Apelin-VEGF-C mRNA delivery as therapeutic for the treatment ofsecondary lymphedema

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  Figure 1. Reduced APLN expression in human LD. (A) Lymphofluoroscopy of the upper limb LD shows dermal lymph backflow associated with hypervascularization of tortuous initial lymphatics (right panel) compared to normal arm (left panel). (B) Lymphoscintigraphy of woman who developed LD after breast cancer shows reduced but lasting lymphatic drainage and lymph node perfusion. (C) Immunodetection of the lymphatic networks in the LD skin shows dilated lymphatic vessels (scale bar: 50 μm) (n = 8). (D) Quantification of the lymphatic vessel diameters. Data represent mean ± SEM (n = 8). (E) Quantification of the dermis lymphatic density. Data represent mean ± SEM of three biological replicates (unpaired t test; *P < 0.05). (F) Masson’s trichome staining of the human lymphedematous skin shows strong fibrosis (scale bar: 50 μm) (n = 8). (G) Masson’s trichome staining of the human LD subcutaneous AT shows fibrosis (scale bar: 50 μm) (n = 8). (H) Quantitative RT-PCR analysis of the genes involved in fibrosis and VEGF-C maturation in dermolipectomy samples from patients with LD. Data represent mean ± SEM of three biological replicates (paired t test; P = O.O34. (I) Quantitative RT-PCR analysis of the…

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  Figure 2. LD increases in APLN-KO mice. (A) Schematic of the experimental design of secondary LD mice model. Quantification of proximal limb swelling 2 and 4 weeks after surgery on control limb, LD limb from APLN-KO mice and control littermates. Data represent mean ± SEM (n = 9) (two-way ANOVA). (B) Lymphangiography reveals dermal backflow (white arrows) and pathological remodeling of lymphatic vessel after LD in APLN-KO mice 4 weeks post surgery (scale bar: 1 mm). (C) Lyve1 immunodetection of the skin lymphangiogenesis in APLN-KO mice (scale bar: 50 μm). (D) Quantification of lymphangiogenesis in the skin from APLN-KO mice. Data represent mean ± SEM (n = 10–14 mice) (one-way ANOVA). (E) Masson’s trichrome staining of LD in APLN-KO mice (scale bar: 50 μm). (F) Quantification of dermis fibrosis in APLN-KO mice 4 weeks post surgery. Data represent mean ± SEM (n = 5) (one-way ANOVA). (G) SHG signal from deep, collagen-rich layer within dermis (scale bar: 50 μm). (H) Quantification of the relative collagen area. Data represent mean ± SEM (n = 7–8) (one-way ANOVA). Source data are available online for this figure.

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  Figure 3. APLN prevents secondary LD. (A) Schematic of the experimental design of secondary LD model in mice injected APLN lentivector (LV-APLN). Quantification of proximal limb swelling at 7 and 14 days after surgery on the control limb, LD limb (n = 17) or LD treated with APLN lentivector (n = 20). Data represent mean ± SEM (two-way ANOVA). (B) EIA dosage of circulating APLN in plasma of control (n = 5) or APLN-treated mice (n = 5). Data represent mean ± SEM (unpaired t test). (C) Lymphangiography reveals pathological remodeling of lymphatic vessels and dermal backflow in LD that is reversed by LV-APLN (n = 10) (scale bar: 1 mm). (D) Masson’s trichrome staining of the skin from mice with LD treated or not with APLN (scale bar: 50 μm). (E) Quantification of dermis thickness. Data represent mean ± SEM (n = 5) (two-way ANOVA). (F). EIA dosage of circulating VEGF-C in plasma of control (n = 5) or APLN-treated mice (n = 5). (G) SHG signal from deep, collagen-rich layer within dermis (scale bar: 50 μm). (H) Quantification of the relative collagen area. Data represent mean ± SEM (n = 6–8) (two-way ANOVA). (I) Lyve1 immunodetection of the skin lymphangiogenesis in APLN-treated mice (scale bar:…

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  Figure EV1. Effect of APLN vector on skin angiogenesis. (A) Skin section staining with Lyve1 (green) and APJ (red). Scale bar = 50 µm. (B) Skin section staining with vimentin (green) and APJ (red). Scale bar = 50 µm. (C) Skin section staining with CD68 (green) and APJ (red). Scale bar = 50 µm. (D) APLN mRNA expression in skin from ctrl and LD limb. Data represent mean ± SEM (n = 3) (*P < 0.05, unpaired t test). (E) Skin sections of control or LD limb from LV-APLN-treated mice were stained for CD31 (red). DNA was stained with DAPI. Scale bar = 100 µm. (F) Quantification of blood vessels per field according the limb and treatment of mice. Data represent mean ± SEM (n = 9 control and n = 8 APLN-treated mice) (*P < 0.05, **P < 0.01, one-way ANOVA). (G) Vascular permeability assay by Evans blue extravasation. Left ear intradermally injected with 2 µL of control lentivector, right ear injected with 2 µL of APLN lentivector. (H) Quantification of extravasated Evans blue dye. Data represent mean ± SEM (*P < 0.05, unpaired t test).

