Role Of Transglutaminase 2 In Acute Type A Aortic Dissection

BackgroundAcute aortic dissection refers to a family of life-threatening pathologies of the aorta characterized by acute onset and symptoms.Mortality associated with thoracic aortic dissection(TAD)has historically been high.The mortality risk associated with TAD depends greatly on the delay between the onset of dissection and diagnosis,the availability of treatment.Early mortality associated with acute Stanford type A TAD and overall in-hospital mortality remains high,although tremendous progress has been made in diagnosing and treating TAD.A better understanding of the molecular,cellular,and genetic mechanisms that cause this disease is necessary for more effective preventative and therapeutic treatment strategies.The key histopathologic feature of TAD is medial degeneration,a process characterized by smooth muscle cell depletion,extracellular matrix degradation,and elastin deficiency and fragmentation.The imbalance between MMPs and TIMPs underlies aortic dissection occurring at any age and at any aortic site.MMPs mainly act as proteases on ECM components.Protein-glutamine y-glutamyltransferase 2(TG2)is a member of the transglutaminase family and is most widely distributed in tissues and cell types.TG2 plays an important role in the extracellular matrix(ECM).TG2 is released into the extracellular environment where it covalently modifies ECM proteins to form homoand heteropolymers to enhance ECM stability.Extracellular TG2 regulates the TGFβ signaling pathway,enhances integrin-mediated signaling,modulates syndecan-4 signaling,and regulates growth factor receptor signaling.Furthermore,TG2 could regulate the expression of MMP9,MMP3,MMP1 and MMP2.It is inferred that TG2 may play an important role in aortic dissection.Materials and MethodsWe did RNA-Seq with the aortic tissues of 10 acute type A aortic dissection patients and 10 control patients,and screened the differentially expressed genes.And we validated the gene expression with qPCR and selected TG2 because of its great difference.We used western blotting and immunofluorescence to explore the expression and distribution of TG2 and MMPs.We developed a cell model of hypoxia to imitate the hypoxic environment in aortic dissection tissues and explored the TG2 expression.We made the mouse model of acute aortic dissection by treating 3-weekold mice with β-aminopropionitrile monofumarate(BAPN,1g/kg/d)for 4 weeks.We treated the mice with cysteamine to inhibit the activity of TG2 and with transretinoic acid to induce TG2 over-expression.And we explored the function of TG2 in aortic dissection by analyzing the survival curves.We evaluated the smooth muscle cell(SMC)distribultion and the integrity of elastic fiber by HE and elastic fiber staining.We used siRNA to knockdown the expression of TG2 in SMCs,and detected the expression of MMPs by qPCR,western blotting and immunofluorescence.And the expression of MMPs was detected in the aortic tissue of mice treated with transretinoic acid.ResultsPart 1:The result of RNA-Seq showed TG2 expression was up-regulated in aortic dissection patients’aortic wall(n=10,P<0.001).The result of qPCR was consistent with RNA-seq(n=53,P<0.0001).But western blotting and immunofluorescence showed TG2 protein was decreased in AAD patients’ aortic wall(western blotting:n=16,P<0.001).The HE staining of human aortic wall showed the sclerotic changes of the Vasa vasorum,which resulted in impairment of Vasa vasorum flow.And the expression of HIF-1α increased in the dissected aortic tissues(n=10,P=0.0387).Both results revealed that the aortic dissection tissues underwent hypoxia.And transmission electron microscope showed the autophagosomes were accumulated in the SMCs of AAD patients.And Western blotting showed that the expression of Beclinl(n=16,P=0.0035)and LC3Ⅱ/LC3I(n=4,P=0.0012)both increased in aortic dissection tissues.These results suggested that the aortic dissection tissues were enduring autophagy.And we imitated the hypoxia environment with SMCs.Hypoxia could induce autophagy and the down-regulation of TG2 expression in SMCs(n=6,P=0.0003).And we treated the SMCs with 3-MA to inhibit autophagy before hypoxia,we found the down-regulation of autophagy accompanied with the upregulation of TG2(n=6,P<0.0001).Part 2:BAPN could induce mice to develop aortic dissection.HE and elastic fiber staining showed the elastic fiber fragment,SMC depletion,and inflammatory infiltration in the dissected aorta.Western blotting showed the expression of TG2 decreased in the dissected aorta of mice(n=6,P<0.0001).The survival of mice treated with cysteamine+BAPN had no difference with the mice treated with BAPN only(n1=n2=40,P=0.8853).But,the survival of mice treated with trans-retinoic acid+BAPN increased a lot when compared with the mice treated with BAPN only(n1=n2=30,P=0.0016).And the structure abnormality of aorta was relieved in transretinoic acid+BAPN mice.Part 3:Western blotting showed that the expression of MMP2,MMP3 and MMP9 increased in aortic dissection tissues(MMP2:n=8,P=0.0014;MMP3:n=10,P=0.01;MMP9:n=11,P=0.0015).And immunofluorescence showed that TG2 distribution was negatively correlated to the MMP9 distribution.When TG2 was knockdown by siRNA,the expression of MMP3 and MMP9 increased in the SMCs(MMP3:n=6,P=0.0015;MMP9:n=4,P=0.0025).These results suggested that MMPs were the downstream signaling molecules of TG2.What’s more,the trans-retinoic acid+BAPN mice who had up-regulation of TG2(n=8,P<0.0001),had a down-regulated expression of MMP2 and MMP3 in aortic tissue when compared with the mice treated with BAPN only(MMP2:n=6,P=0.0031;MMP3:n=6,P<0.0001).Conclusions TG2 plays an important role in the development of acute type A aortic dissection.The dysfunction of the Vasa vasorum contributed to long-standing ischemia or malnutrition of the aortic media,which result in the autophagy of SMCs in aortic media.And TG2 was degraded by autophagy.TG2 was up-regulated if autophagy was inhibited when SMCs suffering hypoxia.Up-regulating TG2 could downregulate the expression of MMPs and decrease the morbidity and mortality of aortic dissection.