NF-κB: pivotal mediator or innocent bystander in atherogenesis? (original) (raw)

Oxidative stress. The various risk factors for atherosclerosis, including hyperlipidemia, hypertension, and diabetes, have in common the generation of intracellular oxidative stress. In respiring cells, small amounts of oxygen are reduced to reactive oxygen species. These reactive oxygen intermediates, produced in mitochondria, peroxisomes, and the cytosol, are scavenged by cellular defending systems, including enzymatic and nonenzymatic antioxidants. A state of moderately increased levels of intracellular reactive oxygen species is referred to as oxidative stress. Cells respond to this stress by increasing the levels of antioxidants and altering the intracellular reduction-oxidation (redox) state.

NF-κB is one of the transcription factors that may be controlled by the redox status of the cell (reviewed in ref.9). Indeed, generation of reactive oxygen species may be a common step in all of the signaling pathways that lead to IκB degradation and NF-κB nuclear accumulation. Support for this concept comes from a variety of studies showing that the diverse agents that can activate NF-κB also elevate levels of reactive oxygen species and that chemically distinct antioxidants, as well as overexpression of antioxidant enzymes, can inhibit NF-κB activation. However, a direct role of reactive oxygen species in signaling to NF-κB remains to be proven. It is unlikely that either NF-κB or IκB is directly activated by oxidation. Most of the evidence suggests that oxidants induce, and antioxidants diminish, some aspect of the signaling events leading to the phosphorylation, ubiquitination, and degradation of IκB. Understanding how redox processes influence the NF-κB system is an important unresolved issue that may provide novel mechanistic insights into vascular dysfunction.

Dyslipidemia. As with oxidative stress, the presence of elevated and modified lipoproteins is strikingly associated with atherogenesis. The accumulation of LDL in the arterial intima is an early event in lesion formation and continues as lesions advance. When LDL particles become trapped in an artery, they undergo a progressive oxidation in a localized microenvironment that is relatively sequestered from plasma antioxidants. The nature of the cellular signals that lead to the generation of the oxidized lipids is not resolved, but once oxidized, the LDL particles can be internalized by macrophages by means of the scavenger receptors on the surfaces of these cells. Removal of oxidized LDL is an important part of the protective function of macrophages, because it minimizes the effects of modified LDL on endothelial cells and smooth muscle cells. The internalization of oxidized LDL leads to the accumulation of lipid peroxides and facilitates the accumulation of cholesterol esters, resulting in the formation of foam cells.

Several studies suggest that elevated levels of native LDL, as well as oxidized LDL, act as pro-oxidant signals regulating vascular cell gene expression. Short-term exposure of monocytes to oxidized LDL activates NF-κB and induces the expression of target genes, although longer exposure may suppress these responses (10). Nonmodified (native) LDL and minimally oxidized LDL stimulate endothelial cells to produce a series of NF-κB–dependent chemokines and adhesion molecules (11). Similarly, components of oxidized LDL such as lysophosphatidylcholine induce expression of mononuclear leukocyte adhesion molecules and can activate NF-κB in endothelial cells (12). Production of these chemokines may help expand the inflammatory response by stimulating proliferation of resident macrophages and the recruitment of new monocytes into lesions. Additionally, the inflammatory cytokines present in lesions can increase binding of LDL to endothelium and smooth muscle cells and increase the expression of the LDL receptor, leading to further inflammation.

To study LDL oxidation in vivo, Calara et al. (13) injected human LDL particles into a rat model and observed that these lipoproteins localized in the arterial wall, where they underwent oxidative modification accompanied by an activation of the endothelial NF-κB system and expression of NF-κB–dependent genes (13). Similar studies show that VLDL causes arterial activation of NF-κB and increased expression of NF-κB–dependent genes (14). Collectively, these studies suggest that both LDL and VLDL may promote atherosclerosis in vivo in vascular cells, at least in part, by activating NF-κB in vascular cells.

