Unique Structural Functional Markers Lymphatic versus BEC

LECs share some markers expressed by most BECs, including factor VIII, anti-thrombin 3 and MHC I, receptors for low-density lipoprotein (LDL-R), and angiotensin-converting enzyme (ACE). However, some studies suggest that LEC may only modestly express some other classical BEC markers. For example, Weibel-Palade bodies and their constituents (von Willebrand factor/factor VIII-related antigen, alpha-1 fucosyltransferase VI, P-selectin) are expressed in LEC [1], but may exhibit quantitative differences based on anatomical location and species. Similarly, levels of expression of 5'-nucleotidase and 5'-nucleotidase/alkaline phosphatase have also been suggested as markers that can qualitatively distinguish LECs and BECs [1]. Extracellular matrix (e.g., heparan sulfate proteoglycan, collagen type IV) proteins are clearly less densely expressed on LECs compared to BECs. However, on the basis of these structural markers alone, BECs and LECs cannot be clearly distinguished. Another structural marker, Pathologische Anatomie Leiden-Endothelium (PAL-E), a caveolae-related marker associated with leaky vessels, does appear to be a highly specific BEC marker and is absent on LECs. Unlike these markers, which were preliminarily used to distinguish BECs from LECs, the more important and highly specific/selective

LEC markers [1] now include podoplanin, Prox-1, LYVE-1, and VEGF-R3 (Flt-4), which are described below.

Podoplanin

Podoplanin is expressed by LECs and has been used as an LEC specific marker. It was originally described as a 43kDa mucoprotein on kidney podocytes (hence the name podoplanin), but is not entirely specific for LEC; podoplanin is also densely expressed in lung alveolar epithelium, osteoblasts, and several other cell types. The main function of podoplanin in LEC is not known, but it may help to regulate LEC vessel structure, or govern solute exchange in lymphatics. Podoplanin has a large extracellular domain with 6 O-glycosylation sites and a shorter cyto-plasmic tail with two sites for phosphorylation that may be important in regulating its function. In disease, the altered expression of podoplanin has been reported during the lymphatic proliferation seen in IBD, cancer, and Kaposi's sarcoma (KS).

Prox-1

The transcription factor Prox-1 is an important regulator of lymphatic system development, but also regulates development of several tissues including the lens, retina, and liver [2]. Prox-1 is probably the most important determinant for endothelial progression/commitment to the lymphatic endothelial phenotype. The activation of Prox-1 appears to help commit lymphatic progenitors both by activating LEC transcripts and by suppressing BEC transcripts. Some of LEC-specific transcripts that are activated by Prox-1 activity include integrins a9 and a1, desmoplakins I/II, adducin-g, plakoglobin, matrix GIa protein, TIMP-3, macrophage mannose receptor, alpha-actinin-2 associated LIM protein, IL-7, SDF-1b, and the cell cycle regulators, Cdkn-1b and Cdkn-1c.

Interestingly, Petrova et al. [3] report that Prox-1 trans-fection into BEC led to the induction of numerous lymphatic endothelial specific genes including p57kip2 and VEGF-R3. Prox-1 expression in BEC also suppressed 40% of BEC-specific genes, including STAT-6, laminin, VEGF-C, neu-ropilin-1, ICAM-1, MCP-1, IL-6, and P-selectin. It is interesting to note that VEGF-C, which is a potent LEC growth factor (see later discussion), is a normal transcript for BEC, which may be suppressed when cells commit to the LEC lineage to avoid a persistent autocrine growth stimulation. The suppression of some of these genes may also reflect Prox-1 dependent inhibition of STAT-6 (which is required to sustain BEC transcripts such as MCP, IL-6, and P-selectin). When Prox-1 is expressed in nonendothelial cells it upregulates message for cyclins E1 and E2 and activates the cyclin E promoter, but does not induce LEC genes. Therefore Prox-1 is necessary for the LEC phenotype, but does require a prior commitment to the endothelial lineage, a prerequisite for the final "programming" in the lymphatic endothelial lineage [3].

