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Original Article| Volume 133, ISSUE 8, P2074-2084, August 2013

Vascular Endothelial Growth Factor-d Modulates Caliber and Function of Initial Lymphatics in the Dermis

  • Sophie Paquet-Fifield
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Sidney M. Levy
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia

    Jack Brockhoff Reconstructive Plastic Surgery Research Unit, Royal Melbourne Hospital and Department of Anatomy and Cell Biology, The University of Melbourne, Parkville, Victoria, Australia

    Department of Surgery, Royal Melbourne Hospital, The University of Melbourne, Parkville, Victoria, Australia
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  • Author Footnotes
    6 Current address: Department of Respiratory Medicine, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.
    Teruhiko Sato
    Footnotes
    6 Current address: Department of Respiratory Medicine, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.
    Affiliations
    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Ramin Shayan
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia

    Jack Brockhoff Reconstructive Plastic Surgery Research Unit, Royal Melbourne Hospital and Department of Anatomy and Cell Biology, The University of Melbourne, Parkville, Victoria, Australia

    Department of Surgery, Royal Melbourne Hospital, The University of Melbourne, Parkville, Victoria, Australia
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  • Tara Karnezis
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Natalia Davydova
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Cameron J. Nowell
    Affiliations
    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Sally Roufail
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Gerry Zhi-Ming Ma
    Correspondence
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
    Affiliations
    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • You-Fang Zhang
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia
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  • Steven A. Stacker
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia

    Department of Surgery, Royal Melbourne Hospital, The University of Melbourne, Parkville, Victoria, Australia

    Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia
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  • Marc G. Achen
    Correspondence
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
    Affiliations
    Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia

    Department of Surgery, Royal Melbourne Hospital, The University of Melbourne, Parkville, Victoria, Australia

    Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia
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  • Author Footnotes
    6 Current address: Department of Respiratory Medicine, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.
      The lymphatic vasculature is important for skin biology as it maintains dermal fluid homeostasis. However, the molecular determinants of the form and function of the lymphatic vasculature in skin are poorly understood. Here, we explore the role of vascular endothelial growth factor-d (Vegf-d), a lymphangiogenic glycoprotein, in determining the form and function of the dermal lymphatic network, using Vegf-d-deficient mice. Initial lymphatic vessels in adult Vegf-d-deficient mice were significantly smaller than wild-type but collecting lymphatics were unaltered. The uptake/transport of dextran in initial lymphatics of Vegf-d-deficient mice was far less efficient, indicating compromised function of these vessels. The role of Vegf-d in modulating initial lymphatics was further supported by delivery of Vegf-d in skin of wild-type mice, which promoted enlargement of these vessels. Vegf-d-deficient mice were subjected to cutaneous wounding to challenge lymphatic function: the resulting wound epithelium was highly edematous and thicker, reflecting inadequate lymphatic drainage. Unexpectedly, myofibroblasts were more abundant in Vegf-d-deficient wounds leading to faster wound closure, but resorption of granulation tissue was compromised suggesting poorer-quality healing. Our findings demonstrate that Vegf-d deficiency alters the caliber of initial lymphatics in the dermis leading to reduced functional capacity.

      Abbreviations

      K14
      cytokeratin 14
      LEC
      lymphatic endothelial cell
      pw
      post wounding
      Vegf
      vascular endothelial growth factor
      Vegfr
      Vegf receptor

