Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin.

Podoplanin is a small transmembrane protein required for development and function of the lymphatic vascular system. To investigate the effects of interfering with its function, we produced an Fc fusion protein of its ectodomain. We found that podoplanin-Fc inhibited several functions of cultured lymphatic endothelial cells and also specifically suppressed lymphatic vessel growth, but not blood vessel growth, in mouse embryoid bodies in vitro and in mouse corneas in vivo. Using a keratin 14 expression cassette, we created transgenic mice that overexpressed podoplanin-Fc in the skin. No obvious outward phenotype was identified in these mice, but surprisingly, podoplanin-Fc-although produced specifically in the skin-entered the blood circulation and induced disseminated intravascular coagulation, characterized by microthrombi in most organs and by thrombocytopenia, occasionally leading to fatal hemorrhage. These findings reveal an important role of podoplanin in lymphatic vessel formation and indicate the potential of podoplanin-Fc as an inhibitor of lymphangiogenesis. These results also demonstrate the ability of podoplanin to induce platelet aggregation in vivo, which likely represents a major function of lymphatic endothelium. Finally, keratin 14 podoplanin-Fc mice represent a novel genetic animal model of disseminated intravascular coagulation.

Tumor lymphangiogenesis and metastasis to lymph nodes induced by cancer cell expression of podoplanin.

The membrane glycoprotein podoplanin is expressed by several types of human cancers and might be associated with their malignant progression. Its exact biological function and molecular targets are unclear, however. Here, we assessed the relevance of tumor cell expression of podoplanin in cancer metastasis to lymph nodes, using a human MCF7 breast carcinoma xenograft model. We found that podoplanin expression promoted tumor cell motility in vitro and, unexpectedly, increased tumor lymphangiogenesis and metastasis to regional lymph nodes in vivo, without promoting primary tumor growth. Importantly, high cancer cell expression levels of podoplanin correlated with lymph node metastasis and reduced survival times in a large cohort of 252 oral squamous cell carcinoma patients. Based on comparative transcriptional profiling of tumor xenografts, we identified endothelin-1, villin-1, and tenascin-C as potential mediators of podoplanin-induced tumor lymphangiogenesis and metastasis. These unexpected findings identify a novel mechanism of tumor lymphangiogenesis and metastasis induced by cancer cell expression of podoplanin, suggesting that reagents designed to interfere with podoplanin function might be developed as therapeutics for patients with advanced cancer.

Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions.

Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.

The lymphatic system in health and disease.

The lymphatic vascular system has an important role in the regulation of tissue pressure, immune surveillance and the absorption of dietary fat in the intestine. There is growing evidence that the lymphatic system also contributes to a number of diseases, such as lymphedema, cancer metastasis and different inflammatory disorders. The discovery of various molecular markers allowing the distinction of blood and lymphatic vessels, together with the availability of a increasing number of in vitro and in vivo models to study various aspects of lymphatic biology, has enabled tremendous progress in research into the development and function of the lymphatic system. This review discusses recent advances in our understanding of the embryonic development of the lymphatic vasculature, the molecular mechanisms mediating lymphangiogenesis in the adult, the role of lymphangiogenesis in chronic inflammation and lymphatic cancer metastasis, and the emerging importance of the lymphatic vasculature as a therapeutic target.

New insights into the molecular control of the lymphatic vascular system and its role in disease.

The cutaneous lymphatic system plays an important role in the maintenance of tissue fluid homeostasis, in the afferent phase of the immune response, and in the metastatic spread of skin cancers. However, the lymphatic system has not received as much scientific attention as the blood vascular system, largely due to a lack of lymphatic-specific markers and to the dearth of knowledge about the molecular regulation of its development and function. The recent identification of genes that specifically control lymphatic development and the growth of lymphatic vessels (lymphangiogenesis), together with the discovery of new lymphatic endothelium-specific markers, have now provided new insights into the molecular mechanisms that control lymphatic growth and function. Moreover, studies of several genetic mouse models have set the framework for a new molecular model for embryonic lymphatic vascular development, and have identified molecular pathways whose mutational inactivation leads to human diseases associated with lymphedema. These scientific advances have also provided surprising evidence that malignant tumors can directly promote lymphangiogenesis and lymphatic metastasis, and that lymphatic vessels play a major role in cutaneous inflammation and in the cutaneous response to UVB irradiation.

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.