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(Circulation. 1998;98:1707-1713.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Nuclear Factor-B Is Selectively and Markedly Activated in Humans With Unstable Angina Pectoris

Michael E. Ritchie, MD

From the Division of Cardiology and Cardiovascular Research Center, University of Cincinnati College of Medicine, and Division of Cardiology, Veterans Administration Medical Center, Cincinnati, Ohio.

Correspondence to Michael E. Ritchie, MD, University of Cincinnati College of Medicine, 231 Bethesda Ave, ML 0542, Cincinnati, OH 45267-0532. E-mail Michael.Ritchie{at}uc.edu

	Abstract

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Background—Nuclear factor-B (NF-B) resides inactive in the cytoplasm of lymphocytes, monocytes, endothelial cells, and smooth muscle cells, where, after stimulation, it transcriptionally activates interleukins, interferon, tumor necrosis factor-, and adhesion molecules. Because acute inflammation may play a role in coronary artery plaque rupture, it was hypothesized that NF-B activation correlated with coronary artery disease (CAD) activity.

Methods and Results—Evidence of NF-B activation in the circulation of 102 consecutive patients without an acute myocardial infarction who were undergoing cardiac catheterization was determined. Of these, 19 had unstable angina (USA) and were within 24 hours of the last episode of chest pain. The remaining 83 were being evaluated for stable angina (53), valvular heart disease (8), atypical chest pain (12), or congestive heart failure (10). Evidence of NF-B activation was determined by electromobility shift assays (EMSAs) with the NF-B binding-site–specific probe and nuclear proteins isolated from the buffy coat of blood obtained at the beginning of the procedure. Specificity of this DNA-protein interaction was confirmed by competition and supershift EMSAs. Analyses showed that 17 of 19 patients with USA had marked activation of NF-B. Despite a significant number of patients with severe CAD (69%), only 2 of the 83 without USA showed marked NF-B activation. A lack of NF-B activation was not due to a lack of functional cell/protein because NF-B was appropriately activated by lipopolysaccharide ex vivo in all patients. NF-B activation was not a nonspecific response of all transcription factors because neither Sp1 or Oct1 was activated in patients with activated NF-B. There was no relationship between drugs used, hemodynamic status, or other clinical characteristics and state of NF-B activation.

Conclusions—These data show that NF-B is specifically and significantly activated in unstable angina pectoris and is not affected by severity of CAD or medical therapy. Furthermore, because NF-B is activated before a clinical event, it may be mechanistically involved in the plaque disruption that produces acute coronary artery syndromes.

Key Words: angina • inflammation • NF-kappa B • coronary disease

	Introduction

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Atherosclerosis is currently considered to be an exaggerated response of the vessel wall to injury characterized by inflammation and fibrocellular proliferation.^{1 2} This view is supported by the demonstration of abundant macrophages and T lymphocytes in atherosclerotic plaques that accumulate because of adhesion molecule expression on monocytes, endothelial cells, and leukocytes.^{3 4 5} Once within the plaque, these cells release growth and chemotactic factors (eg, platelet-derived growth factor and fibrinogen growth factor) and are involved in the local oxidation of products such as LDL, the combination of which leads to smooth muscle cell proliferation and foam cell production.⁶ Plaque-related T cells also express late activation antigens characteristic of an active or chronically active inflammatory state.^{7 8}

Inflammation may also play a role in plaque disruption. A recent study showed that in patients dying of acute myocardial infarction, the immediate site of acute plaque rupture was always marked by an inflammatory response characterized by invasion of macrophages, lymphocytes, and smooth muscle cells expressing the same HLA-DR antigens. This suggested that an acute inflammatory reaction had occurred and that there was "cross talk" between cells.⁹ Other studies have shown that C-reactive protein and amyloid A protein, measures of systemic inflammation, are elevated in patients with unstable angina and are predictive of subsequent unstable coronary artery events in patients with both stable and unstable coronary artery disease.^{10 11 12}

