Pathogenic Role And Mechanism Study For Atypical Chemerin Receptor CCRL2 In Atherogenesis

Atherosclerosis is a multifactorial and chronic systemic inflammatory disease that begins with the accumulation of lipids in the arterial wall, subsequently progresses to complex vulnerable plaques liable to rupture, which probably gives rise to luminal narrowing of arteries and insufficient blood supply to the tissue and organ. Upon plaque rupture and thrombus formation, these most common forms of cardiovascular disease manifest as acute coronary syndrome(ACS), myocardial infarction or stroke. Atherogenic recruitment of leukocytes involves a sequence of rolling, firm adhesion, lateral migration and transendothelial diapedesis and is mostly controlled by chemokines and chemokine receptors. Using gene-deficient mice and antagonists, valuable insights into chemokine ligand-receptor axes and their specific contributions to atherogenesis were obtained. So far, there have been many chemokine receptor antagonists which were selected as a clinical candidates for treatment of atherosclerosis based on their potency and favorable pharmacokinetic properties.Studies on human and animals not only reveal a broad expression of CCRL2 especially in endothelial cells, macrophages and dendritic cells in mice but also suggest a potential role of this chemotactic systems in the pathogenesis of several diseases. CCRL2 has a ligand called chemerin and chemerin has two additional receptors CMKLR1 and GPR1. Recently evidences suggest that chemerin and CMKLR1 are present in mouse and human atherosclerotic plaques. However, whether CCRL2 participates the development of atherosclerosis is still unknown.ObjectiveHere we use the apolipoprotein E-deficient(Apo E-/-) mouse model of atherosclerosis to investigate the influence of a global CCRL2 deficiency on atherogenesis and to clarify the mechanism by which CCRL2 and its ligand chemerin promotes atherosclerosis.Methods1. We isolated m RNA and protein from the aortas of Apo E-/- male mice on high fat diet for 0, 4, 8 and 12 weeks and detected the expression of CCRL2 and chemerin in aorta by real-time-PCR and Western blot and chemerin level in serum by ELISA.2. A fter 4, 16 and 24 weeks on high fat diet, we detected atherosclerotic lesion size in the whole aorta and the area of aortic arch and descending aorta by Sudan IV staining in CCRL2-/-Apo E-/- mice compared to CCRL2+/+Apo E-/- control mice. Furthermore, the plaque size of aortic root was analyzed using oil red O staining. We also measured the weight and the plasma lipid contents of two groups through obtaining the venous blood of hyperlipidemia mice and compared the mice with or without HFD.3. After 12 weeks on high fat diet, we examined accumulation of macrophages, dendritic cells, T cells and vascular smooth cells in the aortic plaques in CCRL2-/-Apo E-/- mice compared to CCRL2+/+Apo E-/- control mice by aortic root immunofluorescence staining, which we can used different combinations of biomarkers such as macrophages(MOMA-2), dendritic cells(CD11c), T cells(CD3), vascular smooth cells(αSMA). We also analyzed collagen content of the atherosclerotic plaque by frozen section combined Masson staining to examine whether CCRL2 deficiency affect the stabilization of atherosclerotic lesions. To detect the effect of CCRL2 on macrophage phenotype, we used different combinations of biomarkers to define the M1 phenotype such as MCP-1、IL-1β and IL-12 and M2 phenotype was identified by IL-10、CD163 and CD304. The m RNA expression of M1 and M2 markers were determined to detect whether CCRL2 deficiency affect polarization of macrophages. Finally, peritoneal macrophages were isolated from two groups to investigate the effect of CCRL2 deletion on foam cell formation. Foam cells were induced by ox-LDL to exhibit extensive oil red O droplets.4. We use frozen section with aortic root immunofluorescence photomicrographs of aortic root from Apo E-/- mice with 12 weeks high fat diet mice to detect whether CCRL2, chemerin, CMKLR1 express in plaque and examine the cellular localization in atherosclerotic plaque between CCRL2 and inflammatory cells