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Cardiovascular Research 1999 44(1):156-165; doi:10.1016/S0008-6363(99)00175-3
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Copyright © 1999, European Society of Cardiology

Temporal and spatial distribution of interleukin-1β in balloon injured porcine coronary arteries

Janet Chamberlain*, Julian Gunn, Sheila Francis, Cathy Holt and David Crossman

Cardiovascular Medicine, Section of Medicine, Division of Clinical Sciences (NGH), University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK

* Corresponding author. Tel: +44-114-271-4004; fax: +44-114-261-9587 j.chamberlain{at}sheffield.ac.uk

Received 15 March 1999; accepted 4 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Interleukin-1β (IL-1β) is a proinflammatory cytokine with a wide range of biological activities. We determined the distribution of IL-1β following percutaneous transluminal coronary angioplasty (PTCA) of porcine arteries, and the presence of caspase-1 (required for the activation of IL-1β). Methods: Oversized balloon angioplasty was performed in Yorkshire White pigs and the vessels excised at selected intervals (1, 6, 18 h, 3, 7, and 14 days) post-PTCA. IL-1β and caspase-1 were then identified using reverse transcription polymerase chain reaction (RT-PCR), in situ RT-PCR, and immunohistochemistry. Results: IL-1β protein was detected in the inflammatory infiltrate up to 3 days after PTCA. Luminal endothelial cells contained IL-1β at 1 h, with peak expression at 3–7 days. Adventitial capillaries were IL-1β-positive at all timepoints. IL-1β was detected in adventitial cells at 3 days, with reduced levels at 7 and 14 days. At 7 days, neointimal cells were also IL-1β positive. No IL-1β was detected in non-PTCA control arteries. RT-PCR demonstrated IL-1β mRNA expression to be induced at 1 h, and absent at 3 days. In situ RT-PCR revealed this expression to be distributed throughout the arterial layers at 6 h, but localized to the adventitia at 18 h, with a baseline expression in the adventitial layer of non-PTCA controls. Caspase-1 was detected in luminal endothelial cells from 6 h, in adventitial cells from 3 days, and in neointimal cells from 7 days post-PTCA. This expression colocalized with IL-1β, indicating the potential for the IL-1β present to become activated. Conclusions: We conclude that IL-1β is synthesised, in conjunction with caspase-1, by endothelial, inflammatory, and adventitial cells early (within 3 days) after PTCA, with decreased levels at later timepoints, suggesting that it has a key role to play in the early stages of healing following PTCA.

KEYWORDS IL-1β=interleukin-1β; ICE=interleukin-1 converting enzyme/caspase-1; PTCA=percutaneous transluminal coronary angioplasty; VSMC=vascular smooth muscle cell; IEL=internal elastic lamina; ABC=avidin biotin complex; DAB=3,3'diaminobenzidine tetrahydrochloride; DBA lectin=Dolichos biflorus lectin; {alpha}SMA={alpha}-smooth muscle actin; PCNA=proliferating cell nuclear antigen

This article is referred to in the Editorial by B.S. Oemar (pages 17–19) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Interleukin-1β (IL-1β) is an inflammatory cytokine which mediates a variety of processes in host defence, inflammation, and response to injury [1–4]. It is primarily a product of activated macrophages, but can also be produced by a number of other cell types including monocytes, neutrophils, fibroblasts, T-lymphocytes, endothelial, and vascular smooth muscle cells. IL-1β is synthesised as a large 31 kD precursor molecule, which requires specific cleavage to a 17.5 kD form for biological activity. Caspase-1/interleukin-1β converting enzyme (ICE), an intracellular protease and a member of the cysteine protease family, specifically performs this cleavage [5].

Due to alternate splicing of its RNA, there are five isoforms of (human) caspase-1 (ICE{alpha}, β, {gamma}, {delta}, {varepsilon}) [6]. Of these, it is believed that only the ICE{alpha} isoform directly cleaves pro-IL-1β, though there is no data for ICE{varepsilon}. However, all the caspase-1 isoforms are involved with the cleavage of the caspase-1 precursor, which is required for activation and secretion of IL-1β. Caspase-1 has a debated role in apoptosis [7,8], although the cleavage of pro-IL-1β by caspase-1 may appear to be quite separate to this role [9,10].

