Figure 4.
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 −log10 of the adjusted P value (list of marker genes for the enrichment analysis in Data S4). 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 log2 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.

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 −log10 of the adjusted P value (list of marker genes for the enrichment analysis in Data S4). 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 log2 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.

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