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Supplemental Digital Content is available in the text., Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Background: Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Results: Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1;Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Conclusion: Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks. Refereed/Peer-reviewed

Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1/Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1−/− mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a−/− mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild-type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks.

Tumour angiogenesis and lymphangiogenesis are hallmarks of cancer and have been associated with tumour progression, tumour metastasis and poor patient prognosis. Many factors regulate angiogenesis and lymphangiogenesis in cancer including non-coding RNAs which are a category of RNAs that do not encode proteins and have important regulatory functions at transcriptional and post-transcriptional levels. Non-coding RNAs can be encapsulated in extracellular vesicles called exosomes which are secreted by tumour cells or other cells in the tumour microenvironment and can then be taken up by the endothelial cells of blood vessels and lymphatic vessels. The “delivery” of these non-coding RNAs to endothelial cells in tumours can facilitate tumour angiogenesis and lymphangiogenesis. Here we review recent findings about exosomal non-coding RNAs, specifically microRNAs and long non-coding RNAs, which regulate tumour angiogenesis and lymphangiogenesis in cancer. We then focus on the potential use of these molecules as cancer biomarkers and opportunities for exploiting ncRNAs for the treatment of cancer.

While the link between the lymphatic system and the metastatic spread of cancer is centuries old, understanding of the underlying mechanisms is still evolving. Lymphatic vessels provide a route for tumour cells to reach regional lymph nodes (LNs), which is prognostic of distant organ metastasis and poor survival. However, genomic analyses of metastatic cancer now reveal complex patterns of dissemination. The lymphatic endothelial cells lining lymphatics respond to molecular cues from the tumour microenvironment, mediating growth and remodelling of lymphatic vessels at the primary tumour, draining LNs and distant premetastatic niches. Recent studies emphasise that this not only supports metastasis but also influences antitumour immunity. Understanding the complex interactions between tumour cells, the immune system and lymphatics will be essential to inform developing therapeutic and prognostic approaches to cancer.

Chemokines are a family of small protein cytokines that act as chemoattractants to migrating cells, in particular those of the immune system. They are categorized functionally as either homeostatic, constitutively produced by tissues for basal levels of cell migration, or inflammatory, where they are generated in association with a pathological inflammatory response. While the extravasation of leukocytes via blood vessels is a key step in cells entering the tissues, the lymphatic vessels also serve as a conduit for cells that are recruited and localized through chemoattractant gradients. Furthermore, the growth and remodeling of lymphatic vessels in pathologies is influenced by chemokines and their receptors expressed by lymphatic endothelial cells (LECs) in and around the pathological tissue. In this review we summarize the diverse role played by specific chemokines and their receptors in shaping the interaction of lymphatic vessels, immune cells, and other pathological cell types in physiology and disease.

The lymphatic system is a series of vessels that transport cells and excess fluid from tissues to the blood vascular system. Normally quiescent, the lymphatics can grow or remodel in response to developmental, immunological, or cells pathological stimuli. Lymphatic vessels comprise lymphatic endothelial cells (LECs) that can respond to external growth factors by undergoing proliferation, migration, adhesion, and tube and lumen formation into new vessel structures, a process known as lymphangiogenesis. To understand the key gene and signaling pathways necessary for lymphangiogenesis and lymphatic vessel remodeling, we have developed a three-dimensional LEC tube formation assay to explore the role of kinase signaling in these processes. The collagen-overlay-based assay was used with primary human adult dermal LECs to investigate a library of 60 tyrosine kinase (TK) and TK-like genes by siRNA knockdown. Nine candidate genes were identified and characterized for their ability to modify key parameters of lymphatic tube formation, including tube length, area, thickness, branching, and number of blind-ended sacs. Four genes-ZAP70, IRAK4, RIPK1, and RIPK2-were identified as high-confidence hits after tertiary deconvolution screens and demonstrate the utility of the assay to define LEC genes critical for the formation of tube structures. This assay facilitates the identification of potential molecular targets for novel drugs designed to modulate the remodeling of lymphatics that is important for the metastatic spread of cancer and other pathologies.

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C.

Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine–chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis. Significance: The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.

Blood vessels and lymphatic vessels are located in many tissues and organs throughout the body, and play important roles in a wide variety of prevalent diseases in humans. Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that can promote the remodeling of blood vessels and lymphatics in development and disease. Recent fundamental and translational studies have provided insight into the molecular mechanisms by which VEGF-D exerts its effects in human disease. Hence this protein is now of interest as a therapeutic and/or diagnostic target, or as a potential therapeutic agent, in a diversity of indications in cardiovascular medicine, cancer and the devastating pulmonary condition lymphangioleiomyomatosis. This has led to clinical trial programs to assess the effect of targeting VEGF-D signaling pathways, or delivering VEGF-D, in angina, cancer and ocular indications. This review summarizes our understanding of VEGF-D signaling in human disease, which is largely based on animal disease models and clinicopathological studies, and provides information about the outcomes of recent clinical trials testing agonists or antagonists of VEGF-D signaling.

