Lipopolysaccharide induced vascular smooth muscle cells proliferation

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Received: 14 August 2016 Accepted: 30 December 2016 DOI: 10.1111/cpr.12332

REVIEW ARTICLE

Lipopolysaccharide induced vascular smooth muscle cells proliferation: A new potential therapeutic target for proliferative vascular diseases Dehua Jiang1

| Yu Yang2 | Dongye Li1,2

1 Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou, Jiangsu, China 2

Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China Correspondence Dongye Li, MD, PhD, Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou, Jiangsu, China. Email: [email protected]

Abstract Vascular smooth muscle cells (VSMCs) proliferation is involved in vascular atherosclerosis and restenosis. Recent studies have demonstrated that lipopolysaccharide (LPS) promotes VSMCs proliferation, but the signalling pathways which are involved are not completely understood. The purpose of this review was to summarize the existing knowledge of the role and molecular mechanisms involved in controlling VSMCs proliferation stimulated by LPS and mediated by toll-like receptor 4 (TLR4) signalling pathways. Moreover, the potential inhibitors of TLR4 signalling for VSMCs proliferation in proliferative vascular diseases are discussed. KEYWORDS

vascular smooth muscle cells, lipopolysaccharide, proliferation, signaling pathways

1 | INTRODUCTION

clinical atherosclerosis, are the sources of inflammatory responses. Liu

There is clear evidence that vascular smooth muscle cells (VSMCs)