Oxidized lipids in the vessel wall may regulate vascular cell gene expression by activating PPARs. These receptors are ligand-activated transcription factors belonging to the nuclear receptor family. They function as regulators of lipid and glucose metabolism and affect both cellular differentiation and apoptosis. PPARα stimulates β oxidation of fatty acids, while PPARγ elicits adipocyte differentiation and promotes lipid storage. Fatty acids and eicosanoids are natural PPAR ligands, while the hypolipidemic fibrates and the antidiabetic glitasones are synthetic ligands for PPARα and PPARγ, respectively. Components of oxidized LDL may activate PPARγ and regulate macrophage gene expression in ways that lead to foam-cell formation (15). However, several recent findings support the concept that the PPARs also suppress chronic inflammation in the vessel wall. Thus, PPARα-deficient mice exhibit a prolonged response to inflammatory stimuli, and PPAR activators inhibit the expression of a number of proteins (such as IL-2, IL-6, IL-8, iNOS, TNF-α and matrix metalloproteinases) involved in the inflammatory response in vascular cells. Finally, PPARγ-specific agonists inhibit the development of atherosclerosis in LDL receptor–deficient male mice (16). Interestingly, PPARs inhibit the vascular inflammatory response, at least in part, by interfering with multiple steps in the NF-κB signaling pathway.

Hypertension and angiotensin II. Hypertension is an established risk factor for atherosclerosis. Patients with hypertension frequently have elevated levels of angiotensin II (Ang II), the principal product of the renin-angiotensin system. Ang II is a potent vasoconstrictor that may be related to atherosclerosis because of its effect on blood pressure or its effects on smooth muscle cell growth. Additionally, Ang II elicits an inflammatory response in both endothelial cells and vascular smooth muscle cells that appears to be dependent upon the generation of oxidant stress and NF-κB activation. Ang II mediates both the hemodynamic and the inflammatory effects through the Ang II receptor (AT1) (Figure 1). In vascular cells, Ang II generates oxidant stress by the induction of superoxide via NADH oxidase, or by stimulating the generation of reactive oxygen species in the mitochondria. Ang II–induced oxidative stress is associated with NF-κB activation and VCAM-1 induction in endothelial cells (17) and with NF-κB–dependent transcription of IL-6 in vascular smooth muscle cells (18). In the rat vasculature, administration of Ang II activates NF-κB and induces the expression of both IL-6 and VCAM-1 (19). Selective inhibitors of AT1, as well as inhibitors of IκB proteolysis, block these responses. Inflammatory activation of the vessel wall by a dysregulated angiotensin system, potentially mediated by NF-κB, may contribute to the pathogenesis of atherosclerosis.

Diabetes and the effects of advanced glycation end products and the receptor for advanced glycation end products. Diabetes is another major risk factor for atherosclerosis. Diabetes-associated hyperglycemia produces intracellular oxidant stress that can lead to vascular dysfunction. Formation of advanced glycation end products (AGEs) due to elevated nonenzymatic glycation of proteins is accompanied by oxidative reactions that generate oxygen free radicals under hyperglycemic conditions. Once produced, AGEs can alter cellular function by binding to the receptor for AGEs (RAGE) (reviewed in ref.20). Binding of AGEs to RAGE results in intracellular oxidant stress and activation of NF-κB in cultured endothelial cells. Hyperglycemia activates NF-κB and promotes leukocyte adhesion to the endothelium through upregulation of cell surface expression of VCAM-1 and other adhesion proteins (21). Similarly, hyperglycemia induces activation of NF-κB in vascular smooth muscle cells. This may contribute to shifting the tone of the arterial wall, as well as increasing smooth muscle cell proliferation, leading to increased intimal wall thickness. Thus, NF-κB activation is an early event in response to increases in glucose, and may elicit multiple pathways contributing to vascular complications.

