human dermal lymphatic endothelial cells (hlecs)  (Galectin Therapeutics)

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: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4/CXCL12 signaling promotes lymphangiogenesis in TSCC. a Cell proliferation of human lymphatic endothelial cells (HLECs) treated with CXCL12 (100 ng/ml) or vehicle control, assessed by CCK-8 assay. b Wound healing assay showing the effect of CXCL12 on HLEC migration. Representative images (left) and quantification (right). Scale bars, 100 μm. c Tube formation assay of HLECs treated with CXCL12 (100 ng/ml) or vehicle control. Representative images (left) and quantification (right). Scale bars, 100 μm. d Western blot analysis of lymphangiogenic factors (VEGF-C, VEGFR-3, and Prox1) in HLECs treated with CXCL12 (100 ng/ml) or vehicle control. Representative blots (left) and quantification (right). e Western blot analysis of PI3K, p-AKT, and AKT in HLECs treated with CXCL12 (100 ng/ml) or vehicle control. Representative blots (left) and quantification (right). f Tube formation assay of HLECs treated with CXCL12 (100 ng/ml), LY294002 (20 μM), or CXCL12 + LY294002. Representative images (left) and quantification (right). Scale bars, 100 μm. g Schematic representation of the co-culture system used to investigate the interaction between TSCC cells and HLECs. h Transwell migration assay of HLECs co-cultured with CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. Data are presented as mean ± SD from three independent experiments. ***p < 0.001 , ****p < 0.0001 (compared to Control); #### p < 0.0001 (compared to CXCL12)

Article Snippet: Human lymphatic endothelial cells (HLECs) were obtained from PromoCell (Heidelberg, Germany).

Techniques: Control, CCK-8 Assay, Wound Healing Assay, Migration, Tube Formation Assay, Western Blot, Co-Culture Assay, Transwell Migration Assay, Cell Culture, Expressing

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: Autocrine and Paracrine Effects of TSCC-Derived CXCL12 and Generalizability of CXCR4/CXCL12 Signaling. a Transwell migration assay of CAL-27 cells stimulated with their own conditioned medium (CM). b Transwell invasion assay of CAL-27 cells under the same conditions as ( a ). c Tube formation assay of Human Lymphatic Endothelial Cells (HLECs) stimulated with CAL-27 conditioned medium. d Transwell migration assay of HSC-3 TSCC cells. e Transwell invasion assay of HSC-3 cells under the same conditions as ( d ). f Western blot analysis of phosphorylated AKT (P-AKT) and total AKT in HSC-3 cells. g ELISA quantification of CXCL12 levels in serum-free medium (Control) and CAL-27 conditioned medium (CAL-27 CM) after 48 h of culture. Representative images are shown for A-E. Bar graphs represent quantification of migrated/invaded cells or relative length of tubes, presented as mean ± SD from three independent experiments. *** p < 0.001, **** p < 0.0001 (compared to control); ### p < 0.001, #### p < 0.0001 (compared to CM or CXCL12 group); &&& p < 0.001, &&&& p < 0.0001 (compared to CM + IgG group); ns = not significant

Article Snippet: Human lymphatic endothelial cells (HLECs) were obtained from PromoCell (Heidelberg, Germany).

Techniques: Derivative Assay, Transwell Migration Assay, Transwell Invasion Assay, Tube Formation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Control

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