in rat VSMCs, and the results revealed that Ang II and LPS increased

proliferation is a key step in the progression of atherosclerosis, inti-

TLR4 expression of VSMCs in a concentration-dependent manner. In

mal hyperplasia and hypertension.1 VSMCs can be converted from the

addition, Ang II and LPS contribute to atherosclerosis through a pro-

contractile phenotype to the synthetic phenotype under physiolog-

inflammatory effect. Liu concluded that Ang II induced an inflammatory

ical and pathological conditions, such as vascular injury, mechanical

response partly via a TLR4-dependent signalling pathway in VSMCs.6

has examined whether or not Ang II directly induced TLR4 expression

stretch and growth factor stimulation. The VSMCs phenotypic modu-

Inflammation is a major event in the process of atherosclerosis and

lation is beneficial for VSMCs proliferation and a robust factor in the

restenosis after vascular injury.7 LPS, often referred to as endotoxin

development of proliferative vascular diseases.2

from Gram-negative bacteria, is a mitogen of sepsis. It is composed of

In recent years, it has been shown that VSMCs proliferation in vitro

three structural elements: a core oligosaccharide, an O-antigen and a

is stimulated by multiple growth factors, such as angiotensin (Ang) II,

lipid A. Marshall reported that LPS mediation actions could be modu-

platelet-derived growth factor (PDGF)-BB, oxidized low-density lipo-

lated at multiple levels.8 The LPS from different bacteria do the same

protein (oxLDL), lipopolysaccharide (LPS), transforming growth factor-β

role on cells. High levels of LPS have certain toxic effect on cells, while

(TGF)-β and fibroblast growth factor.3 These factors can interact with

low levels of LPS promote cells proliferation. Epidemiological studies

each other in an in vitro environment. For example, oxLDL promotes in-

have indicated that LPS is a risk factor in disorders, such as atheroscle-

flammatory responses in VSMCs in association with increased expres-

rosis9 and diabetes. For example, Bataller has reported that serum levels

4

sion of toll-like receptor (TLR) 4, and oxLDL acts synergistically with

of LPS can predict mortality in patients with alcoholic hepatitis and are

Ang II in inducing VSMCs proliferation.5 Ang II and LPS, predictors of

associated with a poor response to corticosteroids. Jayashree reported

Abbreviations: VSMCs, vascular smooth muscle cells; LPS, lipopolysaccharide; Ang II, angiotensin II; PDGF, platelet-derived growth factor; oxLDL, oxidized low-density lipoprotein; TLR, toll-like receptor; ERK, extracellular signal-regulated kinase; ApoE−/−, apolipoprotein E-deficient; IL-1, interleukin-1; PI3K/Akt, phosphatidylinositol 3-kinase/Akt; MAPKs, mitogen-activated protein kinase; IRAK, interleukin-1 receptor-associated kinase; IFN-γ, interferon-γ; EdU, 5-ethynyl-2-deoxyuridine; Rac1, Ras-related C3 botulinum toxin substrate 1; JNK/SAPKs, c-jun N terminal kinase/ stress-activated protein kinases; miRNAs, microRNAs; NF-κB, nuclear transcription factor kappa κB; CDKs, cyclin-dependent protein kinases; GK, Goto-Kakizaki; PCNA, proliferating cell nuclear antigen; SsnB, sparstolonin B; TNF-α, tumour necrosis factor-alpha α; PPARγ, peroxisome proliferator-activated receptor γ; siRNA, small-interfering RNA.

Cell Proliferation 2017;50:e12332; wileyonlinelibrary.com/journal/cpr https:doi.org/10.1111/cpr.12332

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T A B L E 1 LPS activates TLR4 and promotes VSMCs proliferation in vitro and vascular hyperplasia in vivo Mode

Material

Stimulator

Results and function

Level

Reference

Human

VSMCs

10 ng/mL LPS

Increased TLR4 expression

in vitro

14

Human

VSMCs

25 ng/mL LPS


in vitro

27

Human

VSMCs

100 ng/mL LPS


in vitro

16

Human

VSMCs

100 ng/mL LPS


in vitro

30

Human

VSMCs

100 ng/mL LPS


in vitro

43

Human

Macrovessels Tissue

1 μg/mL LPS


in vitro

11

Sprague-Dawley rats

VSMCs

10 μg/mL LPS

Increased TLR4 expression Cell proliferation

in vitro

24

Wistar rats

VSMCs

10 μg/mL LPS


in vivo and in vitro

33

Wistar rats

VSMCs

(30 μg/mL LPS/10 ng/ mL) IFN


in vitro

22

Wistar and GK rats

Mesenteric VSMCs

100 ng/mL LPS

Cell proliferation

in vitro

50

Mouse

VSMCs

100 ng/mL LPS


in vitro

14

Rabbits

Abdominal aortas

110 ng/kg LPS

Thickened neointima increased TLR4 expression

in vivo

27

Rabbits

Abdominal aortas

500 ng LPS/rabbits

Intimal hyperplasia

in vivo

15

LPS, lipopolysaccharide; VSMCs, vascular smooth muscle cells; TLR4, toll-like receptor 4.

that circulatory levels and activity of LPS were significantly increased

(PI3K/Akt), mitogen-activated protein kinase (MAPK s) and interleu-

in patients with type 2 diabetes. Particular attention has focused on

kin-1 receptor-associated kinase (IRAKs), and all of these pathways

the link between LPS-mediated inflammation and VSMCs prolifera-

involve cell proliferation. The precise molecular mechanisms by which

10–13

tion.

LPS, a widely used exogenous ligand that activates TLR4, can

promote a pro-inflammatory phenotype of VSMCs14 and induce neoin-

these signalling pathways are involved in VSMCs proliferation induced by LPS are in need of further clarification.

timal formation in vitro and in vivo (Table 1). Yang has reported that LPS induces phosphorylation of extracellular signal-regulated kinase (p- ERK) 1/2 expression, as well as interleukin (IL)-1 and IL-6 production in

2.1 | LPS and the PI3K/Akt signalling pathway

human and murine VSMCs.14 Furthermore, LPS-induced p-ERK1/2 ex-

As a major transducer of mitogenic signals in a variety of cells, Akt is

pression was attenuated in TLR4 signalling-deficient mice. Danenberg

activated by arterial injury and contributes to neointimal formation.21

has reported that LPS increases neointimal formation in rabbits after

Akt is a main downstream effector of PI3K-mediated cell survival,

15

It

and the PI3K/Akt signalling pathway is required for VSMCs prolifera-

has been reported that TLR4 expression and nitric oxide production are

balloon and stent injury in association with systemic inflammation.