      Introduction

      The lymphatic vasculature is important for tissue fluid homeostasis, immune function, and absorption of dietary fats, and has a key role in a range of pathologies, including cancer, chronic inflammation, and lymphedema (
      • Baldwin M.E.
      • Stacker S.A.
      • Achen M.G.
      Molecular control of lymphangiogenesis.
      ;
      • Achen M.G.
      • McColl B.K.
      • Stacker S.A.
      Focus on lymphangiogenesis in tumor metastasis.
      ;
      • Cueni L.N.
      • Detmar M.
      The lymphatic system in health and disease.
      ;
      • Alitalo K.
      The lymphatic vasculature in disease.
      ). The growth of lymphatic vessels, lymphangiogenesis, is a feature of wound healing (
      • Paavonen K.
      • Puolakkainen P.
      • Jussila L.
      • et al.
      Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing.
      ;
      • Maruyama K.
      • Asai J.
      • Ii M.
      • et al.
      Decreased macrophage number and activation lead to reduced lymphatic vessel formation and contribute to impaired diabetic wound healing.
      ;
      • Shayan R.
      • Karnezis T.
      • Tsantikos E.
      • et al.
      A system for quantifying the patterning of the lymphatic vasculature.
      ) and facilitates the metastatic spread of tumor cells (
      • Mandriota S.J.
      • Jussila L.
      • Jeltsch M.
      • et al.
      Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.
      ;
      • Skobe M.
      • Hawighorst T.
      • Jackson D.G.
      • et al.
      Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.
      ;
      • Stacker S.A.
      • Caesar C.
      • Baldwin M.E.
      • et al.
      VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.
      ;
      • Shayan R.
      • Achen M.G.
      • Stacker S.A.
      Lymphatic vessels in cancer metastasis: bridging the gaps.
      ;
      • Achen M.G.
      • Stacker S.A.
      Molecular control of lymphatic metastasis.
      ;
      • Tammela T.
      • Alitalo K.
      Lymphangiogenesis: molecular mechanisms and future promise.
      ). In skin, lymphangiogenesis can influence dermal fluid volume and various forms of inflammation (
      • Kajiya K.
      • Sawane M.
      • Huggenberger R.
      • et al.
      Activation of the VEGFR-3 pathway by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis.
      ;
      • Huggenberger R.
      • Ullmann S.
      • Proulx S.T.
      • et al.
      Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.
      ,
      • Huggenberger R.
      • Siddiqui S.S.
      • Brander D.
      • et al.
      An important role of lymphatic vessel activation in limiting acute inflammation.
      ), although the molecular determinants of the structure and function of the lymphatic vasculature in skin are not well understood. The lymphatic vasculature of the dermis primarily consists of initial lymphatics, which absorb fluid from the interstitium, and precollector vessels, which drain the lymph fluid into subcutaneous collecting lymphatics (
      • Oliver G.
      • Alitalo K.
      The lymphatic vasculature: recent progress and paradigms.
      ).
      Lymphangiogenesis is in part controlled by members of the vascular endothelial growth factor (Vegf) family of secreted glycoproteins, in particular VEGF-C (
      • Joukov V.
      • Pajusola K.
      • Kaipainen A.
      • et al.
      A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt-4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.
      ) and VEGF-D (
      • Achen M.G.
      • Jeltsch M.
      • Kukk E.
      • et al.
      Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).
      ), signaling via the receptor tyrosine kinase Vegf receptor (VEGFR)-3 (
      • Makinen T.
      • Veikkola T.
      • Mustjoki S.
      • et al.
      Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.
