Figure 1.
HMEC-1 and TIME express both blood vascular and lymphatic markers.
HMEC-1 and TIME cells were stained for flow cytometry using the indicated antibodies against (A) blood vascular markers and (B) lymphatic markers. The marker region shows the antigen-specific staining and is set based on the species- and isotype-specific negative controls. One representative negative control is depicted here. The numbers in the upper right corners of the histograms are the specific median fluorescence intensities of the given antigen, calculated as detailed in materials and methods. (C) Representative images of TIME cells simultaneously expressing lymphatic and blood vascular markers in the same cell are depicted and indicated with white arrows. The scale bar represents 20 µm.
Figure 2.
HMEC-1 and TIME show aberrant leukocyte-endothelial interactions under physiological shear stress.
(A) Rolling and adhesion of PBMC and PMN on HMEC-1, TIME and HUVEC were analyzed using in vitro flow assay. The results are normalized to HUVEC (100%). (B) Absolute numbers of interacting (firmly adhering and transmigrating) leukocytes and (C) the transmigration percentage (the numbers of transmigrated cells divided by the numbers of interacting cells) on the three endothelial monolayers were determined. Data are shown as mean ± SEM (n = 3 for each assay, each with different leukocyte donors). *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001. (D) Images of representative endothelial monolayers 10 min. after start of PMN transmigration studies are shown. Phase contrast bright cells (representative cells indicated by white arrows) are located on the apical surface of the endothelial cells and phase contrast dark cells (representative cells indicated by white arrow-heads) are situated below the monolayer. Note that all three endothelial types form confluent intact monolayers. Inserts show adhering and transmigrating cells in more detail. Scale bars represent 100 µm.
Figure 3.
Gene expression profiles of HMEC-1 and TIME reveal differences from pure BEC and LEC.
(A) A multidimensional scaling plot (MDS-plot) showing clustering of TIME, HMEC-1, primary BEC and LEC (and prostate tissue as a control) based on the expression of about 19000 genes. (B) Exemplary genes that are differently expressed in HMEC-1 and/or TIME when compared to BEC and LEC in the normalized Dataset A. Data is presented as mean with the middle quartile and minimum/maximum values.
Figure 4.
Identification of BEC and LEC specific genes in different individual- and pooled microarray analyses.
(A) The numbers of BEC and LEC specific genes obtained from analyses of a restricted Dataset A (24 samples, one platform: GPL570) and of an extensive Dataset B (47 samples, three platforms: GPL570, GPL571 and GPL5188) are shown using Venn-diagrams. (B) Similar comparisons were performed between our extensive Dataset B and two individual studies (Hirakawa and Petrova) reporting BEC- and LEC specific genes.
Figure 5.
MCAM and COLEC12 are novel BEC- and LEC- specific markers.
Two new endothelial markers MCAM and COLEC12 (selected from our analysis of Dataset B) were used to stain (A) normal human lymph nodes together with two established vascular markers PV-1 (for BEC) and CLEVER-1 (for LEC). In addition, (B) chronically inflamed tonsils and (C) specimens from bladder cancer and colorectal cancer were stained with the antibodies against the indicated proteins. LYVE-1 and COLEC12 co-staining was done on colorectal cancer specimens, whereas the other stainings represent bladder cancer. MCAM staining colocalized very well with the established BEC marker PV-1 whereas no colocalization could be detected between MCAM and the established LEC markers LYVE-1 and podoplanin. COLEC12-staining on the other hand showed colocalization with LYVE-1 but not PV-1. White arrows point to areas of colocalization. Nuclear counterstaining was performed with DAPI. Scale bars represent 100 µm.