IL-1β has a number of functions in vascular cells, which could be linked to atherogenesis. IL-1β inhibits the growth of endothelial cells [2] and stimulates their production of extracellular matrix proteins [11], affecting procoagulant events [12,13], and leukocyte adherence to the endothelium [14–16]. It also stimulates the proliferation of vascular smooth muscle cells (VSMCs) [17–19], is involved in low density lipoprotein metabolism [20,21], promotes vascular permeability [22], and suppresses vascular contractility [23]. Treatment of porcine arteries with IL-1β induces intimal lesions [24]. IL-1β mRNA has been detected in human atherosclerotic plaque [25], and IL-1β protein in cultured human atheromatous macrophages from carotid arteries [26]. IL-1β mRNA and protein have also been demonstrated in VSMCs, endothelium, and macrophages in atherosclerotic arteries from non-human primates [27,28] and in coronary arteries of patients with ischaemic heart disease [29]. However, to date, the IL-1β profile of response has not been documented following other forms of arterial injury, in particular balloon dilatation.

The aims of this study were, therefore, to identify the temporal and spatial distribution of IL-1β in developing stenotic lesions in a porcine model of percutaneous transluminal coronary angioplasty (PTCA), and to correlate the presence of IL-1β with the expression of caspase-1.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Porcine coronary angioplasty model
Coronary artery injury was induced by oversized balloon angioplasty of both the left anterior descending and the right coronary artery in Yorkshire White pigs (Sus scrofa), 25–30 kg in weight, as previously described [30]. The investigation conforms with all UK Home Office regulations. The site of balloon inflation was selected such that the balloon/artery ratio was 1.25:1, as assessed by quantitative angiography. At selected intervals (1, 6, 18 h, 3, 7, 14 days) post-PTCA, the angioplastied vessels were identified and excised, preserving the adventitia. The circumflex coronary artery was also explanted, as an untreated control, from each animal. Three animals were sacrificed at each timepoint, yielding 35 angioplastied coronary arteries (6 per timepoint) plus 18 control arteries (3 per timepoint). All arteries were serially cross-sectioned into 2–3 mm thick blocks (12–14 per artery) and preserved in liquid nitrogen, or formalin-fixed and embedded into paraffin wax, prior to subsequent analysis.

2.2 Histology/quantitative morphology
Sequential, formalin-fixed, paraffin wax-embedded transverse coronary artery sections, 4 µm thick, were cut and representative sections from each block stained with haematoxylin and eosin (H & E) or Alcian blue, Miller’s elastic and van Gieson’s stains to determine the location and extent of injury. Arterial blocks with maximum balloon injury, defined as maximum disruption in the internal elastic lamina (IEL) [31,32] were identified and, together with sections from undilated arteries (controls), were used for analysis. Sections from apparently undamaged areas of angioplastied artery were not analysed because of the possibility of unseen damage caused by the angioplasty catheter affecting the interpretation of data.

2.3 Reverse transcription polymerase chain reaction (RT-PCR)
RNA was isolated from 3–5 pooled segments of control and angioplastied arteries from each timepoint using the one-step phenol/chloroform method of Chomczynski and Sacchi [33]. RNA was isolated from each artery and timepoint separately, yielding six RNA samples of PTCA and three RNA samples of control artery per timepoint for analysis. Briefly, tissue was ground to a fine powder under liquid nitrogen before being transferred into RNAzol (Biogenesis). RNA was precipitated, the pellets were resuspended in sterile water, and checked for purity and yield by spectrophotometry. IL-1β RNA was identified using RT-PCR with β-actin as a control gene transcript. Total RNA (3 µg) was reverse-transcribed using standard techniques, and RNA:cDNA hybrids used immediately as template for RT-PCR. For PCR, each 25 µl reaction contained 5 µl cDNA. Negative controls, substituting water for RNA and cDNA, and the omission of RT or Taq enzymes, were performed to exclude genomic DNA contamination. Thermal cycling consisted of 1 min denaturation at 95°C, 1 min annealing at 55°C (porcine IL-1β), or 60°C (β-actin), and 1 min extension at 72°C. Primer sequences were as follows: porcine IL-1β, 5' TCA TCG TGG CAG TGG AGA AGC 3' (forward), 5' TCT GGG TAT GGC TTT CCT TAG 3' (reverse), 619 base pair (bp) predicted product size; β-actin, 5' CTC GGT CAG GAT CTT CAT GAG G 3' (forward), 5' TTC TAC AAT GAG CTG CGT GTG G 3' (reverse), 324 bp predicted product size. The identity of both product was confirmed by dideoxy sequencing. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining under UV transillumination. Densitometric analysis of bands was performed using NIH image. Each band was normalised to the relevant β-actin control.