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that LGALS1, which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels.

Many cell types undergo migration during embryogenesis and disease. Endothelial cells line blood vessels and lymphatics, which migrate during development as part of angiogenesis, lymphangiogenesis and other types of vessel remodelling. These processes are also important in wound healing, cancer metastasis and cardiovascular conditions. However, the molecular control of endothelial cell migration is poorly understood. Here, we present a dataset containing siRNA screens that identify known and novel components of signalling pathways regulating migration of lymphatic endothelial cells. These components are compared to signalling in blood vascular endothelial cells. Further, using high-content microscopy, we captured a dataset of images of migrating cells following transfection with a genome-wide siRNA library. These datasets are suitable for the identification and analysis of genes involved in endothelial cell migration and morphology, and for computational approaches to identify signalling networks controlling the migratory response and integration of cell morphology, gene function and cell signaling. This may facilitate identification of protein targets for therapeutically modulating angiogenesis and lymphangiogenesis in the context of human disease.

Summary: Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis. : Lymphatic vessels display organotypic function and develop in an organ-specific manner. Vogrin et al. find that the zebrafish Kdr receptor is indispensible for craniofacial, but not trunk, lymphangiogenesis whereas Flt4 is essential for the latter. Thus, vascular endothelial growth factor (VEGF) receptor signaling pathways are differentially employed in different tissues to drive developmental lymphangiogenesis. Keywords: developmental biology, Kdr, lymphangiogenesis, lymphatic, organotypic, vascular biology, VEGF, VEGF receptor, Vegfd, zebrafish

The progression of cancer is supported by a wide variety of non-neoplastic cell types which make up the tumour stroma, including immune cells, endothelial cells, cancer-associated fibroblasts and nerve fibres. These host cells contribute molecular signals that enhance primary tumour growth and provide physical avenues for metastatic dissemination. This article provides an overview of the role of blood vessels, lymphatic vessels and nerve fibres in the tumour microenvironment and highlights the interconnected molecular signalling pathways that control their development and activation in cancer. Further, this article highlights the known pharmacological agents which target these pathways and discusses the potential therapeutic uses of drugs that target angiogenesis, lymphangiogenesis and stress-response pathways in the different stages of cancer care.

Mutations in SOX18, VEGFC and Vascular Endothelial Growth Factor 3 underlie the hereditary lymphatic disorders hypotrichosis-lymphedema-telangiectasia (HLT), Milroy-like lymphedema and Milroy disease, respectively. Genes responsible for hereditary lymphedema are key regulators of lymphatic vascular development in the embryo. To identify novel modulators of lymphangiogenesis, we used a mouse model of HLT (Ragged Opossum) and performed gene expression profiling of aberrant dermal lymphatic vessels. Expression studies and functional analysis in zebrafish and mice revealed one candidate, ArfGAP with RhoGAP domain, Ankyrin repeat and PH domain 3 (ARAP3), which is down-regulated in HLT mouse lymphatic vessels and necessary for lymphatic vascular development in mice and zebrafish. We position this known regulator of cell behaviour during migration as a mediator of the cellular response to Vegfc signalling in lymphatic endothelial cells in vitro and in vivo. Our data refine common mechanisms that are likely to contribute during both development and the pathogenesis of lymphatic vascular disorders. Refereed/Peer-reviewed

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, the formation of veins and lymphatics. VEGFD has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that during primary angiogenesis Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context specific indispensible and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis.

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.

Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes., Adverse life events have been associated with reduced survival in cancer patients. Here, the authors explore the mechanism responsible and show that chronic stress in mice activates a signalling cascade in macrophages and tumour cells, which results in restructuring of the tumour lymphatic system, promoting metastasis.

Tumor metastasis to lymph nodes is a key indicator of patient survival, and is enhanced by the neo-lymphatics induced by tumor-secreted VEGF-C or VEGF-D, acting via VEGFR-3 signalling. These targets constitute important avenues for anti-metastatic treatment. Despite this new understanding, clinical observations linking metastasis with tumor depth or location suggest that lymphangiogenic growth factors are not the sole determinants of metastasis. Here we explored the influence of tumor proximity to lymphatics capable of responding to growth factors on nodal metastasis in a murine VEGF-D over-expression tumor model. We found that primary tumor location profoundly influenced VEGF-D-mediated lymph node metastasis: 89 % of tumors associated with the flank skin metastasised, in contrast with only 19 % of tumors located more deeply on the body wall (p

Substantial evidence supports important independent roles for lymphangiogenic growth factor signaling and prostaglandins in the metastatic spread of cancer. The significance of the lymphangiogenic growth factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, is well established in animal models of metastasis, and a strong correlation exits between an increase in expression of VEGF-C and VEGF-D, and metastatic spread in various solid human cancers. Similarly, key enzymes that control the production of prostaglandins, cyclooxygenases (COX-1 and COX-2, prototypic targets of Non-steroidal anti-inflammatory drugs (NSAIDs)), are frequently over-expressed or de-regulated in the progression of cancer. Recent data have suggested an intersection of lymphangiogenic growth factor signaling and the prostaglandin pathways in the control of metastatic spread via the lymphatic vasculature. Furthermore, this correlates with current clinical data showing that some NSAIDs enhance the survival of cancer patients through reducing metastasis. Here, we discuss the potential biochemical and cellular basis for such anti-cancer effects of NSAIDs through the prostaglandin and VEGF signaling pathways.

