Identification of The Unique Subtype of Macrophages in Aneurysm Lesions at the Growth Phase

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Figure Legends

Fig. 1. The labeling of macrophages accumulating in aneurysm lesions in rats.

The labeling of macrophages accumulating in aneurysm lesions induced in the surgicallyformed bifurcation site induced in carotid artery of rats. The macroscopic view of the

aneurysm lesion induced in the surgically-formed bifurcation site induced in carotid

artery of rats is shown (a). The macrophages were labeled by the engulfment of liposome

containing fluorescent protein, DiI, were then visualized. The representative images of

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accumulating macrophages in lesions from sections (b) or specimens with tissue

transparency (c) are shown. Dotted lines in (b) and (c) indicate the arterial walls or the

lesions. Scale Bars in (a), (b) or (c); 400 µm, 200 µm or 400 µm.

Fig. 2. Engulfing of DiI-containing liposome specifically by macrophages and the

dissection of macrophages by referencing DiI.

(a) Engulfing of DiI-containing liposome specifically by macrophages. HEK293 cells and

RAW264.7 cells were co-cultured and treated with DiI-containing liposome subjecting to

immunohistochemistry. The images from immunohistochemistry for the macrophage

marker, CD68 (green), the images of DiI (red), the nuclear staining by DAPI (blue) or the

merged images are shown. Scale bar; 50 µm. (b) The dissection of macrophages by lasermicrodissection technique. Macrophages were isolated by laser-microdissection

technique by referencing DiI. The images before or after the dissection are shown. Scale

bar; 10 µm.

Fig. 3. The identification of the unique macrophage subtype in aneurysm lesions at

the growth phase.

(a) The clustering analysis for gene expression profile in macrophages isolated from

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aneurysm lesions at the growth phase. (b) The principal component analysis of gene

expression profile in macrophages isolated from aneurysm lesions, bone-marrow derived

M0 macrophages or M1 macrophages. (c, d) The heat map and the Venn diagram of overexpressed (c) or under-expressed genes (d) in macrophages isolated from aneurysm

lesions compared with that in bone-marrow derived M0 macrophages or M1 macrophages.

Fig. 4. The presence of nerves along arterial walls in bifurcation of intracranial

arteries.

The presence of myelinating nerves along arterial walls at bifurcation sites. The

bifurcation site of anterior cerebral artery – olfactory artery was harvested subjecting to

scanning electron microscopic examination and immunohistochemistry. The images from

the scanning electron microscopic examination (a) and the immunohistochemistry for the

smooth muscle cell marker, a-smooth muscle actin (red in b), the markers for Schwann

cells, S100 (gray in b) or Sox10 (green in b), and merged image with nuclear staining by

DAPI (blue in b) are shown (b). The magnified image corresponding to the square in the

upper panel is shown in the lower panel in (a). Scale bar; 50 µm.

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Table 1. Up-represented terms in macrophages from aneurysm lesions at the growth phase

compared with in vitro-differentiated M0 from Biological Process and the top 15 list from

Molecular Function in gene ontology analysis.

Biological Process

P value

Sensory perception of taste

8.1E-05

Response to pheromone

2.8E-02

Neuropeptide signaling pathway

2.8E-02

Humoral immune response

2.8E-02

Antimicrobial humoral response

7.5E-02

Serotonin receptor signaling pathway

9.8E-02

Sensory perception of bitter taste

1.4E-01

Antimicrobial humoral immune response mediated by antimicrobial peptide

1.4E-01

G protein-coupled serotonin receptor signaling pathway

1.6E-01

Defense response to bacterium

1.6E-01

Molecular Function

P value

Odorant binding

1.4E-33

Pheromone receptor activity

8.2E-09

Neurotransmitter receptor activity

1.2E-06

Gated channel activity

1.8E-06

Channel activity

1.3E-05

Passive transmembrane transporter activity

1.3E-05

Hormone activity

1.9E-05

Ion channel activity

2.0E-05

G protein-coupled amine receptor activity

3.0E-05

G protein-coupled peptide receptor activity

3.0E-05

Postsynaptic neurotransmitter receptor activity

7.9E-05

Peptide receptor activity

7.9E-05

Ligand-gated ion channel activity

2.2E-04

Neuropeptide receptor activity

2.5E-04

Transmitter-gated ion channel activity

2.9E-04

Table 2. Up-represented terms in macrophages from aneurysm lesions at the growth phase

compared with in vitro-differentiated M1 from Biological Process and the top 15 list from

Molecular Function in gene ontology analysis.

Biological Process

P value

Sensory perception of taste

6.3E-04

Response to pheromone

3.4E-02

Neuropeptide signaling pathway

3.4E-02

Serotonin receptor signaling pathway

1.8E-01

Molecular Function

P value

Odorant binding

1.3E-33

Pheromone receptor activity

7.6E-09

Gated channel activity

4.9E-07

Neurotransmitter receptor activity

8.6E-07

Channel activity

6.4E-06

Passive transmembrane transporter activity

6.4E-06

Ion channel activity

9.6E-06

G protein-coupled amine receptor activity

1.4E-05

Hormone activity

1.9E-05

G protein-coupled peptide receptor activity

3.7E-05

Potassium channel activity

7.7E-05

Ligand-gated ion channel activity

7.7E-05

Postsynaptic neurotransmitter receptor activity

8.2E-05

Peptide receptor activity

9.3E-05

Transmitter-gated ion channel activity

1.1E-04

...