lymphatic endothelial cells lecs  (Procell Inc)

Journal: Scientific Reports

Article Title: Exosomes derived from ADSCs suppress the fibrosis process of derma in secondary lymphedema

doi: 10.1038/s41598-025-34280-0

Figure Lengend Snippet: ADSC-Exs promote LECs proliferation, migration, and tube formation. ( A ) Representative images of Transwell invasion and migration assays, tube formation and KVA analysis between LECs treated with PBS (LEC + PBS) and ADSC-derived exosomes (LEC + Exo). Scale bar = 200 μm. ( B–C ) Cell viability of LECs measured at 24 h and 48 h after treatment. Group LEC + PBS was compared with Group LEC + Exo, with n = 3 for each group. Unpaired t tests and no multiple-comparison correction were applied. ( D ) Proliferation of LECs measured at 24 h and 48 h after treatment. Group LEC + PBS was compared with Group LEC + Exo, with n = 3 for each group. Unpaired t tests and no multiple-comparison correction were applied. ( E ) Transwell assays were used to determine the effect of ADSC-Exs on LECs invation and migration. Group LEC + PBS was compared with Group LEC + Exo, with n = 3 for each group. Two-way ANOVA and Sidak’s multiple comparisons test were applied. ( F–G ) Tube formation parameters quantified by KAV about the number of nodes, junctions and total tube length between the two groups. Group LEC + PBS was compared with Group LEC + Exo, with n = 3 for each group. Unpaired t tests and no multiple-comparison correction were applied. *P < 0.05, **P 0.01, ***P < 0.001. LECs: lymphatic endothelial cell; Exo: exosome.

Article Snippet: Lymphatic endothelial cells (LECs) were obtained from Procell Life Science & Technology Co., Ltd. (CP-H026, China) and cultured in endothelial cells medium (CM-H026, Procell, China).

Techniques: Migration, Derivative Assay, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: Mixed junctional state in capillary LV. (A and B) Whole mounts prepared from the upper 200 µm of the human dermis showing PDPN + LVs and the distribution of the junctional protein VE-cadherin (VE-cad). Representative images from n = 8 donors are shown. (A) Blind-ended lymphatic capillaries with oak leaf–shaped LECs (depicted by an asterisk) or more elongated LECs (depicted by an arrow) were observed in human dermis. (B) Images of LVs with oak leaf–shaped LECs joined by button junctions (depicted by an asterisk) or LVs with more elongated, zipper-like LECs (depicted by an arrow), as well as BVs joined with zipper junctions. Scale bars: 20 µm. (C) Whole mounts of human dermis (upper 200 µm) showing the presence of intracellular CCL21 in afferent LVs stained with anti-VE-cadherin or anti-PDPN antibodies. Representative images from n = 3 donors are shown. Scale bars: 20 µm. (D) GO term analysis of genes enriched in the cap2 LEC subset compared with all other LEC subsets. Selected terms for enriched GO biological and cellular processes are shown along with the −log 10 of the adjusted P value (list of marker genes for the enrichment analysis in ). The vertical line represents the adjusted P value set at an FDR of 0.05. (E) Volcano plot of DEGs between cap1 and cap2 LEC subsets. The horizontal line shows the log 2 fold change (FC) threshold set at 0.75. Vertical lines show the significance threshold for adjusted P values set at an FDR of 0.05. (F) Individual UMAP plots showing the expression of specific cap2 LEC genes, i.e., WWTR1 , ITGA9 , PTK2 , YAP1 , ITGAV , FBN1 . (G and H) Whole mounts prepared from the upper 200 µm of the human dermis showing PDPN + LVs expressing (G) the focal adhesion kinase (FAK), and (H) FAK and the chemokine CCL21. Representative images from n = 3 donors are shown (G and H). Scale bars: 20 µm. For the FAK images, a thin stack at the level of the LV was made to visualize FAK expression specifically in LECs. (I) Human dermal LECs were subjected to mechanical stretch (10% strain every 30 s for 18 h) with a bioreactor. Representative immunofluorescence images of control (static) human dermal LECs and human dermal LECs subjected to stretching are shown. VE-cadherin was used to visualize the cell boundary and subsequent elongation. Scale bar: 100 µm. (J) Aspect ratio of control LECs or stretched LECs was quantified and is represented as a box plot. Statistics were computed with the nonparametric Mann–Whitney test. Pooled data were derived from n = 3 independent replicates with 695 cells analyzed in total. ***P < 0.001. (K) RNA of control or stretched human dermal LECs was extracted, and RT-qPCR was performed. The fold change expression levels of CCL21 and LYVE1 between stretched and control samples are depicted. A fold change below one shows reduced gene expression under stretched conditions. Two independent experiments from n = 3 human dermal LEC donors, with four pooled technical replicates for each. Statistics were computed with paired Student’s t test. **P <0.01. (L) Violin plots showing differential expression of CCL21 and LYVE1 in the cap1 and cap2 clusters. Statistics: the P adjusted value is shown. ***P <0.001. FDR, false discovery rate.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: Staining, Marker, Expressing, Immunofluorescence, Control, MANN-WHITNEY, Derivative Assay, Quantitative RT-PCR, Gene Expression, Quantitative Proteomics

