Biomechanical Property And The Expression Of Elastic Fiber Associated Proteins In Aortic Walls Of Stanford A Aortic Dissection

Stanford A thoracic aortic dissection (TAD) occurs as blood flow is redirected fromthe aorta through an intimal tear into the media of the aortic wall and the dissectionsinvolve the ascending aorta and arch regardless of its distal extension. From thebiomechanical viewpoint, the occurance of Stanford A type TAD represents the stress,exerted by hemodynamic forces on the dissectional aortic wall, overcome the tissue'smechanical strength limit. Although the maximum diameter of the aorta involved by TADwas usually utilized as an parameter to roughly estimate the possibility of rupture in TADpatients, the more precise quantitative analysis of the biomechanical properties of eachlayer of the aortic wall was also essential for assessment of the patients' prognosis.However, studies with regard to the biomechanical properties of human aortic wall werestill scarce, and most of them focused on the biomechanical properties of aortic walls ofpatients with abdominal aortic aneurysm (AAA) or ascending thoracic aortic aneurysm(ATAA). Animal experiments showed the difference of biomechanical properties betweenthe proximal and distal aortic wall. In addition, the pathophysiological process of TAD wasdifferent from that of ATAA. As a result, the biomechanical properties of ascending aorticwall in patients with Stanford type A were still uncertain.Elastic fibers (EF) is one of the primary extracellular matrix (ECM) in elastic arteriessuch as the aorta, which accounts for30%of the dry weight of aorta. Previous studiesindicated that the contents and relative proportions of EF and collagen fibers in aorta werethe most important determinant of the biomechanical properties of aortic wall. EF is a kindof ECM counterworking the properties of collagen fibers which determined the failurestress of the aortic wall. It takes responsibility for the expansibility and elastic recoil force,and thus determines the compliance and limit of aortic failure strain. Damage and loss ofthe elastic lamellae in the media of the aortic wall was considered as a characteristicpathological change of Stanford A TAD. Exploring the reconstruction process of EF atdifferent periods after the occurance of TAD is important for clarifying the potentialmolecular mechanisms of biomechanical properties changes presented in the aortic wall ofTAD patients, and is valuable for investigating the potential molecular targets of medicaltherapy for TAD patients. However, previous studies about TAD usually focused on ECMdegradation caused by collagenase/elastase and inflammatory reaction. The synthesis ofmature elastic fiber with biomechanical function in aortic wall is a complex biochemicalprocesses, which involves the participation of various molecules and regulations step by step. Current researches about abnormal EF synthesis with regard to aortic disease usuallyfocus on patients with AAA. As a result of the gradual decreasing of elastic/collagenproportion from proximal to distal aorta, togetherwith the different in pathogenesisbetween TAD and TAA, the synthesis process of elastic fiber and the biomechanicalproperty changes in the ascending aortic wall of TAD patients was still uncertain.The purpose of this study are:1. Investigate biomechanical difference betweenStanford A TAD and the normal aortic tissue via in vitro uniaxial tensile test.2. Explore theexpression level of elastic-fiber-associated molecules in the ascending aortic walls ofStanford A TAD patients and normal aortic walls, and this may give light to the potentialmolecular mechanisms of the biomechanical properties changes presented in the aorticwall of TAD patients.Part1Biomechanical study of ascending aorta andhistomorphometric investigation of mature elastic fibers in theascending aortic wall of Stanford A TAD patients.Object:1.To identify the difference of biomechanical properties of ascending aortic wallsbetween patients with Stanford type A TAD and the normal control.2. To investigate thedensity and distribution of the mature EF in the ascending aortic walls of patients withStanford A TAD and the normal control ones.Methods:1. Strain-stress curve was real-time recorded via uniaxial tensile test, and theelastic modulous-stress curve was calculated according to the high-frequency data acquired.Subsequently, the failure Almansi strain, failure Cauchy stress, and the peak elasticmodulus of the inner layer, external layer, and the full-thick ascending aortic wall in acuteand chronic Stanford A TAD patients and the normal control ones were obtained.2.Therelationship between failure Cauchy stress and the peak elastic modulus of the ascendingaortic wall was analyzed via linear regression, and the correlation coefficient wascalculated.3.The distribution of elastic and collagen fibers in each layer of the ascendingaortic wall in patients with Stanford A TAD and the normal control ones were identifiedthrough HE and VG staining.Results:1. Circumferential and longitudinal failure Almansi strain in each layer of the ascending aortic wall in patients with acute or chronic Stanford A TAD were lower thanthose of specimens in the normal ones. There was no significant difference ofcircumferential or longitudinal εfin each layer of the aortic wall between specimens ofacute and chronic Stanford A TAD and the normal contral.2. Circumferential andlongitudinal failure Cauchy stress of the separate inner and outer layer of the ascendingaortic tissues in patients with acute or chronic Stanford A TAD were significantly lowerthan that of normal controlpeople. There was no significant difference in σffor the innerlayer of the ascending aortic wall between acute and chronic Stanford A TAD groups.However, with regard to the outer layer, the σfin the chronic Stanford A TAD group wassignificantly higher than that in the acute group. The circumferential and longitudinal σfoffull-thick aortic wall in the acute Stanford A TAD group was significant lower than that inthe control group, but there were no significant differences between the chronic and controlgroups.3.Circumferential and longitudinal peak elastic modulus of the separate inner andouter layer of the ascending aortic wall in patients with acute or chronic