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(Stroke. 2004;35:458.)
© 2004 American Heart Association, Inc.

Original Contributions

NF-B Activation and Fas Ligand Overexpression in Blood and Plaques of Patients With Carotid Atherosclerosis

Potential Implication in Plaque Instability

José Luis Martín-Ventura, BS; Luis Miguel Blanco-Colio, PhD; Begoña Muñoz-García, BS; Almudena Gómez-Hernández, BS; Ana Arribas, MD; Luis Ortega, MD; José Tuñón, MD Jesús Egido, MD

From the Vascular Research Laboratory (J.L.M.-V., L.M.B.-C., B.M.-G., A.G.-H., J.E.), Department of Vascular Surgery (A.A.), and Department of Cardiology (J.T.), Fundación Jiménez Díaz, Autonoma University, and Department of Pathology, Hospital Clínico San Carlos (L.O.), Madrid, Spain.

Correspondence to Professor Jesús Egido, MD, Vascular Research Laboratory, Fundación Jiménez Díaz, Ave Reyes Católicos 2, 28040, Madrid, Spain. E-mail jegido{at}fjd.es

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background and Purpose— Apoptosis is present in human atherosclerotic lesions. Nuclear factor-B (NF-B) is involved in the transcriptional regulation of the proapoptotic protein Fas ligand (FasL). We have analyzed NF-B activation and FasL expression in atherosclerotic plaques and peripheral blood mononuclear cells (PBMCs) of patients with carotid stenosis.

Methods— NF-B activation and FasL and active caspase-3 expression were analyzed in 32 human carotid plaques. NF-B activation and FasL mRNA were tested in PBMCs of patients and healthy volunteers. We analyzed whether the NF-B inhibitor parthenolide regulates FasL expression and cytotoxicity in human T cells.

Results— The inflammatory region of plaques showed an increase in NF-B activation (3393±281 versus 1029±100 positive nuclei per mm², P<0.001) and FasL (16±1.4% versus 13±1.8%, P<0.05) and active caspase-3 (3.3±0.6 versus 1.5±0.3%, P<0.05) expression compared with the fibrous area. Activated NF-B and FasL protein were colocalized in plaque cells. In PBMCs obtained from those patients the day of endarterectomy, NF-B activation and FasL expression were significantly increased compared with healthy controls (1.5±0.1 versus 0.5±0.1 and 2.1±0.1 versus 1.2±0.1 arbitrary units, respectively; P<0.001). There was a significant correlation between NF-B activation and FasL expression. In activated T cells, parthenolide decreased NF-B activation, FasL promoter activity, and mRNA expression. Parthenolide also decreased cytotoxicity of activated Jurkat cells on FasL-sensitive cells.

Conclusions— NF-B activation and FasL overexpression occur in PBMCs and atherosclerotic lesions of patients with carotid stenosis. The NF-B-FasL pathway could be involved in the mechanisms underlying plaque instability in humans.

Key Words: apoptosis • atherosclerosis • carotid stenosis

	Introduction

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Studies on coronary arteries of patients suffering myocardial infarction demonstrated that the rupture of atheroma usually takes place in the shoulder region,¹ an area characterized by an increased expression of metalloproteinase-1 and -3.² Although this mechanism has been implicated in plaque rupture in this vulnerable region, others (ie, apoptosis, the innate mechanism by which the organism eliminates unwanted cells) could be involved.

Multiple studies found apoptosis in atherosclerotic coronary, carotid, and aortic arteries.^3–5 Specimens from unstable coronary plaques obtained by atherectomy show an increase in apoptotic vascular smooth muscle cells (VSMCs) and a decrease in the total number of these cells.⁶ A main mechanism in the activation of apoptosis is the Fas/Fas ligand (FasL) system.⁷ Fas is a member of the tumor necrosis factor receptor family and is expressed in human atherosclerotic plaques.⁸ FasL engagement promotes the formation of a signaling complex of molecules linked by protein-protein interactions with the cytoplasmic portion of the receptor, which recruits procaspase-8. Aggregated caspase-8 self-activates, cleaves, and activates procaspase-3 (effector caspase), which degrades proteins involved in cell structure and survival.