      ;
      • Veikkola T.
      • Jussila L.
      • Makinen T.
      • et al.
      Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.
      ;
      • Haiko P.
      • Makinen T.
      • Keskitalo S.
      • et al.
      Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos.
      ) located on the surface of lymphatic endothelial cells (LECs) (
      • Lymboussaki A.
      • Partanen T.A.
      • Olofsson B.
      • et al.
      Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.
      ). VEGF-D can stimulate pathologic lymphangiogenesis, and induce dilation of collecting lymphatic vessels, which facilitate lymph node and/or distant organ metastasis in mouse models of cancer (
      • Stacker S.A.
      • Caesar C.
      • Baldwin M.E.
      • et al.
      VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.
      ;
      • Von Marschall Z.
      • Scholz A.
      • Stacker S.A.
      • et al.
      Vascular endothelial growth factor-D induces lymphangiogenesis and lymphatic metastasis in models of ductal pancreatic cancer.
      ;
      • Achen M.G.
      • Mann G.B.
      • Stacker S.A.
      Targeting lymphangiogenesis to prevent tumour metastasis.
      ;
      • Kopfstein L.
      • Veikkola T.
      • Djonov V.G.
      • et al.
      Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis.
      ;
      • Karnezis T.
      • Shayan R.
      • Caesar C.
      • et al.
      VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium.
      ). Its expression in a range of human cancers can correlate with metastasis and poor patient outcome (
      • Debinski W.
      • Slagle-Webb B.
      • Achen M.G.
      • et al.
      VEGF-D is an X-linked/AP-1 regulated putative onco-angiogen in human glioblastoma multiforme.
      ;
      • Stacker S.A.
      • Williams R.A.
      • Achen M.G.
      Lymphangiogenic growth factors as markers of tumor metastasis.
      ;
      • Achen M.G.
      • McColl B.K.
      • Stacker S.A.
      Focus on lymphangiogenesis in tumor metastasis.
      ). Human VEGF-D has been shown to promote lymphangiogenesis and/or angiogenesis in healthy adult tissues in a range of viral and protein delivery studies (
      • Marconcini L.
      • Marchio S.
      • Morbidelli L.
      • et al.
      c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro.
      ;
      • Byzova T.V.
      • Goldman C.K.
      • Jankau J.
      • et al.
      Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo.
      ;
      • Bhardwaj S.
      • Roy H.
      • Gruchala M.
      • et al.
      Angiogenic responses of vascular endothelial growth factors in periadventitial tissue.
      ;
      • Rissanen T.T.
      • Markkanen J.E.
      • Gruchala M.
      • et al.
      VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses.
      ;
      • Wise L.M.
      • Ueda N.
      • Dryden N.H.
      • et al.
      Viral vascular endothelial growth factors vary extensively in amino acid sequence, receptor-binding specificities, and the ability to induce vascular permeability yet are uniformly active mitogens.
      ;
      • Rutanen J.
      • Rissanen T.T.
      • Markkanen J.E.
      • et al.
      Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart.
      ), is localized to vascular smooth muscle cells associated with large arteries in human tissues (
      • Partanen T.A.
      • Arola J.
      • Saaristo A.
      • et al.
      VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.
      ;
      • Achen M.G.
      • Williams R.A.
      • Minekus M.P.
      • et al.
      Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis.
      ;
      • Rutanen J.
      • Leppanen P.
      • Tuomisto T.T.
      • et al.
      Vascular endothelial growth factor-D expression in human atherosclerotic lesions.
      ), and has been proposed to have a role in vascular homeostasis (
