Impact of Notch3 Activation on Aortic Aneurysm Development in Marfan Syndrome

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, Fbn1mgR/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. Fbn1mgR/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 Fbn1mgR/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.


Introduction
Marfan syndrome (MFS) is a dominantly inherited disorder of connective tissue with prominent abnormalities in the ocular, skeletal, and cardiovascular systems. The leading causes of mortality in patients with MFS are progressive aortic root dilation, aneurysm formation, and aortic dissection and rupture [1]. Although β-blockers have been the primary medical therapy for MFS patients with aortic aneurysms, current therapies for MFS have limited efficacy. Therefore, new approaches are required to identify novel therapeutic targets.
Notch signaling is essential for vessel morphogenesis and function. The notch genes encode large single-transmembrane receptors that mediate communication between neighboring cells that are crucial for cell fate decisions during organ development [18]. In mammals, four Notch receptors (Notch1-4) have been identified, all of which are composed of a large extracellular domain which mediates ligand interaction, a transmembrane domain, and an intracellular domain. Upon the binding of transmembrane ligands from Delta/Serrate/LAG-2 (DSL) family, DLL, and Jagged, Notch receptors undergo proteolytic cleavage by several proteinases, including γ-secretase [18]. This 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). Notch3positive 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. 3 Journal of Immunology Research transcription factor CBF-1 to promote transcriptional activation and cell fate determination [19,20]. In vascular smooth muscle cells (SMCs), the Notch2 and Notch3 receptors predominate [21][22][23][24]. Notch signaling is critical for the regulation of SMC contractile differentiation and extracellular matrix protein synthesis both in vitro and in vivo [21,25,26]. Studies by Crosas-Molist et al. showed that expression of SMC contractile protein markers was higher in the aortas of Marfan patients compared to healthy controls [27]. By using a murine model of MFS, Fbn1 mgR/mgR mice, we have demonstrated that premature differentiation of SMCs contributes to aortic aneurysm pathology in MFS [28]. However, the role of Notch receptors in aneurysm formation and SMC differentiation in MFS remains unclear.
In this study, we observed increased Notch3 expression and activation in the aorta of Fbn1 mgR/mgR mice. We investi-gated the impact of Notch3 in aortic aneurysm development in MFS. We found that inhibition of Notch signaling moderated VSMC contractile phenotype transition and attenuated aortic aneurysm development in Fbn1 mgR/mgR mice.

Material and Methods
2.1. Mice. Heterozygous FBN1 mutant mice (Fbn1 mgR/+ ) in a mixed C57Bl/6J:129 SvEv background were mated to generate homozygous mutant mice (Fbn1 mgR/mgR ) and wild-type (WT) littermates [28]. Both male and female mice were included in the study. Genotyping of mice was performed at postnatal day 7 (PD7) by PCR [28,29]. All experiments were carried out in accordance with the guidelines of the University of Nebraska Medical Center Animal Care Committee for the use and care of laboratory animals. All mice were maintained in the pathogen-free animal facility. Human TAA samples (n = 10) were provided from the NIH GenTac Biobank. Age-and gender-matched control aortic samples (n = 10) were obtained from Nebraska Organ Recovery System.

2.2.
Next-Generation RNA-Sequencing and Real-Time RT-PCR. RNA from ascending thoracic aorta of WT and Fbn1 mgR/mgR mice was extracted using TRIzol reagent (Thermo Fisher Scientific). The mRNA was converted to cDNA by reverse transcription, and sequencing adaptors were ligated to the ends of cDNA fragments. Following amplification by PCR, the RNA-Seq library was sequenced using an Illumina NextSeq500 sequence analyzer (Illumina, san Diego, CA). The sequence reads were processed with a series of software [30][31][32]. The values of fragments per kilobase of transcript per million mapped reads (FPKM) were used for statistical comparison. The significant differential expression genes were defined by q value ≤ 0.05 [33]. A list of normalized differentially expressed genes were visualized with heatmap. For real-time RT-PCR, aortic RNA was reverse transcribed into cDNA using iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.). Real-time RT-PCR was performed using SsoAdvanced Universal SYBR® Green Supermix according to the manufacturer's instruction (Bio-Rad Laboratories, Inc.) on an ABI StepOne machine (Thermo Fisher Scientific). Fold differences were calculated using mRNA expression normalized to 18 s rRNA and analyzed using the ΔΔCt relative quantification method.

