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.

No abstract

Naïve macrophages (Mφ) polarize in response to various environmental cues to a spectrum of cells that have distinct biological functions. The extreme ends of the spectrum are classified as M1 and M2 macrophages. Previously, we demonstrated that Notch1 deficiency promotes Tgf-β2 dependent M2-polarization in a mouse model of abdominal aortic aneurysm. The present studies aimed to characterize the unique set of genes regulated by Notch1 signaling in macrophage polarization. Bone marrow derived macrophages isolated from WT or Notch1 +/− mice (n = 12) were differentiated to Mφ, M1 or M2-phenotypes by 24 h exposure to vehicle, LPS/IFN-γ or IL4/IL13 respectively and total RNA was subjected to RNA-Sequencing (n = 3). Bioinformatics analyses demonstrated that Notch1 haploinsufficiency downregulated the expression of 262 genes at baseline level, 307 genes with LPS/IFN-γ and 254 genes with IL4/IL13 treatment. Among these, the most unique genes downregulated by Notch1 haploinsufficiency included fibromodulin ( Fmod ), caspase-4 , Has1 , Col1a1 , Alpl and Igf . Pathway analysis demonstrated that extracellular matrix, macrophage polarization and osteogenesis were the major pathways affected by Notch1 haploinsufficiency. Gain and loss-of-function studies established a strong correlation between Notch1 haploinsufficiency and Fmod in regulating Tgf-β signaling. Collectively, our studies suggest that Notch1 haploinsufficiency increases M2 polarization through these newly identified genes.

Introduction: Our previous studies demonstrated that loss of Notch1 signaling reduces the incidence of abdominal aortic aneurysm (AAA) by preventing the influx of pro-inflammatory M1 macrophages and that Notch1 deficiency promotes M2-polarization. In the present study, we determined if inhibition of Notch signaling could reverse the pre-existing pro-inflammatory M1 macrophages to anti-inflammatory M2 phenotype. Methods and Results: Raw cells (264.7 murine macrophage cell line) were treated with various concentrations of LPS (5, 10, 100 ng/ml) and IFN-γ (10 ng/ml) for 3h to polarize naïve macrophages into M1-phenotype. After washing, these cells were treated with either vehicle or DAPT (10 ng/ml or 25 ng/ml); a potent Notch inhibitor for 6, 12, 24 or 48h and later used for gene expression studies or functional assays. LPS pretreatment significantly increased the gene expression of M1-genes; Il6 , Il12, Cd38, Fpr2 and iNOS , whereas the expression of M2 genes; Egr2 and c-Myc was significantly decreased. Replacement of LPS with vehicle alone lowered the expression of Il6 and Il12 within 6-12h, but remained significantly higher than basal levels. Expression of iNOS , Cd38 and Fpr2 remained unchanged in response to vehicle in the LPS-pretreated macrophages. DAPT treatment further reduced the gene expression of Il6 and Il12 significantly and iNOS moderately as compared to vehicle-treated macrophages. Interestingly, Notch inhibition increased the expression of Egr2 and c-Myc genes at 24 and 48h. Similar trends with regard to M1 and M2 genes were observed in bone marrow derived macrophages from Apoe -/- and WT mice treated with LPS followed by vehicle or DAPT. Phagocytosis assay showed increased uptake of zymosan bioparticles in the macrophages treated with LPS. Replacement of LPS with vehicle for 24h did not affect the phagocytosis significantly. DAPT treatment decreased phagocytic activity of these macrophages as compared to vehicle-treated macrophages. Conclusions: Our data suggest that Notch inhibition reverses the M1-polarized macrophages towards M2-like macrophages. These novel insights in the functions of Notch signaling may have potential implications in chronic inflammatory diseases including AAA and atherosclerosis.