T1α/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

Abstract

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

“…Sequences corresponding to the silencing and the control RNA were manufactured by Invitrogen (Carlsbad, CA), and transfection protocol was performed exactly as described in Ref. 18 using Lipofectin.…”

“…For double transfections with small interfering RNA (siRNA) and plasmid, cells were transfected at a higher confluency (60%), and Lipofectin was used (instead of PrimeFect I reagent), with a combination of 100 nM siRNA and 2.5 g of plasmid per 100-mm dish, following the same protocol described for siRNA transfections in Ref. 18, allowing mixture to transfect the cells for 4 h.…”

“…The protocol for pull-down assays was based on our previous method described in Ref. 18, with slight modifications. Basically, cells plated onto p100 dishes were control siRNA-or podoplanin siRNA-transfected and incubated for 48 h. Cells were then starved overnight in EBM-2-0.5% FBS media and left untreated or treated with 50 ng/ml VEGF (or left in complete media without starvation).…”

“…Moreover, it is reported that in developing mouse embryo, interaction between podoplanin and platelets from the cardinal vein is required for the differentiation of the lymphatic system from the blood vascular system (36). We have previously reported that in human lung lymphatic microvascular endothelial (HMVEC-LLy) cells, early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression (18). Moreover, we (17) reported in abstract form that podoplanin is required for a full migratory response to VEGF in these cells and that complete reorientation of the microtubule-organizing center (MTOC) in response to VEGF in scratch wound assays, necessary for directional migration, is achieved only if the cells express podoplanin.…”

Developmental and pathological lymphangiogenesis: from models to human disease

“…Podoplanin is upregulated in the invasive front of many human carcinomas and promotes collective cell migration by Wlopodia formation via the downregulation of the activities of small Rho family GTPases (Wicki et al 2006). In contrast to the role in tumor cells, siRNA mediated knockdown of Podoplanin in lung LECs prevented activation of RhoA during capillary morphogenesis and impaired localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane (Navarro et al 2008). …”