LYVE-1

LYVE-1 (lymphatic vascular endothelial hyaluronan receptor) is a homolog of CD44 that shares approximately 41% homology with CD44 [4]. LYVE-1 participates in endocytic processing of hyaluronic acid (HA) in LECs, like CD44 in BECs, but it may also perform other functions. In lymphatic vessels, LYVE-1 on the cell surface appears to colocalize with HA, which lines the lymphatic lumen where it has been suggested to help modulate leukocyte/immune cell trafficking. The exact role of the hyaluronan matrix for lymphatic function is still not well understood, but may be important to signaling, lymphocyte migration, or differentiation [5]. Compared to CD44, LYVE-1 may exhibit greater specificity for HA than CD44 [4]. LYVE-1 is expressed on the cell surface as a 60-kDa protein; 20kDa of its mass appears to be produced by dense glycosylation. LYVE-1 expression appears to be generally restricted to lymphatic endothelium within the spleen, lymph nodes, heart, lung, and fetal liver. In adult tissues, LYVE-1 is low in the liver, muscle, bone marrow, and appendix and is generally absent in BECs, hematopoietic cells, and lymphocytes. However, while LYVE-1 is expressed mainly by LECs, some LYVE-1 expression is retained by BECs in the spleen and liver, in syncytiotrophoblasts and in macrophages [2, 5, 6].

The growth factors and growth factor receptor involved in LEC proliferation, maturation, and survival include VEGFs-C and D and the VEGF receptor, Flt-4 (VEGF-R3). Flt-4 was perhaps the first specific lymphatic endothelial marker described and plays an important role in the development of the embryological capillary system; in the adult, Flt-4 action regulates mainly the growth and maintenance of lymphatic vessels. Flt-4 is a 210-kDa receptor for VEGFs-C and D that is expressed at high levels on LECs, but usually not on BECs. Some exceptions when Flt-4 is induced on BECs include wound healing and expression in some tumor blood vessels and in fenestrated endothelium (e.g., bone marrow, spleen and hepatic sinusoids, kidney glomeruli, and endocrine gland endothelium). Flt-4 is also expressed in some nonendothelial cells (e.g., dendritic cells). Flt-4 is a member of the class III receptor tyrosine kinases like Flt-1 and Flk-1/KDR that have seven Ig-like domains and 12 potential glycosylation sites. At least two Flt-4 isoforms are known that are derived from a single transcript.

Animals made genetically deficient in Flt-4 are ultimately embryonic lethals because of defective fluid drainage (chylous ascites), and some abnormal vascular assembly. In human disease, primary (hereditary) lym-phedema can be produced by single amino acid substitutions in Flt-4 (VEGFR-3) that inactivate the receptor kinase activity and prevent the maintenance/development of a normal lymphatic system.

VEGFs C and D

VEGFs C and D are closely related growth factors produced by several tissues, such as lung, skeletal muscle, colon, small intestine, tumor cells, and BECs (umbilical vein ECs). VEGF-C is a potent ligand for Flt-4, but also binds Flk-1/KDR (VEGF-R2) but not VEGF-R1, and induces lymphangiogenesis and LEC migration. Interestingly, although thought to be an LEC-specific growth factor, viral induction of VEGF-C is one of the most powerful stimuli for angiogenesis and lymphangiogenesis [7]. Although both VEGF-C and D can bind Flk-1/KDR and Flt-4, experiments with Flt-4 specific forms of these VEGFs show that Flt-4 binding alone promotes lymphatic vessel growth. VEGF-D is also a ligand for Flt-4 (and VEGF-R2), but not VEGF-R1 (Flt-1). In mice VEGF-D appears to be a selective ligand for Flt-4 [8]. Stimulation of lymphatic endothelial cells with VEGF-C is a strong stimulus for the increased expression of Ang-2 (via VEGFR-2) and in lymphatic development, angiopoetin-2 (Ang-2) appears to control lymphatic development, since Ang-2 knockout mice show errors in the maturation of lymphatic vessels. Interestingly, Ang-1 appears to correct the lymphatic, but not the angiogenesis defects, suggesting that Ang-2 acts as a Tie-2 agonist in the former setting, but as an antagonist in the latter setting. Interestingly, LEC have been shown to express Ang-2, and it has been suggested that the lack of peri-cytes/support cells in lymphatic microvessels could be related to this LEC Ang-2 expression.

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