Search

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we uncover molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR–Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies for overcoming resistance to AAT., Through three-dimensional CRISPR screening, He et al. report that functional inhibition of BET family of proteins BRD2/3/4 shows mitigating effects on blood endothelial cell (EC) survival and/or proliferation upon VEGFA blockade. An interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs through CDC25B phosphatase, is potentially involved with EC resistance to anti-angiogenic therapy.

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.

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a(−/−) phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.

Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk-/- embryos. Ryk-/- mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.

Remodelling of lymphatic vessels in tumours facilitates metastasis to lymph nodes. The growth factors VEGF-C and VEGF-D are well known inducers of lymphatic remodelling and metastasis in cancer. They are initially produced as full-length proteins requiring proteolytic processing in order to bind VEGF receptors with high affinity and thereby promote lymphatic remodelling. The fibrinolytic protease plasmin promotes processing of VEGF-C and VEGF-D in vitro, but its role in processing them in cancer was unknown. Here we explore plasmin’s role in proteolytically activating VEGF-D in vivo, and promoting lymphatic remodelling and metastasis in cancer, by co-expressing the plasmin inhibitor α2-antiplasmin with VEGF-D in a mouse tumour model. We show that α2-antiplasmin restricts activation of VEGF-D, enlargement of intra-tumoural lymphatics and occurrence of lymph node metastasis. Our findings indicate that the fibrinolytic system influences lymphatic remodelling in tumours which is consistent with previo...

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.

The receptor tyrosine kinases (RTKs) are a well-characterized family of growth factor receptors that have central roles in human disease and are frequently therapeutically targeted. The RYK, ROR, PTK7 and MuSK subfamilies make up an understudied subset of WNT-binding RTKs. Numerous developmental, stem cell and pathological roles of WNTs, in particular WNT5A, involve signalling via these WNT receptors. The WNT-binding RTKs have highly context-dependent signalling outputs and stimulate the β-catenin-dependent, planar cell polarity and/or WNT/Ca2+ pathways. RYK, ROR and PTK7 members have a pseudokinase domain in their intracellular regions. Alternative signalling mechanisms, including proteolytic cleavage and protein scaffolding functions, have been identified for these receptors. This review explores the structure, signalling, physiological and pathological roles of RYK, with particular attention paid to cancer and the possibility of therapeutically targeting RYK. The other WNT-binding RTKs are compared with RYK throughout to highlight the similarities and differences within this subset of WNT receptors.

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their diversity as a cell population. However, the developmental origins of this diversity are poorly understood. Here, we identify a rostral-caudal gradient of Wnt-responsiveness that delineates the specification of the two main interneuron subclasses. Caudallysituated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. Parvalbumin (PV)-expressing basket cells, by contrast, originate mostly from the rostral MGE where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through beta-catenin, Wnt signaling transmitted via the non-canonical receptor, Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain-of-function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting Ryk signaling acts in a graded fashion. These data reveal a complex and important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of mature cortical interneuron subtypes.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (Apc(Min/+)) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

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

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.

Recently developed cancer immunotherapy approaches including immune checkpoint inhibitors and chimeric antigen receptor (CAR) T cell transfer are showing promising results both in trials and in clinical practice. These approaches reflect increasing recognition of the crucial role of the tumor microenvironment in cancer development and progression. Cancer cells do not act alone, but develop a complex relationship with the environment in which they reside. The host immune response to tumors is critical to the success of immunotherapy; however the determinants of this response are incompletely understood. The immune cell infiltrate in tumors varies widely in density, composition, and clinical significance. The tumor vasculature is a key component of the microenvironment that can influence tumor behavior and treatment response, and can be targeted through the use of anti-angiogenic drugs. Blood vascular and lymphatic endothelial cells have important roles in the trafficking of immune cells and controlling the microenvironment, and modulate the immune response. Improving access to the tumor through vascular alteration with anti-angiogenic drugs may prove an effective combinatorial strategy with immunotherapy approaches, and might be applicable to many tumor types. In this review we briefly discuss the host’s immune response to cancer and the treatment strategies utilizing this response, before focusing on the pathological features of tumor blood and lymphatic vessels and the contribution these might make to tumor immune evasion.

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.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

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.

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

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.

