Identification of differentially expressed genes
We isolated BECs and LECs from cultures of human dermal microvascular endothelial cells (HDMECs) using magnetic microbeads and antibodies against the lymphatic endothelial cell surface marker podoplanin (
Breiteneder‐Geleff et al., 1999;
Mäkinen et al., 2001b). The purities of the isolated BEC and LEC populations were confirmed to be >99%, as assessed by immunofluorescence using antibodies against VEGFR‐3 and podoplanin (data not shown). The isolated cells were cultured for a couple of passages, and RNA was extracted, labeled and used for hybridization with oligonucleotide microarrays containing sequences from ∼12 000 known genes, i.e. ∼1/3 of the total number of all predicted human transcripts.
Consistent with their known lymphatic‐specific gene expression patterns
in vivo and
in vitro, podoplanin, desmoplakin I/II and the macrophage mannose receptor I were found specifically in the LECs (
Breiteneder‐Geleff et al., 1999;
Ebata et al., 2001;
Irjala et al., 2001); therefore, further characterization of the gene expression profiles was carried out. About 300 genes were found to be differentially expressed between the LECs and BECs, when using a reproducible signal log
2 ratio of 1.0 (2‐fold difference) in the replicate analyses (most of these genes are also expressed in other cell types). The microarray data were validated by northern blotting for 29 of the selected genes (see
Figure 1A for examples; a full list of the differentially expressed genes is available as
Supplementary data at
The EMBO Journal Online).
We detected the most striking differences between the BECs and LECs in the expression of pro‐inflammatory cytokines and chemokines [interleukin (IL)‐8, IL‐6, mono cyte chemotactic protein‐1] and receptors (UFO/axl, CXCR4, IL‐4R) as well as genes involved in cytoskeletal and cell–cell or cell–matrix interactions (see
Table I for other examples). The expression of pro‐inflammatory cytokines predominantly in the BECs can be explained, at least partially, by the fact that the STAT6 transcription factor, which is activated in response to IL‐4 (
Ihle, 2001), is expressed specifically in the BECs, as detected by western blotting and immunofluorescence (
Figure 1B and
C).
Cadherins are a family of membrane receptors that mediate the formation of stable cell–cell junctions via homophilic cell adhesion. The cytoplasmic domains of cadherins interact with β‐catenin, plakoglobin (γ‐catenin) and p120
ctn, which link them to the actin cytoskeleton via α‐actinin, vinculin, ZO‐1, ZO‐2 and spectrin (
Provost and Rimm, 1999). We found that BECs expressed significantly higher levels of β‐catenin (
Figure 2A and
B) and vinculin (data not shown), whereas plakoglobin was present mostly on the LECs (
Figure 2C and
D). Staining of LECs and BECs also revealed striking differences in the organization of the actin cytoskeleton. BECs displayed numerous stress fibers, which in LECs were almost totally absent, and instead a cortical distribution of actin was observed (
Figure 2E and
F).
Integrins, which are important mediators of cell adhesion (
Giancotti and Ruoslahti, 1999), consist of α and β subunits, which bind extracellular matrix proteins, while the cytoplasmic domains interact with the cytoskeleton and with proteins involved in signal transduction. Integrin α5, which acts as a subunit of the fibronectin receptor, was mainly expressed in the BECs. In contrast, integrins α1 and α9, which provide subunits for the receptors for laminin and collagen and for osteopontin and tenascin, respectively, were expressed in the LECs (
Figures 1A,
2G and
H). In human skin, antibodies against integrin α9 stained specifically lymphatic capillaries, while blood vessel endothelia were negative (
Figure 2I–K). In addition, integrin α9 was detected in arterial smooth muscle cells, as reported previously (
Palmer et al., 1993; data not shown). Interestingly, mice lacking integrin α9β1 were reported to develop respiratory failure due to the accumulation of a milky pleural (presumably lymphatic) effusion and to die within 6–12 days after birth (
Huang et al., 2000).
Prox‐1 expression reprograms the BEC transcriptional profile
In the microarray analysis, the Prox‐1 homeobox transcription factor was found to be expressed specifically in the LECs, and this result was confirmed by northern blot analysis and immunostaining (
Figure 3A–C). Despite the fact that the Prox‐1 gene was discovered nearly 10 years ago, Prox‐1 target genes have not been identified so far. We used adenoviral gene transfer of Prox‐1 in primary endothelial cells to address the question of Prox‐1‐induced gene expression. In order to eliminate the gene expression changes caused by the adenoviral infection, we used AdLacZ (encoding β‐galactosidase) to infect control cells.
