Michael Detmar

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1−/− embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild‐type embryos, those of Prox1‐null embryos did not co‐express any lymphatic markers such as VEGFR‐3, LYVE‐1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.

T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

Within the vascular system, the mucin‐type transmembrane glycoprotein T1α/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic‐specific homeobox gene Prox1. In this study, we examined the role of T1α/podoplanin in vascular development and the effects of gene disruption in mice. T1α/podoplanin is first expressed at around E11.0 in Prox1‐positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1α/podoplanin−/− mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1α/podoplanin is also expressed in the basal epidermis of newborn wild‐type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1α/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA‐mediated inhibition of T1α/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1α/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors.

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.

Vascular permeability factor/vascular endothelial growth factor:A multifunctional angiogenic cytokine

VPF/VEGF is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On the one hand, VPF/VEGF stimulates the endothelial cells lining nearby microvessels to proliferate, to migrate and to alter their pattern of gene expression. On the other hand, VPF/VEGF renders these same microvascular endothelial cells hyperpermeable so that they spill plasma proteins into the extravascular space, leading to profound alterations in the extracellular matrix that favor angiogenesis. These same principles apply in tumors, in several examples of non-neoplastic pathology, and in physiological processes that involve angiogenesis and new stroma generation. In all of these examples, microvascular hyperpermeability and the introduction of a provisional, plasma-derived matrix precede and accompany the onset of endothelial cell division and new blood vessel formation. It would seem, therefore, that tumors have made use of fundamental pathways that developed in multicellular organisms for purposes of tissue defense, renewal and repair. VPF/VEGF, therefore, has taught us something new about angiogenesis; namely, that vascular hyperpermeability and consequent plasma protein extravasation are important--perhaps essential--elements in its generation. However, this finding raises a paradox. While VPF/VEGF induces vascular hyperpermeability, other potent angiogenic factors apparently do not, at least in sub-toxic concentrations that are more than sufficient to induce angiogenesis (Connolly et al., 1989a). Nonetheless, wherever angiogenesis has been studied, the newly generated vessels have been found to be hyperpermeable. How, therefore, do angiogenic factors other than VPF/VEGF lead to the formation of new and leaky blood vessels? We do not as yet have a complete answer to this question. One possibility is that at least some angiogenic factors mediate their effect by inducing or stimulating VPF/VEGF expression. In fact, there are already clear example of this. A number of putative angiogenic factors including small molecules (e.g. prostaglandins, adenosine) as well as many cytokines (e.g. TGF-alpha, bFGF, TGF-beta, TNF-alpha, KGF, PDGF) have all been shown to upregulate VPF/VEGF expression. Further studies that elucidate the crosstalk among various angiogenic factors are likely to contribute significantly to a better understanding of the mechanisms by which new blood vessels are formed in health and in disease.

Tumor Lymphangiogenesis : A Novel Prognostic Indicator for Cutaneous Melanoma Metastasis and Survival

Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. We investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.

Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.

Early during development, one of the first indications that lymphangiogenesis has begun is the polarized expression of the homeobox gene Prox1 in a subpopulation of venous endothelial cells. It has been shown previously that Prox1 expression in the cardinal vein promotes and maintains the budding of endothelial cells that will form the lymphatic vascular system. Prox1‐deficient mice are devoid of lymphatic vasculature, and in these animals endothelial cells fail to acquire the lymphatic phenotype; instead, they remain as blood vascular endothelium. To investigate whether Prox1 is sufficient to induce a lymphatic fate in blood vascular endothelium, Prox1 cDNA was ectopically expressed by adenoviral gene transfer in primary human blood vascular endothelial cells and by transient plasmid cDNA transfection in immortalized microvascular endothelial cells. Transcriptional profiling combined with quantitative real‐time reverse transcription‐polymerase chain reaction and Western blotting analyses revealed that Prox1 expression up‐regulated the lymphatic endothelial cell markers podoplanin and vascular endothelial growth factor receptor‐3. Conversely, genes such as laminin, vascular endothelial growth factor‐C, neuropilin‐1, and intercellular adhesion molecule‐1, whose expression has been associated with the blood vascular endothelial cell phenotype, were down‐regulated. These results were confirmed by the use of specific antibodies against some of these markers in sections of embryonic and adult tissues. These findings validate our previous proposal that Prox1 is a key player in the molecular pathway leading to the formation of lymphatic vasculature and identify Prox1 as a master switch in the program specifying lymphatic endothelial cell fate. That a single gene product was sufficient to re‐program the blood vascular endothelium toward a lymphatic phenotype corroborates the close relationship between these two vascular systems and also suggests that during evolution, the lymphatic vasculature originated from the blood vasculature by the additional expression of only a few gene products such as Prox1.

Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.

In mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with important implications for future studies of vascular development and function.

Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.

Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo. Furthermore, VEGF-C induced chemotaxis of macrophages in vitro and in vivo, revealing a potential function of VEGF-C as an immunomodulator. Taken together, our results identify VEGF-C as multifunctional factor involved in regulating tumor lymphangiogenesis, angiogenesis, and immune response.

Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma–associated herpesvirus

Kaposi sarcoma is considered a neoplasm of lymphatic endothelium infected with Kaposi sarcoma–associated herpesvirus. It is characterized by the expression of lymphatic lineage–specific genes by Kaposi sarcoma tumor cells. Here we show that infection of differentiated blood vascular endothelial cells with Kaposi sarcoma–associated herpesvirus leads to their lymphatic reprogramming; induction of ∼70% of the main lymphatic lineage–specific genes, including PROX1, a master regulator of lymphatic development; and downregulation of blood vascular genes.