Vascular endothelial growth factor-D (VEGF-D), a secreted angiogenic and lymphangiogenic glycoprotein, enhances tumor growth and metastasis in animal models, and its expression correlates with metastasis and poor patient outcome in some cancers – it is therefore considered a target for novel anti-cancer therapeutics. The definition of the structure of the complex of VEGF-D bound to its receptors would be beneficial for design of inhibitors of VEGF-D signaling aimed at restricting the growth and spread of cancer. In addition, there is interest in using VEGF-D protein for therapeutic angiogenesis and lymphangiogenesis in the settings of cardiovascular diseases and lymphedema, respectively. However, VEGF-D has proven difficult to express and purify in a highly bioactive form due to a tendency to exist as monomers rather than bioactive dimers. Here we describe a protocol for expression and purification of mature human VEGF-D, and a mutant thereof with reduced glycosylation, potentially suitable for preclinical therapeutic and structural studies, respectively. The degree of glycosylation in mature VEGF-D was reduced by eliminating one of the two N -glycosylation sites, and expressing the protein in Lec3.2.8.1 cells which had reduced glycosylation capacity. Mature VEGF-D and the glycosylation mutant were each enriched for the biologically active dimeric form by optimizing the separation of dimer from monomer via gel filtration, followed by conversion of remaining monomers to dimers via treatment with cysteine. The glycosylation mutant of VEGF-D intended for structural studies preserved all the cysteine residues of mature VEGF-D, in contrast to previous structural studies, exhibited comparable receptor binding to mature VEGF-D and might facilitate structural studies of the VEGF-D/VEGFR-3 complex.

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.

Lymph node metastasis, an early and prognostically important event in the progression of many human cancers, is associated with expression of VEGF-D. Changes to lymph node vasculature that occur during malignant progression may create a metastatic niche capable of attracting and supporting tumor cells. In this study, we sought to characterize molecules expressed in lymph node endothelium that could represent therapeutic or prognostic targets. Differential mRNA expression profiling of endothelial cells from lymph nodes that drained metastatic or nonmetastatic primary tumors revealed genes associated with tumor progression, in particular bone morphogenetic protein-4 (BMP-4). Metastasis driven by VEGF-D was associated with reduced BMP-4 expression in high endothelial venules, where BMP-4 loss could remodel the typical high-walled phenotype to thin-walled vessels. VEGF-D expression was sufficient to suppress proliferation of the more typical BMP-4–expressing high endothelial venules in favor of remodeled vessels, and mechanistic studies indicated that VEGF receptor-2 contributed to high endothelial venule proliferation and remodeling. BMP-4 could regulate high endothelial venule phenotype and cellular function, thereby determining morphology and proliferation responses. Notably, therapeutic administration of BMP-4 suppressed primary tumor growth, acting both at the level of tumor cells and tumor stromal cells. Together, our results show that VEGF-D–driven metastasis induces vascular remodeling in lymph nodes. Furthermore, they implicate BMP-4 as a negative regulator of this process, suggesting its potential utility as a prognostic marker or antitumor agent. Cancer Res; 71(20); 6547–57. ©2011 AACR.

Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that promotes tumor growth and metastatic spread in animal models of cancer. Expression of VEGF-D in prevalent human cancers was reported to correlate with lymph node metastasis and patient outcome—hence, this protein is a potential target for novel anticancer therapeutics designed to restrict tumor growth and spread. Here, we define the binding site in VEGF-D of a neutralizing antibody, designated VD1, which blocks the interaction of VEGF-D with its cell surface receptors vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 and is being used for the development of therapeutic antibodies. We show by peptide-based mapping and site-directed mutagenesis that the VD1 binding site includes the five residues 147NEESL151 and that immunization with a synthetic peptide containing this motif generates antibodies that neutralize VEGF-D. The tertiary structure of VEGF-D indicates that the 147NEESL151 epitope is located in the L2 loop of the growth factor, which is important for receptor binding. Mutation of any of these five residues influences receptor binding; for example, mutations to E148, which abolished binding to VD1, impaired the interaction with VEGFR-2 but enhanced binding to VEGFR-3. This structure/function study indicates that the VD1 binding epitope is part of the receptor binding site of VEGF-D, identifies a region of VEGF-D critical for binding of receptors and explains why VD1 does not bind other members of the VEGF family of growth factors.

