Dfc320

The aortic roots of ldlr^−/− mice fed a high fat, high cholesterol diet for 24 weeks were dissected and embedded in Tissue Tek OCT^TM (Sakura Finetek, Staufen, Germany). Within the aortic root, serial cryostat sections (6 μm, CM3050S, Leica Microsystems, Wetzlar, Germany) at the level of all 3 cusps were prepared. Lesion areas were analyzed for LCN2, endothelial cells (CD31), alpha smooth muscle cells (αSMA), monocytes/macrophages (MOMA-2) as well as the M1 macrophage marker inducible nitric oxide synthase (iNOS) and counterstained with hematoxylin (Carl Roth, Karlsruhe, Germany) for immunohistochemistry or 4',6-diamidino-2-phenylindole (DAPI, Life Technologies) for immunofluorescence, respectively. Visualization was performed with the appropriate VECTASTAIN ABC reagents for biotinylated antibodies and either ImmPACT DAB or VIP Peroxidase (HRP) Substrate (all from Vector Laboratories) according to the manufacturer’s protocol. For immunofluorescence Alexa Fluor 488 and 555 conjugated secondary antibodies were used (Life Technologies). Staining for collagen (Picrosirius Red) were assessed under polarized light. Pictures were captured using a DM4000 B microscope with a DFC320 camera for immunohistochemistry and a DFC350FX camera for immunofluorescence and QWin software (all Leica Microsystems).

First, we tested the cytotoxic effects of Ipilimumab or Nivolumab on co-cultures of cancer cells (or HFC cells) with human lymphocytes. Cells were plated in 96-well flat-bottom plates at 10,000 cells/well for 16 h. Human peripheral blood mononuclear cells (hPBMCs) were added at the effector:target ratio 5:1 in the absence or presence of Ipilimumab or Nivolumab (200 nM), and incubated for one day under standard growth condition [17 (link)]. This dose was chosen on the basis of previous cellular experiments performed to test the antitumor and cardiotoxic effects of Ipilimumab or Nivolumab [15 (link),16 (link),17 (link),18 (link)] and was lower than those reached in the patient serum/plasma administered with the conventional therapeutic doses of 10 mg/Kg. Controls included cancer cells or cardiomyocytes incubated in the absence of effector cells or in the presence of the antibodies, used alone. After the incubation with antibodies, hPBMCs were removed and adherent cancer cells or cardiomyocytes were washed twice and counted by trypan blue. Cell survival was expressed as percent of viable cells tested with Nivolumab or Ipilimumab compared to untreated cells (negative control). Cells were also imaged through a Leica Microsystems integrated microscope (DFC320).

About 500 mg of each fresh biopsy was digested with collagenase [23 (link)], and the mean adipocyte diameter was determined by computerized image analysis [24 (link)] with Leica software (Leica QWin V3; Leica Microsystems, Wetzlar, Germany). The cell suspension was placed between a siliconized glass slide and a cover slip and transferred to the microscope (X5 objective, DM6000B, Leica Microsystems). Twelve random visual fields were photographed with a CCD-camera (DFC320, Leica Microsystems). Uniform microspheres, 98 μm in diameter (Dynal, Invitrogen Corporation, Oslo, Norway), served as reference. Mean adipocyte volume was calculated with the Goldrick formula [25 (link)].

Histological analysis and immunohistochemistry staining were performed as previously described [38 (link)]. OCT-embedded common carotid arteries were cut systematically in serial 5-μm cross sections using a cryotome (Leica CM3050S, Leica Microsystems, Germany). Analysis was carried out in the injured left common carotid artery, whereas the contralateral served as a control. For morphometric analysis, sections were stained with hematoxylin and eosin (HE). For immunofluorescence analysis, sections were stained with a rabbit anti-rat CD31 antibody (1:250, Abcam, USA) and visualized using an Alexa Fluor 647 mouse-anti-rabbit IgG secondary antibody (1:500, eBioscience). For immunohistochemistry analysis, sections were stained with a rabbit anti-rat CD31 antibody (1:150, Abcam, USA) and visualized using a mouse-anti-rabbit secondary antibody (1:500, Sigma). Negative controls using IgG controls matching in species and concentration were run in parallel. Pictures were taken with a microscope (Leica CM3050S, Leica Microsystems, Germany) and a digital camera (DFC 320, Leica Microsystems) at ×100 magnification. Morphometric analysis was performed per sample followed by computer-assisted morphometric analysis (ImageJ, NIH, USA). Subsequent morphometric analyses were performed in a double-blinded manner. Four animals per group and 3 samples per animal were analyzed.