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In: J. Biol. Chem., Vol. 284, Issue 6, 3935-3945, February 6, 2009

A Mechanism of Sialylation Functionally Silences the Hyaluronan Receptor LYVE-1 in Lymphatic Endothelium

Thomas D. Nightingale, Matthew E. F. Frayne, Steven Clasper, Suneale Banerji, and David G. Jackson

The major lymphatic endothelial hyaluronan receptor LYVE-1, a Link superfamily glycoprotein similar to the hyaluronan-binding/inflammatory leukocyte homing receptor CD44, was initially implicated in hyaluronan (HA)-mediated cell adhesion and lymph-borne hyaluronan metabolism. However, the apparently normal phenotype of Lyve-1 knock-out mice and the recent demonstration that the receptor undergoes cytokine-induced endocytosis independent of HA uptake have cast doubt on such functions. Here we present new data that reconcile these anomalies by showing that LYVE-1 is functionally "silenced" in a cell-specific fashion by autoinhibitory glycosylation. We demonstrate that LYVE-1 transfected in HEK 293T fibroblasts and Jurkat T cells is competent to bind HA, whereas the endogenous receptor in cultured lymphatic endothelial cells or the receptor transfected in Chinese hamster ovary and HeLa cells is not. Moreover, through a combination of mutagenesis and functional analysis in HEK 293T fibroblasts and glycosylation-defective Chinese hamster ovary cell lines, we reveal that the inhibitory mechanism is reversible and is exerted by terminal sialylation, most likely through {alpha}2-3 or {alpha}2-6 linkage to O-glycans. Finally, we provide evidence that the mechanism operates in vivo by showing that native LYVE-1 in primary lymphatic endothelial cells is extensively sialylated and that HA binding can be reactivated by neuraminidase treatment of the soluble ectodomain. These results reveal unexpected complexity in the regulation of LYVE-1 function and raise the possibility that this receptor, like CD44, may become active after appropriate unmasking in vivo.

In: Genes & Dev. 2008. 22: 3282-3291

Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity

Nicole C. Johnson, Miriam E. Dillard, Peter Baluk, Donald M. McDonald, Natasha L. Harvey, Sharon L. Frase, Guillermo Oliver

The activity of the homeobox gene Prox1 is necessary and sufficient for venous blood endothelial cells (BECs) to acquire a lymphatic endothelial cell (LEC) fate. We determined that the differentiated LEC phenotype is a plastic, reprogrammable condition that depends on constant Prox1 activity for its maintenance. We show that conditional down-regulation of Prox1 during embryonic, postnatal, or adult stages is sufficient to reprogram LECs into BECs. Consequently, the identity of the mutant lymphatic vessels is also partially reprogrammed as they acquire some features typical of the blood vasculature. siRNA-mediated down-regulation of Prox1 in LECs in culture demonstrates that reprogramming of LECs into BECs is a Prox1-dependent, cell-autonomous process. We propose that Prox1 acts as a binary switch that suppresses BEC identity and promotes and maintains LEC identity; switching off Prox1 activity is sufficient to initiate a reprogramming cascade leading to the dedifferentiation of LECs into BECs. Therefore, LECs are one of the few differentiated cell types that require constant expression of a certain gene to maintain their phenotypic identity.

accompanying editorial: Genes & Dev. 2008. 22: 3232-3235

Master and commander: continued expression of Prox1 prevents the dedifferentiation of lymphatic endothelial cells

M. Gabriele Bixel, Ralf H. Adams

Cell differentiation occurs mostly during a specific developmental time window and is irreversible. The homeobox-containing transcription factor Prox1 is a master regulator of lymphatic endothelial cell differentiation in the embryo. A study by Johnson et al. (3282–3291) published in this issue of Genes & Development now shows that continued expression of Prox1 is required to maintain lymphatic endothelial cell identity even in adult mice. These findings indicate that Prox1 is essential for the differentiation and function of the lymphatic vasculature throughout life.

In: American Journal of Pathology. 2008;173:1891-1901

Transgenic Induction of Vascular Endothelial Growth Factor-C Is Strongly Angiogenic in Mouse Embryos but Leads to Persistent Lymphatic Hyperplasia in Adult Tissues

Marja Lohela, Hanna Heloterä, Paula Haiko, Daniel J. Dumont and Kari Alitalo

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.

In: Cardiovasc Res. 2008 May 1;78(2):315-23. Epub 2007 Dec 7

VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis

Andrew V. Benest, Steven J. Harper, Seppo Yla Herttuala, Kari Alitalo and David O. Bates

Aims: Vascular endothelial growth factor-C (VEGF-C) has been shown to stimulate both angiogenesis and lymphangiogenesis in some but not all models where VEGF-C is over-expressed. Our aim was to investigate the interaction between lymphangiogenesis and angiogenesis in adult tissues regulated by VEGF-C and identify evidence of polarized growth of lymphatics driven by specialized cells at the tip of the growing sprout.

Methods and results: We used an adult model of lymphangiogenesis in the rat mesentery. The angiogenic effect of VEGF-C was markedly attenuated in the presence of a growing lymphatic network. Furthermore, we show that this growth of lymphatic vessels can occur both by recruitment of isolated lymphatic islands to a connected network and by filopodial sprouting. The latter is independent of polarized tip cell differentiation that can be generated all along lymphatic capillaries, independently of the proliferation status of the lymphatic endothelial cells.

