Molecular Mechanism And Prognosis Evaluation Of Type A Acute Aortic Dissection

Part I:Analysis and verification of the differentially expressed genes in aorta tissue in patients with acute aortic dissectionAbstractAim:Acute aortic dissection (AAD) is a devastating cardiovascular condition and represents the most common aortic emergency; however, themolecualr mechanisms of AAD remain poorly understood. Our present study aims to analyze the genes differentially expressed in aortic tissue between patients with AAD and the controlsby usinggenome-widetranscriptomic microarray strategyand verification at protein level after bioinformatics analysis, in order to explore possible new genes and pathways involved in the molecular mechanisms of AAD.Methods:The ascendingaortic tissue was collected from patientswith AAD (n=8) and controls of healthy male organ donors (n=8). Four sample from AAD patients and 4 from the control were selected for gene expression profile analysis with AffymetrixGeneChip Human Genome U133 Plus 2.0array and the differentially expressed genes were analyzed. Bioinformatics were then used to analyze the biologic function of the differentially expressed genes and the key pathways and genes associated with AAD were selected from bioinformatics analysis. The aorta tissue protein was extracted from the sample (8 AAD patients and 8 controls) and the expression of the differentially expressed genes was compared between AAD patients and the control by Western blotting at the protein level. Transform growth factor-P(TGF-β) was the reported pathway associated with AAD and the expressions of SMAD2 and SMAD3, the major components of TGF-β pathway, were compared between AAD patients (n=8) and the control (n=8) by using Western blotting.Results:(1) We found 129 differentially expressed genes with more than 2- foldchanges by gene microarray, of which 83 were upregulated and 46 were downregulated in patients with AAD compared with controls. Bioinformatics analysisshowed a significant change in two pathways:thefocal adhesion pathway (FN1, ITGA9, PAK7, COL6A3, FLNC) and the actin cytoskeleton regulationpathway (FN1, ITGA9, PAK7, PIP5K1B, RRAS2). Among these differentially expressed genes, ITGA9 is the coding gene of the integrin a9 subunit and plays an important part role in the two pathways. The expression of ITGA9 gene was down regulated by 50% in AAD patients as compared with controls; (2) The Western blotting analysis confirmed the significant down-expression of ITGA9 in aortic tissue from AAD patients as compared to the controls (P=0.003); (3) The Western blotting analysis found the expression of p-SMAD2 was downregulated in AAD patients compared with the controls (P<0.05) while the expression of SMAD2, SMAD3, and p-SMAD3 were comparable between AAD patients and the control (all P>0.05).Conclusion:(1) The gene microarray analysis of aortic tissue reveals a significant difference between AAD patients and the control. Bioinformatics analysis reveals that the most affected pathways in AAD arefocal adhesionand actin cytoskeleton regulation, and ITGA9 is a key component in the both pathways. The verification of down-expression of ITGA9 in aortic tissue in AAD patients on the protein level suggests that ITGA9 might be an important gene involved in AAD and further study is needed to clarify the role ITGA9 played in pathogenesis of AAD. Moreover, the key component of traditional TGF-β pathway, p-SMAD2, was downregualted, however, the other components of TGF-β pathway did not change significantly. Whether SMAD2 interacts with ITGA9 in the pathogenesis of AAD remains to be further studied in the future. (2) The Western blotting analysis confirmed the significant down-expression of ITGA9 in aortic tissue from AAD patients as compared to the controls (P=0.003); (3) The Western blotting analysis found the expression of p-SMAD2 was downregulated in AAD patients compared with the controls (P<0.05) while the expression of SMAD2, SMAD3, and p-SMAD3 were comparable between AAD patients and the control (all P>0.05).Conclusion:(1) The gene microarray analysis of aortic tissue reveals a significant difference between AAD patients and the control. Bioinformatics analysis reveals that the most affected pathways in AAD arefocal adhesionand actin cytoskeleton regulation, and ITGA9 is a key component in the both pathways. The verification of