The Role And Mechanism Of TLR7 In The Development Of Thoracic Aortic Aneurysm

Thoracic aortic aneurysms(TAA)is a degenerative process of thoracic aortic wall.It is a life-threaten disease with high mortality in the world.TAA is symptom silent and often been found when dissection or rupture occurs.Clinically,TAAs will be repaired through surgery or endovascular technology due to lack of specific treatment.Therefore,an increasing number of studies aim to investigate the potential mechanism behind TAA development and to develop potential therapeutic strategies.Over the past two decades,researchers began to understand the molecular mechanisms underlying aneurysm formation.Recently,TAA is considered to be caused by gene mutation,such as Marfan syndrome(Fibrillin-1 mutation).It can also be inherited in an autosomal dominant manner.There also have several sporadic cases,but the cause of it is still unknown.However,studies have now shown that inflammatory infiltrates are present in the aorta of both syndromic TAA,familial TAA and sporadic TAA.Most important,T cells and macrophages predominate in the aorta wall.These suggest that aortic inflammatory cells infiltration play a key role in the development of TAA.Inflammation is a component of the immune system.Innate immunity and adaptive immunity play an important role in the initiation and development of aneurysm.Toll-like receptors(TLRs)are classic pattern recognition receptors that connect innate immunity with adaptive immunity.In recent years,researchers have found that TLRs were closely related to cardiovascular diseases,such as atherosclerosis,ischemia/perfusion injury.Some TLRs have been proved to play a pathogenic role in abdominal aortic aneurysm(AAA),such as TLR2 and TLR4.However,the role of TLRs in TAA is rarely been reported.We found that the expression of TLR7 on the ATA of smooth muscle cell(SMC)specific knockout(Tgfbr1^sm-ko)mice was increased,suggesting that TLR7 may associated with TAA.TLR7 is one of classic TLRs that can be activated by exogenous and endogenous RNA.TLR7 activation and its signature cytokine Type 1 interferons(IFNs)are closely associated with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.Now,more and more studies are targeting TLR7 for the treatment of inflammatory diseases.We suspect that TLR7may play a role in TAA by modulating immune response.In order to explore the role and mechanism of TLR7 in TAA,we use real-time PCR and immunofluorescence staining to confirm the up-regulation of TLR7 in Tgfbr1^sm-komice ATA.Then TLR7 knockout mouse were challenged with BAPN and Ang II to determine the effects of TLR7 deficiency on TAA formation and investigate the potential mechanism.This project was expected to find a new cognition of the mechanism for TAA and provide a new target for the prevention and treatment of TAA.Chapter I:The change of TLR7 expression in smooth muscle cell-specific Tgfbr1 deficiency induced thoracic aortic aneurysmObjective:To investigate the expression of TLR7 in thoracic aortic aneurysm of smooth muscle cell specific Tgfbr1 deficiency mice.Methods:The smooth muscle cell Tgfbr1 deficiency(Tgfbr1^sm-ko)was induced by Cre-loxp system.Ultrasound was used to monitor the diameter of ascending thoracic aorta(ATA)in mice.Masson staining was used to evaluate pathological changes of ATAs.Gelatin/In situ zymography were utilized to detect MMP-2/MMP-9 activity.Immunohistochemistry staining and flow cytometry were used to analyze the infiltration of inflammatory cells in ATA.Nanostring and real time-PCR were used to detect TLR7 expression in ATA tissue.Immunofluorescence staining was used to detect TLR7 and its ligand8-OHG in ATA of Tgfbr1^sm-komice and human TAA samples.Results:Once smooth muscle cell Tgfbr1 was deleted,mice rapidly developed aortic dilatation and aortic rupture.Masson staining of Tgfbr1^sm-komice ATA showed medial loss,intima/medial tear,intramural hematoma,which were similar to the pathological characteristics of TAA patients.Gelatin zymography indicated that MMP-2 activity in ATA