TNFα increases the inflammatory response to vascular balloon injury without accelerating neointimal formation

Atherosclerosis 179 (2005) 51–59

TNF␣ increases the inflammatory response to vascular balloon injury without accelerating neointimal formation Ashley M. Millera,1 , Allan R. McPhadenc , Anthony Prestona , Roger M. Wadswortha , Cherry L. Wainwrighta,b,∗ b

a Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G4 0NR, UK School of Pharmacy, The Robert Gordon University, Schoolhill, Aberdeen AB10 1FR, Scotland, UK c Department of Pathology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK

Received 22 January 2004; received in revised form 6 September 2004; accepted 8 October 2004 Available online 23 December 2004

Abstract There is now clear evidence for a contributory role of inflammatory processes to restenosis following vascular balloon injury and stent implantation. The aim of the present study was to study the effects of TNF␣, administered locally in vivo immediately following balloon angioplasty, on the leukocyte adhesive response and extent of neointimal formation in a rabbit model of subclavian artery injury. Initial in vitro studies were performed with normal isolated artery rings to assess the vascular adhesive response to TNF␣ or IL-1␤. Pre-incubation with either cytokine prior to addition of 51 Cr-labelled leukocytes enhanced the adhesion of leukocytes to the artery in both a time- and concentration-dependent manner. Although both cytokines induced an increase in the expression of the adhesion molecules ICAM-1 and VCAM-1, only antibodies to ICAM-1 blocked the enhanced adhesion induced by the cytokines. In artery segments retrieved from rabbits that had previously undergone subclavian artery angioplasty either 24 h or 8 days previously, there was an injury-induced increase in adhesion of leukocytes assessed ex vivo. In segments obtained from rabbits that received a 15 min local infusion of TNF␣ (2 ng/min) to the injured artery immediately after the angioplasty procedure, leukocyte adhesion assessed ex vivo was further significantly enhanced. The pro-adhesive effect of TNF␣ was associated with an increased expression of both ICAM-1 and VCAM-1. However, TNF␣ administration did not alter the extent of neointimal formation observed 8 days after injury. These findings suggest that while TNF␣ may play a role following vascular injury, it does not act alone to induce neointimal formation. Thus anti-inflammatory strategies targeted at multiple cytokines may be more appropriate than targeting a single cytokine to reduce the response to vascular injury. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Leukocyte adhesion; TNF␣; IL-1␤; Thrombin; Leukocyte adhesion molecules; Neointimal formation; Balloon angioplasty

1. Introduction There is now substantial evidence that inflammatory processes play an important role in the response to vascular wall injury, which ultimately results in the formation of neointima following both balloon angioplasty [1] and stent implantation [2]. The infiltration of leukocytes [3], and particularly neu∗

Corresponding author. Tel.: +44 1224 262450; fax: +44 1224 262555. E-mail address: [email protected] (C.L. Wainwright). 1 Present address: Cancer Center Karolinska, Karolinska Hospital/Institute, 171 76 Stockholm, Sweden. 0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.10.019

trophils [4], has been identified as an early event after balloon injury to the vessel wall, while recent studies by us and others with leukopenic rabbits have highlighted a critical role for inflammatory cells in neointimal thickening of balloon injured arteries [5,6]. Damaged endothelial cells and smooth muscle cells retrieved from experimental restenotic lesions [7] and peripheral leukocytes sampled after clinical angioplasty [8] all show increased expression of various adhesion molecules, including ICAM-1, VCAM-1, E-selectin, P-selectin and the leukocyte integrins. However, the demonstration that mice deficient in P-selectin develop less neointima following vascular injury than wild types, while ␤-integrin deficient mice