2.4 In situ RT-PCR
Paraffin-embedded sections, 4 µm thick, were cut under RNase free conditions and used for in situ RT-PCR analysis. Sections were dewaxed in xylene and rehydrated through a series of graded alcohols to RNase-free water before being digested with 0.5 mg/ml proteinase K for 20 min at 37°C. Digestion was stopped by washing in 100 mmol/l sodium chloride/100 mmol/l Tris, pH 7.6. The sections were then dehydrated in alcohol prior to treatment with 1 U/µl RNase-free DNase and 2 U/µl RNasin overnight at 37°C. DNase was removed by washing in RNase-free water, followed by dehydration of the sections in 100% alcohol prior to the RT-PCR reaction. Twenty-five µl of RT-PCR mix [300 µmol/l each dNTP, 1xEZ buffer (Perkin Elmer), 2.5 mmol/l Mn(OAc)2 (Perkin Elmer), 20 µmol/l dig-UTP (Boehringer Mannheim), 0.1 U/µl rTth DNA polymerase (Perkin Elmer), 0.45 µmol/l each primer] was added to each section, and the sections were then coverslipped (Hybaid Easiseal coverslips). Thermal cycling consisted of a 60 min reverse transcription at 70°C, 2 min initial denaturation at 94°C, 30 cycles of 1 min denaturation at 94°C and 1 min combined annealing and extension at 60°C, before a final step of 10 min at 60°C. The sections were then washed in a solution of 0.5% (w/v) bovine serum albumin in 0.1xSSC (0.15 M NaCl, 0.015 M Na-citrate, pH 7.0) for 5 min at 37°C, followed by an antibody-blocking step of 15 min incubation in 5% (v/v) normal sheep serum. Dig-UTP incorporation was detected by incubating the sections with anti-digoxygenin alkaline phosphatase Fab fragments (Boehringer Mannheim) for 30 min at room temperature, and visualised by incubation with nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate substrate (Biogenix) for 10 min. The sections were then counterstained with nuclear fast red and examined under light microscopy.

Several controls were applied with this technique. Negative controls involved incubating the sections with both DNase and RNase prior to the thermocycling reaction, or the omission of rTth enzyme. The positive control involved the omission of the DNase pretreatment step, to allow the amplification of genomic DNA.

2.5 Immunostaining for IL-1β and caspase-1
Sequential, 4 µm thick paraffin-embedded sections were immunohistochemically stained for IL-1β or caspase-1 using standard techniques. Briefly, sections were dewaxed and rehydrated, endogenous peroxidases blocked by incubation with 3% hydrogen peroxide, and nonspecific antibody binding blocked by incubation with a 1% (w/v) dried milk/phosphate buffered saline (PBS) solution prior to incubation with the primary antibody. For IL-1β immunostaining, a microwave oven antigen-unmasking step, (10 mmol/l citrate buffer, pH 6, 2x5 min, 700 W) was also required. Primary antibodies were applied for 2 h at room temperature (22°C), using 0.5% (w/v) dried milk/PBS as diluent. Sections were washed with PBS prior to incubation with the appropriate biotinylated secondary antibody, followed by avidin biotin complex (ABC). Finally, antibody binding was visualized with 3,3'diaminobenzidine tetrahydrochloride (DAB) and counterstained with Carazzi’s haematoxylin.

2.6 Immunostaining for phenotypic/proliferation markers
Sequential sections from each timepoint following PTCA were stained to identify the phenotype of cells containing IL-1β. Mac-387, {alpha}-smooth muscle actin ({alpha}SMA), vimentin, and desmin were used as markers for macrophages/granulocytes, VSMCs, and fibroblasts/myofibroblasts respectively. As specific luminal endothelial cell markers are not available for porcine tissue, lectin from Dolichos biflorus (DBA lectin, horse gram) was used to identify luminal/adventitial capillary endothelial cells. Proliferating cell nuclear antigen (PCNA) staining was performed to identify proliferating cells. Standard techniques were employed, using ABC to amplify the signal, and DAB to detect the antigen, as described above. For mac-387 staining, a trypsinization antigen unmasking step [0.1% (w/v) trypsin in Tris buffer pH 7.8, 10 min, 37°C] was required prior to incubation with the primary antibody. For PCNA staining, a microwave antigen-unmasking step, as described above, was required. Primary antibody incubations were performed for 1 h at room temperature (22°C), with the exception of PCNA, which was performed overnight at 4°C. For DBA lectin staining, sections were incubated with 10 µg/ml of horse radish peroxidase-conjugated lectin (Sigma) for 30 min. Positive staining was then visualized using DAB substrate. All slides were counterstained with Carazzi’s haematoxylin.

2.7 Antibodies
The primary antibodies used are listed: goat anti-human IL-1β (R and D Systems, cat no. AB-201-NA, recognising both active and inactive forms, 1:50 dilution); rabbit anti-human pan-caspase-1 (R105, gift from D. Miller, Merck, 1:50 dilution); mac-387 (Dako, 1:100 dilution); mouse anti-human {alpha}SMA (Dako, 1:150 dilution); goat anti-human vimentin (Sigma, 1:150 dilution); rabbit anti-human desmin (Sigma, 1:150 dilution); mouse anti-PCNA (Dako, 1:125 dilution). Secondary antibodies were: biotinylated goat anti-rabbit IgG (H+L), biotinylated horse anti-mouse IgG (H+L), and biotinylated rabbit anti-goat IgG (H+L) (all from Vector). For the tertiary layers, an Elite ABC kit (Vector) and a StreptABComplex/AP kit (Dako) were used.

