Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a TEM analysis of control and recombinant AIBP-treated hLECs. Arrows depict caveolae. b Quantification of caveolae in ( a ). n = 35 (control) and n = 36 (AIBP) cells. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. c TEM analysis of caveolae in ECs of the cardinal vein from control and apoa1bp2 −/− zebrafish. Arrows indicate closed caveolae and arrowheads show open caveolae. d Quantification of caveolae in cardinal vein ECs. n = 27 (control) and n = 29 ( apo1bp2 −/− ) cells. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. e Scheme illustration of 4 hydroxy-tamoxifen (4OHT)-induced ubiquitous mEos2-APOA1 expression. f Representative images of control and mEos2-APOA1 expressed embryos after 4OHT treatment. Embryos were imaged at 2 dpf. The white dashed line demarcates the control animals. The results are representative of 3 independent repeats. g Photoconversion and vascular circulation of mEos2-APOA1. The head regions of embryos were exposed to UV light for 1 min to induce photoconversion. h Analysis of mEos2-APOA1 secretion. The specified tail region was imaged, and the maximum RFP-to-GFP signal ratio within the ISV lumen was quantified. n = 8 intersegmental vessels from 2 embryos. Data are presented as mean ± SEM, analyzed using one-way ANOVA with Tukey’s post-hoc test. i Maxi-projection confocal images of Prox1 + cells in the apoa1bp −/− ; fli1a:egfp zebrafish with control (mEos2-APOA1) and mEos2-APOA1 overexpression (ubi:Gal4-ERT2+mEos2-APOA1) at 4 dpf and immunostained with GFP (green) and Prox1 (red) antibodies. Arrowheads show the Prox1 + LECs in TD. j Quantitative data of Prox1 + LEC in TD (7 somites). n = 10 ( mEos-APOA1 ) and n = 11 ( ubi:Ert2-Gal4; mEos-APOA1 ) embryos. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. CV cardinal vein. Scale bar: 400 nm in a and 500 nm in c ; 100 µm in ( f , g , i ). Source data are prov i ded as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Control, Recombinant, Expressing, Over Expression

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a − d Effect of MβCD on VEGFR3 phosphorylation. a hLECs were growth factor-starved, and treated with 10 mM MβCD for 5, 15, and 30 min, and the resulting cells were further stimulated with 100 ng/mL VEGFC. The resulting cells were lysed and blotted using CAV-1, VEGFR3, GAPDH antibodies. b Quantitative analysis of panel a. Mean ± SD, n = 3 repeats; two-way ANOVA with Dunnett’s post-hoc test. c , hLECs were treated as in panel a. and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. d Quantitative analysis of ( c ). n = 3 repeats. Data are presented as mean ± SD and were analyzed using one-way ANOVA with Tukey’s post-hoc test. e , f Effect of AIBP treatment on VEGFR3 distribution in caveolar fractions. e hLECs were incubated with either recombinant AIBP or vehicle control in EBM2 supplemented with 10% FBS for 2 h, and the cells were subjected to sucrose gradient ultracentrifugation. n = 3 repeats. The resulting fractions were collected for Western blot analysis as indicated. Tx treatment; cav: caveolar fraction; n.c non-caveolar fraction. f Quantitative data of ( e ). Mean ± SD; two-way ANOVA with Sidak’s post-hoc test. n = 3 repeats. g , h Effect of AIBP and HDL co-treatment on VEGFR3 signaling. g hLECs were growth factor-starved and treated with HDL, AIBP, or HDL and AIBP in combination, and further stimulated with VEGFC. The resulting cells were lysed and immunoblotted as indicated. h Quantitative data of ERK and AKT activation. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 repeats. Ctrl: control. i Maxi-projection confocal images of Prox1 + and pErk1/2 + cells in the apoa1bp −/− ; fli1a:egfp zebrafish at 36 hpf following immunostaining using GFP, pErk1/2, and Prox1 antibodies. Dorsal (DA) aorta and cardinal vein (CV) were imaged. Arrows show the Prox1 + LECs with pErk1/2 expression. j Quantitative data of pErk1/2 intensity in Prox1 + LECs. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. n = 146 (control) and n = 166 ( apoa1bp −/− ) cells. Scale bar: 50 µm. Source data are provided as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Incubation, Recombinant, Control, Activation Assay, Immunostaining, Expressing