such as macrophages(CD68), dendritic cells(CD11c), T cells(CD3) or vascular cells such as endothelial cells(CD31) and smooth cells(αSMA).5. We first analyzed CCRL2 expression in the area of aortic arch and descending aorta and analyzed the inner surface of endothelial cells by CCRL2 en face staining. Then we use partial carotid ligation(PCL) model to detect CCRL2 expression in the left carotid artery(LCA) exposed to disturbed-flow compared to control right carotid artery(RCA) exposed to stable-flow ather ligation for 0 and 24 hours. We aslo analyzed CCRL2 expression in the inner surface of endothelial cells by en face staining. We finally showed that CCRL2-/-Apo E-/- mice and CCRL2+/+Apo E-/- mice on high fat diet for 2 weeks in PCL model to define whether the deletion of CCRL2 affect atherosclerotic plaque formation induced by D-flow.6. CCRL2+/+Apo E-/- mice and CCRL2-/-Apo E-/- mice were treated with LPS and leukocytes were labeled with a kind of cell membrane fluorescent probe DIOC6 in vivo to confirm whether CCRL2 knockout affects leucocyte rolling and adhesion in the acute vascular inflammation model for femoral artery in vivo. Next male Apo E-/- mice at 8 weeks old were irradiated with a dose of 10 Gly and transplanted with 5×106 bone marrow cells from either CMKLR1+/+Apo E-/- or CMKLR1-/-Apo E-/- mice then were ready for the acute vascular inflammation model. We want to detect whether CMKLR1+ leucocyte take participate in leucocyte rolling and adhesion with vessel endothelium at this model.7. Next we also detect the chemerin contents and CMKLR1 positive macrophages in plaque by immunofluorescence in CCRL2-/-Apo E-/- compared to CCRL2+/+Apo E-/- to detect whether there are interaction between CCRL2 and chemerin and whether CCRL2 affects the CMKLR1+ macrophage infiltration in plaque. To confirm whether there is CCRL2/chemerin/CMKLR1 axis, immunofluorescence staining of the aortic root sections of the atherosclerotic aorta of CCRL2+/+Apo E-/- and CCRL2-/-Apo E-/- mice on HFD for 4 weeks was performed using antibodies against CCRL2, chemerin, and CMKLR1. Finally we detect serum chemerin level between this two groups to examine whether CCRL2 can regulate the serum chemerin level.Results1. Results showed that comparing to the Apo E-/- mice on chew diet, CCRL2 m RNA expression reached to its peak level at 4 weeks on HFD and then remained significantly higher levels at 8 weeks and 12 weeks. Consistently, chemerin m RNA expression reached to its peak level at 4 weeks and remained significantly higher levels at 8 weeks, but decreased to normal level at 12 weeks on high fat diet compared to Apo E-/- mice on chew diet. Western Blot was used to determine CCRL2 protein levels in the aortas of Apo E-/- male mice. Consistent to the RNA expression, CCRL2 protein level increased at 4 weeks reached to its peak level at 8 weeks, and remained higher levels till 12 weeks as compared with Apo E-/- mice on chew diet. Serum levels of total chemerin measured by ELISA were significantly elevated in Apo E-/- mice on HFD for 12 weeks compared with WT mice.2. After 4, 16, 24 weeks on high fat diet, the plaque size of total aorta was reduced by 32.6%, 27.2%, 35.2% respectively in CCRL2-/-Apo E-/- mice compared to CCRL2+/+Apo E-/- mice. Effect of CCRL2 deletion on plaque formation was also compared at different locations of aorta. The plaque size in the area of aortic arch after 4, 16, 24 weeks of HFD feeding was reduced by 38.8%, 37.8%, 28.6% respectively, in CCRL2-/-Apo E-/- mice compared to CCRL2+/+Apo E-/- mice. However, the plaque size in the descending aorta after 4 and 16 weeks of HFD feeding did not decreased when CCRL2 was deleted. In contrast, after 24 weeks of HFD feeding, the plaque size in the area of descending aorta was decreased by 40.8%. Total lesion size in aortic root was decreased by 47.9% with 16 weeks HFD. There were no significant differences in serum levels of total cholesterol(TC), triglycerides(TG), high density lipoprotein(HDL), low density lipoprotein(LDL) between CCRL2+/+Apo E-/- and CCRL2-/-Apo E-/- mice before and after HFD. Additionally, CCRL2 deletion did not affect body