The caspase-1 antibody used in this study is not commercially available and has not been fully characterised. Therefore, the specificity of the R105 caspase-1 antibody clone was confirmed by Western blot analysis of porcine VSMCs. A single distinct band corresponding to the 45 kD unprocessed form of caspase-1 was visualized. No nonspecific bands were observed (data not shown).

2.8 Immunostaining controls
For all immunohistochemistry experiments, negative controls were performed. These consisted of sections incubated with the omission of primary antibody (substituting antibody diluent or the appropriate non-immune IgG in each case). Only negative control sections stained with non-immune IgG gave some slight nonspecific (noncellular, extracellular matrix) staining. For dual staining experiments, sections were incubated without the first and/or second primary antibody.

2.9 Examination and analysis of immunostaining
The proportion of luminal endothelial, inflammatory, neointimal, adventitial cells and adventitial vessels that were positively stained for IL-1β or caspase-1 was determined and graded arbitrarily from 0 to +++ as follows: 0=negative; +=<40% cells stained positive; ++=approximately 40–60% positive; +++=>60% positive. Only distinct, cellular staining was scored as "positive". Staining which did not have a clear cellular location was deemed "negative", to avoid any confusion with nonspecific staining seen with the use of polyclonal antibodies. Scoring was performed by four observers (JC, JG, SEF, CMH).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Detection and localization of IL-1β
IL-1β mRNA, assessed by RT-PCR, was induced 1 h after PTCA, with a peak (12-fold) induction detectable at 6 h post-PTCA. IL-1β mRNA was not detected 3 days after PTCA, or in non-angioplastied control arteries (Fig. 1). In situ RT-PCR, however, revealed a baseline expression (approximately <25%) of IL-1β mRNA in the adventitia of non-angioplastied arteries (Fig. 2a). At 6 h, this expression was widespread throughout the artery wall, including the media, adventitia, and inflammatory cells [Fig. 2b and c]. At 18 h, IL-1β mRNA expression was only detected in the adventitia and inflammatory cells. No IL-1β mRNA was detected at 3 days post-PTCA. The identity of arterial and inflammatory cells expressing IL-1β in these sections was confirmed by comparison with sequential sections stained immunohistochemically for phenotypic markers. Specificity of the in situ reaction was confirmed by the fact that mRNA expression is undetectable in the negative controls (template removed by incubation with both RNase and DNase, or the reaction performed with the omission of rTth enzyme) (Fig. 2d), and increased IL-1β expression (amplifying both genomic DNA and mRNA) in the positive control.


Figure 1
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  		Fig. 1 (a) RT-PCR analysis of IL-1β mRNA expression in porcine coronary arteries after PTCA, with β-actin loading control. (b) Graphical representation of IL-1β mRNA levels, relative to β-actin mRNA, 6 h post-PTCA and in non-injured control arteries. Data is representative of three independent RT-PCR reactions.

 

Figure 2
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  		Fig. 2 IL-1β in situ RT-PCR of noninjured control arteries (a) showing low level, expression localized to the adventitia. 6 h after PTCA, expression is widespread throughout all arterial layers (b), and the inflammatory cells (c). No expression was detected in sections in the absence of rTth DNA polymerase (d). Scale bar=20 µm.

 
IL-1β protein, detected immunohistochemically, was present in luminal endothelial cells (where present) at 1 h after PTCA, with a peak in expression at 3–7 days (Fig. 3). IL-1β was also detected in the endothelial cells of adventitial vessels, reaching a peak at 3 days. Luminal endothelial cells of non-angioplastied arteries did not contain IL-1β (Fig. 3), though it was detected in the endothelial cells of some adventitial vessels. Adventitial cells, presumably fibroblasts, did not contain IL-1β until 3 days after PTCA. Positively stained adventitial fibroblasts were localized mainly around the area of breach in the IEL (Fig. 4), more strikingly directly under thrombus. This staining pattern persisted, though was less intense, at 7 and 14 days after PTCA. Colocalization of IL-1β-positive adventitial cells with {alpha}SMA-positive adventitial cells was not seen. All adventitial cells were vimentin-positive (data not shown). IL-1β was detected in the neointima of all angioplastied arteries at 7 and 14 days, colocalizing with {alpha}SMA. No IL-1β was detected in the medial layer. Between 1 h and 3 days after PTCA, IL-1β was also present in the luminal and adventitial inflammatory cell infiltrate. Most IL-1β-positive inflammatory cells were monocytes/granulocytes. Most cells, of all phenotypes, which were IL-1β-positive were also PCNA-positive, as were cells surrounding IL-1β-positive stained cells (data not shown).