Many of the genes involved in the acute inflammatory response that are pivotal in the atherogenic process are potently activated by the transcription factor NF-B (nuclear factor-B). NF-B resides inactive and bound to the inhibitory protein I-B in the cytoplasm of many cell types, including T lymphocytes, monocytes, macrophages, endothelial cells, and smooth muscle cells.^{13 14 15} Numerous stimulants, including lipopolysaccharide (LPS), interleukin-1 (IL-1), and tumor necrosis factor- (TNF-) alter I-B, causing nuclear translocation of NF-B. Once translocated, NF-B transcriptionally activates genes involved in the immune and inflammatory response: IL-1, IL-6, and IL-8; interferon; TNF-; and endothelial cell adhesion molecule-1, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1. NF-B also regulates expression of the genes encoding itself and I-B and is thus capable of autoregulation. Interestingly, anti-inflammatory levels of acetylated (ASA) and nonacetylated (NSA) salicylic acid in culture block NF-B availability for transcriptional activation.¹⁶ This observation may account for the need for high doses of ASA and the equal effectiveness of similar levels of NSA in the treatment of inflammatory disorders such as rheumatoid arthritis, the additional reduction of coronary artery events in patients taking high-dose aspirin, and the low incidence of coronary artery events in arthritic patients taking salicylates.^{17 18 19 20} Coupled with the view that acute inflammation mediates plaque rupture, these data may also provide the rationale for the reduction in subsequent coronary events associated with ASA use in patients with high levels of C-reactive protein.¹¹

On the basis of these data, it was hypothesized that activation of NF-B mediated coronary atherosclerotic plaque rupture in humans. Results of analyses designed to begin to test this hypothesis are detailed in this article.

	Methods

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White Cell Isolation and Preparation of Nuclear Proteins
Standard methods were used.^{21 22} Four milliliters of blood was withdrawn from a femoral artery and placed in an EDTA-containing tube. Half of the blood was incubated with the known NF-B inducer LPS (100 ng/mL) at 37°C for 45 minutes. The remainder was incubated at 37° without the reagent. Two milliliters of each sample was diluted with 2 mL of Tris-buffered saline (TBS), pH 7.5, poured over 3 mL of Ficoll-Paque (Pharmacia) and centrifuged at 400g (1500 rpm) for 35 minutes at 18°C. Serum was removed and the buffy coat isolated and placed in a separate centrifuge tube. The white cells were resuspended in 10 mL of TBS and centrifuged at 1500g (3000 rpm) for 5 minutes. The supernatant was poured off, and the cells were resuspended in 1 mL of TBS and subjected to 30 seconds of centrifugation in a microcentrifuge. The washed and pelleted cells were resuspended in 400 µL of buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF) and allowed to swell on ice for 15 minutes. Twenty-five microliters of 10% NP-40 was added, and the solution was subjected to a vigorous vortex for 10 seconds. The cells were again centrifuged for 30 seconds, and the pellet was isolated and resuspended in buffer C (20 mmol/L HEPES, pH 7.9, 0.4 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF). This solution was then shaken vigorously for 15 minutes at 4°C. Any precipitated proteins were then removed by centrifugation for 5 minutes at 4°C in a microcentrifuge. The nuclear protein–containing supernatant was removed and quantified by standard Bradford assay.²³

Electromobility Shift Assay
Standard methods of electromobility shift assay (EMSA) analysis were used.^{21 22} Double-stranded oligonucleotides containing the consensus binding sequences of NF-B, Sp1, and Oct1 are commercially available (Promega). Oligonucleotides were radiolabeled with -³²P with the use of T4 polynucleotide kinase by standard methods and purified over a column, and the purified product was quantified by ß-counter.²⁴ Competition assays were performed with a described molar excess of unlabeled NF-B consensus oligonucleotide and an unrelated oligonucleotide of similar length or mutant NF-B oligonucleotide (22-mer). Antibodies for supershift analyses were obtained from Santa Cruz Biotechnology. EMSAs were performed with a 0.5x TBE polyacrylamide gel. Five micrograms of nuclear protein was incubated for 20 minutes at room temperature in a 20-µL solution containing 50% glycerol, 2 µL of Poly dIdC, 2 µL 10x gel mobility shift buffer (200 mmol/L HEPES, 15 mmol/L MgCl, 10 mmol/L EDTA, 600 mmol/L KCl, 10 mmol/L DTT), and a large molar excess of radiolabeled probe with 10 000 to 20 000 cpm activity. This solution was then electrophoresed at 160 mV for 2 hours at 4°C. The gel was removed and dried and an autoradiogram obtained. After a 24-hour exposure, shifted bands were quantified by scanning laser densitometer. As reported, consistent and reproducible use of a molar excess of probe at the same degree of radioactivity exposed to film for a defined period of time results in relatively accurate quantification.^{22 25 26} Values were set as arbitrary units (AU).