Conclusion: These results both demonstrate a dependence of VEGF-C-mediated angiogenesis on lymphatic vascular networks and indicate that the mechanism of VEGF-C-mediated lymphangiogenesis is different from that of classical angiogenic mechanisms.

In: Nature Methods Published online: 6 April 2008; | doi:10.1038/nmeth.1198

Spheroid-based engineering of a human vasculature in mice

Abdullah Alajati, Anna M Laib, Holger Weber, Anja M Boos, Arne Bartol, Kristian Ikenberg, Thomas Korff, Hanswalter Zentgraf, Cynthia Obodozie, Ralph Graeser, Sven Christian, Günter Finkenzeller, G Björn Stark, Mélanie Héroult & Hellmut G Augustin

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

In: Cancer Science Volume 99 Issue 3 Page 441-450, March 2008

Sentinel lymph node as a target of molecular diagnosis of lymphatic micrometastasis and local immunoresponse to malignant cells

Hiroya Takeuchi, Masaki Kitajima and Yuko Kitagawa

The sentinel lymph node (SLN) is defined as the lymph node(s) first receiving lymphatic drainage from the site of the primary tumor. The histopathological status of SLN is one of the most significant predictors of recurrence and overall survival for most clinical stage I/II solid tumors. Recent progress in molecular techniques has demonstrated the presence of micrometastatic tumor cells in SLN. There is now a growing body of data to support the clinical relevance of SLN micrometastasis in a variety of solid tumors. Increasing the sensitivity of occult tumor cell detection in the SLN, using molecular-based analysis, should enable a more accurate understanding of the clinical significance of various patterns of micrometastatic nodal disease. The establishment of metastasis to SLN might not be simply reflected by the flow dynamics of lymphatic fluid that drains from the primary site to the SLN, and the transportation of viable cancer cells. Recent studies have demonstrated that primary tumors can actively induce lymphangiogenesis and promote SLN metastasis. Moreover chemokine receptors in tumor cells may facilitate organ-specific tumor metastasis in many human cancers and some experimental models. In contrast, recent clinical and preclinical studies regard SLN as the first lymphoid organ to respond to tumor antigenic stimulation. SLN dramatically show morphological, phenotypical and functional changes that indicate immune suppression by tumor cells. The immune suppression in SLN results in failure of prevention or eradication of tumor metastasis. The mechanism of immunomodulation remains unclear; however, several regulatory molecules produced by tumor cells and tumor-associated macrophages or lymphocytes are likely to be responsible for inducing the immune suppression in SLN. Further studies may develop a novel immunotherapy that overcomes tumor-induced immune suppression and can prevent or eradicate SLN metastasis.

In: Cancer Research 68, 1100-1109, February 15, 2008

Profound but Dysfunctional Lymphangiogenesis via Vascular Endothelial Growth Factor Ligands from CD11b+ Macrophages in Advanced Ovarian Cancer

Bong-Hyun Jeon, Cholsoon Jang, Jinah Han, Raghu P. Kataru, Lianhua Piao, Keehoon Jung, Hye Ji Cha, Reto A. Schwendener, Kyu Yun Jang, Kwan-Sik Kim, Kari Alitalo and Gou Young Koh

Severe ascites is a hallmark of advanced ovarian cancer (OVCA), yet the underlying mechanism that creates an imbalance between peritoneal vascular leakage and lymphatic drainage is unknown. Here, we identified and characterized peritoneal lymphatic vessels in OVCA mice, a model generated by implantation of human OVCA cells into athymic nude mice. The OVCA mice displayed substantial lymphangiogenesis and lymphatic remodeling, massive infiltration of CD11b+/LYVE-1+ macrophages and disseminated carcinomatosis in the mesentery and diaphragm, and progressive chylous ascites formation. Functional assays indicated that the abnormally abundant lymphatic vessels in the diaphragm were not conductive in peritoneal fluid drainage. Moreover, lipid absorbed from the gut leaked out from the aberrant mesenteric lymphatic vessels. Our results indicate that vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF-A from CD11b+ macrophages are responsible for producing OVCA-induced dysfunctional lymphangiogenesis, although other cell types contribute to the increased ascites formation. Accordingly, the combined blockade of VEGF-C/D and VEGF-A signaling with soluble VEGF receptor-3 and VEGF-Trap, respectively, markedly inhibited chylous ascites formation. These findings provide additional therapeutic targets to ameliorate chylous ascites formation in patients with advanced OVCA.

