Smooth muscle cell-specific <i>Notch1</i> haploinsufficiency restricts the progression of abdominal aortic aneurysm by modulating CTGF expression

<div><p>Aims</p><p>Infiltration 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.</p><p>Methods and results</p><p>Cell-specific <i>Notch1</i> haploinsufficient mice, generated on <i>Apoe</i>^<i>-/-</i> background using Cre-lox technology, were infused with angiotensin II (1000 ng/min/kg) for 28 days. <i>Notch1</i> haploinsufficiency in myeloid cells (n = 9) prevented the formation of AAA attributed to decreased inflammation. Haploinsufficiency of <i>Notch1</i> 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 <i>SMC-Notch1</i>^<i>+/-</i><i>;Apoe</i>^<i>-/-</i> mice compared to <i>Apoe</i>^<i>-/-</i> 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 <i>SMC-Notch1</i>^<i>+/-</i><i>;Apoe</i>^<i>-/-</i> mice. Expression of connective tissue growth factor (CTGF), a matrix-associated protein that modulates the synthetic VSMC phenotype, increased in the abdominal aorta of <i>Apoe</i>^<i>-/-</i> mice and in the adventitial region of the abdominal aorta in human AAA. <i>Notch1</i> haploinsufficiency decreased the expression of Ctgf in the aorta and <i>in vitro</i> cell culture system. <i>In vitro</i> 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.</p><p>Conclusions</p><p>Our data suggest that lack of Notch1 in SMCs limits dilation of the abdominal aorta by maintaining contractile SMC-phenotype and preventing matrix-remodeling.</p></div

<i>SMC-specific Notch1</i> haploinsufficiency dependent regulation of SMC phenotype is associated with changes in CTGF expression.

<p>Representative images of IHC staining of abdominal aorta from respective experimental groups with antibodies against smMHC (<b>A-D</b>) and osteopontin (<b>F-I</b>) as markers of contractile or synthetic-phenotype marker of SMCs, respectively. IHC staining with antibody against Mmp12 (<b>K-N</b>), and Ctgf (<b>P-S</b>) performed on cross sections of abdominal aortae isolated from <i>Apoe</i>^<i>-/-</i> cell-specific <i>Notch1</i> haploinsufficient <i>Apoe</i>^<i>-/-</i> mice after 28 days AngII treatment. <b>Scale bars = 50</b> μ<b>m</b>. The IHC for respective immunostaining was quantified using ‘Fiji’ version of image J (<b>E, J, O, T</b>). Bar graphs represent fold change in gene expression of <i>Ctgf</i> (<b>W</b>), <i>Tgf-β1</i> (<b>X</b>), <i>Tgf-β2</i> (<b>Y</b>), <i>Smad2</i> (<b>Z</b>) and <i>Smad3</i> (<b>AA</b>) using mRNA obtained from aortae of <i>Apoe</i>^<i>-/-</i> and cell-specific <i>Notch1</i> haploinsufficient mice. The results were standardized to <i>Rpl13a</i> and reported as ratio to saline-treated mice (mean ± SEM, n = 3 for each group). (<b>AB</b>) Fold change in <i>Ctgf</i> expression in SMCs isolated from Apoe^<i>-/-</i> and <i>Notch1</i>^<i>+/-</i><i>;Apoe</i>^<i>-/-</i> mice at basal levels and in the presence of TNF-α. ***<i>P</i><0.001; **<i>P</i><0.01; and *<i>P</i><0.05 (ordinary ANOVA followed by a Bonferroni-Holm multiple comparisons test).</p

Cell-specific <i>Notch1</i> haploinsufficiency in vascular smooth muscle cells and macrophages protects against extensive structural damage and collagen deposition in the abdominal aortic wall in response to AngII (28 days) treatment.

<p><b>(A-D)</b> Cross-section of representative aortae stained with hematoxylin and eosin (HE) staining showing lumen and intraluminal thrombus. Panel (<b>E-H</b>) Representative photomicrographs showing transmural inflammatory cell infiltration. (<b>I-L</b>) Representative images of modified Verhoeff Van Gieson staining demonstrating the extent of elastin fragmentation; (<b>M-P</b>) Collagen contents in the abdominal aorta of experimental groups visualized in blue using trichrome staining. <b>Scale bar = 50</b> μ<b>m</b> in <b>A-P</b>. (<b>Q-S</b>) Bar graphs represent gene expression of <i>Col1α1</i>, <i>Col1α2 and Col3α1</i> in the aortae of the <i>Apoe</i>^<i>-/-</i> and cell-specific <i>Notch1</i> haploinsufficient <i>Apoe</i>^<i>-/-</i> mice after 28 days AngII treatment. The results were standardized to <i>Rpl13a</i> and reported as ratio (mean ± SEM, n = 3 for each group) to saline-treated mice. <i>***P<0</i>.<i>001</i> (ordinary ANOVA followed by a Bonferroni-Holm multiple comparisons test).</p

Schematic diagram of the study.