Patient Selection and Definitions
All human studies were approved by the University of Cincinnati Medical Center Institutional Review Board, which is responsible for human studies at both the University of Cincinnati Hospital and the Veterans Administration Medical Center of Cincinnati, Ohio. All patients were from the Veterans Administration Medical Center of Cincinnati. Between November 1994 and July 1995, blood samples from 102 consecutive patients undergoing cardiac catheterization were obtained by catheterization laboratory personnel, coded, and transferred to the laboratory, where isolation of nuclear proteins and EMSAs were performed by research personnel who were unaware of each patient's history. Codes were broken after quantification of EMSA autoradiograms. Clinical characteristics evaluated included age; systolic blood pressure and heart rate at time of cardiac catheterization; clinical diagnosis/reason for cardiac catheterization; presence of diabetes mellitus, hyperlipidemia, hypertension, or significant coronary artery disease, defined as angiographically significant (70% stenosis) disease in 1 epicardial artery; and the use of aspirin, heparin, cholesterol-lowering agents, or ACE inhibitors. Unstable angina was defined as chest pain accompanied by ECG changes of ischemia requiring intravenous medical therapy to control symptoms. Blood samples for analyses of the influence of ongoing infection/inflammatory disorders were obtained from patients in the medical intensive care unit or on medical floors who had been diagnosed with bacterial endocarditis, urinary tract infection, pneumonia, bronchitis, alcoholic and infectious hepatitis, cirrhotic liver disease, peptic ulcer disease, bacterial peritonitis, and arthritis.

	Results

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EMSA Detects NF-B Activation In Vivo
Because NF-B can only bind its DNA recognition site after translocation to the nucleus, NF-B activation can be easily and accurately determined by EMSA, a fact that has been successfully exploited by others for in vitro analyses.^{14 15 16} Accordingly, the ability of EMSAs to detect NF-B activation in vivo was determined. As shown in Figure 1, incubation of an NF-B binding-site–specific probe with nuclear proteins isolated from the buffy coat of human blood results in a single shifted band that is detected in unstimulated cells (lanes 3 and 7) and is markedly induced by LPS (lanes 1 and 5). The identity of this band as NF-B was confirmed by competition and supershift (lanes 2 and 6) analyses.

View larger version (56K): [in this window] [in a new window]   Figure 1. EMSA with site-specific probe and nuclear proteins isolated from the buffy coat of human blood. Probes for the NF-B (N) and the Oct1 (O) binding sites were used. Stimulation (Stim) was with LPS. Antibody (AB) used for supershift analysis is specific for the p50 (DNA binding) subunit of NF-B. Low-level NF-B binding activity is present under basal conditions ("-" Stim) and is specifically induced by LPS ("+" Stim). The shifted band representing NF-B is completely supershifted by the antibody to the p50 subunit.