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do not [9], suggests a more important role for vascular adhesion molecules in the response to injury. Balloon angioplasty causes local generation of a number of chemical stimuli, including pro-inflammatory cytokines, from platelets, leukocytes, damaged endothelium and smooth muscle cells. IL-1␤ protein [10] and TNF␣ mRNA and protein expression [11] are both increased in arterial smooth muscle cells following injury. TNF␣ has multiple biological effects, including induction of the expression of cell surface adhesion molecules, release of other inflammatory mediators (particularly IL-1␤) and growth factors, stimulation of acute-phase protein secretion and induction of endothelial cell apoptosis. Recently, neutralization of the effects of free TNF␣ by TNF soluble receptor (TNFsr) has been shown to accelerate functional endothelial recovery following de-endothelialization [12], suggesting a detrimental role for this cytokine on endothelial cell regrowth. In contrast, a more recent study has shown that a polyclonal antibody to TNF␣ was unable to reduce neointimal formation, despite suppressing macrophage infiltration into the vessel wall by 60–75%, suggesting that TNF␣ does not directly influence neointimal formation [13]. However, since these findings may be due to redundancy within the pro-inflammatory cytokine pathways, whereby another cytokine may convey the signal for neointimal formation in the absence of an action of TNF␣, a precise role for TNF␣ in the complex setting of post-angioplasty restenosis cannot be confirmed. What is required to clarify our understanding of the role of TNF␣ following injury is a direct demonstration that TNF␣ per se can encourage neointimal formation by a mechanism involving inflammatory cell infiltration into the vascular wall. Thus the aim of the present study was to examine the effects of TNF␣, administered locally in vivo to the blood vessel wall immediately after balloon angioplasty, on the inflammatory response and the extent of neointimal formation in a rabbit model of subclavian artery injury. In order to identify an appropriate concentration and duration of application of TNF␣ for the in vivo studies, a series of in vitro experiments were performed in which the effects of TNF␣ were compared with those of IL-1␤, on leukocyte adhesion and adhesion molecule expression in artery segments.

2. Methods 2.1. Leukocyte adhesion studies Leukocyte adhesion to segments of rabbit subclavian artery was assessed by a method described by us previously [14]. Briefly, male New Zealand white rabbits (2–2.5 kg) were euthanised with sodium pentobarbitone (Sagatal® ; 60 mg/ml i.v.) containing heparin (1000 U/ml). Blood was extracted from the pulmonary artery into sterile 20 ml syringes containing 2 ml of 3.8% sodium citrate and sedimented with 6% dextran solution at room temperature for 2 h. The leukocyte rich upper layer was then centrifuged at 300 × g for 5 min

and the resultant cell pellet lysed with distilled water and layered onto 1 ml of Histopaque® (1.077 g/ml density). A second centrifugation for 20 min at 300 × g yielded a leukocyte pellet which was re-suspended in Hanks Balanced Salt Solution (HBSS). Isolated cells were counted using an automated cell counter (Medonic Cell Analyser CA460, Sweden) and leukocyte yield was adjusted to 1 × 106 cells/ml by adding an appropriate amount of HBSS. Leukocytes were then labelled with 51 Cr (185 kBq; specific activity 37 MBq/ml, Na2 51 CrO4 in 0.9% saline, half-life 27.7 days) for 1 h at 37 ◦ C, washed twice and re-suspended in HBSS. Following blood removal from the pulmonary artery both left and right subclavian arteries were removed and cut into 2–3 mm rings. The first ring from both arteries immediately adjacent to the aortic arch was discarded and the next five rings from both left and right arteries were used for the study. Each artery ring was placed into a separate eppendorf tube containing Krebs–Henseleit solution (composition in mM: NaCl, 118.3; NaHCO3 , 25.0; glucose, 11.1; KCl, 4.7; CaCl2 , 2.5; KH2 PO4 , 1.2 and MgSO4 , 1.2) and continuously aerated with 95% O2 /5% CO2 and maintained at 37 ◦ C until the adhesion studies were performed. The adhesion of leukocytes to artery segments was assessed by placing 5 ␮l of labelled leukocytes on the luminal surface of the segment and incubating in a humidified chamber (37 ◦ C) for 30 min. Following washing of the segments with HBSS to remove any non-adherent cells, the segments were counted in a gamma counter (Packard Bell Multiprias). Leukocyte adhesion (i.e., cells that remained adherent after washing) was expressed as the percentage of the cells added to the segment, determined from the count obtained from a 5 ␮l aliquot of labelled cells. 2.2. Immunocytochemical analysis Following in vitro adhesion studies, all artery rings were processed for immunocytochemical assessment of adhesion molecule expression. Artery segments were fixed in neutral buffered formal saline, processed and embedded in paraffin wax. All samples were given a code before morphological processing, which was only broken when all analyses were complete. Four sections were cut from each artery ring and were stained using antibodies directed against ICAM-1 and VCAM-1 and the streptavidin-biotin-peroxidase method [15]. Briefly, sections were deparaffinized in xylene and taken down to water. The slides were immersed in a 3% aqueous solution of hydrogen peroxide for 10 min (to destroy endogenous peroxidase activity), washed in tris-buffered saline (TBS) and blocked with a 1:5 dilution (in TBS) of rabbit serum for 15 min. Excess serum was drained off and the section incubated with the primary antibody for 24 h at 4 ◦ C at a pre-determined optimum dilution of 1:200 containing 10% normal rabbit serum. Slides were then washed in TBS and biotinylated anti-goat IgG secondary antibody was added for 30 min (1:200 dilution with secondary antibody diluent containing 5% normal rabbit serum). After washing in TBS, peroxidase-labelled streptavidin diluted 1:200