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). The conserved amino acids are shown in blue. b Co-immunoprecipitation of endogenous VEGFR3 and CAV-1 in hLECs. Lysates from two 10 cm confluent plates of hLECs were combined, then equally divided for immunoprecipitation using VEGFR3 antibody or control protein A beads. The samples were subsequently immunoblotted for CAV-1 and VEGFR3. c , d VEGFR3 AAA loses its binding to CAV-1. c hLECs were transfected with control EGFP, VEGFR3-EGFP (R3), or VEGFR3 AAA -EGFP (R3 AAA ) using lentivirus-mediated gene transduction. After 72 hours, the resulting cells were lysed and immunoprecipitated with GFP antibody conjugated to agarose beads and immunoblotted using GFP and CAV-1 antibodies. d The input lysates were immunoblotted using GFP, CAV1, or GAPDH antibody as indicated. e Localization of VEGFR3 and VEGFR3 AAA in caveolae. hLECs were transduced with VEGFR3-APEX2 or VEGFR3 AAA -APEX2 Lenti-viral particles, and after 72 h, cells were fixed with 2.5% glutaraldehyde, stained using DAB substrate kit, and pelleted for TEM analysis. An enlarged view of a single caveola, highlighted with a white box, is shown in the top left corner of each image. f – h hLECs were transduced using lentivirus, and the resulting cells were growth factor starved and treated with 100 ng/mL VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. R3/R3 AAA -EGFP denotes detection using GFP antibody. Quantitative data of VEGFR3 activation ( g ), ERK activation ( i ), and AKT activation ( j ) were shown. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 independent repeats in g , i , j . Endg: endogenous. Scale bar: 400 nm. Source data are provided as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Binding Assay, Immunoprecipitation, Control, Transfection, Transduction, Staining, Activation Assay

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) Schematic diagram of transwell secretome experiment. ( B) TEER measurements of hLEC monolayers after 48 hours of treatment with dermal fibroblast secretomes with [NHDF(G)] and without growth factors [NHDF(B)]. ( C) Representative immunofluorescence images of VE-cadherin, claudin-5, and ZO-1 on hLECs following treatment with NHDF secretomes. (D) Quantification of VE-cadherin, claudin-5, and ZO-1 expression levels using ImageJ (FIJI). (E) Quantification of lymphatic markers LYVE-1 and PROX1 expression in hLEC monolayers treated with NHDF secretomes. (F) FITC–Dextran (4 kDa) transport assay across hLECs after NHDF secretome treatment. Data are presented as mean ± SEM from n = 6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the permeability (transport) assay ( n = 3, representative), statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Immunofluorescence, Expressing, Transport Assay, Two Tailed Test, Permeability

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) Schematic diagram of transwell co-culture experiment. (B ) Schematic diagram of cells seedings and co-culture. (C) TEER measurements of hLEC monolayers after 48 hours of co-culture with dermal fibroblast with [NHDF(G)] and without growth factors [NHDF(B)]. (D) Representative immunofluorescence images of VE-cadherin, and ZO-1 on hLECs following co-culture with NHDFs. (E) Quantification of VE-cadherin and ZO-1 expression levels using ImageJ (FIJI). (F) FITC–Dextran (4 kDa) transport assay across hLECs after co-culture with NHDFs. Data are presented as mean ± SEM from n = 6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the permeability (transport) assay ( n = 5, representative), statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Co-Culture Assay, Immunofluorescence, Expressing, Transport Assay, Two Tailed Test, Permeability

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) Quantitative analysis of ZO-1 distribution and organization in hLEC monolayers using JAnaP after 48 hours of co-culture with NHDFs. (B) Immunoblot analysis of total ZO-1 (220 kDa) and VE-cadherin (120 kDa) protein expression levels in hLECs following co-culture. (C) RT-qPCR analysis of mRNA expression levels of ZO-1 and VE-cadherin in hLECs under the same conditions. Data are presented as mean ± SEM from n > 2 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****).

