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Cardiovascular Research 2007 75(1):195-204; doi:10.1016/j.cardiores.2007.03.013
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Copyright © 2007, European Society of Cardiology

Nf-{kappa}b and AP-1 activation is associated with late lumen loss after porcine coronary angioplasty and antiproliferative beta-irradiation

Carolin Deinera,1, Erdenechimeg Shagdarsurenb,1, Peter L. Schwimmbeckc, Peter Rosenthald, Christoph Loddenkempere, Ursula Rauchf, Matthias Pauschingerf, Rainer Dietzb, Heinz-Peter Schultheissf, Ralf Dechendb and Klaus Pelsf,*

aInstitute of Veterinary Physiology, Faculty of Veterinary Medicine, FU Berlin, Berlin, Germany
bDepartment of Cardiology, Charité — Campus Buch Franz Volhard Klinik, HELIOS Klinikum Berlin, Germany
cDepartment of Cardiology, Klinikum Leverkusen, Leverkusen, Germany
dDepartment of Radiation Therapy and Oncology, Charité — Campus Benjamin Franklin, Berlin, Germany
eDepartment of Pathology, Charité — Campus Benjamin Franklin, Berlin, Germany
fDepartment of Cardiology, Charité — Campus Benjamin Franklin, Berlin, Germany

* Corresponding author. Department of Cardiology, Charité — Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Tel.: +49 30 8445 2068; fax: +49 30 8445 4648. klaus.pels{at}charite.de

Received 10 December 2005; revised 5 March 2007; accepted 9 March 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objective Despite the success of antiproliferative therapies, restenosis remains a common problem after percutaneous transluminal coronary angioplasty (PTCA). Longer-term clinical results of brachytherapy (intracoronary radiation), the lack of long-term clinical results after implantation of drug eluting stents, and the occurrence of late thrombosis after both procedures leave room for skepticism. Neointimal proliferation is not substantially inhibited at late time points after brachytherapy, and late lumen loss with a "catch-up" proliferation can occur. We hypothesized that the transcription factors nuclear factor-{kappa}B (NF-{kappa}B) and activator protein-1 (AP-1) are involved in these processes. We addressed the role of these mediators in a porcine model of coronary restenosis.

Methods Thirty-nine pigs underwent PTCA in two major coronary arteries. One of the two balloon-injured arteries was randomly assigned to receive immediate 20 Gy beta-irradiation (Brachy group) using a noncentered source train (90Sr/Y Beta-Cath, Novoste). Animals were sacrificed after 1 day, 14 days, or 28 days. Proliferating cells were labeled prior to euthanasia.

Results At late time points, lumen area was significantly smaller and the inflammatory response was more pronounced in the Brachy group than in the PTCA group. These findings coincided with sustained activation of MMP-9 and transcription factors like NF-{kappa}B and AP-1. Initially, cell proliferation was reduced in the Brachy group; however, at late time points, differences between the two treatment groups were no longer significant.

Conclusions Brachytherapy initially inhibits cell proliferation; however, cellular and molecular inflammatory processes (e.g. activation of NF–{kappa}B) are enhanced within the arterial wall. This proinflammatory side effect may be responsible for the observed delayed proliferation and the resulting lumen loss.