Immunohistochemistry and Verhoeff-Van Gieson (VVG)
Connective Tissue Staining. After perfusion-fixation with 10% neutral-buffered formalin, mouse ascending aortic tissues were embedded in paraffin and cut into 4 μm sections. For immunohistochemical staining, tissue sections were incubated with anti-Notch3 antibody (Santa Cruz Biotechnology, Dallas, TX) with a dilution of 1 : 250 for 32 min at  2.5. High Frequency Ultrasound. Transthoracic ultrasound of WT and Fbn1 mgR/mgR mice with or without DAPT treatment were performed with Vevo 3100 High Resolution In Vivo Micro-imaging system (VisualSonics, Toronto, Ontario, Canada) equipped with an integrated isoflurane-based anesthesia system. These studies were performed at 5, 8, and 12 weeks of age. Short-axis scans of the ascending aorta were performed using B-mode ultrasonography with the RMV 707 Scanhead. The aortic diameters were measured by Mmode in systole. Three independent measurements were obtained for each mouse. To define user variability, all echocardiographic studies were performed by 2 experienced individuals, and results were compared for agreement.

Isolation of Mouse SMC and Cell Culture. WT and
Fbn1 mgR/mgR mice (n = 6/group) were anesthetized and underwent laparotomy at PD28. Mouse thoracic aortas were isolated and minced. SMC isolation was described previously [28]. The cells were grown to confluence and passed after trypsinization with 0.25% trypsin. Aortic SMCs were maintained in vascular cell basal medium (ATCC, Manassas, VA) with 5% FBS. To examine the effect of DAPT, cells were incubated with serum-free medium and treated with 20 μM DAPT for 48 h. DMSO treatment was used as vehicle control. Cells were harvested for protein extraction.

Western Blot Analysis.
Aortic proteins were extracted as previously described [29]. Briefly, the protein from the aortic tissue and cells was extracted with RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA). The protein concentration of aortic proteins was standardized with a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA

Notch3 Expression and Activation Were Increased in the Aorta of Both Humans and Mice with MFS. RNA-sequencing (RNA-Seq) analysis with aortic samples from WT and
Fbn1 mgR/mgR mice at PD28 (n = 4) was performed. The RNA-Seq data revealed an increased expression of Notch3 with upregulation of contractile proteins in Fbn1 mgR/mgR mice (Figure 1(a)). This is consistent with our previous findings that aortic SMCs in MFS were prematurely differentiating into a contractile phenotype, i.e., upregulation of α-actin, SM22α, and calponin [28]. Increased Notch3 expression in the aortas of Fbn1 mgR/mgR mice was confirmed with realtime PCR (Figure 1(b)). Immunohistochemical analysis of aortic tissue showed the protein levels of Notch3 were also increased in Fbn1 mgR/mgR mice compared to WT controls at PD28 (Figures 1(c)-1(e)). Notch3 is a receptor that is composed of a large extracellular domain, a transmembrane domain, and an intracellular domain. Upon receptor-ligand binding at the cell surface, Notch3 undergoes proteolytic cleavage by proteinases, including γ-secretase [18]. This cleavage results in Notch3 activation and the release and translocation of the Notch3 intracellular domain (N3ICD) into the nucleus to promote transcriptional activation. We next tested activation of Notch3 in the aorta of WT and Fbn1 mgR/mgR mice at PD28 by Western blot analysis. We found that active Notch3 (N3ICD) was significantly higher in Fbn1 mgR/mgR mice compared to WT controls (Figures 2(a) and 2(b)). Furthermore, we sought to determine whether increased Notch3 activation is also seen in aortic samples from Marfan patients. The levels and activation of Notch3 in human aortic tissue were examined. Western blot analysis displayed that active Notch3 in the aorta was significantly higher in patients with MFS compared to matched controls (Figures 2(c) and 2(d)). Immunohistochemistry results showed that Notch3 levels were higher in Marfan patients than controls (Figures 2(e)-2(g)). These 7 Journal of Immunology Research findings indicate that increased activation of Notch3 may play a role in the pathogenesis of human MFS and mouse model of MFS.

Inhibition of Notch3 Activation Preserved Elastic Fiber
Integrity. Previous studies of Fbn1 mgR/mgR mouse aortas demonstrated aortic elastic fiber disorganization and fragmentation [28,34]. To determine the role of Notch3 activation in aortic elastin degradation and aneurysm formation of Fbn1 mgR/mgR mice, WT and Fbn1 mgR/mgR mice were treated with a γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglucine t-butyl ester (DAPT). The treatment started at PD10 and stopped at PD42. The effects of DAPT treatment on aortic histology were evaluated by VVG staining. At PD42, while aortic lamellae in WT mice were intact and normal with treatment of DMSO or DAPT (Figures 3(a) and 3(c)), aortas of DMSO-(vehicle) treated Fbn1 mgR/mgR mice showed elastic fiber disruption and medial hypertrophy (Figures 3(b)). However, DAPT-treated Fbn1 mgR/mgR mice displayed a markedly lesser degree of aortic medial hypertrophy and elastin fragmentation (Figures 3(d) and 3(e)).

