1. Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating S1P1/Gi/PLC/Ca2+ signaling pathways
by Chang Min Yoon, Bok Sil Hong, Hyung Geun Moon, Seyoung Lim, Pann-Ghill Suh, Yoon-Keun Kim, Chi-Bom Chae, and Yong Song Gho
Blood
Volume 112(4):
August 15, 2008
©2008 by American Society of Hematology

2. S1P induced migration and the formation of capillary-like tube structure of human lymphatic endothelial cells.
S1P induced migration and the formation of capillary-like tube structure of human lymphatic endothelial cells. (A) After 4 hours of incubation with S1P or VEGF-C, migrated HLECs were stained and counted in 3 random fields. (B) S1P or VEGF-C was added to serum-starved HLECs for 24 hours, followed by additional incubation for 12 hours with 0.5 μCi ( MBq) [3H]-thymidine in HE-SFM. Results are expressed as the percentage [3H]-thymidine incorporation of the control versus S1P- or VEGF-C–treated HLECs. (C) HLECs were laid on a 24-well, GFR Matrigel-coated plate and incubated with S1P or VEGF-C for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image. (D,E) Effect of S1P on the migration (D) and capillary-like tube formation (E) of HMVECs-dLy, lymphatic endothelial cells derived from dermal skins, was examined as described in panels A and C, respectively. (F) Cultured HLECs and HMVECs-dLy were fixed, and immunostained using antibodies against Prox-1 (green), LYVE-1 (red), and podoplanin (green). The nuclei were counterstained by Hoechst (blue). Scale bars represent 50 μm. Note that HLECs and HMVECs-dLy were not immunolabeled by isotype-matched control IgG antibodies (data not shown). All values are expressed as means (± SD). Data are representative of 3 independent experiments with similar results. * and ** indicate statistically significant differences (P < .05 and P < .01, respectively).
Chang Min Yoon et al. Blood 2008;112:
©2008 by American Society of Hematology

3. S1P promoted in vivo lymphangiogenesis.
S1P promoted in vivo lymphangiogenesis. Ten days after subcutaneous injection of Matrigel containing none, VEGF-C (1 μg), or S1P (0.4 μg), in C57BL/6 mice (5 mice per group), the Matrigel was removed, fixed, embedded in paraffin, sectioned at 4 μm, and immunostained. (A) Cross-sections of Matrigel were stained by H&E. Scale bars represent 50 μm. (B,C) Lymphatic vessels in Matrigel were immunostained for podoplanin (red). Representative photographs and the number of podoplanin-positive lymphatic vessels per field are shown in panels B and C, respectively. Scale bars represent 200 μm. (D) Lymphatic vessels in Matrigel were immunostained for Prox-1 (green) and podoplanin (red). Arrows show sprouting lymphatic vessels. Scale bars represent 50 μm. (E,F) Proliferating lymphatic endothelial cells in Matrigel were double immunostained for PH3 (green) and LYVE-1 (red). Representative photographs are shown in panel E. Scale bars represent 20 μm. The number of LYVE-1+ (□) or LYVE-1+/PH3+ (■) vessels per field were counted (F). (G) Uptake of injected FITC-dextran (2000 kDa) into newly formed lymphatic vessels was visualized using confocal microscope. Scale bars represent 100 μm. All values are expressed as means (± SEM). ** indicates statistically significant difference (P < .01).
Chang Min Yoon et al. Blood 2008;112:
©2008 by American Society of Hematology

4. S1P-induced in vitro lymphangiogenesis was mediated through the S1P1/Gi protein.
S1P-induced in vitro lymphangiogenesis was mediated through the S1P1/Gi protein. (A) Total RNA (100 ng) from HUVECs (□) or HLECs (■) was amplified using primers for S1P receptors and β-actin. For quantification, the targets were normalized to β-actin as an internal standard. (B) After a 2-hour preincubation with 100 ng/mL PTX, HLECs were treated with 100 ng/mL S1P and 100 ng/mL PTX for an additional 4 hours. The migrated HLECs were stained and counted in 3 random fields. (C) HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P and 100 ng/mL PTX for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image. All values are expressed as means (± SD). Data are representative of 3 independent experiments with similar results. NS and ** indicate no significant difference and a statistically significant difference (P < .01), respectively.
Chang Min Yoon et al. Blood 2008;112:
©2008 by American Society of Hematology

