The Role Of Cathepsin S In Remodeling Of Aortic Wall And Its Related Mechanisms

Acute Stanford Type A aortic dissection is a severe cardiovascular emergency that is potentially catastrophic.Furthermore,it is associated with a high rate of mortality and morbidity soon after symptom onset in historically untreated patients.It remains a life-threatening event and includes a broad range of degenerative,genetic,and structural features that can be complicated by potentially fatal and incompletely understood pathological mechanisms that result in aortic dissection.Therefore,early diagnosis and timely pretreatment are absolutely necessary.With increasing knowledge of AAD,researches on biomarkers are still interesting.D-dimer levels are considered useful for ruling out the possibility of AAD within 24 h of symptom onset,and for predicting in-hospital death.Some previous studies have revealed that,in aortic diseases,the expression pattern of genes regulating cell survival,apoptosis,extracellular matrix(ECM)remodelling,and cell proliferation are all altered.This may result in the major histopathological characteristics of aortic diseases:degradation,disruption of elastic fibres and collagen,and abnormalities of vascular smooth muscle cells(VSMCs)within the media.Although Type A dissections arise from various risk factors,the final common pathway for its development is tunica media dysfunction.Cathepsin S is unique amongst the cysteine cathepsin family due to restricted tissue expression,associated with antigen presenting cells localised in lymph and spleen,as well as other immune cells such as macrophages.Biochemically,cathepsin S is also different from many family members in its ability to retain activity at a neutral pH.As such,these traits highlight cathepsin S as an ideal target for disease treatment,as with its highly restricted expression,therapeutic inhibition should minimise potential side effects.Furthermore,its enhanced stability at a neutral pH over other family members highlights its increased potential for involvement in extracellular proteolytic activities.Although studies have shown that cathepsin S was involved in abdominal aortic aneurysm(AAA)process,but the relationship between cathepsin S and type A dissection with dissection of the role is still unknown,its molecular mechanism or related pathways are still poorly understood.In this study,we investigate the role of Cathepsin-S in destabilization of the aortic wall and to evaluate whether Cathepsin-S could be a novel biomarker in acute aortic dissections(AAD)relating to perioperative complications,moreover,to establish an economical animal model and investigate the role of Cathepsin-S in destabilization of the aortic wall and evaluate the expression of the TAD-related proteins.Furthermore,we endeavored to prove that Cathepsin-S inhibitor regulates the differentiation of vascular smooth muscle cells(VSMCs)during the formation of aortic dissection.We also studied the contribution the PI3K/AKT/p53 signaling pathway has towards this process.Part I Stanford Type A Dissection:Cathepsin-S is central to destabilization of the aortic wall and might be a novel biomarker for predicting perioperative complicationsObjective To investigate the role of Cathepsin-S in destabilization of the aortic wall and to evaluate whether Cathepsin-S could be a novel biomarker in acute aortic dissections(AAD)relating to perioperative complications.Method A total of 162 patients with acute Stanford Type A dissection and 32 organ donators were studied.Hematoxylin-eosin(H.E),Victoria Blue and Masson staining were performed.Immunohistochemistry and western blotting of Type A dissection tissue was performed to assess Cathepsin-S levels.Furthermore,Cathepsin-S,D-dimer and Cystatin-C levels were detected in serum samples and an ROC curve analysis was conducted for these biomarkers.Correlation analysis was used to evaluate the correlation between intraoperative data,postoperative data and Cathepsin-S levels.Results Histological examination showed the degradation of elastin and collagen in acute Type A dissection and Cathepsin-S was stained strongly in vascular smooth muscle cells.Cathepsin-S was markedly expressed in the tissue of patients with AAD,as indicated by western blotting(p<0.05),while serum Cathepsin-S and D-dimer levels were significantly higher in patients