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Growth Factors, December 2007; 25(6): 417–425
A system for quantifying the patterning of the lymphatic vasculature RAMIN SHAYAN1,2,3, TARA KARNEZIS1, EVELYN TSANTIKOS1,2, STEVEN P. WILLIAMS1, ANDREW S. RUNTING1, MARK W. ASHTON3, MARC G. ACHEN1, MARGARET L. HIBBS1, & STEVEN A. STACKER1 1
Melbourne Tumor Biology Branch, Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia, Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Victoria, Australia, and 3Jack Brockhoff Reconstructive Plastic Surgery Research Unit, Royal Melbourne Hospital and Department of Anatomy and Cell Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Victoria, Australia
2
(Received 20 December 2007; revised 14 January 2008; accepted 22 January 2008)
Abstract 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.
Keywords: Lymphatics, lymphangiogenesis, VEGF-D, lymphedema, quantification
Introduction The lymphatic vasculature consists of distinct types of lymphatic vessels with absorptive or transport functions (Skobe and Detmar, 2000; Baldwin et al. 2002). The blind-ended “initial” or “capillary” lymphatics lack a distinct vessel wall and absorb lymph fluid and cellular infiltrate from the peripheral tissues, before draining into more deeply located pre-collector lymphatic vessels (Skobe and Detmar 2000; Scavelli et al. 2004). These pre-collector lymphatics have welldefined vessel walls and valves that direct lymph flow to the collecting lymphatics, the subcutaneous, muscular conduits that transport lymph to lymph nodes and ultimately back to the venous system
(Scavelli et al. 2004; Muthuchamy et al. 2003). Cancer cells may also metastasise via the lymphatics (Stacker et al. 2002), and alterations to normal lymphatic function can result in lymph fluid accumulation (lymphedema) (Baldwin et al. 2002). In addition, lymphatic vessels give rise to tumours such as lymphangioma and Kaposi’s sarcoma (Fukunaga 2005) and have been implicated in asthma, psoriasis, rheumatoid arthritis, transplant rejection, and other inflammatory conditions (Baluk et al. 2005; Alitalo et al. 2005). Animal models have been developed to explore molecular mechanisms underlying such diseases (Stacker et al. 2002; Karkkainen et al. 2001; Wirzenius et al. 2007), and lymphatic markers
Correspondence: S. A. Stacker, Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia. Tel: 61 3 93413155. Fax: 61 3 93413107. E-mail:
[email protected] ISSN 0897-7194 print/ISSN 1029-2292 online q 2007 Informa UK Ltd. DOI: 10.1080/08977190801932550
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418 R. Shayan et al. (Breiteneder-Geleff et al. 1999; Banerji et al. 1999; Wigle and Oliver 1999) have been identified for the detection of lymphatics in animal tissues and human samples (Karpanen et al. 2001; Stacker et al. 2001; Van der Auwera et al. 2006). In the past, the analysis of lymphatic vessels in tissue has been largely restricted to measuring the abundance of immunohistochemically stained lymphatics, in two-dimensional tissue slices (Van der Auwera et al. 2006). Yet this approach does not take into account the distinct types of lymphatic vessels located at different tissue depths, and other anatomically and structurally important features of the lymphatic vasculature, such as branching, blind ended sacs (BES) or vessel loops; and does not easily allow patterning parameters such as vessel dimensions and inter-vessel spacing to be assessed (Skobe and Detmar 2000; Scavelli et al. 2004; Makinen et al. 2005). As lymphatic morphology and patterning are intimately related to function, these parameters could each influence the capacity for fluid or cellular absorption, and the magnitude or direction of lymph flow within the lymphatic vasculature (Skobe and Detmar 2000; Scavelli et al. 2004; Muthuchamy et al. 2003). It is therefore important to quantify the different features of the distinct types of lymphatic vessels in situ, within normal and pathological tissues, to pinpoint their role in these conditions. Further, the recent major focus on embryonic lymphatic development (Karkkainen et al. 2000; Karkkainen et al. 2004) requires a methodology enabling screening and detailed characterisation of functionally important developmental alterations that result from engineered genetic mutations. This will facilitate exploration of the roles of protein growth factors, cell surface receptors and other signaling molecules in lymphatic development and biology (Karkkainen et al. 2000; Karkkainen et al. 2004). Here, we describe such an approach, combined with an accessible user-friendly computer interface that is useful for analyzing lymphatic vessels during development and in disease, and for monitoring the efficacy of experimental therapeutics that target the lymphatic vasculature (Skobe and Detmar 2000; Baldwin et al. 2002; Adams and Alitalo 2007).
