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 Castanospermine site 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 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 18325633 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 MedChemExpress Clavulanate (potassium) 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 with control tissue. B) Notch1 in VSMCs of the aortic media was significantly decreased in both TAA and TAD tissues compared with control tissue (scale bar = 25 mm, insets 6.25 mm). C) NICD was rarely detected in vascular smooth muscle cells (VSMCs) of the aortic media in TAA and TAD tissues. D) Hes1 was rarely detected in VSMCs of the aortic media in TAA and TAD tissues (scale bar = 50 mm). doi:10.1371/journal.pone.0052833.gwith DTAAD. Specifically, the Notch signaling pathway was downregulated in medial VSMCs but activated in CD34+ stem cells, Stro-1+ stem cells, fibroblasts, and macrophages. Our findings suggest that impaired Notch signaling in VSMCs may contribute to the apoptosis and depletion of VSMCs thatcharacterize DTAAD. The activation of Notch signaling in Stro1+ stem cells, CD34+ stem.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 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 18325633 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 with control tissue. B) Notch1 in VSMCs of the aortic media was significantly decreased in both TAA and TAD tissues compared with control tissue (scale bar = 25 mm, insets 6.25 mm). C) NICD was rarely detected in vascular smooth muscle cells (VSMCs) of the aortic media in TAA and TAD tissues. D) Hes1 was rarely detected in VSMCs of the aortic media in TAA and TAD tissues (scale bar = 50 mm). doi:10.1371/journal.pone.0052833.gwith DTAAD. Specifically, the Notch signaling pathway was downregulated in medial VSMCs but activated in CD34+ stem cells, Stro-1+ stem cells, fibroblasts, and macrophages. Our findings suggest that impaired Notch signaling in VSMCs may contribute to the apoptosis and depletion of VSMCs thatcharacterize DTAAD. The activation of Notch signaling in Stro1+ stem cells, CD34+ stem.
