block
vasoconstrictor-mediated inhibition of MLCP.
Other relaxing factors secreted by endothelial cells include
hydrogen peroxide, prostaglandins, and epoxyeicosatrienoic acids
(EETs). In addition, perivascular adventitial adipocytes (PVAs)
have also been shown to secrete factors that influence contractility (reviewed in
111
). These cytokines, collectively known as adi-
pokines, are both vasoactive and pro- and antiinflammatory, and
include cytokines TNF-α, IL-6, chemokines (IL-8 and monocyte
chemoattractant protein [MCP-1]) and hormones (leptin, resistin,
and adiponectin).
111,112
Proliferation
Vascular smooth muscle cell proliferation is important in early
vascular development and in repair mechanisms in response to
injury. However, excessive VSMC proliferation contributes to pathology, not only in vascular proliferative diseases such as atherosclerosis but also, ironically, as a consequence of the intervention
procedures used to treat these occlusive atherosclerotic diseases
and their complications, including postangioplasty restenosis, vein
bypass graft failure, and transplant failure.
Vascular smooth muscle cell proliferation can be regulated by
myriad soluble and insoluble factors that activate a variety of intracellular signaling pathways such as MAPK or Janus kinase/signal
transducers, tyrosine phosphorylation, and mitogen-activated pro-
114,115
teins.
Regardless of the initial proliferative stimulus, these signaling pathways ultimately converge onto the cell cycle
The four distinct phases of the cell cycle are: (1) Gap 1 (G1) in which
factors necessary for DNA replication are assembled; (2) DNA replication or S phase; (3) Gap 2 (G2) in preparation for mitosis; and
(4) mitosis or M phase. Restriction points in the cell cycle exist at
transitions between G1/S and G2/M. Progression through the cell
cycle phases is regulated by cyclin-dependent kinases (CDKs)
and their regulatory cyclin subunits. Cyclins D/E and CDK2, 4, and
5 control G1, cyclin A and CDK2 control the S phase along with
the DNA polymerase cofactor PCNA, and cyclins A/B and CDK1
control the M phase. Cyclin-dependent kinases such as p27
CIP1
p21
bind to and inhibit the activation of cyclin-CDK complexes
(see
Fig. 3-7). Activities of these enzymes depend upon phosphory-
lation status of CDKs, levels of expression of cyclins, and nuclear
translocation of cyclin-CDK complexes. One regulatory protein
is survivin, which competitively interacts with the CDK4/p16
complex to form a CDK4/survivin complex, thus inducing CDK2/
cyclin E activation and S-phase entry and cell cycle progression.
Transcription factors that transactivate CDKs and CDKIs also mediate cell cycle progression. It is known that p53, GAX, and GATA-6
induce p21
CIP1
expression, leading to G1 phase arrest, and E2F transcription factors control the G1/S transition regulated by the retinoblastoma protein Rb, the product of the rb tumor suppressor gene.
Rb exerts its negative regulation on the cell cycle by binding to
E2F transcription factors, rendering them ineffective as transcription factors. When the Rb/E2F complex is phosphorylated by CDKs
in early G1, the complex is dissociated, leaving E2F available to activate genes required for S-phase DNA synthesis.
ing that the HDAC inhibitor trichostatin A blocks proliferation by
induction of the cell cycle inhibitor p21
protein phosphorylation, leading to subsequent cell cycle arrest at
the G1/S phase.
117
In addition to cell cycle regulatory proteins, telomerase activity
is required for VSMC proliferation. Telomeres are noncoding DNA
TTAGGG repeat sequences at the ends of chromosomes that cap
and stabilize chromosomes against degradation, recombination, or
118
fusion.
including telomerase, that synthesize new telomeric DNA in cells
Associated with telomeric DNA are protein complexes,
with high proliferative potential. Telomerase consists of an RNA
component and two protein components, one of which is telomerase reverse transcriptase (TERT), the catalytic component and limiting factor for telomerase activation. When telomerase expression
is low, telomere attrition with each mitotic cycle results in chromosome shortening and instability, replicative senescence, and
growth arrest. In VSMCs, posttranslational phosphorylation of TERT
is linked to telomerase activation, and levels of telomerase expression and activity correlate with proliferation.
erase activation and telomere maintenance have been associated
with excessive VSMC proliferation in both animal and human
vascular injury and disease;
118
disruption of telomerase activity
reduces this proliferative response.