to image the fluorescently labelled lymphatics (Figure 1a – c). To accurately quantify lymphatic vessel patterning, we developed the Lymphatic Vessel Analysis Protocol (LVAP), a plug-in designed for quantifying lymphatic vessels using ImageJ (Abramoff et al. 2004), software commonly used for highthroughput image analysis, on multiple computer platforms (Supplemental Figure 1a and b). Due to the complex architecture of whole-mount-stained lymphatic vessels, the design of LVAP incorporates a strong user-operated component in preference to a fully automated system. LVAP takes into account important morphologic and patterning characteristics (number of BES, lymphatic branches and loops, vessel diameter, density, and inter-lymphatic vessel distance (ILVD)), to study lymphatic vessels. We have selected several animal models for validation of LVAP, in which the lymphatic vasculature is altered due to physiological or pathological stimuli. In order to demonstrate the utility of LVAP in distinguishing distinct features of different types of lymphatic vessels, the program was used to characterise the normal adult and embryonic skin lymphatics in the mouse. Our analysis demonstrated increased branching and loop formation in the embryonic lymphatics, compared with the regular lymphatic capillaries in the adult skin (Figure 1a – d). To determine if LVAP may also be used to study abnormal lymphatic vessels as a result of developmental defects and/or inflammation, we investigated a naturally occurring mutant, the “motheaten-viable” (Me v/Me v) mouse (Shultz et al. 1984), in which a genetic alteration in a signaling molecule involved in immune function (SHP-1) (Tsui et al. 1993), results in systemic inflammation and in inflamed, “motheaten” ears (Supplemental Figure 2a and b). Quantification analysis of fluorescently labelled lymphatics in the motheaten ears (Figure 1e) revealed abnormalities in several morphological and patterning parameters (Figure 1f). While the average density of lymphatics and numbers of BES were not statistically different compared with wild-type mice, the motheaten mouse ears did have statistically significant increases in lymphatic vessel width, numbers of lymphatic vessel branching points and loop structures, compared with wild-type mice (Figure 1f).
Results A computer-aided system for quantifying lymphatic vessel patterning In order to perform detailed in situ three-dimensional visualisation of intact lymphatics in relation to other vessels, we analysed whole-mount-stained (unsectioned) tissue specimens, using lymphatic-specific markers (see Methods). Due to their different anatomical locations within the tissue, adjustable upright microscopes with the capacity to visualise different specific focal depths within tissues, were used
Use of LVAP in pathological models The remodeling of blood vessels and lymphatics is a key component of several human pathologies (Adams and Alitalo, 2007). Two important examples are tumourigenesis and wound healing. The protein growth factors vascular endothelial growth factor (VEGF)-C (Joukov et al. 1996) and VEGF-D (Achen et al. 1998) drive the proliferation of lymphatic vessels (lymphangiogenesis), and have been used to create animal models in which changes to lymphatic vessels
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Figure 1. Analysis of lymphatics in the skin of adult mice and embryos. (a) Immunofluorescent whole-mount staining of adult mouse ear capillary lymphatics in a wild-type animal using anti-LYVE-1 antibody (green). (b) Fluorescent labelling of pre-collector (open arrow) and collecting lymphatics (filled arrow) using podoplanin labelling (green) in the skin of an adult wild-type mouse ear. (c) Embryonic (E18.5) skin lymphatics fluorescently labelled using anti-LYVE-1 antibody (green). (d) Quantification of branching points, lymphatic loops, blind endings sacs (BES), inter-lymphatic vessel distance (ILVD), lymphatic vessel width and lymphatic vessel density, as determined using LVAP, in lymphatic capillary, pre-collector and embryonic vessel networks in wild-type mice. (e) Fluorescent labelling of abnormal ear skin lymphatics in the “motheaten viable” (Me v/Me v) mutant mouse using anti-LYVE-1 antibody (green). (f) Quantification of lymphatic vessel characteristics by LVAP as measured in (a) wild-type and (e) motheaten viable (Me v/Me v) mice. Bar, 500 mm; asterisks, lymphatic loop; circle, blind ending sac. Asterisks above bar graphs indicate statistical significance (*p value ,0.05; **p value ,0.01). Error bars represent SEM.