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in TBS was added to the slides for a further 30 min. After washing, slides were immersed for 10 min in 0.05% 3,3 -diaminobenzidinetetrahydrochloride solution followed by 0.5% copper sulphate solution for a further 10 min to enhance the colour of the brown reaction product. The slides were then placed in haematoxylin for 15 s to counterstain the nuclei, differentiated in 0.5% acid alcohol, placed in Scot’s tap water (NaHCO3 0.35% plus MgSO4 2% in water) to ‘blue’, dehydrated in xylene and mounted in DPX. Staining was quantified using a five-point grading scale where 0 = no staining, 0.5 = focal staining of the endothelium but with areas of endothelium which were unstained, 1 = mild staining of the endothelium with all cells being positive, 1.5 = uniform, moderate staining of the endothelial layer and 2 = intense staining of the whole endothelium. The slides were given a code and all slides graded blind by one investigator (A.R. McP) at one time.

performing the adhesion assay was also carried out. A further set of experiments compared the effects of pre-incubation of vessel segments with TNF␣ (1 ng/ml) for 15 min followed by assessment of adhesion 4 h later, with continuous 4 h incubation prior to performing the adhesion assay. To determine which adhesion molecules were responsible for the cytokine-induced elevation in leukocyte adhesion, artery rings were challenged with either IL-1␤ (1 ng/ml) or TNF␣ (1 ng/ml) for 4 h, washed twice and then incubated with antiICAM-1, anti-VCAM-1, anti-E-selectin and anti-P-selectin antibodies (1/200, 1/50 or 1/20 dilutions) prior to performing the leukocyte adhesion study. Control rings included an artery ring receiving no mediator or antibody treatment, and artery rings receiving mediator challenge but no antibody treatment. Following assessment of adhesion artery segments were subjected to immunohistochemical analysis of adhesion molecule expression as described above.

2.3. Animal model of vascular balloon injury

2.4.2. In vivo assessment of the effects of TNF␣ on leukocyte adhesion, adhesion molecule expression and neointimalformation Following balloon angioplasty of the left subclavin artery in vivo, the guidewire was removed from the balloon catheter to allow infusion of TNF␣ through the inner guidewire channel. The balloon catheter was withdrawn so that the tip of the catheter was just inside the subclavian artery and the balloon inflated to 4 atm to prevent back-flow. A tourniquet was applied distally to minimise the flow down the foreleg. An infusion of 0.9% saline (vehicle control; n = 11) or TNF␣ (10 ng/ml, chosen from the data obtained in the in vitro studies; n = 12) was then delivered locally over a 15 min infusion period at a constant rate of 200 ␮l/min (total perfused volume 3 ml, 30 ng TNF␣), allowing for a 0.5 ml dead space within the catheter. After completion of the drug infusion the tourniquet was removed, the balloon deflated and the catheter withdrawn. The groin wound was sutured and the animals allowed to recover. The rabbits were euthanised either 24 h (six controls, six TNF␣) or 8 days (five controls, six TNF␣) after surgery and the left (injured) and right (noninjured) subclavian arteries and leukocytes harvested to perform the adhesion assay and immunohistochemical analysis as described above. In addition, two additional rings were removed from each artery for morphological analysis of neointimal formation. These rings were immediately immersed in neutral buffered formal saline, processed and embedded in paraffin wax, at which point these segments were allocated a code which remained unbroken until all quantitative analysis had been performed. Two sections (4 ␮m) were cut from each artery ring and stained with haematoxylin and eosin. Qualitative scoring of leukocyte presence (number of adherent or infiltrating leukocytes) within the vessel wall was performed using a scoring system (0 = normal, 1 = mild, 2 = moderate and 3 = severe). Quantitative analysis of neointimal area (defined as the area bounded by the lumen and the internal elastic lamina) was performed by computerised planimetry (Scion Image).