KEYWORDS Restenosis; Inflammation; Proliferation; Brachytherapy; Lumen loss; NF-{kappa}B; AP-1; Transcription factors; Porcine; Coronary arteries; Beta-irradiation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
Restenosis remains a common problem after percutaneous transluminal coronary angioplasty (PTCA) with or without stent placement. As an antiproliferative intervention, intracoronary radiation therapy has been approved by American and European regulatory agencies for treatment of in-stent restenosis. The effectiveness of this antiproliferative therapy has been demonstrated in experimental and major randomized clinical studies [1–6]. Late stent thrombosis, the "edge effect", and "geographic miss" initially proved to be clinical problems, but were resolved by long-term dual antiplatelet treatment, higher radiation doses, and overlapping radiation sources proximal and distal to the target lesion [7,8]. Despite the initial success of antiproliferative radiation therapy, the longer-term clinical results leave room for skepticism. Whether or not radiation-induced arteriopathy results in late restenosis is unknown. Clinical and animal studies show that late thrombosis remains a clinical issue unless dual antiplatelet treatment is sustained for good. Neointimal proliferation is not substantially inhibited and late lumen loss with a "catch-up" proliferation can occur [7–10]. Implantation of drug eluting stents (DES) is the currently most promising therapy for the treatment of coronary artery disease and restenosis. However, recent data suggest that safety concerns for the use of DES exist and warrant further investigation [11]. Late stent thrombosis is a major concern after implantation of DES and occurs especially in high risk patients (diabetes mellitus, renal failure, low ejection fraction) even after the recommended dual antiplatelet therapy and even years after DES implantation [12,13]. The molecular mechanisms involved in the pathophysiology after a local antiproliferative therapy are incompletely understood and difficult to investigate in stented tissue.

Transcription factors are increasingly suspected to be involved in atherosclerosis and restenosis [14,15]. Nuclear factor-{kappa}B (NF-{kappa}B) is known to regulate expression of proinflammatory cytokines, chemokines, and adhesion molecules, and activator protein-1 (AP-1) is also suspected to contribute to restenosis via regulation of various pathways, including cell proliferation, migration, and inflammation.

Whether or not antiproliferative therapies for prevention of coronary restenosis, namely DES and intraluminal irradiation, stimulate these pathways, is unknown [15]. We, therefore, addressed mechanisms related to these molecular and cellular mediators possibly contributing to complications of antiproliferative interventions in a porcine model of coronary restenosis.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1 Animal model
All studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, revised 1996). Thirty-nine domestic crossbred pigs (weight 22 to 27 kg) received daily antiplatelet therapy including aspirin (325 mg) and clopidogrel (300 mg loading dose on the day before intervention; 75 mg daily dose) until sacrifice over the entire observation period. Animals were sedated with ketamine (30 mg/kg) and azaperone (4.8 mg/kg) intramuscularly. For general anesthesia, ketamine (8 mg/kg) and xylazine (0.5 mg/kg) were repeatedly administered intravenously. Following a previously described protocol, cardiac catheterization was performed using a femoral artery approach according to standard procedures. Animals received periprocedural heparin. In each pig, percutaneous transluminal coronary angioplasty (PTCA) was performed in two major coronary arteries by three times inflation of standard balloons, 20 mm in length and 3.5 or 4 mm in diameter, depending on angiographically evaluated vessel size (to ensure a balloon to artery ratio of 1.3–1.5:1). One of the two balloon-injured arteries was randomly assigned to receive immediate radiation treatment with a beta-emitting source train. A non-centered Beta-Rail delivery catheter (40 mm, {emptyset} 2.13 mm; Novoste, Norcross, USA) was introduced over a flexible 0.014-inch guidewire (Wizdom ST Floppy, Cordis Corporation, Miami, USA) and positioned at the angioplasty site, ensuring a proximal and distal overhang. The delivery catheter was afterloaded with a 40 mm source train of 16 90Sr/Y seeds (Novoste Beta-Cath). Dwell times were adapted to the applied balloon size to deliver 19.9 to 22.2 Gy to a depth of 2 mm from the center of the source. Animals were divided into 3 groups (n=12 each; three animals died before completion of the study), and accordingly sacrificed after 1 day, 14 days, or 28 days. To label proliferating cells, six animals of each group received a 50 mg/kg intravenous dose of 5-bromo-2'-deoxyuridine (BrDU; Roche, Mannheim, Germany) 1 h prior to euthanasia.