Vascular endothelial growth factor-D (VEGF-D) is a secreted glycoprotein that promotes growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis), and can induce remodeling of large lymphatics. VEGF-D enhances solid tumor growth and metastatic spread in animal models of cancer, and in some human cancers VEGF-D correlates with metastatic spread, poor patient outcome, and, potentially, with resistance to anti-angiogenic drugs. Hence, VEGF-D signaling is a potential target for novel anti-cancer therapeutics designed to enhance anti-angiogenic approaches and to restrict metastasis. In the cardiovascular system, delivery of VEGF-D in animal models enhanced angiogenesis and tissue perfusion, findings which have led to a range of clinical trials testing this protein for therapeutic angiogenesis in cardiovascular diseases. Despite these experimental and clinical developments, our knowledge of the signaling mechanisms driven by VEGF-D is still evolving--here we explore the biology of VEGF-D, its signaling mechanisms, and the clinical relevance of this growth factor.

Shayan R, Karnezis T, Murali R, Wilmott J S, Ashton M W, Taylor G I, Thompson J F, Hersey P, Achen M G, Scolyer R A & Stacker S A (2012) Histopathology 61, 702–710 Lymphatic vessel density in primary melanomas predicts sentinel lymph node status and risk of metastasis Aims: Important prognostic factors in patients with cutaneous melanoma include primary tumour thickness/depth of invasion, ulceration and mitotic rate, and the presence of tumour cells in regional lymph nodes. More recently, features of stromal components, such as blood and lymphatic vessel density, have been suggested as additional indicators of metastatic potential. Our aim was to investigate the relationship between tumour lymphatic vessels and lymph node metastasis. Methods and results: Metastasizing (n = 11) and non-metastasizing (n = 11) primary melanoma samples matched for depth/thickness, mitotic rate and ulceration were examined for lymphatic vessel density (LVD) in the primary tumour, using an antibody to podoplanin. Significant differences were found between LVD (vessels/unit area) in the peripheral (5.73 ± 0.67) versus central (1.72 ± 0.42) regions of the metastasizing tumour group (P

SummaryLymphatic metastasis is facilitated by lymphangiogenic growth factors VEGF-C and VEGF-D that are secreted by some primary tumors. We identified regulation of PGDH, the key enzyme in prostaglandin catabolism, in endothelial cells of collecting lymphatics, as a key molecular change during VEGF-D-driven tumor spread. The VEGF-D-dependent regulation of the prostaglandin pathway was supported by the finding that collecting lymphatic vessel dilation and subsequent metastasis were affected by nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of prostaglandin synthesis. Our data suggest a control point for cancer metastasis within the collecting lymphatic endothelium, which links VEGF-D/VEGFR-2/VEGFR-3 and the prostaglandin pathways. Collecting lymphatics therefore play an active and important role in metastasis and may provide a therapeutic target to restrict tumor spread.

The theory that angiogenesis could support tumor growth and therefore be a target for cancer therapy was explored in publications by Judah Folkman in the 1970s. This theory was then fostered by Folkman for over 2 decades until, via modern molecular and cell biology techniques, it was vindicated in animal tumor models, and subsequently with the successful clinical trials of a humanized monoclonal antibody which neutralizes vascular endothelial growth factor (VEGF). In the process of proving the "anti-angiogenesis" theory, researchers in vascular, cancer, and developmental biology have gained insight into how the vascular network operates at the molecular level. Following on from the wave of activity that accompanied the study of angiogenesis, some of the molecular mechanisms controlling the related process of lymphangiogenesis have now been identified. The growth of lymphatic vessels was found to be controlled by proteins related to VEGF, namely VEGF-C and VEGF-D, which are associated with a number of human tumor types. The experimental inhibition of this process in animal models has suggested that lymphangiogenic growth factors facilitate the metastatic spread of tumor cells via lymphatics. Unlike the anti-angiogenesis strategy, anti-lymphangiogenesis is yet to be tested clinically; however, the notion that anti-lymphangiogenesis may be beneficial for cancer therapy is supported by extensive data from animal models and clinicopatholgical data. History may show that anti-angiogenesis provided the vantage point from which anti-lymphangiogenesis was seen as a viable concept for targeting tumors and other pathological conditions.