Figure 3
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  		Fig. 3 Immunohistochemical detection of IL-β and caspase-1 protein expression in porcine coronary arteries after PTCA. Luminal endothelial cells contain IL-1β (a) and caspase-1 (c) at 3 days post-PTCA. IL-1β (b) and caspase-1 (d) are absent in luminal endothelial cells of control, non-angioplastied arteries. Scale bar=10 µm.

 

Figure 4
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  		Fig. 4 Immunohistochemical detection of IL-β and caspase-1 protein expression in porcine coronary arteries after PTCA. Adventitial cells contain IL-1β (a) and caspase-1 (b) at 3 days post-PTCA. IL-1β is absent from the adventitia of non-injured control vessels (c). Scale bar=10 µm. (d) H & E stained section, 3 days post-PTCA, outlining the area depicted in pictures A and B.

 
In all cases, negative control sections did not stain for IL-1β. The immunostaining controls showed that no endogenous biotins, free radicals, phosphatases, or peroxidases were present in these sections as the detection techniques for these (ABC, DAB, New Fuschin) all failed to give a positive stain result when the primary antibody was omitted. Therefore, any positive staining seen is solely the result of binding of the primary antibody to the sections. Table 1 shows the histological scoring of IL-1β-stained sections.


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  		Table 1 Representation of immunohistochemical staining for IL-1β protein in arterial sections taken from porcine coronary arteries at selected time intervals after PTCAa

 
3.2 Detection and localization of caspase-1
Caspase-1, detected immunohistochemically, appeared to colocalize to IL-1β-positive stained cells. Caspase-1 was present in luminal endothelial cells (where present) at 6 h after PTCA, and at all subsequent timepoints (Fig. 3), reaching a peak at 14 days. Caspase-1 was also present in the endothelial cells of adventitial vessels, reaching a peak at 18 h. Luminal endothelial cells of non-angioplastied arteries did not contain caspase-1. Caspase-1 staining peaked at 3 days after PTCA in the adventitia. Positively stained adventitial fibroblasts were localized mainly around the area of breach in the IEL, especially when thrombus was present (Fig. 4). This staining pattern persisted, though was less intense, at 7 and 14 days after PTCA. Caspase-1 was detected in the neointima of all angioplastied arteries at 7 and 14 days. Caspase-1 localized to the media of all arteries, including non-angioplastied controls, though the staining intensity did not alter significantly at any particular timepoint. Between 1 h and 3 days after PTCA, caspase-1 was present in the luminal and adventitial inflammatory cell infiltrate. Table 2 shows histological scoring of the timecourse sections for caspase-1.


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  		Table 2 Representation of immunohistochemical staining for caspase-1 protein in arterial sections taken from porcine coronary arteries at selected time intervals after PTCAa

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The main findings of this observational study are the rapid induction of IL-1β and caspase-1 following PTCA. IL-1β mRNA is present in coronary arteries within 1 h, and persists for at least 18 h, after PTCA in a porcine model. mRNA is expressed throughout the artery wall after PTCA, with some expression also detected in inflammatory cells. IL-1β protein, detected immunohistochemically, is localized to the luminal endothelium with peak expression between 3 and 14 days post-PTCA. Adventitial cells, presumed to be fibroblasts, located near to thrombi or the breach in IEL produce IL-1β by 3 days post-PTCA. IL-1β protein is not detectable in medial SMCs. Whenever present, infiltrating inflammatory cells express IL-1β.

IL-1β has previously been described in luminal and adventitial vessel endothelial cells and macrophages in coronary arteries from patients with ischaemic heart disease [29], in foam cells, SMCs, and endothelium of diet-induced iliac artery atherosclerotic plaques in monkeys [25], in endothelium and neointima of a rat vein graft model [34], and in atherectomy specimens from a rabbit model of restenosis [35]. However, this is the first study to investigate the temporal and spatial distribution of IL-1β in developing stenotic lesions following PTCA.

Following PTCA, IL-1β protein is localized to inflammatory cells and luminal endothelium (where present) within 1 h. Inflammatory cells also express IL-1β mRNA at these early timepoints. It is likely that luminal endothelial cells will also express IL-1β mRNA, although all the sections examined in situ were denuded of their endothelium and, therefore, we are unable to confirm this hypothesis. Such early expression of IL-1β may induce further cytokine expression and promote the cascade of signal pathways culminating in intimal hyperplasia. IL-1β stimulates vascular endothelium to produce more IL-1β in an autocrine loop [36]. This localized IL-1β may act to alter many endothelial cell functions. Endothelial cells exposed to IL-1β express adhesion molecules, leading to adherence of leukocytes on their surface [14–16]. The IL-1β detected within endothelial cells would also be capable of acting on neighbouring VSMCs, inducing adhesion molecule expression and enhancing leukocyte adhesion [37,38]. The adherence of leukocytes to the endothelium has previously been shown to be important in the development of atherosclerosis [39], and may have a role in restenosis. IL-1β also acts on the endothelium to impair the fibrinolytic process, causing thrombosis and coagulation [12,13,40]. In addition, the synthesis of prostaglandins and platelet activating factor by endothelial cells and VSMCs are stimulated by IL-1β in vitro [41,42].

