The Role Of CTRP9 In The Formation Of Abdominal Aortic Aneurysm And Its Mechanism

BackgroundAbdominal Aortic aneurysm(AAA),a complex and fatal blood vessel disease,is the most common type of aneurysm clinically.AAA mainly occurs between the bifurcation of the renal artery and the bifurcation of the iliac artery,and is a degenerative vascular disease with permanent and irreversible local dilation.AAA is characterized by abnormal dilation of the abdominal aorta to more than 50%of its normal segment diameter,or dilation of more than 30mm.Risk factors for the development of AAA include advanced age,sex,smoking,genetic factors,coronary artery disease,hypertension,and cholesterol levels.With the diameter of aneurysm increasing,the risk of rupture hemorrhage increases greatly.However,as patients have no symptoms before aortic rupture and the risk is very high after aortic rupture,prevention of the occurrence and progression of AAA has become an important research direction at present.Currently,AAA lacks specific drug therapy and open surgery is the only widely available treatment.Extensive research is still needed to develop safe therapies,such as new drugs to mitigate aneurysm dilation and rupture.The pathogenesis of AAA includes a variety of pathological manifestations,including vascular extracellular matrix degradation,immune cell infiltration,inflammatory response,oxidative stress,apoptosis of vascular smooth muscle cells(VSMC),VSMC phenotypic transformation and so on.Extracellular matrix(ECM)components are mainly composed of macromolecules such as collagen,proteoglycan,elastin and glycoprotein,which form a complex network structure by connecting and interweaving with each other to maintain arterial wall tension and a certain degree of elasticity.Among them,MMP2(Matrix metalloproteinase)and MMP9 are overexpressed in aneurysm wall,and MMPs expressed by VSMC have high collagen degradation ability in aneurysm lesions.Therefore,MMP2 and MMP9 may play a synergistic role in the process of aneurysm degeneration.The location of VSMC makes them an ideal cell type for initiating medial degeneration.Migration of SMC from mid-membrane to intima involves a highly regulated elastin degradation process.Mast cells are another inflammatory cell type recently discovered in human AAA lesions.In addition to proinflammatory cytokines,many substances or enzymes produced by mast cells are thought to be involved in the development of AAA.Studies have shown that the level of superoxide in smooth muscle cells of AAA specimens is increased,and the deposition of a large number of reactive oxygen species(ROS)in cells can activate MMPs and promote the degradation of ECM.Existing studies have shown that inhibition of MMPs activity can reduce the development of AAA,particularly MMP2 and MMP9,highlighting that MMP2 and MMP9 may be drug targets for the treatment of AAA.Complement C1q/Tumor necrosis factor related protein 9(C1q/TNF-related protein 9,CTRP9)is composed of signal peptide,variable region of N-terminal,collagen structure of 56 G-X-Y repeats and c-terminal globule structure homologous to immune complement C1q.Studies have shown that CTRP9 levels are significantly reduced in patients with coronary atherosclerotic heart disease(CAD).With the deepening of research on CTRP9,studies show that CTRP9 is also involved in a variety of pathophysiological processes of various diseases,such as anti-inflammatory and antioxidant activities,inhibit VSMC proliferation and intima formation in mice after vascular injury,regulate glucose and lipid metabolism,and improve endothelial cell function.However,the role of CTRP9 in AAA and related mechanisms have not been reported and studied.Therefore,it is of great significance to explore the role and biomolecular mechanism of CTRP9 in AAA and find effective new targets of AAA for the prevention,diagnosis