The metastatic spread of tumor cells is the most lethal aspect of cancer and often occurs via the lymphatic vasculature. Both experimental tumor models and human clinicopathologic data indicate that growth of lymphatic vessels (lymphangiogenesis) near solid tumors is often associated with lymph node metastasis. Changes in the adhesive properties of lymphatic endothelium near tumors may also facilitate metastatic spread via the lymphatics. Lymphangiogenic growth factors have been identified that promote formation of tumor lymphatics and metastatic spread of tumor cells to lymph nodes. These include the secreted glycoproteins vascular endothelial growth factor-C (VEGF-C) and VEGF-D, which act via their cognate receptor tyrosine kinase VEGF receptor-3 (VEGFR-3) located on lymphatic endothelial cells. Other signaling molecules that have been reported to promote lymphangiogenesis and/or lymphatic metastasis in cancer include VEGF-A, platelet-derived growth factor-BB, and hepatocyte growth factor. However, the quantitative contribution of these proteins to tumor lymphangiogenesis and lymphatic metastasis in different tumor types requires further investigation. In addition, chemokines are thought to play a role in attracting tumor cells and lymphatic vessels to each other. Moreover, it has recently been shown that lymphangiogenic growth factors secreted from a primary tumor can induce lymphangiogenesis in nearby lymph nodes, even before arrival of tumor cells, which may facilitate further metastasis. This article provides an overview of the molecular mechanisms that control lymphatic metastasis and discusses potential therapeutic approaches for inhibiting this process in human cancer.

Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (AdVEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangio-genesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can he safely applied in adults.

Distant organ metastasis is the most important factor in determining patient survival in cancer. This is thought to occur via the body's own systems for transporting fluid and cells, the blood vascular and lymphatic systems. Cancer cells may exploit these vascular systems by expressing growth factors, which alter the normal pattern of angiogenesis and lymphatic vessel growth (lymphangiogenesis), thus creating conduits for tumour metastasis. With respect to lymphatic metastasis, techniques which allow the mapping of a tumour's lymphatic drainage and sampling of the 'sentinel node' from the regional lymph node group provide crucial prognostic information, determine further treatment and offer a window into tumour-host immune interactions. Aberrant drainage patterns so identified are both clinically significant, and highlight important anatomical and molecular complexities not explained by existing models of lymphatic development or anatomy. The molecular controls of tumour lymphangiogenesis and factors determining which lymphatic vessel subtypes are induced may be targets for novel therapeutics designed to restrict cancer metastasis. Furthermore, analyses of these control mechanisms will enhance our understanding of the interactions between the tumour cells and the lymphatic vasculature. For many years, disparate groups of clinical researchers and basic scientists have been working to unravel the mysteries of the lymphatic system. This review aims to summarize these contributions, in terms of the history, identification, structure and function of lymphatic vessels in cancer and the role they play in tumour metastasis. Current ideas about the roles of lymphangiogenic growth factors, their signalling pathways in lymphatic metastasis and therapeutic opportunities to restrict this spread will also be explored.

Endothelial cells line the vessels which transport fluid and cells throughout the body. Although much attention has been paid to these cells in the context of the blood vascular system, endothelial cells also line lymphatic vessels. Recent progress in identifying growth factors which drive the development of lymphatic vessels and molecular markers specific for lymphatics has expanded our understanding of the role the lymphatic system plays in human pathology. Techniques for purifying populations of lymphatic endothelial cells also allow the in vitro analysis of this unique surface to explore its role in tumour metastasis, immune cell function and fluid transport. This review provides a synopsis of the recent data pertaining to the purification and culture of lymphatic endothelial cells, and the interaction of tumour cells with lymphatic endothelium.

The metastatic spread of tumor cells is the most lethal aspect of cancer and can occur via various routes, including the lymphatic vasculature. Studies of tumor models in animals and clinicopathological data have indicated that growth of lymphatic vessels (lymphangiogenesis) in the vicinity of solid tumors may contribute to lymphatic metastasis. Research over the past 5 years has identified a range of lymphangiogenic growth factors that could conceivably play a role in promoting tumor lymphangiogenesis and lymphatic metastasis. The most extensively studied signaling system that promotes lymphangiogenesis in tumors involves the secreted lymphangiogenic proteins vascular endothelial growth factor-C (VEGF-C) and VEGF-D, and their cognate receptor on lymphatic endothelium VEGF receptor-3 (VEGFR-3). More recent studies have identified other signaling molecules that can also promote lymphangiogenesis in vivo, including hepatocyte growth factor and members of the fibroblast growth factor, angiopoietin, platelet-derived growth factor and insulin-like growth factor families of secreted proteins. This article provides an overview of the molecular mechanisms that control lymphangiogenic signaling, emphasizing the more recently identified lymphangiogenic growth factors and the roles they may play in cancer biology. Molecular approaches for inhibiting lymphangiogenic signaling in cancer, designed to restrict tumor metastasis, are also examined.