2.2 Histopathologic processing and morphometry
Porcine hearts were harvested immediately after sacrifice. Hearts of BrDU injected animals were perfusion fixed with 4% paraformaldehyde at 100 mm Hg via pressure tubing placed in the ascending aorta [16]; the remaining six hearts from each group were prepared without perfusion fixation. The angioplasty site (PTCA), the angioplasty + brachytherapy site (Brachy), and the corresponding uninjured major coronary artery (Control) were dissected free from the heart, subdivided into multiple segments, and either snap frozen in liquid nitrogen, embedded in paraffin (fixed arteries), or embedded in OCT compound (fresh arteries). For each paraffin embedded arterial segment, a minimum of 8 sections were stained with hematoxylin and Elastica van Gieson and examined by two investigators blinded to the treatment of the arteries in order to define the respective cross section with the most severe luminal narrowing for further analyses. Morphometric analysis was performed on digitalized images employing the software DP-Soft (version 3.2, Olympus Optical Co. GmbH, Hamburg, Germany). Following a previously described protocol, lumen area, external elastic lamina (EEL) area, and total artery area were measured on Elastica van Gieson stained slides [16]. The EEL area was defined as the area within the innermost layer of the EEL (inner boundary). The adventitia area was defined as the dense connective tissue area between the inner EEL boundary and the loose periadventitial connective tissue and was calculated by subtracting EEL area from total artery area. Due to distinct interruptions of the internal elastic lamina post-balloon angioplasty, it was impossible to consider (neo)intima and media as discrete entities and were, hence, subsumed under a combined intima plus media area (I + M). I + M area values were calculated by subtracting lumen area from EEL area.

2.3 Immunohistochemistry
Immunohistochemical labeling of proliferating cells, T cells, and macrophages was carried out on adjacent cross-sections of paraffin embedded arteries, whereas staining for matrix metalloproteinase-9 (MMP-9) was carried out in OCT embedded arteries. Staining of proliferating cells: After a 5 min incubation with 3% hydrogen peroxide and a 15 min pretreatment with HCl followed by a 7 min digest with 0.1% trypsin, slides were incubated with a 1:25 mouse anti-BrDU monoclonal antibody (Amersham Biosciences Europe GmbH, Freiburg, Germany) for 2 h at 37 °C. A ready-to-use biotinylated horse anti-rabbit/mouse antibody was applied for 30 min followed by a 30 min incubation with ABC Reagent (RTU Vectastain universal Elite ABC Kit, Vector Laboratories Inc., Burlingame, CA, USA). Horseradish peroxidase activity was visualized with 3-amino-9-ethyl carbazole (AEC Substrate Kit, DAKO Cytomation GmbH, Hamburg, Germany). Porcine small bowel served as positive control tissue. Staining of macrophages and T cells: Slides were pretreated with 0.5% proteinase K (Roche, Mannheim, Germany) at 37 °C for 5 min and blocked with 1:10 rabbit serum (DAKO) before applying a 1:25 mouse anti-human macrophages antibody or a 1:25 mouse anti-CD3 antibody (Serotec, Düsseldorf, Germany), respectively, for 120 min at 37 °C. APAAP kit solutions containing rabbit anti-mouse antibody and mouse anti-alkaline phosphatase (AP) antibody plus AP were applied following the manufacturers instructions (DAKO), initially for 30 min and thereafter for another 10 min. AP activity was visualized with Vector Blue Substrate Kit (Vector) or Fast Red (DAKO). Incubations with PBS containing 1% bovine serum albumin and isotype-matched immunoglobulins were used as negative controls for all immunostainings. Hematoxylin or Nuclear Red (DAKO) was used as nuclear counterstain. MMP-9 staining: Ice-cold acetone-fixed cryosections (6 µm) were air-dried and immersed in TBS (0.05 M Tris buffer and 0.15 M NaCl, pH 7.6). The sections were blocked with 10% donkey serum (Jackson Laboratories, UK) and incubated with a 1:500 rabbit polyclonal antibody against human MMP-9 (Chemicon, Hampshire, UK) for 60 min. After washing in TBS, sections were incubated with a 1:500 Cy-3-conjugated donkey anti-rabbit secondary antibody (DAKO). Slides were analyzed under a Zeiss Axioplan-2 microscope (Carl Zeiss, Jena, Germany) and were digitally photographed using an AxioVision-3 multichannel image processing system (Carl Zeiss).