Growth of VSMC is initiated by exposure of the cells to proproliferative signals. Classical growth factors activate RTKs, either
directly or via GPCR-mediated transactivation.
in VSMCs binding to RTKs include PDGF, bFGF, IGF-1, TGF-β, EG F,
113
116
116
It is worth not-
CIP1
and suppression of Rb
118
Importantly, telom-
116,117
Growth factors
(Fig. 3-7).
KIP1
and
INK4a
117
35
CH
3
VASCULAR SMOOTH MUSCLE
36
CH
3
Cyclin A
CDK1
Cyclin A
CDK2
Cyclin B
CDK1
G
2
PCNA
GPCR
RTKIntegrins
NADPH
Oxidase
FIGURE 37 Model for cell cycle regulation
FAK
Rho
Ras
Rac
in Vascular smooth muscle cells (VSMCs).
Mitogens activate growth factor receptor
tyrosine kinase (RTKs), G protein-coupled
receptors (GPCRs), NADPH oxidase, and integrins
to stimulate extracellular signal regulated
kinase (ERK), phosphatidylinositol 3-kinase
ERK
G
0
ROSPI3K
(PI3K) and Rho/Rac pathways, which converge
onto cell cycle components, especially cyclin
D, to regulate proliferation. Cyclin regulatory
subunits and cyclin-dependent kinases (CDKs)
catalytic subunits form holoenzymes that
are phase-specific for the four phases of the
Cyclin D
M
CDK4,5
p21
p53
GAX
cell cycle: G1, deoxyribonucleic acid (DNA)
replication or S phase, G2, and mitosis or M
phase. Endogenous cyclin-dependent kinase
inhibitors (CDKIs), including p21, p27, and
p57, inactivate cyclin/CDKs and therefore
G
1
Cyclin E
S
CDK2
p27
p57
GATA-6
block cell cycle progression and proliferation.
Other cell cycle regulators include the tumor
suppressor p53 and the transcription factors
GAX and GATA-6 that stimulate CDKI p21
CIP1
and induce cell cycle arrest. Cooperating with
cyclin/CDKs is proliferating cell nuclear antigen
(PCNA) for transition through G1 and S phases.
Cell Cycle Arrest
Senescence
E2F
Hyperphosphorylation of the retinoblastoma
protein (pRb) releases elongation factor 2 F
(E2F), allowing cell cycle progression through
the G1 phase restriction point and expression
of genes required for DNA synthesis. Activation
of p53 or Rb pathways results in cell cycle
arrest and senescence. (Adapted from Fuster JJ,
Transcription of Cell Cycle Regulatory Genes
E2F
PPP
pRb
pRb
Fernandez P, Gonzalez-Navarro H, et al: Control of
cell proliferation in atherosclerosis: insights from
animal models and human studies. Cardiovasc Res
86:254–264, 2010; and Dzau VJ, Braun-Dullaeus
RC, Sedding DG: Vascular proliferation and
atherosclerosis: new perspectives and therapeutic
strategies. Nat Med 8:1249–1256, 2002.)
and hypoxia-inducible factor (HIF), and mitogens that activate
GPCRs include hormones such as Ang II,
LDL. Activation of these receptors stimulates sequential signaling cascades mediated by Ras, p70
MEK/ERK, or MAPKK/p38MAPK, which induce cyclin D1 expres-
115
sion.
Src homology 2–containing protein tyrosine phospha-
15
endothelin, or oxidized
S6K
, Rac/NADPH/ROS, PI3K/Akt,
tase 2 (SHP2), a member of the non-receptor protein tyrosine
phosphatase family, dephosphorylates tyrosine residues on target
proteins in response to growth factors, hormones, and cytokines.
119
In VSMCs, SHP2 is a positive mediator of IGF-1- and LPA-induced
MAPK signaling pathways; SHP2 has negative effects on EGF- and
Ang II-induced Akt signaling, implicating SHP2 in modulating cell
cycle progression, growth, and migration.