In: American Journal of Pathology. 2008;172:486-494; Published online before print January 10, 2008

Endosialin (Tem1) Is a Marker of Tumor-Associated Myofibroblasts and Tumor Vessel-Associated Mural Cells

Sven Christian, Renate Winkler, Iris Helfrich, Anja M. Boos, Eva Besemfelder, Dirk Schadendorf and Hellmut G. Augustin

Endosialin (Tem1) has been identified by two independent experimental approaches as an antigen of tumor-associated endothelial cells, and it has been claimed to be the most abundantly expressed tumor endothelial antigen, making it a prime candidate for vascular targeting purposes. Recent experiments have challenged the endothelial expression of endosialin and suggested an expression by activated fibroblasts and pericytes. Thus, clarification of the controversial cellular expression of endosialin is critically important for an understanding of its role during tumor progression and its validation as a potential therapeutic target. We have therefore performed extensive expression profiling analyses of endosialin. The experiments unambiguously demonstrate that endosialin is expressed by tumor-associated myofibroblasts and mural cells and not by endothelial cells. Endosialin expression is barely detectable in normal human tissues with moderate expression only detectable in the stroma of the colon and the prostate. Corresponding cellular experiments confirmed endosialin expression by mesenchymal cells and indicated that it may in fact be a marker of mesenchymal stem cells. Silencing endosialin expression in fibroblasts strongly inhibited migration and proliferation. Collectively, the experiments validate endosialin as a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. The data warrant further functional analysis of endosialin during tumor progression and its exploitation as marker of tumor vessel-associated mural cells, expression of which may reflect the non-normalized phenotype of the tumor vasculature.

In: The FASEB Journal; Published online before print January 7, 2008 as doi: 10.1096/fj.07-100529

Identification of the surface-accessible, lineage-specific vascular proteome by two-dimensional peptide mapping

Christoph Roesli, Viviane Mumprecht, Dario Neri, and Michael Detmar

The formation of blood vessels (angiogenesis) and of lymphatic vessels (lymphangiogenesis) actively contributes to cancer progression and inflammation. Thus, there has been a quest for identifying the molecular mechanisms that control lymphatic and blood vessel formation and function. Membrane and extracellular matrix proteins can serve as suitable targets for imaging and/or therapeutic targeting; however, conventional proteomic technologies often fail to identify them systematically due to insolubility in water and low abundance of membrane proteins. To circumvent this problem, we applied a gel-free proteomics methodology termed two-dimensional peptide mapping (2D-PM) to cultured blood vascular (BECs) and lymphatic (LECs) endothelial cells. 2D-PM comprises biotinylation of surface-accessible proteins, their selective enrichment, separation by HPLC, and analysis by mass spectrometry. We identified 184 proteins that were specifically or predominantly expressed by LECs and 185 proteins specifically expressed by BECs, whereas 377 additional proteins were equally detected in both cell types. For representative proteins, the differential, lineage-specific expression was confirmed by Western analyses of cultured cells and by differential immunofluorescence analyses of tissue samples. Our results identify the surface-accessible, vascular lineage-specific proteome, and they also reveal 2D-PM as a powerful technology for the large-scale screening of lineage-specific protein expression.—Roesli, C., Mumprecht, V., Neri, D., Detmar, M. Identification of the surface-accessible, lineage-specific vascular proteome by two-dimensional peptide mapping.

In: J. Clin. Invest. 118(1): 40-50 (2007) published January 2, 2008

Adrenomedullin signaling is necessary for murine lymphatic vascular development

Kimberly L. Fritz-Six, William P. Dunworth, Manyu Li and Kathleen M. Caron

The lymphatic vascular system mediates fluid homeostasis, immune defense, and tumor metastasis. Only a handful of genes are known to affect the development of the lymphatic vasculature, and even fewer represent therapeutic targets for lymphatic diseases. Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through the calcitonin receptor–like receptor (calcrl) when the receptor is associated with a receptor activity–modifying protein (RAMP2). Here we report on the involvement of these genes in lymphangiogenesis. AM-, calcrl-, or RAMP2-null mice died mid-gestation after development of interstitial lymphedema. This conserved phenotype provided in vivo evidence that these components were required for AM signaling during embryogenesis. A conditional knockout line with loss of calcrl in endothelial cells confirmed an essential role for AM signaling in vascular development. Loss of AM signaling resulted in abnormal jugular lymphatic vessels due to reduction in lymphatic endothelial cell proliferation. Furthermore, AM caused enhanced activation of ERK signaling in human lymphatic versus blood endothelial cells, likely due to induction of CALCRL gene expression by the lymphatic transcriptional regulator Prox1. Collectively, our studies identify a class of genes involved in lymphangiogenesis that represent a pharmacologically tractable system for the treatment of lymphedema or inhibition of tumor metastasis.

In: Oncogene 2007 Dec 3 [Epub ahead of print]

p19/Arf and p53 suppress sentinel lymph node lymphangiogenesis and carcinoma metastasis

A Ruddell, K S Kelly-Spratt, M Furuya, S S Parghiand C J Kemp

The ability of tumor cells to metastasize is increasingly viewed as an interaction between the primary tumor and host tissues. Deletion of the p19/Arf or p53 tumor suppressor genes accelerates malignant progression and metastatic spread of 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced squamous cell carcinomas, providing a model system to address mechanisms of metastasis. Here, we show that benign pre-metastatic papillomas from wild-type mice trigger lymphangiogenesis within draining lymph nodes, whereas there is no growth of primary tumor lymphatic vessels. Lymph node lymphangiogenesis is greatly accelerated in papilloma-bearing p19/Arf- or p53-deficient mice, which coincides with the greater propensity of these tumors to progress to carcinomas and to metastasize. The extent of accumulation of B cells within the tumor-draining lymph nodes of wild-type mice predicted the level of lymph node lymphangiogenesis and metastatic potential. Arf or p53 deficiency strongly accelerated lymph node immune cell accumulation, in a manner that was associated with the extent of lymph node lymphatic sinus growth. This immune cell accumulation and lymph node lymphangiogenesis phenotype identifies host anti-tumor responses that could drive metastatic spread of cancers via the lymphatics.

