CCL7 Promoted The Development Of Angiotensin Ⅱ-induced Abdominal Aortic Aneurysm And Its Mechanism

Background:Abdominal aortic aneurysm（AAA）is a chronic inflammatory vascular disease characterized by persistent and local expansion of the abdominal aorta.Once ruptured,the mortality rate is more than 80%.It is a high-risk disease,but there is no effective treatment at present.The pathological process of AAA mainly includes abundant inflammatory cells infiltration in the media and adventitia,vascular smooth muscle cell（VSMC）apoptosis in the media,endothelial dysfunction and extracellular matrix remodeling caused by elastic fiber rupture and collagen deposition.Macrophages play a key role in the formation of AAA by secreting proinflammatory cytokines,matrix metalloproteinases and oxygen free radicals.Chemokine 7（CCL7）is a member of chemokine family.It can chemotactic macrophages,mast cells,eosinophils and other inflammatory cells,playing an important role in a variety of inflammatory diseases.However,Whether CCL7 plays a role in AAA and its possible mechanism have not been reported so far.Objective:In this study,we established the AAA model induced by angiotensin Ⅱ（Ang Ⅱ）and explored the role of CCL7 in the formation of AAA and its possible molecular mechanism at the in vivo and in vitro levels.Furthermore,the role of CCL7 neutralizing antibody in the formation of AAA was studied by intraperitoneal injection of CCL7neutralizing antibody in Ang Ⅱ-induced AAA model mice.Methods:1.8-10 weeks old apo E knockout(ApoE^-/-)male mice were subcutaneously implanted with Alzet osmotic micropump and continuously given Ang Ⅱ（1000ng/kg·min）or saline for 28 days.At one day（baseline）before the implantation of the micropump and the 28 day,the superior renal artery was examined by small animal ultrasound imaging system to obtain the maximum luminal diameter and area of the abdominal aorta.AAA was defined as an increase of more than 50%in maximum abdominal aortic diameter from baseline.During the experiment,the weight and blood pressure of mice were measured every week,and the survival of mice was observed every day.At the end of the experiment,the mice were sacrificed,the plasma and aorta tissues were collected respectively.The plasma was used to detect the level of CCL7,and the abdominal aorta was used to extract RNA and protein.Some mice were perfused with 10%formalin through left ventricle to fix the aorta in diastolic state,then the abdominal aorta was separated and embedded in paraffin for subsequent tissue section staining.2.C57BL/6J male mice aged 8-10 weeks were selected.The mice were sacrificed by cervical dislocation,and the bilateral tibia and fibula were isolated under aseptic conditions.The bone marrow cavity of mice was washed with sterile PBS solution to collect and obtain bone marrow cells.Bone marrow derived macrophages（BMDM）were obtained from 1640 RPMI medium supplemented with 10%fetal bovine serum（FBS）and induced by recombinant mouse macrophage colony stimulating factor（M-CSF）for 7 days.BMDM cells were co-cultured with recombinant CCL7（rmccl7）for 24 hours to observe the effects of CCL7 on proliferation,migration and differentiation of BMDM cells and intracellular signaling molecules.q RT-PCR was used to study the effect of CCL7 on the corresponding receptors of BMDM cell membrane.BMDM cells were co-cultured with CCR1 specific antagonist（BX471）,JAK2 specific inhibitor（Fedratinib）and STAT1 specific inhibitor（Fludrabine）for 1hour,respectively.Then rm CCL7 was added into the culture medium to explore the possible intracellular mechanism of CCL7 on the proliferation,migration and differentiation of BMDM cells.3.8-10 weeks old male ApoE^-/-mice were subcutaneously implanted with Alzet osmotic micropump and continuously given Ang Ⅱ（1000ng/kg·min）.The mice were randomly divided into three groups with 10 mice in each group:（1）CCL7-n Ab group:CCL7 neutralizing antibody was intraperitoneally injected 20ug/time（dissolved in200ul PBS）every 3 days;（2）control Ig G group:control Ig G antibody was intraperitoneally injected 20ug/time（dissolved in 200ul PBS）every 3 days;（3）PBS group:PBS was intraperitoneally injected 200 ul/time every 3 days.The experiment lasted for 28 days.At one day（baseline）before the implantation of the micropump and the 28 day,the superior renal artery was examined by small animal ultrasound imaging system to obtain the maximum diameter and area of the abdominal aorta.During the experiment,the weight and blood pressure of