can be studied (Karpanen et al. 2001; Stacker et al. 2001; Karkkainen et al. 2004; Mandriota et al. 2001; Baldwin et al. 2005). We previously used a mouse flank tumour model that secreted VEGF-D (Stacker et al. 2001), to show that local lymphangiogenesis at the primary tumour was associated with spread of tumour cells to lymph nodes. We have shown that mice harboring these tumours have high circulating
levels of VEGF-D (data not shown), however, it is not known whether this may have an effect on the systemic lymphatic network. Here, using the ear lymphatics (Figure 2a and b) to represent the systemic lymphatic network in the same flank tumour model, we have examined whether the LVAP can detect subtle systemic changes to the lymphatic vasculature that may occur in the presence of high systemic levels
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A system for quantifying the patterning of the lymphatic vasculature of VEGF-D. We also analysed a similar tumour model which secreted VEGF-C. Overall, we found minimal effects as a result of the high circulating levels of VEGF-D (Figure 2a and c), despite significant local lymphangiogenesis in the primary tumours (Stacker et al. 2001). In contrast, the enhanced circulating levels of VEGF-C resulted in alterations to ear lymphatics, in particular by inducing a small but significant increase in the lymphatic vessel width (Figure 2b and c), and consistent with this, the ILVD was diminished reciprocally in these mice (Figure 2c). This experiment is consistent with the reported effects of VEGF-C (Wirzenius et al. 2007), and demonstrates the ability of the LVAP to detect subtle variations in the lymphatic vasculature. In order to evaluate the utility of LVAP to examine models of ‘local’ effects of lymphangiogenic stimuli, the lymphatic vessels in two pathological models were studied. The same VEGF-D-secreting tumour cells as described above were grown in the mouse ear, and the resulting lymphatic vessels were compared with those generated in a simple ear-wound model (Saaristo et al. 2006) (Figure 2d– h). To investigate lymphangiogenic activity in a three-dimensional tissue specimen using the LVAP, the plug-in parameters were extended to address lymphatic vessel sprouting and loops, which are significant features in the formation of the neolymphatics (see Methods). The average number of lymphatic sprouts in the tumours (Figure 2d, e, and h) was significantly higher than at the edges of the wounds (Figure 2f – h), whereas no lymphatic sprouts were observed in healthy ear tissue (Figure 1a). In addition, the number of ‘sprout-tips’ per sprouting neolymphatic vessel (see Figure 2g for example of sprouttip) was also significantly higher in tumours than in wounds (Figure 2d– h). Interestingly, however, the length of sprouts was greater in the wound-generated lymphatics (Figure 2 h). While the tumours had significantly higher numbers of lymphatic loops, the lymphatics in the wound healing model grew in a more orderly fashion (Figure 2e, g, and h). Therefore, the LVAP system highlighted differences between lymphatic sprouting in two different pathological models, which may have biological and functional significance. These represent differences that could be overlooked using standard methods of quantification and twodimensional tissue analysis.