      • Achen M.G.
      • Williams R.A.
      • Minekus M.P.
      • et al.
      Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis.
      ;
      • Rutanen J.
      • Leppanen P.
      • Tuomisto T.T.
      • et al.
      Vascular endothelial growth factor-D expression in human atherosclerotic lesions.
      ).
      The role of VEGF-D in the development of the lymphatic vasculature in mammals is thought to be subtle (
      • Koch M.
      • Dettori D.
      • Van Nuffelen A.
      • et al.
      VEGF-D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis.
      ), with the only alteration reported to date in Vegf-d-deficient mice under nonpathological conditions being a reduction in the abundance of pulmonary lymphatics (
      • Baldwin M.E.
      • Halford M.M.
      • Roufail S.
      • et al.
      Vascular endothelial growth factor d is dispensable for development of the lymphatic system.
      ). A study in Xenopus laevis tadpoles showed that knockdown of VEGF-D transiently impaired LEC migration and suggested a modifier role in the regulation of embryonic lymphangiogenesis (
      • Ny A.
      • Koch M.
      • Vandevelde W.
      • et al.
      Role of VEGF-D and VEGFR-3 in developmental lymphangiogenesis, a chemicogenetic study in Xenopus tadpoles.
      ). These findings are in contrast to Vegf-c, which has been shown to have a key role in lymphatic development, being required for sprouting of the first lymphatic vessels from embryonic veins (
      • Karkkainen M.J.
      • Haiko P.
      • Sainio K.
      • et al.
      Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.
      ).
      Lymphangiogenesis occurs during cutaneous wound healing, and involves the sprouting of new VEGFR-3-positive lymphatic vessels from pre-existing lymphatics at the wound edge. These new lymphatics grow deep into the granulation tissue, where they appear after the establishment of blood vessels (
      • Paavonen K.
      • Puolakkainen P.
      • Jussila L.
      • et al.
      Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing.
      ;
      • Shimamura K.
      • Nakatani T.
      • Ueda A.
      • et al.
      Relationship between lymphangiogenesis and exudates during the wound-healing process of mouse skin full-thickness wound.
      ;
      • Martinez-Corral I.
      • Olmeda D.
      • Dieguez-Hurtado R.
      • et al.
      In vivo imaging of lymphatic vessels in development, wound healing, inflammation, and tumor metastasis.
      ). The functional significance for wound healing of these lymphatics, which later regress (
      • Paavonen K.
      • Puolakkainen P.
      • Jussila L.
      • et al.
      Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing.
      ), is unclear. In contrast, the lymphatics in intact skin adjacent to cutaneous wounds are known to have a role in draining some of the wound-derived fluid (
      • Shimamura K.
      • Nakatani T.
      • Ueda A.
      • et al.
      Relationship between lymphangiogenesis and exudates during the wound-healing process of mouse skin full-thickness wound.
      ).
      Given that VEGF-D is a lymphangiogenic growth factor and that the mouse vegfd gene is strongly expressed in developing skin (
      • Achen M.G.
      • Williams R.A.
      • Minekus M.P.
      • et al.
      Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis.
      ), Vegf-d could have a role in development and function of the dermal lymphatic vasculature. Here, we test the hypothesis that Vegf-d modulates the structure and function of dermal lymphatics by monitoring the morphology of the lymphatic network, and the function of the lymphatics during cutaneous wound healing, in Vegf-d-deficient mice. We report that Vegf-d deficiency modulates the caliber and function of initial lymphatics in the dermis.