and treatment of AAA.Objectives1.To explore whether CTRP9 is related to the formation of abdominal aortic aneurysm;2.To explore the pathophysiological mechanism of CTRP9 on abdominal aortic aneurysm;3.To explore the effect of CTRP9 on smooth muscle cells in abdominal aortic vascular wall and related mechanisms.Methods1.Construct knockout miceUse CRISPR/Cas9 technology to obtain CTRP9 knockout mice from C57BL/6 background mice.After genotype identification,the homozygous interbred.2.Establish animal modelsAfter peritoneal injection of 1%pentobarbital anesthesia,an incision of approximately 2 cm was made along the linea alba.The abdominal aorta was dissected from surrounding tissues,and then the abdominal aorta was covered with 0.5 mol/L CaCl2 infiltrated cotton swabs.After 20 minutes,the cotton swabs were removed,and the same position was covered with PBS infiltrated cotton swabs.After 10 minutes,the mice were taken out,and the abdominal cavity was repeatedly irrigated with 0.9%sterile normal saline with a 1 mL syringe for 2～3 times.Then the abdominal muscles and skin of the mice were sutured with surgical thread,and the incision was closed.In the control group,the abdominal aorta was covered with 0.9%sterile normal saline.Part I:male WT mice aged 8 to 10 weeks and weighing 20 to 25 g were randomly divided into normal group and AAA group.Part Ⅱ:male WT mice and CTRP9-/-mice aged 8 to 10 weeks and weighing 20 to 25g were randomly divided into normal group and AAA group,respectively.Ultrasound imaging and measuring the maximum diameter of the aorta:after the experiment,vascular ultrasound was performed on 4 groups of mice to obtain B-mode images of abdominal aortic vessels.Then various tissues of mice were obtained and the maximum diameter of abdominal aorta of mice was measured.3.HE,Masson and VVG stainingThe morphological changes of abdominal aortic wall in each group were observed after staining.4.Immunohistochemical stainingThe relative contents of CTRP9,F4/80,a-SMA,Mast cell tryptase,MMP2,MMP9 and Collagen Ⅰ in the abdominal aorta tissues of mice in each group were detected by immunohistochemical staining.5.Cell cultureThe cells were cultured in DMEM high glucose medium+10%fetal bovine serum+100 U/mL penicillin-streptomycin medium in a constant temperature and humidity incubator at 37℃and 5%CO2.6.Establish cell model(1)Inflammatory stimulation Ang Ⅱ was successively added into VSMC according to the concentration gradient(0,100 nmol/L,500 nmol/L,l μmol/L,2 μmol/L).(2)After transfection of siCTRP9,1 μmol/L Ang Ⅱ was added into the corresponding wells to stimulate VSMC for 24 hours.VSMC were divided into four groups:siNC,siCTRP9,Ang Ⅱ+siNC,Ang Ⅱ+siCTRP9.After transfecting siCTRP9,the cells were pretreated with AMPK agonist AICAR for 1 h,followed by 1 μmol/L Ang Ⅱ stimulation for 24 h.7.DCFH-DA detection of ROS in VSMCAdd 250 μL DCFH-DA solution diluted at the ratio of 1:500 into each well,incubate for 30 min,rinse with PBS for 3 times,and observe ROS under manual inverted fluorescence microscope.8.Gelatinase spectrometryThe activities of MMP2 and MMP9 in abdominal aorta and VSMC were measured.9.Western blotThe expression levels of CTRP9,MMP2,MMP9,Collagen Ⅰ,Collagen Ⅲ,p-AMPK/total AMPK,SIRT1 were detected by Western blot in abdominal aorta and VSMC.10.Statistical analysisGraphPad Prism 8.0 was used for statistical analysis.The statistical results were expressed in the form of mean ± SEM.P<0.05 means that the difference was statistically significant.Results1.The expression of CTRP9 decreased significantly in AAA modelImmunohistochemical staining and Western blot analysis showed that the expression of CTRP9 in the abdominal aorta of WT AAA group was significantly reduced compared with the control group.Compared with the control group,double immunofluorescence staining of AAA tissues showed that α-SMA-expressing