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Evaluation of organ-specific lymphatic vessels As lymphatics are integral to disease states in many organs (Skobe and Detmar, 2000; Alitalo et al. 2005), we compared lymphatic morphology and patterning in whole-mounted tissue specimens from normal healthy organs that are relevant to some important diseases in which lymphatics are implicated. We analysed the gastrointestinal and respiratory tracts, and the skin, which are prone to develop metastatic cancers and inflammatory disorders, involving the lymphatic system (Stacker et al. 2002; Baluk et al. 2005; Alitalo et al. 2005). We found significant variations in the nature of the lymphatics in the different organs. The characteristic pattern of skin lymphatic capillaries (Figure 3a) is distinct from the more uniform, truncated form of lymphatic vessel, the lacteal, in each villus of the small-bowel mucosa (Figure 3b), whilst there is a dense, highly branched and looped pre-collecting lymphatic network deeper within the bowel mucosa (Figure 3c). Dual staining with the blood vessel marker PECAM-1 also reveals the intimate relationship between the blood and lymphatic vascular networks (Figure 3b). In contrast, the trachea boasts few lymphatic loops, branching points or BES and the lymphatics in this tissue are arranged circumferentially between the cartilaginous tracheal rings (Figure 3d– f). Both the width and the ILVD of the lymphatics in the trachea differ significantly from those in the skin, however the overall lymphatic density was not statistically different across the three tissues assessed (Figure 3f).
Discussion We have developed a computer-aided approach for quantitative evaluation of the morphology and patterning of the lymphatic vasculature. When applied to fluorescently labelled lymphatics in whole tissue specimens, this method allows the comparison of a number of parameters, which describe the form, and relate to the function, of lymphatic vessels. The methodology was validated on healthy and pathological tissues, including models in which lymphatics were subjected to a spectrum of stimuli. This methodology allows a more comprehensive and quantitative analysis of the lymphatic vasculature than previous approaches
R Figure 2. Analysis of the lymphatics with LVAP in tumour and wound healing models. Low power image of the lymphatics in the anterior ear skin of mice bearing (a) VEGF-D or (b) VEGF-C-secreting flank tumours, labelled with anti-LYVE-1 antibody (green). (c) Quantitative comparison of ear skin lymphatic vessels from mice bearing VEGF-D or VEGF-C-secreting flank tumours, using LVAP. (d) Low and (e) highpower representative images of whole-mounted, fluorescently labelled lymphatics (anti-LYVE-1) (green) in an ear tumour; and (f) low and (g) high-power representative images of whole-mounted, fluorescently labelled skin lymphatics in an ear wound. Open arrow indicates a sprout with a single tip, filled arrows indicate sprout with multiple tips. (h) Comparative quantification of average number of sprouts, tips per sprout, and lymphatic loops formed, and average sprout length in the tumour and wounding models, using the LVAP. Bar, 500 mm; asterisks, lymphatic loops; dotted line, wound edge; T, tumour. Asterisks above bar graphs indicate statistical significance (*p value ,0.05); error bars represent SEM.
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Figure 3. Comparison of lymphatics in different organs using LVAP. (a) Low power representative image of whole-mount fluorescent labelled lymphatics in wild-type adult mouse ear skin, using anti-LYVE-1 antibody (green). Whole-mount gastrointestinal (small bowel) mucosa depicting fluorescent-labelled lymphatics in (b) high-power, side view of lacteal in villus (filled arrows) and pre-collectors (open arrows); and (c) low-power view of small bowel mucosa stained using anti-LYVE-1 antibody (green) to show lymphatics; and anti-PECAM-1 antibody (red) to label blood vessels. (d) Low and (e) high-power images of whole-mount fluorescent labelling of lymphatics in the respiratory tract (trachea) mucosa using anti-LYVE-1 antibody (green) (open arrows show cartilaginous tracheal rings). (f) Comparative quantification of average number of branching points, loops, BES and average lymphatic vessel width, ILVD and lymphatic vessel density in ear skin, gastrointestinal tract (small bowel from (c)), and trachea. Bar, 500 mm; Asterisks above bar graphs indicates statistical significance (*p value ,0.05; **p value ,0.01); error bars represent SEM.
that are based on counting lymphatic vessels in two-dimensional tissue slices (Stacker et al. 2002; Van der Auwera et al. 2006) or are semi-quantitative in nature. Another advantage of our approach is that subtle lymphatic abnormalities, which may have previously been overlooked, can now be identified and evaluated. Importantly, the LVAP system also
allows the user to store a record of each individual vessel parameter count performed, aiding validation and allowing retrospective comparisons of data as knowledge in the field of lymphatics, including definitions of vessel structures, continues to evolve. The LVAP will also be useful for quantifying the results of in vitro assays of lymphangiogenesis
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Supplemental Figure 1. (a) ‘Screen grab of ‘Lymphatic Vessel Analysis Protocol’ plug-in and Image J tool bar (Abramoff et al. 2004). (b) ‘Screen grab of ‘Lymphatic Vessel Analysis Protocol’ graph plug-in layover on picture, to enable quantification of whole-mounted, fluorescently labelled lymphatic vessels.
