Additional file 1 of Immunoregulatory and neutrophil-like monocyte subsets with distinct single-cell transcriptomic signatures emerge following brain injury

Additional file 1: Figure S1. Dil-Liposome localizes in white blood cells, and is distributed in Iba1 + cells of liver and spleen, but not the brain. (A-B) Representative confocal images for brain cortex showing Dil-LPM (red) inside blood vessels (CD31 + , white) and not in microglia (Iba-1 + , green) or brain parenchyma. (E–G) Dil-LPM is not detected in the brain of naïve mice at 1 h (F) or 24 h after injection (G). (H) Representative confocal images showing Dil-LPM (red) staining in Iba1 + (green) cells in kidney, (I) liver parenchyma (J) and spleen. n = 5/group, *P < 0.05; **P < 0.01; ****P < 0.0001. One-way ANOVA with Bonferroni post hoc

mRNA sequence profile of WT and KO ECs.

<p>(A) Genome-wide differential expression analysis via RNA-sequencing indicates KO ECs differening gene expressions that predominantly effect the extracellular matrix, immune response, wound healing, cell proliferation, cell adhesion and vascular development. (B—E) Overall ontological analysis of both upregulated and downregulated genes between WT and KO ECs. (F) Differential expression of seven genes significant in vascular development was further assessed using qRT-PCR which confirms an increase in Ang-1, MCP-1, and MMP2 in KO RNA transcripts using PCR. *P<0.05; **P<0.01; represented as mean ± SEM.</p

EphA4 EC-specific ablation increases collateral formation in the post-natal murine brain.

<p>(A) KO mice have increased total collaterals in P1, P7, P21 and adult mice. (B) average tortuosity is increased in all KO groups except adult. (C) KO mice have more MCA—ACA collaterals but there is no significant difference in average diameter of collaterals in the mice. (E—H) VP of P1 and adult WT and KO brains. *P<0.05; ***P<0.001; n = 5–9 per group; represented as mean ± SEM.</p

Plantar reperfusion and collateral remodeling post- hindlimb ischemia.

<p>(A) Laser Doppler images of blood flow pre and post ischemia in WT and KO mice. (B) Analysis shows significant increase in plantar blood flow perfusion at 7d post hindlimb ischemia or femoral artery ligation (FAL) in KO mice compared to WT. (C) KO mice also have a significant reduction in toe necrosis compared to WT mice at 7 days post-FAL. (D) The number of CD31-positive arterioles were are in increased in serial sections of both un-injuured and injured adductor muscles in the absence of EC-specific EphA4. (E) Average arteriole diameter in the injured aductor muscle is increased in KO mice compared to WT and KO un-injured tissue at 7d post-FAL. (F-I) Representative high magnification (x20) images from un-injured and injured adductor muscles at 7d post-FAL using CD31 immuno-staining and -fluorescence. Scale bar = 100μm. (J-M) Brightfield images of WT (J and K) and KO (L and M) adductor muscles at 7d post-FAL. *P<0.05; **P<0.01; n = 5-8/group; represented as mean ± SEM. FA = Femoral artery.</p

Vessel Painting and co-labeling of post-natal murine pial vessels.

<p>(A) Inverted VP image of a mouse brain showing selective labeling of the cerebral arteriole network using vessel painting. (B) Identification of pial collaterals using vessel painting (VP, red) and double labeling of with anti-SMA shows, VP does not label SMCs. (C) Confocal image showing cross section a vessel painted pial arteriole, immunotained for SMA, confirming that VP does not label SMCs. (D) Quantification of EphA4 on pial collaterals, represented as mean fluorescence intensity. (E—M) Double labeling of psot-natal day (P)1, P7 and P21 brains using VP (red) and anti-EphA4 (green). EphA4 is expressed on VP-labeled collaterals at P1, P7 and minimally at P21. *P<0.05; n = 5 per group; represented as mean ± SEM.</p

EphA4 EC-specific ablation increases EC proliferation, migration and tube formation.

<p>Panel shows WT (A, A1) and KO (B, B1) BrdU analysis of ECs stained with DAPI (blue) and BrdU (red). (C) KO ECs have significantly increased BrdU incorporation compared to WT ECs. WT (D, D1) and KO (E, E1) EC scratch. (F) KO have greater migration rate 24 hours post scratch compared to WT EC. (G, G1) WT and (H, H1) KO ECs were seeded on growth factor-reduced matrigel to form tubes. (I) Vascular index (number of tubes x tube length) is enhanced in KO ECs compared to WT. ***P<0.001; n = 16–20 wells/group; represented as mean ± SEM.</p

PI3K inhibition attenuates endothelial cell functions following EphA4 ablation.

<p>(A) Western blot analysis indicates increased p-Akt expression in KO ECs compared to WT ECs. Expression is reduced in KO ECs after LY294002 treatment. (B) Densitometric analysis shows a three-fold increase of p-Akt in KO ECs. KO treated with LY294002 have identical p-Akt expression as WT vehicle control. (C—E) In addition treating KO ECs with LY294002 significantly reduced BrdU incorporation, migration and vascular index, respectively. ***P<0.001; n = 16–20 wells/group; represented as mean ± SEM.</p

Phenotype and genotype of littermates from EphA4f/f and Tie2::Cre breeding paradigm.

<p>(A—F) Reporter labeling of Tie2::Cre is specific to blood vessels. Panel F further confirms eGFP expression specifically to blood vessels. (G) Genotyping using PCR shows a flox primers of about 290bp and a bottom Cre band. Lane 1 and 2: EphA4^f/f/Tie2::Cre (KO); Lane 3 and 4: EphA4^f/f (WT). Vessel painting of surface pial arterioles of WT (H) show increased numbers in KO (I) mice. Panel J and K are WT and KO endothelial cells cultured from brain endothelial cell progenitor cells. ECs are labeled with CD31 (green) and DAPI (blue). (L) ECs from KO mice do not express EphA4 RNA transcripts.</p