An important integration point in growth factor signaling is
mTOR (mammalian target of rapamycin), which regulates protein
synthesis, cell cycle progression, and proliferation.
target of rapamycin is a protein kinase that regulates translation
initiation through effectors p70
S6K
and eIF4E, leading to protein syn-
117
Mammalian
thesis necessary for cell division. Rapamycin, an immunosuppressive macrolide antibiotic, inhibits mTOR downstream signaling
cascades, with reductions in protein synthesis leading to cell cycle
arrest.
116
In VSMCs, rapamycin inhibits the mTOR/p70
S6K
signaling
axis, promotes a VSMC differentiated, contractile phenotype by regulating transcription of contractile proteins, and induces expression of the antiproliferative CDKIs p21
cycle progression.
117
Use of rapamycin (sirolimus)-coated coronary stents is highly effective in reducing the postangioplasty restenosis rate in interventional cardiology.
Ion channels for Ca2+, M g2+, and K+ are also activated by growth
factors and mediate proliferation. Transient increases in Ca
CIP
and p27
120
KIP
to inhibit cell
2+
concentration, together with subsequent Ca2+ binding to its
intracellular receptor CaM, are universally required for prolifera-
121
tion.
The mechanism for the Ca2+ sensitivity of this G1-to-S transition involves the Ca
and activation of CDK2 to promote G1/S transition and VSMC
proliferation (reviewed in
expression of cyclin D1 and CDK4 and decrease activation of
CIP1
p21
and p27
independent pathway.
2+
-dependent binding of CaM to cyclin E
122,123
). Elevated levels of Mg2+ increase
KIP1
through an ERK1/2-dependent, p38 MAPK-
124
Changes in VSMC K+ channel expression profiles and activity are linked to cell cycle progression,
implicating these ion channels as “internal timers” of VSMC
cell division.
intracellular Ca
intermediate-conductance Ca
nels, the predominant Ca
ing VSMCs.
and K
of these Ca
proliferation and attenuates vascular disease/injury–induced
remodeling in rodents.
125
Growth factor–induced release of Ca2+ from
2+
storage organelle activates and up-regulates
126
In addition, voltage-gated K+ channels KV1.3
128
3.4
are up-regulated in proliferating VSMCs. Blockade
V
2+
-activated and voltage-gated K+ channels inhibits
2+
-activated K+ (IKCa)-type K+ chan-
2+
-sensitive K+ channel in proliferat-
129
Signals from insoluble ECM-activated integrins and from soluble growth factor mitogens converge and jointly regulate upstream
cytoplasmic signaling networks to mediate expression of cyclin D1
and cyclin E and associated CDK4/6 and CDK2 in the G1 phase,
the part of the cell cycle most affected by extracellular stimuli.
In addition, joint RTK/integrin complex signaling networks impact
G1 phase regulation by inhibiting p21
CIP1
and p27
KIP1
, resulting in
Rb phosphorylation and induction of E2F-dependent genes, with
progression to autonomous stages of the cell cycle (S, G2, and M)
that are independent of external stimuli.
127
130
As noted previously, Notch proteins are also important
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
regulators of VSMC proliferation (reviewed in
induced repression of p27
sion of p21
CIP1
, as well as up-regulation of Akt signaling, an
KIP1
and Notch3/HRT1-induced repres-
22
). Notch4/HRT-
anti-apoptosis pathway, result in promotion of VSMC proliferation.
Furthermore, Notch1 is critical in mediating neointimal formation and remodeling after vascular injury.
Peroxisome proliferator-activated receptors (PPARs), nuclear
hormone receptors with regulatory roles in lipid and glucose
metabolism, are beneficial in VSMCs by targeting genes for cell
cycle progression, cellular senescence, and apoptosis to inhibit
proliferation and neointimal formation in atherosclerosis and
postangioplasty restenosis (reviewed in
suppresses G1-to-S progression by inducing expression of p16
(a CDKI), thereby inhibiting phosphorylation of Rb.
roliferative effect is mediated by repression of telomerase activity
by inhibiting E2F binding sites in the TERT promoter.