In: Cancer Research 67, 11528-11535, December 15, 2007

Significance of Tumor-Associated Stroma in Promotion of Intratumoral Lymphangiogenesis

Pivotal Role of a Hyaluronan-Rich Tumor Microenvironment

Hiroshi Koyama, Nobutaka Kobayashi, Michihiko Harada, Michiko Takeoka, Yoshiko Kawai, Kenji Sano, Minoru Fujimori, Jun Amano, Toshio Ohhashi, Reiji Kannagi, Koji Kimata, Shun’ichiro Taniguchi and Naoki Itano

Stromal cells, together with extracellular matrix components, provide a tumor microenvironment that is pivotal for cancer cell growth and progression. In our previous study using a conditional transgenic mouse model of breast cancer, the overproduction of hyaluronan, a major extracellular constituent, accelerated tumor angiogenesis through stromal cell recruitment. This finding led us to investigate the role of hyaluronan in the lymphatic vessel system. Here, we have found that microenvironmental hyaluronan promoted tumor lymphangiogenesis concurrently with the formation of stromal structures. Additionally, lymphatic vessels frequently penetrated and accumulated into stromal compartments, and up-regulation of vascular endothelial growth factor-C and -D was detected at tumor-stromal interfaces. To assess the contribution of stromal cells to lymphangiogenesis in vivo, we established tumor-associated fibroblasts from hyaluronan-overproducing mammary tumors and implanted them together with carcinoma cells from control tumors or MCF-7 human breast carcinoma cells in nude mice. Carcinoma cells grew rapidly in association with marked stromal reactions and lymphangiogenesis. Without the stromal cells, however, the tumors developed slowly with less stroma and lymphatic vessels. These findings underline the significance of tumor-associated stroma in the promotion of intratumoral lymphangiogenesis and suggest a pivotal role for the hyaluronan-rich tumor microenvironment.

In: Cancer Research 67, 11528-11535, December 15, 2007

Endostatin Overexpression Inhibits Lymphangiogenesis and Lymph Node Metastasis in Mice

Gaëlle Brideau, Markus J. Mäkinen, Harri Elamaa, Hongmin Tu, Gunnar Nilsson, Kari Alitalo, Taina Pihlajaniemi and Ritva Heljasvaara

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and control mice, nor in the rate of conversion of benign papillomas to malignant squamous cell carcinomas (SCC). We did find, however, that endostatin regulates the terminal differentiation of keratinocytes because the SCCs in the J4 mice were less aggressive and more often well differentiated than those in the control mice. We observed an inhibition of tumor angiogenesis by endostatin at an early stage in skin tumor development, but more strikingly, there was a significant reduction in lymphatic vessels in the papillomas and SCCs in association with elevated endostatin levels and also a significant inhibition of lymph node metastasis in the J4 mice. We showed that tumor-infiltrating mast cells strongly expressed vascular endothelial growth factor-C (VEGF-C), and that the accumulation of these cells was markedly decreased in the tumors of the J4 mice. Moreover, endostatin inhibited the adhesion and migration of murine MC/9 mast cells on fibronectin in vitro. Our data suggest that endostatin can inhibit tumor lymphangiogenesis by decreasing the VEGF-C levels in the tumors, apparently via inhibition of mast cell migration and adhesion, and support the view that the biological effects of endostatin are not restricted to endothelial cells because endostatin also regulates tumor-associated inflammation and differentiation, and the phenotype of epithelial tumors.

In: Nature Medicine 13, 1458 - 1466 (2007); Published online: 2 December 2007

Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation

Tuomas Tammela, Anne Saaristo, Tanja Holopainen, Johannes Lyytikkä, Anna Kotronen, Miia Pitkonen, Usama Abo-Ramadan, Seppo Ylä-Herttuala, Tatiana V Petrova & Kari Alitalo

Surgery or radiation therapy of metastatic cancer often damages lymph nodes, leading to secondary lymphedema. Here we show, using a newly established mouse model, that collecting lymphatic vessels can be regenerated and fused to lymph node transplants after lymph node removal. Treatment of lymph node–excised mice with adenovirally delivered vascular endothelial growth factor-C (VEGF-C) or VEGF-D induced robust growth of the lymphatic capillaries, which gradually underwent intrinsic remodeling, differentiation and maturation into functional collecting lymphatic vessels, including the formation of uniform endothelial cell-cell junctions and intraluminal valves. The vessels also reacquired pericyte contacts, which downregulated lymphatic capillary markers during vessel maturation. Growth factor therapy improved the outcome of lymph node transplantation, including functional reconstitution of the immunological barrier against tumor metastasis. These results show that growth factor–induced maturation of lymphatic vessels is possible in adult mice and provide a basis for future therapy of lymphedema.

