Oncostatin M receptor β deficiency attenuates atherogenesis by inhibiting JAK2/STAT3 signaling in macrophages

Abstract

Oncostatin M (OSM) is a secreted cytokine mainly involved in chronic inflammatory and cardiovascular diseases through binding to OSM receptor β (OSMR-β). Recent studies demonstrated that the presence of OSM contributed to the destabilization of atherosclerotic plaque. To investigate whether OSMR-β deficiency affects atherosclerosis, male OSMR-β^-/-ApoE^-/- mice were generated and utilized. Here we observed that OSMR-β expression was remarkably upregulated in both human and mouse atherosclerotic lesions, which were mainly located in macrophages. We found that OSMR-β deficiency significantly ameliorated atherosclerotic burden in aorta and aortic root relative to ApoE-deficient littermates and enhanced the stability of atherosclerotic plaques by increasing collagen and smooth muscle cell content, while decreasing macrophage infiltration and lipid accumulation. Moreover, bone marrow transplantation of OSMR-β^-/- hematopoietic cells to atherosclerosis-prone mice displayed a consistent phenotype. Additionally, we observed a relatively reduced level of JAK2 and signal transducer and activator of transcription (STAT)3 in vivo and under Ox-LDL stimulation in vitro. Our findings suggest that OSMR-β deficiency in macrophages improved high-fat diet-induced atherogenesis and plaque vulnerability. Mech-anistically, the protective effect of OSMR-β deficiency on atherosclerosis may be partially attributed to the inhibition of the JAK2/STAT3 activation in macrophages, whereas OSM stimulation can activate the signaling pathway.

Keywords: Janus kinase 2/signal transducer and activator of transcription 3; atherosclerosis; inflammation.

Fig. 1.

Enhanced OSMR-β expression in the atherosclerotic plaques of ApoE^−/− mice and patients with CHD. A: The expression of OSMR-β in the coronary arteries of normal donors and patients with CHD (n = 4, *P < 0.05 versus donor). B: The expression of OSMR-β in aortas from ApoE^−/− mice fed NC or a HFD (n = 4, *P < 0.05 versus control). C, D: Double immunofluorescence staining for OSMR-β (red) and Mac3 (macrophage, green) in the coronary arteries of normal donors and patients with CHD and in the aortic sinuses from ApoE^−/− mice fed NC or a HFD (scale bar = 50 μm). The quantification was carried out by normalizing the fluorescence intensity of the OSMR-β-positive area with the Mac3-positive area in the atherosclerotic plaque. E: OSMR-β expression in BMDMs upon 15 μg/ml Ox-LDL stimulation for 24 h. *P < 0.05 versus control group.

Fig. 2.

Deletion of OSMR-β ameliorated the development of atherosclerosis. A: Genotyping of OSMR-β^−/−ApoE^−/− mice and ApoE^−/− littermates. B, C: En face analysis of aortas from OSMR-β^−/−ApoE^−/− mice and ApoE^−/− littermates fed NC or a HFD; aortas were stained with Oil Red O (n = 10). D, E: Left panel, representative images of the aortic sinus or brachiocephalic arteries from OSMR-β^−/−ApoE^−/− and ApoE^−/− mice stained with H&E. Right panel, quantification of the atherosclerotic lesion area (n = 6). *P < 0.05 compared with ApoE^−/−.

Fig. 3.

OSMR-β ablation reduces the area of necrotic core and enhances plaque stability. A, B: Left panel, representative images used to analyze the necrotic area in aortic roots and brachiocephalic arteries from OSMR-β^−/−ApoE^−/− and ApoE^−/− mice. Right panel, quantitation of the necrotic area (n = 6). C–E: Histological analysis of plaque stability in the aortic sinus. C: PSR staining for the detection of collagen content. D: Immunofluorescence analysis of SMA for the detection of SMC content. E: Immunofluorescence analysis of CD68 for the detection of macrophage infiltration. F: Oil Red O staining for the detection of lipid accumulation. *P < 0.05 versus ApoE^−/−.

Fig. 4.

OSMR-β deficiency decreases atherosclerosis-induced inflammation. A: Real-time PCR analysis of pro-inflammatory cytokine expression in the whole aortas of OSMR-β^−/−ApoE^−/− and ApoE^−/− mice compared with CD68 expression (n = 3). B: Detection of MCP-1, TNF-α, IL-6, and IL-1β expression in the serum with ELISA (n = 6). *P < 0.05 compared with ApoE^−/−.

Fig. 5.

OSMR-β deficiency inhibits the activation of JAK2/STAT3 signaling. A: Western blot analysis of the expression of phosphorylated and total JAK2, STAT3, STAT1, and STAT5 in the whole aortas of OSMR-β^−/−ApoE^−/− and ApoE^−/− littermates. Quantitation of relative phosphorylated protein expression after normalization to each total protein expression, respectively (n = 3). B: Immunofluorescence costaining of phosphorylated STAT3 (green) and CD68 (red) in atherosclerotic plaques (scale bar = 50 μm). C: Western blot analysis of phosphorylated and total JAK2 and STAT3 in peritoneal macrophages from OSMR-β^−/−ApoE^−/− and ApoE^−/− littermates upon 15 μg/ml Ox-LDL treatment for 24 h. The results present phosphorylated protein expression compared with the total protein expression, respectively (n = 3). D: The JAK2 and STAT3 expression in macrophages from ApoE^−/− mice upon PBS or OSM treatment. *P < 0.05 compared with control group.

Fig. 6.

The absence of OSMR-β from bone marrow-derived cells attenuates atherogenesis. A: DNA was isolated from whole blood for genotyping to confirm genome reorganization with PCR. B: Left panel, Oil Red O staining for en face analysis of atherosclerotic plaques in aortas from OSMR-β^−/−ApoE^−/−→ApoE^−/− (n = 8) and ApoE^−/−→ApoE^−/− (n = 10) mice. Right panel, analysis of the atherosclerotic lesion area (n = 10 or 8, each group). C: Left panel, H&E staining of the aortic root from OSMR-β^−/−ApoE^−/−→ApoE^−/− mice and ApoE^−/−→ApoE^−/− mice. Right panel, quantification of the atherosclerotic lesion area (scale bar = 500 μm, n = 6). D: Oil Red O staining of peritoneal macrophages from OSMR-β^−/−ApoE^−/−→ApoE^−/− mice and ApoE^−/−→ApoE^−/− mice treated with Ox-LDL. E: Western blot analysis of CD36 and ABCA-1 expression in OSMR-β^−/−ApoE^−/−→ApoE^−/− and ApoE^−/−→ApoE^−/− mice. F: Real-time PCR analysis of pro-inflammatory cytokine expression in macrophages from OSMR-β^−/−ApoE^−/−→ApoE^−/− mice and ApoE^−/−→ApoE^−/− mice treated with Ox-LDL (n = 3). *P < 0.05 compared with ApoE^−/−→ApoE^−/− group.