M.G.A. and S.A.S. are supported by Senior Research Fellowships from the National Health and Medical Research Council of Australia (NHMRC) and the Pfizer Foundation, respectively, and by a Program Grant from the NHMRC. B.K.M. is a recipient of a Melbourne Research Scholarship from the University of Melbourne. We thank Tony Burgess for helpful comments and acknowledge researchers whose work could not be cited here due to space restrictions. M.G.A. and S.A.S. declare that they have ownership of a company with interests related to the topic discussed in this article.

Understanding the complex process of tumor metastasis is a problem which has challenged both clinician and scientist for well over 100 years. Defining molecular markers which reflect the metastatic potential of a tumor has also proved elusive. Recently, members of the vascular endothelial growth factor (VEGF) family of glycoproteins have been demonstrated to be potent mediators of both blood vessel and lymphatic vessel formation in the context of tumor biology. Experimental studies in animal models combined with extensive clinicopathological data provide a compelling case indicating that members of the VEGF family play a key role in the formation of metastases in a broad range of solid tumors. The question of whether VEGF signaling pathways can now serve as therapeutic targets alone, or in combination with other forms of anti-cancer agents, needs to be addressed.

The vascular endothelial growth factor (VEGF) family of secreted glycoproteins are critical inducers of angiogenesis (growth of blood vessels) and lymphangiogenesis (growth of lymphatic vessels). These proteins are attractive therapeutic targets for blocking growth of blood vessels and lymphatics in tumors and thereby inhibiting the growth and spread of cancer -- in fact, the first VEGF inhibitor has recently entered the clinic for treatment of cancer. In addition, the VEGFs are being considered for stimulation of angiogenesis in the context of ischemic disease and lymphangiogenesis for treatment of lymphedema. These therapeutic possibilities have focused great interest on the molecular regulation of VEGF family members. Much has been learned in the past five years about the mechanisms controlling the action of the VEGFs, including the importance of hypoxia, proteolysis, transcription factors and RNA splicing. An understanding of these mechanisms offers broader opportunities to manipulate expression and activity of the VEGFs for treatment of disease.

The dysfunction or proliferation of lymphatic vessels (lymphangiogenesis) is linked to a number of pathological conditions including lymphedema and cancer. The recent discovery and characterisation of the lymphangiogenic growth factors vascular endothelial growth factor-C (VEGF-C) and VEGF-D and of their receptor on lymphatic endothelial cells, VEGFR-3, has provided an understanding of the molecular mechanisms controlling the growth of lymphatic vessels. In addition, other genes and protein markers have been identified with specificity for lymphatic endothelium that have enhanced the characterization and isolation of lymphatic endothelial cells. Our growing understanding of the molecules that control lymphangiogenesis allows us to design more specific drugs with which to manipulate the relevant signalling pathways. Modulating these pathways and other molecules with specificity to the lymphatic system could offer alternative treatments for a number of important clinical conditions.

Vascular endothelial growth factor (VEGF) C and VEGF-D stimulate lymphangiogenesis and angiogenesis in tissues and tumors by activating the endothelial cell surface receptor tyrosine kinases VEGF receptor (VEGFR) 2 and VEGFR-3. These growth factors are secreted as full-length inactive forms consisting of NH2- and COOH-terminal propeptides and a central VEGF homology domain (VHD) containing receptor binding sites. Proteolytic cleavage removes the propeptides to generate mature forms, consisting of dimers of the VEGF homology domain, that bind receptors with much greater affinity than the full-length forms. Therefore, proteolytic processing activates VEGF-C and VEGF-D, although the proteases involved were unknown. Here, we report that the serine protease plasmin cleaved both propeptides from the VEGF homology domain of human VEGF-D and thereby generated a mature form exhibiting greatly enhanced binding and cross-linking of VEGFR-2 and VEGFR-3 in comparison to full-length material. Plasmin also activated VEGF-C. As lymphangiogenic growth factors promote the metastatic spread of cancer via the lymphatics, the proteolytic activation of these molecules represents a potential target for antimetastatic agents. Identification of an enzyme that activates the lymphangiogenic growth factors will facilitate development of inhibitors of metastasis.

Impaired lymphatic drainage in human limbs causes the debilitating swelling termed lymphoedema. In mammals, known growth factors involved in the control of lymphangiogenesis (growth of new lymph vessels) are vascular endothelial growth factors-C and -D (VEGF-C/D). Here we characterize a model of lymphangiogenesis in which the tail of lizards is regenerated without becoming oedematous. Three weeks after the tail is shed (autotomy), there are a small number of large diameter lymphatic vessels in the regenerated tail. Thereafter, the number increases and the diameter decreases. A functional lymphatic network, as determined by lymphoscintigraphy, is established 6 wk after autotomy. The new network differs morphologically and functionally from that in original tails. This lymphatic regeneration is associated with an up-regulation of a reptilian homologue of the VEGF-C/D protein family (rVEGF-C/D), as determined by Western blot analysis using a human reactive VEGF-C polyclonal antibody. Regenerating lizard tails are potentially useful models for studying the molecular basis of lymphangiogenesis with a view to developing possible treatments for human lymphoedema.