Stanford A TADwere significantly lower than that of specimens of the normal control group. There was nosignificant difference in Mpof the inner aortic wall between acute and chronic Stanford ATAD groups. However, the Mpof the outer aortic wall in the chronic Stanford A TADgroup was significantly higher than that in the acute group. The circumferential andlongitudinal Mpof full-thick aortic wall in the acute Stanford A TAD group was significantlower than that in the control group, but there were no significant differences between thechronic and control groups, and Mpin chronic group was significant higher than that in theacute group.4. The comparison of biomechanical difference between the inner and outerlayer of the ascending aortic wall indicated that the εf, σf, and Mpof the outer layer in TADpatients as well as normal ones were significantly higher than those of the inner layer.Comparison of the biomechanical difference between the circumferential and longitudinalspecimens of the same aortic layer in TAD patients and the normal control ones indicatedthat there was no significant difference in εfbetween the two directions. However, the σfand Mpof the aortic tissue in the circumferential direction was significantly higher thanthose in the longitudinal direction.5. Results of linear regression indicated that σfwashighly positive correlated with and Mpfor each layer of the specimens in thecircumferencial and longitudinal directions.6. Results of morphological studydemonstrated the characteristic damage and a lower density of elastic fibers in the aorticwalls of TAD patients as compared with specimens from the normal control. Conclusion:1.The utmost expansionary of the ascending aortic wall in TAD patients wassignificantly lower than that of normal aortic wall. There was no significant difference inthe utmost expansionary of each layer of the ascending aortic wall between the acute andchronic TAD patients.2. Generally speaking, the σf, and Mpof ascending aortic wall in theacute and chronic TAD patients were significantly lower than those of normal people.However, the two parameters in the chronic TAD patients were compensated to someextent and this process was mostly presented in the outer layer of the aortic wall.3. Theouter layer of aorta in both TAD patients and normal people was a kind of moreexpandable and strong connective tissue as compared with the inner layer.4. Thecircumferential tissue intensity of ascending aorta was significant higher than that in thelongitudinal direction, however, there was no significant difference in expandabilitybetween these two directions, which intimated that there was directional difference in thebiomechanical properties of ascending aorta.5. Results of histological study indicated thatelastic fibers in the aortic wall of TAD patients were seriously damaged and the densitywas significantly lower than that in the normal aortic walls.Part2A study with regard to the tissue level of elastic fibreassociated molecules in the wall of ascending aorta in Stanford ATAD patients.Objective: To investigate the expression level of EFAM in each layer of the ascendingaortic specimens of patients with Stanford A TAD so as to supply experimental informationabout potential ECM molecular mechanisms related to the alteration of aorticbiomechanical property in TAD patients.Methods:1. RT-PCR and real-time quantitative RT-PCR were used to evaluate the mRNAexpression levels of tropoelastin, Microfibrillar associated proteins, and Loxl-1in eachlayer of the ascending aorta;2. Immunohistochemistry was used to investigate thedistribution of EFAM-related proteins in the ascending aortic wall;3. Western blot analysiswas carried out to compare the protein levels of EFAM in each layer of ascending aortaderived from normal people and TAD patients. Results:1. The mRNA and protein level of the soluble Te increased significantly in bothacute and chronic TAD groups as compared with the control group, and this differencewas more significantly in the adventitia tissue. As compared with chronic TAD patients,the mRNA expression level of Te was higher in acute TAD patients. Hoever, there was nosignificant difference in the protein level of Te between acute and chronic TAD patients.2.The mRNA and protein level of microfibrillar associated molecules in the full-thick aorticwalls of TAD patients were not significantly higher than that of normal control group. Inthe inner layer, the expression levels of these molecules were also not significantly higherin the TAD patients as compared with the normal people. In the outer layer, however, theexpression level of Emilin-1, Fibulin-4, Fibulin-5, and MAGP-1were significantly higherin the ascending aorta of both acute and chronic TAD patients, as compared with thecontrols.3. The expression level of DDR-2, a molecular marker of fibroblast, wassignificantly increased in each layer of both acute and chronic TAD patients, as comparedwith the controls, and this difference was more significant in outer aortic walls.4. ThemRNA and protein level of Loxl-1, a key enzyme being responsible for the hinge of Te andthe maturation of elastic fibers, in each layer of the ascending aortic walls in acute andchronic TAD patients were significantly lower than thost in controls.Conclusion: The sunthesis and expression of EFAM in the aortic walls after the occuranceTAD was mainly lociated in the adventitia tissue. The inner layer, however, did notpresented active expression of these EFAMs. When analyzing from the components ofsunthesised molecules, the tissue level of soluble EFAMs, which constitute mature elasticfibers, demonstrated imbalanced expression in the TAD patients. In brief, the tissue levelof Te, which is the main component of amorphous, was significantly higher in the TADgroup, as compared with the control group. However, the tissue level of microfibrillarassociated molecules and Loxl-1, which were very important for the assembly and stabilityof mature elastic fibers, showed either no significant change or decreased. The imbalancedexpression of these proteins in TAD patients may eventually result in the significantdecrease in the content of mature elastic fiber with biomechanical functions in theascending aortic walls of acute and chronic TAD patients, as compared with the controlpatients.