Homocysteine and NF-κB activation. Increased plasma levels of homocysteine metabolism are an independent risk factor for atherosclerosis and thromboembolic disease. It is postulated that homocysteine induces endothelial dysfunction, leading to a prothrombotic environment (reviewed in ref.22). Although the mechanisms by which this occurs are uncertain, attempts have been made to establish in vitro model systems to evaluate the effects of homocysteine on cultured cells. This approach suggests that homocysteine creates oxidative stress by altering the redox thiol status of the cell (23). A consequence of these effects is the activation of NF-κB, possibly by a homocysteine-generated reactive oxygen species. Exposure of cultured vascular smooth muscle cells to physiologic levels of homocysteine leads to a marked increase in vascular smooth muscle proliferation in vitro, an effect that is due in part to increased expression of cyclin D1 (22). Activation of NF-κB may contribute to the mitogenic effects of homocysteine, since NF-κB activates cyclin D1 expression (24) and NF-κB activity is important for proliferation of vascular smooth muscle cells. Additionally, homocysteine has proinflammatory effects on both endothelial and smooth muscle cells. Homocysteine increases leukocyte rolling, adherence, and transmigration in mesenteric venules by upregulation of adhesion molecules and suppression of endothelial-derived nitric oxide (NO) (25). This suggests that homocysteine inhibits the important role of endothelial NO in preventing endothelial dysfunction. In cultured aortic smooth muscle cells, homocysteine leads to both an increase in NO production and a NF-κB–mediated increase in the expression of inducible NO synthase (iNOS) (22). Upregulation of iNOS in smooth muscle cells may generate oxidative stress and contribute to the inflammatory response that characterizes the atherogenesis associated with hyperhomocysteinemia.

Infectious agents. Atherosclerosis is considered to be a chronic inflammatory disease of the arterial wall. There is increasing interest in the possible role of microbial agents as stimuli that trigger the arterial inflammation. There is a correlation between atherosclerosis and the presence of at least two classes of microorganisms: herpesviruses, such as cytomegalovirus (CMV), and Chlamydia pneumonia (reviewed in ref.26). Briefly, there is seroepidemiologic evidence that supports the involvement of both types of infectious agents in atherosclerosis, and preliminary findings that treatment with antibiotics might reduce recurrent coronary events. In studies localizing these infectious agents to human arterial lesions, both types of organisms are identified in atheromas in coronary arteries, as well as in other vessels. Additionally, both pathogens can produce lesions in experimental animals and can infect vascular cells, generating products that cause both endothelial and smooth muscle cells to become dysfunctional. In endothelial cells these changes result in increased expression of leukocyte adhesion molecules, cytokines, and procoagulants; smooth muscle cells are stimulated to proliferate and produce cytokines.

CMV infection is associated with atherosclerosis, as well as restenosis and allograft vasculopathy. In vascular smooth muscle cells, CMV infection generates intracellular reactive oxygen intermediates and activates NF-κB (ref.27; see also Hiscott et al., this Perspective series, ref.28). These initial signaling events may be necessary for expression of viral components and viral replication. Additionally, virally induced NF-κB may increase the expression of cellular genes, such as cytokines and the adhesion molecule ICAM-1, that are involved in the inflammatory response. In endothelial cells, CMV infection is associated with a modulation of adhesion molecule expression (29). Additionally, herpesviruses can act as prothrombotic agents by activating the coagulation cascade (reviewed in ref.30). These virally mediated changes may exacerbate the host inflammatory response associated with atherosclerotic lesion formation.

There is provocative evidence for Chlamydia as a trigger of arterial inflammation. The chlamydial heat shock protein 60 (HSP 60) is found in human atheroma (31). It activates NF-κB and induces the expression of a series of leukocyte adhesion molecules in endothelial cells and stimulates the production of TNF-α and matrix metalloproteinase expression in macrophages. Thus, consistent with the hypothesis that NF-κB plays a key integration role in atherosclerosis, some microbial products activate NF-κB and initiate a series of dysfunctional changes in vascular cells that can contribute to the pathophysiology of the atheroma.