Pharmacological inhibition of Notch signaling regresses pre-established abdominal aortic aneurysm

CITATION: Sharma, N. et al. 2019. Pharmacological inhibition of Notch signaling regresses pre-established abdominal aortic aneurysm. Scientific Reports, 9. doi:10.1038/s41598-019-49682-0The original publication is available at https://www.nature.com/srep/ENGLISH ABSTRACT: Abdominal aortic aneurysm (AAA) is characterized by transmural infiltration of myeloid cells at the vascular injury site. Previously, we reported preventive effects of Notch deficiency on the development of AAA by reduction of infiltrating myeloid cells. In this study, we examined if Notch inhibition attenuates the progression of pre-established AAA and potential implications. Pharmacological Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPT) was administered subcutaneously three times a week starting at day 28 of angiotensin II (AngII) infusion. Progressive increase in pulse wave velocity (PWV), maximal intra-luminal diameter (MILD) and maximal external aortic diameter (MEAD) were observed at day 56 of the AngII. DAPT prevented such increase in MILD, PWV and MEAD (P < 0.01). Histologically, the aortae of DAPT-treated Apoe−/− mice had significant reduction in inflammatory response and elastin fragmentation. Naked collagen microfibrils and weaker banded structure observed in the aortae of Apoe−/− mice in response to AngII, were substantially diminished by DAPT. A significant decrease in the proteolytic activity in the aneurysmal tissues and vascular smooth muscle cells (vSMCs) was observed with DAPT (P < 0.01). In human and mouse AAA tissues, increased immunoreactivity of activated Notch signaling correlated strongly with CD38 expression (R2 = 0.61). Collectively, we propose inhibition of Notch signaling as a potential therapeutic target for AAA progression.Publisher’s versio

Novel Markers to Delineate Murine M1 and M2 Macrophages.

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages

Comparison of common and distinct genes of classically activated and alternatively activated macrophages.

<p>Fold-change (FC) vs. FC plot of M1 vs. M0 on the x-axis and M2 vs. M0 on the y-axis highlighting common up-regulated genes in red (81 genes, 105 probes), common down-regulated genes in blue (125 genes, 172 probes), M1 up- and M2 down-regulated genes in purple (57 genes, 78 probes) and M2 up- and M1 down-regulated genes in green (33 genes, 45 probes). Arrows indicate distinct M1 or M2 genes. Red lines represent a ±2FC cut-off. The black line indicates the <i>x = y</i> diagonal expected if all gene probes were up- or down-regulated to the same extent in M1 and M2 macrophages (FC M1 vs. M0 = FC M2 vs. M0). Previously reported markers of classical and alternative macrophages are included for reference in small, italicized font. Genes representing classical macrophages include Nos2, Tnfa, IL-1b and IL12b. Genes representing alternative macrophages include Arg1, Mrc1 (CD206), Retnla (Fizz-1), and Chi3l3 (Ym1). The genes labeled in larger bold font, CD38, Fpr2 and Gpr18 or c-Myc and Egr2 were selected as candidate M1- or M2-selective markers, respectively.</p

Identification and validation of novel classically activated M1 markers CD38, Fpr2 and Gpr18.

<p>Expression of (<b>A</b>) CD38, (<b>B</b>) Formyl peptide receptor 2 (Fpr2) and (<b>C</b>) G-Protein Coupled Receptor 18 (Gpr18) was determined by cDNA microarray in BMDMs stimulated for 24 hours in M0, M1, and M2 (n = 2–3 mice, 3 replicates/mouse/condition) conditions. Gene expression is represented as fold change relative to unstimulated M0 condition (FC ± SEM), multiple corrected t-test. Expression of (<b>D</b>) CD38, (<b>E)</b> Fpr2, (<b>F</b>) and Gpr18 was determined by Real-Time PCR in BMDMs stimulated for 24 hours in M0, M1, and M2 (n = 8, 6 mice, 1–2 replicates/mouse/condition) conditions. Gene expression expressed as fold-change relative to unstimulated M0 condition (FC ± SD); ANOVA followed by multiple comparison post-hoc t-test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Expression of CD38 and Egr2 protein in M0, M1 and M2 macrophages.