The secreted adhesive glycoprotein vitronectin (VTN) is a multifunctional component of plasma and the extracellular matrix. A high-yielding, inexpensive, low endotoxin source of bioactive recombinant human vitronectin (rhVTN) is highly desirable for in vitro use in diverse cell culture systems ranging from basic research settings to clinical-grade production of human cells. We describe modifications to a previously reported heparin-based affinity chromatography procedure that improve yield and achieve efficient removal of endotoxin from washed and urea-solubilized human VTN inclusion bodies following standard autoinduction of expression in Escherichia coli. This simple procedure makes accessible the low-cost expression and purification of large quantities of bioactive rhVTN using basic equipment and facilitates its use in a spectrum of endotoxin-sensitive applications.

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.

Vascular endothelial growth factor (VEGF)-D is a secreted glycoprotein that induces angiogenesis and lymphangiogenesis. It consists of a central domain, containing binding sites for VEGF receptor-2 (VEGFR-2) and VEGFR-3, and N- and C-terminal propeptides. It is secreted from the cell as homodimers of the full-length form that can be proteolytically processed to remove the propeptides. It was recently shown, using adenoviral gene delivery, that fully processed VEGF-D induces angiogenesis in vivo, whereas full-length VEGF-D does not. To better understand these observations, we monitored the effect of VEGF-D processing on receptor binding using a full-length VEGF-D mutant that cannot be processed. This mutant binds VEGFR-2, the receptor signaling for angiogenesis, with approximately 17,000-fold lower affinity than mature VEGF-D, indicating the importance of processing for interaction with this receptor. Further, we show that members of the proprotein convertase (PC) family of proteases promote VEGF-D processing, which facilitates the VEGF-D/VEGFR-2 interaction. The PCs furin and PC5 promote cleavage of both propeptides, whereas PC7 promotes cleavage of the C-terminal propeptide only. The finding that PCs promote activation of VEGF-D and other proteins with roles in cancer such as matrix metalloproteinases, emphasizes the importance of these enzymes as potential regulators of tumor progression and metastasis.

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.

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.

Recent discovery of new members of the vascular endothelial growth factor (VEGF) family has generated much interest as to which members may be best suited for therapeutic angiogenesis in various tissues. In this study we evaluated angiogenic responses of the different members of the VEGF family in vivo using adenoviral gene transfer. Adenoviruses (1 x 10(9) plaque-forming units [pfu]) encoding for VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-C(deltaNdeltaC) and VEGF-D(deltaNdeltaC) (deltaNdeltaC are proteolytically cleaved forms) were transferred locally to the periadventitial space of the rabbit carotid arteries using a collar technique that allows efficient local transfection of the periadventitial tissue. Expression of the transfected VEGFs was confirmed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Seven days after the gene transfer maximum neovessel formation was observed in VEGF-A-, VEGF-D-, and VEGF-D(deltaNdeltaC)-transfected arteries. VEGF-C(deltaNdeltaC) also showed angiogenic activity whereas VEGF-B was not effective in inducing angiogenesis. Pericytes were detected around the neovessels, which also frequently showed the presence of intraluminal erythrocytes. Infiltration of inflammatory cells in response to VEGF-D and VEGF-D(deltaNdeltaC) was less prominent than that caused by other VEGFs. In line with the absence of lymphatics in the normal carotid arteries no significant evidence of lymphatic vessel formation was seen in response to any of the studied VEGFs in the periadventitial space. The results help to define possibilities for local angiogenic therapy around blood vessels and support the concept that angiogenic effects may be tissue-specific and depend both on the growth factor ligands and the target tissues. It is concluded that VEGF-A, VEGF-D, and VEGF-D(deltaNdeltaC) are the best candidates for therapeutic angiogenesis when delivered around large arteries.