In: Cancer Research 67, 10840-10848, November 15, 2007

Placental Growth Factor-1 Attenuates Vascular Endothelial Growth Factor-A–Dependent Tumor Angiogenesis during ß Cell Carcinogenesis

Tibor Schomber, Lucie Kopfstein, Valentin Djonov, Imke Albrecht, Vanessa Baeriswyl, Karin Strittmatter and Gerhard Christofori

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the ß cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic ß cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the ß tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

In: Blood, 1 November 2007, Vol. 110, No. 9, pp. 3158-3167.

VEGF-A produced by chronically inflamed tissue induces lymphangiogenesis in draining lymph nodes

Cornelia Halin, Nadja E. Tobler, Benjamin Vigl, Lawrence F. Brown, and Michael Detmar

Lymphangiogenesis is involved in tumor cell metastasis and plays a major role in chronic inflammatory disorders. To investigate the role of lymphangiogenesis in inflammation, we induced and maintained delayed-type hypersensitivity (DTH) reactions in the ears of mice and then analyzed the resulting lymphangiogenesis in the inflamed tissue and draining lymph nodes (LNs) by quantitative fluorescence-activated cell sorting (FACS) and by immunofluorescence. Long-lasting inflammation induced a significant increase in the number of lymphatic endothelial cells, not only in the inflamed ears but also in the ear-draining auricular LNs. Inflammation-induced lymphangiogenesis was potently blocked by systemic administration of a vascular endothelial growth factor (VEGF)-A neutralizing antibody. Surprisingly, tissue inflammation specifically induced LN lymphangiogenesis but not LN angiogenesis. These findings were explained by analysis of both VEGF-A protein and mRNA levels, which revealed that VEGF-A was expressed at high mRNA and protein levels in inflamed ears but that expression was increased only at the protein level in activated LNs. Inflammation-induced lymphangiogenesis in LNs was independent of the presence of nodal B lymphocytes, as shown in B cell-deficient mice. Our data reveal that chronic inflammation actively induces lymphangiogenesis in LNs, which is controlled remotely, by lymphangiogenic factors produced at the site of inflammation.

In: Human Molecular Genetics 2007 Oct.15

Genetic causes of vascular malformations

Pascal Brouillard and Miikka Vikkula

Vascular malformations are localized defects of vascular development. They usually affect a limited number of vessels in a restricted area of the body. Although most malformations are sporadic, inheritance is observed, enabling genetic analysis. Usually, sporadic forms present with a single lesion whereas multiple lesions are observed in familial cases. The last decade has seen unraveling of several causative genes and beginning of elucidation of the pathophysiological pathways involved in the inherited forms. In parallel, definition of the clinical phenotypes has improved and disorders such as Parkes-Weber syndrome, first thought to be sporadic, is now known to be part of a more common inheritable phenotype. In addition, the concept of double-hit mechanism that we proposed earlier to explain the incomplete penetrance, variable expressivity and multifocality of lesions in inherited venous anomalies is now becoming confirmed, as some somatic mutations have been identified in venous, Glomuvenous and cerebral cavernous malformations. It is thus tempting to suggest that familial forms of vascular malformations follow paradominant inheritance and that sporadic forms, the etiopathogenic causes of which are still unelucidated, are caused by somatic mutations in the same genes.

In: BMC Bioinformatics 2007, 8:418, October 2007

HeliCis: a DNA motif discovery tool for colocalized motif pairs with periodic spacing

Erik Larsson, Per Lindahl and Petter Mostad

Background Correct temporal and spatial gene expression during metazoan development relies on combinatorial interactions between different transcription factors. As a consequence, cis-regulatory elements often colocalize in clusters termed cis-regulatory modules. These may have requirements on organizational features such as spacing, order and helical phasing (periodic spacing) between binding sites. Due to the turning of the DNA helix, a small modification of the distance between a pair of sites may sometimes drastically disrupt function, while insertion of a full helical turn of DNA (10–11 bp) between cis elements may cause functionality to be restored. Recently, de novo motif discovery methods which incorporate organizational properties such as colocalization and order preferences have been developed, but there are no tools which incorporate periodic spacing into the model.

Results We have developed a web based motif discovery tool, HeliCis, which features a flexible model which allows de novo detection of motifs with periodic spacing. Depending on the parameter settings it may also be used for discovering colocalized motifs without periodicity or motifs separated by a fixed gap of known or unknown length. We show on simulated data that it can efficiently capture the synergistic effects of colocalization and periodic spacing to improve detection of weak DNA motifs. It provides a simple to use web interface which interactively visualizes the current settings and thereby makes it easy to understand the parameters and the model structure.

Conclusion HeliCis provides simple and efficient de novo discovery of colocalized DNA motif pairs, with or without periodic spacing. Our evaluations show that it can detect weak periodic patterns which are not easily discovered using a sequential approach, i.e. first finding the binding sites and second analyzing the properties of their pairwise distances.

In: GENES & DEVELOPMENT 21:2433-2447, 2007

Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis

Bruno Larrivée, Catarina Freitas, Marc Trombe, Xiang Lv, Benjamin DeLafarge, Li Yuan, Karine Bouvrée, Christiane Bréant, Raquel Del Toro, Nicolas Bréchot, Stéphane Germain, Françoise Bono4, Frédérique Dol, Filip Claes, Christian Fischer, Monica Autiero, Jean-Léon Thomas, Peter Carmeliet, Marc Tessier-Lavigne, and Anne Eichmann

Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target.