tion. Inhibition of the PI3K/Akt signalling pathway has been shown to

increased by LPS in human aortic VSMCs.16 Moreover, TLR4 is strongly

limit neointimal formation in rat vascular injury models. Hattori22 re-

expressed in human atherosclerotic vessels, and antagonizing TLR4 is

ported that LPS plus interferon-γ (LPS/IFN) stimulation of VSMCs led

− −

shown to reduce atherosclerosis in apolipoprotein E-deficient (ApoE / )

to PI3K/Akt pathway activation. Hattori examined the phosphoryla-

mice.17 Furthermore, downstream effectors of TLR4 have been shown

tion of Akt (p-Akt) and total Akt for 4 hours in VSMCs after LPS/IFN

to participate in VSMCs proliferation.18 Studies involving TLR signalling

stimulation, and showed that expression of p-Akt and total Akt was

have mainly focused on pathways activated in macrophages, mono-

time-dependent. Pre-incubation with LY294002, a PI3K/Akt signal-

19,20

In this review, we will summa-

ling pathway inhibitor, significantly down-regulate p-Akt expression

rize the remarkable effects and molecular mechanisms of LPS in VSMCs

cytes and vascular endothelial cells.

in VSMCs. These data indicated that the PI3K/Akt signalling pathway

proliferation. In addition, the potential inhibitors of TLR4 signalling for

could be activated in VSMCs stimulated by LPS.

VSMCs proliferation in proliferative vascular diseases will be discussed.

Phosphorylation of Ser473 and Thr308 sites is essential for Akt activation, which is involved in VSMCs proliferation associated with

2 | MECHANISMS AND POTENTIAL SIGNALLING PATHWAYS

inflammatory stimuli, such as AngII and PDGF in vitro and mechanical vascular injury in vivo. A previous study has suggested that LPS induced lung fibroblast proliferation through TLR4 signalling and the PI3K/Akt signalling pathway.23 Our previous study has shown p-Akt

It is well-known that LPS-stimulated TLR4 activation involves many

expression after LPS stimulation in rat VSMCs.24 In our experiments,

signal cascade reactions; including phosphatidylinositol 3-kinase/Akt

VSMCs were stimulated with LPS for 24 hours before cell counts and

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the 5-ethynyl-2-deoxyuridine (EdU) staining assays. These results

pre-treatment with SB203580, SP600125 and PD98059. These re-

showed that pre-incubation with LY294002 partially blocked VSMCs

sults suggest that ERK1/2, SAPK/JNK and p38 MAPK are different

proliferation stimulated by LPS. Moreover, Western blotting demon-

regulatory signalling mechanisms. Lin also reported that LPS admin-

strated that the levels of p-Akt (308) and p-Akt (473) protein expres-

istration affects TLR4 expression in vivo. Indeed, intravenous injec-

sion were up-regulated and achieved a peak 60 minutes after LPS

tions of LPS significantly elevated neointimal hyperplasia and TLR4

stimulation. Taken together, these results suggest that the PI3K/Akt

expression in New Zealand White rabbit aortas induced by balloon

pathway mediates VSMCs proliferation after LPS stimulation.

injury compared with the control group. However, whether or not the MAPKs signalling pathway is directly required for VSMCs proliferation stimulated by LPS warrants further investigation.

2.2 | LPS and the Rac1 signalling pathway The previous study has summarized the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) in cardiovascular and cerebrovascular diseases.25 It has been reported that Rac1 promotes pulmonary artery 26

VSMCs proliferation,

and is essential in mediating VSMCs prolifera-

2.4 | LPS and the IRAKs signalling pathway It is well-known that activity of IRAK1 and IRAK4 kinases is indispensable in TLR signalling.31 Barthwal32 reported that IRAK1 mediates

tion induced by PDGF in rats. It has been reported that LPS treatment

VSMCs proliferation and neointimal formation via the protein kinase

rapidly induces the activation of Rac1 in a time-dependent manner in

C-ε-IRAK1-ERK signalling pathway. Our previous studies have inves-

VSMCs.27 Because Rac1 is involved in cell proliferation, we evaluate

tigated the effect of IRAK1 and IRAK4 kinase activities on neointimal

24

whether Rac1 is also required for LPS-induced VSMCs proliferation.

