Molecular characterization of lymphatic endothelial cells

Abstract

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.

Fig. 1.

Selective expression of vascular markers in human skin vasculature. (A) Double immunofluorescent staining for LYVE-1 (red) and a PAL-E, a marker of blood vessels (green), in a 50-μm thick section of human foreskin. The stainings are mutually exclusive, indicating high specificity of LYVE-1 antibody for lymphatic vessels. Note high lymphatic vessel density. (B–D) Fluorescent staining for LYVE-1 (red), CD31 (green), or both together (merged), respectively, demonstrates that all LYVE-1⁺ vessels are also CD31⁺. (E and F) Double-staining for CD34 (red) and PAL-E (green) revealed identical expression pattern in blood vessels (E), whereas LYVE-1⁺ lymphatic vessels (green) do not express CD34 (F). (G and H) Smooth muscle α-actin (red) was colocalized with PAL-E (green) in blood vessels (G) but was absent from LYVE-1⁺ vessels (H). Arrows point to the lymphatics; arrowheads point to blood vessels; dots indicate dermal-epidermal junction. (Bar = 100 μm.)

Fig. 2.

Purification of lymphatic and blood microvascular endothelium. (A) Primary culture consisting mainly of ECs, a few fibroblasts, and keratinocytes, with LYVE-1-coated magnetic beads attached to the subpopulation of ECs. (B) After the second purification step, all cells are stained with the CD31 antibody at cell junctions, indicating a pure endothelial population. (C and D) Confluent layers of lymphatic (C) and blood vascular (D) ECs at passage 7. (Bar = 25 μm.)

Fig. 3.

Selective effects of VEGF-C on survival and tube formation by LECs in a collagen gel. Cells either received no treatment (A and B) or were exposed to exogenous FGF-2 (C and D), VEGF (E and F), or VEGF-C (G and H). (Bar = 25 μm.)

Fig. 4.

Quantitative assessment of LEC and BEC network formation in the collagen gel. Total length of cell cords formed after 48 h was measured. Data are expressed as total cord length (in μm ± SEM) per field from at least 15 fields. Data are pooled from four experiments. The unpaired Student's t test was used for statistical analyses. LEC control vs. FGF, P = 0.5; vs. VEGF, P < 0.001; vs. VEGF-C, P < 0.001. BEC control vs. FGF, P = 0.5; vs. VEGF, P < 0.001; vs. VEGF-C, P < 0.05.

Fig. 5.

Differential expression of vascular markers by cultured LECs and BECs. (A) Western and Northern analyses of LYVE-1 expression. Two major bands of LYVE-1 protein (≈70 and 200 kDa) and RNA (2.0 and 2.6 kb) were expressed in LECs. Hybridization with a β-actin cDNA probe was performed as a loading control for the Northern analysis. For Western analyses, equal amounts of proteins were loaded. (B) Western analyses of CD31, VEGFR-2, VEGFR-3, and plakophilin expression. Positions of molecular mass markers are shown in kDa.

Fig. 6.

Quantitative assessment of differential gene expression.