Myeloid-specific knockout of Notch-1 inhibits MyD88- and TRIF-mediated TLR signaling pathways by regulating oxidative stress-SHP2 axis, thus restraining aneurysm progression

Construction of Notch-1-knockout mice

ApoE^−/−/Lyz2-Cre^+/− mice and ApoE^−/−/Notch-1^flox/flox mice were constructed from ApoE^−/− mice purchased from Xiang'an Hospital of Xiamen University, specifically as follows: Transgenic vectors containing the Lyz2-Cre gene were constructed, the fertilized eggs were implanted into the uterus of the female mouse, and then the Lyz2-Cre^+/− mice were screened. Based on the Notch-1 gene sequence, specific primers were designed to insert two loxP sites, constructing transgenic vectors. Transgenic vectors should also include elements such as selective marker genes, which were used for in vitro DNA recombination. Then recombinant DNA was transfected into mouse embryonic stem cells for hybridization and recombination, and Notch-1^flox/flox embryonic stem cells were injected into normally developing mouse embryos. The embryos were implanted into the uterus of the female mouse. Finally, Notch-1^flox/flox mice were screened. All mice were housed in SPF rooms with a 12 h/12 h light-dark cycle, and were given adequate diet and water.

Establishment of ApoE^−/− chimeric mouse model (ApoE-KO/Notch-1^MAC-KO)

18 Male 12-weeks old ApoE^−/− mice were given gentamicin sulfate (320,000 U/L) in the drinking water from 1 week before transplantation. On the day of transplantation, ApoE^−/− mice were subjected to total body irradiation by X-ray at a total dose of 10 Gy (0.8 Gy/min, twice at an interval of 4 h). The mice were divided into the ApoE-KO/Notch-1^WT group and the ApoE-KO/Notch-1^MAC-KO group.9 mice per group. Bone marrow cells of KO mice were prepared into mononuclear cell (MNC) suspension with serum-containing RPMI1640 medium, and the cell concentration was adjusted to 5 × 10⁷ MNCs/mL. Following irradiation for 4–6 h, 5 × 10⁶ MNCs were injected via the caudal vein for hematopoietic reconstitution. 6 weeks later, the successfully chimeric mice were pumped with angiotensin II at 1000 ng/kg/min to induce aneurysm formation (AAA group), while the remaining mice were pumped with normal saline (CTL group).

Sampling

All mice were sacrificed by decapitation 28 d later, and the aortic tissue and blood were promptly harvested for further analysis. The tissues of four mice in each group were fixed with 4% paraformaldehyde followed by histological observation, while those of the remaining mice in each group were stored at −80°C for later molecular assays. During sampling, the adipose tissue of vascular adventitia was peeled off, the morphology of aorta was observed, and the maximum diameter of vessel was measured.

Histological observation

After drying, the sections were mounted with gum, and the change in tissue morphology was observed under an optical microscope. Following dehydration with gradient ethanol, the sections were stained with Weigert's iron hematoxylin solution, water-washed and differentiated with acidic ethanol differentiation solution. After washing, the sections were added with Masson staining solution, washed again, stained with Ponceau acid fuchsin staining solution, washed with phosphomolybdic acid solution, added with aniline blue dye, and washed with weak acid solution. Finally, the sections were dehydrated with gradient ethanol, transparentized with xylene and mounted with neutral gum, followed by microscopic observation.

Immunofluorescence and immunohistochemical staining

After complete drying, the 4 μm-thick paraffin sections were deparaffinized with xylene, rehydrated with gradient ethanol, and incubated with proteinase K solution at room temperature for 20 min, followed by incubation with primary antibodies at 4°C overnight and with corresponding fluorescence-based HRP-labeled secondary antibodies (1:500) at room temperature for 1 h. After washing with PBS, the sections were stained with DAPI or hematoxylin. Finally, the macrophage infiltration and the distribution of Notch-1 in aortic aneurysms were observed under a fluorescence or bright-field microscope, and the fluorescence was excited at the corresponding wavelength and merged during multiple staining. The results were photographed and recorded.

Western blotting

The protein was extracted from tissues of ApoE^−/− mice using RIPA lysis buffer and PMSF. Specifically, the tissues were lysed with lysis buffer containing the protease inhibitor PMSF (PMSF: RIPA=1:100) on ice for 30 min to extract the protein. Then the protein was separated by SDS-PAGE, transferred onto a PVDF membrane, and incubated with antibodies against iNOS, IL-10, Arginase-1, GAPDH, p22, p47, p-MyD88, p-TRIF, p-NF-κB, IFN-γ, IL-1β and TNF-α (1:1000) at 4°C overnight. Finally, the membrane was incubated again with HRP-labeled goat anti-rabbit or goat anti-mouse IgG secondary antibodies at room temperature for 2 h, exposed using the Ultra High Sensitivity ECL Kit and FluorChem Q, and quantified by AlphaView software.

