The Role of DAMPs and PAMPs in Inflammation-mediated · increasing inflammation and in the development; progression and transition of stable to unstable plaque and binding of HMGB-1

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The Role of DAMPs and PAMPs in Inflammation-mediated

Vulnerability of Atherosclerotic Plaques

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0664.R2

Manuscript Type: Review

Date Submitted by the Author: 29-Dec-2016

Complete List of Authors: Rai, Vikrant; Creighton University School of Medicine, Department of Clinical & Translational Science Agrawal, Devendra; Creighton University School of Medicine, Department of Clinical & Translational Science

Is the invited manuscript for consideration in a Special

Issue?:

IACS Sherbrooke 2016 special issue Part 2

Keyword: Inflammation, Atherosclerotic Plaque, DAMPs, PAMPs, Plaque Vulnerability

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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The Role of DAMPs and PAMPs in Inflammation-mediated Vulnerability of Atherosclerotic

Plaques

Vikrant Rai and Devendra K. Agrawal

Department of Clinical and Translational Science, Creighton University School of Medicine,

Omaha, NE 68178

*Corresponding author

Devendra K. Agrawal, Ph.D. (Biochem), Ph.D. (Med. Sciences), MBA, MS (ITM), FAAAAI,

FAHA, FAPS, FIACS

Professor and Chairman, Department of Clinical & Translational Science

The Peekie Nash Carpenter Endowed Chair in Medicine

Senior Associate Dean for Clinical & Translational Research

Creighton University School of Medicine

CRISS II Room 510, 2500 California Plaza

Omaha, NE, 68178, USA

Tel: (402) 280-2938

Fax: (402) 280-1421

E-mail: [email protected]

Running title: DAMPs, PAMPs and plaque vulnerability

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Abstract

Atherosclerosis is a chronic inflammatory disease resulting in the formation of the

atherosclerotic plaque. Plaque formation starts with the inflammation in fatty streak and

progress through atheroma, atheromatous plaque, and fibroatheroma leading to

development of stable plaque. Hypercholesterolemia, dyslipidemia, hyperglycemia are the

risk factors for atherosclerosis. Inflammation, infection with viruses and bacteria, and

dysregulation in the endothelial and vascular smooth muscle cells leads to advanced plaque

formation. Death of the cells in the intima due to inflammation results in secretion of

damage-associated molecular patterns (DAMPs) such as high mobility group box 1 (HMGB1),

receptor for advanced glycation end products (RAGE), alarmins (S100A8, S100A9, S100A12,

and oxidized low-density lipoproteins), and infection with pathogens leads to secretion of

pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides, lipoteichoic

acids, and peptidoglycans. DAMPs and PAMPs further activate the inflammatory surface

receptors such as TREM-1 and TLRs and downstream signaling kinases and transcription

factors leading to increased secretion of pro-inflammatory cytokines such as tumor necrosis

factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (IFN)-γ and matrix metalloproteinases

(MMPs). These mediators and cytokines along with MMPs render the plaque vulnerable for

rupture leading to ischemic events. In this review, we have discussed the role of DAMPs and

PAMPs in association with inflammation-mediated plaque vulnerability.

Keywords: Inflammation; Atherosclerotic arterial plaque; DAMPs; PAMPs; Oxidized-LDL;

Plaque vulnerability

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1. Introduction

Atherosclerosis, a chronic disease characterized by the deposition of excessive lipids

in the arterial intima is associated with inflammation, hyperglycemia, hyperlipidemia, and

dysregulation of the angiotensin system (Xu et al. 2016). Aggregation and oxidation of the

deposited lipids in the intima leads to the generation of modified lipid moieties (minimally-

oxidized lipid, oxLDL) further causing the chronic stimulation of the innate and adaptive

immune response. Induction of endothelial cells and vascular smooth muscle cells leads to

expression of adhesion molecules, chemoattractants, and growth factors. Interaction of

these factors with receptors on monocytes results in homing, migration, and differentiation

of these monocytes into macrophages and dendritic cells, and secretion of pro-

inflammatory cytokines involving the toll-like receptors (TLRs) (Bentzon et al. 2014) (Figure

1).

Atherosclerotic plaques consist of thick sclerotic collagen-rich fibrous cap and a lipid-

rich atheromatous mass. Increases in plaque size, intra ‑ and extracellular lipid

accumulation, intra-plaque rupture, and inflammation renders the stable plaque unstable.

Vulnerable plaques are described as the plaque prone to rupture and susceptible for

thrombus formation and ischemic events (Butcovan et al. 2016). Infiltration of the

inflammatory cells and secretion of the pro-inflammatory cytokines enhances the immune

response and inflammation in the plaque. Increased level of inflammation in the plaque has

been associated with plaque vulnerability (Ridker and Luscher 2014). Vulnerable plaques

are characterized by the presence of a thin inflamed fibrous cap, a lipid core, necrotic core,

increased neovascularization, inflammation, changes in vessel media and adventitia,

intraplaque hemorrhage, and positive vascular remodeling (Otsuka et al. 2014; Virmani et al.