2.4 Cell quantification
Immunohistochemically stained macrophages, T cells, and proliferating cells in the arterial wall were quantified by means of a computer-assisted protocol described elsewhere [17].

2.5 Electrophoretic mobility shift assay (EMSA)
Tissue preparation for EMSA was performed as described earlier [18]. Nuclear extracts (5 µg) were incubated in binding reaction medium with 0.5 ng of 32P-dATP end-labeled oligonucleotide, containing the nuclear factor-kappa B (NF-{kappa}B) binding site from the MHC-enhancer (H2K, 5'-GATCCAGGGCTGGGGATTCCCCATCTCCACAGG) and then super-shifted with antibodies against the NF-{kappa}B subunits p50 and p65, indicating that p50 and p65 are the active members of the NF-{kappa}B family in the vessel wall (data not shown). For activator protein-1 (AP-1), double-stranded oligonucleotides containing the consensus sequence for AP-1 (Santa Cruz Biotechnology, Santa Cruz, CA; 5'-CGCTTGATGACTCAGCCGGAA-3') were radiolabeled with {gamma}-32P with the use of T4 polynucleotide kinase by standard methods and purified over a column. The DNA-protein complexes were analyzed on a 5% polyacrylamide gel, dried, and autoradiographed. In competition assays, 50 ng of unlabeled H2K or AP-1 oligonucleotides were used.

2.6 Statistical analysis
Quantitative data were expressed as mean±SEM. If SDs were equal within the populations, an unpaired two-tailed student t-test was used to compare the treatment groups; if SD values differed significantly, an unpaired two-tailed alternate t-test (Welch) was used. A value of P<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
Thirty-six of 39 pig coronary interventions were successfully performed. These animals recovered uneventfully. Two animals died from ventricular fibrillation acutely and one from pericardial tamponade on day 5.

Representative photographs in Fig. 1 give an idea of lumen area, intima + media (I + M) area, external elastic lamina (EEL) area, and adventitia area development in uninjured Control (1A), PTCA (1B), and Brachy (1C) vessels at day 28. Also given is quantification of the results (Fig. 1D, E). In both treatment groups, lumen area was increased immediately after angioplasty (day 1: Control 3.46±0.57 mm2 vs. PTCA 5.41±0.57 mm2 and Brachy 5.77±0.69 mm2, p<0.05; Fig. 1D). This luminal gain was followed by a lumen loss in both intervention groups at day 14. At day 28, the lumen in the Brachy group was significantly reduced compared to PTCA and Control (p<0.05). However, this late lumen loss was not due to an excessive growth of the I+M, but due to shrinkage of the artery (negative arterial remodeling) as expressed by a reduction of the EEL area (EEL area day 28: PTCA 5.15±0.37 mm2 vs. Brachy 3.65±0.44 mm2, p<0.05; Fig. 1E). This negative remodeling at day 28 was associated with a significant adventitial thickening (adventitia area: PTCA 2.71±0.1 mm2 vs. Brachy 5.69±0.83 mm2, p<0.05).


Figure 1
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  		Fig. 1 Representative photomicrographs of uninjured Control (A), PTCA (B), and Brachy vessels (C) at day 28 (Elastica van Gieson; white scale bar=2 mm). Please note the distinct differences in lumen area and vessel wall dimensions. Also given is quantification of the results (D, E). D: Both coronary interventions initially increased lumen area. At day one, immediately after angioplasty, lumen area differed significantly between uninjured Controls (black bars) and PTCA (grey bars; p<0.05) or Brachy vessels (light grey bars; p<0.05), followed by a lumen loss reaching Control vessel dimensions at day 14. At day 28, the lumen area was significantly reduced in the Brachy group compared to PTCA (*p<0.05 PTCA vs. Brachy) and Control vessels (p<0.05 Control vs. Brachy). E: This late lumen loss in the Brachy group coincided with a significant decrease in external elastic lamina (EEL) area (*p<0.05 PTCA vs. Brachy) and a significant increase in adventitia area (*p<0.05 PTCA vs. Brachy; p<0.05 Control vs. Brachy). Combined intima + media (I + M) area did not differ notedly between balloon-treated groups.

