The Inhibitory Effect Of Chemerin-9 On Experimental Abdominal Aortic Aneurysm In Mice

BackgroundAbdominal aortic aneurysm(AAA),a life-threatening arterial degenerative disease,is defined as the development of irreversible localized aortic dilatation that exceeds 50%of the normal diameter.At present,the prevalence of AAA is estimated at 8%among men over 65 years old.The diameter of aneurysm progressively exceeds and the risk of aneurysm rupture elevated with increasing age.The mortality rate of AAA is over 80%after ruptured.Currently,AAA is one of the serious clinic diseases with high mortality that threatens older people’s health.Providing adequate protection against rupture of aneurysms is the purpose of AAA treatment.Surgical repair is the only effective treatment to inhibit AAA rupture.When the AAA diameter of male and female patients exceeds 5.5cm and 5cm,open surgery or endovascular aneurysm repair can be performed.However,early surgical intervention for patients with small AAAs whose diameter does not reach the operative indications has no obvious advantage in terms of survival rate.Currently,the treatment of small AAAs is limited to control cardiovascular risk factors and regularly monitor by imageological examination.There is no effective drug that can limit the development of small aneurysms.Therefore,the discovery of novel,efficient clinical therapeutic drug that inhibit AAA progression has immense significance.The formation of AAA is a complex and dynamic pathophysiological process.It is reported that the increased infiltration of macrophages and lymphocytes on the aneurysm is the most prominent pathological feature in the development of AAA.These inflammatory cells secrete matrix metalloproteinases(MMPs)and various proinflammatory chemokines,such as interleukin(IL)-1 β,IL-6 and tumor necrosis factor(TNF)-α,which can damage the elastic fibers of the media,increase the depletion of vascular smooth muscle cells(VSMCs),and reduce the compliance of artery walls,which makes the artery progressively dilation and rupture under the pulsive blood flow.In addition,proinflammatory chemokines recruit more inflammatory cells to the injured site to initiate inflammatory cascades,and aggravate the damage of aortic wall.Inflammation runs through the whole process of the formation,development and rupture of AAA.Hence,intervening these inflammatory mechanisms is beneficial to delay the progression of AAA.Chemerin,a coding product of tazarotene inducible gene 2(TIG2),is mainly secreted by liver,platelet,white adipose tissue and perivascular adipose tissue.As a multifunctional adipocytokines,chemerin can promote inflammatory responses,adipogenesis and angiogenesis,which plays an important role in cardiovascular disease,tumor and diabetes.Chemokine-like receptor 1(CMKLR1),as the main receptor of chemerin,is mainly expressed in adipocytes,endothelial cells,macrophages and natural killer cells.Previous reports demonstrated that chemerin binding to CMKLR1,was involved in atherosclerosis,rheumatoid arthritis and inflammatory bowel disease(IBD)by mediating inflammatory responses.Therefore,we speculated that the levels of chemerin and CMKLR1 may be associated with AAA progression.Chemerin-9,comprising of nine amino acids from the C-terminus of chemerin,has a high affinity for CMKLR1.In vivo,chemerin-9 exerts an anti-inflammatory effect by targeting CMKLR1,thus restraining the development of several diseases,including atherosclerosis,Alzheimer’s disease,and pancreatogenic diabetes.However,the role of chemerin-9 in AAA has not yet been reported.In this study,we will evaluate the levels of chemerin and CMKLR1 in AAA and explore the effects of chemerin-9 on murine AAA models to provide new therapeutic drug for the clinic treatment of AAA.Objective1.To evaluated the levels of chemerin in human AAA serum and detect the expression of chemerin and CMKLR1 in human AAA tissue.2.To evaluated the levels of chemerin in mouse AAA serum and detect the expression of chemerin and CMKLR1 in mouse AAA tissue.3.To explore the effect of chemerin-9 on AAA model in mice,and to provide a theoretical basis for the future study of AAA.Methods1.The expression of chemerin and CMKLR1 in AAA patients:1)From October 2019 to May 2020,human blood samples were collected from 18 patients with AAA who were diagnosed by computer tomography(CT)and magnetic resonance imaging(MRI)at Shandong Provincial Hospital and 10 age-matched healthy volunteers.10 human AAA specimens were obtained from patients undergoing open repair at Shandong Provincial Hospital.As a control,6 age-matched normal human aorta samples were obtained from multiorgan donors.Patients with aortitis,connective tissue disorders,or ruptured aneurysms were excluded.This study was approved by the Human Research Committee of Shandong Provincial Hospital affiliated with Shandong University,and the procedures conformed to the ethical guidelines of the Declaration of Helsinki.2)The expression of chemerin in human serum was detected by ELISA.The levels of mRNA and protein of chemerin and CMKLR1 in human AAA tissue were detected by qRT-PCR and Western blotting.The expression of chemerin and CMKLR1 in human AAA tissue were detected by immunofluorescence double staining.2.The expression of chemerin and CMKLR1 in Ang Ⅱ-induced AAA models:1)Establishment and grouping of murine AAA models:40 male ApoE-/-mice were randomly divided into sham and AAA groups(n=20 per group).Treatments were administered by subcutaneously implanted micro-osmotic pumps in the dorsum of the neck for 28 days.Mice in the sham group were infused with saline;those in the AAA group,with Ang Ⅱ.At 28 days after pump implantation,B-ultrasound