Role And Mechanism Of Oncostatin M Receptor ? In Atherogenesis

Objective:High-fat-diet(HFD)induced systemic OSMR-?-/-ApoE-/-(Double KO),ApoE-/-(ApoE knockout)mice and isolated primary peritoneal macrophages under ox-LDL stimulation were used as experimental subjects to explore the underlying mechanism of OSMR-? deficiency on the development of HFD induced atherosclerosis of mouse models both in vivo and in vitro.Methods:(1)Experimental mouse were randomly separated into four groups,which were ApoE-/-NC,ApoE-/-HFD,OSMR-?-/-ApoE-/-NC and OSMR-?-/-ApoE-/-HFD respectively.(2)Male mouse of 8-week-old were fed with HFD or NC for up to 28 weeks before sacrificed.After anesthetization with pentobarbital Sodium,open the chest and rapidly separate the entire aorta(from the root of aorta to the branch of iliac artery),followed by dissection of adipose tissue from the vascular adventitial,take photos and then fixed in formalin.The lower third cardiac apex were removed and the upper third were reserved to obtain the aortic sinus.(3)Paraffin-embedded tissue slices were used to perform pathological and immunofluorescent staining.Oil Red O staining was performed to analyze the en face atherosclerotic lesions of the aorta.The aortic roots and brachiocephalic arteries were stained with hematoxylin and eosin(H&E)for morphological analysis of atherosclerotic plaques,picrosirius red(PSR)to evaluate collagen deposition.Immunofluorescence were performed to detect the expression of OSMR-? within the plaque,transcriptional and protein levels of pro-inflammatory cytokines,activated levels of relative signaling transducers.(4)Studies of primary peritoneal macrophages:Bone marrow-derived macrophages eluted from the peritoneum of ApoE-/-mice were harvested under sterile conditions.After serum starvation for 24 h,the macrophages were incubated with 15?g/ml Ox-LDL for 24 h.Finally,the cells were fixed and stained with Oil O Red to asses the lipid accumulation capacity of macrophages.(5)Bone marrow transplantation:Male ApoE-/-recipient mice were irradiated with a total dose of 11 Gy prior to transplantation.Donor bone marrow was isolated from male OSMR-?-/-ApoE-/-or ApoE-/-mice by flushing femurs and tibias and injected to each irradiated mouse.After a HFD for 16 weeks,the mice were sacrificed for the following experiments.(6)Human specimens:Atherosclerotic plaques were collected from the right coronary artery of patients with CHD who undergoing heart transplantation.Control samples were obtained from normal donors who were unsuitable for transplantation without cardiovascular diseases.Vascular tissues were under procedures similar to animal operation to detect the expression and location of OSMR-? in the artery.Results:(1)Double-immunofluorescence staining with OSMR-? and Mac3 revealed revealed an increased OSMR-P expression,which located mainly in the macrophages of the atherosclerotic lesions of patients with CHD and the HFD-induced ApoE-/-mouse model.(2)En face analysis of atherosclerotic lesions by Oil Red O and H&E staining indicat ed that OSMR-? deficiency(ApoE-/-background)attenuated the development of HFD induced atherosclerosis,plaque areas of aorta root and brachiocephalic arteries were both decreased.(3)OSMR-? deficiency enhanced the stability of atherosclerotic plaques,presented as total necrotic core area was significantly smaller in OSMR-?-/-ApoE-/-mice(H&E staining of aorta root and brachiocephalic arteries),the collagen and SMC content covering the fibrous cap were increased(PSR and immunofluorescent staining),lipid accumulation decreased(Oil O Red staining)and macrophage infiltration reduced(immunofluorescent staing with anti-CD68).(4)The protective role of OSMR-? deficiency in atherogenesis was dependent on its anti-inflammation effect,Real-time PCR analysis showed that multiple pro-inflammatory genes were significantly decreased.The level of inflammatory factors in the serum was also down regulated,measured with ELISA kit.(5)Loss of OSMR-? inhibited the JAK2/STAT3 signal pathway.Western blot showed the phosphorylated level of both factors were decreased with the immunofluorescent staining revealed the activation of STAT3 were clearly impaired.(6)In vitro experiments of primary peritoneal macrophages stimulation with ox-LDL were consistent with in vivo results.Bone marrow transplantation further confirmed the conclusion by displaying an acquired loss of OSMR-? improved the HFD induced atherosclerosis with reduction of foam cell formation which was possibly attributed to the intervention of LDL flux of macrophages by facilitating its extracellular transportation.