or lymphatic tube formation (for example see Supplemental Figure 3a and b). In designing the LVAP, we chose to examine parameters that reflect the definitive structural features of lymphatic vessels and those subject to change during physiological or pathological challenges. When the lymphatics respond to altered tissue demands or stimulatory growth factors, the capacity for fluid absorption and transport, and the dimensions of the lymphatics and surrounding interstitial space can change (Skobe and Detmar 2000; Wirzenius et al. 2007; He et al. 2005). In developmental defects, signals which specify the correct branching, caliber or spacing of vessels can become deranged (Karkkainen et al. 2001; Makinen et al. 2005). In pathological conditions such as wound healing (Saaristo et al. 2006) and tumour formation (Karpanen et al. 2001; Stacker et al. 2001; Mandriota et al. 2001; Stacker et al. 2004; Achen et al. 2006), the expression of lymphangiogenic growth factors can cause abnormal proliferation, and possibly differentiation, of lymphatic vessels resulting in a mixture of vessel changes, including vessel sprouting and vessel dilatation
Supplemental Figure 2. (a) Macroscopic pictures of adult wild-type mouse ear. (b) Macroscopic picture of swollen motheaten ear of agematched “motheaten viable (Me v/Me v)” mutant mouse.
(Wirzenius et al. 2007; He et al. 2005). These features of lymphatics are assessed in the current version of the LVAP program, which has the capacity to be updated in the future, as our understanding of, and capacity to manipulate, lymphatics evolves. The LVAP program has a number of applications for examining lymphatic vessels. As a tool for research it will facilitate the analysis of lymphatic defects (such
Supplemental Figure 3. (a) Representative image of tube formation by lymphatic endothelial cells in culture. (b) Quantitative comparison of lymphatic endothelial cell culture in vitro, pictured in Supplemental Figure 3a, compared to in vivo lymphatic vessel formation (as seen in Supplementary Figure 1b), using LVAP. (2mu*p value ,0.05); error bars represent SEM. Bar, 200 mm.
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424 R. Shayan et al. as lymphedema) in mice with experimentally generated genetic mutations (Karkkainen et al. 2001; Karkkainen et al. 2000). This could include large populations of mutant mice generated in functional genetic screens. Finally, the capacity to evaluate local and systemic lymphatic responses and complimentary in vitro assays, makes the LVAP valuable as an efficient and reproducible platform, with which to comprehensively analyze the therapeutic agents that target lymphatics, in animal models of disease.
Methods Mouse mutant, tumour and wounding experiments 293EBNA-1 cells stably transfected to express VEGFC or VEGF-D, were xenografted subcutaneously to the flank or ear of SCID/NOD mice (Stacker et al. 2001). Animals were sacrificed on reaching the tumour size limit stipulated by the guidelines of the Ludwig Institute Animal Ethics Committee, and serum samples were taken, before harvesting both ears. Embryos used were embryonic day 18.5. Homozygous Motheaten viable (Me v /Me v (C57BL/6)) mutant mice (Shultz et al. 1984) and wild-type, age-matched littermates were obtained from The Walter and Eliza Hall Institute of Medical Research. Whole-mounted tissues were imaged after fluorescent labelling with lymphatic and/or bloodvessel markers. Ear wounds were generated using an ear skin punch, as described (Saaristo et al. 2006), and the wound-edge imaged circumferentially.
Fluorescent lymphatic labelling Skin from embryos and whole ears from adult mice were dissected and fixed (6 h) in 4% paraformaldehyde, before incubation in blocking solution (1% BSA, 5% goat serum in 0.3% Triton in PBS), followed by primary antibody (16 h, 48C) (Makinen et al. 2005). Samples were washed (6 h) then incubated with fluorescently conjugated secondary antibodies (16 h, 48C), before mounting in Vectashield (Vector Laboratories). Samples were imaged and analysed using a Nikon Eclipse 90i upright fluorescent microscope and Nikon DXM1200c digital camera.