formation in Wistar rats. In in vivo studies, inhibition of IRAK1 and

As seen in our previous study,24 VSMCs were stimulated with LPS for

IRAK4 attenuated neointimal formation and fibrotic remodelling after

24 hours. Cell counts and the EdU staining assays revealed that pre-

injury, which was monitored by elastic-van Gieson staining. Inhibition

treatment with NSC23766, a novel selective Rac1 inhibitor, partially

of IRAK1 and IRAK4 also attenuated cell proliferation as characterized

abolished VSMCs proliferation stimulated by LPS, suggesting that

by EdU incorporation. In in vitro studies, VSMCs were stimulated with

Rac1 mediated VSMCs proliferation. Glutathione-S-transferase pull-

LPS for 24 hours before the EdU staining assay. It was shown that

down assays demonstrated that Rac1 activity is up-regulated after LPS

pre-incubation with N-(2-morpholinylethyl)-2-(3-nitrobenzoylamido)-

stimulation. Moreover, Rac1 activity was essential for LPS-induced p-

benzimidazole, an IRAK1/4 inhibitor, partially blocked VSMCs prolif-

Akt expression. Inhibition of Rac1 partially reduced p-Akt (308) and

eration stimulated by LPS. Furthermore, the IRAK1 and IRAK4 kinase

p-Akt (473) protein expression in VSMCs. These results suggest that

activities were suppressed by pre-incubation with an IRAK1/4 inhibi-

Rac1 signalling pathway mediates LPS-induced VSMCs proliferation.

tor in vivo and in vitro.33–35 These results suggest that IRAK1/4 participates in VSMCs proliferation and neointimal formation. Whether

2.3 | LPS and the mitogen-activated protein kinases signalling pathway

or not the IRAK1 and IRAK4 signalling pathway is directly required for VSMCs proliferation stimulated by LPS via the TLR4 signalling pathway warrants further investigation.

As a classical signalling pathway, MAPKs are involved in cardiovascular disorders.28 MAPKs are phosphorylated on serine and tyrosine residues by dual-specific kinases. The kinase family has three mem-

2.5 | LPS and MicroRNAs

bers, including ERKs, c-jun N terminal kinase/stress-activated protein

MicroRNAs (miRNAs) are small, long chain, non-coding RNAs with

kinases (JNK/SAPKs) and the protein kinase, p38. Downstream effec-

regulatory effects on biological process, such as cell proliferation and

tors of MAPKs, including ERK1/2, JNK1/2 and p38MAPK, are rapidly

cell apoptosis. It has been reported that TLR induces differential ex-

activated by balloon injury in rat carotid arteries.29 The proliferative

pression of miRNAs and immune responses against infections.36 Study

effect of the MAPKs pathway in VSMCs is an increasing concern.

has indicated that the expressions of miRNAs are changeable stimu-

Kim investigated the effects of TLR4 signalling and the involvement

lated by LPS via TLR signalling pathways in mouse macrophages.37 In

of MAPKs in VSMCs induction by LPS.30 Compared with the control

recent years, miRNAs are involved in VSMCs phenotypic modulation

group, the levels of p-ERK and p-p38 MAPK expression were elevated

and proliferation in some diseases.38 Li39 has reviewed that some

in response to LPS stimulation, and the level of p-JNK expression

miRNAs regulate VSMCs functions in atherosclerosis. For example,

was transiently increased at early time points in the LPS-stimulated

miR-21 has been shown to have a role in VSMCs proliferation, and

27

reported that LPS-promoted TLR4 expres-

inhibition of miR-21 decreases the proliferation of VSMCs. Zhang40

sion in VSMCs was associated with activation of intracellular MAPKs

has reported that the expression of ssc-miR-146a-5p and ssc-miR-

groups. Meanwhile, Lin

signalling pathway. Western blotting demonstrated that the levels of

221-5p is up-regulated in pig skeletal muscle after LPS stimulation.