The lymphatic vasculature plays a critical role in the regulation of body fluid volume and immune function. Extensive research into the molecular mechanisms that control blood vessel growth has led to identification of molecules that also regulate development and growth of the lymphatic vessels. This is generating a great deal of interest in the molecular control of the lymphatics in the context of embryogenesis, lymphatic disorders and tumor metastasis. Studies in animal models carried out over the past three years have shown that the soluble protein growth factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, and their cognate receptor tyrosine kinase, VEGF receptor-3 (VEGFR-3), are critical regulators of lymphangiogenesis. Furthermore, disfunction of VEGFR-3 has recently been shown to cause lymphedema. The capacity to induce lymphangiogenesis by manipulation of the VEGF-C/VEGF-D/VEGFR-3 signaling pathway offers new opportunities to understand the function of the lymphatic system and to develop novel treatments for lymphatic disorders.

The capacity of an adenovirus encoding the mature form of vascular endothelial growth factor (VEGF)-D, VEGF-D Delta N Delta C, to induce angiogenesis, lymphangiogenesis, or both was analyzed in 2 distinct in vivo models. We first demonstrated in vitro that VEGF-D Delta N Delta C encoded by the adenovirus (Ad-VEGF-D Delta N Delta C) is capable of inducing endothelial cell proliferation and migration and that the latter response is primarily mediated by VEGF receptor-2 (VEGFR-2). Second, we characterized a new in vivo model for assessing experimental angiogenesis, the rat cremaster muscle, which permits live videomicroscopy and quantitation of functional blood vessels. In this model, a proangiogenic effect of Ad-VEGF-D Delta N Delta C was evident as early as 5 days after injection. Immunohistochemical analysis of the cremaster muscle demonstrated that neovascularization induced by Ad-VEGF-D Delta N Delta C and by Ad-VEGF-A(165) (an adenovirus encoding the 165 isoform of VEGF-A) was composed primarily of laminin and VEGFR-2-positive vessels containing red blood cells, thus indicating a predominantly angiogenic response. In a skin model, Ad-VEGF-D Delta N Delta C induced angiogenesis and lymphangiogenesis, as indicated by staining with laminin, VEGFR-2, and VEGFR-3, whereas Ad-VEGF-A(165) stimulated the selective growth of blood vessels. These data suggest that the biologic effects of VEGF-D are tissue-specific and dependent on the abundance of blood vessels and lymphatics expressing the receptors for VEGF-D in a given tissue. The capacity of Ad-VEGF-D Delta N Delta C to induce endothelial cell proliferation, angiogenesis, and lymphangiogenesis demonstrates that its potential usefulness for the treatment of coronary artery disease, cerebral ischemia, peripheral vascular disease, restenosis, and tissue edema should be tested in preclinical models.

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

The generation of new lymphatic vessels through lymphangiogenesis and the remodelling of existing lymphatics are thought to be important steps in cancer metastasis. The past decade has been exciting in terms of research into the molecular and cellular biology of lymphatic vessels in cancer, and it has been shown that the molecular control of tumour lymphangiogenesis has similarities to that of tumour angiogenesis. Nevertheless, there are significant mechanistic differences between these biological processes. We are now developing a greater understanding of the specific roles of distinct lymphatic vessel subtypes in cancer, and this provides opportunities to improve diagnostic and therapeutic approaches that aim to restrict the progression of cancer.

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc-driven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.

The vascular endothelial growth factor (VEGF) family of soluble protein growth factors consists of key mediators of angiogenesis and lymphangiogenesis in the context of tumor biology. The members of the family, VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PIGF), play important roles in vascular biology in both normal physiology and pathology. The generation of a humanized neutralizing antibody to VEGF-A (bevacizumab, also known as Avastin) and the demonstration of its benefit in numerous human cancers have confirmed the merit of an anti-angiogenesis approach to cancer treatment and have validated the VEGF-A signaling pathway as a therapeutic target. Other members of the VEGF family are now being targeted, and their relevance to human cancer and the development of resistance to anti-VEGF-A treatment are being evaluated in the clinic. Here, we discuss the potential of targeting VEGF family members in the diagnosis and treatment of cancer.