PPAR isotype, PPARγ, also blocks G1-to-S cell cycle transition by
preventing degradation of p27
phosphorylation and suppression of E2F-regulated genes responsible for DNA replication.
KIP1
131
Similar to PPARα, PPARγ also inhib-
its telomerase activity in VSMCs by inhibition of early response
gene Ets-1-dependent transactivation of the TERT promoter.
131
). Activation of PPARα
132
INK4a
This antip-
133
Another
, resulting in inhibition of pRb
131
Thiazolidinediones (TZD), PPARγ agonists used clinically in the
treatment of type 2 diabetes mellitus, decrease VSMC proliferation
and prevent atherosclerosis in murine models of the disease.
Cyclic adenosine 3′,5′-monophosphate (cAMP) and cGMP are
second messengers in myriad signal transduction pathways.
131
134
In
VSMCs, cAMP serves as an antagonist both to mitogenic signaling pathways (by inhibiting MAPK, PI3 kinase, and mTOR signaling axes) and to cell cycle progression (by down-regulating cyclins
or up-regulating CDKI p27
KIP1
). An additional antiproliferative effect
is due to down-regulation of S-phase kinase-associated protein-2
(Skp2) mediated by inhibition of FAK phosphorylation and adhesion-dependent signaling. Skp2 is a ubiquitin ligase subunit that
targets p27
proliferation.
KIP1
for proteasomal degradation, thus promoting VSMC
135
A more recently appreciated pathway that controls VSMC growth
involves miRNAs. The potential involvement of these molecules
was first noted in balloon-injured rat carotid arteries, where several miRNAs, including miR-21, are up-regulated compared with
control arteries (reviewed in
136
). Cell culture models show that
miR-21 is a pro-proliferative and anti-apoptotic regulator of VSMCs,
with target genes phosphatase and tensin homology deleted from
chromosome 10 (PTEN), programmed cell death 4 (PDCD4), and
Bcl-2. miR-21 has opposite effects on PTEN and Bcl-2: overexpression down-regulates PTEN and up-regulates Bcl-2. PTEN modulates
VSMCs through PI3K and Akt signaling pathways, while Bcl-2 mediates its downstream signaling through AP-1.
Finally, cell-cell junctions, as described above for cadherins
and gap junction connexins, and cell-matrix contacts can greatly
influence VSMC proliferation (reviewed in
115
). Normally, resident
VSMCs, surrounded by and binding to polymerized collagen type
1 fibrils through α2β1 integrins, exhibit low proliferation indices,
are arrested in the G1 phase of the cell cycle, and are refractory
to mitogenic stimuli. In this quiescent state, levels of cell cycle
regulatory proteins are modulated to inhibit the G1/S transition:
cyclin E and CDK2 phosphorylation is inhibited, while CDKIs are
up-regulated and suppress cyclin E/CDK2 activity. Additionally,
S6K
p70
, a potent stimulator of mitogenesis and a regulator of
KIP1
p27
, is suppressed. In contrast, VSMCs on monomeric collagen
matrices are responsive to growth factor signals which result in
increased cyclin E–associated kinase activity and cell proliferation.
These differential responses of VSMCs to structurally distinct forms
of collagen type 1 are reflected in the differential regulation of cell
cycle proteins and the differential response to mitogenic stimuli.
Therefore, perturbations or degradation of the collagen matrix, as
found in sites of monomeric collagen in vascular lesions, result in
altered VSMC proliferation, response to mitogens, and neointimal
formation.
76
Migration
Smooth muscle migration is an essential element of wound
repair, but unchecked migration and proliferation can contribute to neointimal thickening and development of atherosclerotic
plaques. A number of promigratory and antimigratory molecules
regulate VSMC migration, including peptide growth factors, ECM
components, and cytokines.
enced by physical factors such as shear stress, stretch, and matrix
stiffness. PDGF-BB, bFGF, and S1P are among the most potent promigratory stimuli in the vascular system. Intracellular signaling cascades initiated by these growth factors act in concert with those
activated by integrin receptor interaction with matrix to mediate
the migratory response. Matrix surrounding the migrating cell must
be degraded by MMPs to allow a pathway into which the cell can
protrude. Important promigratory matrix components include collagen I and IV, osteopontin, and laminin. Matrix interactions can
also be antimigratory, as with the formation of stable focal
adhesions, activation of TIMPs, and heparin.