with AAD than in controls(p<0.05).Moreover,the trend of higher serum Cathepsin-S levels was consistent with the observations in tissues.ROC curve analysis confirmed the sensitivity and specificity of serum biomarkers.Elevated Cathepsin-S serum levels were significantly correlated with postoperative hospital stay(LOS)and mechanical ventilation(MV)time.Conclusion Cathepsin-S could be a key factor in the progressive destabilization of the aortic wall in vascular remodelling,which evolves into acute dissection.Furthermore,this may be a valuable tool for predicting perioperative complications and guiding intervention strategies in patients with AAD.Part ? The role of cathepsin S in C57BL/6 mice thoracic aortic dissection model which induced by ?-AminopropionitrileObjective The underlying pathological mechanisms of this disease remain elusive because there are no appropriate animal models,limiting discovery of effective therapeutic strategies.We want to establish an economical animal model and investigate the role of Cathepsin-S in destabilization of the aortic wall and evaluate the expression of the TAD-related proteins.Method We treated 10 C57BL/6 mouse with ?-aminopropionitrile monofumarate(BAPN),an irreversible inhibitor of lysyloxidase,for 4 wks.10 mouse were control group,and without treatment of BAPN.The body weight and the change of body weight were examinate.Immunohistochemistry and western blotting of mice aortic tissue were performed to assess Cathepsin-S levels.Furthermore,TAD-related proteins levels were detected in tissue samples and evaluate the correlation of mechanisms between human beings and mouse,and investigate the preliminary mechanism of TAD.Results BAPN treatment induced formation of aortic dissections in C57BL/6 mice.Moreover,BAPN induced TAD formation with effects on body weight.Histological examination showed Cathepsin-S was stained strongly in vascular smooth muscle cells,and higher expression in TAD mouse by Western blotting.Expression of TAD-related proteins were compared in control and BAPN-treated C57BL/6 mice.In the BAPN-treated group,compared with the control,MMP2 was significantly upregulated,while MMP3 and MMP9 were downregulated.COLlal and ?-SMA expression were dramatically decreased with BAPN treatment,while CTGF was not changed.These results revealed that BAPN-induced TAD was associated with typical ECM degradation,possibly via MMP2,and loss of SMC leading to decreased a-SMA,effects consistent with previous observations in humans and mouse models.Conclusion We have generated a reliable and convenient TAD model in C57BL/6 mice for studying the pathological process and exploring therapeutic targets of TAD.Part ? Cathepsin S Inhibition Attenuates Vascular smooth muscle cells remodelling via PI3K/AKT pathway mediated by p53 and p21Objective We endeavored to prove that Cathepsin S inhibitor regulates differentiation of vascular smooth muscle cells(VSMCs)during the formation of aortic dissection.We also studied the contribution the PI3K/AKT signaling pathway has towards this process.Methods We treated mouse aortic VSMCs with 0.1?M regimens of Ang ? for 12h(duration and dosages)in vitro.Z-FA-FMK was used to inhibit the expression of Cathepsin S in VSMCs.First,we identify the selection of cathepsin S inhibitor,then,we study the PI3K/AKT signaling pathway mediated by p53/p21 with treatment of cathepsin S inhibitor by Western blotting.Finally,we determined expression levels of VSMCs phenotype marker proteins(a-SMA and OPN)by Western blotting.Results Ang ? induced the phenotypic switching of VSMCs.When the cathepsin S was blocked by pre-treatment with Z-FA-FMK,the expression of p-AKT,p85 and p53.were up-reguated.Western blotting showed that the expression of ?-SMA decreased,while the OPN increased and the arrangment of the smooth muscle cells of the media was dysregulated,Which we verified in vitro.Conclusion We have shown that cathepsin S inhibitor affects the PI3K/AKT pathway in mouse aortic VSMCs and promotes the phenotypic switching of VSMCs.Our data supports the involvement of the PI3K/AKT signaling pathway amongst this.These outcomes may offer another mechanism of Ang ?-induced VSMCs phenotypic modulation during the formation of AD.Our study suggests that the cathepsin S inhibitor may be an attractive target for strategies aiming to prevent the formation of AD.