Objective: Vascular endothelial growth factor-D (VEGF-D) is a recently characterized member of the VEGF family, but its expression in atherosclerotic lesions remains unknown. We studied the expression of VEGF-D and its receptors (VEGFR-2 and VEGFR-3) in normal and atherosclerotic human arteries, and compared that to the expression pattern of VEGF-A. Methods: Human arterial samples ( n =39) obtained from amputation operations and fast autopsies were classified according to the stage of atherosclerosis and studied by immunohistochemistry. The results were confirmed by in situ hybridization and RT-PCR. Results: We found that while VEGF-A expression increased during atherogenesis, VEGF-D expression remained relatively stable only decreasing in complicated lesions. In normal arteries and in early lesions VEGF-D was mainly expressed in smooth muscle cells, whereas in complicated atherosclerotic lesions the expression was most prominent in macrophages and also colocalized with plaque neovascularization. By comparing the staining profiles of different antibodies, we found that proteolytic processing of VEGF-D was efficient in the vessel wall. VEGFR-2, but not VEGFR-3, was expressed in the vessel wall at every stage of atherosclerosis. Conclusions: Our results suggest that in large arteries VEGF-D is mainly expressed in smooth muscle cells and that it may have a role in the maintenance of vascular homeostasis. However, in complicated lesions it was also expressed in macrophages and may contribute to plaque neovascularization. The constitutive expression of VEGFR-2 in arteries suggests that it may be one of the principal mediators of the VEGF-D effects in large arteries.

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.

Purpose: Signaling pathways initiated by the epidermal growth factor receptor (EGFR) play important roles in the response to ionizing radiation. In this study the consequences of inhibiting the EGFR on the response of A431 cells (human vulvar squamous cell carcinoma cells that overexpress EGFR) to radiation, were investigated in vitro and in vivo , using the selective EGFR-tyrosine kinase inhibitor, ZD1839 ("Iressa"). Methods and Materials: The effect of ZD1839 on proliferation, apoptosis, and clonogenic survival after radiation was determined in vitro . For in vivo studies, athymic nude mice with established subcutaneous A431 xenografts (∼100 mm 3 ) were treated with either a single 10 Gy fraction or 4 daily 2.5 Gy fractions of radiation with or without ZD1839 (75 mg/kg/day intraperitoneally for 10 days) to determine effects on tumor growth delay. Results: Treatment of A431 cells with ZD1839 in vitro reduced proliferation, increased apoptosis, and reduced clonogenic survival after radiation. Strikingly greater than additive effects of ZD1839 in combination with radiation on tumor growth delay were observed in vivo after either a single 10 Gy fraction (enhancement ratio: 1.5) or multiple 4 × 2.5 Gy fractions (enhancement ratio: 4). ZD1839 reduced tumor vascularity, as well as levels of vascular endothelial growth factor (VEGF) protein and mRNA induced by stimulation with epidermal growth factor (EGF), suggesting a possible role of inhibition of angiogenesis in the effect. Conclusions: Inhibiting EGFR-mediated signal transduction cascades with ZD1839 potentiates the antitumor effect of single and multiple fractions of radiation. These data provide preclinical rationale for clinical trials of EGFR inhibitors including ZD1839 in combination with radiation.

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.

Members of the related to tyrosine kinase (RYK) receptor subfamily display a small extracellular region featuring a Wnt inhibitory factor domain, a single transmembrane helix with a strong propensity to self-associate, and an intracellular region exhibiting several abnormal protein sequence motifs that contribute to a protein tyrosine kinase domain. These sequence deviations are characteristic and strongly associated with a lack of detectable intrinsic protein kinase activity, implying that RYK receptors form a subfamily of pseudokinases within the receptor-type protein tyrosine kinase group. RYK subfamily members are receptors for the Wnt family of acylated glycoproteins during embryonic development and in postnatal axon pathfinding. Emerging details of Wnt/RYK-triggered β-catenin-dependent and β-catenin-independent signaling cascades regulate metazoan cell polarity and fate patterning, survival, proliferation, drug resistance, axon pathfinding and topographic mapping, migration, and the selection of appropriate target sites by cell processes. Sequential Notch-like proteolytic events that target distinct sites in RYK contribute in significant but mechanistically ill-defined ways to signaling via the coupled release of an extracellular fragment with predicted Wnt-binding activity and generation of a cytoplasmic/nuclear fragment that regulates cell fate in the mammalian central nervous system. These exciting discoveries, together with early insights into the involvement of RYK in mammalian pathology, indicate that RYK, the products of its proteolysis, and its protein partners represent attractive candidates for therapeutic targeting in a wide variety of animal and human conditions.

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.