SUPPLEMENTARY FIGURES
Supplementary Figure 1. Effect of psychosine and related lysolipids on endothelial cell
proliferation. MAE cells were seeded at 2,500 cells/cm2. After overnight incubation, cells were
incubated with vehicle or increasing concentrations of psychosine (1--D-galactosylsphingosine)
(), glucopsychosine (1--D-glucosylsphingosine) (), or N-acetyl-D-sphingosine () for 72 h.
Then, cells were trypsinized and counted in a Burker chamber. Data are the mean ± S.D. of 3
determinations.
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Supplementary Figure 2. (A) Confluent 1G11 cells were wounded with a 1.0-mm-wide rubber
policeman and incubated with vehicle or increasing concentrations of psychosine. At the indicated
time points after wounding, the percentage of cells at the edge of the wound showing membrane
ruffles were counted. Insets: representative images of 1G11 cell monolayers treated with vehicle
or 100 M psychosine photographed 3 h after wounding. Arrows point to membrane ruffles. Data
are the mean  S.D. of 3-4 determinations. (B) BAE cell monolayers grown on type I collagen gel
were incubated with FGF2 plus VEGF in the presence of increasing concentrations of psychosine.
After 24 h, cells invading the gel in a plane of focus beneath the cell monolayer surface were
quantified by computerized image analysis. Data are the mean  S.D. of 3-4 determinations. Insets:
representative images showing the numerous BAE cells invading the gel beneath the growth
factor-stimulated cell monolayer almost absent in 100 M psychosine-treated cells. (C) FGF2-TMAE cell aggregates were embedded in fibrin gel in the presence of increasing concentrations of
psychosine. After 48 h, sprouts were quantified by computerized image analysis. Inset:
representative image of endothelial cell sprouting from an untreated FGF2-T-MAE cell aggregate.
Data are the mean ± S.E.M. of 5 determinations.
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Supplementary Figure 3. Psychosine inhibits endothelial cell spreading. BAE cells were seeded on
FN-coated coverslips in the presence of vehicle (black symbols) or 50 M psychosine (open
symbols). (A) After 6 h, adherent cells were observed under a phase contrast inverted microscope
(a, c) and stained with rhodamine-phalloidin (b, d). Also, the percentage of FN-adherent cells that
showed morphologic spreading was calculated at the indicated time points after seeding (B). At
variance with cell spreading, psychosine had only a very limited effect on BAE cell adhesion to FN
(-10 % in respect to vehicle-treated cells at 360 min after seeding, C). Data are the mean  S.D. of 3
determinations.
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Supplementary Figure 4. GALC in murine endothelial cells. (A) Cell extracts (50 µg) of
immortalized endothelial cell lines (ECs) established from murine brain (MBE cells), dermis (SIE
cells), lung (1G11 cells) and aorta (MAE cells) were evaluated for GALC activity using a fluorometric
assay. In this assay, the GALC substrate LRh-6-GalCer is converted to LRh-6-Cer and the two
fluorescent compounds are separated by thin layer chromatography and visualized under an
ultraviolet lamp. Brain extracts from P36 wt and twi/twi mice were used as positive and negative
controls, respectively. (B) Confocal immunofluorescence analysis of GALC expression in the
microvascular endothelium of the brain of P36 wt mice. Frontal cortex sections were stained with
anti-GALC antibody (in green) and biotin-conjugated Bandeiraea Simplicifolia BSI-B4 lectin (in red)
and analysed under a confocal laser scanning microscope. Confocal immunofluorescence images
are representative of two independent experiments in which three individual mice were analyzed.
No signal was observed when the primary antibody was omitted (not shown). Scale bar, 10 μm.
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Supplementary Figure 5. Angiogenic growth factor expression in the brain of twitcher mice.
Brains from wild type (wt), heterozygous carrier (twi/+) and homozygous (twi/twi) mice were
analyzed at P12, P24 and P36 for the expression of FGF2, VEGF and angiopoietin-2 by quantitative
RT-PCR. Data, normalized for actin expression, are the mean ± SD of 7 animals per group. *,
P<0.01.
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Supplementary Figure 6. Morphologically altered microvessels in the brain of twitcher mice.
Cerebral cortex FVIII/vWF+ blood vessels in P36 twi/twi brain show a dilated morphology (B) when
compared to wt brain (A). Scale bar, 12.5 m.
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Supplementary Figure 7. Vessel permeability of visceral organs in twitcher mice. A) Formalin-fixed, paraffin-embedded organs (including liver,
kidney and lungs) from wild type (wt) and homozygous twitcher (twi/twi) mice at P39 were sectioned at a thickness of 2 µm, dewaxed, hydrated, and
stained with hematoxylin and eosin (H&E). (B, C) Vessels of P39 wt and twi/twi mice were biotinylated by intracardiac injection of sulfo-NHS-LCbiotin. Then liver, kidney and lungs were harvested and fixed overnight at 4°C in 4% paraformaldehyde and embedded in 6% agarose. Agaroseembedded samples were cut at a thickness of 100 μm using a vibratome, and free-floating sections were stained with either CD31 antibody to
analyze vessel density (B) or with Alexa Fluor 488-conjugated streptavidin to detect vessel permeability (C). Z-stack images from 100 µm sections
were acquired with an epifluorescence microscope equipped with ApoTome optical sectioning device. Sulfo-NHS-LC-biotin diffuses through the
endothelial barrier and accumulates in the extravascular environment in all organs examined. Scale bar, 100 μm.
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