      Results

      Initial lymphatics in the dermis are significantly smaller in Vegf-d-deficient mice

      We monitored dermal lymphatic vessels in adult male Vegf-d-deficient mice (hemizygous vegfd−/-males; note the vegfd gene is located on the X chromosome (
      • Jenkins N.A.
      • Woollatt E.
      • Crawford J.
      • et al.
      Mapping of the gene for vascular endothelial growth factor-D in mouse and man to the X chromosome.
      )) on pure C57Bl/6 genetic background at three distinct anatomical locations—tail, ear, and flank skin. Dermal lymphatic vessels, predominantly initial lymphatics and precollectors, were immunostained for the LEC marker LYVE-1 (Figure 1 and Supplementary Materials and Methods online). Initial lymphatics in the dermis were significantly smaller in Vegf-d-deficient mice than in wild-type littermate controls at all the anatomical locations analyzed. The average size of these vessels in the tails of Vegf-d-deficient mice (taking into account the endothelium and the lumen as assessed in tissue sections) was 34% smaller than in wild-type mice (Figure 1a and b), and, similarly, the width of the initial lymphatics was 34% smaller in the ears of Vegf-d-deficient mice, as assessed in tissue whole mounts (Figure 1c and d). Further, there was a clear alteration in the size distribution of initial lymphatics in the flank dermis, with larger initial lymphatics (>480μm2 in size as measured in tissue sections) being far more abundant in wild-type than in Vegf-d-deficient mice (Figure 1e and f). We found that there was no significant difference in the number of endothelial cell nuclei per initial lymphatic vessel in the tails of Vegf-d-deficient and wild-type mice (as detected by immunohistochemistry on tissue sections) (Figure 1g), indicating that these vessels are smaller in Vegf-d-deficient mice because the endothelial cells lining them are smaller, not because there are fewer endothelial cells.
      Figure thumbnail gr1
      Figure 1Initial lymphatics in the dermis are significantly smaller in adult vascular endothelial growth factor-d (Vegf-d)-deficient mice. (a) Initial lymphatics (red) in dermis (white bar) are visualized by LYVE-1 staining in tail of wild-type (Wt) mouse, and (b) sizes of LYVE-1-positive vessels are compared in Wt and Vegf-d-deficient (Ko) mice (n=6). (c) Whole-mount LYVE-1 staining reveals abundant initial lymphatics (green) in the ear of Wt mouse, and (d) width of these vessels is compared (n=9 for Wt; 13 for Ko). (e) Initial lymphatics (arrowheads) in the dermis are detected in flank skin of Wt and Ko mice by LYVE-1 staining, and (f) abundance of different size classes of these vessels is compared (n=11 for Wt; 10 for Ko). (g) Abundance of 4′6-diamidino-2-phenylindole (DAPI)-stained endothelial cell nuclei in initial lymphatics in tissue sections of dermis from Wt and Ko tails. (h) Density of initial lymphatics in the dermis of the tail and flank in Wt and Ko mice. (i) An epifascial collecting lymphatic vessel (red) is visualized in the tail of Wt mouse by podoplanin staining, and (j) sizes of collecting lymphatics are compared (n=6 for Wt; 5 for Ko). (k) The density (left) and size (right) of PECAM-1-positive blood vessels in the dermis of flank skin are compared (n=5 for Wt; 6 for Ko). Graphs show mean±SEM, and statistical analysis was by Student t-test; *P<0.05; **P<0.01.
      There was no difference in spatial distribution of initial lymphatics in the dermis of the tail or flank (i.e., in the depth of these vessels within the tissue) in Vegf-d-deficient compared with wild-type mice (data not shown), nor in the density of these lymphatic vessels (Figure 1h). The alteration in the size of initial lymphatics in Vegf-d-deficient mice exhibited specificity in terms of lymphatic subtypes given there was no difference in the size, or location, of the major epifascial lymphatic collecting vessels in the tails of these mice compared with wild-type controls, as assessed by immunostaining for the LEC marker podoplanin (Figure 1i and j). There were no significant differences in overall density or size of blood vessels in the dermis of the flank in Vegf-d-deficient compared with wild-type mice, as assessed by staining for PECAM-1 (Figure 1k).

      Initial lymphatics of Vegf-d-deficient mice are functionally compromised

      The functional capacity of initial lymphatics in the dermis to absorb and transport macromolecules was assessed in the tail by intravital lymphangiography. FITC–dextran was injected at the tip of the tail, and its uptake and transport in the network of initial lymphatics monitored over time by fluorescence microscopy (see Figure 2a and b, and Materials and Methods). The peak fluorescence intensity arising from the FITC–dextran was similar in initial lymphatics of Vegf-d-deficient and wild-type tails; however, the fluorescence intensity increased much slower in Vegf-d-deficient lymphatics (Figure 2c and d). The difference between the fluorescence intensity in the initial lymphatics of Vegf-d-deficient mice compared with wild-type was statistically significant over a prolonged period (i.e., ∼40seconds) during the experiment. These findings indicate that uptake and/or transport of dextran was far less efficient in Vegf-d-deficient mice than in wild-type controls.
      Figure thumbnail gr2
      Figure 2Compromised uptake/transport of FITC–dextran in lymphatics of vascular endothelial growth factor-d (Vegf-d)-deficient mice. Intravital lymphangiography was carried out in the tails of mice to monitor uptake/transport of FITC–dextran in the initial lymphatic network of the dermis. Schematic representation of approach (a): FITC–dextran was injected at tail tip and fluorescence measured inside initial lymphatics over time at three locations (denoted 1, 2, and 3) 2.5cm from tip. (b) Representative image of initial lymphatics in Vegf-d-deficient (Ko) mouse 2minutes after injection of FITC–dextran. (c) Fluorescence intensity measured over time in initial lymphatics of wild-type (Wt) and Ko mice (n=4 for Wt; 3 for Ko). (d) Table shows measurement parameters from analysis in c. Scale bar in b represents 500μm; “AU” in c and d denotes arbitrary units; * in c and d denotes P<0.05 as assessed by Student’s t-test.
      Perturbation in lymphatic function can lead to swelling of tissue; however, there was no visual or histological evidence of altered interstitial fluid volume in the skin of adult Vegf-d-deficient mice. Hence, although the uptake and/or transport of macromolecules in the initial lymphatics of these mice were compromised, this was not sufficient to induce detectable edema or lymphedema under nonpathological conditions.

