The main functions of the lymphatic vascular system are to maintain the fluid balance in the interstitial spaces, to provide a highway for circulating leukocytes, and to take up and transport larger particles such as chylomicrons and bacteria. These important functions of lymphatic vessels in health and disease are well known, but have rarely been studied experimentally (review: [
4]). Malformations of lymphatic vessels, such as lymphangiomas, are associated with a failure in lymph transport and high morbidity of the patients [
24]. The etiology of lymphangiomas is unknown, which may not be surprising, because even the mechanisms of normal embryonic development of lymphatic vessels are still a matter of debate (review: [
2]). Some authors assume that lymphatic vessels are exclusively derived by sprouting from the venous system, whereas others suggest that there is an additional origin from mesenchymal lymphangioblasts [
25]. A few "lymphangiogenesis" genes have been identified in patients presenting with congenital lymphedema, due to hypoplasia or dysplasia of the lymphatic vascular system. Milroy lymphedema in some, but not all, affected families is due to mutations in the VEGFR-3 gene [
26,
27], which encodes the receptor for the lymphangiogenic growth factors VEGF-C and -D [
3]. Lymphedema-distichiasis is linked to the forkhead transrciption factor FOXC2 [
28], and the lymphedema-hypotrichosis-telangiectasia syndrome is caused by mutations in the transcription factor SOX18 [
29]. However, besides lymphangioma, there are approximately 40 syndromes that are associated with abnormal development of the lymphatic vascular system [
30]. The isolation and characterization of LECs from normal and malformed tissues will facilitate the characterization of the diseases, as a first step for diagnosis and therapy.
Isolation and characterization of LECs
Isolation studies have concentrated on LECs from foreskin and not from other tissues. Characterization of LECs from different healthy or diseased tissues has not been carried out. We have started to address this question and isolated LECs from normal and diseased tissues with commercially available antibodies. The expression and distribution of blood and lymph vessel markers was analyzed at RNA level with micro-arrays and at protein level by FACS analysis, immunocytology and ELISA. The main results of our studies show that
(i) LEC markers vary among the types of tissue used for cell culture
(ii) microvascular endothelial cells from the myometrium are a homogenous population of BECs, but positive for LYVE-1
(iii) LECs from lymphangiomas compared to those from foreskin have numerous markers in common, but show decreased LYVE-1 and increased VEGFR-3 expression
(iv) Prox1 is by now the most reliable marker of LECs.
Our data are in line with and extend previous studies demonstrating that LECs can be isolated from neonatal foreskin and identified by their ability to express markers like VEGFR-3, podoplanin and LYVE-1 [
5,
13,
14].
Human dermal microvascular endothelial (HDMECs) have a high capacity to express lymphatic markers, and we observed that BECs seem to make up the minority of cells after primary isolation. This has also been observed in studies where these cells were used for immortalization [
23]. It is not known by now, if directly after the initial isolation of the cells from foreskin with anti-CD31 antibodies by the supplier LECs are the numerical dominant cell type, or if during further expansion and splitting of the cultures the population of LECs preferentially increases. In initial experiments with HDMECs we found that the percentage of podoplanin
+ cells is very high (60–90%). Further characterization of the cells by FACS analyses showed that some batches can be divided into three groups: podoplanin-negative, moderately positive and strongly positive. However, most of the batches contain only two groups: podoplanin
- and podoplanin
+ (data not shown). Approximately 50% of the podoplanin
+ cells are also positive for LYVE-1, and these are the same cells, which are also positive for VEGFR-3. At the moment it remains unclear, if podoplanin
+/LYVE-1
- cells represent an intermediate cell-type, and if only the podoplanin
+/LYVE-1
+/VEGFR-3
+ cells can be regarded as primary LECs from neonatal foreskin.