An in vivo rabbit model of balloon angioplasty previously developed in this laboratory was used in this study [7]. All studies were performed under the guidelines of the UK Animals (Scientific Procedures) Act 1982, with an appropriate project licence. 23 male NZW rabbits were sedated with Hypnorm® (fluanisone/fentanyl citrate mixture; 0.3 ml/kg i.m.) and anaesthesia induced and maintained with a mixture of 2% nitrous oxide and 1.5–2% halothane in oxygen. All animals were given pre-operative antibiotic cover (100 mg ampicillin i.m.) and heparin (500 units i.v.). The left femoral artery was exposed by blunt dissection and a 3.0 mm overthe-wire balloon angioplasty catheter (CR Bard, Eire) containing a 0.014 in. steerable guide wire was introduced into the artery. The catheter was advanced into the left subclavian artery under fluoroscopic control (Siemens Memoskop and Siremobil 2), ensuring the balloon was positioned with the centre point about 1cm from the first rib. The contrast-filled balloon was then inflated twice to 10 atm for 30 s. A third inflation to 8 atm was performed and the balloon was withdrawn by half its own length to ensure endothelial damage. The balloon was deflated, withdrawn and the wound to the femoral artery was sutured. All animals were given 0.15 mg i.m. buprenorphine (Vetergesic® ) immediately after surgery as analgesic cover and allowed to recover from anaesthetic. 2.4. Experimental protocols 2.4.1. In vitro assessment of the effect of IL-1␤ and TNF-␣ on leukocyte adhesion and adhesion molecule expression To identify an optimim concentration of cytokine for subsequent in vivo studies, artery segments were incubated with either IL-1␤ (0.01–10 ng/ml) or TNF␣ (0.01–10 ng/ml) for 4 h prior to performing the adhesion assay. A time course study, in which artery rings were pre-incubated with IL-1␤ (1 ng/ml) or TNF␣ (1 ng/ml) for 15, 30 min, 1 or 4 h prior to

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2.5. Statistical analysis All data are expressed as the mean ± standard error of the mean. The n number for each experiment refers to the number of animals from which artery segments were used. For the leukocyte adhesion studies data is expressed as percentage of leukocyte adhesion. Data from the studies where the effect of each mediator on adhesion was examined were compared using one-way ANOVA followed by Dunnett’s Multiple Range Test. Data from the experiments to determine the effects of antibodies on adhesion were compared by a Student’s paired t-test. For the immunocytochemical studies, statistical significance was determined by a non-parametric Wilcoxon Test. For analysis of neointimal areas, values are given in mm2 and differences between control and TNF␣ treated groups were analysed using one-way ANOVA followed by a Tukey’s test. A value of P < 0.05 was considered to be statistically significant. 2.6. Materials Heparin was purchased from Leo Laboratories Ltd., Buckinghamshire, UK. Sagatal® was purchased from Rhˆone M´erieux Ltd., Harlow, Essex, UK. Sodium citrate, sodium chloride, acetic acid (glacial, 100%) and sodium acetate were all purchased from BDH Laboratories, Poole, Dorset, UK. Harris Haematoxylin, Dextran (MW approx. 500,000), Hank’s Balanced Salt Solution, Histopaque 1077, potassium phosphate and bovine serum albumin were all purchased from Sigma, Poole, Dorset, UK. 51 Chromium sulphate radioisotope was obtained from Amersham International Plc, Little Chalfont, Buckinghamshire, UK. Neutral Buffered Formal Saline was purchased from Rimon Laboratories Ltd., UK. Recombinant Human IL-1␤ and Recombinant Human TNF ␣, Goat Polyclonal Anti-human ICAM-1, VCAM-1, E-Selectin and Sheep polyclonal Anti-human P-Selectin were all purchased from R&D Systems Europe Ltd., Abingdon, Oxfordshire, UK. Rabbit serum was supplied by Scottish Antibody Production Unit, Law Hospital, Carluke, Lanarkshire, UK. Biotinylated anti-goat IgG antibody was obtained from Vector Laboratories, Burlinghame, CA, USA and streptavidinPOD was obtained from Boehringer Mannheim, GmbH, Germany. IL-1␤ and TNF ␣ were reconstituted in phosphate buffered saline (10 mM potassium phosphate and 150 mM sodium chloride, pH 7.4) containing 0.1% bovine serum albumin. All antibodies were reconstituted in sterile water.