p-ERK1/2, p-SAPK/JNK and p-p38 MAPK were up-regulated after

Wang has reported that the expression of miR-152 is down-regulated

LPS stimulation. LPS-induced TLR4 mRNA expression was reduced by

in LPS-stimulated VSMCs. The over-expression of miR-152 decreases

pre-treatment with SP600125 (an SAPK/JNK inhibitor) or PD98059

VSMCs proliferation stimulated by LPS.41 As new regulators of TLR,

(an ERK1/2 inhibitor), but not by SB203580 (a p38MAPK inhibitor).

further investigations should focus on the potential function of miR-

LPS-induced TLR4 protein expression was significantly reduced by

NAs in VSMCs proliferation induced by LPS.

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2.6 | LPS and other signalling pathways

phenotype-related proteins in VSMCs induced by LPS were investigated. Compared to control Wistar rats, the expression of proliferat-

As a classical transcription factor, nuclear transcription factor kappa B

ing cell nuclear antigen (PCNA), a marker of cell proliferation, was

(NF-κB) activation plays a critical role in LPS-induced VSMCs prolif-

increased, whereas the expression of calponin was decreased in GK

eration.22 Our previous study has involved the TLR4-mediated NF-κB

rats. Mesenteric VSMCs from diabetic GK rats exhibited increased

signalling pathway by microarrays analysis in rats with carotid artery

levels of p-ERK1/2 expression compared to controls. Furthermore,

balloon injury.34 It has been reported that VSMCs proliferation and

LPS enhanced p-ERK1/2 levels to a greater extent in GK rats’ VSMCs

neointimal formation in balloon-injured rat carotid arteries are as-

than controls. In addition, high glucose levels significantly increased

sociated with activation of cyclin-dependent protein kinases (CDKs)

PCNA and decreased calponin expression in mesenteric VSMCs in

and are cyclin-dependent,42 suggesting that CDKs regulated prolif-

a time-dependent manner. These results suggest that mesenteric

erative vascular diseases. James43 compared the genes of quiescent

VSMCs from GK rats undergo phenotypic modulation. However, the

human coronary artery smooth muscle cells with cells treated by LPS.

precise mechanisms underlying VSMCs phenotypic modulation and

Treatment of VSMCs with LPS resulted in up-regulation of several cell

the direct relationship with proliferation in GK rat mesenteric VSMCs

cycle-dependent genes such as CDK5 and CDK10, while several CDK

have not been described. Thus, future experiments are needed to de-

inhibitors such as p27, p21 and p16 were repressed. Whether or not

termine whether or not LPS regulates VSMCs proliferation and rel-

CDKs and CDK inhibitory proteins are directly required for VSMCs

evant signalling pathways in diabetic rats.

proliferation stimulated by LPS warrants further investigation.

In all, we discuss some relevant signalling pathways activated by TLR4 stimulated with LPS in VSMCs. How TLR4 is activated by LPS in

2.7 | LPS and phenotypic modulation

VSMCs is rarely investigated. Lin27 has reported that nicotinamide adenine dinucleotide phosphate oxidase activation, mRNA stabilization

Phenotypic modulation of VSMCs from the contractile phenotype to

and MAPKs signalling pathways play critical roles in LPS-enhanced

the synthetic phenotype has been shown to be regulated in different

TLR4 expression in HASMCs. Our previous study has revealed that

stimulations.44 Bae has described the regulatory mechanism under-

PI3K/Akt and Rac1 are involved in TLR4 expression in VSMCs stim-

lying PDGF-induced phenotypic modulation of VSMCs. It has dem-

ulated by LPS.24 However, whether internalization or translocation of

onstrated that the levels of p-Akt, p-ERK1/2 and p-p38 MAPK are

TLR4 is activated by LPS in VSMCs needs further investigation. By the

up-regulated after PDGF stimulation in VSMCs, and are significantly

way, whether or not LPS activates these signalling pathways via non-

reduced by pre-treatment with U0126 (an ERK inhibitor), LY294002

TLR4 in VSMCs needs to be discussed.