Extraction of bone marrow-derived macrophages and phagocytosis assay with fluorescent microspheres

The mice were sacrificed and thoroughly disinfected with 75% alcohol. The tibia and femur of mice were isolated under sterile conditions, placed in a cell culture dish containing 75% alcohol, washed with PBS 2–3 times, and transferred to a cell culture dish containing complete medium (1% penicillin-streptomycin + 10% fetal bovine serum + DMEM basal medium). Then both ends of the tibia and femur were cut with ophthalmic scissors, and rinsed with complete medium pipetted with a 1 mL syringe to harvest the bone marrow cells from one end of the bone into a 50 mL sterile centrifuge tube, and the operation was repeated several times until the bones became white. After 5-fold red blood cell lysis buffer was added to the centrifuge tube, the cells were pipetted repeatedly, and left to stand for 15 min, followed by centrifugation at 1000 rpm. After the supernatant was discarded, the cells were resuspended with an appropriate amount of DMEM and filtered through a 200-mesh strainer, followed by centrifugation at 1,000 rpm for 10 min, and the supernatant was discarded. The above operation was repeated twice. Finally, the supernatant was discarded, and the cells were resuspended with DMEM containing 10 ng/mL M-CSF to induce differentiation into macrophages. The resulting macrophages were then treated with the SHP2 inhibitor PHPS1.

The macrophages were incubated with 10 μm fluorescent silver YG microspheres, and pre-incubated in 2.1% BSA (Polysciences; 4.55 × 10⁶ beads/mL) for 24 h. Then they were washed fully with PBS, fixed with 2% paraformaldehyde in PBS, counterstained with 4’,6-diamidino-2-phenylindole and 1,1’-octacosyl-3,3,3’,3’-tetramethylindoline according to the manufacturer's instructions, and mounted on slides with Vectashield mounting media.

Statistical analysis

Data were analyzed using GraphPad Prism 9.0 and described by mean ± standard deviation. The difference was compared by the Student’s t-test between two groups, and by one-way analysis of variance among groups. ^*P < 0.05 was considered statistically significant. All assays were repeated three times independently.

Data availability

Our data can be obtained by contacting the corresponding author.

Notch-1 in macrophage was implicated in AAA

To assess the role of Notch-1 in macrophages in the development of AAA, aneurysm mouse models were established by bone marrow transplantation, and the mice were divided into the ApoE-KO/Notch-1^WT group and ApoE-KO/Notch-1^MAC-KO group. As observed by two-dimensional B-mode high-resolution ultrasonography, ApoE-KO/Notch-1^MAC-KO group exhibited a significantly smaller lumen diameter than the ApoE-KO/Notch-1^WT group (P < 0.01), which was also verified by the quantitative analysis. The lumen of the artery refers to the lumen in which blood flows through the artery, and the space inside the artery. In AAA, the lumen of the artery refers to the space surrounded by the dilated aortic wall. These results implied that knockout of Notch-1 in macrophages can reduce the aortic dilation and the lumen diameters in ApoE-KO/Notch-1^WT mice. Besides, these mice were infused with angiotensin II to induce AAA. Abdominal aorta sections (including the aneurysm) were surgically harvested, and representative aortic images are shown in Figure 1. It was found that the aortic diameters increased, suggesting that the AAA mouse models were established successfully. Furthermore, smaller aortic diameters were found in the CTL group than the AAA group (P < 0.05, P = 0.0211).

Figure 1. Specific deficiency of notch-1 in macrophage suppressed the progression of AAA. (A) representative aortic images with identical magnification and quantitative analysis; (B) two-dimensional B-mode high-resolution ultrasonography and quantitative analysis. N = 9, ^*P < 0.05, ^**P < 0.01, apoE-KO/Notch-1^MAC-KO group vs. apoE-KO/Notch-1^WT group.

The results of Masson staining, EVG staining and immunohistochemical staining showed that the macrophage-positive area and the area of aneurysms in the ApoE-KO/Notch-1^MAC-KO group significantly decreased compared with the ApoE-KO/Notch-1^WT group (P < 0.01), while the SMC-positive area was not significantly different between the two groups (P > 0.05, P = 0.8394), indicating that macrophages play a major role in the regulation of AAA (Figure 2).

Figure 2. Notch-1-cKO in macrophages inhibited the elastic lamina degradation and macrophages infiltration in AAA. (A) representative image for Masson’s staining, α-SMA staining, CD68 immunostaining, and Verhoeff-van Gieson staining; (B) statistical data for AAA areas, macrophages, smooth muscle cells as well as ratio of aortic dissection. N = 9, ^*P < 0.05, ^**P < 0.01, apoE-KO/Notch-1^MAC-KO group vs. apoE-KO/Notch-1^WT group.