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: Valve population is composed of two subclusters with different gene expression. (A) Subclustering analysis of the valve cluster revealed two valve subclusters, corresponding to the LECs on the upstream sides of the valve leaflets, and LECs on the downstream sides of the valve leaflets. (B) Volcano plot of DEGs between the upstream valve LEC and downstream valve LEC clusters. The horizontal line shows the log 2 fold change (FC) threshold set at 0.75. Vertical lines show the significance threshold for adjusted P values set at an FDR of 0.05. (C) Individual UMAP plots showing the expression of DEGs in upstream valve LECs ( GJA1 , ITGA9 , NEO1 , CD24 ) and in downstream valve LECs ( GJA4 , CLDN11 , FOXC2 , ANGPT2 ). (D and E) Confocal analysis of valves present in the upper 200 µm of human dermis confirmed (D) the differential expression of FOXC2 and CD24 by LECs on different sides of the valve leaflet, and (E) the colocalization of CD24 and NEO1 by LECs on the same valve leaflet side. Representative images from n = 2–3 independent experiments (donors) are shown in D and E. Scale bars: 20 µm. (F) Schematic illustration of the structure of the valves and the positioning of the LECs within the valves, based on . (G) Schematic illustration of bulk RNA-seq of human dermal LECs subjected to laminar shear stress (4 dyn/cm 2 ), oscillatory-like shear stress (laminar shear stress at 4 dyn/cm 2 that reverses direction by 180° every 4 s), or static conditions for 48 h. The top 50 DEGs in the laminar and oscillatory conditions are shown in and . (G) Pearson correlation plot of the bulk RNA-seq described in E. (H) PCA plot of the samples described in G analyzed at three different conditions (static, oscillatory, and laminar). (I) Similarity of LEC clusters upstream and downstream of valves with the bulk RNA-seq of human dermal LECs subjected to static conditions; laminar or oscillatory flow was determined by the correlation of the top 100 DEGs of valve LEC clusters with the bulk RNA-seq dataset. FDR, false discovery rate.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: Gene Expression, Expressing, Quantitative Proteomics, RNA Sequencing, Shear