When a cell begins to migrate, a number of coordinated events
must take place in a cyclic fashion
nisms that regulate migration have mostly been studied in fibroblasts, but recently many have been confirmed in VSMCs. Migration
requires specialized signaling domains at the front and rear of the
cell. When confronted with a migratory stimulus, the cell senses the
gradient and establishes polarity. Plasma membrane in the form of
lamellipodia is then extended in the direction of movement. This
process is controlled by reorganization of the actin cytoskeleton
just under the protruding membrane. New focal complexes are
formed in the lamellipodia via cytoskeletal remodeling and integrin interaction with the matrix. The cell body begins to contract,
powered by engagement and phosphorylation of myosin II, and
focal adhesions in the rear of the cell become detached, leading
to retraction of the “tail” of the cell. Finally, adhesion receptors are
recycled by endocytosis and vesicular transport. Successful migration is thus dependent on proper temporal and spatial activation of many molecules, most of which are related to cytoskeletal
elements.
Much is known or inferred about the signaling mechanisms
activated by PDGF in migrating cells.
PDGFRs, receptor autophosphorylation creates binding sites for
phospholipase Cγ, which mobilizes calcium; PI3K, which forms the
membrane-targeting lipid PIP
Nucleation of new actin filaments at the leading edge is initiated
by binding of nucleation promoting factors verprolin-homologous
protein (WAVE) and Wiskott-Aldrich's syndrome protein (WASP)
to actin-related protein ARP2/3; phosphorylation of the actin binding coronin; and dissociation of actin capping proteins, many of
which are regulated by PIP
promoted by formins (mDia1 and mDia2), which act on the plus
end of actin filaments in coordination with profilin. Regulation
of mDia proteins is largely via conformational changes induced
by the small G-proteins RhoA and cdc42. Profilin increases nucleotide exchange on G-actin monomers, thus enhancing actin
polymerization. Severing of existing actin filaments is a consequence of activation of gelsolin and cofilin, which limit filament
length and initiate turnover of existing filaments. Rac also regulates actin reorganization in the lamellipodium, perhaps by activation of p21-activated kinase (PAK)-mediated phosphorylation
of actin binding proteins. The result of these complicated, coordinated events is protrusion of lamellipodia in the direction of the
detected migratory stimulus (see
Once lamellipodial protrusion has occurred, it is necessary for
the cell to create new contacts with the matrix and dissolve ones
no longer needed. These nascent focal contacts provide traction
for eventual contraction of the cell body and propulsion of the
cell forward.
137
Very little is known about focal adhesion composition in VSMCs, but signaling at focal adhesions is coordinated by
integrin interaction with the matrix, integrin clustering, activation
of a series of protein tyrosine kinases including integrin-linked
137
The extent of migration is also influ-
138
(Fig. 3-8). Signaling mecha-
137
When PDGF-BB binds to
; and Ras, which activates MAPKs.
2
. Extension of new actin filaments is
2
Fig. 3-8).
37
CH
3
VASCULAR SMOOTH MUSCLE
38
Lamellipodium
Trailing Edge
CH
3
Focal Contact
F-Actin
Rac
CaM
Ca
Myosin II
2
Paxillin
PA K
c-Src
MLCK
FAK
Integrins Integrins
Col Col Col
FIGURE 38 Summary of signaling and effectors molecules leading to remodeling of actin cytoskeleton at the leading edge and in focal contacts in
migrating vascular smooth muscle cells (VSMCs). In response to promigratory stimuli and activation of multiple intracellular signaling pathways (details given in
text), cells extend lamellipodia and form new focal contacts, areas of dynamic actin turnover. Coordination of actin dynamics depends upon multiple actin binding
and associated proteins for actin filament nucleation and extension (actin-related protein [Arp2/3], WAVE, Wiskott-Aldrich's syndrome [WASP], mDia, profilin) and
actin filament depolymerization (cofilin) and filament capping and severing (gelsolin), remodeling events regulated by small G-proteins Rho, Rac, and cdc42 and Rhoactivated protein kinase (ROCK). Myosin II activation by Ca2+/calmodulin (CaM)/myosin light chain kinase (MLCK) and p21-activated kinase (PAK) generates traction
forces on the matrix to move the cell forward. In turn, matrix components exert tractile forces by matrix/integrin binding-induced phosphorylation of focal contact
components such as paxillin, focal adhesion kinase (FAK) and c-Src, which induce actomyosin motor protein interaction to move the cell forward. (Adapted from
Gerthoffer WT: Mechanisms of vascular smooth muscle cell migration. Circ Res 100:607–621, 2007.)