Effect of Notch-1 on macrophage polarization

The results of Western blotting revealed that knockout of Notch-1 decreased the expression of iNOS (an M1 macrophage marker) (P < 0.01), but increased the expressions of IL-10 and Arginase-1 (M2 macrophage markers) (P < 0.01). It can be seen that Notch-1 may promote M1 macrophage polarization, while knockout of Notch-1 promotes M2 macrophage polarization. Besides, the immunofluorescence results showed that the number of iNOS-expressing macrophages in Notch-1-knockout mice decreased after M1 polarization, i.e., M1 polarization was impaired after the knockout of Notch-1. In contrast, the number of Arginase-1-expressing macrophages in Notch-1-knockout mice increased after M2 polarization (Figure 3).

Figure 3. Notch-1 cKO in macrophages promote the M2 polarization in AAA. (A) Immunofluorescence staining results showed the Notch-1 deficiency in macrophages inhibited the expression of iNOS (M1 marker) and increased the expression of arginase-1 (M2 marker) and statistical data; (B) macrophages’ deficiency of Notch-1 decreased the expression of iNOS and increased the expression of arginase-1 and IL-10 in AAA tested by western blot and the quantitative analysis. N = 6, ^*P < 0.05, ^**P < 0.01, apoE-KO/Notch-1^MAC-KO group vs. apoE-KO/Notch-1^WT group.

Myeloid-specific knockout of Notch-1 regulated oxidative stress and SHP2 protein expression

Oxidative stress and inflammatory response are typical characteristics of aneurysms, in which SHP2 plays a variety of roles in different cells, different environments and different stages. Therefore, the role of SHP2 in the inflammatory response or inflammatory environment was explored. It was found that compared with those in the CTL group, the protein expressions of p22 and p47 significantly decreased in the Notch-1^MAC-KO mice, accompanied by an increase in the protein expression of SHP2 (P < 0.01). The above findings suggest that Notch-1 can exert a pro-inflammatory effect through the oxidative stress-SHP2 axis (Figure 4).

Figure 4. Notch-1 cKO in macrophages increased the SHP2 and suppressed the ROS in AAA. (A) DHE staining results showed the macrophages’ deficiency of Notch-1 suppressed the ROS production and its statistical data. N = 9; (B) macrophages’ deficiency of Notch-1 suppressed the expression of Notch-1, p22, p47 and consistent with increased expression of SHP2 in AAA tested by western blot. ^*P < 0.05, ^**P < 0.01, apoE-KO/Notch-1^MAC-KO group vs. apoE-KO/Notch-1^WT group.

Myeloid-specific knockout of Notch-1 suppressed p-MyD88/TRIF pathway-mediated inflammatory responses

MyD88/TRIF pathway, which activates NF-κB accompanied by an excessive expression of inflammatory factors, is the major and key pathway accelerating aneurysm progression. Then more inflammatory cells, especially M1 macrophages, accumulate toward the aneurysm, thus accelerating aneurysm progression [13–16]. The results of Western blotting showed that after treatment with angiotensin II, the MyD88/TRIF/NF-κB signaling pathway was activated, thus inducing an inflammatory response in the artery. In Notch-1^MAC-KO mice, the phosphorylation levels of the MyD88/TRIF/NF-κB signaling pathway-related proteins were significantly inhibited, accompanied by significant decreases in the protein expressions of inflammatory factors IFN-γ, IL-1β and TNF-α (P < 0.01) (Figure 5).

Figure 5. Notch-1 deficiency inhibited the activation of MyD88/TRIF/NF-κB signals. (A) Macrophages’ deficiency of Notch-1 inhibited the phosphorylated-MyD88, TRIF, and NF-κB and there statistical data; (B) Notch-1 cKO decreased the expression of IFN-γ, IL-1β, TNF-α expression in AAA tested by western blot. N = 6, ^*P < 0.05, ^**P < 0.01, apoE-KO/Notch-1^MAC-KO group vs. apoE-KO/Notch-1^WT group.

Myeloid-specific knockout of Notch-1 enhanced phagocytic capacity of macrophages by promoting the SHP2 signaling pathway

To verify the effect of myeloid-specific knockout of Notch-1 on the phagocytic capacity of macrophages, the phagocytosis assay with fluorescent microspheres was designed. The results showed that the number of fluorescent microspheres phagocytosed in the ApoE-KO/Notch-1^MAC-KO group was significantly larger than that in the ApoE-KO/Notch-1^WT group. However, after the addition of PHPS1, the number of fluorescent microspheres phagocytosed decreased significantly in both groups, without significant differences (Figure 6).

Figure 6. Monocytes/macrophages-specific knockout of Notch1 promotes phagocytic capacity by promoting the SHP2 signaling pathway. (A) Immunofluorescent spheroid phagocytosis experimental results; (B) Macrophage engulfs fluorescent spheroid count statistics. N = 3, ^**P < 0.01, WT group vs. KO group, WT+PHPS1 group vs. KO+PHPS1 group.