cells co-expressed CTRP9,which suggested that CTRP9 within VSMC may play a critical role in AAA.2.CTRP9-/-aggravated the development of AAA in miceAll CTRP9-/-were homozygous,which verified that CTRP9 had been knocked out in mice.By measuring the diameter of abdominal aorta and the ultrasonic images of blood vessels,the abdominal aorta dilated more significantly in the CTRP9-/-AAA group compared with the WT AAA group.3.CTRP9-/-aggravated pathological changes of aortic wallHE staining,Masson staining and VVG staining were used to observe the morphological changes of abdominal aortic wall in each group.The results showed that there was no significant difference in the morphology of the abdominal aorta in the control group.In WT AAA group,the abdominal aorta was significantly dilated,the middle membrane was ruptured,the extracellular matrix was degraded significantly,and elastic fibers lost their integrity.Compared with WT AAA group,the abdominal aorta in CTRP9-/-AAA group was dilated more significantly,the rupture of middle membrane was more serious,and the degradation of extracellular matrix was more obvious,obviously damaging the integrity of elastic fibers.4.CTRP9-/-promoted the infiltration of macrophages and mast cells,and reduced the content of VSMCImmunohistochemical staining showed that compared with WT AAA group,the relative contents of F4/80 and mast cell tryptase in the abdominal aorta of CTRP9-/-AAA group were significantly increased,while the relative contents of α-SMA were significantly decreased.5.CTRP9-/-promoted degradation of ECMImmunohistochemical staining showed that CTRP9-/-AAA group could increase the relative contents of MMP2 and MMP9 in abdominal aorta,while decrease the relative contents of Collagen Ⅰcompared with WT AAA group.Western blot was used to detect tissue proteins from abdominal aorta.Compared with WT AAA group,CTRP9-/-AAA group could promote protein expression levels of MMP2 and MMP9 in abdominal aorta,and reduce protein expression levels of Collagen Ⅰ and Ⅲ6.CTRP9 expression in VSMC decreased under Ang Ⅱ stimulationThe protein expression level of CTRP9 decreased gradually with the increase of Ang Ⅱconcentration,and the decrease of CTRP9 was most significant at 1 μmol/L Ang Ⅱconcentration.7.Interference with CTRP9 promoted the expression and activity of MMP in VSMC and reduced the expression of collagenInterference with CTRP9 promoted the expressions of MMP2 and MMP9 in VSMC,enhanced the activities of MMP2 and MMP9,and decreased the expressions of Collagen I andⅢ.First,siCTRP9 was used to interfere CTRP9 in VSMC.Compared with Ang Ⅱ group,AngⅡ+siCTRP9 promoted ROS production,promoted the expressions of MMP2 and MMP9,enhanced the activities of MMP2 and MMP9,and decreased the expressions of Collagen Ⅰ andⅢ.8.CTRP9 regulated MMP expression through AMPK/SIRT1 signaling pathwayIn vivo,compared with the control group,the protein expression levels of AMPK and SIRT1 in WT AAA group were significantly decreased,and CTRP9-/-AAA could further decrease AMPK and SIRT1.In vitro,interference with CTRP9 reduced the expression of AMPK and SIRT1.Compared with Ang Ⅱ.Ang Ⅱ+siCTRP9 decreased AMPK and SIRT1 more significantly.After the addition of AICAR,Western blot showed that CTRP9 regulated the expression of MMP2 and MMP9 through AMPK/SIRT1 signaling pathway.Conclusions(1)The expression of CTRP9 was significantly decreased in wild-type mice AAA and VSMC under inflammatory stimulation.(2)CTRP9 knockout promote the formation of AAA by promoting inflammatory response,smooth muscle cell reduction,MMP activation and extracellular matrix degradation,and destroy the systolic properties and integrity of smooth muscle in vascular wall.(3)CTRP9 knockdown promoted the activation of MMP and reduce the production of collagen in VSMC.(4)CTRP9 regulated the activation of MMP through AMPK/SIRT1 axis in vitro.