down-expression of ITGA9 in aortic tissue in AAD patients on the protein level suggests that ITGA9 might be an important gene involved in AAD and further study is needed to clarify the role ITGA9 played in pathogenesis of AAD. Moreover, the key component of traditional TGF-β pathway, p-SMAD2, was downregualted, however, the other components of TGF-β pathway did not change significantly. Whether SMAD2 interacts with ITGA9 in the pathogenesis of AAD remains to be further studied in the future.Part Ⅱ:Impact of ITGA9 on the rat vascular smooth muscle cell phenotypeAbstractAim:Aortic medial degeneration is regarded as the key mechanism of aortic dissection; however, the precise mechanism remains poorly understood. Vascular smooth muscle cell (VSMC) is the main component of aortic media for maintaining normal structure and function of aorta. VSMC has two phenotypes:contractile phenotype and contractile phenotype. Previous studies have shown that VSMC phenotypic switching is involved in the aortic medial degeneration. In our first section, gene microarray and Western blotting analysis reveal thatITGA9, the coding gene of the subunit of integrin a9, was downregulated in aortic tissue in AAD patientsas compared with controls.Because the ITGA9 is expressed on cytomembraneand plays a critical role in both the cell adhesionand actin cytoskeleton regulation pathways, we hypothesized that ITGA9 might be involved in the pathogenesis of AAD. The present section aims to investigate the impact of ITGA9 on the phenotype transformation of rat VSMCs and to explore the possible role of ITGA9 in the pathogenesis of AADMethods:Primary rat aortic VSMC was cultured by tissue adherence method and the expression of ITGA9 in rat primary VSMC was tested by Western blotting analysis. Cultured rat VSMC was transfected with a fluorescent oligonucleotide probe to confirm whether rat VSMC could be transfected by small molecular RNA and short interfering RNA (siRNA) targeting ITGA9 was designed and used to transfect rat VSMC to knock down the expression of ITGA9. The expression of phenotype-dependent markers of VSMC including contractile VSMC markers (SM22a and a-SMA) andsynthetic VSMC biomarkers (SMemb and OPN)was analyzed after siRNA targeting ITGA9 and VSMC proliferation and migration ability was tested after siRNA targeting ITGA9 by cell scarification test. Adenovirus-ITGA9 recombinant vector was constructed and packaged to infect synthetic VSMC induced by PDGF. The expression of contractile VSMC markers (SM22a and a-SMA) and synthetic VSMC biomarker (SMemb) were analyzedafter overexpressing ITGA9 by Western blotting analysis.Results:(1) Primary rat arterial VSMC was successfully cultured by tissue adherence method and was identified by immunofluorescence; (2) Rat VSMC could express ITGA9 with high amount by Western blotting analysis, which laid foundation for further study by interfering ITGA9 expression; (3) Rat VSMC could be efficiently transfected by fluorescence labeled small molecular RNA; (4) Three designed siRNA targeting ITGA9 were transected VSMC, one of which could successfully knock down the expression of ITGA9; (5) After knocking down the expression of ITGA9, SM22a and a-SMA, the markers of contractile VSMC were decreased while OPN and SMemb, the markers of synthetic VSMC, were increased, and cell scarification test showed an increased proliferation and migration ability in siRNA-ITGA9 treated VSMC, which suggested the VSMC phenotype switches to synthetic phenotype; (7) The recombinant adenovirus vector was successfully constructed with a titre of 109 PFU/mL after virus packageand could overexpress ITGA9 in rat VSMC. After overexpression of ITGA9 in rat synthetic VSMC induced by PDGF, SM22a was significantly increased (P=0.017) and a-SMA was also increased (P=0.119), while SMemb was decreased (P=0.104) although they did not reach statistical difference.Conclusion:ITGA9 could affect the VSMC phenotype and downregualted ITGA9 may take part in the pathogenesis of AAD via inducing transformation of VSMCfrom contractilephenotype towards synthetic phenotype. Our results suggested abnormal expression of ITGA9 may be involved in the molecular mechanisms of AAD.PartⅢ:Impact of D-dimer levels upon admission on in-hospital and long-term outcome in patients with type A acute aortic dissectionAbstractAim:As a degradation product of cross-linked fibrin, the