Induction of pro-angiogenic signaling by a synthetic peptide derived from the second intracellular loop of S1P3 (EDG3)‏ by Tamar Licht, Lilia Tsirulnikov, Hadas Reuveni, Talia Yarnitzky, and Shmuel A. Ben-Sasson Blood Volume 102(6):2099-2107 September 15, 2003 ©2003 by American Society of Hematology

Screening of synthetic peptides derived from the second intracellular (i2) loops of S1P1 and S1P3. Screening of synthetic peptides derived from the second intracellular (i2) loops of S1P1 and S1P3. (A) Sequences of i2 loop–derived peptides and their activities in aortic ring assay. (B) Penetration into the cell. HUVECs were incubated with biotin-tagged KRX-725 for 10 and 30 minutes. Nonbiotinilated KRX-725 was used as control. Fixed and permeabilized cells were reacted with FITC-conjugated extravidin. Visualization was done using confocal microscope (axiovert M135; Zeiss). Micrographs were taken under × 63 objective. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

The effect of KRX-725 and S1P on aortic ring sprouting. The effect of KRX-725 and S1P on aortic ring sprouting. (A) Aortic rings from C57BL/6 mice embedded in collagen matrix were exposed to KRX-725 (10 and 20 μM), S1P (100 nM), and vehicle (0.1% DMSO) for 9 days. The rings were then fixed and stained with crystal violet (0.02%) to illustrate sprouting. Representative micrographs of each arm of the experiment are shown. Micrographs were taken under × 4 objective. (B) Statistical morphometric analysis of the sprout length and thickness of 4 repeats from panel A. Mean ± SEM is presented relative to the control rings (100%), treated with vehicle. * indicates statistical significance (P < .05, Wilcoxon test) of treatment versus control. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

KRX-725 and S1P induce Gi-dependent ERK phosphorylation in human endothelial and vascular smooth muscle cells. KRX-725 and S1P induce Gi-dependent ERK phosphorylation in human endothelial and vascular smooth muscle cells. (A) Expression of S1P3 in HA-VSMCs and HUVECs. Lysates of cells were subjected to Western blotting with anti-EDG3 NT antibody. (B) Levels of phosphorylated ERK1/2 (pERK) and ERK2 (ERK) in HA-VSMCs exposed to either S1P (1 μM, 10 minutes), the control peptide KRX-723 (20 μM, 3 hours), KRX-725 (20 μM, 3 hours), or vehicle (0.1% DMSO, 3 hours). (C) Effect of siRNAs directed against the sequence of S1P1 or S1P3 on ERK phosphorylation induced by KRX-725. HUVECs were transfected with S1P1, S1P3, and nonrelevant (NR) 21-nucleotide siRNA. ERK phosphorylation was detected following overnight starvation and KRX-725 stimulation (20 μM, 1 hour). S1P3 protein level was assessed in the same assay conditions. (D) Phospho-ERK1/2 and ERK1/2 levels in HUVECs exposed to vehicle (0.1% DMSO, 3 hours), S1P (1 μM, 10 minutes), VEGF (10 ng/mL, 10 minutes), or KRX-725 (20 μM, 3 hours). Samples were preincubated with PTX (500 ng/mL) 3 hours prior to addition of the stimulant. Cell lysates were subjected to Western blotting with antiphospho-ERK (pERK) and reprobed with anti-ERK2 (ERK) antibodies. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

KRX-725-induced internalization of S1P3. KRX-725-induced internalization of S1P3. HUVECs were grown overnight with 2% c-FCS and incubated with vehicle (0.1% DMSO), S1P (1 μM), and KRX-725 (20 μM) for 45 minutes. Cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and immunostained with anti-EDG1 and EDG3-NT antibodies, followed by Cy3- and FITC-labeled secondary antibodies, respectively. Visualization was done using confocal microscope using × 63 objective. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

Detailed structure of sprouts induced by KRX-725. Detailed structure of sprouts induced by KRX-725. (A) Sprouts induced by KRX-725 (20 μM) or VEGF (25 ng/mL) in Sprague-Dawley rat aortic rings. After 10 days, the rings were fixed with formaldehyde, stained with crystal violet (0.02%), and examined microscopically. Representative micrographs of 6 repeats are depicted. (B) H&E staining of cross-section (5 μm) of paraffin-blocked BALB/c aortic rings incubated with KRX-725 (20 μM, 7 days). Micrographs were taken under × 40 objective. L indicates lumen; AO, aortic ring. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