Preliminary titration experiments showed that infection of human microvascular endothelial cells with AdProx‐1 or AdLacZ led to nuclear expression of the adenovirus‐encoded protein in >90% of the cells by 24 h (data not shown). To investigate the changes in gene expression induced by Prox‐1, we first used human cDNA filter arrays, which contain ∼1000 genes known to be important for general cellular pathways as well as genes specifically implicated in the regulation of cardiovascular function or hematopoiesis. AdProx‐1 regulated the expression of 50 of these genes (see
Supplementary data I available at
The EMBO Journal Online), which was confirmed by northern blotting for 10 out of 11 selected genes. When compared with genes expressed differentially in LECs and BECs, 15 genes (i.e. ∼30%) modulated by Prox‐1 were found to be differentially expressed between cultured LECs and BECs, suggesting that Prox‐1 is a major regulator of the lymphatic endothelial cell identity.
We next asked whether the introduction of Prox‐1 into BECs (where it is absent) can modify the transcriptional program of these cells towards the lymphatic endothelial phenotype. AdLacZ did not significantly alter the expression of BEC‐ or LEC‐specific transcripts in oligonucleotide microarray analysis. In contrast, AdProx‐1 increased the expression of many LEC‐specific mRNAs, such as VEGFR‐3, p57
Kip2, desmoplakin I/II and α‐actinin‐ associated LIM protein (
Figure 3D;
Table II). Surprisingly, Prox‐1 also suppressed the expression of ∼40% of genes characteristic for the BECs, such as the transcription factor STAT6, the UFO/axl receptor tyrosine kinase, neuropilin‐1 (NRP‐1), monocyte chemoattractant protein‐1 (MCP‐1) and integrin α5 (
Figure 3D; data not shown; see
Table II and
Supplementary data II for other examples). These results on gene expression analysis are in agreement with the
in vivo studies of lymphatic vessels. For example, VEGFR‐3 and desmoplakin I/II are found in the lymphatic endothelium (
Kaipainen et al., 1995;
Ebata et al., 2001), and we found that the VEGF co‐receptor NRP‐1, which was suppressed by Prox‐1 in the BECs, is expressed in a subset of blood vessels, but not in lymphatic vessels in mouse skin (
Figure 2L–N).
Prox‐1 induces the expression of cyclin E1 and cyclin E2 in various cell types
In addition to suppressing BEC‐specific genes, overexpression of Prox‐1 in BECs resulted in the upregulation of a group of genes linked to cell cycle S‐phase progression, such as cyclin E1 and cyclin E2, histone H2B, proliferating cell nuclear antigen (PCNA) and dehydrofolate reductase (
Figure 4A; data not shown). Increased levels of PCNA were also observed in AdProx‐1‐infected BECs by immunofluorescence (
Figure 4B). In transient transfection experiments, Prox‐1, but not a Prox‐1 mutant containing two amino acid substitutions in its DNA‐binding domain, stimulated the activity of the
cyclin e promoter, whereas the activity of the control construct was not modified (
Figure 4C). Because transcription factors of the E2F family are major regulators of S‐phase progression and the
cyclin e promoter contains an E2F binding site (
Ohtani et al., 1995;
Botz et al., 1996), we also tested whether Prox‐1 can transactivate a synthetic reporter construct 6xE2F‐luc, which contains six consensus E2F binding sites. As with the results obtained with the
cyclin e promoter, Prox‐1 strongly transactivated 6xE2F‐luc reporter (
Figure 4D). Co‐transfection of cyclin inhibitors p16
INK4a and p27
Kip1a, which act by increasing the concentration of transcriptionally inactive retinoblastoma (Rb)–E2F complexes, completely abolished transactivation of 6xE2F‐luc reporter by Prox‐1, further confirming the specificity of the Prox‐1 effect.
In order to study whether the Prox‐1‐induced changes of gene expression are cell type specific, we analyzed changes in gene expression after AdProx‐1 or AdLacZ infection of two additional endothelial cell types, namely human coronary artery endothelial cells (CAECs) and saphenous vein endothelial cells (SAVECs), as well as human amniotic epithelial cells (HUACs) as an example of a non‐endothelial cell type. In all these cell types, AdProx‐1 strongly upregulated levels of cyclins E1 and E2, histone H2B and PCNA. However, AdProx‐1 induced VEGFR‐3 only in CAECs and SAVECs, and not in HUACs (
Figure 5; data not shown). These data suggest that the capacity of Prox‐1 to induce cell proliferation is distinct from its role as a regulator of the lymphatic endothelial phenotype, as the latter most likely requires the presence of other endothelial‐specific transcriptional co‐activators.