FasL expression in activated T cells implicates 2 different signals: activation of protein kinase C pathway and release of intracellular Ca²⁺ stores.⁹ These signals can be mimicked by treatment of cells with phorbol-myristate-acetate (PMA) and ionomycin. These agents induce activation of transcription factors involved in the regulation of FasL expression in T cells, among them nuclear factor-B (NF-B).¹⁰ NF-B is found as a trimer consisting of p50, p65, and IB subunits in the cytosol. The release of IB from the trimer results in the migration of the p50/p65 heterodimer to the nucleus,¹¹ where binding to specific sequences activates genes involved in the immune, inflammatory, or acute-phase response. The increment of NF-B activation in peripheral blood mononuclear cells (PBMCs) has been related to cardiovascular diseases, and patients with unstable angina have elevated NF-B activation in PBMCs.¹²

Here, we have analyzed NF-B activation and FasL expression in atherosclerotic plaques and PBMCs of patients with carotid stenosis. Moreover, we have studied NF-B regulation of FasL expression and cytotoxicity in activated T cells in vitro.

	Methods

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Patients
Thirty-two consecutive patients undergoing carotid endarterectomy at our institution were included in the study, and informed consent was obtained before enrollment. Blood samples were collected from 18 available patients before surgery and from 12 healthy volunteers matched by age and sex. The study was approved by the ethics committee of Fundacion Jimenez Diaz in accordance with institutional guidelines.

Histological Analysis
Tissue Sampling
Specimens were collected and stored in paraformaldehyde and later in ethanol until paraffin embedded. The region of the bifurcation of the common carotid artery was chosen. We studied atherosclerotic plaques in 2 different areas: shoulder and cap. The shoulder region was defined as the plaque area at both sides of the lipid core and the fibrous cap as the rim over the atheroma.

Immunohistochemistry
Paraffin-embedded carotid arteries were cross sectioned into 4-µm-thick pieces, dewaxed, and rehydrated. Monoclonal anti-human macrophages (HAM-56, DAKO), rabbit anti-human T lymphocytes (CD3, DAKO), monoclonal anti- smooth muscle actin (HHF-35, Sigma), and rabbit anti-human Fas and FasL (M-20 and N-20, respectively, Santa Cruz Biotechnology) were applied overnight. A rabbit anti-human anti-active caspase-3 antibody (G7481, Promega) was used as a marker of cell apoptosis.^13,14 Secondary antibodies were applied for 1 hour, ABComplex/HRP was then added for 30 minutes, and sections were stained with 3,3'-diaminobenzidine (DAKO), counterstained with hematoxylin, and mounted in Pertex (Medite). For colocalization studies, immunofluorescence for FasL was carried out on slides directly after performing immunohistochemistry for macrophages and VSMCs. Negative controls using the corresponding IgG were included to check for nonspecific staining.

Southwestern Histochemistry
The distribution and DNA binding activity of NF-B in situ were detected with a digoxigenin-labeled double-stranded DNA probe with a specific NF-B consensus sequence as described.^15,16 Competition assays with 100-fold excess of unlabeled probe were used as negative controls. For colocalization studies, immunohistochemistry for FasL was carried out directly from the final wash of Southwestern histochemistry.

Quantification
Computer-assisted morphometric analysis was performed with the Olympus semiautomatic image analysis system with Micro Image software. Preparations were digitized via an Olympus microscope (BH-2), at 400 magnification, connected to a CCD video camera as described.¹⁷ Results are expressed as percentage of positive staining per mm² for immunohistochemistry and nuclei staining as positive per mm² for Southwestern.

Studies in Circulating Mononuclear Cells
Isolation of PBMCs
Blood (20 mL) was obtained from 18 available patients before endarterectomy and from 12 healthy controls. PBMCs were obtained as described.¹² Approximately 95% of cells were mononuclear cells (flow cytometry, not shown).

Electrophoretic Mobility Shift Assay
Protein extracts from mononuclear cells were prepared and quantified as described.¹² NF-B consensus oligonucleotide (5'- AGTTGAGGGGACTTTCCCAGGC-3') was ³²P-end-labeled with 10 U of T4 polynucleotide kinase (Promega). Quantification was performed by densitometric analysis with the ImageQuant densitometer program, yielding results in arbitrary units (a.u.).

RNA Extraction, Reverse Transcriptase Polymerase Chain Reaction
Total RNA was obtained by the Trizol method (Life Technologies) and quantified by absorbance at 260 nm. Then, 100 ng RNA was applied to the access reverse-transcriptase polymerase chain reaction (RT-PCR) system (Promega). The following primers were used: hFasL: antisense, 5'-TCAGCACTGGTAAGATTG-3'; sense, 5'-TCTGGAATGGGAAGACAC-3' (1 minute at 51°C for annealing, 28 cycles, 286 bp); and GADPH: antisense, 5'-AATGCA-TCCTGCACCACCAA-3'; and sense, 5'-ATACTGTTAC-TTATACCGATG-3' (1 minute at 58°C for annealing, 25 cycles, 515 bp). DNA products were analyzed on 1% agarose gel. Quantification was performed with ImageQuant.

