AGEN-D-14-00089 R1
Supplementary Material
Supplementary Figure Legends
Suppl. Fig. 1 Albumin-alginate microparticles for sequential slow-release of VEGF-A, FGF-2, and HGF
In vitro growth factor release rate evaluated every other day for 6 weeks for VEGF-A (A), FGF-2 (B), and HGF (C). Amounts are given as ng growth factor released per day per mg of albumin-alginate microparticles. n= 3-6 samples/group.
Suppl. Fig. 2 Vascular density and macrophage infiltration in plugs stimulated with VEGF-A or F+H
Examples of vessel density (A: CD31, green, 20x) and macrophage infiltration (B: F4/80+, green, MRC-1, red, 20x) at 3 week post-implantation of matrigels containing empty microparticles (control), or microparticles containing VEGF-A (VEGF) or the combination of FGF-2 and HGF (F+H). Scale bar = 50 µm; Arrows point to rare double positive M2-like macrophages in control and VEGF-stimulated plugs.
Suppl. Fig. 3 Circulating levels of monocyte subpopulations
Flow cytometric analyses of circulating levels of M1 or M2-like monocytes during angiogenic stimulation in mouse matrigel model. Mononuclear cells were identified among CD45+ circulating cells (A, gate R1, F4-80+ CD45+), and further analyzed for high or low Ly6C expression (B, gate R2, F4/80+ LY6Clow M2-like monocytes; gate R3, F4/80+ LY6Chigh M1-type monocytes). IgG controls, for both CD45 and F4-80, show specificity of gate R1 (C). Quantification of circulating monocyte levels in control and F+H-stimulated mice at 3 week post-implantation, results given as % of CD45+ circulating cells (D). n=3-5 mice/group.
Suppl. Fig. 4 Expression analyses of fibroblast and blood vessel markers in matrigel plugs
Relative expression levels of the fibroblast marker FSP-1 (A) and the vascular endothelial marker CD31 (B) in matrigel plugs injected with empty microparticles (control), or microparticles containing FGF-2, HGF, or the combination F+H. Whereas the growth factor therapies reduced the level of fibroblast infiltration, the ingrowth of vessels was increased at 3 weeks following implantation. One-way ANOVA comparisons vs control: *, p<0.05; **, p<0.01; ***, p<0.001. n= 7 mice/group.
Suppl. Fig. 5 Blockage of VEGFR2 signaling does not alter macrophage infiltration but abolishes the link between macrophages and vessel density
Quantification F4/80+ macrophage infiltration in matrigel plugs evaluated at 3 weeks post-implantation in the absence or presence of DC101 (A). Absence of correlation between macrophage infiltration and vessel density in F+H stimulated matrigel plugs of animals treated with DC101 (B). Improved correlation between macrophage infiltration and vessel density in VEGF stimulated plugs after treatment with DC101 (C). Control plugs (filled circles, n=5), F+H plugs (open circles, n=5), F+H plugs treated with DC101 (filled triangles, n=5). VEGF plugs (open circles, n=6), VEGF plugs treated with DC101 (filled triangles, n=6). One-way ANOVA comparisons vs control: *, p<0.05; **, p<0.01.
Supplementary Methods
In vitro growth factor release
Lyophilised albumin-alginate microparticles were loaded with growth factors by imbibition[13], using 1 µg growth factor per mg microparticle (approximately 35’000 particles), during a 1h incubation at +4°C. The growth factor-loaded microparticles were either used for therapy or evaluated for growth factor release as follows: microparticles were resuspended at 4 mg microparticles/mL in extracellular fluid mimetic release buffer (EFM-RB; 5 mM KCl, 125 mM NaCl, 20 mM Hepes, 1.5 mM MgCl2, 1.5 mM CaCl2, pH 7.4) and incubated under continuous rotation (6 rpm) for 40 days at 37°C. Every other day the tubes were centrifuged (300g, 8 min) to pellet the microparticles. A sample of the supernatant was collected and stored at -80°C. The initial volume in the test tube was restored by addition of fresh EFM-RB to simulate unlimited diffusion conditions. The growth factor release was quantified by ELISA according to the manufacturer’s instructions (HGF and VEGF-A, RD systems; FGF-2, Invitrogen). Data are presented as mean amount (ng) of growth factor released per day per mg of microparticles (n=3).
RT-PCR
Matrigel plugs were rapidly excised and placed in RNAlater buffer and snap-frozen for storage prior to mechanical homogenization. RNA was extracted using a Qiagen RNA isolation kit (Qiagen Inc.). The quantity and quality of RNA was assessed with NanoDrop™ ND 1000 Spectrophotometer (NanoDrop Technologies). Reverse transcriptase (RT) was performed during 1h at 37°C using 0.5 µg total RNA in the presence of 1.25 µg hexameric random probes (oligo pd(N)6, 100 pmol/µl), 10 mM dNTP, 20 U RNAse out, and 100 U reverse transcriptase (Moloney Murine Leukemia Virus Reverse Transcriptase, Life Technologies). Real-time PCR was performed on a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) using a commercially available mix containing Taq DNA polymerase, SYBR-Green I, and MgCl2 (FastStart DNA Master SYBR Green I kit; Roche). The following primers were used:
Target gene: / sense / antisensemurine FSP-1 / 5’-TCA GGC AAA GAG GGT GAC AAG-3’ / 5’-AGG CAG CTC CCT GGT CAG T -3’
murine CD31 / 5’- GCG CAG GAC CAC CTG TTA GT-3’ / 5’-CCT GCA ATT TGA ATC CGG AC -3’
murine 18S / 5’-GTG GAG CGA TTT GTC TGG TT-3’ / 5’-CGC TGA GCC AGT CAG TGT AG-3’
For each condition 3-6 tissue samples were analyzed and for each sample 0.5µg of RNA was reverse transcribed in duplicate. Data were analyzed using the Light Cycler software and reported as expression level normalized to 18S.
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