Fig. 1. Stimulated adhesion of leukocytes to rabbit subclavian artery by IL1␤ and TNF␣ assessed in vitro. For the concentration studies (top panel), artery segments were challenged with IL-1␤ or TNF␣ for 4 h at the concentrations indicated, prior to incubation with 51 Cr-labelled leukocytes. For the time course studies (middle panel), artery rings were incubated with either IL-1␤ (1 ng/ml) or TNF␣ (1 ng/ml) for the various times indicated. The bottom panel shows the effect of pre-incubation with TNF␣ (1 ng/ml) for either 15 min or 4 h, with adhesion assessed after 4 h in both cases. The leukocyte adherence was expressed as percentage of total leukocytes added. Data are mean ± S.E.M. of six experiments. * P < 0.05; ** P < 0.01, compared to the control.

3. Results 3.1. The in vitro effects of TNF ␣ and IL-1␤ on the adherence of leukocytes and adhesion molecule expression in rabbit subclavian artery segments TNF␣ and IL-1␤ both produced a concentration- and timedependent increase in the adhesion of leukocytes (Fig. 1a

and b). Adhesion was significantly different from control (21 ± 5%) following incubation with 1 ng/ml IL-1␤ (38 ± 4%; P < 0.05) or 10 ng/ml TNF␣ (47 ± 4%; P < 0.01) for 4 h. A significant increase in the adhesion of leukocytes was observed after stimulation of the artery with either cytokine (1 ng/ml) for 0.5 h and reached a maximum after 4 h. However, when segments were incubated with TNF␣

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Fig. 3. The effects of preincubation of artery segments with 1 ng/ml TNF␣ or IL-1␤ for 4 h on endothelial staining intensity for ICAM-1 and VCAM-1. * P < 0.05 compared to staining in artery segments not challenged with either cytokine.

3.2. The effects of in vivo TNF␣ administration on injury-induced leukocyte adhesion, adhesion molecule expression and neointimalformation Fig. 2. Effects of anti-ICAM-1 (top panel) and anti-VCAM-1 (bottom panel) antibodies on IL-1␤- and TNF␣-stimulated subclavian artery ring adhesiveness for leukocytes. Subclavian artery rings were pre-incubated with no cytokine or antibody treatment (control), 1 ng/ml IL-1␤ or TNF␣ alone for 4 h, or 1 ng/ml IL-1␤ for 4 h followed by washing and incubation for 20 min with antibody at the indicated dilutions prior to assessing adhesion. Data are mean ± S.E.M. of six experiments. *** P < 0.001, compared to segment treated with cytokine alone.

(1 mg/ml) for 15 min, and then TNF removed from the incubation medium for a further 4 h before assessing adhesion, the increase in adhesion observed was similar to the adhesion seen following 4 h of continuous stimulation with TNF␣ (Fig. 1c). Pre-treatment of both TNF␣- and IL-1␤-stimulated subclavian artery rings with anti-ICAM-1 antibody significantly inhibited the adhesion of leukocytes to the artery in a concentration-dependent manner, when compared to adhesion in the presence of the cytokine alone (Fig. 2a). In contrast, no significant inhibition of either TNF␣- or IL-1␤stimulated adhesion was observed using anti-VCAM-1 antibody (Fig. 2b). Immunocytochemical staining of control artery sections (no treatment) with antibodies to ICAM-1 and VCAM1 revealed some specific staining in the endothelium with a low level of background non-specific staining in the medial and adventitial layers. Semi-quantification of the intensity of staining demonstrated that incubation of artery rings with 10 ng/ml TNF␣ or IL-1␤ for 4 h significantly increased the intensity of endothelial staining for both ICAM-1 and VCAM-1 (Fig. 3) to the same extent.