Presence of vascular smooth muscle and endothelial cells in aortic ring sprouts induced by KRX-725, S1P, and VEGF. (A) Involvement of vascular smooth muscle cells in sprouts. Presence of vascular smooth muscle and endothelial cells in aortic ring sprouts induced by KRX-725, S1P, and VEGF. (A) Involvement of vascular smooth muscle cells in sprouts. Aortic rings of BALB/c mice were incubated with vehicle (0.1% DMSO), KRX-725 (20 μM), S1P (200 nM), and VEGF (20 ng/mL) for 8 days. The rings were then fixed with paraformaldehyde and stained with FITC-conjugated alpha smooth muscle actin antibody (left column), followed by 0.02% crystal violet (right column). (B) Involvement of endothelial cells in sprouts. Aortic rings of FVB/N-TgN(TIE2GFP)287Sato mice were incubated with vehicle (0.1% DMSO), KRX-725 (20 μM), S1P (200 nM), and VEGF (20 ng/mL) for 8 days. Fluorescent micrographs were taken prior to fixation and staining. Fluorescence emanates from expression of green fluorescent protein (GFP) under endothelial-specific promoter (tie-2). Each micrograph is a representative of 9 repeats. Micrographs were taken under × 10 objective. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

Sprout formation induced by KRX-725 and S1P is mediated via the Gi-MEK-ERK pathway. Sprout formation induced by KRX-725 and S1P is mediated via the Gi-MEK-ERK pathway. (A) Aortic rings from C57BL/6 mice were maintained for 10 days with vehicle (0.1% DMSO), KRX-725 (20 μM), S1P (200 nM), and VEGF (10 ng/mL) in the presence or the absence of PTX (200 ng/mL). Representative micrographs of rings of each arm of the experiment are shown. (B) Morphometric analysis of sprout length of 4 rings for each group described in panel A; mean ± SEM is presented relative to the control rings (100%). * indicates statistical significance (P < .05, Wilcoxon test) of treatment without PTX versus treatment with PTX. (C) Aortic rings from BALB/c mice were maintained for 7 days with vehicle (0.1% DMSO), KRX-725 (20 μM), and S1P (200 nM) in the presence or the absence of the MEK inhibitor U0126 (10 μM). Representative micrographs of rings from 9 repeats of each arm of the experiment are shown. (D) Morphometric analysis of sprout length of 4 rings in each group described in panel C; mean ± SEM is presented relative to the control rings (100%). * indicates statistical significance (P < .05, Wilcoxon test) of treatment without U0126 versus treatment with U0126. Original magnification of all micrographs is × 4. Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

KRX-725 and S1P synergize with protein angiogenic growth factors in sprout formation. KRX-725 and S1P synergize with protein angiogenic growth factors in sprout formation. BALB/c aortic rings were cultured with vehicle (0.1% DMSO), VEGF (10 ng/mL), SCF (30 ng/mL), and bFGF (20 ng/mL) alone or in combination with S1P (200 nM) and KRX-725 (20 μM) for 10 days. The rings were then fixed, stained with 0.02% crystal violet, and examined microscopically. (A) Representative micrographs of rings of each arm of the experiment. Original magnification × 4. (B) Morphometric analysis of sprout length from 4 repeats; mean ± SEM is presented relative to the control rings (100%). * indicates statistical significance (P < .05, Wilcoxon test) of the combined treatments with KRX-725 or S1P versus the controls (vehicle or protein factors alone). Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology

Synergistic effect of KRX-725 and bFGF in the corneal pocket assay. Synergistic effect of KRX-725 and bFGF in the corneal pocket assay. (A) Hydron pellets containing bFGF (80 ng), KRX-725 (10 μg), or myristoylated control peptide (10 μg) were implanted into micropockets within the corneas of C57BL/6 mice. Blood vessel formation was assessed by stereoscopy. Angiogenic response after 8 days is presented (representative pictures from 4 repeats in each experimental group). Arrows indicate weakly visible vessels. (B) Quantitation of the response at days 4 and 8 after implantation, using the formula (vessel length) × (clock hour of vessel sprouting from limbus) × 2 π/(pellet distance from limbus). Tamar Licht et al. Blood 2003;102:2099-2107 ©2003 by American Society of Hematology