DAPT Treatment Delayed Aortic Expansion and
Improved Survival of Fbn1 mgR/mgR Mice. The ascending aortic diameters of WT or Fbn1 mgR/mgR mice receiving DAPT or DMSO were measured with high-frequency ultrasound at 5, 8, and 12 weeks of age (Figure 4(a)). Aortic diameters of DMSO-treated WT mice were significantly smaller than those of DMSO-treated Fbn1 mgR/mgR mice at all time points (Figure 4(b)). At the 5-week time point, aortic diameters in DAPT-treated Fbn1 mgR/mgR mice were significantly smaller than those in DMSO-treated Fbn1 mgR/mgR mice (Figures 4(b) and 4(c)). The aortic diameters in DAPTtreated mice did not differ from DMSO-treated mice at 8or 12-week time points. However, by 8 weeks of age, 3 out of 10 Fbn1 mgR/mgR DMSO-treated mice died from aortic rupture. Furthermore, survival studies were performed in WT and Fbn1 mgR/mgR mice treated with DAPT or DMSO (Figure 4(d)). The mice were followed up until death or to 32 weeks. Mice surviving to 32 weeks were euthanized. WT mice survived a normal lifespan; 2 out of 15 WT mice treated with DAPT died after 30 weeks of age. The mean survival of DMSO-treated Fbn1 mgR/mgR mice (n = 15) was 138:4 ± 13:6 days, while Fbn1 mgR/mgR mice treated with DAPT (n = 15) survived 199:9 ± 10:5 days. This result demonstrated that DAPT treatment significantly prolonged the lifespan of Fbn1 mgR/mgR mice. Taken together, these data suggest that inhibition of Notch3 activation could play a role in preventing the early developmental abnormalities that occur in the Marfan aorta.

DAPT Treatment Reversed Abnormal SMC Phenotypic
Switch in Fbn1 mgR/mgR Mice. Our previous studies demonstrated that aortic SMCs of Fbn1 mgR/mgR mice prematurely switched to a more contractile phenotype which contributed to aneurysm formation [28]. We sought to determine whether pharmacological inhibition of Notch3 activation in Fbn1 mgR/mgR mice had effect on SMC differentiation. The levels of a SMC marker, α-actin, in the aorta were examined by Western blot (Figure 5(a)). While DAPT treatment inhibited aortic Notch3 activation in Fbn1 mgR/mgR mice, it also reduced the expression of α-actin (Figures 5(a) and 5(b)). To confirm the effect of Notch3 activation on SMC differentiation, aortic SMCs were isolated from WT and Fbn1 mgR/ mgR mice. Consistent with the results from mouse aorta, N3CID levels were significantly higher in SMCs from Fbn1 mgR/mgR mice than in SMCs from WT mice (Figures 6(a) and 6(b)). Furthermore, DAPT treatment reduced Notch3 activation and expression of contractile markers, α-actin and SM22α, in SMCs from Fbn1 mgR/mgR mice. These results indicate that contribution of enhanced Notch3 activity to aneurysm formation in Fbn1 mgR/mgR mice may be associated with aortic SMC differentiation. Taken together, these data suggest that inhibition of Notch3 activation could play a role in preventing the developmental abnormalities that occur in the Marfan aorta.

