Omentum

We interrogated the 16 new (or proxy; r² > 0.8) CAD risk SNPs for cis eQTL expression in multiple tissues: the ASAP study^{31 (link)} used tissue biopsies taken from patients undergoing carotid endartectomy (plaque n = 117) or valve surgery (liver n = 152, aorta media n = 117, aorta adventitia n = 103 and mammary artery n = 88). Expression data were generated using the Affymetrix HG-U133 plus 2.0 array (plaque) or the Affymetrix ST1.0 Exon array (liver, aorta and mammary artery); in the MuTHER study^{32 (link)}, RNA levels were measured in LCLs (n = 826), skin (n = 705) and fat biopsies (n = 825) from 850 female twins (one-third monozygotic and two-thirds dizygotic) from the TwinsUK resource using the Illumina HumanHT-12v3 array. We assessed genotype with gene expression associations, using an additive linear model (within a 1-Mb window); in Cardiogenics^{5 (link)}, monocytes and macrophages were collected from healthy subjects and individuals with CAD, and RNA was profiled with the Illumina Human Ref-8 array. eQTL analysis was undertaken in 459 healthy individuals from Cambridge, UK, using an additive linear model (1-Mb window); in the Massachusetts General Hospital study^{33 (link)} of liver, omentum and subcutaneous adipose tissue among subjects undergoing Roux-en-Y gastric bypass surgery, eQTL analysis was performed with a linear regression model using a 1-Mb window.
In loci with significant cis-eQTL signal(s) (P < 1 × 10⁻⁴), we also identified the most strongly associated cis-eQTL SNP (eSNP) for the corresponding transcript and then performed conditional analyses, including in the regression model, with either the lead eSNP or the lead CAD-associated SNP. On the basis of the conditional analysis, we determined whether the same variant underlies both gene expression regulation and disease.
Finally, we interrogated the lead SNPs in the 16 new CAD susceptibility loci for allelic expression imbalance effects in LCLs, fibroblasts and monocytes (n = 188; Cardiogenics), as described in ref. 34 (link).

For in vivo homing experiments, CMPTX-labeled SKOV3ip1 ovarian cancer cells (4 × 10⁶) were pretreated (30 min) with inhibitory antibodies (R&D Systems) to CXCR1 (MAB330), IL-6R (MAB227) or mouse IgG control (MAB002). Mice were pretreated with 100 μg per kg body weight TIMP-1–specific (R&D Systems AF970) or goat IgG (R&D Systems AB108C) antibodies 30 min before cancer cell injection. Labeled SKOV3ip1 cells were injected intraperitoneally into female athymic nude mice. The omentum was removed 20 min later, digested in 1% (vol/vol) NP-40, and fluorescence was measured using a plate reader. In vitro homing was assessed by preparing a Matrigel plug in chamber slides. The plugs consisted of growth-factor–reduced Matrigel and human omental adipocytes in SFM containing an inhibitory antibody or a goat IgG control, in triplicate. Inhibitory antibodies (R&D Systems) to the following proteins were used at the following concentrations: IL-6 (AB206NA) and IL-8 (AB208NA), 50 ng ml⁻¹; MCP-1 (AB279NA), 100 μg ml⁻¹; MMP-9 (EMD Chemicals, IM09L), 6 μg ml⁻¹; TIMP-1, 100 ng ml⁻¹. CMFDA-labeled SKOV3ip1 cells (3 × 10⁶) were added to a culture dish containing the plugs in 6 ml SFM. The plate was then incubated at 37 °C for 30 min. Plugs were removed, and fluorescence was measured using a plate reader. In vitro adhesion to equal portions (wt/wt) of full human omentum was carried out in low adhesion plates. Omentum was preincubated in a TIMP-1 inhibitory antibody or a control goat IgG antibody (100 μg ml⁻¹). CMFDA-labeled SKOV3ip1 cells (4 × 10⁶) were pretreated (30 min) with inhibitory antibodies to CXCR1, IL-6R or mouse IgG control. Cancer cells were added to the full omentum (separate wells for each treatment) and allowed to adhere for 20 min at 50 r.p.m. and 37 °C on a rotary shaker. Omentum was then digested in 1% NP-40, and fluorescence was measured using a plate reader.