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TRAIL attenuates the development of atherosclerosis in apolipoprotein E deficient mice

V Watt, J Chamberlain, T Steiner, S Francis... - Atherosclerosis, 2011 - Elsevier TRAIL (tumour necrosis factor-related apoptosis inducing ligand) is most often reported to induce apoptosis in tumour cells. It is expressed in artery walls but its role and regulation in vascular pathologies is little studied. We aimed to measure the effect of genetic deletion of ...

The pathogenesis of atherosclerosis is a complex process involving inflammation, cell proliferation, apoptosis and necrosis. The tumour necrosis factor (TNF) superfamily of cytokines includes tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10); the primary ligand involved in apoptosis signalling in this family. TRAIL is a 40 k Da type II transmembrane protein that induces apoptosis by interaction with two death receptors (TRAIL R1 and R2) [1]. In addition, there are 3 decoy receptors for TRAIL: TRAIL R3, R4 and the soluble receptor osteoprotegerin, which, when bound to the ligand, do not induce apoptosis [2] and [3]. TRAIL exists in soluble and membrane bound forms and its expression has been detected in a variety of human tissues [4]. Lymphocytes, monocytes and neutrophils have been shown to express TRAIL, and soluble TRAIL (s TRAIL) is detectable in humans in both health and disease [5] and [6].

Following TRAIL ligand receptor binding, downstream signalling leads to apoptosis by the caspase-dependent and mitochondrial pathways [7]. However, TRAIL signalling can also promote cell survival and proliferation via induction of transcription factors NF-кB and c-jun, the mitogen-activated protein kinase ERK1 and the phosphatidylinositol 3-kinase P13K/serine/threonine kinase Akt pathway signalling which is of central importance to angiogenesis and endothelial cell survival [8] and [9]. Whilst TRAIL can induce apoptosis of many malignant and transformed cell lines, there are conflicting data surrounding the effects of TRAIL on normal human cells, including vascular smooth muscle cells (VSM Cs) and endothelial cells. Published data have shown that TRAIL can induce apoptosis of human VSMC and endothelial cells [10] and [11] yet, under conditions of trophic factor withdrawal, TRAIL promotes their survival and proliferation [8], [12] and [13]. These conflicting data have led to uncertainty regarding the role of TRAIL in vascular biology and disease.

TRAIL ligand is detectable in stable and unstable human atherosclerotic plaques and levels of s TRAIL are lower in patients with coronary disease compared to disease-free controls [14] and [15]. These findings have led to the suggestion that TRAIL may have a protective role in the atherosclerotic process.

Recent data indicate that TRAIL promotes VSMC proliferation and neointimal formation after arterial injury induced using a femoral cuff in mice [16]. Using the Apo E / mouse model of atherogenesis, Secchiero et al. showed that administration of recombinant human TRAIL to diabetic Apo E / mice led to a greater VSMC lesion content and a reduced overall lesion size [17]. These findings indicating that TRAIL is pro-proliferative contrast with those of Sato et al. who demonstrated induction of VSMC apoptosis by TRAIL-expressing T cells isolated from patients with ACS [11]. Given these conflicting data, we sought to further clarify the role of TRAIL in atherogenesis by genetic deletion of TRAIL in Apo E / mice, feeding of a Western diet and assessment of atherosclerosis.

