Sustained Focal Vascular Inflammation Accelerates Atherosclerosis in Remote Arteries

Supplemental Digital Content is available in the text.


Aortic injury of the abdominal aorta
Surgery was performed under isoflurane anaesthesia using a dissection microscope (Leica, Wetzar, Germany). Prior to surgery, fur was removed, and the surgical field was disinfected. After a midline incision of the abdomen, the aorta, renal, and iliac arteries were exposed and isolated. Subsequently, two ligatures (surgical silk, size 4-0, Aragó, Zaragoza, Spain) were placed around the aorta, below the renal arteries and above the iliac bifurcation, respectively. A 30G syringe was introduced into the abdominal aorta followed by a total infusion of 1mL saline buffer, that was administered in 3 boluses of 300-350L each, to remove the endothelial cells (Supplemental Figure  I). Finally, the aortic puncture was repaired, the ligatures were removed, the muscle and skin were sutured (5-0 Vicryl, Ethicon Inc., Somerville, NJ, USA) and the animals were allowed to recover. Each surgery lasted 30min. Shamoperated animals underwent the same surgical procedure but without injuring the endothelial layer.

In vivo MRI protocol
In vivo MRI of the arterial vessel wall was performed using a Philips Achieva MR scanner (Philips Healthcare, Best, The Netherlands) equipped with a clinical gradient system (30 mT m −1 , 200 mT m −1 ms −1 ). All animals were scanned twice. For the first imaging session, mice were scanned 30min after intravenous administration of gadofosveset (Gd-albumin, 0.03mmol/kg). After a washout period of 24h, mice underwent a second imaging session 1h after intravenous administration of an elastinspecific gadolinium-based MR contrast agent (Gd-elastin;; 0.2mmol/kg). Both contrast agents were obtained from Lantheus Medical Imaging (North Billerica, MA, USA). In each mouse, two vascular segments were imaged at each time point. First, the abdominal aorta was imaged using a single-loop surface coil (diameter=47mm) and secondly, the brachiocephalic artery (BCA) was imaged using a single-loop surface coil (diameter=23mm). For imaging the aorta, mice were placed in a supine position while for imaging the brachiocephalic artery (BCA) mice were placed in a prone position. Anaesthesia was induced with 5% and maintained with 1-2% isoflurane mixed in medical oxygen. For visualization of the vasculature contrast-enhanced angiography images were acquired. The maximum intensity projection (MIP) images were used to plan the subsequent late gadolinium enhancement (LGE) and T1 mapping scans. A 2D-Look-Locker sequence was planned perpendicular to the aorta and BCA to determine the optimal inversion time (TI) for blood signal nulling. 3D LGE fast gradient echo sequence was used for visualization of contrast agent uptake. 3D T1 mapping was performed using a Modified-Look-Locker sequence that employs non-selective inversion pulses with inversion times ranging from 20 to 2000ms, followed by eight segmented readouts for eight individual images. The two interleaved imaging trains employed to achieve higher temporal resolution result in a set of 16 images per slice with increasing inversion times. MRI acquisition parameters are summarised in supplemental tables 1 and 2. N=5-9 mice were analysed per group.
Subsequently, samples were cleared from red blood cells and platelets using a lysis buffer (FACS lysing solution, BD Bioscience). Finally, samples were fixed in 1% paraformaldehyde. All samples were analysed using a FACSCanto flow cytometer and a FACSDiva software (BD Biosciences) and analysed using FlowJo Version 10 software (Ashland, US). N=4-6 animals were analysed per group.

Cytokine quantification in serum (Luminex assay)
Mouse serum samples were assayed for IL-6, IL-5, CCL-2, GM-CSF, IFNγ, IL-1ß, TNFα and VEGF using a Mouse Magnetic Luminex Assay according to the manufacturer instructions (R&D Systems, Minneapolis, US). Blood was collected and allowed to clot for 1h at room temperature before centrifuging for 20min at 2000xg.
Then serum was aliquoted and stored at -20 o C until was used for the cytokine quantification. 6-9 animals were analysed per group.

Statistical analysis
The sample sizes included in the full study were calculated based on pilot MRI data acquired at 12 weeks in injured and non-inured mice showing that detection of changes in plaque burden between these groups when using MRI with Gd-Albumin required 3-6 animals per group (133.5 ± 27.70 mm3 vs 201.1 ± 9.317 mm3). When using the Gd-elastin 5-7 animals per group were required (