presence of D-dimer indicates activation of the coagulation and fibrinolytic systems. Previous studies have shown D-dimer is a useful marker for complementary initial diagnosis or exclusion of acute aortic dissection (AAD). However, few studies reported the prognostic value of D-dimer level in patients with AAD. The present study aimed to evaluate the association of admission D-dimer levels with both in-hospital and long-term clinical outcomes in patients with type A AAD.Methods:This is a single-center, prospectively observational study and enrolled consecutive patients with type A AAD confirmed by computed tomographic (CT) scanning admitted to Fuwai hospital between March 2010 and June 2011. Those who presented with intramural haematoma, penetrating aortic ulcer, traumatic aortic injury, iatrogenic dissection, pregnancy, Marfan syndrome, and familial aortic dissection were excluded for analysis. D-dimer levels were measured upon admission and baseline characteristics as well as treatment during hospitalization were collected. The primary endpoints were in-hospital and long-term all-cause mortality. D-dimer levels were compared between survivors and non-survivors and the in-hospital and long-term mortalities were compared among groups according to admission D-dimer quartiles. The association of admission D-dimer levels with in-hospital and long-term mortality was analyzed with univariate and multivariate Cox regression analysis. Due to the significant impact of surgical treatment on both in-hospital and long-term outcome, patients were divided into operation treatment group and non-operation treatment group, and the outcome was compared between the 2 groups stratified by admission D-dimer level (>6.10 and ≤6.10 μg/mL).Results:A total of 375 patients were diagnosed as AAD by CT imaging between March 2010 and June 2011. Of those,225 patients were Stanford type A and 150 were Stanford type B. After exclusion of 13 patients with incomplete data or lost to follow up,212 patients with type A AAD were analyzed. The median length of hospitalization was 14 days (interquartile range 9-20 days) and the median length of follow-up was 18.8 months (interquartile range 6.7-24.4 months). The mean age was 48.5±11.5 years, with male predominance (75.9%). The in-hospital and long-term all-cause mortality rates were 12.7% and 12.4%, respectively. Compared with the survivors, the non-survivors had significantly higher D-dimer levels (median 6.8 vs.2.5 μg/mL, P<0.001), higher WBC counts (median 12.1 vs.9.9×109/L, P<0.001), and creatinine level (median 102.5 vs.87.3 μmol/L, P<0.001), but received less surgical treatment (14.0% vs.74.7%, P<0.001). When divided into four groups according to admission D-dimer quartiles (Q1:≤1.06 μg/mL; Q2:1.07～2.82 μg/mL; Q3:2.83～6.10μg/mL; Q4:>6.10μg/mL), patients in Q4 (>6.10 μg/mL) had the highest in-hospital mortality (30.2%) and long-term mortality (24.3%) among groups (P<0.001 and P=0.046, respectively). After multivariate adjustment, the D-dimer level in Q4 (>6.10 μg/mL) was an independent risk factor for in-hospital mortality (hazard ratio [HR] 6.12,95% confidence interval [CI] 1.35 to 27.89, P=0.019); however, this was not an independent predictor for long-term mortality. Because surgical treatment was the main factor associated with in-hospital and long-term mortality, the impact of D-dimer on clinical outcomes was analyzed according to surgical intervention. In patients who received surgery, both in-hospital and long-term mortality rates were comparable between patients with D-dimer>6.10 and≤6.10 μg/mL. However, in those receiving conservative therapy, patients with admission D-dimer levels>6.10 μg/mL had significantly higher in-hospital (53.3% vs.18.5%, P=0.001) and long-term mortalities (57.1% vs.15.9%, P=0.004) than those with admission D-dimer levels≤6.10 μg/mL.Conclusion:(1) Surgical treatment is associated with reduced risk of in-hospital and long-term mortality in patients with type A AAD; (2) Elevated admission D-dimer levels (>6.10 μg/mL) might be a predictor for increased risk of in-hospital mortality, and urgent-emergent surgery might be needed in patients with elevated D-dimer levels upon admission. However, D-dimer levels on admission failed to predict long-term mortality; (3) Admission D-dimer level may provide more prognostic value for type A AAD patients with conservative therapy.