An increase in capillary vessels has been documented in atherosclerosis [43] and after experimental PTCA [44], and the present study has demonstrated IL-1β expression in the endothelial cells of these capillaries. The degree of neovascularization has been correlated with the degree of injury following PTCA [44] and with intimal hyperplasia [45]. Neovascularization after PTCA may be the result of activated endothelial cells. IL-1β identified in adventitial capillaries may be involved in neovascularization since it stimulates the production of other cytokines and growth factors [46], e.g., fibroblast growth factor and transforming growth factor-β, which are known to induce angiogenesis [45,47–50].

IL-1β expression by adventitial capillaries may also be responsible for the large inflammatory cell infiltrate in the adventitia. As with luminal endothelium, IL-1β would stimulate adhesion molecule expression, resulting in the adherence and recruitment of inflammatory cells through the capillary vessel wall and into the adventitia. Adventitial inflammation surrounding atherosclerotic vessels is common, and has been correlated with the severity of intimal disease [51–54].

The polyclonal antibody used to detect IL-1β in this study recognises both processed and unprocessed IL-1β. However, staining for IL-1β shows a clear cellular localization for this protein, and appeared to colocalize with caspase-1. As IL-1β cannot be activated or secreted without caspase-1 [9,10], the apparent colocalization of these two proteins suggests that the IL-1β detected in this study is likely to be active. Like IL-1β, caspase-1 can be detected in the inflammatory infiltrate within 1 h after PTCA, in the endothelium by 6 h, and is still present in the artery wall (endothelium, neointima, media, and adventitia) at 14 days, suggesting that IL-β is active during all stages of healing.

In conclusion, this study has shown that IL-1β is present in luminal endothelial cells, adventitial fibroblasts, and inflammatory cells in coronary arteries within hours after PTCA. Locally generated IL-1β may, therefore, contribute to the vascular response to balloon injury. Inhibiting the action of IL-1β after PTCA may reduce the response to this injury and, thus may have therapeutic implications for restenosis.

Time for primary review 20 days.


