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. 2021 Jun 8;12:3460. doi: 10.1038/s41467-021-23546-6

Fig. 2. Administration of VEGFC mRNA-LNPs induces local lymphatic growth in mice in vivo.

Fig. 2

a–b Analysis of lymphatic morphology in the ear of Prox1GFP lymphatic reporter mice injected with 1 µg of Poly(C) or VEGFC mRNA-LNPs. Representative images 22 days (a) and 60 days (b) after the ear treatment of 15 (a) and 5 (b) mice in each group are shown by whole-mount fluorescent stereo microscopy (upper panels; bars, 1000 µm (a) and 500 µm (b)) and Prox1-GFP signal and LYVE1 immunostaining of slides processed by paraffin-based histology are shown (lower panels; bars, 50 µm). Arrows indicate Prox1-GFP and LYVE1 double positive lymphatic vessels. c Assessment of the time-dependent effect of intradermal administration of 1 µg of Poly(C) or VEGFC mRNA-LNPs in the ear at days 5, 12, 17, 22, 35, and 60. Quantitative data for the length of lymphatic network, average lymphatic vessel diameter, and number of branching points per field of view are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears of 5–15 mice in each group (two-tailed, paired T-test, for lymphatic network length P = 0.0005 after 22 days for 15 mice and P = 0.0005 after 60 days for 10 mice; for average lymphatic vessel diameter P = 0.000005 after 22 days for 15 mice and P = 0.0002 after 60 days for 10 mice; for number of branching points P = 7.48 × 10−7 after 22 days for 15 mice and P = 0.0001 after 60 days for 10 mice). d Monitoring the dose-dependent effect of Poly(C) or VEGFC mRNA-LNPs (0.04, 0.2, and 1 µg) 20 days after intradermal treatment of the ear. Quantitative data for the length of lymphatic network, average lymphatic vessel diameter, and number of branching points per field of view are represented as mean and SEM from LNP complex-injected ear of 6–17 mice in each group. (two-tailed Wilcoxon signed-rank test for lymphatic network length P = 7.04 × 10−5 for 17 mice, two-tailed, paired T-test for average lymphatic vessel diameter P = 4.61 × 10−7 for 17 mice and two-tailed, paired T-test for number of branching points P = 6.84 × 10−7 for 17 mice when injected with 1 µg of Poly(C) or VEGFC mRNA-LNP). e Monitoring the effect of intradermal injection into ears of Prox1GFP mice with 1 µg of Poly(C) or VEGFC mRNA-LNPs shown by flow cytometry analysis. Quantitative data for GFP positive cell number are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears of 6 mice in each group, 22 days after mRNA-LNP injection (two-tailed, paired T-test, P = 0.0010 for 6 mice). f Monitoring the effect 1 µg of locally injected Poly(C) or VEGFC mRNA-LNPs on lymphatic growth in Prox1GFP mice in the diaphragm 22 days after intraperitoneal injection, in the lungs 22 days after intratracheal treatment, and in the musculus gastrocnemius 22 days after intramuscular injection. Quantitative data for number of the lymphatics are shown as mean and SEM of Poly(C) or VEGFC mRNA-LNP-injected organs of 3 mice in each group. Asterisks indicate P < 0.05 compared with control. (two-tailed, unpaired T-test, diaphragm: P = 0.0354 for 3 mice, lung: P = 0.0222 for 3 mice, two-tailed, paired T-test m. gastrocnemius: P = 0.0145 for 3 mice).