Impact of Notch3 Activation on Aortic Aneurysm Development in Marfan Syndrome

Abstract

Background: The leading cause of mortality in patients with Marfan syndrome (MFS) is thoracic aortic aneurysm and dissection. Notch signaling is essential for vessel morphogenesis and function. However, the role of Notch signaling in aortic pathology and aortic smooth muscle cell (SMC) differentiation in Marfan syndrome (MFS) is not completely understood.

Methods: RNA-sequencing on ascending aortic tissue from a mouse model of MFS, Fbn1^mgR/mgR , and wild-type controls was performed. Notch 3 expression and activation in aortic tissue were confirmed with real-time RT-PCR, immunohistochemistry, and Western blot. Fbn1^mgR/mgR and wild-type mice were treated with a γ-secretase inhibitor, DAPT, to block Notch activation. Aortic aneurysms and rupture were evaluated with connective tissue staining, ultrasound, and life table analysis.

Results: The murine RNA-sequencing data were validated with mouse and human MFS aortic tissue, demonstrating elevated Notch3 activation in MFS. Data further revealed that upregulation and activation of Notch3 were concomitant with increased expression of SMC contractile markers. Inhibiting Notch3 activation with DAPT attenuated aortic enlargement and improved survival of Fbn1^mgR/mgR mice. DAPT treatment reduced elastin fiber fragmentation in the aorta and reversed the differentiation of SMCs.

Conclusions: Our data demonstrated that matrix abnormalities in the aorta of MFS are associated with increased Notch3 activation. Enhanced Notch3 activation in MFS contributed to aortic aneurysm formation in MFS. This might be mediated by inducing a contractile phenotypic change of SMC. Our results suggest that inhibiting Notch3 activation may provide a strategy to prevent and treat aortic aneurysms in MFS.

Figure 1

Genes were differentially expressed in the aorta of WT and Fbn1^mgR/mgR (mgR) mice including Notch3, Fbn1, and α-actin. (a) Heatmap of the differentially expressed genes in the aorta of WT and mgR mice at PD28 (n = 4/group) was generated using data from RNA-Seq analysis. (b) Notch3 mRNA expression in the aorta of WT (n = 8) and mgR (n = 8) mice at PD28 was analyzed by real-time PCR. The bar graph (b) shows relative expression of Notch3 and 18 s rRNA. Immunohistochemical staining of Notch3 in the aortic sections from (c) WT and (d) mgR mice at PD28 (n = 4–6/group), respectively. Positive staining is shown in brown (DAB). Notch3-positive cells were quantitated by Definiens Tissue Studio software. Brown chromogen intensity is shown in (e) bar graph (n = 4–6/group). ^∗P < 0.05 compared to WT controls; Student's t-test.

Figure 2

Notch3 activation was increased in the aorta of Marfan patients and Fbn1^mgR/mgR (mgR) mice. Western blot analysis of active Notch3 (N3ICD) levels was performed on aortic protein from WT and mgR mice at PD28 (a) (n = 8/group) and Marfan patients and normal controls (n = 10/group) (c). The bar graphs show relative N3ICD levels in mouse (b) and human aortic tissue (d), respectively. ^∗P < 0.05 compared to WT controls; ^†P < 0.01 compared to normal controls; Student's t-test. Immunohistochemical staining of human Notch3 in the aortic sections from normal control (e) and Marfan patients (f) (n = 6/group). Positive staining is shown in brown (DAB). Notch3-positive cells were counted (cells/high power field, 40x). (g) The values reflect the mean ± SE. ^†P < 0.01 compared to normal control; Student's t-test.

Figure 3

DAPT treatment, which inhibits Notch activation, prevented elastin fragmentation. (a–d) Verhoeff-van Gieson (VVG) staining of elastic fibers of the ascending aorta from WT and Fbn1^mgR/mgR (mgR) mice at PD42 treated with DMSO (vehicle control) or DAPT; WT mice treated with DMSO (a) or DAPT (c); mgR mice treated with DMSO (b) or DAPT (d) at PD42. (e) Elastin breaks per field under 40x magnification in DMSO- or DAPT-treated WT and mgR mice (n = 5 aorta/groups). ^∗P < 0.05, ^ǂP < 0.01, and ^†P < 0.001, ANOVA with Tukey-Kramer post hoc test.

Figure 4

DAPT treatment delayed aortic expansion and improved survival of Fbn1^mgR/mgR (mgR) mice. (a) Timeline of mouse experiments: WT and mgR mice were treated with DAPT (10 mg/kg) or DMSO by subcutaneous injection started at PD10 (start) and stopped at PD42 (stop). (b) Aortic diameters of mgR mice and WT littermates receiving DAPT or DMSO (n = 10/group) were measured by ultrasound (US) at 5, 8, and 12 weeks of age. The aortic diameters of DMSO-treated mgR mice were larger compared with WT controls at all time points (^†P < 0.01 compared to WT controls). DAPT treatment inhibited aortic dilation of mgR mice at the 5-week time point (^∗P < 0.01 compared to DMSO-treated mgR mice). (c) Representative ultrasound images of ascending aorta of mice at 5-week time point. (d) Life table analysis of WT (n = 10/group) and mgR (n = 15/group) mice treated with DAPT or DMSO from PD10 to PD42. Survival rate of mgR mice treated with DAPT was improved compared with DMSO-treated mgR mice (P < 0.01).

Figure 5

DAPT treatment blocked Notch3 activation and reversed SMC phenotype in the aorta of Fbn1^mgR/mgR (mgR) mice. (a) Aortic protein from WT and mgR mice treated with DMSO and DAPT (n = 5-6/group) was extracted at PD 42. The levels of N3ICD and the smooth muscle cell differentiation marker, α-actin, were examined by Western blot analysis. (b) The bar graph shows relative active Notch3 (N3ICD) and α-actin levels in the aortic tissue. ^∗P < 0.01 and ^†P < 0.001; ANOVA with Tukey-Kramer post hoc test.

Figure 6

DAPT treatment inhibited Notch3 activation and reversed SMC phenotype of aortic SMCs from Fbn1^mgR/mgR (mgR) mice. Aortic SMCs were isolated from WT and mgR mice and treated with DMSO or DAPT (20 mM) for 48 hrs. Protein from cells was extracted. (a) The levels of Notch3 activation (N3ICD), α-actin, and SM22α were examined by Western blot analysis. (b) The bar graph shows relative N3ICD, α-actin, and SM22α levels in the cells. ^∗P < 0.05 compared to DMSO-treated mgR SMCs.