      Cutaneous wound healing in Vegf-d-deficient mice

      The process of cutaneous wound healing involves accumulation of fluid, which must ultimately be drained from the wound region. It has been proposed that lymphatic vessels, particularly in intact skin surrounding cutaneous wounds, drain a proportion of the wound-derived fluid (
      • Shimamura K.
      • Nakatani T.
      • Ueda A.
      • et al.
      Relationship between lymphangiogenesis and exudates during the wound-healing process of mouse skin full-thickness wound.
      ). Therefore, we tested the functional capacity of the dermal lymphatic network of Vegf-d-deficient mice by generating full-thickness excisional wounds (6mm in diameter) on the upper dorsal back. At day 7 post wounding (pw), there was pronounced edema in the epithelium in Vegf-d-deficient wounds, in contrast to wild-type (Figure 3a). The fluid accumulation occurred between the keratinocytes in the basal layer of the epithelium. The epithelium was also much thicker, which was predominantly owing to the edema given that there was no alteration in the number of cells across the width of the epithelium (Figure 3b). The density of nuclei in the basal layer of the wound epithelium was lower in Vegf-d-deficient than in wild-type mice, consistent with the observed edema (Figure 3b). The edema in the epithelium was no longer apparent at day 10 pw, indicating it was restricted to the early phase of the healing process. We did not detect edema in the granulation tissue of Vegf-d-deficient wounds.
      Figure thumbnail gr3
      Figure 3Altered wound healing in vascular endothelial growth factor-d (Vegf-d)-deficient mice. Wild-type (Wt) and Vegf-d-deficient (Ko) mice were subjected to 6-mm-diameter full-thickness wounds on back. (a) Hematoxylin and eosin staining of epithelium over wounds (white bars) revealed edema (arrowheads) and thickening in Ko mice at day 7 post wounding (pw). (b) Thickness of epithelium (top left), number of cells across depth of epithelium (top right), and density of nuclei in the basal layer of the epithelium (bottom) are compared in Wt and Ko mice (n=4) at day 7 pw. (c) Representative wounds are shown macroscopically, and (d) wound closure was monitored by measuring the length of the wound and crust, along anterio-posterior axis, with callipers (n≥4 for all time points). (e) Abundance of smooth muscle actin-positive (SmA+) myofibroblasts in granulation tissue (GT) at days 7 and 10 pw (n=4). (f) Thickness of GT (indicated by white bars in photographs that show trichrome staining at day 10 pw) in wounds of Wt and Ko mice is compared in graph (n=4). Scale bar in a corresponds to 100μm and in c to 2mm. In f, “D” denotes days pw and black scale bars correspond to 100μm. Graphs show mean±SEM, and statistical analysis was by Student t-test; *P<0.05; **P<0.01.
      Interestingly, wounds closed faster in Vegf-d-deficient mice. By day 6 pw, the fibrin clot had resolved whereas the wound area of control mice was still covered by a large fibrin crust until day 10 pw. Further, the rate at which the wound edges approached each other between days 1 and 10 pw was accelerated in Vegf-d-deficient mice (Figure 3c and d). We observed that smooth muscle actin-positive myofibroblasts were 4-fold more abundant at day 7 pw in the granulation tissue of Vegf-d-deficient mice than in wild-type (Figure 3e), but were of comparable abundance at day 10 pw. It is highly likely that the faster rate of wound closure in Vegf-d-deficient mice is owing to the increase in myofibroblasts, given that these cells enhance the contraction of wounds (