<p>Flow cytometry staining of surface CD38 and intracellular Egr2 (<b>A</b>), CD38 and iNOS (<b>C</b>) or Egr2 and Arginase-1 (<b>E</b>) in BMDM 24 hours post-differentiation in M0, M1 and M2 conditions (representative data of three independent experiments). Flow plots correspond to the CD11b gate. <b>B.</b> Quantification of <b>A</b>, showing the proportion of M0, M1 and M2 macrophages with CD38⁺Egr2^- (putative M1) or CD38^-Egr2⁺ (putative M2) phenotype at 24 hours (n = 3–6 replicates from two independent experiments). <b>D.</b> Quantification of <b>C</b>, showing the proportion of M0, M1 and M2 macrophages with CD38⁺ or iNOS+ phenotype at 24 hours (n = 3 replicates, representative of three independent experiments). <b>F.</b> Quantification of <b>E</b>, showing the proportion of M0, M1 and M2 macrophages with Egr2⁺ or Arg-1⁺ phenotype at 24 hours (n = 3 replicates representative of three independent experiments). Percentage of CD38⁺ cells in iNOS⁺ (<b>G</b>) or TNF-α⁺ (<b>H</b>) BMDM differentiated for 24 hours in M1 conditions. Percentage of Egr2⁺ cells in Arg-1⁺ (<b>I</b>) or CD206⁺ (<b>J</b>) BMDM differentiated for 24 hrs in M2 conditions. **p<0.01, ****p<0.0001.</p

Discrimination of M1 and M2 macrophages via CD38 and Egr2 flow cytometry assay.

<p>A. Experimental design. BMDMs were differentiated for 24 hours in M1 conditions and tagged with CD11b-V450. Another set of BMDM was differentiated in M2 conditions and tagged with CD11b-PE. The M1 and M2 populations were then mixed prior to staining with surface CD38 and intracellular Egr2. CD38⁺Egr2^- (putative M1) and CD38^-Egr2⁺ (putative M2) gates were drawn and the percentage of cells in each gate that originated from M1 (V450 tagged) or M2 (PE tagged) cultures was calculated. B. Representative flow cytometry data of CD38 and Egr2 staining in tagged M1 and M2 populations as indicated in A. C. Quantification of the results from B (n = 3, representative of three independent experiments). D. M0, M1 and M2 cells were stained with either CD11b-V450 or CD11b-PE and subsequently stained with CD38 or Egr2 to confirm that CD11b-V450 or -PE tagging did not alter CD38 or Egr2 staining. ****p<0.0001.</p

Macrophage signature in classically activated M1 and alternatively activated M2 macrophages.

<p>Fold Change (FC) vs. Mean Expression Value (MEV) plot of microarray data highlighting 2 FC or higher up-regulated genes (red, p≤0.05) or down-regulated genes (blue, p≤0.05) in <b>(A)</b> classically activated M1 macrophages (compared to M0 condition) or <b>(B)</b> alternatively activated M2 macrophages (compared to M0 condition). Red lines represent a ± 2FC cut-off. The numbers above and below the lines indicate the number of probes (some genes are represented by several probes and, hence, as several dots) above or below the 2 FC cut-off, respectively. Genes previously associated with classical and alternative macrophage phenotypes are labeled with corresponding gene names. Genes associated with (<b>A</b>) classically activated macrophages include Nos2, CCR7, TNFa, Inhba, IL12b, IL-6, and IL-1b, and CD86. Genes associated with (<b>B</b>) alternatively activated macrophages include Arg1, Fn1, Egr2 (human), Mrc1/CD206, Chi3l3/Ym1 and Retnla/Fizz-1.</p

Identification and validation of alternatively activated M2 markers c-Myc and Egr2.

<p>Expression of (<b>A</b>) Early Growth Response Protein-2 (Egr2) and (<b>B</b>) c-Myc was determined using cDNA microarray in BMDMs stimulated for 24 hours in M0, M1, and M2 (n = 2–3 mice, 2 replicates/mouse/condition) conditions. Gene expression is represented as fold change (FC ± SEM) relative to unstimulated M0 condition; multiple comparison corrected t-test. Expression of <b>(C)</b> Early Growth Response Protein-2 (Egr2) and <b>(D) c-</b>Myc expression was measured via RT-PCR and expressed as mean relative expression (±SD) in M0, M1, and M2 (n = 8, 6 mice, 1–2 replicates/mouse/condition) BMDMs. Gene expression is expressed as FC ± SD of unstimulated M0 condition; ANOVA followed by multiple comparison post-hoc t-test; **p<0.01, ***p<0.001, ****p<0.0001. Data shown are from one experiment representative of two independent experiments.</p