In Vitro Studies
Reagents
RPMI-1640, penicillin, and streptomycin were obtained from BioWhitaker. Fetal bovine serum was from Gibco. FuGENE 6 transfection reagent was from Roche. The remaining reagents were obtained from Sigma.

Cell Cultures
Fas-sensitive A20 and Fas-resistant A20R murine B lymphoma cells were from Dr Jürg Tschopp (Lausanne University). A20R cells were generated by continuous culture in the presence of FasL and have downregulated Fas receptor expression. Jurkat (ATCC), A20, and A20R cells were cultured in RPMI-1640 supplemented with 10% decomplemented fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Northern Blot Analysis
Total RNA (20 µg) was denatured and electrophoresed on a l % agarose–formaldehyde gel and transferred to nylon membranes as described.¹⁶ The cDNA probes for human FasL and GAPDH were obtained from preparative RT-PCR. Quantification was performed with ImageQuant.

Cell Transfections
Reporter fusion plasmid containing 1.2 kb FasL promoter and the firefly luciferase gene (pFasL-LUC) were from Dr Douglas Green (La Jolla Institute for Allergy and Immunology). Transfection of Jurkat cells was carried out by the FuGENE 6 method following manufacturer’s instructions with 3 µg human FasL promoter and 0.5 µg Renilla luciferase reporter vector pRL-TK. Cells were harvested and lysed in 100 µL lysis buffer. Finally, Renilla and firefly luminescences were measured.

FasL Bioactivity
FasL killing activity was assessed by incubating 1x10⁵ to 1x10³ effector Jurkat cells with 1x10⁵ bromodeoxyuridine-labeled target A20 or A20R cells. Higher effector-to-target ratios were abandoned because Jurkat cells became overconfluent at effector-to-target ratios >1. Release of bromodeoxyuridine was determined after 20 hours of coincubation with a cellular DNA fragmentation enzyme-linked immunosorbent assay (Boehringer Mannheim). As positive control, Jurkat cells were replaced by 10 ng/mL recombinant human FasL. Cell death induced in Fas-sensitive cells by recombinant FasL was considered to be 100%.

Statistical Analysis
Statistical analysis was performed with GraphPAD InStat (GraphPAD Software). Spearman’s correlation coefficient analysis was used to assess associations between values. Results are expressed as mean±SEM and were analyzed by analysis of variance and Student’s t test (P<0.05 was considered significant).

	Results

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NF-B Activation, FasL and Active Caspase-3 Expression in Human Atherosclerotic Plaques
We studied cell composition of the plaques and observed that the percent of inflammatory cells (macrophages and T lymphocytes) was significantly greater in the shoulder than in the cap (16±2% versus 4±1% and 2.6±1.1% versus 0.5±0.2%, respectively; P<0.001), whereas the percent of VSMCs displayed the opposite pattern (13±2 versus 34±2%, P<0.001). No differences were observed in the total number of cell-positive staining when both regions were compared. NF-B activation in situ was significantly higher in the shoulder than in the cap (3393±281 versus 1029±100 nuclei staining positive per mm², P<0.001) (Figure 1A). We performed negative controls, and there was no nuclear staining in any of the cases (not shown). Moreover, FasL immunostaining was significantly higher in the shoulder than in the cap (16±1.4% versus 13±1.8%, P<0.05) (Figure 1B). Fas expression was also higher in the shoulder although it did not reach statistical significance (19±2% versus 14±2%; P=NS; not shown).

View larger version (124K): [in this window] [in a new window]   Figure 1. NF-B activation and FasL and active caspase-3 expression in human carotid atherosclerotic sections. Shoulder region showed increased NF-B activation (A), FasL expression (B), and active caspase-3 expression (C). Squares display a magnification (x400). D, Schematic of human carotid plaque showing atheroma region (*) and cap region (**). Shoulder region is also shown ().

Finally, we used an antibody that selectively recognizes the active form of caspase-3. This technique stains apoptotic cells because activation of caspase-3 is required for the execution phase of apoptosis in mammalian cells. We showed an augmented expression of this marker of apoptosis when comparing the shoulder with the cap region (3.3±0.6% versus 1.5±0.3%; P<0.05; Figure 1D), indicating that apoptosis was present mainly in the inflammatory region of the plaques.

To determine which cells contribute to FasL expression, we performed double immunohistochemistry/immunofluorescence to colocalize these proteins with macrophages and VSMCs. We showed that both cells express FasL (Figure 2A and 2B).