Growth of cultured LECs depends on the presence of VEGF-C, which, in mixed cultures, is supplied by the BECs [
3]. After separation of LYVE-1
+ or podoplanin
+ cells we cultured them in the presence of VEGF-C as recommended before. However, selective stimulation of VEGFR-2 by VEGF-E is obviously also sufficient to grow and expand LECs in culture [
31]. The activity profile of another family of endothelial growth factors, the angiopoietins (e.g. Ang-1 and Ang-2) is still controversially discussed. Angiopoietins bind the Tie2 receptor, and may activate or inhibit signal transduction in a cell-type and tissue-specific manner [
32,
33]. Angiopoietins are essential for the hierarchical organization of the blood vascular tree, but the expression of Tie2 in lymph vessels of the human has remained unclear. We have observed Tie2 expression in LECs sorted with anti-podoplanin antibodies from HDMECs, as well as Tie1 and Tie2 in LECs from lymphangioma patients. Mice deficient in Ang-2 show defects in the patterning and function of the lymphatic vasculature, and a lack of lymph nodes, and they develop chylous ascites [
33]. Overexpression of Ang-1 in the skin of adult mice induces lymphangiogenesis, which is associated with VEGFR-3 up-regulation in LECs. Interestingly, proteolytically processed VEGF-C, which binds VEGFR-2 and -3, induces Ang-2 expression in LECs
in vitro via VEGFR-2 signalling, indicating a new interaction between the VEGF and angiopoietin family members, which may regulate the hierarchy of the lymphvascular tree [
34].
Lymphangioma endothelial cells
Lymphangiomas are disfiguring neoplasias of childhood and may also manifest or enlarge rapidly in adulthood [
35]. More than 95% of lymphangiomas occur in the soft tissues of the head, neck and axilla, with less than 5% occurring in the abdominal cavity. The prevalence of lymphatic malformations is 1.2 – 2.8‰ [
36]. Macroscopically lymphangiomas are solitary, multicystic masses. The lining of the cysts is smooth and they have thin walls. Histological criteria for lymphangiomas are: 1) lymphatic spaces lined by endothelium, 2) fascicles of smooth muscle in the septa, and 3) lymphoid aggregates in the delicate collagenous stroma [
37]. Immunoelectron microscopic studies have demonstrated up-regulation of CD31 and CD34 and show type IV collagen expression in lymphangiomas [
38]. The vascular endothelial marker PAL-E is confined to blood vessels in lymphangiomas. VEGFR-3 mRNA has been localized to lymphangioma LECs [
8]. The detection of transcripts for VEGF-C, VEGFR-2 and VEGFR-3 in endothelial cells from different lymphangiomas by in situ hybridization was reported before [
39]. No expression of these genes was found in adjacent tissue or in normal lymphatic vessels.
With immunohistological methods, we have recently been able to identify VEGFR-3 and Prox1 in CD31-positive LECs of lymphangiomas [
22].
Here we have measured the amount of VEGFR-3 protein in lysates from different endothelial cell types and found significantly higher expression in LECs derived from the two lymphangioma patients as compared to foreskin LECs. Significant amounts of VEGFR-3 and its intra-cellular signalling cascade (shc, PLCγ, p44MAPK) were also detectable at RNA level. Increased VEGFR-3 signalling may be a major course for aberrant lymph vessel formation since the opposite, lymphatic hypoplasia, can be observed in VEGFR-3 mutated patients with Milroy's disease [
26,
27]. The reasons or regulatory pathways for VEGFR-3 up-regulation are not known. Direct (mutations) or indirect mechanisms have to be considered, e.g. an increase of Ang-1 expression, as discussed above. However, we did not detect Ang1 or Ang2 expression in lymphangioma LECs at significantly higher levels as compared to HUVECs. Because lymphangioma tissue mainly consists of LECs and stromal cells, the ligands for VEGFR-3, VEGF-C and VEGF-D, may be derived from either of the cell types, acting in an auto- or paracrine mode, respectively. However, we have not found any measurable VEGF-C protein in the conditioned media obtained from stromal cells of the two patients. Also, we did not observe significant levels of VEGF-C and -D in the LECs. Other growth factors may be involved in the development of lymphangiomas. We have observed high expression of FGF-12 and TGF-α in lymphangioma LECs, but the significance of this finding remains to be studied. In contrast to BECs, LECs seem to be highly responsive to stimulation with hepatocyte growth factor (HGF) [
15], but we could not detect significant expression of the HGF receptor, c-Met, in lymphangioma LECs as compared to HUVECs. We have found high levels of VEGFR-2 in the LECs from the lymphangioma patients as compared to HUVECs (Table
2). This is a surprising finding because usually VEGFR-2 mRNA levels of BECs and LECs are equal, or they are lower in LECs [
12,
13]. We did not find significant mRNA levels of the ligands PlGF, and VEGF-B in the LECs and only in patient-A there was high expression of VEGF-A, which may indicate an autocrine loop. Further studies with the cells
in vitro and
in situ will be necessary to identify the underlying mechanisms involved in LEC hyperplasia and dysplasia.