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: Bulk RNA-seq of human dermal LECs subjected to laminar or oscillatory shear stress. (A) Selected pathways from Enrichr analysis (GO terms) of DEGs upregulated in the LECs on the upstream sides of valve leaflets or upregulated in LECs on the downstream sides of valve leaflets (list of DEGs used for the enrichment analysis in ). The vertical line represents the adjusted P value set at an FDR of 0.05. (B) Flow cytometry analysis of CD31 and PDPN expression of the three human dermal LEC donors used for bulk RNA-seq. Cells were gated on single cells, followed by viable cells, and the expression histograms shown in this panel. (C) Volcano plots of significant DEGs when comparing laminar or oscillatory shear stress to static control. (D) Heatmap of expression levels of selected genes in the bulk RNA-seq study. FDR, false discovery rate.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: RNA Sequencing, Shear, Flow Cytometry, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: CD24 expression in murine and human dermal LECs in vitro and in vivo . (A) RNA expression in FPKM in three donors of human dermal LECs subjected to laminar (lam) shear stress, oscillatory (osc) shear stress, or static conditions (data from the bulk RNA-seq). (B) CD24 is not expressed at the protein level in in vitro –cultured human dermal LECs as assessed by flow cytometry. Representative flow cytometry plot of donor 431Z006.2. Donors 433Z032.3 and 439Z007.2 neither expressed CD24. (C) CD24 expression in mouse dermal imLECs. Representative flow cytometry plot of three experiments. (D and E) Whole mounts of Prox1- eGFP mouse ear showing expression of (D) CD24 in lymphatic valves visualized with Prox1 (high in valves) and (E) in combination with the cell adhesion molecule CD31. Representative images from five independent experiments. Scale bars: (D) 100 µm, (E) 25 µm. (F and G) Quantification of ITGA9 + valves was performed in 6-image Tilescans acquired in an ear skin area containing predominantly collectors (coll: CD31 + aSMA + ) or pre-collectors (precoll: CD31 + aSMA − ). (F) Representative image of a Tilescan (left) and of ITAGA9 + valves in CD31 + aSMA − pre-collecting (middle) and CD31 + aSMA + collecting vessels (right). Scale bars from left to right: 150, 100, 50 µm. (G) Quantifications of valves in the pre-collector or collector area of ear skin whole mounts from adult WT ( Cd24 +/+ ) and Cd24 −/− mice ( n = 8 WT and n = 7 Cd24 −/− ). (H and I) Lymphatic drainage assay: adult WT and Cd24 −/− mice were injected with a near-infrared dye conjugate intradermally in the ear skin. Clearance of the tracer was monitored over 24 h by IVIS imaging. (H and I) (H) Average dye clearance plots and (I) calculated half-lives in WT and Cd24 − /− mice (data from one experiment with four to five mice per group are shown). ns, not significant; FPKM, fragments per kilobase of transcript per million mapped reads.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: Expressing, In Vitro, In Vivo, RNA Expression, Shear, RNA Sequencing, Cell Culture, Flow Cytometry, Injection, Imaging

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: ( A ) Human embryonic slides corresponding with different embryonic developmental Carnegie stages (CS12, CS15, CS18, and CS22) were hybridized with fluorescence probes for KIF11 (white), PROX1 or PDPN (green), and FLT4 (red). Magnified regions marked with a square box in overview images contain the precardinal veins (PCVs), cardinal veins (CVs), and jugular lymphatic sacs (JLSs), and arrows point to regions of coexpression between KIF11 and lymphatic endothelial marker expression. HP, hepatic primordia. Scale bar: 200 μm. ( B ) ChIP-Seq analysis in cultured human dermal LECs identified an intronic region of KIF11 bound by FOXC2 and NFATC1 (top panel) and PROX1 binding 1.3 kb upstream of KIF11 promoter (bottom panel). ( C ) LECs were treated with siRNA PROX1 or siRNA FOXC2 , and KIF11 expression was evaluated by qPCR. Bars represent mean relative expression ± SEM from n = 3 experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Two-tailed unpaired Student’s t test.

Article Snippet: Human dermal LECs (HDLECs) (PromoCell, C-12217 or C-12216) were cultured and maintained in endothelial cell growth medium MV2 (PromoCell, C-22022) with FCS-based supplement mix (C-39226) and recombinant human VEGFC 50 ng/mL (R&D Systems, 2179-VC-025).

Techniques: Fluorescence, Marker, Expressing, ChIP-sequencing, Cell Culture, Binding Assay, Two Tailed Test

