D2-40 is a monoclonal antibody directed against human podoplanin, a transmembrane mucoprotein that is expressed in lymphatic endothelial cells. D2-40 reacts with an O-linked sialoglycoprotein (MW 40K) found on lymphatic endothelium, but does not react with blood vessel endothelium8
. Thus, D2-40 has been used as lymphatic endothelial marker to study lymph-angiogenesis in physiological and pathological tissue samples and has been proved to be helpful in determining lymphatic invasion in tumor tissues9-12
. Using D2-40 as a lymphatic marker, we examined D2-40/podoplanin expressions in the human placenta. Complementary immunostaining of blood vessels was obtained with established endothelial marker CD31. VEGFR-3 expression, a lymphatic-selective endothelial marker, was also examined for comparison. Interestingly, we found compartmental differences for D2-40 (podoplanin), CD31, and VEGFR-3 distribution in the placental villous tissue. D2-40 localized in the stroma exhibited a network plexiform distribution, CD31 was found in endothelium of villous core fetal vessels, and VEGFR-3 was localized to both trophoblasts and fetal vascular endothelium. The network pattern for D2-40 (podoplanin) expression in the placental stroma was additionally confirmed by examining D2-40 (podoplanin) expression in the 1st, 2nd, and 3rd trimester placental tissue sections (). We found evidence for abundant expression of podoplanin in the normal placental tissue.
Podoplanin/D2-40 is a mucin-type transmembrane glycoprotein which was originally found on the surface of rat glomerular epithelial cells (podocytes), loss of podoplanin has been linked to the flattening of foot processes that occurs in glomerular diseases13
. Podoplanin shows features of a membrane mucoprotein with conserved O-glycosylation sites. Although the precise biological function of podoplanin is not clear, podoplanin is known to be one of the most highly expressed lymphatic-specific transcripts in human lymphatic endothelial cells14
. Therefore, D2-40/podoplanin expression has been used to define lymphatic vascular density in tumor tissues associated with lymphatic metastasis and poor prognosis. One study has also shown that heavily O-glycosylated mucoproteins probably function as counter-receptors for adhesion molecules like selectins which mediate cell attachment15
, indicating mucoproteins may have important cell adhesion functions. While the placenta has no proven lymphatic vessels, the placenta has a high density blood vascular supply constantly undergoing dynamic angiogenesis throughout the first, second and early third trimesters. A well-functioning, fully developed placenta ensures a healthy fetus and a successful pregnancy. Although the biological function of podoplanin in the placenta is not known, based on the assigned role of this glycoprotein in interstitial fluid balance, placental podoplanin may support fetal vessel angiogenesis during placenta development within the villous stroma.
Placental villous tissue contains abundant mesenchymal and matrix channels, especially in the immature intermediate villi. Placental villous stroma is a unique channel-like structure16
. It consists of a network of cells and fibers with fetal vessels. Within the stroma, it forms a fluid compartment with Hofbauer cells suspended in the interspaces16
. Mesenchymal and matrix channels provide a path for Hofbauer cells to patrol the villous core. The distribution of D2-40/podoplanin in the stroma suggests that a lymphatic-like conductive network might exist in the human placenta. In addition to lymphatic endothelial cells and kidney podocytes, podoplanin is also expressed in germ cells, mesothelial cell, stromal reticular cells and many types of tumor tissues17
. At present, we did not know as to what cells (Hofbauer cells/macrophages or fibroblasts) produce or secrete podoplanin in the placenta, even though we did notice D2-40 positive cells within villous stroma. Further studies to identify the cell type(s) that express and produce D2-40/podoplanin would be worth pursuing. Nonetheless, the network plexus pattern of D2-40/podoplanin in the placenta villous core stroma is highly unique, since none of the matrix proteins (including laminin α1, α2, α3, α5, β1, β2, and γ1 chains and extradomain A, extradomain B, and oncofetal fibronectin) showed a similar distribution pattern as to podoplanin18
. Although D2-40/podoplanin is a marker for lymphatic endothelium, our data suggest that cells other than lymphatic endothelial cells express D2-40 within the placenta.
Interestingly, we found reduced D2-40/podoplanin expression in placentas from women with preeclampsia compared to those from normotensive pregnant controls. Reduced D2-40 expression is demonstrated by both immunostaining of villous tissue sections and by total protein expression of snap frozen placental tissue homogenates. It is well known that abnormal angiogenesis occurs in the placenta during preeclampsia. This has been demonstrated by reduced expression and production of placental growth factor (PlGF) and vascular endothelial growth factor (VEGF-A) and increased expressions and production of anti-angiogenic factors of sFlt-1 and sEng19, 20
. Defective trophoblast invasion and angiogenesis are believed to contribute significantly to the placental pathophysiology in preeclampsia.
Increased VEGFR-3 expression is another finding of the present study. VEGFR-3 is also considered a marker for lymphatic endothelium. In contrast to D2-40, we found that VEGFR-3 expression is localized in trophoblast cells and in villous core vessel endothelium. The pattern of VEGFR-3 expression is similar to LYVE-1 expression in the placental villous tissue as we previously reported4
. In addition, we also found increased VEGFR-3 expression in preeclamptic placentas compared to those from normotensive controls. The reason for increased VEGFR-3 expression in preeclamptic placentas is not clear. However, this could be linked to the hypoxia condition that is believed to be associated with placental pathophysiology in preeclampsia. Although direct evidence of VEGFR-3 up-regulation by hypoxia in the placenta is lacking, an in vitro
study did show that hypoxia could promote VEGFR-3 expression in MCF7 and A549 adenocarcinomal cells21
. In addition, hypoxia-driven vascular development requiring the activity of VEGFR-3 has also been demonstrated during mouse embryonic stem cell differentiation22
. Increased VEGFR-3 expression could be a compensative mechanism to overcome increased oxidative stress and promote placental angiogenesis in the preeclamptic placenta.
At present, the outcome of reduced D2-40 in preeclamptic placenta is not clear. However, reduced D2-40 expression in quantity and quality may contribute to placental dysfunction in preeclampsia. Reduced D2-40 expression may lead to failure of interstitial fluid conduction, and subsequently increase in interstitial fluid accumulation, pressure, and edema. Poor placental tissue perfusion in preeclampsia might provoke higher perfusion pressures, which are pathognomonic for preeclampsia. Whether reduced D2-40/podoplanin sialoglycoprotein expression and/or dysregulation of the protein function contribute to improper trophoblast invasion, abnormal placental angiogenesis, and maladaptive immunity in preeclamptic placenta warrant further investigation.