Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression
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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.
KeywordsAtherosclerosis SYK Monocytes Macrophages Progenitors Proliferation Egress
Atherosclerosis involves the accumulation of lipids and inflammatory cells in the arterial intima forming plaques. Macrophages ingest lipids and elaborate inflammatory mediators that promote plaque progression and destabilization . During atheroma initiation, macrophages accumulate primarily through infiltration of Ly6Chigh monocytes. In established plaques, however, macrophages renew through local proliferation rather than recruitment . It is unknown whether prolonged interference with monocyte recruitment to established plaques can limit macrophage accumulation.
Spleen tyrosine kinase (SYK), a cytosolic signaling protein, binds to various receptors and adaptors containing immunoreceptor tyrosine-based activation motifs (ITAM). Integrin signaling in myeloid cells, for example, requires the ITAM-bearing DAP12 adaptor and subsequent SYK activation to induce cell adhesion [9, 16]. We showed that treatment with the orally available SYK inhibitor fostamatinib reduced inflammatory cell recruitment to the vessel wall and infiltration to sites of inflammation in low density lipoprotein receptor deficient (Ldlr−/−) mice. Consequently, these animals developed less atherosclerosis . Of note, fostamatinib demonstrated significant improvement in ACR20 scores over placebo in two recent phase 3 trials with rheumatoid arthritis (RA) patients [5, 17]. The long-term effects of this SYK inhibitor on cardiovascular events in this high risk study population with chronic inflammation and accelerated atherosclerosis [4, 14] remains unknown.
The present study compared the efficacy of SYK inhibition in early and established atherosclerosis. We used Apoe−/− mice, which develop highly inflammatory atherosclerosis characterized by hypercholesterolemia-associated monocytosis . The myelopoietic growth factors GM-CSF and IL-3 stimulate monocyte production in atherosclerosis . Their receptors share a common beta chain (CBS) that associates with SYK . We hypothesized that SYK inhibition by fostamatinib would reduce monocytosis and cell adhesion, and provide us with a tool to lower monocyte contribution to the plaque in early and late atherosclerosis.
6-week-old female Apoe−/− mice (B6.129P2-Apoetm1Unc/J) were purchased from Charles River (Calco, Italy) and consumed a high cholesterol diet (1.25 % cholesterol; D12108 mod., Ssniff GmBH, Soest, Germany) ad libitum for 8 and 20 weeks, respectively, as indicated. Fostamatinib disodium (Astra Zeneca, UK) without carrier was incorporated at 0.3 % (w/w) into the diet as previously described . 1 mg BrdU per mouse (BD Bioscience, San Jose, CA, USA) was injected intravenously 2 h before euthanasia. Intravital microscopy of mesenteric veins was performed 3 h after intraperitoneal stimulation with TNFα. Ly6Chigh monocytes were mobilized into peripheral blood by intravenous injection of 300 ng CCL2 (R&D System, Minneapolis, MN, USA). 1 μm large green fluorescent beads (Fluoresbrite YG plain microspheres, Polysciences Inc., Eppelheim, Germany), diluted 1:4 in sterile PBS, were injected intravenously for in vivo cell labeling and tracking. Mice were housed under specific pathogen-free conditions and procedures approved by the Animal Care Committee of the University of Freiburg and the Regional Council (G-12/083).
Aortic roots were embedded in OCT Tissue Tek (Sakura Finetek, Tokyo, Japan) for sectioning and staining with specific antibodies that are listed in the supplemental method section. Abdominal aortas were pinned for Oli-red O en face staining.
Flow cytometry and cell sorting
Cells isolated from the blood, spleen, bone marrow and aorta were stained with specific antibodies and dyes that are listed in the supplemental method section. The APC Brdu Flow Kit detected intracellular BrdU incorporation (BD Bioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Intracellular phospho-SYKTyr519/520 (Cell Signaling Technology, Boston, MA, USA) was evaluated in fixed and permeabilized (ebioscience, San Jose, CA, USA) bone marrow and blood cells. Data were recorded on a BD Facs Canto II (BD Bioscience, San Diego, CA, USA). Monocytes and macrophages were sorted with a BD Facs Aria III (BD Bioscience, San Diego, CA, USA).