In: Clinical Cancer Research 13, 3696-3705, June 15, 2007

[Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 Is a Highly Efficient Radiotherapeutic for Glucagon-Like Peptide-1 Receptor–Targeted Therapy for Insulinoma

Andreas Wicki, Damian Wild, Daniel Storch, Christian Seemayer, Martin Gotthardt, Martin Behe, Stefan Kneifel, Michael J. Mihatsch, Jean-Claude Reubi, Helmut R. Mäcke and Gerhard Christofori

Purpose: Although metabolic changes make diagnosis of insulinoma relatively easy, surgical removal is hampered by difficulties in locating it, and there is no efficient treatment for malignant insulinoma. We have previously shown that the high density of glucagon-like peptide-1 receptors (GLP-1R) in human insulinoma cells provides an attractive target for molecular imaging and internal radiotherapy. In this study, we investigated the therapeutic potential of [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4, an 111In-labeled agonist of GLP-1, in a transgenic mouse model of human insulinoma.

Experimental Design: [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 was assessed in the Rip1Tag2 mouse model of pancreatic ß-cell carcinogenesis, which exhibits a GLP-1R expression comparable with human insulinoma. Mice were injected with 1.1, 5.6, or 28 MBq of the radiopeptide and sacrificed 7 days after injection. Tumor uptake and response, the mechanism of action of the radiopeptide, and therapy toxicity were investigated.

Results: Tumor uptake was >200% injected activity per gram, with a dose deposition of 3 Gy/MBq at 40 pmol [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4. Other GLP-1R–positive organs showed ≥30 times lower dose deposition. A single injection of [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 resulted in a reduction of the tumor volume by up to 94% in a dose-dependent manner without significant acute organ toxicity. The therapeutic effect was due to increased tumor cell apoptosis and necrosis and decreased proliferation.

Conclusions: The results suggest that [Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a promising radiopeptide capable of selectively targeting insulinoma. Furthermore, Auger-emitting radiopharmaceuticals such as 111In are able to produce a marked therapeutic effect if a high tumor uptake is achieved.

In: Nephron Exp Nephrol 2007;106:e32-e36, published online June 6 2007

The Glomerular Transcriptome and Proteome

Christer Betsholtz, Liqun Hea, Minoru Takemotoa, Jenny Norlina, Ying Suna, Jaakko Patrakkaa, Karl Tryggvasona

Histopathology provides the current basis for classification and diagnosis of glomerular disorders. Molecular profiling methods, such as microarray analysis of mRNA expression, have rapidly emerged over the past years and are now applicable to minute amounts of tissue material, such as glomeruli from embryos or adult experimental animals, or from human renal needle biopsies. This review summarizes current efforts aiming at the determination of the glomerular transcriptome and proteome during development, in the healthy adult, and in disease. These studies are encouraging and show that comprehensive molecular profiling of the kidney glomerulus will most likely provide significant new insights into the normal structure and function of the glomerular filter, the molecular mechanisms of glomerular development, the diagnosis and classification of glomerular disease, and the pathogenic mechanisms underlying the stepwise breakdown of glomerular filter function that accompanies several common systemic disorders.

In: Blood, 1 June 2007, Vol. 109, No. 11, pp. 4777-4785


Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment


Stefan Amatschek, Ernst Kriehuber, Wolfgang Bauer, Barbel Reininger, Paul Meraner, Alois Wolpl, Norbert Schweifer, Christian Haslinger, Georg Stingl, and Dieter Maurer


The discovery of marker proteins of human blood (BECs) and lymphatic endothelial cells (LECs) has allowed researchers to isolate these cells. So far, efforts to unravel their transcriptional and functional programs made use of cultured cells only. Hence, it is unknown to which extent previously identified LEC- and BEC-specific programs are representative of the in vivo situation. Here, we define the human BEC- and LEC-specific in vivo transcriptomes by comparative genomewide expression profiling of freshly isolated cutaneous EC subsets and of non-EC skin cells (fibroblasts, mast cells, dendritic cells, epithelial cells). Interestingly, the expression of most of the newly identified EC subset-discriminating genes depends strictly on the in vivo tissue environment as revealed by comparative analyses of freshly isolated and cultured EC subsets. The identified environment-dependent, EC subset-restricted gene expression regulates lineage fidelity, fluid exchange, and MHC class II–dependent antigen presentation. As an example for a BEC-restricted in vivo function, we show that non-activated BECs in situ, but not in vitro, assemble and display MHC class II protein complexes loaded with self-peptides. Thus, our data demonstrate the key importance of using precisely defined native ECs for the global identification of in vivo relevant cell functions.

