Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6Chigh monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe−/− mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6Chigh monocytes and macrophages. SYK inhibition limited Ly6Chigh monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe−/− mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression. Electronic supplementary material The online version of this article (doi:10.1007/s00395-016-0535-8) contains supplementary material, which is available to authorized users.

1 Supplemental Material Supplemental Methods Study design. This study investigates SYK inhibition by Fostamatinib in early and advanced atherosclerosis. Fostamatinib disodium (Astra Zeneca, UK) without carrier was incorporated at 0.3% (w/w) into high cholesterol diet (HCD) pellets (1,25% cholesterol; D12108 mod., Ssniff GmbH, Soest, Germany) as previously described [7]. Different color dyes were used to mark control and drug diet. To study SYK inhibition in early atherosclerosis 6 week old female Apoe -/mice consumed a HCD ad libitum for 8 weeks with or without Fostamatinib. To study effects in established atherosclerosis 6 week old female Apoe -/mice consumed a HCD for 8 weeks before being randomized to HCD supplemented with fostamatinib or HCD alone for another 12 weeks.
Animal procedures and tissue processing. Mice were anesthetized with isofluran for retroorbital bleedings, intravenous injection of 1m Fluoresbrite YG microspheres (230l of a 1:4 dilution in sterile PBS, Polysciences Inc., Eppelheim, Germany), intravenous injection of 300ng CCL2 (R&D System, Minneapolis, MN, USA) and intravenous injections of BrdU 1mg/mouse (0.1 ml, BD Bioscience, San Jose, CA, USA) 2 hours prior to sacrifice. For intravital microscopy mice were stimulated by intraperitoneal injection of 10mg/kg TNF (R&D System, Minneapolis, MN, USA) 3 hours before anesthesia by intraperitoneal injection of 87.5mg/kg Ketamine plus 12.5mg/kg Xylazine. 50l of rhodamine 6G (1mg/ml) were injected intravenously to label leukocytes before the peritoneal cavity was cut open and the mesentery was carefully spread out on a glas cover slide thereby exposing mesenteric veins. Leukocyte rolling and adhesion within 100m long and 50-80m wide vein segments was recorded over 30sec with an AxioScope Vario and analyzed with the Axiovision Software (Carl Zeis Inc., Oberkochen, Germany). Mice were euthanized with CO2. Spleens, femurs and aortas were excised after vascular perfusion with 10ml NaCl 0.9%. Minced spleen and flushed bone marrow suspensions were strained through a 40 μm-nylon mesh (BD Biosciences). Blood and splenic cell suspensions were lysed in RBC lysis buffer (Biolegend, San Diego, Ca, USA). The aortic sinus was embedded in OCT (Sakura Tissue Tek, Torrance, CA), abdominal aortas were fixed in 10% formalin for en face staining. In order to obtain aortic cell suspensions for flow cytometry aortas were excised and opened longitudinally for pinning and brief enzymatic digestion of the intima. Plaques scraped and picked from the thoracoabdominal aorta and the plaque laden aortic root plus ascending aorta were collected, minced and digested in 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I, 60 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) for 45minutes at 37°C while shaking (750rpm). Cells were counted with a Hemocytometer (Neubauer Chamber).
Cholesterol assay. Total cholesterol levels were measured in heparinized plasma after a > 6 hour fasting period using the Lab Assay Cholesterol (code 294-65801, Wako) according to the manufacturer's instructions. The colorimetric assay was measured with a SpectraMAX Plus at 600nm wavelength (Serial P02918, Molecular Devices).
Histology. Frozen sections of the aortic roots were stained for lipids with Oil-Red-O (Sigma Aldrich, cat# 00625-25G) and macrophages with anti-Mac3 (rat anti-mouse, BD Pharmingen, clone M3/84, cat#553322). For immunofluorescent co-staining we used the Ki-67 (clone SP6, Abcam, #ab16667) and TUNEL kits (Promega, #G3250) according to the manufacturer's instructions. Abdominal aortas were fixed in 10% buffered formalin solution (Th. Geyer GmbH & Co, #2137) over night and washed in PBS prior to incubation with 85% propylene glycol for 2 minutes (Carl Roth, #0340.3). Aortas were stained with 0.5% Oil Red O for 4 hours at room temperature and washed with 85% propylene glycol and PBS. External fat was removed before aortas were cut longitudinally and pinned down on a silicon-elastomere plate (Factor II, cat#2186). En face Oil Red O staining and washing was repeated. Lesion and macrophage areas were quantified by blinded investigators with a computer-assisted image analysis software (Image-Pro Plus 5.1.2 Media Cybernetics). Table   Supplemental Fig. 1 Fostamatinib inhibits hypercholesterolemia-associated extramedullary myelopoiesis. Quantification of common myeloid progenitor (CMP) cell number and rate of proliferation as determined by BrdU incorporation as well as the number of macrophage dendritic cell progenitors (MDP) in the spleens of control (grey) and fostamatinib 0.3% (white) treated Apoe -/mice (n=10 per group) after 8 weeks of HCD. Results are presented as mean±SEM. *p≤0.05.

Supplemental Fig. 2 Fostamatinib lowers lesional macrophage content after fluorescent bead administration.
Apoe -/mice received fluorescend beads intravenously on day 4 after the start of HCD and were randomized to continued HCD with or without fostamatinib 0.3% on day 10. Mice were sacrificed 8 weeks thereafter for histologic analysis of aortic root lesions. Quantification of macrophage (Mac3) content in aortic root lesions of control (grey) and fostamatinib 0.3% (white) treated Apoe -/mice (n=8 per group). Results are presented as mean±SEM. *p≤0.05, t-test.