and PD98059 respectively. Pharmacologic inhibition of ERK and Akt blocks PDGF-induced phenotypic modulation of VSMCs. Taken together, PDGF regulates VSMCs phenotypic modulation.45 We re-

3 | POSSIBLE THERAPEUTIC TARGETS

cently published our findings on IRAK4 and modulation of the VSMCs phenotype in diabetic rats.46 The result showed that an IRAK1/4

To date, some experimental animal models and clinical study have

inhibitor increased LPS-mediated smooth muscle 22α protein and

suggested that TLRs have a crucial role in cardiovascular diseases.51

mRNA expression, which define the contractile phenotype of VSMCs.

A previous study has investigated the role of TLR4 on atherosclero-

Whether or not LPS directly regulates VSMCs phenotypic modulation

sis stimulated by LPS in ApoE−/− mice and reported that TLR4 may

involved in VSMCs proliferation is unknown.

be a therapeutic target in atherosclerosis. Thus, regulation of TLR4 signalling may be a novel therapeutic approach for treating these

2.8 | LPS and signalling pathways in diabetic rats

diseases.52 However, it remains uncertain whether treatment with a TLR4 inhibitor may attenuate atherosclerosis and proliferative vas-

We and others have discussed the proliferative effect and underly-

cular diseases. Because TLR4 is predominated in VSMCs stimulated

ing mechanism of LPS on VSMCs in non-diabetic rats. A previous

by LPS, TLR4 has been employed as the primary pharmacological

study has showed that the circulatory LPS levels and activities were

approach in experimental models of VSMCs proliferation and prolif-

increased in patients with type 2 diabetes.47 Maria has reported high

erative vascular diseases. Pharmacological approaches of TLR4 sig-

glucose-induced TLR4 activation in VSMCs.48 Wang49 has concluded

nalling inhibition include TLR4 antagonists, neutralizing antibodies,

that TLR4 may become a new potential pharmacological target for

small molecules, natural chemicals and agents. Table 2 summarizes

treating diabetic retinopathy. Thus, we have detected DNA synthe-

the current applications of TLR 4 inhibition mainly at the experimen-

sis and neointimal formation after catheter balloon injury in Goto-

tal level.

Kakizaki (GK) and matched control Wistar rats. After balloon injury, DNA synthesis and neointimal formation in GK rats were more apparent than in Wistar rats.34 In a recent study, mesenteric VSMCs were isolated from GK rats and age-matched control Wistar rats.50

3.1 | Chemicals Several studies have been performed using inhibitors of TLR4 acti-

Mesenteric VSMCs were synchronized before stimulation with LPS

vation. TAK-242, a small-molecule agent, has been shown to inhibit

for 15 minutes. The effects of VSMCs phenotypic modulation and

LPS-induced inflammation by targeting TLR4 signalling. TAK-242 also

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T A B L E 2 TLR4-targeting therapeutic strategies

Therapeutic strategies

Model and effect

References number

Chemicals A promising therapeutic agent for sepsis

54,55

Reduces macrophages recruitment to atherosclerotic plaques in vivo and murine peritoneal foam cell formation in vitro

56

Attenuates the development of cardiac hypertrophy

57

Reduces myocardial ischaemia-reperfusion injury

58

Prevents LPS-induced loss of vascular contractility

59

Inhibits TNF-α production in cultured VSMCs

60

Sparstolonin B

Inhibits VSMCs proliferation

61

Kolaviron

Decreases VSMCs proliferation

62

Hydroxysafflor yellow A

Attenuates VSMCs proliferation

63

Lithospermic acid

Reduces VSMCs proliferation

64

Atorvastatin

Inhibits phenotype modulation in VSMCs

65

Rosiglitazone

Atenuates intmal hyperplasia ameliorates LPS- induced infammaton in VSMCs

66, 67

Probucol

Suppressed LPS-accelerated atherosclerosis

68

Anti-TLR4 Antibody

Antagonizes inflammatory responses in VSMCs

67

NI-0101

Significantly suppress cytokine release in rheumatoid arthritis patients

69

Inhibits inflammatory responses in VSMCs

67

TAK-242 (CLI-095)