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2006). Further, the serum inflammatory markers and the oxidized LDL, cholesterol,

apolipoprotein B, homocysteine levels and the matrix metalloproteinases (MMPs) levels

have been discussed as the serum markers of vulnerabilities (Benedek et al. 2016; Chan and

Watts 2006; Koening et al. 2011; Minamisawa et al. 2016; Mittal et al. 2014; Sreckovic et al.

2016).

Increased inflammation and lipid accumulation render the plaque unstable. Exposure

of the macrophages to apoptotic bodies and necrosis in the plaque resulting in the necrotic

core formation leads to the release of damage-associated molecular patterns (DAMPs) such

as high mobility group box 1 (HMGB1). Increased HMGB1 and pro-inflammatory cytokines

secreted from pro-inflammatory cells feedbacks the cycle and further increases the pro-

inflammatory cytokines and HMGB1 (Butcovan et al. 2016). Increased HMGB1 enhances the

inflammation through TLRs, receptor for advanced glycation end products (RAGE), triggering

receptors expressed on myeloid cells (TREM)-1, and increased pro-inflammatory cytokines

production such as interleukin (IL)-6, IL-1β (beta), and tumor necrosis factor alpha (TNF-α)

(Andersson and Tracey 2011; Pelham and Agrawal 2014) (Figure 2). Since increased

inflammation renders the stable plaque unstable; these mediators of inflammation may play

a potential role in vulnerability of atherosclerotic plaque. The role of TREM-1 and TLRs

involving NF-κB and pro-inflammatory cytokines TNF-α in the pathogenesis of unstable

plaque has been documented (Gargiulo et al. 2015; Rai et al. 2016; Rao et al. 2016a; 2016b).

Along with inflammation, the role of infection of the arterial intima and the presence

of viruses and bacteria and their products has been discussed in association with the plaque

development, progression and vulnerability (Lanter et al. 2014; Rosenfeld and Campbell

2011; Zimmer et al. 2015). Inflammation of the intima leads to damage of the endothelial

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and vascular smooth muscle cells and production of DAMPs such as HMGB-1, RAGE,

endogenous receptor ligand for advanced glycation end products (EN-RAGE), and alarmins

(calgranulins). The deposition of the lipids and oxLDL causes activation of innate and

adaptive immunity leading to increased production of inflammatory cytokines and homing

of inflammatory cells. Infection of the arterial intima with viruses and bacteria results in the

production of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides

(LPS), peptidoglycans, and lipoteichoic acid (Chen et al. 2013; Kim et al. 2013; Thankam et al.

2016) (Figures 2 and 3). Here, we have discussed the role of DAMPs and PAMPs and their

interaction with inflammatory mediators in the pathogenesis of inflammation-mediated

plaque vulnerability.

2. Damage-associated molecular Patterns

2.1 High Mobility Group Box 1

HMGB1 is a highly conserved protein released by the injured or necrotic cells, and by

the immune cells in response to inflammation and infection (Andersson and Tracey 2011).

HMGB1 plays a role in sterile infection as well as in infectious inflammation. Exposure of the

monocytes, macrophages, dendritic cells, endothelial cells, and platelets to pro-

inflammatory cytokines such as TNF-α, IL-1β, and interferon-gamma (IFN-γ), microbe-

associated molecular patterns (MAMPs), and PAMPs leads to the secretion of DAMPs such

as HMGB-1. Further, the secretion of HMGB-1 is increased by a feed-forward loop by the

secreted HMGB-1, and due the damage and apoptosis of the cells in intima and exposure of

the monocytes with apoptotic bodies (Andersson and Tracey 2011). Increased HMGB-1

induces the production of inflammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-6) contributing a

potential role in the progression of atherosclerosis and plaque vulnerability (Su et al. 2015)

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(Figure 2). The development of advanced plaque in the rabbit model of atherosclerosis with

the administration of HMGB-1 and TNF-α suggests the role of pro-inflammatory cytokines

and HMGB-1 in increasing the inflammation and development of advanced plaque (Kim et

al. 2016).

Metabolic syndrome, a low-grade chronic inflammatory state, affects 35% of US

adults and consists of increased blood pressure, high blood sugar, excess body fat around

the waist, and abnormal cholesterol or triglyceride levels. It is associated with increased risk

for cardiovascular disease, and increased expression of the pathogen recognition receptors,

toll-like receptors (TLRs). The increased levels of TLRs (TLR2 and 4) may be due to the

increased circulating levels of PAMP-binding proteins (soluble CD14 and lipopolysaccharide

binding protein), and the DAMPs (HMGB-1) (Jialal et al. 2014; Jialal et al. 2015). The

increased levels of DAMPs and PAMPs contribute to TLRs mediated pro-inflammatory

response, a major factor associated with the pathogenesis of atherosclerosis.

Hypercholesterolemia promotes pro-inflammatory cytokine production and plaque

destabilization by inducing IL-1β, IL-8, TNF-α, S100A8, S100A12 (ENRAGE), and MMP1

through Syk/PI3K and NF-κB signaling (Corr et al. 2016; Gargiulo et al. 2015) (Figures 2 and

3). Hyperglycemia regulates the release of HMGB-1 from leukocytes which facilitates the

thrombus formation (Yamashita et al. 2012). Activated platelets have a potential role in

increasing inflammation and in the development; progression and transition of stable to

unstable plaque and binding of HMGB-1 with platelets involving RAGE further enhance the

inflammation and progression of plaque (Ahrens et al. 2015).