Effect of Unstable Coronary Syndromes on NF-B Activation
EMSAs were used to determine NF-B activation in the circulation of 102 consecutive patients without acute myocardial infarction undergoing cardiac catheterization. The baseline characteristics of these patients are shown in Table 1. Nineteen patients had unstable angina and were within 24 hours of their last episode of pain. The remainder were referred for cardiac catheterization for evaluation of stable angina (53), valvular disease (8), atypical chest pain (12), or heart failure (10). Analyses showed that 17 of 19 patients with unstable angina had marked activation of NF-B. Despite the expected high incidence of significant coronary artery disease (75%) in the remainder of the patients, only 2 showed marked NF-B activation. Interestingly and importantly, these 2 patients (both with stable exertional angina pectoris) developed acute coronary artery syndromes 1 and 6 hours later and required emergent percutaneous revascularization. Low levels of activated NF-B were seen in the rest of the patients, with discernible individual variability. These results are summarized in Figures 2 and 3.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Patients (n=102)

View larger version (74K): [in this window] [in a new window]   Figure 2. EMSAs with nuclear proteins isolated from 3 patients with stable (S) angina and 3 with unstable angina (U) with NF-B as probe. Stimulation (Stim) was with LPS. Patients with stable angina have low levels of NF-B that are appropriately induced with LPS. Those with unstable angina have marked activation of NF-B in the circulation in the basal state. There is a significant difference in the level of NF-B activation in the basal state between patients with stable and those with unstable angina (note shifted bands in - lanes).

View larger version (12K): [in this window] [in a new window]   Figure 3. NF-B levels of activation of patients with stable angina pectoris (SAP) or unstable angina pectoris (USA) extrapolated from NF-B binding availability derived from EMSAs. NF-B levels were quantified by scanning laser densitometer and are shown as arbitrary units (AU). Each dot represents an individual patient.

Figure 2 shows EMSA data from 6 individuals, 3 with stable angina and 3 with unstable angina, who are representative of the patients tested; these data were analyzed on 2 separate gels performed on 2 different days. These gels illustrate the markedly higher level of NF-B binding availability, and therefore NF-B activation, under basal (unstimulated, or "-") conditions in patients with unstable angina than in those with stable angina and demonstrate the low but variable levels of NF-B activation in patients with stable angina. This figure also shows that the low level of NF-B activation in stable patients was not due to a lack of NF-B or some related problem, because stimulation of these cells ex vivo with LPS resulted in marked induction of NF-B (note the increase in binding in "+" lanes of stable patients). Furthermore, this figure demonstrates consistency and reproducibility of the assay, suggesting that accurate quantification is possible. Figure 3 is a graphic representation of the level of NF-B activation in the tested patients and shows in a quantitative fashion the difference between those with marked NF-B activation (arbitrarily defined as NF-B binding activity >2 AU) and those without.

Clinical Characteristics
The clinical characteristics of those with stable and unstable angina are shown in Table 2. Of note are the similarities in age, hemodynamic status, and high incidence of significant coronary artery disease. There are stark differences in NF-B levels between the groups, with NF-B levels being higher in those with unstable angina. Those with unstable angina also have a significantly higher percentage incidence of systemic heparinization. This is not surprising and reflects their clinical condition. Although this difference suggests a possible relationship between heparinization and NF-B activation, the presence of anticoagulation after administration of intravenous heparin does not appear to compel activation of NF-B. This conclusion is based on the fact that in the absence of unstable angina, heparinization was not associated with activation of NF-B. Also, the 2 stable patients who had marked NF-B activation before plaque rupture did not have anticoagulation. The most important evidence that heparin does not lead to increased NF-B activation is that of all patients in the study who received heparin, only half showed elevation of NF-B. Specifically, 16 (19%) of 83 patients shown in Table 3 with low NF-B levels were heparinized. Seventeen (89%) of 19 patients (Table 3) with high NF-B levels were heparinized. Hence, of the 33 patients receiving heparin intravenously, 48% (16 of 33) had low NF-B levels and 52% (17 of 33) had high NF-B levels. All those with high levels had unstable angina. Therefore, there is no relationship between NF-B activation and use of intravenous heparin.