<p>The dashed arrows depict the mechanistic pathways to be tested in the study. We propose that myeloid specific <i>Notch1</i> haploinsufficiency prevents aneurysm development by modulating macrophage polarization. SMC-specific <i>Notch1</i> haploinsufficiency interferes with the progression of aneurysm by modulating CTGF-dependent SMC-phenotype. (Mφ: macrophages, SMC: smooth muscle cells; CTGF, connective tissue growth factor, opn: osteopontin,vim: vimentin).</p

Gain- and loss-of-function studies suggest direct involvement of Notch1 in regulating CTGF expression.

<p><b>(A-B)</b> Bar graphs represent fold change in gene expression of CTGF in the human aortic smooth muscle cells <b>(A)</b> and mouse embryonic fibroblast <b>(B)</b> treated with DAPT for 24 hours followed by TGF-β agonist or inhibitor SB431542 [activin receptor-like kinase (ALK5; the TGF-beta type I receptor inhibitor] treatment for 24 hours. <b>(C-F)</b> Graphs represent fold change in the expression of Notch1, CTGF, Notch3 and smoothelin in the human aortic smooth muscle cells 48 hours post transfected with Notch1 <i>intracellular domain</i> (NICD) overexpressing plasmid or dominant negative mastermind like proteins (dnMAML). (<b>G-J)</b> Graphs represent fold change in the expression of Notch1, CTGF, Notch3 and smoothelin in the human aortic smooth muscle cells 48 hours post transfected siRNAs for Notch1/Notch3.The qPCR data were standardized to <i>RPL13a</i> and reported as ratio (mean ± SEM, n = 3 for each group) to empty plasmids for NICD/dnMAML or non-specific siRNA. <i>***P<0</i>.<i>001</i>, <i>**P<0</i>.<i>01</i>, <i>*P<0</i>.<i>05</i> (ordinary ANOVA followed by a Bonferroni-Holm multiple comparisons test). (EP = empty plasmid).</p

Cell-specific <i>Notch1</i> haploinsufficiency differentially interferes with AAA development in AngII-induced mouse model.

<p>Representative aortae isolated from <i>Apoe</i>^<i>-/-</i> mice treated with saline (<b>A</b>), AngII (<b>B</b>), <i>SMC-Notch1</i>^<i>+/-</i><i>;Apoe</i>^<i>-/-</i> mice (<b>C</b>;) and <i>myeloid-Notch1</i>^<i>+/-</i><i>;Apoe</i>^<i>-/-</i> mice (<b>D</b>;) treated with AngII (28 days). Images were taken using Zeiss Stemi 2000-C microscope. <b>Scale bar, 1 mm</b>. (<b>E-F</b>), Quantitative measurement of maximal aortic width (mm) in different groups. Each symbol represents an individual animal. Mean and SEM are shown. (<b>G</b>) Survival graph showing the mortality of individual mice in each group during 28 day AngII infusion. <i>***P<0</i>.<i>001</i> (post-Bonferroni test with Holm correction).</p

CTGF expression in human AAA correlates strongly with synthetic SMC-phenotype.

<p><b>(A-D)</b> Immunohistochemical staining for CTGF on the normal and aneurysmal human abdominal aorta sections. <b>(E-P)</b> Micrographs showing immunofluorescence staining for CTGF <b>(green; E, I, M)</b> and fibroblast marker TE-7 <b>(red; E, J, N)</b> on the tissue sections obtained from medial layer of healthy human controls <b>(E-H)</b> or aneurysmal <b>(I-L)</b> or non-aneurysmal aorta <b>(M-P)</b> from same human AAA. Merged images of CTGF and TE-7 staining from respective tissues <b>(yellow; G, K, O)</b> and nuclear staining <b>(DAPI; H, L, P)</b> are shown. <b>Scale bars = 50 μm</b>.</p