Increased HMGB-1 is associated with induction of matrix metalloproteinases (MMPs)

and matrix degradation resulting in collagen loss and blockade of HMGB-1 results in

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decreased inflammation and MMP activity (Kohno et al. 2012). Increased MMP activity is

associated with plaque vulnerability and blocking MMPs may be a novel strategy and a

promising target for stabilizing vulnerable plaques in patients with carotid stenosis (Rao, et

al. 2014). Further, the TREM-1 (Rao et al. 2016a) and TLR4 (Gargiulo et al. 2015) mediated

regulation of MMPs via TNF-α in carotid plaque vulnerability, and the role of TREM-1,

HMGB-1 and RAGE axis in inflammation has been discussed (Thankam et al. 2016)

suggesting the association of these mediators in inflammation mediated plaque

vulnerability.

2.2 Receptor for Advanced Glycation End-products

RAGE is a pattern recognition receptor and mediates the innate immune response.

RAGE in expressed on immune cells, T and B-lymphocytes, and dendritic cells, and is

involved in immediate inflammatory responses. The ligand for the RAGE is advanced

glycation end products (Kierdorf and Fritz 2013). The non-enzymatic reaction between

reducing sugars with free amino groups on proteins, lipids, or nucleic acids results in the

formation of a heterogeneous group of complex structures called as advanced glycation end

products (AGEs). Further, glucose and (or) fructose metabolism intermediate derived

glyceraldehydes-derived AGEs are called as toxic AGEs (TAGE) and play a role in

development and progression of plaque (Takeuchi 2016). AGE formation and accumulation,

a normal process of aging is accelerated by hyperglycemia in diabetes making it a major risk

factor for atherosclerosis (Xu et al. 2016). Increased concentration of AGEs, oxidative stress,

and inflammation facilitates the lipid deposition leading to development and progression of

atherosclerosis (Figure 2). AGEs play a potential role in the pathogenesis of atherosclerosis

via engagement with RAGE present on the various cells found in the atherosclerotic lesion.

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The binding of the AGEs (commonly carboxymethyl lysine and methylglyoxal-derived

hydroimidazolone-1) with RAGE results in increased monocyte activation and macrophage

migration, thereby increasing the inflammation in the arterial wall (Heier et al. 2015)

(Figures 2 and 3). Further, AGEs-RAGE engagement also increases the lipid accumulation in

macrophages by regulating cholesterol uptake, esterification, and efflux, thereby increasing

the foam cells and atherogenesis (Xu et al. 2016).

RAGE also increases the lipid deposition and foam cell formation in smooth muscle

cells by smooth muscle cell priming and increasing the uptake of minimally oxidized low-

density lipoprotein (Chellan et al. 2016). Further, as discussed earlier, increased deposition

of lipids, oxidative stress, and inflammation are the characteristics of unstable plaque. RAGE

being a mediator of these factors plays a potential role in the destabilization of plaque.

Vascular smooth muscle cell and endothelial cell dysfunctions are associated with

atherogenesis and unstable plaque, and RAGE plays a role in AGEs-induced vascular smooth

muscle cell (VSMC) dysfunction (Nam et al. 2016). This suggests that RAGE is a potential

mediator of plaque vulnerability.

Endothelial dysfunction is associated with atheroma formation. Endothelial

progenitor cells are essential for re-endothelialization and neovascularization, and the

paucity and dysfunction of these cells have been associated with the pathogenesis of

atherosclerosis (Chen et al. 2016; Yamashita et al. 2012). Hyperactivity of JNK signaling

pathway mediated by AGEs-RAGE engagement results in increased reactive oxygen species

and oxidative stress leading to cellular apoptosis and inhibition of cell proliferation (Chen et

al. 2016). The transition of stable to unstable plaque is also mediated by increased

inflammation by homing of inflammatory cells and pro-inflammatory cytokines.

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Inflammatory mediators such as HMGB1, TLRs, TREM-1, S100A8, S100A9, and S100A12 play

a potential role in increasing inflammation through inflammatory cytokines in the plaque,

thereby increasing the vulnerability (Oesterle and Bowman 2015) (Figures 2 and 3).

RAGE is the receptor for not only AGEs, HMGB-1 (Bangert et al. 2016), TLRs

(Medeiros et al. 2014), and TREM-1 (Zysset et al. 2016) but also for the calcium binding

S100/calgranulin proteins (S100A8, S100A9, and S100A12) (Oesterle and Bowman 2015),

and plays a potential role in inflammation-induced atherosclerosis and destabilization of

plaque (Figure 3). This suggests that RAGE axis is a mediator for inflammation-induced

development and progression of plaque as well as instability. Further, it has also been

documented that soluble RAGE (sRAGE) works as the decoy receptor and decrease the RAGE

signaling, thereby decreasing the inflammation. Increasing levels of sRAGE have been

associated with the protective effect on inflammation but not on arterial wall thickness and

stiffness, suggesting a potential therapeutic target and the role of RAGE in plaque

vulnerability (Heier et al. 2015).

