Article Figures & Data
Figures
- Figure 1.
rFKBPL protein inhibits angiogenesis in vitro and ex vivo. A, transient transfection of an FKBPL cDNA construct inhibits migration of wounded HMEC-1 monolayers compared with empty vector controls. Representative images of wounded monolayers and overexpression of FKBPL following transfection. The histogram shows the wound size relative to wound size at time = 0 h ± SEM; n = 3. Significance was determined by ANOVA. B, inhibition of HMEC-1 wound closure (compared with time-matched control) after exposure to a range of concentrations of rFKBPL. Data points show means ± SEM; n = 3. C, inhibition of HMEC-1 tubule formation in Matrigel following exposure to increasing concentrations of rFKBPL; data are corrected to a sham-treated control. Data points are means ± SEM; n = 3. D, microvessel sprouting from rat aortic rings incubated with increasing concentrations of rFKBPL (left). Quantitative determination of vessel length and number of vessels after 7 days compared with time-matched controls (right). Data points are means ± SEM; n = 3.
- Figure 2.
FKBPL inhibits angiogenesis in vivo and prevents the growth of DU145 human tumor xenografts. A, rFKBPL (5 ng injected directly into the sponge on alternate days) inhibited β-FGF (10 ng)–induced angiogenesis in C57 black mice. About 14 days after implant, there was a marked decrease in vessel density and cellular ingrowth in rFKBPL-treated sponges (left; arrows indicate vessels containing bright eosin-stained erythrocytes). Graph shows quantification of microvessel density in β-FGF alone or β-FGF + rFKBPL-injected sponges. Each symbol represents the average number of vessels per 40× field, with 10 fields counted blindly in 5 sponges; n = 5 mice/sponges per treatment group. B, immunohistochemistry showing FKBPL expression in DU145 tumors grown in SCID mice after injection with a cDNA construct expressing FKBPL. C, DU145 tumors grown in SCID mice were intratumorally injected twice weekly for the duration of the experiment with 25 μg of a cDNA construct expressing either FKBPL or endostatin (as a positive control), or pcDNA3.1 empty vector (as a negative control). Graph shows tumor volume over time ± SEM; n = 4–7 mice per condition. D, Kaplan–Meier survival curves; *, significance was determined by the log-rank test.
- Figure 3.
The active domain within FKBPL resides between aa-34–58 and a 24mer-based peptide, AD-01, spanning this domain inhibits angiogenesis and is more potent than rFKBPL. A, wound size of HMEC-1 monolayers 7 hours after transfection with truncated DNA constructs; n = 3. B, inhibition of migration (left; compared with time-matched controls) and tubule formation (right; compared with time-matched controls) of HMEC-1 cells after treatment with AD-01/AL-57 peptides across a range of concentrations. Data points are means ± SEM; n = 3. C, inhibition of microvessel sprouting from rat aortic rings (compared with time-matched controls ± SEM) incubated for 7 days with a range of concentrations of AD-01/AL-57; n = 3. D, AD-01 inhibited β-FGF–induced angiogenesis in the sponge assay in vivo. Microvessel densities in implanted sponges treated with in β-FGF alone (10 ng) or β-FGF + AD-01 (0.35 μg or 0.11 ng). Each symbol represents the average number of vessels per 40× field, with 10 fields counted blindly in each sponge; n = 5 mice/sponges for β-FGF alone; n = 3 mice/sponges for AD-01 treatment.
- Figure 4.
Systemic delivery of AD-01 inhibits blood vessel development and tumor growth. A, growth curves for DU145 xenografts or MDA-231. B, with or without daily intraperitoneal injection of a range of doses of AD-01. Data points are means ± SEM; n = 5–8 mice per treatment group and Kaplan–Meier survival curves with time-to-tumor tripling volume as the end point for survival; *, significance was determined by the log-rank test. C, intravital images (compressed Z-stacks) of DU145 xenografts showing blood vessels at 7 and 14 days after the start of treatment with AD-01 (0.3 mg/kg/d) or PBS control intraperitoneally (left). Number of vessel branch points or average vessel diameter (μm) at 7 and 14 days after initiating treatment with AD-01 (0.3 mg/kg/d i.p.) or PBS ± SEM; n = 5 mice per treatment group, 4 fields of view per tumor and 30 vessels per field. Significance was determined by the 2-tailed t test.
- Figure 5.
Systemic delivery of AD-01 inhibits DU145 tumor growth in combination with docetaxel. A, fold change in tumor volume following treatment with AD-01 (0.3 and 0.003 mg/kg/d i.p.) in combination with docetaxel (20 mg/kg once every 15 days in 3 cycles) compared with PBS alone, AD-01 alone, and docetaxel alone controls ± SEM; n = 5–7 mice per treatment group. B, Kaplan–Meier survival curves with time-to-tumor doubling as the end point for survival; *, significance was determined by the log-rank test. C, hematoxylin and eosin staining of FFPE tissue sections taken from control animals and animals exposed to high-dose AD-01 (0.3 mg/kg/d) at the end of the experiment; no obvious toxicity was observed.
- Figure 6.
FKBPL is secreted and is dependent on CD44 for its antiangiogenic activity. A, immunoprecipiation (IP) Western blots using conditioned medium from HMEC-1, L132, and MDA-231 cells 24 hours after plating. FKBPL was immunoprecipiated using an anti-FKBPL antibody and then run on a Western blot and probed for FKBPL. Control medium (no cell exposure) or immunoprecipitation with an IgG control were used as negative controls. Whole cell lystates were used as positive controls. B, inhibition of migration of HMEC-1 compared with time-matched control 72 hours after transfection with nontargeted siRNA compared with CD44-targeted siRNA in untreated cells (top) and treated (10−9 mol/L AD-01; bottom) ± SEM; n = 5. C, migration of tumor cells after treatment with AD-01 (left) or rFKBPL (right) across a range of concentrations (± SEM; n = 3). D, representative gel showing inhibition of Rac activity in HMEC-1 cells after treatment with AD-01 (10−9 mol/L) for 10 min/60 min prior to serum/HA activation. CTRL, control.
Additional Files
Supplementary Data
Supplementary Figures S1-S10; Supplementary Table S1.
Files in this Data Supplement:
- Supplementary Data - Supplementary Figures S1-S10; Supplementary Table S1.
Volume 17, Issue 5