 
Cell proliferation by BrDU uptake is shown in Fig. 2. PTCA initiated cell proliferation, while the Brachy group exhibited no cell proliferation at day 1. However, by day 14 and day 28, the overall extent of proliferation was similar in both treatment groups. Separate analysis of the two arterial wall compartments (I + M and adventitia area) revealed that at day 28 the minority of cross sectional proliferating cells in the PTCA group were found in the adventitia (I + M area 40±8.1 cells, adventitia 18.7±3.91 cells; {approx}32% in adventitia), whereas in the Brachy group the adventitia was relatively more involved (I + M area 21.3±6 cells, adventitia 20.5±4.85 cells; {approx}50% in adventitia).


Figure 2
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  		Fig. 2 BrdU staining (nuclear brown staining) in Control vessels (A), PTCA (B), or Brachy vessels (C) at day 28 (black scale bar=100 µm). Quantification of proliferating cells (D) showed that in PTCA (grey bars) and in Brachy vessels (light grey bars) proliferation was more pronounced than in Controls (black bars), increasing over time. At day 1, the Brachy group showed significantly less proliferating cells per cross section than the PTCA group (*p<0.05), at days 14 and 28 there were no significant differences. Control vs. PTCA was always significant (p<0.05), Control vs. Brachy was significant at days 14 and 28 (p<0.05).

 
T cell and macrophage infiltration is shown in Fig. 3. Both treatments induced T-cell infiltration at day 14. In the Brachy group, the T cell infiltration was sustained and remained high through day 28, while the T cell count in the PTCA group returned to values of uninjured vessels (PTCA 12.83±4.26 cells vs. Brachy 72.67±10.61 cells; p<0.05; Fig. 3D). Macrophage infiltration showed a different pattern (Fig. 3E–H). Both treatments induced an immediate and pronounced macrophage infiltration. Whereas in the PTCA group macrophages were back to baseline at day 14, the Brachy group showed continued macrophage infiltration up to day 28 (day 14: PTCA 22.67±6.31 cells vs. Brachy 222.17±50.45 cells; p<0.05; day 28: PTCA 16.33±2.16 cells vs. Brachy 144.67±23.3 cells; p<0.05).


Figure 3
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  		Fig. 3 Immunohistochemistry for T cells (red staining, A–C) and macrophages (blue staining, E–G) in Control vessels (A, E), PTCA (B, F), or Brachy vessels (C, G) at day 28 (black scale bar=100 µm). T cell infiltration was absent at day 1, but pronounced at day 14 in PTCA and Brachy groups (D). At day 28, the T cell number decreased to initial values in the PTCA group, but remained high in the Brachy group (*p<0.05 PTCA vs. Brachy; Control vs. Brachy also significant (p<0.05)). In contrast, macrophages were present yet at day one after intervention (H), and PTCA and Brachy both had profound macrophage infiltration. At day 14, macrophages were back to baseline in the PTCA group, but were elevated in the Brachy group and remained high at day 28 (*p<0.05 PTCA vs. Brachy at days 14 and 28). Control vessels differed significantly from both treatment groups at all time points evaluated (p<0.05).

 
We performed EMSA for relevant transcription factors (NF-{kappa}B and AP-1) shown in Fig. 4. We found that PTCA as well as PTCA + brachytherapy induced these transcription factors at all time points (p<0.01). However, the effect was significantly greater in the Brachy group, compared to PTCA (p<0.05) and Control (p<0.01). In Fig. 5 we show an NF-{kappa}B/AP-1 dependent target gene implicated in arterial remodeling, namely matrix metalloproteinase-9 (MMP-9). The protein was upregulated in both interventions, albeit to a greater degree with the Brachy regimen.