View larger version (66K): [in this window] [in a new window]   Figure 2. Colocalization studies. Localization of cells of lesion expressing FasL: (A) FasL (left) and VSMCs (right); (B) FasL (left) and macrophages (right). Squares show in detail the double staining; FasL is light grey, and macrophages and VSMCs are dark grey. C, Colocalization of NF-B activation (positive nucleus in dark grey) and FasL (light grey).

We studied the simultaneous localization of FasL and NF-B. As shown in Figure 2C, colocalization of NF-B and FasL was observed, suggesting that in vivo activation of this factor is involved in the transcriptional regulation of FasL.

Studies in Circulating Mononuclear Cells
To assess whether the activation of circulating mononuclear cells could provide information about the state of atherosclerotic patients, NF-B activation and FasL expression were evaluated in PBMCs obtained from patients’ blood samples taken the day of surgery. NF-B activation was significantly increased in PBMCs of patients with carotid atherosclerosis compared with healthy controls (1.5±0.1 versus 0.5±0.1 a.u.; P<0.001; Figure 3A).

View larger version (35K): [in this window] [in a new window]   Figure 3. NF-B activation and FasL mRNA expression in PBMCs of patients with atherosclerosis. NF-B activation (A) and FasL mRNA expression (B) in 18 patients tested and 12 healthy controls. C, Correlation of NF-B and FasL in PBMCs.

Because of the limited amount of mRNA obtained from PBMCs, FasL mRNA was tested by RT-PCR. We observed that FasL expression was significantly augmented in PBMCs of patients with carotid atherosclerosis compared with healthy controls (2.1±0.1 versus 1.2±0.1 a.u.; P<0.001; Figure 3B).

Interestingly, there was a significant correlation between NF-B activation and FasL expression (r=0.64; P<0.005), indicating an in vivo association between these proteins (Figure 3C).

In Vitro Studies
NF-B Inhibition Regulates FasL Expression
Previous studies demonstrated that PMA plus ionomycin induces NF-B activation and FasL mRNA expression in Jurkat cells.¹⁰ Jurkat cells were preincubated with parthenolide and then incubated 2 hours with PMA/ionomycin. Parthenolide decreased NF-B activation in a dose-dependent manner (3.9±0.3 PMA/ionomycin versus 1.1±0.4 parthenolide 10 µmol/L; P<0.05; Figure 4A).

View larger version (23K): [in this window] [in a new window]   Figure 4. NF-B activation and FasL expression in activated T cells in vitro treated with parthenolide. Effect of parthenolide on (A) NF-B activation, (B) FasL promoter activity, and (C) FasL mRNA expression (*P<0.05 vs PMA/ionomycin; n=3).

Jurkat cells were transfected with a luciferase reporter construct containing the FasL promoter. Parthenolide treatment significantly reduced FasL reporter activity (23.8±2.8 PMA/ionomycin versus 9.3±1.4 parthenolide 10 µmol/L; P<0.05; Figure 4B) and FasL mRNA expression induced by PMA/ionomycin (3.6±0.4 PMA/ionomycin versus 0.6±0.5 parthenolide 10 µmol/L; P<0.05; Northern blot; Figure 4C), indicating that NF-B regulates FasL expression in activated T cells.

Parthenolide Decreases Cytotoxicity of Activated T Cells
To understand the physiological relevance of FasL regulation in T cells, we studied whether diminution of FasL expression induced by parthenolide can regulate the cytotoxicity of activated T cells. Apoptosis induced by activated Jurkat cells on Fas-sensitive A20 cells was prevented by parthenolide (Figure 5), whereas apoptosis induced by activated Jurkat cells was not present in Fas-resistant A20R cells. Apoptosis in A20 cells was inhibited by neutralizing antibodies to FasL, indicating that this protein was required for apoptosis induced by activated T cells (not shown).

View larger version (12K): [in this window] [in a new window]   Figure 5. NF-B inhibition decreases cytotoxicity of Jurkat cells. A20 or A20R cells were cocultured with Jurkat cells preincubated with parthenolide. BrdU release by apoptotic cells to culture was measured (*P<0.05 vs PMA/ionomycin; n=4).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The Fas/FasL system is one of the most important mechanisms in the activation of apoptosis. Fas-mediated apoptosis is involved in the maintenance of peripheral tolerance and in the termination of an ongoing immune response. Aberrant expression of Fas and FasL is dangerous and leads to severe diseases. Fas expression has been detected in VSMCs of human atherosclerotic plaques and colocalized with apoptotic cells, implicating the Fas/FasL system in the control of viability of the plaque cells.⁸ FasL is present in human coronary atheroma, whereas little immunoreactive FasL was observed in nonatherosclerotic tissue.¹⁸ We have observed that these proteins are expressed mainly in inflammatory regions of atherosclerotic lesions. We showed the presence of active caspase-3 (a protease related with apoptosis induced by FasL) in this vulnerable region. Furthermore, extensive macrophage apoptosis has been shown at the site of plaque rupture in autopsy specimens from people who suffered sudden coronary death.¹⁹ Caspase-3 has been detected in human atherosclerotic plaques,²⁰ although an antibody that recognizes both activated and unactivated forms of this protein was used. However, this is the first time that activated caspase-3 was detected in atherosclerotic lesions.