LN LN
ROCK
N
RhoA
Leading Edge
WAVE
Arp2/3
WASP
Rac
mDia
cdc42
Promigratory Stimuli:
Growth factors
ECM
F-Actin
Myosin II
Cofilin
Profilin
PDGF-BB
bFGF
S1P
Collagens I and IV
Osteopontin
Laminin
MLCK
CaM
2
Ca
PLCy
Gelsolin
mDia
PDGFR
PIP
PI3K
2
MAPKs
Ras
kinase (ILK), FAK and Src, and interaction with the cortical F-actin
cytoskeleton. Phosphorylation of focal adhesion components
including FAK and paxillin occurs during VSMC migration, as does
turnover of focal adhesion proteins by membrane-type metalloproteinases. Regulation of focal adhesion turnover is also intimately
related to the microtubular network.
The final major event in cell migration is contraction of the cell
body. Similar to contraction in differentiated cells, cell body contraction is initiated through calcium-mediated activation of MLCK
and MLC phosphorylation following matrix interaction. RhoA and
Rho kinase may also play a role because pharmacological inhibition of Rho kinase blocks migration of VSMCs.
suggests that myosin II generates traction forces on the matrix, and
the matrix in turn regulates myosin II activation.
139
Current theory
137
Much research remains to fully understand the mechanisms
underlying VSMC migration, but the potential for identifying new
targets for prevention of restenosis and plaque formation is obvious.
Inflammation
As noted earlier, VSMCs can assume an inflammatory phenotype
that is found primarily in atherosclerotic lesions. These cells are
found in the media of the vessel wall and express both markers
of differentiation and inflammatory genes such as VCAM-1 and
exhibit activated NF-κB signaling.
development of this inflammatory phenotype is oxidized LDL, but
ECs activated by disturbed flow also contribute to inflammatory
changes in VSMC by secreting proinflammatory cytokines.
140
One of the primary stimuli for
14
Oxidized LDL and other cytokines like IL-1β and TNF-α stimu-
late VSMC expression of chemokines such as MCP-1, TNF-α, and
chemokine (C-X-C motif) ligand 1 (CXCL1), as well as adhesion
molecules such as VCAM-1, ICAM-1, and CCR-2, the receptor for
MCP-1. Because many of these molecules activate NF-κB, exposure to one of them often induces the expression of others, resulting in propagation of a positive feedback signaling mechanism
to enhance the local inflammatory response. The end result is
recruitment and adhesion of T cells and monocytes to smooth
muscle cells (SMCs) in the vessel wall.
Proinflammatory gene expression in VSMC, as in other cell
types, is largely a consequence of posttranscriptional regulation
of inflammatory gene expression by the stress-activated protein
kinase p38MAPK and transcriptional regulation by proinflammatory transcription factors such as NF-κB and STAT1/3. Both of
these pathways are activated by ROS, which have been shown to
be increased in inflammatory regions of plaques as a result of
macrophage infiltration as well as direct stimulation of VSMCs by
cytokines. Stimulation of cytokine receptors activates p38MAPK,
which controls proinflammatory protein levels by MAPKAPK-2
mediated phosphorylation of adenylate uridylate–rich elements
(AREs) binding proteins such as tristetraprolin (TTP), thus promoting mRNA stability of TNF-α.
