Objective Activation of inflammatory pathways plays a critical role in the development of abdominal aortic aneurysms (AAA). Notch1 signaling is a significant regulator of the inflammatory response; however, its role in AAA is unknown. Methods and Results In an angiotensin II (AngII)-induced mouse model of AAA, activation of Notch1 signaling was observed in the aortic aneurysmal tissue of Apoe−/− mice and a similar activation of Notch1 was observed in aneurysms of humans undergoing AAA repair. Notch1 haploinsufficiency significantly reduced the incidence of AAA in Apoe−/− mice in response to AngII. Reconstitution of bone marrow-derived cells from Notch1+/−; Apoe−/− mice (donor) in lethally irradiated Apoe−/− mice (recipient) decreased occurrence of aneurysm. Flow cytometry and immunohistochemistry demonstrated that Notch1 haploinsufficiency prevented the influx of inflammatory macrophages at the aneurysmal site by causing defects in macrophage migration and proliferation. Additionally, there was an overall reduction in the inflammatory burden in the aorta of the Notch1+/−;Apoe−/− mice as compared to the Apoe−/− mice. Lastly, pharmacologic inhibition of Notch1 signaling also prevented AAA formation and progression in Apoe−/− mice. Conclusion Our data suggest that decreased levels of Notch1 protect against the formation of AAA by preventing macrophage recruitment and attenuating the inflammatory response in the aorta.

BackgroundThe progression of abdominal aortic aneurysm (AAA) involves a sustained influx of proinflammatory macrophages, which exacerbate tissue injury by releasing cytokines, chemokines, and matrix metalloproteinases. Previously, we showed that Notch deficiency reduces the development of AAA in the angiotensin II–induced mouse model by preventing infiltration of macrophages. Here, we examined whether Notch inhibition in this mouse model prevents progression of small AAA and whether these effects are associated with altered macrophage differentiation.Methods and ResultsTreatment with pharmacological Notch inhibitor (DAPT [N‐(N‐[3,5‐difluorophenacetyl]‐L‐alanyl)‐S‐phenylglycine t‐butyl ester]) at day 3 or 8 of angiotensin II infusion arrested the progression of AAA in Apoe−/− mice, as demonstrated by a decreased luminal diameter and aortic width. The abdominal aortas of Apoe−/− mice treated with DAPT showed decreased expression of matrix metalloproteinases and presence of elastin precursors including tropoelastin and hyaluronic acid. Marginal adventitial thickening observed in the aorta of DAPT‐treated Apoe−/− mice was not associated with increased macrophage content, as observed in the mice treated with angiotensin II alone. Instead, DAPT‐treated abdominal aortas showed increased expression of Cd206‐positive M2 macrophages and decreased expression of Il12‐positive M1 macrophages. Notch1 deficiency promoted M2 differentiation of macrophages by upregulating transforming growth factor β2 in bone marrow–derived macrophages at basal levels and in response to IL4. Protein expression of transforming growth factor β2 and its downstream effector pSmad2 also increased in DAPT‐treated Apoe−/− mice, indicating a potential link between Notch and transforming growth factor β2 signaling in the M2 differentiation of macrophages.ConclusionsPharmacological inhibitor of Notch signaling prevents the progression of AAA by macrophage differentiation–dependent mechanisms. The study also provides insights for novel therapeutic strategies to prevent the progression of small AAA.

AimsInfiltration of macrophages and apoptosis of vascular smooth muscle cells (VSMCs) promote the development of abdominal aortic aneurysm (AAA). Previously, we demonstrated that global Notch1 deficiency prevents the formation of AAA in a mouse model. Herein, we sought to explore the cell-specific roles of Notch1 in AAA development.Methods and resultsCell-specific Notch1 haploinsufficient mice, generated on Apoe-/- background using Cre-lox technology, were infused with angiotensin II (1000 ng/min/kg) for 28 days. Notch1 haploinsufficiency in myeloid cells (n = 9) prevented the formation of AAA attributed to decreased inflammation. Haploinsufficiency of Notch1 in SMCs (n = 14) per se did not prevent AAA formation, but histoarchitectural traits of AAA including elastin degradation and aortic remodeling, were minimal in SMC-Notch1+/-;Apoe-/- mice compared to Apoe-/- mice (n = 33). Increased immunostaining of the contractile SMC-phenotype markers and concomitant decreased expression of synthetic SMC-phenotype markers were observed in the aortae of SMC-Notch1+/-;Apoe-/- mice. Expression of connective tissue growth factor (CTGF), a matrix-associated protein that modulates the synthetic VSMC phenotype, increased in the abdominal aorta of Apoe-/- mice and in the adventitial region of the abdominal aorta in human AAA. Notch1 haploinsufficiency decreased the expression of Ctgf in the aorta and in vitro cell culture system. In vitro studies on SMCs using the Notch1 intracellular domain (NICD) plasmid, dominant negative mastermind-like (dnMAML), or specific siRNA suggest that Notch1, not Notch3, directly modulates the expression of CTGF.ConclusionsOur data suggest that lack of Notch1 in SMCs limits dilation of the abdominal aorta by maintaining contractile SMC-phenotype and preventing matrix-remodeling.

The progression of abdominal aortic aneurysm (AAA) involves a sustained influx of proinflammatory macrophages, which exacerbate tissue injury by releasing cytokines, chemokines and matrix metalloproteinases (MMPs). While multiple factors are involved in disease pathogenesis, the critical stimulus is the differentiation and infiltration of naïve macrophages (Mϕ) towards classical (M1) activation. In contrast, Mϕ can also be programmed to M2-macrophages which inhibit the inflammatory response and promote tissue repair. Previously, we showed that Notch deficiency reduces the development of AAA in the angiotensin II (AngII)-induced mouse model by preventing infiltration of Mϕ. Because of its regulatory roles in macrophage differentiation, we examined if Notch inhibition in a mouse model prevents progression of small AAA and if these effects are associated with Mϕ-differentiation. Notch inhibition (DAPT) started at day 3 or 8 of AngII infusion arrested the progression of AAA in Apoe -/- mice as demonstrated by a decreased luminal diameter and aortic width. The abdominal aortae treated with DAPT showed decreased MMPs expression and presence of elastin precursors including tropoelastin. Marginal adventitial thickening observed in DAPT-treated Apoe -/- mice was not associated with increased total macrophage content. Instead, DAPT-treated abdominal aortae showed increased expression of Cd206 positive M2-macrophages and decreased expression of Il12 positive M1-macrophages. Increased expression of IL12 positive M1 macrophages and its strong correlation with active Notch1 signaling (NICD) was also observed in the infrarenal aortae from AAA patients. Notch1 deficiency promoted M2-differentiation of Mϕ by upregulating transforming growth factor ( Tgf)-β2 expression in bone marrow-derived Mϕ at basal levels and in response to IL4. Protein expression of Tgf-β2 and its downstream pSmad2 also increased in DAPT-treated Apoe -/- mice, indicating a potential link between Notch and Tgf-β2 signaling in the M2-differentiation of Mϕ. Overall, Notch inhibition stabilizes the progression of AAA by macrophage-differentiation-dependent mechanisms and provides insights for novel therapeutic strategies to prevent the progression of small AAA.