A marked increase in leukocyte adhesion, assessed ex vivo 24 h after balloon angioplasty, was observed in injured arteries from saline-treated rabbits (Fig. 4) compared to the non-injured contralateral right artery. By 8 days after the angioplasty procedure this enhanced adhesion was still evident, but much less marked. This increased adhesive response to injury was accompanied by an increase in the expression of both VCAM-1 and ICAM-1 in the injured artery, compared to the non-injured artery (Fig. 5). In arteries from rabbits that received locally-delivered TNF␣ immediately following angioplasty the injury-induced enhanced adhesive response was further elevated (43.8 ± 1.8%) compared to controls

Fig. 4. The effect of balloon angioplasty, with and without subsequent TNF␣ administration, on leukocyte adhesion assessed ex vivo 24 h and 8 days after the angioplasty procedure. * P < 0.05 compared to non-injured artery. # P < 0.05 compared to injured artery in the saline-treated group.

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both groups (Fig. 7). Evidence of leukocyte infiltration was observed in three rabbits from each treatment group culled after 24 h and only one rabbit from each treatment group culled after 8 days. Furthermore, the extent of leukocyte infiltration was similar in all rabbits (data not shown), indicating that TNF␣ did not increase leukocyte recruitment to the injured tissue.

4. Discussion

Fig. 5. The effect of balloon angioplasty, with and without subsequent TNF␣ administration, on endothelial staining for VCAM-1 (upper panel) and ICAM-1 (lower panel). * P < 0.05 compared to contralateral non-injured artery. # P < 0.05 compared to corresponding artery segment in saline-treated rabbits.

(34.5 ± 1.0%; P < 0.05) in the 24 h group (Fig. 4). However, this effect was not maintained 8 days after angioplasty. In the 24 h group the expression of ICAM-1 in TNF-treated injured vessels was significantly greater than the expression seen in the contralateral non-injured artery, and also significantly greater than expression in the saline-treated injured arteries (Figs. 5 and 6). In contrast, while the expression of VCAM1 was greater in TNF-treated injured arteries compared to saline-treated injured arteries, the expression of VCAM-1 in the non-injured right arteries from the TNF-treated rabbits was also significantly elevated. In vessels harvested 24 h after the angioplasty procedure, due to the early time point there was no evidence of neointimal formation in either group of rabbits, despite clear injury to the blood vessel evidenced by rupture of the internal elastic lamina and endothelial disruption. However, in both saline and TNF␣ treated rabbits from which blood vessels were harvested 8 days after the procedure there was significant neointimal formation. The size of the neointima was similar in

Previous studies have shown that inhibition of TNF␣ with TNF soluble receptor accelerates functional recovery of the vascular endothelium following balloon angioplasty [12], suggesting that locally expressed TNF␣ inhibits endothelial recovery. In contrast, monoclonal antibodies to TNF␣ failed to reduce the degree of neointimal formation, despite evidence that TNF␣ levels within injured arterial tissue is elevated some 100,000-fold for at least 6 days after the induction of injury [13]. However, considering the redundancy that exists within the cytokine pathways, it is possible that any beneficial effects of blocking the actions of TNF␣ may be overridden by other cytokines generated at the site of injury. With this in mind, we sought to determine the effects of TNF␣ administration directly to the vessel wall immediately after the angioplasty procedure in an attempt to demonstrate a direct link between a functional pro-adhesive response and an acceleration of neointimal formation. Our findings show that, while TNF␣ enhances adhesion of leukocytes to both normal endothelium of artery segments in vitro and previously injured artery segments ex vivo, this has no effect on the amount of neointima detected 8 days after balloon injury. This suggests that while TNF␣ may play an important role in initiating the inflammatory response to vascular injury, this alone is insufficient to activate the entire process of neointimal formation. 4.1. The effects of TNF ␣ on leukocyte adhesion to artery segments in vitro The in vitro studies demonstrated a time- and concentration-dependent effect of TNF␣ on the adhesion of leukocytes to intact arterial segments. For comparison we also assessed the effects of IL-1␤, which we found to exert very similar effects. These findings are consistent with those observed in the adhesive response of endothelial cell monolayers and cultured smooth muscle cells to the two cytokines [16–19]. Although the effect of TNF␣ clearly increased in line with duration of exposure, with a maximum effect observed after 4 h of incubation, we found that a 15 min exposure to TNF ␣, with subsequent assessment of the adhesion of leukocytes 4 h later, resulted in enhanced adhesion similar to 4 h continuous exposure. This suggests that at early time points the increased adhesive response may be due to an increase in the avidity of the adhesion molecules [20] or translocation of adhesion molecules from intracellular stores