2.3 EN-RAGE (S100A12)

EN-RAGE is a member of the S100 protein (calcium-binding proteins) family (Foell et

al. 2003). EN-RAGE is an endogenously produced inflammatory ligand of the RAGE and Toll-

like receptor 4 and endogenous receptor ligand for AGEs. The binding of RAGE with EN-

RAGE activates inflammatory cascades involving NF-κB and inflammatory cytokines

mediating inflammation and atherosclerosis (Ligthart et al. 2014). The significant association

between the increased levels of S100A12 and atherosclerotic cardiovascular disease

(Shiotsu et al. 2011) and with carotid atherosclerosis (Abbas et al. 2012) has been reported.

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Abbas et al. (2012) reported the association of the highest increase in S100A12 plasma

levels with early symptoms (2 months), whereas only the modest increase in

S100A8/S100A9 plasma levels at 2 to 6 months but not later. The increased mRNA levels of

S100A8, S100A9, and S100A12 were also associated with the carotid plaque development.

Further, the TLR2 and TLR4 activation increases the mRNA expression of S100A8, S100A9,

and S10012 and increases the release of interleukin-1β and interferon -γ from thrombin-

activated platelets leading to significantly enhanced expression of S100A12 (Figure 3). This

suggests the pathogenic role of S100A12 in association with other inflammatory mediators

in the early phase of development.

However, the pleiotropic role of S10012A has also been reported. Higher levels of

S100A12 were found to be associated with plaque rupture and percutaneous coronary

intervention, but no stimulation of pro-inflammatory cytokines and inhibition of MMP-2,

MMP-9, and MMP-3 via Zn2+

sequestration suggest the protective role of S100A12 in

advanced atherosclerotic plaque (Goyette et al. 2009). Contrarily, reduced amount of

infiltrating inflammatory cells, diminished intimal and medial vascular calcification, smaller

necrotic cores, normalization of RAGE expression and delayed type hypersensitivity with a

S100 protein binding immuno-modulatory compound (ABR-215757) by attenuating the

S100A12 expression suggest its protective role on plaque instability, but atherogenic role of

S100A12 (Yan et al. 2013). Furthermore, the strong association between neutrophil-derived

human S100A12 with the increased risk of coronary heart disease suggests the potential

role of S100A12 in atherosclerosis and plaque vulnerability (Ligthart et al. 2014; Liu et al.

2014).

2.4 Alarmins

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2.4.1 S100A8 and S100A9 (calgranulins)

Alarmins S100A8 and S100A9 are the members of calcium-binding protein family

calgranulins. S100A8 and S100A9 are markers of inflammation, and play a potential role in

the pathogenesis of various inflammatory diseases (Vogl et al. 2014). In inflammatory states,

S100A8 and S100A9 are expressed primarily by myeloid cells such as dendritic cells and by

non-myeloid cells such as endothelial cells and vascular smooth muscle cells (VSMCs), and

modulate the inflammatory processes through TLR4 and RAGE (Figure 3). LPS increases the

expression of S100A8 and S100A9 in VSMCs. Increased levels of S100A8 and S100A9 have

been associated with increased cardiovascular events and atherosclerosis (Averill et al.

2012). The accumulation of AGEs is associated with the increase in S100A8, S100A9, and IL-

1β expression thereby increasing the inflammation (Nakajima et al. 2015). Further, the

increased expression of S100A8 and S100A9 on monocytes, macrophages, and the human

arterial wall has been associated with atherogenesis and rupture prone-plaque (Ionita et al.

2009; McCormick et al. 2005). Increased levels of S100A8 and S100A9 in the area of

atherosclerotic plaque and thrombus are believed to be derived from the activated platelets

(Lood et al. 2016).

Unstable plaque is characterized by the thin fibrous cap. Thinning of the fibrous cap

is mediated by the degradation of the collagen content of the extracellular matrix by the

matrix metalloproteinases (MMPs) resulting in plaque rupture and ischemic events (Butoi et

al. 2016; Newby 2015; Rao et al. 2015). Further, it has also been documented that S100A8

and S100A9 are the inducers of MMPs (Moz et al. 2016; Schelbergen et al. 2012; van Lent et

al. 2012) (Figure 3). Hence, increased alarmins with age and inflammation in the arterial

plaque may induce MMPs and may mediate the collagen loss and plaque rupture. These

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results suggest that S100A8 and S100A9 increases inflammation and induces MMPs

mediating thinning of the fibrous cap and plaque rupture. Higher levels of these alarmins

have been correlated with plaque rupture (Ionita et al. 2009; Schiopu and Cotoi 2013; Xia et

al. 2016). However, studies have also reported that S100A8 and S100A9 inhibit MMPs

(Isaksen and Fagerhol 2001) and may suppress innate immune response in dendritic cells

(Averill et al. 2011). Therefore, further studies are needed to correlate the level of alarmins

and MMPs activation in stable plaque rendering them unstable. Furthermore, higher levels

of alarmins have been associated with plaque vulnerability, and studies are needed to

determine the predictive power of alarmins in at-risk patients (Averill et al. 2011; Ionita et

al. 2009; Isaksen and Fagerhol 2001; Schiopu and Cotoi 2013; Xia et al. 2016).

