Lymphatic Endothelial Cells

Lymphatic Endothelial Markers

A number of markers have been identified that distinguish blood from lymphatic vascular endothelium (Table 1). These include podoplanin, a glomerular podocyte membrane mucoprotein; Prox-1, a homeobox gene product involved in regulating early lymphatic development; and LYVE-1, a lymphatic endothelial receptor for the extracellular matrix/lymphatic fluid glycosaminoglycan, hyaluro-nan. None of these markers are expressed entirely specifically by LECs. LECs also express vascular endothe-lial growth factor-3 (VEGFR-3); with the exception of fen-estrated blood vascular endothelium, VEGFR-3 is expressed exclusively by lymphatics in normal adult tissues. However, it is widely expressed in embryonic blood vascular endothe-lium and is reexpressed in tumor blood vessels. In wound healing VEGFR-3 is confined to lymphatic endothelium (quiescent and regenerating), and is not expressed in blood vessels.

5'-Nucleotidase has also been used to distinguish lymphatic from blood vascular endothelium, and several apparently lymphatic-specific monoclonal antibodies have been reported, although none appear to have found widespread use. Finally, since lymphatic capillaries lack a continuous basement membrane, lack of immunoreactivity for extracellular matrix components (collagen IV, laminin, collagen XIII) has also been used to distinguish them from capillaries of the blood vascular system. However, this is unlikely to be reliable in tumors, since angiogenic blood vessels also appear to be partially or completely devoid of a basement membrane.

Lymphatic Endothelial Growth Factors

Two growth factors have been identified that induce growth of new lymphatic capillaries, and both belong to the vascular endothelial growth factor (VEGF) family. These are VEGFs -C and -D. Members of the VEGF family are highly conserved secreted glycoproteins that regulate vascu-logenesis, hematopoiesis, angiogenesis, lymphangiogenesis, and vascular permeability and are implicated in many physiological and pathological processes. The VEGF family comprises VEGFs-A, -B, -C, -D and placental growth factor (PlGF). Of the three VEGF tyrosine kinase receptors identified thus far (VEGFR -1, -2, and -3), VEGFR-1 binds VEGFs-A and -B as well as PlGF, VEGFR-2 binds VEGFs-A, -C, and -D, and VEGFR-3 binds VEGF-C and -D. VEG-FRs differ with respect to mechanisms of regulation and patterns of expression. For example, VEGFRs-1 and -2 are expressed almost exclusively by vascular endothelial cells and hematopoietic precursors, whereas VEGFR-3 is widely expressed in the early embryonic vasculature but becomes restricted to lymphatic endothelium at later stages of development and in postnatal life. VEGF-C also binds neuropilin-2, a receptor for class III semaphorins, which regulate chemorepulsive guidance of developing axons. Neuropilin-2 appears to be required for normal lymphatic development.

VEGFs-C and -D display a high degree of similarity to VEGF-A in their so-called VEGF homology domain, including conservation of the eight cysteine residues

Table I Characteristics of Lymphatic and Blood Vascular Endothelium.

Lymphatic capillaries

Blood capillaries

Morphological features

Lumen diameter

10-50 mm

10 mm

Pericytes

Absent

Present

Basement membrane

Absent or discontinuous

Continuous

Anchoring filaments

Present

Absent

Intercellular junctions

Overlapping, narrow

Well developed

Weibel-Palade bodies

Present

Present

Caveolae

Present

Present

Molecular markers

CD31/PECAM-1

+

+

CD34

+ (low)

+ (high)

Von Willebrand factor

+

+

VE-cadherin

+

+

VEGFR-1

+

+

VEGFR-2

+

+

VEGFR-3

+ (high)

+ (low)

Tie-2

-/+

+

PAL-E

-

+

LYVE-1

+

-

Podoplanin

+

-

Prox-1

+

-

Mannose receptor

+

-

CCBP2/D6

+

-

characteristic of the VEGF family. However, VEGFs-C and -D also contain N- and C-terminal extensions that are removed by cell-associated proteolytic processing following secretion. Processing increases the affinity of the ligands for VEGFR-3 and also allows them to bind to VEGFR-2. In addition to their lymphangiogenic effect, under certain conditions VEGFs-C and -D also stimulate angiogenesis. The respective roles of VEGFR-2 and -3 in mediating the lym-phangiogenic and angiogenic effects of VEGF-C and -D are incompletely understood.

Other molecules that have been implicated in the formation of the lymphatic vascular system include angiopoietin-2, the adaptor protein SLP76 and the tyrosine kinase syk, both of which are expressed primarily in hematopoietic cells, the transcription factors Prox-1 and net, and chemokines (CCL21/SLC) and their receptors (CCBP2/D6). a9 integrin has also been implicated.

Isolation and Culture of Lymphatic Endothelial Cells

Although many attempts have been made in the past to isolate and culture lymphatic endothelial cells from a variety of species, all of these studies have described isolation of the cells from large lymphatic vessels and have employed crude mechanical methods of cell separation. Identification of cell surface markers that allow easy distinction between lymphatic and blood vascular endothelium has recently led to the development of superior techniques for the isolation of pure endothelial cell populations. LECs have been isolated by positive selection using antibodies to podoplanin, VEGFR-3, or LYVE-1, and by a negative selection with antibodies to CD34. Molecular characterization of these cells has revealed many important differences in their molecular profiles. Of major importance is the observation that LECs express Prox-1, since Prox-1 appears to be a master switch for the lymphatic phenotype. In the adult organism, lymphatic endothelium has been shown to expresses VEGFR-2 and -3. Cultured LECs express VEGFR-1, -2, and -3, while BECs express VEGFR-1 and -2 and low levels of VEGFR-3. Many of the characteristic in vivo patterns of gene expression are altered with time in culture. For example, LYVE-1 is progressively lost from cultured LECs.

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