Activated in fibroblasts in DTAAD patientsFibroblasts 
can proliferate rapidly in response to injury and contribute to tissue repair [23]. Furthermore, fibroblasts are important in maintaining Epigenetics aortic tensile strength and preventing aortic dilatation and rupture in response to aortic injury. Notch signaling has been shown to be involved in fibroblast-mediated tissue repair [24]. Thus, we examined changes in fibroblast levels in the diseased [24] aortic wall and the activation of Notch signaling in fibroblasts. Using ER-TR7 as a fibroblast marker, we detected significantly 
more fibroblasts in the adventitia of TAA and TAD tissues than in control tissue (P,0.001) (Fig. 5). Additionally, NICD was detected in most aortic fibroblasts in TAA and TAD tissues (35.2 in control; 69.2 in TAA [P = 0.009 vs. control]; 65.5 in TAD [P = 0.02 vs. control])Notch signaling is downregulated in aortic medial VSMCs of DTAAD patientsWe examined the activation of Notch signaling in different types of cells found within the aortic wall. First, we assessed Notch signaling in medial VSMCs. Immunofluorescence double staining experiments showed that both DLL1/4 Notch Epigenetics ligand and theNotch Signaling in Aortic Aneurysm and DissectionFigure 1. Overall activation of Notch signaling is increased in the aortic wall of DTAAD patients. A) Western blot studies showed that the expression of Notch1 (transmembrane/intracellular region NTM, ,120 kDa) in human aortic tissue was significantly increased in TAA tissue compared with control tissue, and NICD was significantly increased in TAA and TAD samples compared with control. B) Quantitative real-time RT-PCR showed increased expression levels of Notch1 in TAA and TAD samples compared with control. C) Western blot studies showed that the expression of Hes1 in human aortic tissue was significantly increased in TAA and TAD samples compared with control. doi:10.1371/journal.pone.0052833.g(Fig. 5A); Hes1 was also highly expressed in most fibroblasts (Fig. 5B). These findings indicate the activation of Notch signaling in fibroblasts of the aortic wall in DTAAD patients.Notch signaling is activated in macrophages in DTAAD patientsMacrophages have been shown to play a critical role in aortic destruction and AAD development [25,26]. Moreover, Notch1 positively regulates IL-6 expression in activated 23727046 macrophages [26]. Thus, we examined Notch activation in macrophages in TAA and TAD. Using CD68 as the marker for macrophages, we found significantly more macrophages in the aortic wall in TAA and TAD tissues than in control tissue (P,0.001). Moreover, NICDwas detected in most macrophages in TAA and TAD tissues (35.8 in control; 70.4 in TAA [P,0.001 vs. control]; 77.2 in TAD [P,0.001 vs. control]) (Fig. 6). Furthermore, Hes1 was also expressed by most macrophages (Fig. 6B). These results suggest activation of the Notch signaling pathway in macrophages of the aortic wall in TAA and TAD patients.DiscussionThe Notch signaling pathway is a versatile regulator of cell growth and differentiation [27,28,29], as well as cardiovascular development [5,6] and repair [30]. In this study, we have shown a complex pattern of Notch signaling in the aortic tissue of patientsNotch Signaling in Aortic Aneurysm and DissectionFigure 2. Notch signaling is downregulated in aortic medial VSMCs in DTAAD patients. A) Immunofluorescence double staining showed that DLL1/4 in VSMCs of the aortic media was significantly decreased in both TAA and TAD tissues compared wit.Activated in fibroblasts in DTAAD patientsFibroblasts can proliferate rapidly in response to injury and contribute to tissue repair [23]. Furthermore, fibroblasts are important in maintaining aortic tensile strength and preventing aortic dilatation and rupture in response to aortic injury. Notch signaling has been shown to be involved in fibroblast-mediated tissue repair [24]. Thus, we examined changes in fibroblast levels in the diseased [24] aortic wall and the activation of Notch signaling in fibroblasts. Using ER-TR7 as a fibroblast marker, we detected significantly more fibroblasts in the adventitia of TAA and TAD tissues than in control tissue (P,0.001) (Fig. 5). Additionally, NICD was detected in most aortic fibroblasts in TAA and TAD tissues (35.2 in control; 69.2 in TAA [P = 0.009 vs. control]; 65.5 in TAD [P = 0.02 vs. control])Notch signaling is downregulated in aortic medial VSMCs of DTAAD patientsWe examined the activation of Notch signaling in different types of cells found within the aortic wall. First, we assessed Notch signaling in medial VSMCs. Immunofluorescence double staining experiments showed that both DLL1/4 Notch ligand and theNotch Signaling in Aortic Aneurysm and DissectionFigure 1. Overall activation of Notch signaling is increased in the aortic wall of DTAAD patients. A) Western blot studies showed that the expression of Notch1 (transmembrane/intracellular region NTM, ,120 kDa) in human aortic tissue was significantly increased in TAA tissue compared with control tissue, and NICD was significantly increased in TAA and TAD samples compared with control. B) Quantitative real-time RT-PCR showed increased expression levels of Notch1 in TAA and TAD samples compared with control. C) Western blot studies showed that the expression of Hes1 in human aortic tissue was significantly increased in TAA and TAD samples compared with control. doi:10.1371/journal.pone.0052833.g(Fig. 5A); Hes1 was also highly expressed in most fibroblasts (Fig. 5B). These findings indicate the activation of Notch signaling in fibroblasts of the aortic wall in DTAAD patients.Notch signaling is activated in macrophages in DTAAD patientsMacrophages have been shown to play a critical role in aortic destruction and AAD development [25,26]. Moreover, Notch1 positively regulates IL-6 expression in activated 23727046 macrophages [26]. Thus, we examined Notch activation in macrophages in TAA and TAD. Using CD68 as the marker for macrophages, we found significantly more macrophages in the aortic wall in TAA and TAD tissues than in control tissue (P,0.001). Moreover, NICDwas detected in most macrophages in TAA and TAD tissues (35.8 in control; 70.4 in TAA [P,0.001 vs. control]; 77.2 in TAD [P,0.001 vs. control]) (Fig. 6). Furthermore, Hes1 was also expressed by most macrophages (Fig. 6B). These results suggest activation of the Notch signaling pathway in macrophages of the aortic wall in TAA and TAD patients.DiscussionThe Notch signaling pathway is a versatile regulator of cell growth and differentiation [27,28,29], as well as cardiovascular development [5,6] and repair [30]. In this study, we have shown a complex pattern of Notch signaling in the aortic tissue of patientsNotch Signaling in Aortic Aneurysm and DissectionFigure 2. Notch signaling is downregulated in aortic medial VSMCs in DTAAD patients. A) Immunofluorescence double staining showed that DLL1/4 in VSMCs of the aortic media was significantly decreased in both TAA and TAD tissues compared wit.