141
Many other inflammatory gene
mRNAs, including MCP-1, IL-1β, IL-8, intercellular adhesion molecule 1 (ICAM-1), and VCAM-1, also contain AREs. It should be
noted that ARE binding proteins can both stabilize and destabilize mRNA: HuR protects ARE-containing transcripts from degradation, but AUF1 destabilizes its targets. p38MAPK can also regulate
inflammatory protein expression by translational regulation via
Данная книга находится в списке для перевода на русский язык сайта https://meduniver.com/
activation of MAPK signal-integrating kinase-1 (Mnk-1), which
phosphorylates the translation initiation factor eIF-4E and
enhances its affinity for the mRNA cap.
142
Transcriptional regu-
lation of proinflammatory gene expression is largely a consequence of activation of the NF-κB pathway. Commonly, the p65-p50
heterodimer is the transactivating factor that binds to NF-κBcontaining elements to increase proinflammatory gene transcription. Regulation of gene expression by STATs is a consequence
of the canonical tyrosine kinase receptor activation of JAK, and
subsequent phosphorylation of STAT followed by translocation to
the nucleus.
Another major environmental factor that contributes to maintenance of the VSMC proinflammatory phenotype is the matrix
milieu in which cells exist. In atherosclerotic plaques, VSMCs begin
to secrete collagen I and collagen III, but also, as a result of NF-κB
activation, express MMP-1, MMP-3, and MMP-9, which degrade collagen fibrils to the monomeric form, thus promoting an inflammatory
phenotype, as evidenced by an increase in VCAM-1 expression.
similar response is seen to osteopontin, which is also increased
in atherosclerosis.
are mediated by binding to specific integrins, most likely α5β1 or
14
αvβ3.
The nonintegrin matrix receptor CD44, which binds to hyal-
143
The effects of these matrix proteins on VSMCs
75
A
uronic acid in the matrix, has also been implicated in the transition
to the proinflammatory phenotype, as shown by its ability to stimulate VCAM-1 expression.
144
Senescence, Apoptosis, and Autophagy
In response to aging and oxidative stress, cells that have accumulated damaged organelles/proteins/DNA due to limitations in DNA
repair or antioxidant mechanisms rely on two processes to avoid
replication and passing the damage to daughter cells: permanently
arresting the cell cycle (senescence), or programmed cell death,
including apoptosis (self-killing) or autophagy (self-eating).
Senescent cells are permanently arrested in the G1 phase of
the cell cycle and exhibit specific senescence-associated markers such as β-galactosidase, heterochromatin foci, and accumulation of lipofuscin granules. Unlike quiescent cells, senescent cells
are not responsive to growth factors.
146
Multiple stresses, including
DNA-damaging radiation or chemicals, mitochondrial dysfunction,
and oxidant stress, can invoke two types of senescence programs:
stress-induced premature senescence (SIPS) and replicative senescence associated with accelerated telomere uncapping or short-
147
ening.
These diverse stimulatory pathways converge onto two
effector pathways: the tumor suppressor protein p53 and the Rb
pathways; p53 is normally targeted to proteasome-mediated degradation by mouse double minute 2 MDM2). Mitogenic stress or
DNA damage suppresses MDM2 activity, resulting in p53-mediated
activation of the CDKI p21 and cell cycle arrest.
145
In the second
pathway, stress or damage activates Rb, which then binds to and
inhibits E2F, a transcription factor required for the G1 phase/S
phase transition to cell cycle progression (see
Fig. 3-7). These two
senescence pathways exhibit cross-talk at the level of p53 and can
overlap death pathways. Senescent cells release degradative proteases, growth factors, and inflammatory cytokines, which impact
on neighboring cells.
In VSMCs, DNA damage caused by ROS (e.g., superoxide, hydrogen peroxide, hydroxyl radicals) incites rapid (within days) SIPS.
There are increased levels of ROS in all diseased layers of an atherosclerotic lesion, particularly in the plaque itself,
cent VSMCs have been identified in injured arteries and in the
intima of atherosclerotic plaques.
148
Many of the changes in senescent VSMCs are reminiscent of
changes indicative in age-related vascular disease, implicating cellular senescence in vascular pathologies.
148
Therefore, a model
for how senescence contributes to vascular disease emerges.
