Fig 2: Lymphatic function assessment in mice following a specific knockdown of LDLR in murine endothelial cells. Two weeks after the intraperitoneal injection of an adeno-associated virus type 1 (AAV1) containing a shRNA targeting LDLR, skin draining lymph nodes were harvested, digested and analyzed by flow cytometry to assess lymphatic endothelial cell (LEC)-specific knockdown of LDLR expression in mice. Membrane-bound LDLR expression was measured on (A) CD45-CD31+Podoplanin+ and (B) CD45+ cells. Fluorescence minus one (FMO) control was used for LDLR expression, as depicted by the dotted line in the first histogram. White histogram, shSCR and dark histogram, shLldlr. LDLR expression was also determine by immunoblotting in (C) aorta isolated from female mice and (D) in liver from wild-type and Pcsk9-/- male (full dot) and female (empty dot) mice. ShSCR in black and ShLdlr in grey. Total plasmatic cholesterol was measured following FPLC in each liproprotein subfraction in (E) wild-type and (F) Pcsk9-/- female (dotted lines) and male (solid lines) mice treated with shSCR (black lines) and shLdlr (grey lines). Lymphatic contraction capacity was assessed by fluorescent in vivo imaging in (G) wild-type and (H) Pcsk9-/- mice. Black histogram, ShSCR and grey histogram, ShLdlr. n=3-9. Statistics **p < 0,01.

Fig 3: LDLR regulation in human LEC. (A) Cell surface expression of LDLR was measured by flow cytometry after incubating human LEC with (ctl) or without FBS-containing media for 24 h. (B) Human LEC were incubated with Dil-LDL for 4 h and LDL uptake was measured by flow cytometry (dotted line, no LDL; grey line, Dil-LDL). (C) The percentage of LDL internalization compared to control as well as (D) cell surface expression of LDLR was then quantified by flow cytometry. Human LEC were incubated with exogenous human recombinant PCSK9 (6.5 µg/mL) for 16 h and LDLR protein expression was detected and quantified by (E) immunoblotting,(F) flow cytometry and (G) LDLR messenger RNA by qPCR (H) Immunofluorescence confirmed LDLR expression in human LEC. Representative scatterplot of red and green pixel intensities of cholera toxin subunit B (red) and anti-LDLR (green) in lymphatic endothelial cells and a reconstruction of colocalized voxels (scale bar, 20 µM). (I) Protein expression of VEGFR-3 was measured in human LEC by flow cytometry following a 16 h incubation with 6.5 µg/mL human recombinant PCSK9. n=3-9. Statistics: *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 4: Western blotting. Effect of D. odorifera leaves on the protein levels of SCAP, SREBP-2, HMGCR, and LDLR in the liver.

Fig 5: Human LEC-LDLR modulation at the mRNA level. Human LEC were transfected for 48 h with 25 nM of non-targeting siRNA (called siCtl) or siLDLR. The siRNA off-target effect of was verified by two siRNA sequences, herein called siLDLR2 and siLDLR3. Messenger RNA expression was assessed by qPCR in human LEC treated with either (A) siLDLR2 or (B) siLDLR3 or their respective control (siCtl). LDLR protein expression was measured by (C) immunoblotting and (D) flow cytometry. (E) LDLR, PROX-1, PDPN (podoplanin) and VEGFR-3 mRNA expression in human LEC was assessed by qPCR. (F) Representative scatterplot of red and green pixel intensities of cholera toxin (red) and anti-LDLR (green) in lymphatic endothelial cells and a reconstruction of colocalized voxels was performed for immunofluorescence experiments (scale bar, 20 µM). Protein expression of (G) VEGFR-3, (H) LYVE-1 and (I) podoplanin were measured by flow cytometry. (J) Percentage of cells in the S phase of the cell cycle. n = 3-10. Statistics: *p < 0.05, ****p < 0.0001.