Figure 4
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  		Fig. 4 EMSA for NF-{kappa}B (A–C) and AP-1 (E–G) are given. Compared to Control vessels, NF-{kappa}B and AP-1 were activated in PTCA and Brachy groups at day one, 14, and 28 after intervention (two- to nine-fold induction, D and H). However, the activation was greater in the Brachy group and remained high at day 28.

 

Figure 5
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  		Fig. 5 Representative (red) staining for matrix metalloproteinase 9 (MMP-9) at day 28. Control section (A) shows no staining. PTCA (B) revealed moderate staining. The Brachy section (C) revealed pronounced staining. Background autofluorescence (green) indicates elastic membranes.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
The major findings in this study were that late lumen loss was greater in the Brachy group compared to the PTCA group. Cell proliferation did not account for this late lumen loss, as the BrDU uptake was similar in both treatment groups at later time points. I + M area measurements substantiated this observation, since both treatment groups did not differ in I + M growth. Instead, a significant decrease in EEL area was observed in the Brachy group (negative arterial remodeling), which coincided with adventitial thickening. An initial inflammatory reaction was observed in both treatment groups, however, at late time points infiltration of inflammatory cells was substantially higher in the Brachy group. EMSA results confirmed these late inflammatory reactions, since NF-{kappa}B and AP-1 were still activated as late as day 28 in the Brachy group. Interestingly, MMP-9, a molecule shown to be associated with compensatory arterial enlargement and aneurysm formation in other studies, was upregulated.

Our data suggest that brachytherapy engenders a delayed wound healing response featuring prolonged inflammation. In contrast to irradiation of tumors, beta-irradiation in coronary vessels has never been shown to be a direct causal link to inflammation. An indirect relationship can be postulated involving platelet activation. We did not focus on platelets in our study, however, platelets were very likely participants in our observations. Salame and colleagues found that endovascular irradiation resulted in incomplete endothelial recovery, impaired resolution of intramural hemorrhage, and an increase in platelet recruitment [19]. Their data have important implications for delayed wound recovery and potential thrombogenicity at a later time point. Platelets could also have contributed to transcription factor activation, particularly NF-{kappa}B. Platelet factor 4 (PF4) has been shown to induce E-selectin in endothelial cells via NF-{kappa}B activation and platelet-type 12-lipoxygenase has also been shown to activate NF-{kappa}B [10,20]. Platelet-endothelial cell interactions have been shown to cause endothelial cells to produce the monocyte chemoattractant protein-1 (MCP-1) via an NF-{kappa}B-dependent mechanism thereby mediating a proinflammatory effect [21]. Platelets also release substances that promote tissue repair and influence the reactivity of vascular and other blood cells in angiogenesis and inflammation [22]. Furthermore, platelet-associated signalling events lead to vascular inflammatory and immune-related events. Taken together, there is ample experimental evidence suggesting a relationship between irradiation and inflammation via a platelet-NF-{kappa}B-pathway.

Witzke and colleagues studied in-stent restenosis of coronary artery bypass vein grafts following beta-brachytherapy [23]. They observed a sparse cellular component, myofibroblasts, and histiocytes in their sections. The histiocytes were lipid-laden cells suggesting an atherosclerotic process featuring ample inflammation after brachytherapy. A study of Kollum et al. provides further indirect evidence for a causal irradiation-inflammation relationship. In their study, they drew attention to delayed re-endothelialization and T-cell infiltration following intracoronary irradiation in the porcine model [24]. This group's findings suggest incomplete healing of the irradiated vessels. The infiltrating T cells exhibited an increased degree of apoptosis which was interpreted as resolving inflammation. Similar to their study, we also observed increased T cell and macrophage infiltration. However, in our study, inflammatory infiltration was sustained until day 28 in the Brachy group, whereas the infiltration abated in the PTCA group. Kollum et al. conducted their study only to day 14, while we contribute an additional later time point. Perhaps this explains the discrepant result. Although our study is not an interventional study aiming to prove a direct causal relationship, we demonstrate that – compared to PTCA and Control –inflammation is induced by brachytherapy and sustained until late time points, and are in agreement regarding the long-term inflammation and delayed wound healing engendered by irradiation, as our most prominent finding was unbridled inflammation with strong NF-{kappa}B and AP-1 activation as long as 1 month after the brachytherapy treatment.

