Primary Research On The Contents And Molecular Structures Of Glycosaminoglycans In Aortic Dissection

Background:Aortic dissection(AD) is one of the most common catastrophe involving the aortae, carrying high morbidities and mortalities.For the improvements of human life,age growing of the society and wide use of imaging studies such as transesophageal ultrasound, MRA and CTA,aortic dissections have been detected more and more requently in recent years,with its incidence rising.Improvements on techniques of traditional open surgeries have reduced operation-related morbidities and mortalities,while developments of endovascular theories and techniques in recent years have utilized their minimal invasion instead of the huge trauma of open surgery,furtherly reduced the morbidities and mortalities and improved the treatments of aortic dissections.Although all of the above have benefited patients suffering aortic dissection,many of them died or disabled from ruptures or major morbidities before final diagnosis and surgery made,which was determined by the characteristics of the disease itself.Thus,it is very important for us to achieve the prevention,diagnosis and treatment of aortic dissection at the level of pathogenesis.Although aortic dissection could be initialized by many factors(e.g.hemodynamics and histopathology) together,we still don't know the exact pathogenesis now.Abnormal structures of the aortae are usually regarded as the basis of its pathogenesis,while abnormal hemodynamics as a trigger factor.Glycosaminoglycans(GAG) are important biomacromolecules and their important biological activities are attracting more and more people's attention.It is difficult to determine GAG for their polyanion,large molecular weight and inabsorption by UV,so there are little reports on them.With rapid advancements on biomedical detecting techonology in recent years,researches on GAG have gradually become more popular.We have observed many abnormal changes with GAG in many diseases such as orthopedics and tumors,while the abdominal aortic aneurysms are mainly focused on when it involves the aortic diseases.So far we have found four kind of GAG in human aortae,including hyaluronic acid (HA),heparin sulfate(HS),chondroitin sulfate(CS) and dermatan sulfate(DS),all of which are important for maintaining the integrity of normal structures and functions of the aortae.Since structures usually determine functions,we should first determine the changes on the contents and structures of GAG in aortic dissections before elucidating their roles in the pathogenesis of the disease.However,we haven't known the exact contents of GAG in aortic dissection and there's little information about their structures.Based on the above backgrounds,we'll elucidate the changes on the contents and fine structures of GAG in aortic dissection in this study,which may benefit us on exploring the pathogenesis of the disease.Objectives:1.To elucidate the total contents of GAG in the aortae of aortic dissections and discuss the influences exerted by age and hypertension and so on.2.To elucidate the relative amounts of HA,HS,CS and DS in the aortae of aortic dissections and discuss the influences exerted by age and hypertension and so on.3.To elucidate the changes in fine structures of HA,HS,CS and DS in the aortae of aortic dissections.4.To discuss the likely roles of abnormal contents and molecular structures of GAG in the pathogenesis of aortic dissection.Methods:1.Two groups were set up in this study.Ten ascending aortae in the experimental group were collected from patients with aortic dissections and seven specimens in the control group were collected from age-matched normal human ascending aortae at the same level.2.Histopathological stains:The basic structures of the aortae in the experimental group and the control group were observed by HE stain and VB stain and the distribution of GAG in the aortae were observed by AB-PAS stain.3.GAG in the aortic specimens were released by hydrolysis with combined papain and NaOH,and then extracted by ethanol precipitation and freeze-dried for further analytical uses.4.HA,HS,CS and DS were separated and purified respectively through DEAE Sephacel by ion exchange chromatography,with 0.1～1.2 M NaCl as stepwise gradient elutions.Then the separated GAGs were identified by cellulose acetate electrophoresis comparing with standard GAG.5.After the total