5. S1P-induced lymphangiogenesis was mediated through the S1P1.
S1P-induced lymphangiogenesis was mediated through the S1P1. (A) HLECs were infected with retroviruses carrying S1P1 and S1P3 shRNA expression vectors in pRS plasmid, after which stably transfected cells were obtained by selection with puromycin. pRS plasmid was used as control. After a 4-hour incubation with 100 ng/mL S1P, migrated HLECs were stained and counted in 3 random fields (□). HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image (■). (B,C) Effect of S1P1 antagonist or S1P1 control molecule on the S1P-induced migration (B) and capillary-like tube formation (C) of HLECs was examined as described in panel A. (D,E) Ten days after subcutaneous injection of Matrigel in C57BL/6 mice (5 mice per group), the Matrigel was removed, fixed, embedded in paraffin, sectioned at 4 μm, and immunostained using antibodies specific for Prox-1 (green) and podoplanin (red). (D) Representative photographs of untreated control mice, and mice treated with S1P (0.4 μg) in the absence or presence of S1P1 antagonist (100 μM) or S1P1 control molecule (100 μM). Scale bars represent 50 μm. (E) The number of Prox-1+/podoplanin+ lymphatic vessels per field was counted. All values are expressed as means plus or minus SD (A-C) and means plus or minus SEM (E). In panels A-C, data are representative of 3 independent experiments with similar results. ** indicates a statistically significant difference (P < .01).
Chang Min Yoon et al. Blood 2008;112:
©2008 by American Society of Hematology

6. S1P-induced in vitro lymphangiogenesis was mediated through the PLC/Ca2+ pathway.
S1P-induced in vitro lymphangiogenesis was mediated through the PLC/Ca2+ pathway. (A) After a 4-hour isncubation with 100 ng/mL S1P containing 10 μM LY294002, 1 mM L-NAME, 10 μM PD98059, 25 μM SB203580, 30 μM BAPTA-AM, or 5 μM U73122, the migrated HLECs were stained and counted in 3 random fields. (B) HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P containing 10 μM LY294002, 1 mM L-NAME, 10 μM PD98059, 25 μM SB203580, 30 μM BAPTA-AM, or 5 μM U73122 for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image. (C) HLECs were loaded with fura-2/AM for 30 minutes. The cells were resuspended in Ca2+-free Locke solution, transferred to a quartz cuvette, and exposed to 100 ng/mL S1P with 100 ng/mL PTX, 10 μM LY294002, 1 mM L-NAME, 10 μM PD98059, 25 μM SB203580, 30 μM BAPTA-AM, or 5 μM U Relative intracellular Ca2+ influx was calculated from the tracing. All values are expressed as means (± SD). Data are representative of 3 independent experiments with similar results. ** indicates a statistically significant difference (P < .01).
Chang Min Yoon et al. Blood 2008;112:
©2008 by American Society of Hematology

7. Plausible mechanism for S1P-mediated lymphangiogenesis.
Plausible mechanism for S1P-mediated lymphangiogenesis. Extracellular S1P, which is derived from tumor or inflammatory cells, binds to its receptor S1P1 and stimulates the activation of coupled Gi proteins. Dissociation of active heterotrimeric Gi proteins from activated S1P1 stimulates PLC, which causes the release of intracellular Ca2+, resulting in the stimulation of HLEC migration and differentiation. Extracellular S1P activates lymphatic endothelial cells to induce lymphangiogenesis, as well as vascular endothelial cells to induce angiogenesis, for tumor metastasis or an immune response.
Chang Min Yoon et al. Blood 2008;112:
©2008 by American Society of Hematology