View this table: [in this window] [in a new window]   Table 2. Clinical Characteristics of Patients With Stable Angina Pectoris or Unstable Angina Pectoris

View this table: [in this window] [in a new window]   Table 3. Clinical Characteristics of Patients With High (>2 AU) or Low NF-B Levels

To determine if factors other than the presence of an unstable plaque influenced NF-B activation, patients were divided into those with high (>2 AU) or low (<2 AU) NF-B levels and the clinical characteristics analyzed. Table 3 shows no relationship between NF-B activation and age, presence of diabetes or hypertension, or hemodynamic status. Those with high NF-B levels uniformly had significant coronary artery disease and more commonly used ASA and heparin, with the difference in percentage heparin use being significant. This increased use of heparin in the group with elevated NF-B levels is not believed to be the cause of NF-B activation, for the reasons discussed above.

Specificity of NF-B Activation
To determine if the increase in NF-B activation was a nonspecific inflammatory response, an additional 29 patients with infectious/inflammatory disorders were evaluated. These studies showed that NF-B activation was not activated in the circulation of such patients. Indeed, NF-B levels were nearly undetectable in many patients with infection. Thus, NF-B activation is not a nonspecific response of inflammation. Finally, to determine if the increase in NF-B binding was specific for NF-B or a generic response of all nuclear factors to stimulation, binding of the transcription factors Sp1 and Oct1 was assessed. These analyses showed that Oct1 binding decreases rather than increases in those patients with increased NF-B binding. Binding of Sp1 also did not correlate with increases in NF-B activation. These data suggest that increases in NF-B binding are specific and not a result of generalized increases in transcription factor binding.

Taken together, these data suggest that NF-B is specifically and significantly activated in unstable angina and that activation occurs before the clinical event, indicating that NF-B may be mechanistically involved. Because of the interpatient variability in NF-B activation in those with stable coronary disease, these data further imply that the level of NF-B may be related to coronary artery disease activity. Thus, these data are consistent with the initial hypothesis that NF-B plays a pivotal role in plaque rupture.

	Discussion

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Despite substantial progress in the diagnosis and treatment of the manifestations of coronary artery disease, it is not currently possible to identify a priori those specific patients at risk for subsequent sudden cardiac death, myocardial infarction, or unstable angina.²⁷ This limitation may in part be due to the lack of insight into the mechanism underlying coronary artery disease. Fortunately, recent data suggesting that inflammation plays a role in coronary artery disease may allow for a better assessment of the extent of coronary artery disease activity.^{8 9 10 11}

Inflammation, NF-B, and Coronary Artery Disease Activity
Analyses focusing on the composition and appearance of affected coronary arteries have shown clear evidence of both chronic and acute inflammation, with acute inflammatory conditions commonly predominating at sites of plaque rupture.²⁸ The transcription factor NF-B has been directly linked to induction of genes encoding proteins integral for each event in this inflammatory cascade.¹³ For example, activation of circulating inflammatory cells is dependent on adequate and appropriately timed increases in expression in the interleukins induced by NF-B. NF-B is necessary and sufficient to transcriptionally activate monocyte chemoattractant protein-1, vascular cell adhesion molecules, and intracellular adhesion molecule-1, which appear critical for vascular wall recruitment of inflammatory cells from the circulation.^{29 30 31} NF-B also vigorously induces metalloproteinase genes. Activated NF-B has been found in the intima and media of human atherosclerotic vessels, and the degree of NF-B activation correlates with the extent of atherosclerotic disease progression.^{32 33 34} Factors capable of activating NF-B have been identified adjacent to NF-B within the vessel wall and in the circulation.^{32 33 34 35 36} Thus, activated NF-B and factors that influence its activation, as well as those genes that serve as its targets, are present in atherosclerotic plaques and correlate with the extent of disease progression, which suggests that NF-B may play a role in the acute inflammatory reaction proposed to precipitate the unpredictable rupture of coronary artery plaques.¹¹

A role for NF-B in acute coronary artery syndromes is further suggested by the present study, which demonstrates that NF-B is selectively and markedly activated acutely in the circulation of patients with unstable angina. The intriguing possibility that the degree of NF-B activation correlates with coronary artery disease activity is also raised by the substantial variability in low-level NF-B activation observed in those with stable angina pectoris. These provocative data prompt many additional questions. For example, what is the stimulus for NF-B activation in circulating white cells in these patients? Is it in response to a circulating agent or a locally (site of "plaque rupture") produced substance? Is NF-B activated before the event, as suggested by those (2) patients with evidence of systemic NF-B activation before their clinical events, or is NF-B activated as a consequence of active coronary artery disease?