      • Grinnell F.
      Fibroblasts, myofibroblasts, and wound contraction.
      ). We monitored the resorption of granulation tissue by staining for collagen. Resorption begins at about day 10 pw in the mouse, leads to thinner granulation tissue and is a hallmark of wound maturation (
      • James D.W.
      • Newcombe J.F.
      Granulation tissue resorption during free and limited contraction of skin wounds.
      ). As expected, granulation tissue became significantly thinner from day 10 to 20 pw in wild-type wounds (Figure 3f); however, it did not alter over this time period in Vegf-d-deficient wounds, suggesting compromised wound remodeling.
      We monitored lymphatic vessels in granulation tissue to assess whether an altered lymphatic vasculature within the wounds might have contributed to edema in the epithelium (Figure 4a). There was no statistically significant difference in the overall density of lymphatic vessels in the granulation tissue of Vegf-d-deficient and wild-type mice (Figure 4b, left graph). However, a higher proportion of these lymphatics were small in Vegf-d-deficient granulation tissue (see Figure 4b, right graph, for densities of various size classes of lymphatics). Eighteen percentage of lymphatics in Vegf-d-deficient granulation tissue were in the smallest size class (i.e., detected as being <78μm2 in tissue sections) as opposed to nine percentage for wild-type wounds. These findings indicate subtly altered lymphangiogenesis and/or lymphatic modeling. Levels of messenger RNA for Vegf-c (Figure 4e and Supplementary Materials and Methods online) initially increased during wound healing and returned to basal levels by day 20 pw, and were comparable in Vegf-d-deficient and wild-type wounds. In contrast, Vegf-d messenger RNA levels decreased in wild-type mice throughout wound healing. We also analyzed blood vessels in granulation tissue to assess whether enhanced angiogenesis in Vegf-d-deficient wounds might have contributed to faster wound closure. However, there were no alterations in the size or density of blood vessels in Vegf-d-deficient granulation tissue compared with wild-type (Figure 4c and d), consistent with the finding that levels of messenger RNA for the angiogenic protein Vegf-a (Figure 4e) were not statistically different in Vegf-d-deficient versus wild-type wounds at any of the time points analyzed.
      Figure thumbnail gr4
      Figure 4Analysis of initial lymphatics and blood vessels in the granulation tissue of wounds. (a) Initial lymphatics (arrowheads) in the granulation tissue of wounds from wild-type (Wt) and vascular endothelial growth factor-d (Vegf-d)-deficient (Ko) mice at day 10 post wounding (pw) were identified by staining for LYVE-1, and (b) LYVE-1-positive vessel structures with clearly visible lumens were quantitated for overall density (left graph) and for densities of various size classes of lymphatics (right graph) (n=7 for both graphs). (c) Blood vessels in the granulation tissue were stained for PECAM-1 and (d) quantitated for size (left) and density (right) (n=4). (e) Messenger RNAs for Vegf-d, Vegf-c, and Vegf-a were quantitated in wounds by quantitative reverse transcriptase–PCR. Scale bars in a and c correspond to 100μm; “GT” in panels b and d denotes the granulation tissue; “D” in panel e denotes days pw, with D0 denoting normal (i.e., not wounded) skin. Graphs show mean±SEM, and statistical analysis was by Student’s t-test; *P<0.05; **P<0.01.