contents of GAG of each sample being determined by concentrated sulfuric acid-hydroxydiphenyl method,they were compared between the experimental group and the control group and then the influences exerted by age and hypertension and so on were discussed.6.The extracted GAG were separated by acetate cellulose electrophoresis and stained with 0.02%alcian.Each of the stained bands were cut down and dissolved with dimethylsulfoxide separately,and then their OD at 677 nm were determined and the relative contents of HA,HS,CS and DS were calculated.Then the relative contents of them in the experimental group were compared with these in the control group and the influences exerted by age and hypertension and so on were discussed.7.After all the purified GAG being hydrolyzed into corresponding unsaturated disaccharides with chondroitinase ABC,B and heparinaseⅠ,Ⅱ,Ⅲ,each disaccharide was determined by high performance capillary electrophoresis with standard disaccharides as control.Then the disaccharide compositions of each GAG in the experimental group were compared with these in the control group.Results:1.Histopathological stains:Compared with normal human aortae,the basic structures in aortic dissections changed dramatically.We observed exist of false lumens,fracture and reduction of elastic fibers and hyperplasia of collagen fibers in the media by HE stain and VB stain.Increased acidic GAGs were observed throughout the full aortic wall in aortic dissections by AB-PAS stain.2.The contents of GAG in the aortae in the experimental group and the control group were 6.703±0.495 and 6.415±0.539 mg/g dry defatted tissue respectively and there was no significant difference between the two groups(P>0.05).After influencing factors such as age(grouped by 40 y),history(grouped by 7 d),diameter of the aorta(grouped by 5.5 cm) and co-existed hypertension being discussed,significant decrease in the contents of total GAG with age growing was observed(P<0.05),while the other three factors had no significant impact(P>0.05).3.Significant changes in the percentages of each GAG were observed in the experimental group(CS:62.5%vs 54.7%;DS:9.8%vs 15.8%;HA:10.6%vs 7.8%;HS: 17.1%vs 21.8%),among which CS and HA increased significantly(P<0.01) while DS and HS decreased significantly(P<0.01).Significant decreases in the contents of DS and HS(P<0.05) with age growing were observed and while CS and HA changed insignificantly(P>0.05).The other three factors such as history,diameter of the aorta and co-existed hypertension had no significant effects on the contents of each GAG(P>0.05).4.By HPCE analysis,only one disaccharide named Adi-nonSHA was detected in HA samples in the experimental group,with its content increasing significantly(P<0.05).Five kind of disaccharides such as△di-di(2,6)S_CS/DS,△di-di(2,4)S_CS/DS,△di-di(4,6)S_CS/DS,△di-mono4S_CS/DS and△di-nonS_CS/DS were detected in DS samples in the experimental group,with the absence of△di-mono6S_CS/DS instead of△di-di(4,6)S_CS/DS and the other four kind decreasing significantly(P< 0.05).Six kind of disaccharides such as△di-di(2,6)S_CS/DS,△di-di(2,4)S_CS/DS,△di-di(4,6)S_CS/DS,△di-mono6S_CS/DS,△di-mono4S_CS/DS and△di-nonS_CS/DS were detected in CS samples in the experimental group,with△di-di(2,4)S_CS/DS decreasing significantly(P<0.05) and the other five kind increasing significantly(P<0.05).Seven disaccharides such as△di-tri(2,6,N)S_HS,△di-di(2,N)S_HS,△di-di(6,N)S_HS,△di-di(2,6)S_HS,△di-monoNS_HS,△di-mono2S_HS and a△di-nonS_HS were detected in HS samples,with the last one increasing significantly(P<0.05) and the other six not changed(P>0.05) in the experimental group.Conclusions:1.Although there was no significant difference in the total contents of GAG in the aortae between aortic dissections and normal human,the proportion of each GAG changed dramatically.So we think the proportional imblance of each GAG may play an important role in the pathegenesis and progression of aortic dissection.2.Significant changes in structural compositions of each GAG were observed in aortic dissection,which may lead to weakness of the aortic wall and damage the functional integrity of the aorta.We aslo found that the proportional imbalance of each GAG was mainly resulted from their abnormal disaccharide compositions.Therefore,we consider the abnormal fine structures of GAG may play a key role in the initialization and progression of aortic dissection.