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  Figure 4. APLN controls lymphatic endothelial cell gene expression. (A) Bulk RNA sequencing in HDLEC treated with APLN-conditioned medium. Volcano plot showing log2FC (fold change) values calculated between control and APLN-treated HDLECs for 24 h. Red and blue dots: significantly (P value adjusted < 0.05) up- (log2FC > 0.5) and downregulated genes (log2FC < -0.5), respectively. (B) Heatmap of the top 30 significantly upregulated genes (P value adjusted < 0.05 and log2FC > 0.5). Expression levels are plotted as log10 normalized counts for each sample. Red represents higher FC; Dark blue represents lower FC. (C) Top significantly (FDR < 0.05) enriched Gene Ontology (GO) terms for biological processes of significantly upregulated genes after APLN treatment at 24 h time point. (D, E) qRT-PCR validation of CCBE1 (D) and E2F8 (E) in APLN-stimulated HDLEC. Data represent mean ± SEM (three independent replicates)(unpaired t test). (F) Schematic representation of chromatin immunoprecipitation (Chip) by E2F8. (G) Chip analysis of E2F8 on CCBE1 promoter. Data represent mean ± SEM (three independent replicates) (unpaired t test). (H) Chip analysis of E2F8 on E2F1 promoter. Data represent mean ± SEM…

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  Figure EV2. Gene expression in APLN-stimulated HDLEC. (A–C). RT-qPCR showing the expression of COL1A (A), FBN (B), and ADAMTS3 (C) in APLN-stimulated HDLEC. Data represent mean ± SEM (ns=non significant, unpaired t test).

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  Figure EV3. Stimulation of VEGFR3 phosphorylation by APLN. (A) Representatives phospho-VEGFR3/VEGFR3 and CCBE1 immunoblots of HDLEC treated with APLN +/− siRNA CCBE1. (B) Graphs represent quantification of CCBE1/GAPDH protein ratio from at least three independent experiments. Data represent mean ± SEM (n = 3 independent replicates) (*P < 0.05, two-way ANOVA). (C) Graphs represent quantification of VEGFR3/GAPDH protein ratio from at least three independent experiments. Data represent mean ± SEM (n = 3 independent replicates) (*P < 0.05, two-way ANOVA). (D) Graphs represent quantification of phospho/total protein ratio of VEGFR3 from at least three independent experiments. Data represent mean ± SEM (n = 3 independent replicates) (*P < 0.05, two-way ANOVA).

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  Figure 5. APLN plays a role in LD through Akt/eNOS activation in lymphatic endothelial cells. HDLEC were treated in vitro with conditioned media of NIH3T3 cells infected APLN lentivector. (A) Relative expression of APLN in NIH3T3 evaluated by RT-qPCR on NIH3T3 transduced by APLN lentivector. Data represent pool of Lv-transduced cells. (B) Expression of APLN in conditioned media evaluated by EIA. Data represent pool of Lv-transduced cells. (C) Representative phospho-AKT/AKT and phosphor-Erk/Erk immunoblots of HDLEC treated with FBS, conditioned medium containing VEGF-C or APLN. (D) Graphs represent quantification of phospho/total protein ratio of at least three independent experiments. All graphical data are mean ± SEM. *P < 0.05, two-way ANOVA. (E) Representative images of scratch wound healing assay on HDLEC stimulated by VEGF-C or APLN. (scale bar: 100 μm). (F) Quantification of migration. Data represent mean ± SEM (n = 9) (one-way ANOVA). (G) F-actin and VE-Cadherin immunostaining of HDLEC after APLN treatment reveals no effect on lymphatic endothelial monolayer junctions (n = 3) (scale bar: 25 μm). (H) Representatives phospho-eNOS/eNOS immunoblots of HDLEC treated with FBS, conditioned…

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  Figure 6. APLN-induced vasodilatation of lymphatic vessels is mediated by eNOS signaling. (A) Contractile activity of collecting lymphatic vessels in control mice (n = 7) treated with APLN lentivector (n = 8) and L-NAME (n = 4) was investigated by filming autonomous collecting vessel contraction in vivo (scale bar: 100 μm). (B, C) graphs represent the number of vessel contractions per film (90 s) and the dilatation of collecting lymphatic vessels (differences between maximum and minimum diameter). Data represent mean ± SEM (n = 8) (one-way ANOVA). (D) Schematic of the experimental design of secondary LD mice model. (E) Quantification of proximal limb swelling at 7 and 14 days after surgery on control limb, LD limb (n = 9) or LD treated with APLN lentivector (n = 10) followed or not by treatment with L-NAME (n = 5). Data represent mean ± SEM (two-way ANOVA). (F) Representative images of lymphangiography from mice treated with LV-APLN and L-NAME. (scale bar: 1 mm). (G) Skin sections were stained with Lyve1 (green) to assess the number of lymphatic capillaries in control (n = 9), LV-APLN (n = 9) or LV-APLN + L-NAME (n = 5) treated mice. (scale bar: 50 μm). (H, I) Graphs show the number of…

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  Figure EV4. Effect of L-NAME on dermis fibrosis. (A) Graph represent mean vessel width normalized to the minimum vessel width. (LV-APLN n = 8, Ctrl n = 7 and LV-APLN + L-NAME n = 4). (B) Fibrosis was evaluated by Masson’s trichrome staining of the skin in control mice (n = 9) treated with APLN lentivector (n = 9) and LV-APLN + L-NAME (n = 5). (scale bar: 50 μm). (C) graph display the quantification of dermis thickness. Data represent mean ± SEM (n = 9) (**P < 0.01, two-way ANOVA). (D) Number of total circulating white blood cells, lymphocytes, monocytes and granulocytes in blood from mice 2 weeks after Lentiflash-APLN intradermal injection. Data represent mean ± SEM (n = 5) (ns = non significant, unpaired t test). (E) Number of blood platelet cells from mice 2 weeks after Lentiflash-APLN intradermal injection. Data represent mean ± SEM (n = 5) (ns=non significant, unpaired t test). (F) Representative images of macrophages (F4/80) immunodetection at the site of injection in skin from mice injected with Lf (Lf Luc) or adjuvant (TSSM) (scale bar = 50 μm). (G) Quantification of the number of macrophages at the site of Lf injection or TSSM control. Data represent mean ± SEM (n = 7).