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Fig. 6. Representative morphological sections of non-injured right arteries retrieved from rabbits given either saline administration (a) or local administration of TNF␣ (b) and injured arteries from saline treated (c) and TNF␣ treated (d) rabbits 8 days after balloon angioplasty. Bar represents 100 ␮m. A, adventitia; EC, endothelial cell; IEL, internal elastic lamina; L, lumen; M, media; N, neointima.

or cell junctions to the endothelial surface [21], whereas later increased adhesion seen at the later time points is more likely to be due to de novo synthesis of ICAM-1 stimulated by TNF␣. This is important since we performed these studies to determine an optimum concentration and duration of exposure to TNF for the subsequent in vivo studies, which confirmed that a 15 min exposure of the injured artery wall to TNF␣ enhanced the adhesive response 24 h later.

Both TNF␣ and IL-1␤ increased the expression of ICAM-1 and VCAM-1 in the vascular endothelium, while the adhesive response to both cytokines was only blocked by antibodies to ICAM-1, suggesting that ICAM-1 is largely responsible for the process of adhesion under these conditions. Although other studies have demonstrated an inhibition of leukocyte adhesion with both ICAM-1 and VCAM-1 antibodies [19,22], those studies were performed using monocyte-rich leukocyte suspensions. In contrast, the leukocyte suspensions employed in the present study were comprised primarily of neutrophils [7], which do not express VLA-4 (the ligand for VCAM-1 [23]), which would explain the lack of effect of VCAM-1 antibodies. 4.2. In vivo studies

Fig. 7. The extent of neointima present in injured left subclavian arteries from saline-treated rabbits and rabbits given local administration of TNF␣. The extent of neointima was determined 8 days after the angioplasty procedure and is expressed as a percentage of the total media/neointima area. * P < 0.05 compared to contralateral non-injured arteries.

From the in vivo studies, we have shown that the process of vascular balloon injury results in an enhanced adhesive response to leukocytes ex vivo 24 h after injury, an effect that has partially waned 8 days after angioplasty. Moreover, the expression of both ICAM-1 and VCAM-1 is increased in injured arteries. These findings are consistent with our previous observations [7]. Moreover, we have shown for the first time that administration of TNF␣ to the site of injury immediately after angioplasty markedly increased this adhesive response assessed at 24 h. This effect was associated with an enhanced