2.4.2 Oxidized low-density lipoprotein (oxLDL)

Oxidized LDL is a marker of cardiovascular disease and correlates with its risk and

severity (Fraley and Tsimikas 2006). The oxLDL increases the accumulation of cholesterol

within the foam cells of atherosclerotic lesions (Itabe et al. 2011). Oxidized-LDL enhances

the endothelial dysfunction and foam cell formation and increases the atherogenesis

involving the monocytes, macrophages, and mast cells through increased secretion of TNF-α

and histamine (Chen and Khismatullin 2015; Cipolletta et al. 2005; Singh et al. 2002; Zhu et

al. 2005). Mast cell granules also increase the development of foam cell by its binding with

LDL and increasing the uptake of LDL by macrophage. Reduced progression of

atherosclerosis, lipid deposition, and recruitment of T-lymphocyte and macrophage into the

plaque in LDLr and mast cell-deficient (LDLr−/−

KitW-sh/W-sh

) mouse (Sun et al. 2007) suggest

the role of mast cell-LDL interaction in the progression of atherosclerosis. These studies also

suggest that mast cell deficiency markedly reduces the progression of atherosclerosis. The

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co-activation of macrophage and mast cell by higher circulating levels of oxLDL enhances the

risk of atherosclerosis (Chen and Khismatullin 2015). Since oxLDL can be formed in the liver

as well as in the atherosclerotic lesion, the circulating oxLDL and the oxLDL produced in the

plaque affect the plaque vulnerability differently (Itabe et al. 2011). Niccoli et al. (2007)

reported that neither oxLDL (recognized by monoclonal antibody mAb-4E6) nor

malondialdehyde-modified LDL (recognized by monoclonal antibody mAb-1H11) in the

plaque plays a role in plaque instability; however circulating oxLDL may play a role in plaque

vulnerability. Similar to oxLDL, oxidized-cholesterol was also found to be more

hypercholesterolemic and atherogenic than cholesterol in hamster (Ng et al. 2008).

Hypercholesterolemia, dyslipidemia, and inflammation are risk factor for

atherosclerosis and plaque vulnerability (Gupta et al. 2016). Dyslipidemia is also associated

with the increased expression of inflammatory mediator TREM-1 in advanced plaque (Zysset

et al. 2016). Further, increased TREM-1 expression is associated with unstable plaque and

plaque vulnerability. This suggests that dyslipidemia enhances the inflammation in plaque

through increased surface expression of TREM-1 leading to increased monocytes homing,

production of pro-atherogenic or pro-inflammatory cytokines, and foam cell formation, and

thus enhanced the plaque vulnerability (Rai et al. 2016; Rao et al. 2016a; Zysset et al. 2016)

(Figures 2 and 3). Increased expression of TREM-1 on matured dendritic cells has been

correlated with plaque instability (Rai et al. 2016), and DCs and activated T cells have been

co-localized in atherosclerotic plaque. Further, suppression of the atherogenic potential of

the oxLDL induced DCs and limitation of the pro-inflammatory cytokine (TNF-α, IL-1β, and IL-

6) production with statins suggest the role of oxLDL in atherogenesis and plaque

vulnerability (Frostegard et al. 2016). Similarly, decreased expression of TNF-α and oxLDL-

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induced inflammatory cytokines with soluble-TREM-1 suggests the potential role of TREM-1

and oxLDL in coronary artery disease and atherosclerosis (Dai et al. 2016).

TREM-1 amplifies the TLR4 signaling and enhances the inflammation and immune

reaction (Pelham and Agrawal 2014). TLRs participate in lipid deposition and enhanced

inflammation in atherogenesis. The expression of TREM-1 and TLR4 in macrophage can be

increased by oxLDL leading to increased pro-inflammatory cytokine production. Inhibition of

TREM-1 with short-hairpin RNA and synthetic polypeptide LP-17 leads to decreased

secretion of pro-inflammatory cytokines, suggesting the potential role of oxLDL induced

TREM-1 and TLR4 in atherosclerotic plaque development, progression, and vulnerability (Li

et al. 2016). The role of oxLDL mediated TLR4 activation in macrophages, and association of

increased expression of TLR4 with increased inflammation suggests the correlation of TLR4,

oxLDL, inflammation, and atherosclerosis (Xu et al. 2001). Since, increased expression of

inflammatory mediators (TREM-1, TLRs, and inflammatory cytokines), dyslipidemia, and

inflammation is also associated with unstable plaque; activation of TLR4 with oxLDL may be

correlated with plaque vulnerability (Miller 2005; Rai et al. 2016; Rao et al. 2016a; 2016b).

3. Pathogen Associated Molecular Patterns

Along with inflammation, infection of the intima may also play a role in the

development, progression and plaque destabilization. The presence of bacteria and viruses

in the atherosclerotic area has been documented (Lanter et al. 2014; Rosenfeld and

Campbell 2011; Zimmer et al. 2015). Direct infection of vascular cells, production of

endotoxins, modulating the immune response, secretion of pro-inflammatory cytokines or

acute phase proteins, destruction of the intima or activation of the mediators of

inflammation by these viruses and bacteria are the possible mechanism contributing to

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atherogenesis (Lanter et al. 2014; Rosenfeld and Campbell 2011; Zimmer et al. 2015).

