Supplementary Table 1.
Expression levels of 162 angiogenesis-associated genes.
Supplementary Figure 1.
Graphic illustration of the magnitude of the fold change at two (A) and at 10 days (B)
in angiogenesis-associated gene expression in each of 162 angiogenesis-associated
genes after stroke (for the complete list of genes, please refer to Supplementary
Table 1). ANOVA on ranks revealed that IL-6 deficiency blunted the activation of the
angiogenesis gene network at two days after MCAo. Note that only a subset of genes
was upregulated in wildtype mice, and the genes that were downregulated in wildtype
mice tended to be downregulated less in IL-6-/- mice. Therefore, strictly speaking, the
knockout phenotype is one of ‘diminished response’ rather than of ‘diminished
upregulation’.
Supplementary Figure 2.
Validation of microarray results by quantitative RT-PCR. Abbreviations: Anxa2
(Annexin A2), Thbs1 (Thrombospondin 1), Cxcl4 (chemokine (C-X-C motif) ligand 4),
Dicer1
(Dcr-1
homolog),
C1galt1
(core
1
synthase,
glycoprotein-N-
acetylgalactosamine 3-beta-galactosyltransferase 1), Cx3cl1 [chemokine (C-X3-C
motif) ligand 1], Bai3 (brain-specific angiogenesis inhibitor 3), Kdr (VEGF receptor 2),
Nos3 (endothelial NO synthase). N=3-4 mice per group. *p<0.05 relative to sham,
#p<0.05 between genotypes within the same time point.
Supplementary Figure 3.
(A-F) Regulation of key angiogenesis-related genes at 28 days after 30 min MCAo.
Anxa2
(Annexin
A2),
Cxcl4
(chemokine
(C-X-C
motif)
ligand
4),
Thbs1
(Thrombospondin 1), Kdr (VEGF receptor 2), Nos3 (endothelial NO synthase). N=4-6
animals per group. *p<0.05 versus sham, #p<0.05 between genotypes.
Supplementary Figure 4.
(A-D) Regulation of adherens junction protein-encoding genes at 48 hours after 30
min MCAo. Abbreviations: Jam-1 (junctional adhesional molecule-1), ICAM1 (intercellular adhesion molecule 1), PECAM-1 (platelet endothelial cell adhesion molecule1), vascular endothelial (VE)-cadherin. N=3-4 animals per group. *p<0.05 versus
sham. #p<0.05 between genotypes.
Supplementary Figure 5.
Cerebral angioarchitecture and acute CBF are similar in wildtype and IL-6-/- mice. (A)
The development of left and right posterior communicating arteries (PcomAs) was
determined in India ink perfused brains as follows: 0, absent; 1, present but
hypoplastic; and 2, well formed. ACA: Anterior cerebral artery; BA: Basilar artery;
MCA: middle cerebral artery; P1: P1 segment of the posterior cerebral artery. MannWhitney Rank Sum Test. (B) Number of ACA-MCA anastomoses on the dorsal
hemispheric surface. Analysis of n=8 wildtype (16 hemispheres) and n=7 IL-6-/- mice
(14 hemispheres). Mann-Whitney Rank Sum Test. (C) Acute CBF was measured
within the ischemic striatum (ipsi) and corresponding area of contralateral (contra)
striatum during MCAo (30 minutes after onset of MCAo) using
14
C-iodoantipyrine
tissue equilibration technique. Analysis of n=6 wildtype and n=7 IL-6-/- mice.
Supplementary Figure 6.
Regulation of key angiogenesis-related genes at 48 hours after 60 min MCAo
(‘damaging ischemia model’). Anxa2 (Annexin A2), Cxcl4 (chemokine (C-X-C motif)
ligand 4), Thbs1 (Thrombospondin 1), Edn1 (Endothelin 1), Adamts1 (a disintegrinlike and metallopeptidase), Kdr (VEGF receptor 2), Nos3 (endothelial NO synthase).
N=3 animals per group. *p<0.05 versus sham.
Supplementary Figure 7.
Phenotypic characterization of newly generated cells at four weeks after mild brain
ischemia. (A, C) All animals received S-phase marker BrdU for seven consecutive
days beginning on the day of MCAo. Phenotypic analysis of BrdU+ cells for
expression of activated microglia marker Iba1, astrocyte marker GFAP or for
association with von Willebrand factor (vWF) immunoreactivity (BrdU in vessel sites)
using confocal laser-scanning microscopy (A). The number of newly generated (i.e.
BrdU+) cells in ischemic striatum was significantly reduced in IL-6-/- mice (C). (B, D)
GFAP is a marker of reactive astrocytes which is typically upregulated in and around
the ischemic lesion. GFAP mRNA expression was not reduced in IL-6-/- mice at any
of the time points investigated. *p<0.05 versus sham. N=3 animals per group.
Similarly, at 28 days after MCAo, GFAP protein expression did not differ appreciably
between genotypes. N=6-7 animals per group. Immunostaining demonstrates fewer
newly born macrophages/microglia ([Iba1+/BrdU+ cells], E) and reduced vessel
density as assessed by PECAM-1 immunoreactivity (F) in IL-6-/- mice. Scale bar (in
C) 100 μm, 105 μm (D), 32 μm (E) and 146 μm (F).