Deficiency Of Scavenger Receptor A1 Exacerbates Abdominal Aortic Aneurysm Formation In Mice

Abdominal aortic aneurysm(AAA)is a permanent and progressive expansion of the aorta associated with the complications such as aortic rupture.The AAA mortality rate has decreased by both open and endovascular surgery.However,the morbidity is still on the rise worldwide,since the curative pharmacological treatments for this disorder are still lacking.The pathophysiological features of AAA are characterized as infiltration of inflammatory cells,degradation of extracellular matrixes,and apoptosis of smooth muscle cells.More recently,accumulating evidence indicates that macrophages play a key role in the development of AAA by release of proinflammatory cytokines aggravating aorta expansion and secretion of extracellular matrix protease degrading the vascular wall.Class A1 scavenger receptor(SR-A1)is one of the pattern recognition receptors mainly expressed in macrophages.It is involved in a variety of macrophage biological processes,including immune response,cell adhesion,cytokine release,and clearance of apoptotic cells in the cardiovascular diseases.In the present study,SR-A1 knockout mice were used to generate two AAA models separately to explore the potential effect of SR-A1 on AAA progression and the underlying molecular mechanisms.Firstly,mini-osmotic pumps were implanted subcutaneously and angetensin Ⅱ(Ang Ⅱ)or physiological saline was infused into Apo E-/-;SR-A1-/-and Apo E-/-mice over the course of 28 days.We found that the vascular diameter was significantly increased by two-dimensional ultrasound measurement in Apo E-/SR-A1-/-mice compared with Apo E mice after 28 days of Ang Ⅱ infusion.HE staining showed remarkably larger and wider lumen accompany with more severe elastin degradation by EVG staining in Apo E-/-;SR-A1-/-mice.To further verify the above observations,CaPO4 model was further performed on WT mice and SR-A1-/-mice to induce AAA.The intravascular diameter was monitored by two-dimensional ultrasound before and after the operation weekly for 4 weeks.Compared with wild type group,SR-A1-/-mice exhibited significant enlarged vessel lumen in the surgical area after CaPO4 surgery at 14,21,and 28 days.The external diameter of SR-A1-/-group was significantly increased as well.Histological analysis showed that SR-A1-/-mice displayed significant thiner middle layer of arterial wall and stronger degradation of elastin.Immunohistochemistry showed that there were more macrophages accumulated in lesion area of SR-A1-/-group,compared with SR-A1+/+ group.The mRNA levels of pro-inflammation cytokines,such as TNF-α,IL-6,MCP-1 and iNOS were up-regulated in SR-A1-/-group,compared with those in SR-A1+/+group.There was no significant difference in the levels of anti-inflammatory cytokines between two groups.To further reveal the source of the macrophages in AAA,bone marrow transplantation experiment was performed.After 28 days of CaPO4 surgery treatment,the SR-A1+/+bone marrow-transplanted SR-A1-/-mice showed decrease in arterial diameter whereas the SR-A1-/-bone marrow-transplanted SR-A1+/+mice shows an opposite phenotype.The results suggest that lack of SR-A1 in the recruited macrophages aggravate AAA.To understand the mechanisms SR-A1 deficiency aggravating AAA,we evaluated the changes of matrix metalloproteinases(MMPs)in aneurysm tissue.qRT-PCR revealed that mRNA level of MMP-9 in SR-A1-/-mice was significantly higher than that in SR-A1+/+mice,while level of TIMP-1,an endogenos inhibitor of MMPs,was significantly lower.Immunohistochemistry and western blot experiments further confirmed the abundance of MMP-9 in SR-A1-/-group was stronger than those of SR-A1+/+group.There was no significant difference in MMP-2 level between two AAA groups.Since AAAs are characterised by lossing of vascular smooth muscle cells,we hypothesis that deleption of SR-A1 may affect the number of VSMCs.The results of α-SMA immune-histochemistry showed that the loss of smooth muscle cells was elevated in SR-A1-/-group,compared with SR-A1+/+ group.The level of apoptotsis detected by caspase-3 immunoblotting showed that apoptotsis was exacerbated in SR-A1-/-group,compared with SR-A1+/+ group,indicating that the deletion of SR-A1 may up-regulate the expression of MMP-9 and aggravate apoptosis and phenotype changes of smooth muscle cells.When co-cultured with SR-A1-/-macrophages,CaPO4-treated smooth muscle cells exhibited a lowere expression of contraction markers compared with thoses co-cultured with SR-A1+/+macrophages.Treatment with MCP-1 increased MMP-9 in the SR-A1-/-macrophages compared with SR-A1+/+ group,while the expression of TIMP-1 was lower.Similarly,activity of MMP-9 significantly was augmented in SR-A1-/-group compared with SR-A1+/+group.Number of macrophages in SR-A1-/-group significantly rose in transwell experiments,which was abolished after the application of MMP-9 inhibitor.To investigate altered genes profile after SR-A1 gene knockout in AAA progression,we used gene microarray assay to detect differentially expressed genes in the two groups.It was found that the nuclear transcription factor NR4A1 was significantly reduced after SR-A1 knockout in transcriptional level.Immunohistochemistry and qRT-PCR measurements were consistent with those of the gene microarray assay.The expression level of NR4A1 in blood vessels of two groups significantly increased in aneurysm,with remarkably higher level in SR-A1+/+mice.When NR4A1 was oversexpressed by lenti virus in SR-A1-/-mice,the MCP-1-induced up-regulation of MMP-9 was attenuated.In summary,deletion of SR-A1 may aggravate abdominal aortic aneurysm in mice.SR-A1 deficiency may down-regulate expression of NR4A1 under inflammatory stimuli and promote inflammatory response and secreation of MMP-9 by macrophages.Our results suggest that SR-A1-NR4A1-MMP-9 be an useful target for intervention of AAA.