As another important observation our data show that proliferation is not completely suppressed by brachytherapy, just temporally delayed. We hypothesize that the triggers for proliferation are different in both interventional groups, namely that in the PTCA group proliferation in the intima, media, and adventitia is induced by mechanical forces ("mechano-proliferative" response), whereas in Brachy vessels this early proliferation is effectively inhibited [25] (irradiation-mediated cell cycle arrest), but followed by a delayed proliferative response possibly induced by inflammatory processes within the vessel wall ("inflammato-proliferative" response). Voisard et al. showed in a co-culture model that inflammation triggers reactive proliferation of vascular smooth muscle cells [26]. This mechanism may be a key event in restenosis and arteriosclerosis. Experimental studies indicate a marked activation of inflammatory cells at the site of stent struts which are likely to play a key role in the process of neointimal proliferation and restenosis [27]. Ma and O'Brien showed that antagonism of the alpha4 integrin subunit attenuates the acute inflammatory response to stent implantation thereby inhibiting proliferation and preventing late intimal formation [28]. Farb et al. found that intimal growth accompanied by proliferation is maintained for 6 to 12 months after beta-irradiation [29]. Kaluza et al. observed a late catch-up of proliferation following beta-irradiation in porcine coronary arteries [30]. The authors found that the initial inhibition of neointimal formation was not sustained. Instead, the neointimal areas of irradiated and non-irradiated vessels were similar at 6 months. Although healing occurred in irradiated vessels, Kaluza postulated delayed re-endothelialization and increased inflammation as the trigger. Hence, the delayed proliferation observed in our study in the Brachy group – from almost zero proliferation on day 1 to nearly PTCA proliferation levels on days 14 and 28 – seems to be a consequence of the observed inflammatory processes and the increased transcription factor activity within the vessel wall. However, the causal connection remains to be proven. Moreover, the DNA binding activity of AP-1 does not match the proliferation levels observed, since AP-1 is expected to induce proliferation in vascular smooth muscle cells [31]. However, various other factors are under the control of AP-1, e.g. proinflammatory cytokines and chemokines, and recently MMP-9 expression was also shown to be regulated via AP-1 [32–35].

Our histomorphometric data show that negative arterial remodeling (= reduction of the EEL area) and not the increase in I + M area accounts for the late lumen loss after brachytherapy. Along with this late lumen loss, we observed adventitial thickening ("neoadventitial formation"). In accordance, in the PTCA group proliferating cells at day 28 were primarily found in the I + M, whereas in the Brachy group the adventitia was relatively more affected. Coussement et al. also observed increased lumen loss in irradiated balloon-injured porcine coronaries which was associated with adventitial thickening and fibrosis [36]. Wexberg et al. even indicated a correlation between radiation dosage and adventitial thickening [37]. Andersen et al. found impressive neoadventitial formation 3 weeks after intervention, and hypothesized that one mechanism of restenosis could be circumferential neoadventitial shrinkage, "strangulating" the artery to late luminal narrowing [38]. Physiological mechanisms proposed for adventitial thickening include changes in extracellular matrix (ECM) composition. Normal uninjured coronary arteries consist of a thin adventitial layer with a relatively high percentage of elastin, and balloon injury generally causes formation of a neoadventitia with a relative reduction of elastin content. The role of adventitial collagen is controversially discussed: Kingston et al. reported that a dense collagenous adventitia might prevent constrictive remodeling by acting as an external scaffold, whereas Ward et al. observed that suppressed constrictive remodeling is associated with a decrease in collagen accumulation [39,40]. However, it is possible that the gross amount of adventitial collagen does not determine arterial remodeling, but rather the spatial arrangement and cross-link of collagen fibrils and the changes in collagen type distribution (eg, altered collagen I/collagen III ratio). The observed early inflammatory response of the neoadventitia (macrophage infiltration at day 1 after intervention) is considered to be an initiator of new tissue formation in the arterial wound and important for restructuring of the ECM (removal of accruing ECM fragments) [41]. In the Brachy group we observed a prolonged neoadventitial macrophage infiltration, possibly indicating acceleration of reparative processes in this group. This assumption is supported by the significant increase in local T cell response in the neoadventitia of brachytherapy treated arteries at day 28 after intervention.