mice were measured every week,and the survival of mice was observed every day.At the end of the experiment,the mice were sacrificed,and the aorta was fixed in the diastolic state by left ventricular perfusion with10%formalin.The abdominal aorta was separated under the stereomicroscope,and the surrounding adipose tissue was removed after the abdominal aorta was soaked in 10%formalin.The maximum diameter of the abdominal aorta was measured by Image Pro software under the stereomicroscope.The fixed abdominal aorta tissue was embedded in paraffin for subsequent tissue section staining.4.The abundance of CCL7 in plasma of mice in each group was detected by ELISA.The number of CD68 positive cells in AAA lesion was semi-quantitatively analyzed by immunohistochemical staining.CCL7,α-SMA and Vimentin were performed with immunofluorescence staining.The elastic fibers were analyzed qualitatively by Verhoeff van Gieson/EVG staining,and the collagen deposition was analyzed by modified Masson trichrome staining.5.Statistical analysis:All measurement data were presented in the form of Mean±SEM.Sigma Plot 12.5 was used for statistical analysis.According to different data types and groups,Student t-test or one-way ANOVA were used for analysis and comparison.The survival curve was evaluated by Log Rank test.The difference was statistically significant when P<0.05.Results:1.At the end of the experiment,14 mice in Ang Ⅱ group developed AAA（14/17,82.4%）.Two mice died of AAA rupture（11.8%,2/17）and one mouse died of thoracic and abdominal aortic aneurysm rupture（5.9%,1/17）;all mice in saline group survived.The maximum diameter and area of abdominal aorta in Ang Ⅱ group were significantly higher than those in saline group（P<0.01）.Compared with the saline group,the m RNA and protein levels of CCL7 in aortic tissue of Ang Ⅱ group were significantly increased（P<0.01）,and the plasma concentration of CCL7 was also significantly increased（P<0.01）.Among the CCRs of CCL7,the expression of CCR1 receptor was significantly different（P<0.05）.CCL7 was mainly distributed in VSMC of the medial and adventitial fibroblasts,abundant of macrophages were found in the aneurysmal lesion by immunohistochemistry.2.Transwell assay showed that rm CCL7 could promote the migration of BMDM.q RT-PCR showed that rm CCL7 could promote the expression of adhesion molecules（ICAM,VCAM）in BMDM.These effects were inhibited by CCR1 antagonist（BX471）.rm CCL7 significantly increased the m RNA levels of M1 markers such as i NOS,IL-6,IL-12A,IL-12B and TNF-αin BMDM（P<0.001）,there was no significant change in the m RNA levels of M2 markers such as CD206 and Arg1.Western blot showed that rm CCL7 upregulated JAK2/STAT1 protein in BMDM cells（P<0.05）.The expression levels of i NOS,JAK2 and STAT1 in AAA tissues induced by Ang Ⅱ were also significantly increased（P<0.05）.Pretreatment of BMDM with CCR1 antagonist（BX471）,JAK2 inhibitor（Fedratinib）or STAT1 inhibitor（Fludarabine）inhibited rm CCL7-induced upregulation of M1 type markers.In addition,BMDM pretreated with BX471 significantly inhibited the increase of JAK2/STAT1 protein expression induced by rm CCL7（P<0.05）.3.At the end of the experiment,there was no significant difference in weight,blood pressure,survival curve and mortality in PBS group,Control Ig G group and CCL7-n Ab group.The incidence of AAA in CCL7-n Ab group（3/10,30%）was significantly lower than that in PBS group（8/10,80%）and Control Ig G group（8/10,80%,P<0.05）.Compared with PBS group and Control Ig G group,CCL7-n Ab mice had significant reduction in the maximum luminal diameter（P<0.01）,the maximum luminal area（P<0.01）,and the increase of the maximum external diameter（P<0.05）.The results of pathological and immunohistochemical examination showed that macrophage infiltration in aneurysmal lesion of CCL7-n Ab group was significantly reduced,elastic fiber fracture and collagen deposition were improved significantly.Conclusion:In conclusion,this study found that CCL7 promoted Ang Ⅱ-induced AAA formation,which may play a role through CCR1/JAK2/STAT1 pathway in mediating macrophage infiltration into aortic wall and differentiation into pro-inflammatory phenotype.CCL7-n Ab significantly reduced the incidence of AAA.The possible protective mechanism of CCL7-n Ab is to inhibit the infiltration of macrophages in aortic wall and delay vascular remodeling.Our results suggest that CCL7 may be a potential molecular target for the prevention and treatment of AAA,which has important clinical value.