Different transcription factors have been involved in FasL regulation,¹⁰ among them NF-B. NF-B, which is involved in the transmission of signals from the cytoplasm to the nucleus of cells, is present in human atherosclerotic lesions, whereas little or no activated NF-B was detected in normal vessels.²¹ NF-B activity is greater in complicated than in stable atherosclerotic plaques.²² We showed that activated NF-B is present in the nuclei of cells of atherosclerotic lesions and is activated mainly in the shoulder region of the plaque. This is in accordance with previous articles showing an increment in the expression of some of the molecules that are transcriptionally regulated by NF-B in this vulnerable region.^2,23,24 Interestingly, FasL expression colocalized with NF-B, indicating that this transcription factor can regulate FasL expression in vivo.

The activation of NF-B in circulating mononuclear cells has been related to cardiovascular diseases. Ritchie¹² reported that NF-B activity is elevated in circulating monocytes of patients with unstable angina. We show that NF-B activity and FasL mRNA are increased in PBMCs of patients with carotid atherosclerosis compared with healthy controls. Furthermore, there was a significant correlation between NF-B activation and FasL expression, indicating an in vivo association between these proteins. These data could suggest that the inflammatory cells that show active NF-B and FasL expression in the vulnerable region of the plaque could originally be activated in the circulation before they enter the arterial wall.

Finally, we have analyzed the effect of parthenolide, which specifically inhibits NF-B activation by preventing the degradation of its inhibitory subunit,²⁵ on FasL expression in T cells. Parthenolide prevented renal lesions in experimental glomerulonephritis by inhibiting NF-B activation and expression of regulated genes.²⁶ We have observed that parthenolide inhibits NF-B activation and FasL expression and diminishes cytotoxicity of activated T cells on Fas-bearing cells. The physiological importance of NF-B regulation of FasL expression is due to the fact that it has been demonstrated that inflammatory cells expressing FasL can induce apoptosis of VSMCs expressing Fas through the activation of caspases.²⁷ Furthermore, FasL could be involved in the recruitment of inflammatory cells to the lesion, and overexpression of FasL in arteries of hypercholesterolemic rabbits can accelerate atherosclerotic lesion formation.²⁸ However, Sata and Walsh²⁹ demonstrated that FasL expression by endothelial cells decreases inflammation in atherosclerotic lesions. In pathological conditions, endothelial cells did not express FasL, favoring that inflammatory cells can enter in the vessel wall. It is sensible to think that FasL regulation in the cells present in atherosclerotic lesions could be different, depending on the cell type analyzed. Despite the potential importance of apoptosis in vascular lesions, little is known about the mechanisms that regulate proteins involved in cell viability. Because we have shown that parthenolide inhibits NF-B activation and the coordinate expression of the proapoptotic protein FasL, it can be suggested that agents capable of blocking this pathway could represent a novel therapeutic approach to atherosclerosis. Nevertheless, further studies are needed to clarify this issue.

In conclusion, we have observed the association between NF-B activation and FasL expression in PBMCs. The vulnerable region of atherosclerotic plaques is characterized by an augmented NF-B activation, FasL expression, and, importantly, active caspase-3. Our results suggest that NF-B, through FasL expression, could participate in the mechanisms underlying plaque instability in patients with carotid atherosclerosis.

	Acknowledgments

 
Grant and sources of support are as follows: Fundación Ramón Areces, Fundación Española del Corazón and Fondo de Investigaciones Sanitarias (99/0139), Ministerio de Educación y Ciencia (SAF 2001/0717), Pfizer Spain, Spanish Cardiovascular Network (03/01), and (MH02541A8640). J.L.M.-V. is a fellow of Spanish Fondo de Investigaciones Sanitarias.

	Footnotes

 
The first 2 authors contributed equally to this work.

Received June 25, 2002; revision received September 24, 2003; accepted October 30, 2003.

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