Generation and Characterization of Telomerase-Transfected Human Lymphatic Endothelial Cells with an Extended Life Span

Abstract

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin ␣9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tubelike structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro. The blood and lymphatic vascular systems are anatomically and histologically closely related but distinct, and do not anastomose except at the level of the main lymphatic ducts. The blood vascular system supplies nutrients and oxygen to the body while the lymphatic vasculature is crucial for immune cell traffic and the uptake of extracellular fluid. The formation of new blood vessels from pre-existing blood vessels (angiogenesis) occurs in physiological (eg, postnatal growth, inflammation, and tissue repair) and pathological (eg, tumor recruitment of blood vessels) settings. Lymphangiogenesis is the formation of new lymphatic vessels from pre-existing lymphatic capillaries, and occurs in similar settings. Blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) are quiescent cells that line the lumina of blood and lymphatic vessels.The lymphatic system has traditionally been overshadowed by the greater emphasis placed on the blood vascular system. This has been due in part to the absence of suitable markers that distinguish lymphatic from blood vascular endothelium. In the past few years, this limitation has been overcome. Lymphatic markers include LYVE-1, a lymphatic endothelial receptor for the extracellular matrix/lymphatic fluid glycosaminoglycan hyaluronan; 1 Prox-1, a homeobox gene product involved in regulating early lymphatic development; 2 podoplanin, a glomerular podocyte membrane mucoprotein; 3 and vascular endothelial growth factor re...

Insulin-like growth factors 1 and 2 induce lymphangiogenesis in vivo

“…Human LECs were isolated by outgrowth from commercial dermal microvascular endothelial cells purchased from PromoCell (Heidelberg, Germany) using the method described in Nisato et al (46). Murine dermal LECs were selected from the collagenase digested skin of C57BL͞6 mouse pups (2-3 days old) using a rat anti-mouse LYVE-1 mAb with MACS microbeads and grown on collagen-coated vessels in microvascular endothelial growth medium (EGM-2 MV; Cambrex, East Rutherford, NJ).…”

The C-Type Lectin Macrophage Galactose-Type Lectin Impedes Migration of Immature APCs

“…2, in which we did not observe any MGL-mFc binding to endothelial cells lining small blood vessels in LN. In contrast, HMEC-1 cells, a human endothelial cell line that expresses several lymphatic endothelial markers (18), strongly interacted with MGL-Fc (Fig. 5B).…”

Modeling lymphangiogenesis in a three-dimensional culture system

“…Several lymphatic 2D-culture systems now available use human dermal lymphatic cells isolated either by immuno-purification with Fluorescence-Activated Cell Sorting (FACS) or by magnetic beads 11 , and on occasion employ transformation with human telomerase reverse transcriptase (hTERT-HDLEC) 12 . Thoracic duct endothelial cells from different species are isolated by enzymatic digestion 13,14 .…”