In: The EMBO Journal (2007) 26, 2832–2842; published online May 31, 2007

Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function

Angelika Kren, Vanessa Baeriswyl, François Lehembre, Christoph Wunderlin, Karin Strittmatter, Helena Antoniadis, Reinhard Fässler, Ugo Cavallaro and Gerhard Christofori

Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization, as well as transduction of signals into cells, to promote various aspects of cellular behavior, such as proliferation or survival. Integrins participate in many aspects of tumor biology. Here, we have employed the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis to investigate the role of beta1-integrin in tumor progression. Specific ablation of beta1-integrin function in pancreatic beta cells resulted in a defect in sorting between insulin-expressing beta cells and glucagon-expressing alpha cells in islets of Langerhans. Ablation of beta1-integrin in beta tumor cells of Rip1Tag2 mice led to the dissemination of tumor cell emboli into lymphatic blood vessels in the absence of ongoing lymphangiogenesis. Yet, disseminating beta1-integrin-deficient beta tumor cells did not elicit metastasis. Rather, primary tumor growth was significantly impaired by reduced tumor cell proliferation and the acquisition of cellular senescence by beta1-integrin-deficient beta tumor cells. The results indicate a critical role of beta1-integrin function in mediating metastatic dissemination and preventing tumor cell senescence.

In: Kidney International (2007) 71, 889–900, published online Feb. 28, 2007

Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis

L He, Y Sun, J Patrakka, P Mostad, J Norlin, Z Xiao, J Andrae, K Tryggvason, T Samuelsson, C Betsholtz and M Takemoto

The kidney glomerulus plays a crucial role in blood filtration but the molecular composition and physiology of the glomerulus is not well understood. We previously constructed and large-scale sequenced four mouse glomerular expressed sequence tag (EST) libraries from newborn and adult mouse glomeruli. Here, we compared glomerular EST profiles with whole kidney EST profiles, thereby identifying 497 transcripts corresponding to UniGene clusters that were glomerulus-enriched, that is expressed more abundantly in glomeruli than in whole kidney. These include several known protein-coding glomerulus-specific transcripts critical for glomerulus development and function, but also a large number of gene transcripts, which have not previously been shown to be expressed in the glomerulus, or implicated in glomerular functions. We used in situ hybridization to demonstrate glomerulus-specific RNA expression for six novel glomerular genes and the public Human Protein Atlas to verify glomerular protein expression for another two. The higher mRNA abundance for the eight genes in glomeruli compared with whole kidney was also verified by Taqman quantitative polymerase chain reaction. We surmise that the further characterization of these genes and proteins will increase our understanding of glomerular development and physiology.

In: Cancer Research 67, 5221-5230, June 1, 2007


Distinctive Features Of Angiogenesis And Lymphangiogenesis Determine Their Functionality During De Novo Tumor Development

Alexandra Eichten, William C. Hyun, and Lisa M. Coussens

Kallikrein 4 (KLK4) is a member of the human tissue KLK family. Whereas all other KLKs are secreted proteins with extracellular functions, KLK4 is primarily localized to the nucleus, indicating that it has a different function compared with other members of the KLK family. In addition, KLK4 expression is highly enriched in the prostate and is regulated by androgens. Here, we studied the possible functional role of KLK4 in prostate cancer cells and examined its expression at the protein level in prostate cancer specimens. Consistent with its mRNA expression, KLK4 protein is significantly overexpressed in malignant prostate compared with normal prostate. KLK4 expression is predominantly in the nucleus of basal cells in the prostate epithelium in keeping with its distribution in prostate cancer cells in vitro. Furthermore, adenovirus-mediated expression of KLK4 dramatically induces proliferation of prostate cancer cells, at least in part through significant alterations in cell cycle regulatory gene expression. Consistent with these data, small interfering RNA–mediated knockdown of endogenous KLK4 in LNCaP prostate cancer cells inhibits cell growth. These data identify KLK4 as the first member of the KLK family that is a proliferative factor with effects on gene expression and indicate that it may have an important role in prostate cancer development and progression.     

In: The Journal of Cell Biology, Vol. 177, No. 5, 751-755, published online May 29, 2007

Building blood vessels—stem cell models in vascular biology

Lars Jakobsson, Johan Kreuger, and Lena Claesson-Welsh

Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell–based models for the study of human vascular development.

In: J. Exp. Med., 29 May 2007, 10.1084/jem.20062642 EPub before Print


Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting


Maria Wirzenius, Tuomas Tammela, Marko Uutela, Yulong He, Teresa Odorisio, Giovanna Zambruno, Janice A. Nagy, Harold F. Dvorak, Seppo Yla-Herttuala, Masabumi Shibuya, and Kari Alitalo


Lymphatic vessel growth, or lymphangiogenesis, is regulated by vascular endothelial growth factor-C (VEGF-C) and -D via VEGF receptor 3 (VEGFR-3). Recent studies suggest that VEGF, which does not bind to VEGFR-3, can also induce lymphangiogenesis through unknown mechanisms. To dissect the receptor pathway that triggers VEGFR-3–independent lymphangiogenesis, we used both transgenic and adenoviral overexpression of placenta growth factor (PlGF) and VEGF-E, which are specific activators of VEGFR-1 and -2, respectively. Unlike PlGF, VEGF-E induced circumferential lymphatic vessel hyperplasia, but essentially no new vessel sprouting, when transduced into mouse skin via adenoviral vectors. This effect was not inhibited by blocking VEGF-C and -D. Postnatal lymphatic hyperplasia, without increased density of lymphatic vessels, was also detected in transgenic mice expressing VEGF-E in the skin, but not in mice expressing PlGF. Surprisingly, VEGF-E induced lymphatic hyperplasia postnatally, and it did not rescue the loss of lymphatic vessels in transgenic embryos where VEGF-C and VEGF-D were blocked. Our data suggests that VEGFR-2 signals promote lymphatic vessel enlargement, but unlike in the blood vessels, are not involved in vessel sprouting to generate new lymphatic vessels in vivo.