Discussion
We have shown that Notch3 expression and activation are increased in the aorta of MFS. During aortic development of MFS (Fbn1 mgR/mgR ) mice, increased Notch3 activation contributes to aortic aneurysm formation. Inhibition of Notch3 activation by γ-secretase inhibitor, DAPT, attenuates aortic elastic fiber fragmentation and aortic enlargement and improves mouse survival. The mechanism is due, in part, to mediation of aortic SMC phenotype modulation. These findings indicate that Notch3 is an important regulator of aortic SMC function and differentiation during aortic development and suggest that therapy modulating Notch3 signaling in the aorta may be beneficial in inhibiting aortic aneurysm formation in MFS.
The Fbn1 mgR/mgR mice are one of commonly used murine models of MFS with a hypomorphic mutation of FBN1. Homozygous Fbn1 mgR/mgR mice display clinical features and manifestations similar to classic patients of MFS. They die naturally at an average age of 2-3 months [34]. Previous studies showed that dysregulation of TGF-β activation contributed to pathogenesis of MFS [35,36]. Pharmacological inhibition of TGF-β with TGF-β-neutralizing antibody attenuated aneurysm formation in MFS mice [16]. However, later results with the Fbn1 mgR/mgR mouse model indicated that TGF-β could exert opposite effects on thoracic aortic aneurysm (TAA) pathology that broadly correlated with the early and late stages of TAA progression [37]. It was demonstrated that early treatment (PD16) with TGF-β-neutralizing antibodies exacerbated TAA formation, while later treatment (PD45) had a contrasting beneficial effect [37]. Our previous study also supported that initial consequence of FBN1 mutation was not accompanied by a significant increase in TGF-β activation [28]. Differential contributions of TGF-β signaling to aortic physiology early and later after birth in MFS prompt us to identify new biomarkers and therapies independent of the TGF-β signaling pathway to better understand aortic pathogenesis in MFS.
It is demonstrated that aortic development in mice occurs primarily between embryonic day (ED)14 and 8 Journal of Immunology Research postnatal day (PD)14 and is typically complete by PD28 [38,39]. During this time, aortic SMCs are highly proliferative and express structural matrix proteins that are important for vascular strength and compliance. After that, aortic SMCs shift out of the matrix phase and express the spectrum of contractile proteins to prepare for their unique contractile function. In patients with MFS, aortic tissue and aortic SMCs had increased expression of contractile protein markers (α-actin, SM22α, and calponin-1) and collagen [27]. Our previous studies using a murine model of MFS, Fbn1 mgR/mgR mice, supported these findings [28]. We demonstrated that aortic SMCs in MFS mice prematurely differentiated into a contractile phenotype, which contributed to the pathology in MFS [28]. We also showed that abnormal phenotypic switching of aortic SMC led to reduced elastin synthesis in Marfan mice [28]. However, the mechanisms modulating SMC phenotypic switching and matrix protein production in MFS are not completely understood. In this study, we have found that expression and activation of Notch3 are increased in the aortas of mice and humans with MFS, which is consistent with the findings from other investigators [40,41]. Notch signaling is critical for cell growth regulation and cell fate determination in many cell types, including aortic SMCs [42,43]. The regulation of contractile differentiation and extracellular matrix synthesis by Notch activity has been well studied, but results have shown that Notch signaling has both anti-differentiation and prodifferentiation functions in vascular SMCs, suggesting context dependence and/or spatial-temporal regulation during development [44][45][46]. The Notch signaling pathway plays an important role in the developing cardiovascular system. Notch1 is the primary expressed receptor in endothelial cells. Notch1 mutation is linked to bicuspid aortic valve and plays an important role in aortic root dilation [47,48]. Notch2 and 3 are mainly expressed in vascular SMC and required for mural cell expansion and maturation. Notch4 is an endothelial cell-specific mammalian Notch gene [49]. In addition, Notch ligands, such as Jagged 1, also play important roles in normal arterial development [50][51][52]. Notch3 is predominantly expressed in vascular SMC in humans [25,[53][54][55]. It is a key regulator of vascular SMC phenotypes and is required for arterial identity and vascular SMC maturation [25,53,56]. Expression of the constitutively active Notch3 resulted in cell shape changes and an increase in α-actin [25]. Our studies showed similar results that levels of contractile protein, α-actin, were higher in the aorta of Fbn1 mgR/mgR mice compared to control mice [28]. To determine the impact of increased Notch3 activity on the phenotypic state of VSMC in MFS, we administered DAPT, a γ-secretase inhibitor, to Fbn1 mgR/mgR mice starting at PD10. DAPT inhibited Notch3 signaling by preventing the proteolysis of Notch3. DAPT treatment also reduced the expression of SMC contractile marker, α-actin, in the aorta of Fbn1 mgR/mgR mice, indicating that DAPT treatment could reverse aortic SMC phenotype in Fbn1 mgR/mgR mice. Furthermore, we investigated the effects of DAPT treatment on the aortic development and aneurysm formation/rupture of MFS. Blocking Notch3 activation with DAPT in the early aortic development of Fbn1 mgR/mgR mice attenuated aneu-rysm enlargement and preserved elastic fiber integrity, therefore prolonging the lifespan of Fbn1 mgR/mgR mice.
In summary, our data demonstrate that the early and potentially important SMC phenotypic switch and the associated matrix abnormalities in the Marfan aorta are associated with increased Notch3 activation. Increased Notch3 activation in MFS contributed to perturbation of early aortic development in MFS through inducing contractile phenotypic switch of SMC. The aortic pathology can be attenuated by treatment with a drug that blocks Notch3 activation. Our results suggest that inhibition of the effect of Notch3 signaling in the early aortic development of MFS may be a useful strategy to prevent and treat aortic aneurysms in MFS.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.