Further, the presence of bacteria as biofilm proximal to the internal elastic lamina and in

association with fibrous tissue has been found, and rupture or dispersion of this biofilm is

associated with the increased propensity of plaque rupture (Lanter et al. 2014). Chronic

inflammation plays a potential role in atherogenesis and increased inflammation in the

plaque induces the plaque instability. HMGB-1 secretion from the necrotic core and by

positive feedback enhances this process through secretion of the pro-inflammatory

cytokines. HMGB-1 expression and release can also be increased by bacterial endotoxins

lipopolysaccharide, a known pathogen-associated molecular patterns (PAMPs) (Chen et al.

2013). As discussed above, the interaction of HMGB-1 with TREM-1, TLRs, and RAGE result

in increased secretion of pro-inflammatory cytokines, inflammation, and plaque

vulnerability. Further LPS exposure increases expression of HMGB-1 and inflammation

rendering the plaque more vulnerable (Jaw et al. 2016) (Figure 3). Increased TREM-1

expression on macrophages with LPS, TLR4 mediated TREM-1 expression, LPS mediated

increased TLRs expression, and involvement of HMGB1-RAGE axis and LPS binding protein in

increasing inflammation suggests the potential role of LPS in enhancing the expression of

inflammatory mediators (Arts et al. 2011; Huebener et al. 2015; Murakami et al. 2007; Park

and Lee 2013; Pelham and Agrawal 2014; Wu et al. 2012) (Figure 3). Since these mediators

are involved in the pathogenesis of plaque instability, LPS may play a potential in plaque

destabilization.

An aberrant vascular smooth muscle proliferation result in plaque formation,

however, VSMCs regeneration is essential for the repair of the plaque area (Bennett et al.

2016). Reduced regenerating power of VSMCs renders the plaque unstable and is a

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characteristic feature of unstable plaque. VSMCs apoptosis, cell senescence, and VSMC-

derived macrophage-like cells may promote inflammation, progression, and destabilization

of the plaque (Bennett et al. 2016). VSMCs associated plaque progression and instability is

mediated by the DNA damage products called double-stranded breaks, which promotes cell

senescence, apoptosis, and inflammation (Bennett et al. 2016; Gray et al. 2015). Activation

of Na+/H

+ exchanger-1 by LPS via Ca

2+/calpain results in apoptosis of VSMCs apoptosis and

atherosclerosis and inhibition of Na+/H

+ exchanger-1 leads to attenuation of LPS-accelerated

atherosclerosis and promotes plaque stability, suggesting the potential role of LPS in plaque

destabilization (Li et al. 2014).

Rupture of the plaque is mediated by the destruction of the extracellular membrane

by MMPs (Newby 2008). Newby (2015) reviewed the role of MMPs in atherosclerosis and

concluded the stabilizing effect of MMPs on intimal thickening leading to plaque

stabilization, as well as the destabilizing effect on the plaque by extracellular matrix and

collagen degradation (Newby 2008; Newby 2015). Further, LPS induced stimulation of

monocytes and production of MMP-1 and MMP-9 has been documented in association with

the ischemic events and plaque destabilization (Speidl et al. 2004). Furthermore, both TLR4

and TREM-1 are mediators of inflammation and the role of LPS in enhancing the MMP-9

expression through TLR/NF-κB stimulation, and TREM-1 expression through TLR4 dependent

pathway, in the context of atherosclerosis has been reported (Li et al. 2012; Zheng et al.

2010). The correlation of increased expression of MMP-1, and MMP-9, TREM-1, and

decreased expression of collagen I and collagen III through involvement of TNF-α, epidermal

growth factor, Ets-1, NF-kB, p38-MAPK, JNK-MAPK, and PI3K in relation to plaque

destabilization has also been documented (Rao et al. 2014; Rao et al. 2015; Rao et al. 2016a)

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(Figure 3). These results suggest the potential role of LPS in carotid plaque destabilization

via stimulation of these mediators of inflammation.

The role of PAPMs such as lipoteichoic acid and peptidoglycans has also been

discussed in the literature in relation to the atherogenesis and the plaque vulnerability (Kim

et al. 2013; Laman et al. 2002; Schoneveld et al. 2005). Lipoteichoic acid suppresses the LPS

induced inflammation in the plaque by attenuating the expression of MMP-9, COX-2, Bax,

HSP27, TLR4 and CCR7 through downregulation of NF-κB along with inhibiting the

monocyte/macrophage infiltration (Kim et al. 2013). Peptidoglycan present in

atherosclerotic plaque is a functional LPS analog which induces pro-inflammatory cytokine

production and MMPs via binding to CD14 on macrophages. Significantly increased numbers

of the peptidoglycan positive cells have been correlated with the vulnerable plaque (Laman

et al. 2002). Peptidoglycan stimulates TLR2 and enhances the inflammation and plaque

vulnerability (Schoneveld et al. 2005). Further, amplification of mRNA and protein

expression of IL-6, TLR2 and TLR4 increased the secretion of IL-6, IL-8, and nuclear

translocation of NF-κB in the human umbilical vein endothelial cells incubated with LPS,

lipoteichoic acid, or peptidoglycan in the presence of histamine (Jehle et al. 2000; Talreja et

al. 2004). These findings suggest a synergy between PAMPs and endogenously secreted

histamine in activating the endothelial cells resulting in enhanced production of

inflammatory cytokines (Jehle et al. 2000; Talreja et al. 2004) (Figures 2 and 3).