   Acknowledgements
 
This study was supported by a grant from the University of Sheffield Research Fund.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Dinarello C.A. Biology of interleukin 1. FASEB J (1988) 2:108–115.[Abstract]
  2. di Giovine F.S., Duff G.W. Interleukin 1: the first interleukin. Immunol Today (1990) 11:13–20.[CrossRef][ISI][Medline]
  3. Duff G.W. Peptide regulatory factors in non-malignant disease. Lancet (1989) 1:1432–1435.[ISI][Medline]
  4. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature (1993) 362:801–809.[CrossRef][Medline]
  5. Black R.A., Kronheim S.R., Cantrell M., et al. Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor. J Biol Chem (1988) 263:9437–9442.[Abstract/Free Full Text]
  6. Alnermri E.S., Fernandes-Alnermri T., Litwack G. Cloning and expression of four novel forms of human interleukin-1β converting enzyme with different apoptotic activities. J Biol Chem (1995) 270:4312–4317.[Abstract/Free Full Text]
  7. Henkart P.A. ICE family proteases: mediators of all apoptotic cell death? Immunity (1996) 4:195–201.[CrossRef][ISI][Medline]
  8. Chinnaiyan A.M., Dixit V.M. The cell death machine. Curr Biol (1996) 6:555–562.[CrossRef][ISI][Medline]
  9. Li P., Allen H., Banerjee S., et al. Mice deficient in IL-1β-converting enzyme are defective in production of mature IL-1β and resistant to endotoxic shock. Cell (1995) 80:401–411.[CrossRef][ISI][Medline]
  10. Kuida K., Lippke J.A., Ku G., et al. Altered cytokine export and apoptosis in mice deficient in interleukin-1β converting enzyme. Science (1995) 267:2000–2002.[Abstract/Free Full Text]
  11. Montesano R., Mossaz A., Ryser J.-E., Orci L., Vasseli P. Leukocyte interleukins induce cultured endothelial cells to produce a highly organised glycosaminoglycan-rich pericellular matrix. J Cell Biol (1984) 99:1706–1715.[Abstract/Free Full Text]
  12. Bevilacqua M.P., Pober J.S., Majeau G.R., et al. Recombinant tumour necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparisons with the actions of interleukin 1. Proc Natl Acad Sci USA (1986) 83:4533–4537.[Abstract/Free Full Text]
  13. Stern D.M., Bank I., Nawroth P.P., et al. Self-regulation of procoagulant events on the endothelial cell surface. J Exp Med (1985) 162:1223–1235.[Abstract/Free Full Text]
  14. Bevilacqua M.P., Pober J.S., Wheeler M.E., Cotran R.S., Gimbrone M.A. Interleukin-1 acts on cultured human vascular endothelium to increase adhesion of polymorphonuclear leukocytes, monocytes and related leukocyte cell lines. J Clin Invest (1985) 76:2003–2011.[ISI][Medline]
  15. Schliemer R.P., Rutledge B.K. Cultured human endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin-1, endotoxin and tumour-promoting phorbol diesters. J Immunol (1986) 136:649–654.[Abstract]
  16. Cavender D.E., Haskard D.O., Loseph B., Ziff M. Interleukin 1 increases the binding of human B and T lymphocytes to endothelial cell monolayers. J Immunol (1986) 136:203–207.[Abstract]
  17. Libby P., Warner S.J.C., Friedman G.B. Interleukin-1: a mitogen for human vascular smooth muscle cells that induces the release of growth inhibitory prostanoids. J Clin Invest (1988) 81:487–498.[ISI][Medline]
  18. Libby P., Wyler D.J., Janicka M.W., Dinarello C.A. Differential effects of human interleukin-1 on growth of human fibroblasts and vascular smooth muscle cells. Arteriosclerosis (1985) 5:186–191.[Abstract/Free Full Text]
  19. Bonin P.D., Fici G.J., Singh J.P. Interleukin-1 promotes proliferation of vascular smooth muscle cells in co-ordination with PDGF or a monocyte-derived factor. Exp Cell Res (1989) 181:475–482.[CrossRef][ISI][Medline]
  20. Ku G., Thomas C.E., Akeson A.L., Jackson R.L. Induction of interleukin-1 beta expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol Chem (1992) 267:14183–14188.[Abstract/Free Full Text]
  21. Rasmussen L., Seljelid R. The modulatory effects of lipoprotein on the release of interleukin-1 by human peritoneal macrophages stimulated by WF-1, 3-D polyglucose derivatives. Scand J Immunol (1989) 29:477–484.[CrossRef][ISI][Medline]
  22. Martin S., Murata K., Bukart V., Gillis S., Kolb H. Interleukin-1 and IFN-gamma increase vascular permeability. Immunology (1988) 64:301–305.[ISI][Medline]
  23. Mckenna T.M., Reusch D.W., Simpkins C.O. Macrophage-conditioned medium and interleukin 1 suppress vascular contractility. Circ Shock (1988) 25:187–196.[ISI][Medline]
  24. Shimokawa H., Ho A., Fukumoto Y., et al. Chronic treatment with interleukin-1β induces coronary intimal lesions and vasospastic responses in pigs in vivo. J Clin Invest (1996) 97:769–776.[ISI][Medline]
  25. Wang A., Doyle M., Mark D. Quantitation of mRNA using the polymerase chain reaction. Proc Natl Acad Sci USA (1989) 86:9717–9721.[Abstract/Free Full Text]
  26. Tipping P.G., Hancock W.W. Production of tumour necrosis factor and interleukin-1 by macrophages from human atheromatous plaques. Am J Pathol (1993) 142:1721–1728.[Abstract]
  27. Moyer C.F., Sajuthi D., Tulli H., Williams J.K. Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis. Am J Pathol (1991) 138:951–960.[Abstract]