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expression of ICAM-1 and VCAM-1 in the injured arteries, compared to saline-treated injured arteries. ICAM-1 expression in the TNF␣-treated rabbits was elevated only in the injured artery, whereas VCAM-1 expression was elevated in both injured and non-injured arteries. This latter finding suggests that systemic leakage of TNF␣ following release of the tourniquet was sufficient to incur a systemic up-regulation of adhesion molecules. However, the lack of increased expression of ICAM-1 in non-injured vessels suggests that this systemic response was not common to all adhesion molecules. Since ICAM-1 expression was only increased in the presence of injury, this suggests that the process of injury itself causes the release of a mediator that acts synergistically with TNF␣ to increase ICAM-1 expression. Taken together these data support a role for ICAM-1, rather than VCAM-1, in the increased adhesive response, since the non-injured arteries did not exhibit enhanced adhesion compared to non-injured arteries from saline-treated rabbits, despite the increased VCAM1 expression. Notwithstanding the increased adhesion seen following in vivo TNF␣ administration, there was no evidence of acceleration of the formation of neointima by the cytokine, as shown by the similar degree of neointima in both groups of rabbits at 8 days following angioplasty. These findings suggest that TNF␣ alone is insufficient to promote smooth muscle cell proliferation through a mitogenic action. It could be argued that the rate and magnitude of neointimal formation seen under control conditions cannot be exceeded. However, it has been shown that induction of a systemic inflammatory response with lipopolysaccharide can increase neointimal size [6], demonstrating that neointimal formation can be exaggerated. It could further be argued that the ultimate neointimal area may be increased at a time when the process of neointimal formation is complete (4 weeks in this model). However, time course studies have shown that the phase of smooth muscle cell proliferation is complete within 7 days, with the latter stages of neointimal formation (15–28 after injury) involving extracellular matrix production to add bulk to the lesion [24]. Thus, if TNF␣ were contributing to the proliferative phase we would have expected to see an effect at 8 days. Similarly, there was no evidence of increased infiltration of inflammatory cells into the blood vessel wall. Since inflammatory cell infiltration is well recognised as an early response to injury the present findings suggest that while TNF␣ enhances vascular adhesiveness it does not act alone to induce inflammatory cell transmigration. It is, of course, possible that the dose of TNF␣ (30 ng) delivered to the injured artery was too low to have an effect on leukocyte transmigration, and was thereby unable to contribute to neointimal proliferation. However, only mild development of neointimal hyperplasia was seen in a study by Fukumoto et al. [25], which involved adventitial delivery of a dose as high as 2.5 ␮g TNF␣ to porcine coronary artery over a period of 2 weeks. This suggests that doses of TNF␣ well in excess of those required to induce a proinflammatory response are required to induce a proliferative response and implies a dissociation of these effects.

In its role as a pro-inflammatory cytokine following injury, it is likely that TNF␣ activates other signaling mechanisms that may contribute to the increased leukocyte adhesiveness. For example, TNF␣ (10 ng/ml) has been shown to induce expression at the RNA and protein levels of the chemokines MCP-1 and IL-8, both of which stimulate monocyte adhesion and transmigration, in human umbilical vein endothelial cells (HUVECs) [26]. Furthermore, endothelial progenitor cells (EPCs), a subset of pluripotent stem cells derived from the bone marrow, can be released from the marrow in response to chemokines generated as a result of remote injury. Intravenous infusions of EPCs have been shown to reduce neointimal formation after vascular injury [27]. Recently, it has been demonstrated that cardiomyocytes over-expressing TNF␣ attract the migration of embryonic stem cells in vitro [28] and hence it is possible that TNF␣ may recruit circulating stem cells to sites of vascular injury, which could serve to limit neointimal progression. However, the role of stem cells in the response to injury remains uncertain, since there is evidence that while on the one hand they may promote vascular healing and stabilization of vulnerable atherosclerotic plaques, they may also contribute to increased neointimal area following vascular injury and ultimately restenosis [29]. It has been suggested previously that blockade of TNF␣ can improve functional recovery of the endothelium following injury [12]. In the present study, morphological analysis of injured arteries demonstrated the presence of endothelial damage and partial denudation 24 h after angioplasty. The functional integrity of this endothelium was also examined in artery ring preparations (data not shown) and we found the responses to the endothelium-dependent vasodilator carbachol to be significantly reduced in the injured left subclavian arteries of both the saline and TNF␣-treated groups compared to their respective non-injured control arteries. However, responses to the endothelium-independent vasodilator SIN-1 were the same in injured and non-injured vessels. This demonstrates a functional impairment in endothelial function in the balloon-injured vessels irrespective of TNF␣ treatment. A likely explanation for these differing findings is that the study by Krasinski et al. [12] employed air desiccation as a means of injury, in contrast to the balloon stretch method employed in the present study. In summary, our data support the concept that, while TNF␣ clearly induces a functional response (seen here as an increase in leukocyte adhesion and vascular adhesion molecule expression) in previously injured arteries, this is not translated to an increase in neointimal formation. This agrees with the previous demonstration that a monoclonal antibody to TNF␣ was unable to abrogate neointimal formation in a rabbit atherosclerotic model, despite reducing inflammation [13]. This suggests that while TNF␣ may play a specific role following vascular injury, it may not be appropriate as a single target for the prevention of restenosis and that strategies aimed at multiple cytokines may be the way forward.

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Acknowledgements Ashley M. Miller was the recipient of an A.J. Clark Scholarship from the British Pharmacological Society.

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