Atherogenic stimuli such as Ang II initially stimulate proliferation,
followed by mitogen-induced SIPS or replicative senescence via
telomere uncapping. Inflammatory cytokine/chemokine release
145
147
and senes-
by senescent VSMCs results in ECM degradation. The decreased
cellularity and increased inflammation contribute to plaque
instability.
148
Senescent VSMCs are also implicated in vascular calcification.
They exhibit enhanced expression of osteoblastic genes such as
alkaline phosphatase (ALP), type 1 collagen, and RUNX-2, while
expression of matrix Gla protein (MGP), an anticalcification factor,
is down-regulated.
149
Apoptosis, the controlled activation of proteases and hydrolases within an intact cell's plasma membrane boundary so that
neighboring cells are not affected and an immune response is
not triggered,
150
is an important mechanism for blood vessel
remodeling during proliferative vascular disease and after therapeutic interventions (e.g., angioplasty/stenting of arteries, vein
bypass graft surgery).
induce proliferative episodes in VSMCs within atherosclerotic
lesions (reviewed in
151
Mitogens such as thrombin or PDGF can
152
). Proliferation is counterbalanced by
death-inducing VSMC apoptosis triggered by a variety of proinflammatory mediators, cytokines, oxidized lipids, and free
radicals produced by immune cells within the plaque. These
proinflammatory mediators activate caspases, components
of the extrinsic death receptor pathway (e.g., Fas/CD95 TRAIL
[TNF-related apoptosis-inducing ligand]), and/or cause intrinsic mitochondrial dysfunction in VSMCs under the control of Bcl
family members (reviewed in
153
).
Interactions among mitogenic, apoptotic, and survival signals
produce a variety of lesion characteristics and determine whether
there is a fragile fibrous cap poised for rupture, a lipid-rich necrotic
core, or a fibrotic and calcified core (reviewed in
152
). High percentages of apoptotic VSMCs within atherosclerotic plaques are one of
the major causes of plaque rupture due to decreased cellularity
in the media and thinning of the fibrous cap. In addition, reduced
phagocytotic clearance of apoptotic VSMCs, resulting in necrotic
VSMCs, and low levels of VSMC apoptosis over extended periods
of hyperlipidemia induce viable VSMC release of IL-6 and MCP-1 to
produce chronic inflammation.
thrombin, promoting coagulation.
154
Apoptotic VSMCs also generate
152
Vascular smooth muscle cell apoptosis has also been associated with other lesion characteristics including inflammation,
calcification, thrombosis, and aneurysms (reviewed in
156
) . In vivo,
VSMC apoptosis causes release of cytokines and MCP-1, recruiting
macrophages. Vascular calcification has been associated with
inorganic phosphate–induced VSMC apoptosis and subsequent
generation of VSMC-derived matrix vesicles that serve as the nidus
for calcification (reviewed in
156
). Statins restore the Gas6-mediated
survival pathway and inhibit VSMC calcification by preventing
apoptosis.
In addition to apoptosis, autophagy, a survival process by
which the cell degrades its own components, such as damaged
organelles or long-lived aberrant or aggregated proteins,
145
contributes to pathology in atherosclerotic plaques. Ultrastructural
analysis of VSMCs in the fibrous cap of advanced plaques reveals
characteristics of cells undergoing autophagic degradation.
157
Because autophagy is a survival mechanism and not a death pathway, VSMC autophagy in the fibrous cap may function in plaque
stability and protection from oxidative stress.
157
If oxidative stress
damages lysosomal membranes, lysosome/autophagic vacuole
fusion is impaired and apoptosis ensues.
Stem/Progenitor Cells
The ability of stem cells to differentiate into a variety of cell types
has led to research on the potential efficacy of using pluripotent
embryonic stem cells as a source of VSMCs for regenerative cellbased therapies and tissue engineering in injury/disease repair.
Research on the role of putative resident adult stem cells in bone
marrow and/or unipotent lineage committed VSMC progenitor
cells within the circulating blood, vascular wall, or other peripheral
tissues in the development of the neointima in atherosclerotic
lesions is also ongoing.
158–161
39
CH
3
VASCULAR SMOOTH MUSCLE
Соседние файлы в папке Библиотека им академика М.И. Перельмана