NF-B Activation in the Circulation of Humans With Unstable Angina: Cause or Effect?
A number of agents are capable of activating NF-B, including the cellular products IL-1ß, IL-11, and IL-17 and TNF-, intracellular signaling molecules such as protein kinase C, and other stimuli such as oxygen radicals, transient exposure to oxidized LDL, heparan sulfate, and mechanical stretch.^{37 38 39 40 41} As expected for such a ubiquitous factor, the capacity for these stimuli to activate NF-B is cell-type and milieu specific. This degree of regulation is expanded by the presence of cell-specific intracellular and extracellular negative and positive feedback loops.⁴² For example, in response to endotoxin, TNF-, and IL-1ß, lymphocytes and monocytes produce IL-10 and IB, which significantly limit NF-B activation. In contrast, in human atheroma vascular smooth muscle cells, NF-B–mediated induction of TNF- and IL-1ß further upregulates NF-B, resulting in persistent activation of NF-B.³⁶ Accordingly, it could be postulated that systemic but self-limited stimulation of NF-B initiates the process of persistent NF-B activation in the "primed" vessel wall.

Hence, it is likely that the marked NF-B activation observed in the current study was transient. In support of this conjecture is our recent work showing that NF-B activation in patients with unstable angina persists for <24 hours and is not present in those presenting >24 hours after a myocardial infarction (data not shown). These data would suggest that activation of NF-B is a primary event. However, it is possible that NF-B was activated in circulating cells via contact with an unstable plaque or through release from the plaque of circulating molecules (eg, TNF-) capable of stimulating NF-B. In such a scenario, NF-B activation could either represent an event that occurs after the culmination of the acute coronary episode or one that happens as a consequence of coronary artery disease activity. The abbreviated time course of activation, the selectivity of activation, and the fact that activation occurred in 2 patients before their symptomatic event suggest that the latter possibility (that NF-B activation is a marker of coronary artery disease activity) is more likely. This is further supported by our recent data (data not shown) demonstrating a lack of discernible variability in NF-B activation in venous blood isolates from patients with stable angina pectoris. Thus, it could be proposed that NF-B is transiently activated relative to the extent of coronary artery disease activity and that this activation is stable through the coronary and pulmonary circulations.

These speculations are predicated on the assumption that data derived primarily from cell culture are applicable to in vivo studies in humans. It is important to also consider that mechanisms of NF-B activation in circulating cells in atherosclerosis may differ from those occurring in response to infectious and inflammatory products in culture. For example, NF-B activation in white cells from patients with coronary disease may be primarily self-propagating and not subject to the self-limiting regulation observed in response to endotoxin. NF-B activation by oxidized LDL in vivo may demonstrate a linear dose-related response rather than the abbreviated induction with low-level oxidized LDL that occurs in culture. Alternatively, if coronary artery disease activity reflects atherosclerosis as influenced by an infectious agent, results from culture studies may overlap in vivo analyses to varying degrees, making analyses even more complicated.

Conclusions
The data on NF-B activation presented in this article, combined with its known properties, suggest that NF-B could indeed serve as a marker of disease activity. Additional analyses to confirm and extend these initial observations are currently under way. It is hoped that results stemming from these investigations will lead to the use of NF-B as a clinical marker for coronary artery disease activity and efficacy of therapy.

	Acknowledgments

 
The work in this article was supported in part by a Grant-in-Aid from the American Heart Association–Ohio Affiliate and by a research advisory grant from the Veterans Administration. The author would like to recognize the excellent efforts of research assistants Margaret Collins and Lucy Kim and cardiac catheterization laboratory personnel Sherri Rosser and Steve Vidourek. Sandy Nagel provided quality secretarial support.

Received February 5, 1998; revision received June 17, 1998; accepted June 22, 1998.

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