weights of atherosclerosis-prone mice during the development of atherosclerosis.3. Macrophages accumulation within aortic root plaques was significantly reduced by 58.4% in CCRL2-/-Apo E-/- mice compared to Apo E-/-mice when mice were fed on HFD for 12 weeks. M1 markers were universally downregulated in the atherosclerotic plaque of CCRL2-/-Apo E-/- mice compared to CCRL2+/+Apo E-/- mice. Conversely, expression levels of M2 markers were universally upregulated after CCRL2 deletion. However, accumulation of T cells, DCs, SMCs, collagen contents did not change when CCRL2 was deleted in atherosclerotic prone mice. We examined the effect of CCRL2 deletion on the foam cell formation as peritoneal macrophages can be differentiated into lipid-laden macrophages by taking up ox-LDL. CCRL2 deficiency impaired form cell formation.4. To examine the cellular localization of CCRL2, the markers for vascular wall cells(mainly endothelial cells, vascular smooth muscle cells) and for the atherosclerotic prone cells(macrophages, T cells and dendritic cells) were used. Results show that CCRL2 was found to co-localize with endothelial cells, macrophages, dendritic cells and T cells in atherosclerotic lesions but not co-localize with VSMCs in the atherosclerotic lesion.5. We first examined CCRL2 expression in aortic arch and descending aorta, and found that RNA level of CCRL2 was higher in aortic arch compared with descending aorta and CCRL2 en face staining is same as the RNA expression. To confirm this finding, we induced D-flow in vivo by partial carotid ligation(PCL). After ligation for 24 h, results showed that CCRL2 expression was significantly increased in the left carotid artery(LCA), which was partially ligated and exposed to D-flow for 24 hours, than in the contralateral right carotid artery(RCA) exposed to stable flow(S-flow) as expressed by the RNA ratio of LCA to RCA. To directly visualized CCRL2 expression on the surface of vascular endothelium, carotid arteries were also treated by en face staining after PCL. A profound CCRL2 expression on the endothelium of LCA with PCL was observed. To determine whether CCRL2 is involved in D-flow-induced atherosclerosis, we performed PCL and with HFD 2 weeks. Atherosclerotic lesion size in the LCA under disturbed condition was decreased by 44.72% in CCRL2-/-Apo E-/-mice compared to CCRL2+/+Apo E-/- control mice.6. An in vivo acute vascular inflammation model of femoral artery showed that LPS-induced leucocyte rolling over the endothelium of CCRL2-/-Apo E-/- mice was reduced by 44.8% than CCRL2+/+Apo E-/- mice. After bone marrow transplantation, Apo E-/- recipient mice transplanted with CMKLR1+/+Apo E-/-(WT-WT) or CMKLR1-/-Apo E-/- bone marrow(KO-WT). After injection of LPS, leucocyte rolling over the vascular wall was dramatically decreased by 56% in the femoral arteries of KO-WT mice compared with WT-WT animals.7. By immunofluorescence staining, both chemerin contents and CMKLR1 positive macrophages decreased in CCRL2-/-Apo E-/- mice compared to CCRL2+/+Apo E-/- mice on HFD 12 weeks and there is an elevated in serum chemerin level in CCRL2-/-Apo E-/- mice. CCRL2 was found to co-localize with chemerin and CMKLR1 in the intima interspace of the atherosclerotic aorta root sections of Apo E-/-mice. And our data showed that CCRL2 not only colocalizes with CMKLR1 on endothelium to anchor chemerin but also forms a trio-complex with chemerin and CMKLR1 within the atherosclerotic aorta root, presumably facilitating leucocyte infiltration and cell-cell interaction during plaque formation.Conclusion1. Deletion of CCRL2 reduces plaque size in atherosclerotic-prone mice.2. Deletion of CCRL2 reduces macrophage infiltration and polarity.3. CCRL2 is upregulated in aortas with HFD or PCL model and deletion of CCRL2 decreases carotid plaque formation induced by PCL and HFD4. Vascular endothelial CCRL2 is likely to modulate local concentrations of chemerin to recruit CMKLR1+ monocytes to the subendothelial space of vessel wall.5. Therefore, our data showed that CCRL2 participates the development of atherosclerosis potentially via its interaction with ligand chemerin and CMKLR1.