Eritoran

Rapamycin Natural chemicals

Clinical drugs

Antibody

RNAi TLR4 siRNA

inhibits LPS-induced phosphorylation of MAPKs.53 Ii54 has examined

contractility which could be effectively antagonized by eritoran. They

the therapeutic effect of TAK-242 in a mouse sepsis model. TAK-242

concluded inhibition of TLR4 by eritoran might serve as a novel ther-

is a promising therapeutic agent for sepsis and immune-mediated lung

apeutic concept to prevent cardiac dysfunction and hypotension after

injury.55 TAK-242, also known as CLI-095 or resatorvid, is a small-

endotoxemia. Eritoran has failed in treating sepsis, but perhaps it

molecule inhibitor of TLR4 signalling that acts by binding to the intra-

has positive outcomes in proliferative vascular diseases. However, it

cellular domain of TLR4. Wang examined whether CLI-095 attenuated

is still unclear whether eritoran prevents atherosclerosis in vivo and

the progression of atherosclerosis.56 The results showed that CLI-095

LPS-dependent VSMCs proliferation in vitro. Further investigations

reduced macrophages recruitment to atherosclerotic plaques in vivo

regarding the impact of eritoran on proliferative vascular diseases are

and murine peritoneal foam cell formation in vitro. They concluded

necessary.

that CLI-095 was able to alleviate atherosclerotic plaque in ApoE−/−

Rapamycin is an important pharmacologic agent against athero-

mice by reducing foam cell formation. So CLI-095 may be a candidate

sclerosis by inhibiting VSMCs proliferation and neointimal thickening.

as a new anti-proliferative drug. Eritoran, a TLR4 antagonist, potently suppresses TLR4 signalling pathway. Baumgarten57 have demonstrated that application of the

It has been reported based on a clinical study that rapamycin inhibits tumour necrosis factor-alpha (TNF-α) production in cultured VSMCs obtained from saphenous vein segments.60 Therefore, rapamycin may

TLR4 antagonist eritoran attenuates the development of cardiac hy-

reduce in-stent restenosis via attenuation of inflammatory cytokine

pertrophy. Shimamoto reported that inhibition of TLR4 with eritoran

expression in VSMCs. Due to the established efficacy in the preven-

in an in situ murine model significantly reduced myocardial ischaemia–

tion of stent restenosis, rapamycin might be an interesting therapeutic

reperfusion injury.

58

Meyer has tested for eritoran impact on vascular

agent in cardiovascular disorders. However, whether or not rapamycin

contractility.59 The data demonstrated that LPS up-regulated cyto-

can directly treat VSMCs proliferation stimulated by LPS needs further

kine expression via TLR4 and induced attenuation of smooth muscle

investigation.

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VSMCs.65 Meanwhile, peroxisome proliferator-activated receptor γ (PPARγ) agonist attenuates intimal hyperplasia by inhibiting TLR4-mediated inflammation in VSMCs.66 Moreover, Ji67 tested the role of rosiglitazone in regulating LPS-induced vascular inflammation; the results showed that rosiglitazone exerted a potent anti- inflammatory action via decreasing TLR4 and increasing PPARγ in LPS-induced VSMCs. In addition, administration of probucol suppressed LPS-accelerated atherosclerosis and plaque instability in ApoE−/− mice.68

3.4 | Anti-TLR4 antibody and TLR4 small-interfering RNA (siRNA) As the first monoclonal antibody blocking TLR4 signalling, NI-0101 is well tolerated without safety concern in healthy volunteers. Hatterer F I G U R E 1 Possible mechanisms for lipopolysaccharide (LPS) exerting its proliferative effects on vascular smooth muscle cells (VSMCs) by signalling pathways. Multiple signalling pathways can be activated in VSMCs proliferation. LPS can induce signalling pathways such as PI3K/Akt, MAPKs, IRAK1/4 which further promotes the expression of NF-κB for proliferative effects on VSMCs. Further investigation is required to determine whether the mechanisms of proliferative effects of LPS on VSMCs involve CDK, microRNA and phenotypic modulation, which are not shown in the figure. Moreover, whether internalization or translocation of TLR4 is activated by LPS in VSMCs needs further investigation