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: ( A ) Differential inference contrast images of 14 and 17 hpf sibling ( kif11 +/+ or +/uom131 ) and mutant embryos ( kif11 uom131 ) . Somite defects, brain necrosis, and aberrant tail development observable in 100% of mutants at both time points ( n = 8/8 mutants and n = 0/19 sibling at 14 hpf and n = 8/8 mutants and n = 0/7 sibling at 17 hpf). Scale bar: 100 μm. ( B ) Upper: brightfield images of sibling and mutant embryos at 24 hpf. Brain necrosis and reduced embryo size observable in mutants. Lower: confocal maximum projection images of Tg( fli1a:nEGFP ) embryos showing reduced intersegmental vessel sprouting and vascular development likely consistent with delayed embryonic development ( n = 26/26 mutants and n = 0/88 sibling). Scale bar: 1 mm. ( C ) Brightfield and confocal maximum projection images of DMSO control (upper) and treated (lower) embryos following exposure to 50 μM ispinesib (ISP) at 10–48 hpf. Tg( fli1a:nEGFP;lyve1b:DsRed2) used to visualize ECs, veins, and lymphatic progenitors (PL). ISP treatment caused brain necrosis, pericardial edema, and aberrant tail development ( n = 26/26 ISP-treated, n = 27 1% DMSO). Scale bar: 1 mm. ( D ) Confocal maximum projection images of DMSO control or 50 μM ISP-treated Tg( fli1a:nEGFP;lyve1b:DsRed2) embryos at 72 hpf. ISP treatment 24 –72 hpf; 50 μM ISP-treated embryos showed impaired PL formation at horizontal myoseptum (quantified in E ). Scale bar: 100 μm. ( E ) Zoomed images of DA and PLs from D . ( F ) Bar plots of EC numbers (per somite) in DA and PLs treated with increasing ISP concentrations ( n = 11, 1% DMSO; n = 12, 10 μM ISP; n = 12, 25 μM ISP, n = 9, 50 μM ISP). Reductions in LECs and DA ECs were observed. Unpaired Student’s t test for normally distributed data and Mann-Whitney U test for non-normally distributed data. * P = 0.0261; ** P = 0.0018; *** P = 0.0002; ns, nonsignificant. TD, thoracic duct; DA, dorsal aorta; EC, endothelial cell.

Article Snippet: Human dermal LECs (HDLECs) (PromoCell, C-12217 or C-12216) were cultured and maintained in endothelial cell growth medium MV2 (PromoCell, C-22022) with FCS-based supplement mix (C-39226) and recombinant human VEGFC 50 ng/mL (R&D Systems, 2179-VC-025).

Techniques: Mutagenesis, Control, MANN-WHITNEY

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: ( A ) 5 x 10 4 serum starved cells were plated per fibronectin-coated Transwell chamber and given 10 hours to migrate in the presence of increasing concentrations of ispinesib (0 to 200 nM) before fixation and staining of the nuclei with DAPI. Resulting membranes (2 examples on left) were scored for the movement of nuclei through the Transwell membrane and results represented in a plot (right). Data are shown as SD, n = 3 biological replicates (with 2 technical replicates per biological replicate). Scale bar: 50 μm. ( B ) Single-cell tracking migration assay follows wound closure ability over 15 hours in DMSO (Ctrl) and 50 nM ispinesib-treated cells. Scale bar: 200 μm. ( C ) Number of cells in experimental wound-window area for Ctrl and cells treated with 50 nM ispinesib over time (frames). ( D ) Distance and direction of a subset of cells illustrated as star plots of cell tracks for Ctrl and cells treated with 50 nM ispinesib. ( B – D ) One representative experiment is shown from n = 2. ( E ) 3D spheroid sprouting assay. Sprouting was stimulated in LECs by VEGFC 150 ng/mL either in the presence of DMSO vehicle or several doses of ispinesib (25, 50, 100 nM). Average sprout length in μm (28 spheroids analyzed) and total number of sprouts per spheroid (15 spheroids analyzed) were quantified in each condition. One representative image is shown per condition from n = 2 biological repeats. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. Scale bar: 100 μm.

Article Snippet: Human dermal LECs (HDLECs) (PromoCell, C-12217 or C-12216) were cultured and maintained in endothelial cell growth medium MV2 (PromoCell, C-22022) with FCS-based supplement mix (C-39226) and recombinant human VEGFC 50 ng/mL (R&D Systems, 2179-VC-025).

