| Part one Platelet Activation is involved in Aortic MedialDegenerationResearch significance Aortic medial degeneration（AMD）is the common pathological basis of aortic aneurysm（AA）and aortic dissection（AD）.Intra-aortic blood flow disorder is prone to cause endometrial damage and platelet activation.This part of the study aimed to investigate the status of platelet activation in the AMD process and to determine whether antiplatelet therapy has potential role in alleviating AMD progression.Methods Patients with aortic dissection（Stanford type A,Stanford type B）,aortic aneurysm,and cardic valvular disease（as a control group）in our hospital were enrolled.The detail of hospitalized informations and blood samples were collected.The total number of white blood cells,neutrophil ratio/count,lymaphocyte ratio/count,mononuclear-macrophage ratio/count difference,total platelet count,thrombocytocrit,and platelet mean volume,platelet distribution width and the count of large platelets were analyzed.The relationship between inflammation,platelet activation and AMD was initially assessed.In in vivo study,AMD mice were established by aminopropionitrile（BAPN）oral administration combined with angiotensin II pumping,and clopidogrel was used to inhibit platelet activation.The elastic fibers,collagen accumulation,metalloproteinases（MMP-2,MMP-9）and the apoptotic condition of ASMCs in aortic wall were assessed.Results Compared with the control group,the total number of white blood cells,the ratio of neutrophils,the count of neutrophils,and the count of mononuclear-macrophages in Stanford type A and type B AD groups were increased.In patients with AA,although there was no intravascular rupture and hemorrhage in the aorta,it also showed an increase in the total number of white blood cells,the count of neutrophils,and the count of monocyte-macrophages compared with the control group.Compared with the Stanford type A and type B AD groups,the AA group showed lower total white blood cell count,neutrophil count,and neutrophil ratio;higher lymphocyte count and lymphocyte ratio;There was no significant difference in the ratio and count of mononuclear-macrophages.Compared with the control group,platelet count was significantly lower in the Stanford type A AD group,but not in the Stanford B type AD group and the AA group.Mean platelet volume,platelet distribution width,and large platelets were significantly reduced in the Stanford Type A,Type B AD and AA groups.In the same period,AMD model mice fed with clopidogrel had a significantly longer lifespan than simplely AMD model.Clopidogrel can significantly reduce the aortic elastic fiber destruction,collagen accumulation,ASMCs apoptosis,and MMP-2/MMP-9 expression in AMD mice.Conclusions:（1）In the AMD process,regardless of whether the dissection occurred,there was an increase in the number of white blood cells and the total number of neutrophils/count.The distribution width and average volume of platelets were significantly changed.（2）Treatment with clopidogrel,which depresses platelet activation,can inhibit AMD progression in a mouse model,prolong their survival,and reduce lesions in the aortic media.Part two Platelet-derived mi R-223 is involved in Aortic Smooth Muscle Cells Phenotype ModulationResearch significance Although antiplatelet therapy has been shown to attenuate AMD progression,its mechanism is not clear.Previously studies indicate that platelet activation is closely related to the plasmic content of mi R-223.This section aimed to investigate whether platelet activation participates in the AMD process by mi R-223.Methods The content of plasmic mi R-223 in AD,AA and control patients,and the content of mi R-223 in AD aortic media specimens were analyzed.The expression of mi R-223 in plasma and aortic tissues of the AMD group,clopidogrel+AMD group,clopidogrel group,and control mice was further examined.The Ang II-induced ASMCs phenotype transformed cell model was constructed to detect the expression changes of mi R-223.Ectopic mi R-223 was overexpressed in ASMCs by infection of adenovirus,which encodes mi R-223,and the effect of mi R-223 on proliferation and motility of ASMCs was further examined.The potential targets of mi R-223 were analyzed by databases,and further verified by using western-blot and fluorescein reporter gene experiments.Results The results showed that mi R-223 was significantly elevated in plasma of AMD patients（including AA patients and AD patients）.In the aortic medial tissue,the expression of AD specimens was also significantly higher than that of control specimens;the expression of plasmic mi R-223 in AMD model mice was also significantly increased,and clopidogrel administration inhibited plasmic mi R-223elevation in AMD mice.The expression of mi R-223 in the aorta of AMD mice was also significantly up-regulated,which can also be suppressed by clopidogrel adminitration.In in vitro conditions,Ang II effectly induce the proliferation of ASMCs,decreased the expression ofα-SMA and increased the expression of OPN.However,Ang II cannot regulate the expression of mi R-223 in ASMCs.Overexpression of mi R-223 significantly inhibited the expression of STIM1 and down-regulated the intracellular calcium concentration.Furthermor,mi R-223 can effectively inhibit the fluorescence of the fluorescent assay（containing the STIM1targeting sequence）.Conclusion Antiplatelet therapy in the AMD process can inhibit the mi R-223secretion of platelet.mi R-223 can inhibit the expression ofα-SMA and motility in ASMCs.mi R-223 directly controls the expression of STIM1,reduces the concentration of intracellular calcium.Part three The Deregulation of STIM1 and SOCE Impaired ASMCs Contractility in AMDBackground Micro-array analysis of clinical aortic samples suggested a potential role for STIM1 in the modulation of aortic medial degeneration（AMD）,despite the uncertainty role of STIM1 in normal aortic smooth muscle cells（ASMCs）.Here,we aimed to explore changes in STIM1 expression in AMD,and the possible mechanisms.Methods An AMD model was established using auto-delivery of angiotensin II into Apo E-/-mice.We assessed the effects of SKF96365,a STIM1 inhibitor,in AMD model and in vitro cultured ASMCs.Elastic van Gieson staining was used to visualize elastic fiber injury.Mitochondria changes were viewed by transmission electron microscopy.Cytoplasmic calcium was quantified by measuring fluo-4 staining in a flow cytometer.Mechanical stretching device was used to mimic stretching that ASMCs experience in vivo.Cell apoptosis was determined by using Annexin V/propidium iodide（PI）staining.The expression of STIM1,contractile related proteins（α-smooth muscle actin[α-SMA],myosin light chain[MLC]）,endoplasmic reticulum（ER）stress related proteins（CHOP,ATF-6）and smad2/3 were assessed by Western blotting,immunohistochemistry and immunofluorescence.Results SKF96365 exacerbated aortic injury in the AMD model.SKF96365 reduced cytoplasmic calcium concentration in ASMCs,caused mitochondrial swelling and elevated the expression of ATF-6 and CHOP.SKF96365 decreased the expression of MLC andα-SMA in ASMCs,causing them to be vulnerable to mechanical stretch.SKF96365 suppressed smad2/3 activation after treatment with transforming growth factorβ1（TGFβ1）.Conclusions STIM1 is indispensable in ASMCs.Interfering with STIM1 exaggerated the AMD process by modulating the expression of contractile proteins,inducing ER stress in ASMCs. |
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