Metastasis to regional lymph nodes is an important and early event in many tumors. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and their receptor VEGFR-3, play a role in tumor spread via the lymphatics, although the timing of their involvement is not understood. In contrast, VEGFR-2, activated by VEGF-A, VEGF-C and VEGF-D, is a mediator of angiogenesis and drives primary tumor growth. We demonstrate the critical role for VEGFR-3, but not VEGFR-2, in the early events of metastasis. In a tumor model exhibiting both VEGF-D-dependent angiogenesis and lymphangiogenesis, an antibody to VEGFR-2 (DC101) was capable of inhibiting angiogenesis (79 % reduction in PECAM + blood vessels) and growth (93 % reduction in tumor volume). However, unlike an anti-VEGFR-3 Mab (mF4-31C1), DC101 was not capable of eliminating either tumor lymphangiogenesis or lymphogenous metastasis (60 % reduction of lymph node metastasis by DC101 vs 95 % by mF4-31C1). Early excision of the primary tumors demonstrated that VEGF-D-mediated tumor spread precedes angiogenesis-induced growth. Small but highly metastatic primary human breast cancers had significantly higher lymphatic vessel density (23.1 vessels/mm(2)) than size-matched (11.7) or larger non-metastatic tumors (12.4) thus supporting the importance of lymphatic vessels, as opposed to angiogenesis-mediated primary tumor growth, for nodal metastasis. These results suggest that lymphangiogenesis via VEGF-D is more critical than angiogenesis for nodal metastasis.

Important prognostic factors in patients with cutaneous melanoma include primary tumour thickness/depth of invasion, ulceration and mitotic rate, and the presence of tumour cells in regional lymph nodes. More recently, features of stromal components, such as blood and lymphatic vessel density, have been suggested as additional indicators of metastatic potential. Our aim was to investigate the relationship between tumour lymphatic vessels and lymph node metastasis.Metastasizing (n = 11) and non-metastasizing (n = 11) primary melanoma samples matched for depth/thickness, mitotic rate and ulceration were examined for lymphatic vessel density (LVD) in the primary tumour, using an antibody to podoplanin. Significant differences were found between LVD (vessels/unit area) in the peripheral (5.73 ± 0.67) versus central (1.72 ± 0.42) regions of the metastasizing tumour group (P 0.001), and between LVD in the peripheral areas of metastasizing (5.73 ± 0.67) versus non-metastasizing (4.21 ± 0.37) tumours (P 0.01). No overall difference was found between total average LVD in the two tumour groups or between their vessel morphology.Our results show that LVD is associated with risk of lymph node metastasis. Furthermore, the ratio of peripheral LVD:central LVD is a useful marker of primary melanomas that are likely to metastasize to lymph nodes.

Despite the key roles of lymphatic vessels in homeostasis and disease, the cellular sources of signals that direct lymphatic vascular growth and patterning remain unknown. Using high-resolution imaging in two and three dimensions, we demonstrated that postnatal mouse mammary gland lymphatic vessels share an intimate spatial association with epithelial ducts and large blood vessels. We further demonstrated that the lymphatic vasculature is remodeled together with the mammary epithelial tree and blood vasculature during postnatal mouse mammary gland morphogenesis. Neither estrogen receptor α nor progesterone receptor were detected in lymphatic endothelial cells in the mouse mammary gland, suggesting that mammary gland lymphangiogenesis is not likely regulated directly by these steroid hormones. Epithelial cells, especially myoepithelial cells, were determined to be a rich source of prolymphangiogenic stimuli including VEGF-C and VEGF-D with temporally regulated expression levels during mammary gland morphogenesis. Blockade of VEGFR-3 signaling using a small-molecule inhibitor inhibited the proliferation of primary lymphatic endothelial cells promoted by mammary gland conditioned medium, suggesting that lymphangiogenesis in the mammary gland is likely driven by myoepithelial-derived VEGF-C and/or VEGF-D. These findings provide new insight into the architecture of the lymphatic vasculature in the mouse mammary gland and, by uncovering the proximity of lymphatic vessels to the epithelial tree, suggest a potential mechanism by which metastatic tumor cells access the lymphatic vasculature. Refereed/Peer-reviewed

VEGF-D is a mitogen for endothelial cells that promotes tumor growth and metastatic spread in animal models, and expression of which correlates with lymph node metastasis in some human cancers. It is secreted from the cell as a full-length form with propeptides flanking a central region containing binding sites for VEGFR-2 and VEGFR-3, receptors that signal for angiogenesis and lymphangiogenesis. The propeptides can be cleaved from VEGF-D, enhancing affinity for VEGFR-2 and VEGFR-3 in vitro; however, the importance of this processing in cancer is unclear. To explore the necessity of processing for the effects of VEGF-D in cancer, we use a mutant full-length form that cannot be processed, and show that, in contrast to full-length VEGF-D that is processed, this mutant does not promote tumor growth and lymph node metastasis in a mouse tumor model. Processing of VEGF-D is required for tumor angiogenesis, lymphangiogenesis, and recruitment of tumor-associated macrophages. These observations may be explained by the requirement of processing for VEGF-D to bind neuropilin receptors and activate VEGFR-2. Our results indicate that proteolytic processing is necessary for VEGF-D to promote the growth and spread of cancer, and suggest that enzymes catalyzing this processing could be targets for antimetastatic therapeutics.