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Co-Culture Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) TEER measurements of hLEC monolayers after 48 hours of treatment with NHLF secretomes or fibroblast media. (B) Representative immunofluorescence images of VE-cadherin, and ZO-1 on hLECs following treatment with NHLF secretome. (C) Quantification of VE-cadherin and ZO-1 expression levels using ImageJ (FIJI). (D) FITC–Dextran (4 kDa) transport assay across hLECs after treatment with 10, 25 and 50% NHLF secretome. Data are presented as mean ± SEM from n = 6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the transport assay ( n = 3, representative), statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Immunofluorescence, Expressing, Transport Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) TEER measurements of hLEC monolayers after 4 hours of thrombin treatment followed by 48 hours of co-culture with dermal fibroblast (NHDF(G) ( n = 3 ). (B) Total cell counts ( n = 6 ). (C) FITC–Dextran (4 kDa) transport assay across hLECs after 4 hours of thrombin stimulation and/or NHDF co-culture for 48 hours. (D) Representative immunofluorescence images of ZO-1 and VE-cadherin on hLECs with and without thrombin treatment. Data are presented as mean ± SEM from n = 3-6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the transport assay, statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Co-Culture Assay, Transport Assay, Immunofluorescence, Two Tailed Test

Journal: JCI Insight

Article Title: Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity

doi: 10.1172/jci.insight.136937

Figure Lengend Snippet: (A) Images of primary hLECs infected with GFP expressing M. tuberculosis for 2 to 72 hours. Over time, M. tuberculosis grows and forms large intracellular cords. Nuclei are stained with DAPI (blue) and F-actin is stained by rhodamine phalloidin (red). (B) 3D reconstruction of Z-stacks taken of an intracellular cord from (A). Various angles are shown to confirm that the cord is completely encapsulated within the host cell. (C) Measurement of the intracellular cords over time in hLECs (using the Feret diameter; see Supplemental Figure 1) showing that the cords elongated up to a maximum of 150 μm. The numbers of bacterial clusters analyzed were 418 (2 hours), 233 (24 hours), 814 (48 hours), and 618 (72 hours), obtained from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons tests: ***P < 0.001. (D) Image of A549 cells infected with M. tuberculosis-EGFP for 72 hours showing an intracellular cord looping around the nucleus. Nuclei are stained with DAPI (blue) and F-actin is stained with rhodamine phalloidin (red). (E) Intracellular cord formation after 72 hours was also observed in hLECs infected with representative strains from 3 other M. tuberculosis lineages: N0072 (lineage 1), N0145 (lineage 2), and N0024 (lineage 3). Images displayed in D and E are representative of at least 3 independent experiments. (F) Tissue section of a granuloma present in a human lymph stained for AFB. Zoomed region shows association of M. tuberculosis cords with cells (black boxes). Representative histological sections from human patients after lymph node tissue resection surgery were stained for PDPN, M. tuberculosis, and nuclei (DAPI). Scale bar: 1 mm. White boxes delimit the zoomed regions displayed on the right-hand side. Arrows indicate the presence of M. tuberculosis cords within PDPN+ cells. Scale bar: 20 μm. hLECs, human lymphatic endothelial cells; AFB, acid fast bacilli; PDPN, podoplanin.

Article Snippet: Cells Primary hLECs taken from inguinal lymph nodes (ScienCell Research Laboratories, catalog 2500) were cultured according to the manufacturer’s instructions up to passage 5 as described fully in ref. 19 .

Techniques: Infection, Expressing, Staining