  28. Ross R., Masuda J., Raines E.W., et al. Localization of PDGF-B protein in macrophages in all phases of atherogenesis. Science (1990) 248:1009–1012.[Abstract/Free Full Text]
  29. Galea J., Armstrong J., Gadson P., et al. Interleukin-1β in coronary arteries of patients with ischaemic heart disease. Arterioscler Thromb Vasc Biol (1996) 16:1000–1006.[Abstract/Free Full Text]
  30. Gunn J., Holt C.M., Francis S.E., et al. The effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima formation after porcine coronary angioplasty. Circ Res (1997) 80:520–531.[Abstract/Free Full Text]
  31. Shi Y., Pieniek M., Fard A., et al. Adventitial remodeling after coronary arterial injury. Circulation (1996) 93:340–348.[Abstract/Free Full Text]
  32. Schwartz R.S., Huber K.C., Murphey J.G., et al. Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. J Am Coll Cardiol (1992) 19:267–274.[Abstract]
  33. Chomczynski P., Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate phenol–chloroform extraction. Anal Biochem (1987) 162:156–159.[ISI][Medline]
  34. Faries P.L., Marin M.L., Veith F.J., et al. Immunolocalization and temporal distribution of cytokine expression during the development of vein graft intimal hyperplasia in an experimental model. J Vasc Surg (1996) 24:463–471.[CrossRef][ISI][Medline]
  35. Clausell N., Delima V.C., Molossi S., et al. Expression of tumor necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy. Br Heart J (1995) 73:534–539.[Abstract/Free Full Text]
  36. Warner S.J.C., Auger K.R., Libby P. Interleukin 1 induces interleukin 1, II: recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells. J Immunol (1987) 139:1911–1917.[Abstract]
  37. Wang X.K., Feuerstein G.Z., Clarke R.K., Yue T.L. Enhanced leukocyte adhesion to interleukin-1-beta stimulated vascular smooth muscle cells is mainly through intercellular-adhesion molecule-1. Cardiovasc Res (1994) 28:1808–1814.[Abstract/Free Full Text]
  38. Wang X.K., Feuerstein G.Z., Gu J.L., Lysko P.G., Yue T.L. Interleukin-1-beta induces expression of adhesion molecules in human vascular smooth-muscle cells. Atherosclerosis (1995) 115:89–98.[CrossRef][ISI][Medline]
  39. Cybulski M.I., Gimbrone M.A. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science (1991) 251:788–791.[Abstract/Free Full Text]
  40. Nachman R.L., Hajjar K.A., Silverstein R.L., Dinarello C.A. Interleukin 1 induces endothelial cell synthesis of plasminogen activator inhibitor. J Exp Med (1986) 163:1595–1600.[Abstract/Free Full Text]
  41. Dejana E., Breviaro F., Erroi A., et al. Modulation of endothelial cell function by different molecular species of interleukin 1. Blood (1987) 69:695–699.[Abstract/Free Full Text]
  42. Beasley D., Cohen R.A., Levinsky N.G. Interleukin 1 inhibits contraction of vascular smooth muscle. J Clin Invest (1989) 83:331–335.[ISI][Medline]
  43. Zhang Y., Cliff W.J., Schoefl G.I., Higgins G. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol (1993) 143:164–172.[Abstract]
  44. Chamberlain J., Gunn J., Francis S.E., et al. Increased adventitial capillary density after coronary arterial injury (abstract). Eur Heart J (1996) 17:195.
  45. Edelman E.R., Nugent M.A., Smith L.T., Karnovsky M.J. Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries. J Clin Invest (1992) 89:465–473.[ISI][Medline]
  46. Clinton S.K., Libby P. Cytokines and growth factors in atherogenesis. Arch Pathol Lab Med (1992) 116:1292–1300.[ISI][Medline]
  47. Thomas K.A., Rios-Candelore M.R., Gallego G.G., et al. Pure brain derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1. Proc Natl Acad Sci USA (1985) 82:6409–6413.[Abstract/Free Full Text]
  48. Offner F.A., Feichlinger H., Stadlmann S., et al. Transforming growth factor-β synthesis by human peritoneal mesothelial cells. Induction by interleukin-1. Am J Pathol (1996) 148:1679–1688.[Abstract]
  49. Yue T.L., Wang X.K., Olson B., Feuerstein G. Interleukin-1β (IL-1β) induces transforming growth factor-β1 (TGF-β1) production by rat aortic smooth muscle cells. Biochem Biophys Res Commun (1994) 204:1186–1192.[CrossRef][ISI][Medline]
  50. Roberts A., Sporn M., Assoian R., et al. TGFβ-rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA (1986) 83:4167–4171.[Abstract/Free Full Text]
  51. Schwartz C.J., Mitchell J.R.A. Cellular infiltration of the human arterial adventitia associated with atheromatous plaques. Circulation (1962) 26:73–78.[Abstract/Free Full Text]
  52. Parums D., Mitchinson M.J. Demonstration of immunoglobulin in the neighbourhood of advanced atherosclerotic plaques. Atherosclerosis (1981) 38:211–216.[CrossRef][ISI][Medline]
  53. Parums D., Chadwick D.R., Mitchinson M.J. The localization of immunoglobulin in chronic periaortitis. Atherosclerosis (1986) 61:117–123.[CrossRef][ISI][Medline]
  54. Naftilan A.J. Role of the tissue renin–angiotensin system in vascular remodeling and smooth muscle cell growth. Curr Opin Nephrol Hypertens (1994) 3:218–227.[CrossRef][Medline]

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W. F. Fearon and D. T. Fearon
Inflammation and Cardiovascular Disease: Role of the Interleukin-1 Receptor Antagonist
Circulation, May 20, 2008; 117(20): 2577 - 2579.
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