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Blood, Vol. 113, Issue 1, 233-243, January 1, 2009
Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCβ2 axis
Blood Maeng et al.
113: 233
Supplemental materials for: Maeng et al
Files in this Data Supplement:
- Figure S6. IGF1 had no significant effect on the adhesion of EPCs (JPG, 75 KB)
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EPCs were treated with IGF1 (1, 10, and 100 ng/ml). After 30 min, adhesion was quantified by counting the cells that attached to the fibronectin-coated matrix with optical microscopy at 200× magnification.
- Figure S7. IGF2 enhances adhesion CD34+ and CD34− EPCs (JPG, 24.4 KB)
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(A) and (B) CD34+ and CD34− EPCs were preincubated for 30 min with or without 5 mM M6P and stimulated with 50 ng/ml IGF2. After 30 min, adhesion was quantified by counting the cells that attached to the fibronectin-coated matrix with optical microscopy at 200× magnification.
- Figure S8. IGF2-induced chemotaxis of EPCs is inhibited by G(i) inactivator PTX in dose-dependent manner (JPG, 29 KB)
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EPCs were preincubated for 6h with or without PTX (1, 10, 25, and 50 ng/ml) and stimulated with 50 ng/ml IGF2. After 24 h, chemotaxis was quantified by counting the cells that migrated to the lower side of the filter with optical microscopy at 200× magnification. Data are the mean ± S.E. **, p < 0.01 vs. IGF2 alone.
- Figure S9. IGF2 enhances invasion of EPCs through MMP9 upregulation (JPG, 100 KB)
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(A) EPCs were preincubated for 6 h with or without 50 ng/ml PTX or preincubated for 30 min with or without 5 µM U73122, 5 µM U73343 or 5 µM BAPTA-AM, and then stimulated with 50 ng/ml IGF2. After 24 h, invasion was quantified by counting the invaded cells. (B) Total mRNA was isolated from EPCs treated with IGF2 (50 ng/ml) or left untreated, and MMP or TIMP gene expression was assessed by RT-PCR. Data are the mean ± S.E. **, p < 0.01 vs. IGF2 alone.
- Figure S10. IGF2 increases intracellular Ca2+ mobilization (JPG, 65.7 KB)
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(A) EPCs were preincubated for 6 h with or without 50 ng/ml PTX or preincubated for 30 min with or without 5 mM M6P or 5 µM BAPTA-AM. Cells were stimulated with 50 ng/ml IGF2 and intracellular Ca2+ levels were measured. (B) EPCs were transfected with non-silencing (siCont) or PLCβ2 siRNA (siPLCβ2). EPCs then were stimulated with 50 ng/ml IGF2 and intracellular Ca2+ levels were measured.
- Figure S11. Incorporated GFP+ mBMMNCs in neovasularture have endothelial properties (JPG, 82.8 KB)
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(A) Immunofluorescence on matrigel sections showing the identity of incorporated mature BM-derived GFP+ CD31+, GFP+ VE-cadherin+, GFP+ vWF+ -coexpressing cell into vessels (arrowheads). (B) EGFP CD34+ mouse BMMNCs were isolated using the MACS system and then cultured for seven days. (C) C57BL/6 mice were injected with 0.6 ml Matrigel with or without IGF2 (200 ng) (n = 7 per group). After 1 day, CD34+ mBMMNCs from EGFP transgenic mice (5 × 105/mouse) were infused into the tail vein. At 6 days after infusion, mice were killed and Matrigel plugs were excised. To isolate endothelial cells from matrigel, excised matrigel is minced and digested with collagenase. After blood cells are removed by a single sucrose step gradient centrifugation, endothelial cells are isolated using a magnetic cell sorting (MACS) system using an anti-CD31 antibody. After subculture for 3 day, EC purity was confirmed, the cells were stained with anti–VE-cadherin.
- Figure S12. IGF2 increases in vivo homing of mBMMNC in the retinas and ears of mice (JPG, 73.2 KB)
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(A) Mouse IGF2 (250 ng in 10 µl) was subcutaneously administrated in the right ear and vehicle was injected to the left ear of athymic nude mice (n=6 per group). Imaging assessment of the GFP-positive cells accumulated in IGF2-injected area was performed at 1 h after administration of CD34+ mBMMNCs isolated from EGFP transgenic mice (5 × 105/mouse). The representative images were shown. The IGF2-treated area of the mouse ear showed the increased recruitment of GFP+-mBMMNCs (arrowhead) compared with the vehicle-treated ear (scale bar = 100 µm). (B) The mBMMNCs staying in fields of views (1040 × 1376 µm2) were counted and summarized. (C) Athymic nude mice (n=8 per group) were injected with 200 ng mouse IGF2 into the vitreous of one eye and with vehicle in the contralateral eye. After 30 min, CD34+ mBMMNCs isolated from EGFP transgenic mice (5 × 105/mouse) were injected into the tail vein. After indicated times, retinal vasculature was stained with lectin (BS-1) (red) and retinas were flat-mounted onto glass slides and viewed using fluorescence microscopy. (D) Quantitative assessment of GFP-positive mBMMNCs. **, p < 0.01 vs. each mouse group.
- Figure S13. Proposed model for roles and signaling mechanisms of the IGF2/IGF2R system in EPC homing (JPG, 33.9 KB)
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- Figure S14. IGF2 has no effects on the expression of other homing related receptors or their ligands (JPG, 36.8 KB)
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Total mRNA was isolated from EPCs treated with IGF2 (50 ng/ml) or left untreated, and gene expression was assessed by RT-PCR.
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