Endothelial dysfunction plays a crucial role in plaque development and vulnerability

(Raveendran et al. 2011; Tan et al. 2007). In response to increased inflammation,

endothelial cells may also enhance secretion of the inflammatory cytokines and activation of

mediators of inflammation, thereby contributing to plaque progression and rupture

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(Raveendran et al. 2011; Tan et al. 2007). The synergy between endogenously released

histamine and LPS in increasing the inflammation via increased mRNA expression of H1

receptors resulting in increased production of prostaglandin I2, prostaglandin E2 and IL-6 by

endothelial cells has been reported. This effect was partly attributed to histamine-induced

expression of TLR4 (Raveendran et al. 2011) (Figures 2 and 3). Production of prostaglandin

I2 and prostaglandin E2 can also be increased by stimulating the cyclooxygenase-2 through

LPS and histamine (Tan et al. 2007). These studies suggest that histamine release in

endothelial cells increases inflammation and may contribute to plaque vulnerability. The

role of flagellin-induced activation of NADPH oxidase-4 involving TLR5 and production of

hydrogen peroxide has been discussed. This lead to increased secretion of pro-inflammatory

cytokines and adhesion molecule leading to increased inflammation, reduction in the

adhesion and transendothelial migration of monocytes, and atherosclerosis (Kim et al.

2016). Since monocytes and macrophages play a potential role in inducing inflammation and

plaque destabilization, flagellin-induced inflammation may play a potential role in plaque

vulnerability (Kim et al. 2016).

4. Conclusion

Atherosclerotic plaque development, progression, and vulnerability are affected by

inflammation and infection by viruses and bacteria and their products. Inflammation and

infection in the fatty streak result in homing of inflammatory cells, increased apoptosis and

necrosis, and increased lipid deposition leading to secretion of DAMPs and PAMPs, and

inflammatory cytokines involving surface receptors and downstream signaling molecules

(Lanter et al. 2014; Rosenfeld and Campbell 2011; Thankam et al. 2016; Zimmer et al. 2015)

Increased secretion of the inflammatory cytokines and matrix metalloproteinases mediate

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the transition of stable plaque to unstable, thus increasing the plaque vulnerability (Rao et

al. 2014; Rao et al. 2016a; Rao et al. 2016b). As discussed, these mediators of inflammation

serve as the markers for the assessment of the extent of plaque vulnerability as well as

potential therapeutic targets for the vulnerable plaque. Decreased secretion of

inflammatory cytokines and morphological features of unstable plaque with blocking

HMGB1, the use of sRAGE and sTREM1, and TREM-1 blocking with LP17 and short hairpin

RNA suggest the potential therapeutic options. The pleiotropic effect of EN-RAGE and oxLDL

(plaque vs. circulatory), differential expression of S100A12 and S100A8/A9 with time has

been discussed but needs further research. Further understanding the effect of DAMPs and

PAMPs on VSMCs and endothelial cells, analyzing the protective effect of lipoteichoic acid

by attenuating LPS action, and elaborating the effects of plaque and circulatory oxLDL would

be therapeutically beneficial.

Acknowledgement

This work was supported by research grants R01 HL112597, R01 HL116042, and R01

HL120659 to DK Agrawal from the National Heart, Lung and Blood Institute, National

Institutes of Health, USA. The content of this review article is solely the responsibility of the

authors and does not necessarily represent the official views of the National Institutes of

Health.

Financial and competing interests’ disclosure

The authors have no other relevant affiliations or financial involvement with any

organization or entity with financial interest or financial conflict with the subject matter or

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materials discussed in the manuscript apart from those disclosed. No writing assistance was

utilized in the production of this manuscript.

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Figure legend

Figure 1: Pathogenesis of the development of atherosclerotic plaque. Fatty streaks are the

initial lesions in the process. The inflammation and oxidative stress in the fatty streaks and

lipid deposits lead to the formation of atheroma with increased VSMCs proliferation and

homing of inflammatory cells. Innate immune response results in the production of

inflammatory cytokines and increased inflammation in atheroma. Apoptosis and necrosis of

the cells result in secretion of damage associated molecular patterns (DAMPs), which

further activate the inflammatory mediators resulting in the formation of necrotic core.

Thus, atheroma progresses through atheromatous plaque to fibro-atheromatous plaque

with increased inflammation and inflammatory cytokines. This leads to thining of fibrous

cap, endothelial dysfunction, angiogenesis, VSMCs proliferation and collagen damage

resulting in the destabilization of the plaque to become unstable.

oxLDL - Minimally oxidized-low density lipoproteins; RBCs - red blood cells, VSMCs -

vascular smooth muscle cells.

Figure 2: Progression of atherosclerotic plaque. Increased deposition of the oxLDL and

AGEs induces the innate immune response resulting in increased homing of inflammatory

cells and secretion of inflammatory cytokines, such as IL-6, TNF-α, IFN-γ and IL-1β, leading to

formation of apoptotic bodies and necrotic core and enhanced inflammation in plaque area.