Colony forming cell assay
Bone marrow cells were isolated from the femur and resuspended in Mouse Methylcellulose Complete Medium (R&D, Minneapolis, MN, USA) at 105 cells/ml. Cells were stimulated with 10 ng/ml GM-CSF and 10 ng/ml IL3 (Peprotech, Rocky Hill, NJ, USA) in the presence of the vehicle DMSO 0.1 %, 0.1uM R406 in DMSO 0.1 % and 1μM R406 in 0.1 % DMSO, respectively, as indicated. Cells were incubated at 37 °C and 5 % CO2 for 5 days according to the manufacturer’s instructions and the number of colonies per 35 mm culture plate was counted under the microscope.
Cell culture and transwell assay
Ly6Chigh monocytes isolated from the blood and spleen were stimulated with 30 ng/ml M-CSF (Peprotech, Rocky Hill, NJ, USA) in normal culture media (RPMI-1640, 10 % FCS, NEAA, 1 % PenStrep) as indicated. Ly6Chigh monocytes isolated from the bone marrow were differentiated into macrophages with 30 ng/ml M-CSF in normal culture media over 7 days and then stimulated with 10 μg/ml human DiI-medium oxidized LDL (Kalen Biomedical, Montgomery Village, MD, USA) for 16 h in a minimal medium (2 % FCS in RPMI without growth factor supplements). Foam cell formation was evaluated by microscopy and flow cytometry. Bone marrow monocytes, isolated with the EasySep Mouse Monocyte Isolation Kit (Stemcell Technologies, Cologne, Germany) according to the manufacturer’s instructions, were allowed to migrate for 3 h in a transwell chamber (2 × 105 cells per well; Costar 6.5 mm Transwell inserts with 5 μm pore membrane, Corning, Kennebunk, ME, USA) stimulated by CCL2 20 ng/ml, CCL5 20 ng/ml (R&D System, Minneapolis, MN, USA) or BSA 20 ng/ml as control. Cells in the lower chambers were quantified with an automated cell counter (Z2 analyzer, Beckman Coulter, Inc., Brea, CA, USA).
RNA isolation and real-time PCR
Cells were sorted into RLT/β-mercaptoethanol for subsequent RNA isolation using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The Oviation PicSL WTA System V2 (NuGEN, San Carlos, CA, USA) was used for reverse transcription and cDNA amplification according to the manufacturer’s instructions. Quantitative TaqMan-PCR was run on a Bio-Rad CFX96 Touch Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The specific TaqMan probes used are listed in the supplemental method section.
Results are presented as mean ± SEM. Differences between 2 groups were evaluated by the unpaired Student’s t test if they passed the Kolmogorov–Smirnov normality test or otherwise by the non-parametric Mann–Whitney test as indicated. Differences between more than 2 groups were evaluated by Kruskal–Wallis test with Dunn’s multiple comparison test. p values ≤0.05 denote significant changes.
SYK inhibition attenuates atherosclerotic plaque development in Apoe−/− mice
SYK inhibition reduces medullary and extramedullary myelopoiesis in atherosclerotic Apoe−/− mice
SYK inhibition blocks GM-CSF/IL-3 stimulated myelopoiesis
SYK inhibition attenuates monocyte infiltration and differentiation but not macrophage egress from atherosclerotic lesions
SYK inhibition exerts subtle effects on macrophage foam cell formation and inflammation
SYK inhibition fails to prevent plaque progression
Our study indicates that the treatment of atherosclerosis may require disease stage specific approaches. Interventions directed against monocyte production and recruitment may limit early lesion formation or acute aggravation. In advanced lesions, however, when local proliferation dominates macrophage accumulation, targeting cell division itself or the respective stimulators may prove more effective. This concept, if applicable to humans, has major clinical implications as patients with atherosclerosis usually become symptomatic only after plaques have already formed.
We thank Jennifer Buchholz and Diana Selig for editorial assistance. We thank Esteban Masuda, Rigel Pharmaceuticals Inc. for his critical review.
Compliance with ethical standards
Conflict of interest
This work was supported by internal funds of the Department of Cardiology and Angiology I, University Heart Center Freiburg, and Astra Zeneca to A.Z., the German Research Foundation to I.H. (HI 1573/2-1) and the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School) to K.D.B.
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