Canonical nuclear factor-kappa B signaling in atherosclerosis
LPS, Lipid A, dsRNA

Proinflammatory cytokines
TNFαIL-1βShear stress

LPS

MyD88

TRADD
RIP1IRAK4TRADD
TRAF2

IRAK1
TRAF6

RIP1 Tab3

IRAK1TRAF6

Tab2 Tab3

MyD88

PI3K

IRAK4

TRAF6

PIP3

Tab2

IKKγP

IKKαP

IKKβNucleus
PIκBαp50

p65

P

IκBα

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PIP3Atherosclerosis involves upregulation of the canonical nuclear factor-kappa B (NF-κB) signaling pathway inseveral vascular cell types throughout evolution of the disease.

κB pathway
The canonical NF-κNF-κB comprises a family of heterodimeric transcription factors that reside in the cytoplasm of every cell andupon activation translocate to the nucleus. The canonical pathway is activated by a wide variety of agentsincluding inflammatory stimuli, pathogens, free radicals, stress, and cigarette smoke. These disparate signaling
pathways ultimately lead to phosphorylation of the IKK complex. This complex is composed of a regulatory subunit(IKKγ , also known as NF-κB essential modifier - NEMO) and two similar subunits (IKKα and IKKβ). Typically, the IKK
complex is activated by phosphorylation of IKKβ, which in turn phosphorylates IκBα. Phosphorylated IκBα is degradedby the proteasome pathway and the resulting liberated NF-κB ( p50/p65 dimer, for example) translocates to the nucleus toregulate the expression of almost 400 different genes (some indicated below).

p50

p65

p50

p50

p65

κB and progression of atherosclerosis
NF-κ
Atherosclerosis also involves NF-κB activation through toll like receptors (TLR4 and TLR2). These receptors of innate immunity recognize multiple ligands including
bacterial lipopolysaccharide (LPS); however, in atherosclerosis they are thought to potentially be activated by oxidized products of LDL. Engagement of TLR4 orTLR2 (not shown) initiates similar signaling events. The cytoplasmic portion of the TLRs is complexed to myeloid differentiation primary response gene 88 (MyD88)
via the adaptor MyD88-adaptor-like (MAL) also called TIR-containing adaptor protein (TIRAP). MyD88 recruits IL-1 receptor-associated kinases (IRAKs) and TNF
receptor-associated factors (TRAFs) into the complex. TLR engagement leads to IRAK4 phosphorylation of IRAK1 and dissociation of an IRAK1/TRAF6 complex
that then recruits a protein kinase complex containing TAK1 (transforming growth factor-β-activated kinase-1) and TAK1 binding proteins (TAB2 and TAB3). TAK1then phosphorylates the IKK complex.

p65NF-κBPPIκBαIKKγ (NEMO)
p65 (Rel A)
p300 (HAT)
MAL (TIRAP)

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USAκB and the onset of atherosclerosis
NF-κ
Atherosclerosis develops at large vessel branch points, where turbulent blood flow activates shear stress sensors of endothelial cells.
One such sensor is thought to be platelet endothelial cell adhesion molecule (PECAM1) in complex with VE-cadherin, β-catenin and vascularendothelial cell growth factor receptor-2 (VEGFR2). Shear stress leads to VEGFR2 activation of phosphatidylinositol-3-OH kinase (PI3K), which produces phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 in turn activates the αVβ3 integrin, which then engages with extracellular matrix proteins containing the RGD
binding site. A second round of PI3K activation and PIP3 production ensues, which ultimately leads to phosphorylation of the IKK complex through an unclear RAC
dependent mechanism. NF-κB activation leads to upregulation of several genes implicated in initiating and perpetuating atherosclerosis including 1) enzymes(group IIa secretory phospholipase A2, 5-lipoxygenase, 12-lipoxygenase, and COX2) that convert lipid from low density lipoprotein (LDL) particles into inflammatory
lipids, 2) chemokines (such as monocyte chemoattractant protein-1 (MCP-1)) that attract monocytes and 3) cell adhesion molecules (intercellular adhesion molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1, E-selectin, and P-selectin), which in part enable the monocytes to adhere to endothelial cells and thentraverse into the subendothelial space. Once in the inflamed vessel wall monocytes differentiate into macrophages under the influence of a NF-κB regulated product,macrophage colony-stimulating factor (MCSF). Differentiated macrophages then engulf oxidized LDL and become foam cells.

IKKγ

IKKβAbcam in Japan

RAC

TAK1

IKKαAbcam in Europe

β3

PI3K

TAK1
TRAF2

αV

β3αV

VEGFR2β-catenin

MAL

IRAK1

VE-cadherin

PECAM1

TLR4

TLR4

MD2

CD14

IL-1RAcP

IL-1R

TNFR1

TNFR1

TNFR1

RGD

Abcam in the USA

p300

Macrophages/foam cells and other inflammatory cells accumulating within the atherosclerotic plaque produce a variety of NF-κB dependent proinflammatory
cytokines that enhance plaque formation (tumor necrosis factor α (TNFα), IL-1β, IL-4, IL-12, interferon-γ (IFNγ)). Of these interleukin 1β (IL-1β), and potentially TNFα
are thought to potentiate the inflammatory process, as these cytokines can also activate NF-κB. IL-1β, binds to a heterodimeric complex comprised of the IL-1 receptor(IL-1R) and IL-1R accessory protein (IL-1RAcP). Similar to TLRs, IL-1β signaling is mediated by TRAF6 activation of TAK1, which phosphorylates the IKK complex.
In contrast, signaling by TNF receptor-1 (TNFR1) leads to formation of a complex comprised of TNFR1 associated death domain protein (TRADD) receptor-interacting
protein (RIP) and TRAF2. These three components in concert with TAB2 and TAB3 activate TAK1 to phosphorylate the IKK complex.

CREBTranscription
p50

p65

Proteasomal
degradation

Other NF-κB dependent genes that may influence atherosclerosis are other chemokines (CX3CL1, CCL10, andplatelet factor 4) that attract inflammatory cells to atherosclerotic lesions, matrix metalloproteinase-9, which may
facilitate migration of inflammatory and smooth muscle cells within the plaque, and tissue factor and plasminogenactivator inhibitor 1 (PAI-1), which convey a procoagulant tone to the plaque.

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Produced in collaboration with Dr. Nicholas W. Shworak, Dartmouth Medical School.

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