Vessel wall remodeling is characterized by a disruption in the delicate balance between ECM deposition and degradation, involving matrix metalloproteinases and their inhibitors. Matrix metalloproteinase 9 has been associated with arterial remodeling. Although most papers relate MMP-9 upregulation to arterial enlargement (or even aneurysm formation), Galis et al. showed that targeted disruption of MMP-9 in mice is associated with impaired smooth muscle cell migration and geometrical arterial remodeling [42]. They used a carotid artery ligation model to induce arterial remodeling and found that – at the latest time point investigated – the carotids of MMP-9 knockout mice had significantly larger lumen. Hence, MMP-9 deficiency decreased the late (28 days) lumen loss and reduced intimal thickness in the remodeling mouse carotid artery, suggesting a pivotal role of MMP-9 in late lumen loss. Concordantly, in our study, enhanced lumen loss was associated with abundant MMP-9 expression. This observation raises the hypothesis that increased MMP-9 expression, a target of NF-{kappa}B and AP-1, contributed to the changes we observed. Accordingly, strong NF-{kappa}B and AP-1 activation was found up to 1 month after brachytherapy compared to PTCA and Controls. Moreover, it is conceivable that the increased macrophage content found in Brachy vessels also contributed to the abundance of MMP-9 protein [43].

Our study is necessarily limited by various factors. In their commentary, Fischell and Virmani pointed out differences between the porcine model and humans [44]. Animal models usually do not include vessels with atherosclerosis, and the differential healing rate between species is not the same. Finally, animal models are not statin-treated. Statins have been shown to decrease endothelial activation after irradiation and to decrease the resulting inflammatory and thrombotic responses [45]. In addition, we used only one irradiation dose. Moreover, other forms of radiation therapy – other than the beta-irradiation we studied – may show utility in the future. For instance, external beam radiation was reported as promising for femoral artery lesions [46].

Drug eluting stents (also an antiproliferative therapy) may limit the necessity for brachytherapy in the future for several reasons. However, it is unknown actually if implanted DES may not cause similar problems in the long run. Recent data indicate that there might be an inflammatory reaction associated with DES, either due to the polymers, the drug coatings themselves, or perhaps both. Animal models confirmed the presence of a very late inflammatory response that may induce or at least promote thrombosis. Virmani et al. were the first to describe localized hypersensitivity vasculitis in response to a Cypher coronary stent resulting in an acute myocardial infarction secondary to late in-stent thrombosis [47]. They found pathological evidence for polymer fragments surrounded by giant cells and eosinophils, so that a reaction to the polymer was the most likely mechanism.

For the time being, brachytherapy is still the only evidence-based treatment for in-stent restenosis. Therefore, experimental research in brachytherapy offers an excellent tool to study molecular mechanisms of the pathophysiology of restenosis after an antiproliferative therapy. This research may help to identify potential risks and pitfalls that – partially – also apply to the use of DES and may allow for the establishment of preventive strategies (e.g. anti-inflammatory treatment at later stages after intervention) for a better long term result after coronary intervention [48,49].

Finally, our data suggest that brachytherapy, and possibly DES as well, may warrant revision. Although immediate cell proliferation initially is markedly inhibited by brachytherapy, late proliferation nonetheless occurs, negative arterial shrinkage is observed, the infiltration of inflammatory cells is sustained, and pro-inflammatory as well as pro-proliferative transcription factors are induced.

Time for primary review 27 days


   Notes
 
1 Authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

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