Tara Karnezis

Sox18 induces development of the lymphatic vasculature in mice

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.

Lymphangiogenesis and lymphatic vessel remodelling in cancer

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.

VEGF-D Promotes Tumor Metastasis by Regulating Prostaglandins Produced by the Collecting Lymphatic Endothelium

Lymphatic 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.

Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2

Vascular endothelial growth factor (VEGF)‐D is a secreted glycoprotein that induces angio‐genesis 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 propep‐tides. 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 ~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.—McColl, B. K., Paavonen, K., Karnezis, T., Harris, N. C., Davydova, N., Rothacker, J., Nice, E. C., Harder, K. W., Roufail, S., Hibbs, M. L., Rogers, P. A. W., Alitalo, K., Stacker, S. A., Achen, M. G. Proprotein convertases promote processing of VEGF‐D, a critical step for binding the angiogenic receptor VEGFR‐2. FASEB J. 21, 1088–1098 (2007)

Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination

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.

Early detection of the chemical changes occurring during the induction and prevention of autoimmune-mediated demyelination detected by FT-IR imaging

Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Despite progress in understanding immunogenetic aspects of this disease, the mechanisms involved in lesion formation are unknown. To gain new insights into the neuropathology of MS, we used an innovative integration of Fourier transform infrared (FT-IR) microspectroscopy, bioinformatics, and a synchrotron light source to analyze macromolecular changes in the CNS during the course and prevention of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We report that subtle chemical and structural changes not observed by conventional histology were detected before the onset of clinical signs of EAE. Moreover, trained artificial neural networks (ANNs) could discriminate, with excellent sensitivity and specificity, pathology from surrounding tissues and the early stage of the disease progression. Notably, we show that this novel measurement platform can detect characteristic differences in biochemical composition of lesion pathology in animals partially protected against EAE by vaccination with Nogo-A, an inhibitor of neural outgrowth, demonstrating the potential for automated screening and evaluation of new therapeutic agents.

A role for bone morphogenetic protein-4 in lymph node vascular remodeling and primary tumor growth

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.

A system for quantifying the patterning of the lymphatic vasculature

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.

Lymphatic vessel density in primary melanomas predicts sentinel lymph node status and risk of metastasis

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

Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer

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.—Harris, N. C., Paavonen, K., Davydova, N., Roufail, S., Sato, T., Zhang, Y. ‐F., Karnezis, T., Stacker, S. A., Achen, M. G. Proteolytic processing of vascular endothelial growth factor‐D is essential for its capacity to promote the growth and spread of cancer. FASEB J. 25, 2615–2625 (2011). www.fasebj.org