LPS, bacterial endotoxins and endogenously released substance such as histamine also

enhance inflammatory cytokines and other mediators of inflammation. HMGB-1 released

from these dying cells activates the inflammatory cascade through TREM-1, TRLs and RAGE

leading to enhanced inflammation, inflammatory cytokine secretion, activation of MMPs,

collagen loss, angiogenesis and hemorrhage. VSMCs proliferation, calcification, necrotic core

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and thining of the fibrous cap render the plaque vulnerable to rupture. AGEs - Advanced

glycation end products, HMGB-1 - high mobility group box-1, IL-6 – interleukin-6, IL-1β –

interleukin-1 beta, IFN-γ- interferon-gamma, LPS - lipopolysaccharides, ox-LDL - minimally

oxidized-low density lipoproteins, MMPs - matrix metalloproteinases, RAGE - receptor for

advanced glycation end products, TREM-1 - triggering receptor expressed on myeloid cells,

TLRs - toll-like receptors, TNF-α - tumor necrosis factor- alpha, and VSMCs - vascular smooth

muscle cells.

Figure 3: Role of DAMPs and PAMPs in inflammation-mediated plaque vulnerability.

HMGB1 secreted from necrotic cells and from the interaction of apoptotic bodies with

inflammatory cells interacts with RAGE, TREM-1 and TLRs to activate them. Activation of

these inflammatory receptors initiates a downstream signaling pathway involving NF-κB

resulting in increased secretion of inflammatory cytokines such as IL-6, TNF-α, IFN-γ and IL-

1β. This leads to further increased inflammation, activation of MMPs, collagen loss and

plaque rupture. Accumulation and oxidation of AGEs and their interaction with RAGE and

EN-RAGE, and accumulation of alarmins S100A8 and S100A9 and interaction with TLRs

enhance inflammatotory cytokine production and plaque vulnerability. LPS-induced HMGB-

1, TREM-1, TLRs, and alarmins and peptidoglycans-induced activation of TLRs also increase

inflammatory cytokines and plaque vulnerability. AGEs - Advanced glycation end products,

EN-RAGE - endogenous receptor for advanced glycation end products, ECM - extra cellular

matrix, HMGB-1 - high mobility group box1, IFN-γ - interferon gamma, IL-6 - interleukin-6, IL-

1β - interleukin- 1beta, LPS - lipopolysaccharides, MMPs - matrix metalloproteinases, oxLDL

- minimally oxidized lo density lipoprotein, NF-kB - nuclear-factor kappa beta, RAGE -

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receptor for advanced glycation end products, TLRs - toll-like receptors, TREM1 - triggering

receptor expressed on myeloid cell1, TNF-α - tumor necrosis factor alpha.

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Apoptotic cell

Adhesion molecules, chemoattractants, and growth factors

Angiogenesis

Endothelial cell

Foam cells

Hemorrhage

Lipids

Monocyte

Macrophages

Necrotic core

oxLDL

Platelets

RBCs

Thrombus

VSMCs

Fatty Streak

Atheroma Atheromatous

plaque

Fibro-atheromatous

plaque

Ruptured

plaque

↑VSMCs

proliferation

↑ Homing of inflammatory cell and secretion of

inflammatory cytokines

Endothelial dysfunction, VSMCs proliferation, impaired repair

and plaque rupture

oxLDL accumulation, release of

chemoattractant and growth factors

Increased inflammation, necrotic core formation,

angiogenesis, and hemorrhage

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Fatty Streak

Atheroma

Atheromatous

plaque

Fibro-atheromatous

plaque

Ruptured

plaque

Deposition and oxidation of lipids (oxLDL) and AGEs

Activation of innate and adaptive immunity

Release of adhesion molecules, chemoattractant and growth factors

Increased homing of inflammatory cells

(monocyte/macrophage)

Apoptosis and necrosis

↑inflammatory cytokines (IL-6, TNF-α, IL-1β, etc.)

↑HMGB-1

Stages of plaque development

Pathogenesis of plaque development and rupture

Activation of inflammatory surface receptors (RAGE, TREM-1, TLR2/4) on

macrophages, endothelial cells and VSMCs

Increased inflammatory cytokines, MMPs, necrotic

core formation, angiogenesis, hemorrhage

Collagen damage, thinning of fibrous cap, calcification,

hemorrhage

LPS/endotoxin/ endogenous substances

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LPS

HMGB1

TREM

-1

TLR

s

RAGE

↑ NF-κB

↑ Inflammatory cytokines (TNF-α, IL-1β, IL6, IL-8, IFN-γ)

↑ Plaque vulnerability

↑ MMPs

AGEs

Collagen and ECM degradation

Cytoplasmic Kinases S100A8 and

S100A9

Lipids

Apoptotic bodies

oxLDL

Vascular smooth

muscle cells

Macrophage

Foam cells

Monocytes

Apoptotic cells

Hemorrhage

Necrotic core

Peptidoglycans

Bacteria

↑ MMPs

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Documents

The Role of DAMPs and PAMPs in Inflammation-mediated · increasing inflammation and in the development; progression and transition of stable to unstable plaque and binding of HMGB-1