Antibodies Antibodies used: hamster anti-mouse podoplanin (RDI), 1:1000; rabbit polyclonal anti-mouse LYVE1 (Fitzgerald Industries), 1:1000; rat anti-mouse CD31 (BD Pharmingen), 1:200. Anti-rat IgG Alexa 594, anti-rabbit IgG Alexa 488, and anti-hamster IgG Alexa 488 fluorescent secondary antibodies (Molecular Probes) were used at 1:200 dilution.
Quantification of lymphatic vessels Apical photographs of mouse ears were taken ( £ 4 magnification), opened in the Image J program (Abramoff et al. 2004), and the image ‘initialised’ in the ‘Lymphatic Vessel Analysis Protocol’ (LVAP) plugin. Images were overlaid with a 200 pixel grid using a specific plug-in. (Supplemental Figure 1b). The operator selects a parameter (variable of lymphatic vessel morphology or patterning e.g. vessel width) then, aided by the grid, proceeds systematically through the image. Locating the cursor on the parameter (or “event”) the mouse button is clicked, both counting the event and automatically recording a marker at that point, to avoid re-counting. In the case of the lymphatic vessel width and ILVD, the operator follows the horizontal grid lines from left to right across the screen, and places a marker at the intersection between this line and a lymphatic. The second click on the other side of the vessel demarcates the end of the vessel and this distance is measured. In the case of the ILVD, the markers are placed at the beginning and end of the space between vessels. The lymphatic vessel density was the total number of intersections between a horizontal grid line and a lymphatic vessel throughout the image, divided by the number of lines. All these measurements are made, and the values tallied, before the data are integrated into an Excel spreadsheet for statistical analysis. The number of lymphatic vessel branching points, loops, and BES were calculated using the appropriate buttons in the LVAP tool, in a blinded fashion, and averaged over each image. Averaged field counts for each parameter were collated for all sections and used to obtain averages for each ear assessed, before data for all mice were de-identified and grouped to generate averages for each mouse type, and their respective statistical significance determined via the Student’s T-test (Microsoft Excel). Parameters chosen in wounding experiments were: number of (1) lymphatic sprouts, (2) tips per sprout, (3) loops and (4) sprout length (Figure 2f – h).
Software design The LVAP was designed as a plug-in for ImageJ, a Java-based image analysis application for highthroughput processing and image manipulation on multiple computer platforms (Abramoff et al. 2004). ImageJ is free to download and use, with numerous plug-ins that extend the functionality of the program, which have been contributed by the scientific community. As there were no plug-ins that were sufficient for the needs of this project, two existing plug-ins were modified as required. An on-line tutorial on the use of LVAP, the modified source code and plug-ins are available from the Ludwig Institute for Cancer Research (Melbourne Branch) website: http:// www.ludwig.edu.au/archive.
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Acknowledgements The authors would like to acknowledge Tony Burgess for critical reading of this manuscript, and thank JeeWong Chew for his IT expertise, Kari Alitalo for the generous provision of a VEGF-C cDNA, Janna Taylor and Pierre Smith for assistance with the generation of figures, and Stephen Cody for assistance with microscopy. This work was supported by Program and Project Grants from the National Health and Medical Research Council of Australia (NHMRC). R.S. is supported by the Surgical Scientist Scholarship Program of the Royal Australasian College of Surgeons; E.T. by an Australian Postgraduate Award, M.G.A. and M.L.H. by Senior NHMRC Research Fellowships and S.A.S. by a Foundation Fellowships and a Senior NHMRC Research Fellowship from Pfizer.
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Author Contributions R. Shayan and T. Karnezis concept and method development, experimental work and manuscript preparation; E. Tsantikos and S.P. Williams experimental work and manuscript preparation; A.S. Runting software design and testing; M.W. Ashton, M.G. Achen, M.L. Hibbs, and S.A. Stacker concept and manuscript preparation. Competing Interest Statement S.A. Stacker and M.G. Achen are consultants for Vegenics Ltd.