system was used to examine the maximum inner luminal diameters of the abdominal aortas and calculate the incidence of AAA.2)At 28 days after pump implantation,mice were sacrificed for serum and tissue harvesting.ELISA was used to detect the expression of chemerin in mice serum.qRT-PCR and Western blotting were used to detect the levels of mRNA and protein of chemerin and CMKLR1 in murine AAA tissue.Immunofluorescence double staining was used to assess the positive expression of chemerin and CMKLR1 in murine AAA tissue.3.The effect of chemerin-9 on Ang Ⅱ-induced AAA models:1)Establishment and grouping of murine AAA models:60 male ApoE-/-mice were randomly divided into sham,AAA and chemerin-9 groups(n=20 per group).Treatments were administered by subcutaneously implanted micro-osmotic pumps in the dorsum of the neck for 28 days.Mice in the sham group were infused with saline;those in the AAA group,with Ang Ⅱ;those in the chemerin-9 group,with Ang II and chemerin-9.2)A B-ultrasound system was used to examine the maximum aneurysm diameters of the abdominal aortas at 0,7,14,28 days after pump implantation.The growth curve of the aneurysms was drawn according to maximum aneurysm diameters.3)At 28 days after pump implantation,the incidence of AAA was calculate on the basis of maximum aneurysm diameters.4)At 28 days after pump implantation,mice were sacrificed for tissue harvesting.HE staining was used to detect the morphological changes.EVG staining was used to detect the elastin in the media.Immunohistochemical staining was used to detect theα-SMA positive expression in aortic wall.5)Immunohistochemical staining was perform on the CD68,CD8,B220 and CD31 indicators in aortic wall to observe the infiltration of inflammatory cells and angiogenesis.6)qRT-PCR was perform to detect the expression levels of IL-1β,IL-6 and TNF-αin aortic wall.7)Immunohistochemical staining,qRT-PCR and Western blotting were used to compare the expression differences of MMP-2 and MMP-9 in the abdominal aorta tissues.8)At 28 days after pump implantation,the serum and tissue of mice in AAA and chemerin group were harvested.ELISA was used to detect the expression of chemerin in mice serum.qRT-PCR and Western blotting was used to test the levels of mRNA and protein of chemerin and CMKLR1 in aortic tissue.Immunofluorescence double staining was used to compare the differences of chemerin and CMKLR1 in aortic tissue.Results1.Chemerin and CMKLR1 were upregulated in human AAAELISA results showed that the chemerin levels in the AAA group was significantly higher than those in the normal group.qRT-PCR and Western blotting results showed that the levels of chemerin and CMKLR1 in human AAA tissues were also remarkably elevated.Immunofluorescence double staining was performed to investigate the expression of chemerin and CMKLR1.And the result revealed that the positive expression of chemerin and CMKLR1 in AAA was upregulated compared with that in normal aortic tissue.2.Chemerin and CMKLR1 were elevated in experimental AAAExperimental AAA models was successfully constructed by administering AngⅡ with subcutaneously implanted micro-osmotic pumps.ELISA demonstrated that increased expression of chemerin in the serum of AAA mice.qRT-PCR and Western blotting results exhibited that compared with the sham group,the levels of chemerin and CMKLR1 in the AAA group were also remarkably increased.Similar results were also obtained via immunofluorescence double staining that the expression of chemerin and CMKLR1 in mice with AAA was upregulated.3.Chemerin-9 inhibited AAA enlargement and pathological lesionChemerin-9 remarkably suppressed the enlargement of abdominal aorta and decreased the incidence of AAA according to the B-ultrasound results.HE staining showed that the adventitia of aortic wall became thinner after chemerin-9 administration.EVG staining revealed that chemerin-9 prevented complete degradation and deficiency of elastic laminae in the medial layer.Furthermore,immunohistochemical staining suggested that chemerin-9 effectively reversed the VSMC loss and restored the compliance of vascular wall.4.Chemerin-9 alleviated inflammatory responses and neovascularization in experimental AAAImmunohistochemical staining results detected that chemerin-9 mitigated the infiltration of CD68+macrophages,B220+B cells and CD8+T cells in the aortic wall,and decreased the expression of CD31+neovascularization.The mRNA levels of IL-1β,IL-6 and TNF-α were downregulated after chemerin-9 treatment.In addition,immunohistochemical staining results showed that the positive expression of MMP-2 and MMP-9 was decreased significantly in the chemerin-9 group.qRT-PCR and Western blotting results indicated that chemerin-9 inhibited the mRNA and protein levels of MMP-2 and MMP-95.Chemerin-9 downregulated the levels of chemerin and CMKLR1 in experimental AAAELISA results showed that the circulating chemerin levels in the chemerin-9 group was significantly lower than that in the AAA group.qRT-PCR and Western blotting showed that the mRNA and protein expression of chemerin and CMKLR1 were downregulated after chemerin admonition.And the results of immunofluorescence double staining showed that chemerin-9 suppressed the expression of chemerin and CMKLR1 in aortic wall.Conclusion1.Chemerin and CMKLR1 were obviously elevated in AAA of patients and mice.2.Chemerin-9 could markedly suppress the degradation of elastic fibers,the loss of VSMCs,chronic inflammation and neovascularization,which inhibited the formation and progression of AAA.3.Chemerin-9 may alleviated inflammatory lesions of aortic wall by inhibiting the Chemerin/CMKLR1 axis,which provided theoretical basis for clinic AAA therapy.