TRAIL / mice, on a C57BL/6 background, were the kind gift of Mark Smyth, Peter Mac Callum Cancer Institute, Australia under Amgen materials transfer agreement 200308096. Apo E / mice, also on a C57BL/6 background, were bred in house. All mice were used in accordance with UK legislation (1986 Animals (Scientific Procedures) Act) and were housed in a controlled environment with a 12 h light/dark cycle at 22 C. All animal experiments were approved by the University of Sheffield Project Review Committee and carried out under a UK Home Office Project Licence. Following re-derivation, TRAIL / mice were crossed with Apo E / mice to create a double knockout mouse colony. Genotype was confirmed by polymerase chain reaction. All animals were fed standard chow until they reached 6 weeks of age (20 g). Male TRAIL / /Apo E / mice and TRAIL+/+/Apo E / controls were then fed chow or a cholate-free Western diet (n = 6 8 for each group, Special Diet Services, Essex, UK). All mice were observed daily and weighed weekly to monitor their health. After 8 or 12 weeks of high fat diet, the animals were euthanized by pharmacological overdose of pentobarbitone. Blood was collected by cardiac puncture and the mice were perfusion-fixed with 10% (w/v) buffered formalin. The aortic sinus from each animal was dissected, embedded in paraffin and serially sectioned (5 μm). The thoracic aorta was removed and fixed in 4% (w/v) buffered formalin prior to en face preparation. Aortic sinus sections were stained with Alcian Blue/Elastic Van Gieson for atheromatous lesions. The thoracic aortae were stained with Oil Red O and pinned onto wax.

Immunohistochemistry was carried out on aortic sinus sections. Sections were dewaxed and incubated with VSMC α-smooth muscle actin primary antibody (Dako αSMA Clone 1A4, diluted 1:150) for 1 h, then with a secondary biotinylated goat anti-mouse antibody (Vector Labs), followed by ABC solution (Vector Labs). Immunogenicity was visualized with DAB (Sigma) and sections were counterstained with Carazzi's haematoxylin. Macrophages were detected using F480 and the Mac3 antibody (M3/84 monoclonal antibody 550292 BD Pharmingen). Sections were dewaxed and antigen retrieval was carried out using citrate buffer. Dako serum-free protein block was applied to the sections for 1 h, followed by incubation with the primary antibody (1:50 dilution) for 2 h. A secondary antibody (biotinylated anti-rat Ig G, Vector Labs) was applied for 1 h, the slides were incubated in ABC-alkaline phosphatase (Vector labs) and the Vector Blue stain applied. For TRAIL staining an iso HC DAB kit (Biogenex) was used. Apoptotic cells were detected using Apoptag Fluorescein in situ reagents (Millipore).

Total lesion area or area of positive staining was determined for each section/aorta, respectively, using computer assisted image analysis (NIS-elements/Lucia G software, Nikon UK). Data were generated for mean lesion area and lesion area:cross-sectional area (CSA) ratio of the aortic sinus. For the en face aortic preparations, area of positive staining for Oil Red O is expressed as a percentage of total aortic area. For immunohistochemical quantitation, positive staining Mac3 is expressed as an area proportional to total lesion area. Cells staining positive for TRAIL, αSMA and apoptosis (TUNEL) were counted as a proportion of the total cells in 0.1 mm2 plaque to create a percentage index. A minimum of 5 areas of plaque were counted per section.

Mice were generated from the breeding programme in the expected Mendelian ratio. TRAIL / Apo E / mice fed on chow for both 8 weeks and 12 weeks weighed more than Apo E / controls on chow at the same timepoint (32.81 0.534 vs. 30.31 0.377 g Apo E / chow, all n = 8 at 8 weeks, p < 0.01; 33.58 0.375 vs. 29.43 0.702 g Apo E / chow, all n = 8 at 12 weeks, p < 0.01).

Plasma cholesterol increased as expected with dietary fat content (Supplementary Tables 1 and 2). There was no significant difference in plasma cholesterol between the two mouse strains studied. Mice fed the Western diet had lower mean random glucose levels compared to the mice fed chow. There was no significant difference in plasma glucose or liver enzymes between the two transgenic strains.

TRAIL was expressed throughout the plaque but predominantly in the smooth muscle cell layer (Fig. 1A and B). A few macrophages in the lesions appeared to express TRAIL (Fig. 1C). TRAIL is a secreted molecule and some positive staining was seen in the extracellular matrix. There was no difference in the % of cells staining positively for TRAIL in lesions after 8 weeks compared to 12 weeks on a Western Diet (data not shown). There were no TRAIL positive cells in Apo E / TRAIL / mice.