3.2 | Natural chemicals

reported TLR4 inhibition using NI-0101 in an ex vivo model of rheumatoid arthritis pathogenesis can significantly modulate cytokine release.69 Ji67 has reported that the TLR4 blocker or TLR4 siRNA significantly antagonized the LPS-mediated inflammatory responses in VSMCs. Taken together, whether or not rapamycin, TAK-242, eritoran, PPARγ agonist and atorvastatin can be used directly to treat VSMCs proliferation stimulated by LPS warrants further investigate. It will likely be a long time before these agents, who are currently undergoing testing in animal models, enter human trials.

4 | CONCLUSION AND PERSPECTIVE

Pharmacologic intervention involves multiple signal transduction

Abnormal VSMCs proliferation plays an important role in prolifera-

pathways (mainly PI3K/Akt and ERK). However, which pathway

tive vascular diseases, such as atherosclerosis and restenosis,70 which

plays the major role in anti-proliferative is not determined. Due to

involve all stages of inflammation associated with complex immune

the complexity of signalling pathways involved in regulating VSMCs

responses. LPS is reported to be involved in the modulation of VSMCs

proliferation, it is essential to develop multi-targeted drugs for

proliferation and neointimal formation.71 As a TLR4 agonist, LPS is re-

anti-proliferative effect. As important therapeutic targets, the anti-

ported to be dominated by binding TLR4. Zhang72 has reported TLR4

proliferative effects of drugs have been utilized for inhibiting VSMCs

as potential clinical biomarker for in-stent restenosis in drug-eluting

proliferation. Sparstolonin B (SsnB), a selective TLR4 antagonist with

stent patients, so we figure out that TLR4 may be the main therapeu-

potent anti-inflammatory properties, has protective effects on en-

tic target able to block LPS action.

dotoxin shock. Fan has investigated the role of SsnB in VSMCs pro-

The present in vitro study has demonstrated that LPS induces

liferation and inflammatory responses and found that SsnB reduces

VSMCs proliferation, although the exact mechanism has not been

VSMCs proliferation, and TNF-α and IL-6 expression stimulated by

LPS in VSMCs.61 SsnB significantly inhibited ERK1/2 and Akt signal-

completely elucidated (Figure 1). Whether or not these data obtained via in vitro experiments can explain the mechanism underlying LPS-

ling pathways in VSMCs activated by LPS. Thus, SsnB significantly in-

induced vascular hyperplasia in vivo remains to be determined. Anti-

hibits VSMCs proliferation associated with inflammatory responses.

proliferative approaches by targeting the LPS/TLR4 pathway have

Together, these results suggest that SsnB may be a candidate as a new

shown promising prospects in inhibiting VSMCs proliferation and

anti-proliferative drug. Similarly, Kolaviron,62 Hydroxysafflor yellow

neointimal thickening. These findings further our understanding of the

A63 and lithospermic acid64 also significantly inhibits VSMCs prolifera-

function of LPS in vascular diseases, and suggest TLR4 as a prospec-

tion stimulated by LPS.

tive target for the preventive and therapeutic proliferative vascular diseases.

3.3 | Clinical drugs Atorvastatin can effectively reduce neointimal formation and stent

CONFLICTS OF INTEREST

restenosis via the TLR4/NF-κB signalling pathway. Moreover, Atorvastatin inhibits phenotype modulation of PDGF-BB-induced

The authors declare no conflict of interest.

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ACKNOWLE DG E MEN TS We thank Dr Haochen Xuan for his technical support in developing the figure.

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How to cite this article: Jiang D, Yang Y, Li D. Lipopolysaccharide induced vascular smooth muscle cells proliferation: A new potential therapeutic target for proliferative vascular diseases. Cell Prolif. 2017;50:e12332, https://doi.org/10.1111/cpr.12332