of Tgfbr1^sm-komice was 2.3 times higher than that of control mice(P=0.017),in situ zymography indicated that MMP-2 was mainly located in the area of intima/medial tear.Immunohistochemical staining revealed that Tgfbr1^sm-koATAs contains more CD45⁺cells,which were 1.8 times and 2.8 times higher than that in control mice ATA on d7 and d14,respectively(P=0.017).Flow cytometry also found that a greater number of CD45⁺cells and significantly higher percentage of CD4+T cells were detected in Tgfbr1^sm-koATAs when compare to Tgfbr1^f/fATAs(P=0.01 and P=0.04).Nanostring gene analysis and real-time PCR showed that the expression of TLR7 was up-regulated in Tgfbr1^sm-koATAs,which was 2.8 times(P=0.007)and 10.9 times(P=0.029)higher than that in Tgfbr1^f/fATAs on day 7 and day 14,respectively.Immunofluorescence staining showed that TLR7 was mainly located in the medial layer,and its ligand 8-OHG was also increased in Tgfbr1^sm-koATAs.TLR7 also showed similar expression and located characteristics in ATAs of TAA patients.Summary:1.Smooth muscle cell specific Tgfbr1 deficiency promotes TAA development and aortic rupture;2.Smooth muscle cell specific Tgfbr1 deficiency promotes inflammatory cells infiltrate into ATA;3.The expression of TLR7 was up-regulated in ATA of Tgfbr1^sm-komice.Chapter II:The role and mechanism of TLR7 deficiency in thoracic aortic aneurysm formationObjective:To explore the role and mechanism of TLR7 deficiency on BAPN+Ang II induced TAA in mice.Methods:TLR7 knockout mice(TLR7^-/-)were challenged with BAPN and Ang II to induce TAA.The diameter of mouse ATA was monitored by ultrasound.Gelatin Zymography were used to detect MMP-2/MMP-9 activity.Cytokines and chemokines in TLR7^-/-mice ATAs were detected by Luminex array.Flow cytometry was used to detect the infiltration of inflammatory cells in ATAs.T-bet knockout mice(T-bet^-/-,Th2 cells predominate environment)were also challenged with BAPN+Ang II to investigate the role of Th2 cell in TAA formation.The diameter,MMP-2/MMP-9 activity,cytokines,chemokines and infiltration of inflammatory cells in mouse ATA were also measured like TLR7^-/-mice.In addition,Immunofluorescence staining were utilized to detect ATA SMCs?-actin in T-bet^-/-mice.Necroptosis marker(RIP-3)in IL-13 treated SMCs was detected by western blotting.Results:TLR7^-/-mice treated with BAPN+Ang II result in aortic dilatation and rupture rapidly.Within 4 weeks,100%of TLR7^-/-mice died of aortic rupture.Gelatin zymography indicated MMP-2 activity in TLR7^-/-mice ATA was 1.4 times higher than that in C57BL/6J mice(P<0.05).Luminex assay found that TLR7^-/-mice ATA have higher CCL5(725.2±36.4 vs 1063.1±64.6 pg/ml,P=0.01),Eotaxin(142.9±20.4 vs220.5±17.4 pg/ml,P=0.038)and IL-13(113.6±8.0 vs 129.6±9.7 pg/ml,P=0.016)levels.Flow cytometry showed that more CD4⁺T cells(13.3%±1.49%vs 19.9%±1.18%in CD45⁺cells,P=0.04)and CD4⁺GATA3⁺Th2 cells(4.9%±1.5%vs 2.7%±1.5%in CD4⁺cells,P=0.04)were infiltrated into TLR7^-/-mice ATA.T-be^-/-mice challenge with BAPN+Ang II to established a Th2 predominate environment that confirmed by higher CD4⁺GATA3⁺Th2 cells in T-bet^-/-ATAs(6.3%±2.5%vs 9.8%±3.4%,P=0.029).Within 4 weeks,90%of T-bet^-/-mice died of aortic rupture.Consistent with TLR7^-/-mice,T-bet^-/-mice also have higher MMP-2 activity(30%increase,P=0.02),higher CCL5(709.1±46.4 vs 889.6±114.8 pg/ml,P=0.02)and IL-13(112.5±7.7 vs131.7±6.3 pg/ml,P=0.007)levels in T-bet^-/-ATAs,when compare to C57BL/6J mice.In addition,T-bet^-/-mice ATA have more CD19⁺B cells(9.8%±3.1%vs 13.1%±3.5%in CD45+cells,P=0.017),loss more SMCs.SMCs treated with IL-13 highly express RIP-3,a marker of necroptosis(P<0.05).Summary:1.TLR7 deficiency increased the mortality of BAPN+Ang II induced TAA.2.TLR7 deficiency aggravate BAPN+Ang II induced TAA by promoting Th2 cell polarization and IL-13 secretion.Conclusion:1.The expression of TLR7 was up-regulated in ATA of Tgfbr1^sm-ko TAA model mice.2.TLR7 knockout can aggravate BAPN+Ang II induced TAA.The mechanism is involved in promoting Th2 cells polarization and IL-13 secretion.