Techniques: Staining, Membrane, Single Cell Tracking, Migration, Two Tailed Test

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: ( A ) Human phospho-kinase array showing changes in phosphorylation levels of 43 kinases. Dots have been magnified for selected kinases with decreased (in blue) or increased (in red) phosphorylation. ( B ) Network pathway analysis using STRING and Cytoscape reveals known interactions and the directionality of the phosphorylation change in siRNA KIF11 -treated cells (blue, decreased; red, increased). ( C ) Effect of ispinesib treatment (50 nM) on AKT and MAPK phosphorylation in LECs over time after stimulation with VEGFC (100 ng/mL). DMSO (vehicle) as control. Phosphorylation was analyzed with anti-pAKT S473 and anti-p44/42 MAPK Thr202/Tyr204 (upper panels) and levels of AKT and MAPK protein expression with anti-AKT and MAPK antibodies (lower panels). Molecular mass markers (in kDa) are indicated on the left. Blots are quantified on the right. * P < 0.05, ** P < 0.01, ns, nonsignificant. Two-tailed unpaired Student’s t test. ( D ) FLT4 (VEGFR3), PROX1 , PDPN, FOXC2 , LYVE1, TJP1 (ZO-1) , CLDN5 , and CDH5 gene expression related to GAPDH in LECs treated with siRNA 6 KIF11 for 48 and 72 hours analyzed by qPCR ( n = 3–4 experiments). * P < 0.05, ** P < 0.01, *** P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. ( E ) Western blot analysis and quantification of PROX1 and VEGFR3 expression in LECs treated with siRNA KIF11 for 48 and 72 hours. GAPDH was used as control. ** P < 0.01, *** P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. ( C – E ) Bars represent mean relative expression ± SEM. ( C and E ) One representative image is shown from n = 3–4 experiments.

Article Snippet: Human dermal LECs (HDLECs) (PromoCell, C-12217 or C-12216) were cultured and maintained in endothelial cell growth medium MV2 (PromoCell, C-22022) with FCS-based supplement mix (C-39226) and recombinant human VEGFC 50 ng/mL (R&D Systems, 2179-VC-025).

Techniques: Phospho-proteomics, Control, Expressing, Two Tailed Test, Gene Expression, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Organ-specific features of human kidney lymphatics are disrupted in chronic transplant rejection

doi: 10.1172/JCI168962

Figure Lengend Snippet: ( A ) Violin plots showing upregulation of IFN-inducible genes IFITM2 and IFITM3 in LECs from rejecting allografts. ( B ) UMAP of the scRNA-Seq data showing enrichment of IFN-γ within the T/NK cell cluster. ( C ) UMAP showing enrichment of an IFN-γ signature, including IFNGR1 , IFNGR2 , IFITM2 , and IFITM3 . ( D ) CellPhoneDB interaction map depicting predicted lymphatic-CD4 + T cell crosstalk in rejection. Inhibitory interactions (blue) include PVR and LGALS9; stimulatory interactions (red) are also shown. Node size reflects expression frequency; line intensity indicates interaction strength. Ligands of interest, PVR and LGALS9 , are highlighted. ( E ) Heatmap of immune checkpoint interactions between LECs and effector CD4 + T cells across disease states. Color indicates normalized CellPhoneDB interaction score. All scores were normalized for each ligand-receptor pair. ( F ) Immunofluorescence validation of PVR expression on PDPN + lymphatics (arrowhead) in rejecting kidneys ( n = 2); CD4 + T cell shown in contact (asterisk). Scale bar: 30 μm. ( G ) IFN-γ stimulation of cultured human LECs increases LGALS9 levels at 24 and 48 hours (qPCR; *** P = 0.0002, ** P = 0.0093, respectively) relative to HPRT. ( H ) LGALS9 protein secretion increased at 48 and 72 hours (ELISA; *** P = 0.0002, **** P < 0.0001, respectively) after IFN-γ stimulation of cultured human LECs. qPCR and ELISA experiments were repeated 3 times, and all assays were performed in duplicate, with each dot on the graph representing the mean data obtained for each repeat.

Article Snippet: Adult human dermal LECs (PromoCell, C-12217) were cultured in MV2 medium and treated with recombinant human IFN-γ (50 ng/mL) or unstimulated control medium for 24, 48, or 72 hours.

Techniques: Expressing, Immunofluorescence, Biomarker Discovery, Cell Culture, Enzyme-linked Immunosorbent Assay