The blood and lymphatic vascular networks combine to facilitate immune function and maintain tissue fluid homeostasis in the body. Although these two systems share many common structural and molecular features, recent advances in our understanding of the molecular control of the lymphatics have identified distinct molecular pathways responsible for the formation and function of the lymphatic network. These advances have led to the characterisation of lymphatic-specific markers and growth factors which control lymphatic development and function. Insights gained from in vitro and in vivo studies over the past decade have highlighted the importance of the lymphatic system in human diseases such as lymphedema, inflammatory disorders and cancer. The lymphatic vasculature is an important route for the metastatic spread of tumor cells, and recent studies based on animal models of cancer indicated that lymphangiogenic growth factors, secreted by tumor cells or components of the tumor stroma, can induce formation of new lymphatic vessels in the vicinity of a primary tumor. These studies, as well as clinicopathological data, suggest that this process of tumor lymphangiogenesis can be associated with enhanced metastastic spread – hence tumor lymphangiogenesis is being explored as a therapeutic target for restricting the metastatic spread of cancer.

Lymphatic vessels play an important role in the regulation of tissue fluid balance, immune responses, and fat adsorption and are involved in diseases including lymphedema and tumor metastasis. Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) is necessary for development of the blood vasculature during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the lymphatic vasculature. We analyzed mice deficient in both of the known VEGFR-3 ligands, VEGF-C and VEGF-D. Unlike the Vegfr3(-/-) embryos, the Vegfc(-/-); Vegfd(-/-) embryos displayed normal blood vasculature after embryonic day 9.5. Deletion of Vegfr3 in the epiblast, using keratin 19 (K19) Cre, resulted in a phenotype identical to that of the Vegfr3(-/-) embryos, suggesting that this phenotype is due to defects in the embryo proper and not in placental development. Interestingly, the Vegfr3(neo) hypomorphic mutant mice carrying the neomycin cassette between exons 1 and 2 showed defective lymphatic development. Overexpression of human or mouse VEGF-D in the skin, under the K14 promoter, rescued the lymphatic hypoplasia of the Vegfc(+/-) mice in the K14-VEGF-D; Vegfc(+/-) compound mice, suggesting that VEGF-D is functionally redundant with VEGF-C in the stimulation of developmental lymphangiogenesis. Our results suggest VEGF-C- and VEGF-D-independent functions for VEGFR-3 in the early embryo.

The lymphatic vasculature is critical for immunity and interstitial fluid homeostasis, playing important roles in diseases such as lymphedema and metastatic cancer. Animal models have been generated to explore the role of lymphatics and lymphangiogenic growth factors in such diseases, and to study lymphatic development. However, analysis of lymphatic vessels has primary been restricted to counting lymphatics in two-dimensional tissue slices, due to a lack of more sophisticated methodologies. In order to accurately examine lymphatic dysfunction in these models, and analyse the effects of lymphangiogenic growth factors on the lymphatic vasculature, it is essential to quantify the morphology and patterning of the distinct lymphatic vessels types in three-dimensional tissues. Here, we describe a method for performing such analyses, integrating user-operated image-analysis software with an approach that considers important morphological, anatomical and patterning features of the distinct lymphatic vessel subtypes. This efficient, reproducible technique is validated by analysing healthy and pathological tissues.

The lymphatic system plays a key role in tissue fluid regulation and tumour metastasis, and lymphatic defects underlie many pathological states including lymphoedema, lymphangiectasia, lymphangioma and lymphatic dysplasia. However, the origins of the lymphatic system in the embryo, and the mechanisms that direct growth of the network of lymphatic vessels, remain unclear. Lymphatic vessels are thought to arise from endothelial precursor cells budding from the cardinal vein under the influence of the lymphatic hallmark gene Prox1 (prospero homeobox 1; ref. 4). Defects in the transcription factor gene SOX18 (SRY (sex determining region Y) box 18) cause lymphatic dysfunction in the human syndrome hypotrichosis-lymphoedema-telangiectasia, suggesting that Sox18 may also play a role in lymphatic development or function. Here we use molecular, cellular and genetic assays in mice to show that Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells. Sox18 is expressed in a subset of cardinal vein cells that later co-express Prox1 and migrate to form lymphatic vessels. Sox18 directly activates Prox1 transcription by binding to its proximal promoter. Overexpression of Sox18 in blood vascular endothelial cells induces them to express Prox1 and other lymphatic endothelial markers, while Sox18-null embryos show a complete blockade of lymphatic endothelial cell differentiation from the cardinal vein. Our findings demonstrate a critical role for Sox18 in developmental lymphangiogenesis, and suggest new avenues to investigate for therapeutic management of human lymphangiopathies.