In: Circulation Research. 2007;100:1460-1467;Published online before print May 3, 2007

Enhanced Capillary Formation Stimulated by a Chimeric Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor-C Silk Domain Fusion Protein

Salla Keskitalo, Tuomas Tammela, Johannes Lyytikka, Terhi Karpanen, Michael Jeltsch, Johanna Markkanen, Seppo Yla-Herttuala, Kari Alitalo

Vascular endothelial growth factor (VEGF)-C and VEGF-D require proteolytic cleavage of the carboxy terminal silk-homology domain for activation. To study the functions of the VEGF-C propeptides, we engineered a chimeric growth factor protein, VEGF-CAC, composed of the amino- and carboxy-terminal propeptides of VEGF-C fused to the receptor-activating core domain of VEGF. Like VEGF-C, VEGF-CAC underwent proteolytic cleavage, and like VEGF, it bound to and activated VEGF receptor-1 and VEGF receptor-2, but not the VEGF-C receptor VEGF receptor-3. VEGF-CAC also bound to neuropilins in a heparin-dependent manner. Strikingly, when VEGF-CAC was expressed via an adenovirus vector in the ear skin of immunodeficient mice, it proved to be a more potent inducer of capillary angiogenesis than VEGF. The VEGF-CAC–induced vessels differed greatly from those induced by VEGF, as they formed a very dense and fine network of pericyte and basement membrane–covered capillaries that were functional, as shown by lectin perfusion experiments. VEGF-CAC could prove useful in proangiogenic therapies in patients experiencing tissue ischemia.

In: Circ Res 2007;100 1468-1475 EPub before Print


Distinct Architecture of Lymphatic Vessels Induced by Chimeric Vascular Endothelial Growth Factor-C/Vascular Endothelial Growth Factor Heparin-Binding Domain Fusion Proteins


Tuomas Tammela, Yulong He, Johannes Lyytikka, Michael Jeltsch, Johanna Markkanen, Katri Pajusola, Seppo Yla-Herttuala, and Kari Alitalo


Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF165 or VEGF189 isoform also containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/VEGF–heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo. However, both chimeras induced a distinctly different pattern of lymphatic vessels when compared with VEGF-C. Whereas VEGF-C–induced vessels were initially a dense network of small diameter vessels, the lymphatic vessels induced by the chimeric growth factors tended to form directly along tissue borders, along basement membranes that are rich in heparan sulfate. For example, in skeletal muscle, the chimeras induced formation of lumenized lymphatic vessels more efficiently than wild-type VEGF-C. We conclude that the matrix-binding domain of VEGF can target VEGF-C activity to heparin-rich basement membrane structures. These properties may prove useful for tissue engineering and attempts to regenerate lymphatic vessels in lymphedema patients.


In: Oncogene 2007 26: 2997-3005; advance online publication, November 27, 2006; 10.1038/sj.onc.1210114


Chemokine-mediated migration of melanoma cells towards lymphatics – a mechanism contributing to metastasis


J D Shields, M S Emmett, D B A Dunn, K D Joory, L M Sage, H Rigby, P S Mortimer, A Orlando, J R Levick & D O Bates


The mechanisms that cause tumors such as melanomas to metastasize into peripheral lymphatic capillaries are poorly defined. Non-mutually-exclusive mechanisms are lymphatic endothelial cell (LEC) chemotaxis and proliferation in response to tumor cells (chemotaxis-lymphangiogenesis hypothesis) or LECs may secrete chemotactic agents that attract cancer cells (chemotactic metastasis hypothesis). Using migration assays, we found evidence supporting both hypotheses. Conditioned medium (CM) from metastatic malignant melanoma (MMM) cell lines attracted LEC migration, consistent with the lymphangiogenesis hypothesis. Conversely, CM from mixed endothelial cells or LECs, but not blood endothelial cells, attracted MMM cells but not non-metastatic melanoma cells, consistent with the chemotactic metastasis hypothesis. MMM cell lines expressed CCR7 receptors for the lymphatic chemokine CCL21 and CCL21 neutralizing antibodies prevented MMM chemotaxis in vitro. To test for chemotactic metastasis in vivo tumor cells were xenotransplanted into nude mice ~1 cm from an injected LEC depot. Two different MMM grew directionally towards the LECs, whereas non-metastatic melanomas did not. These observations support the hypothesis that MMM cells grow towards regions of high LEC density owing to chemotactic LEC secretions, including CCL21. This chemotactic metastasis may contribute to the close association between metastasizing tumor cells and peri-tumor lymphatic density and promote lymphatic invasion.


In: Dev Biol. 2007 May 15;305(2):451-459


The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

Jörg Wilting, Kerstin Buttler, Inga Schulte, Maria Papoutsi, Lothar Schweigerer and Jörg Männer


The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1(+)/CD31(+)/CD45(+) cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.