      Initial lymphatics in adult dermis enlarge in response to mouse Vegf-d

      The smaller caliber of initial lymphatics in the skin of Vegf-d-deficient mice suggest that Vegf-d can promote enlargement of initial lymphatic vessels. We tested the capacity of initial lymphatics in the dermis of adult skin to respond to Vegf-d by intradermally injecting 293EBNA-1 cells stably transfected to express recombinant mature mouse Vegf-d (i.e., lacking the N- and C-terminal propeptides in full-length Vegf-d (
      • Baldwin M.E.
      • Roufail S.
      • Halford M.M.
      • et al.
      Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis.
      )), or control cells harboring the expression vector lacking Vegf-d sequence, into the ears of SCID/NOD mice. 293EBNA-1 cells were used as a model system because they do not produce significant quantities of VEGF family members, and they form tumor xenografts that do not induce significant lymphangiogenesis or angiogenesis (
      • Stacker S.A.
      • Caesar C.
      • Baldwin M.E.
      • et al.
      VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.
      )—hence they provide a “clean background” in which to assess the effects, through transfection, of lymphangiogenic or angiogenic growth factors. The mature form of Vegf-d was used instead of the full-length form because it has been shown in studies of human VEGF-D that proteolytic processing to remove the propeptides is required to generate the most bioactive form of the protein (
      • Stacker S.A.
      • Stenvers K.
      • Caesar C.
      • et al.
      Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers.
      ;
      • McColl B.K.
      • Baldwin M.E.
      • Roufail S.
      • et al.
      Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D.
      ,
      • McColl B.K.
      • Paavonen K.
      • Karnezis T.
      • et al.
      Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2.
      ;
      • Harris N.C.
      • Paavonen K.
      • Davydova N.
      • et al.
      Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer.
      ). Furthermore, the degree to which ectopically delivered full-length mouse Vegf-d would have been proteolytically activated in the dermis of the ear was unknown. Cells were mixed with Matrigel before injection to ensure they would be localized around the injection site, and ears were harvested 1 week after injection.
      Staining of whole mounts with LYVE-1 revealed that mouse Vegf-d promoted both the enlargement of initial lymphatic vessels in the dermis (the width of these lymphatics was about 70% larger) as well as sprouting to generate new vessels, at a distance of 1mm from the Matrigel plug (Figure 5a and d). There was an abundance of sprouts from almost all initial lymphatics in this region, but virtually none in ears treated with control cells not expressing Vegf-d. At ∼3mm from the plug, mouse Vegf-d promoted the enlargement of initial lymphatics (the width of these lymphatics was about 40% larger) but not vessel sprouting (Figure 5b and d). Staining for PECAM-1 indicated that Vegf-d increased the size, but not density, of blood vessels at a distance of 1mm from the plug (Figure 5c and e), but neither blood vessel size nor abundance was altered at a distance of 3mm (Figure 5e). The control cells, that did not express Vegf-d, did not induce any significant effects on lymphatics or blood vessels as assessed by comparison with injection of Matrigel alone (data not shown). The findings at 3mm from the plug, that mouse Vegf-d modulates the size, but not abundance, of initial lymphatics in the dermis and does not affect blood vessels, are consistent with the alterations observed in Vegf-d-deficient skin.
      Figure thumbnail gr5
      Figure 5Initial lymphatics in adult dermis are responsive to mouse vascular endothelial growth factor-d (Vegf-d). The ears of adult SCID/NOD mice were intradermally injected with Matrigel containing 293-EBNA-1 cells stably transfected to express mature mouse Vegf-d (Vegf-d) or control cells transfected with Apex expression vector lacking DNA sequence for Vegf-d (Control). After 1week, the ears were harvested, whole-mounted, and stained for LYVE-1 to visualize initial lymphatics (green) in the dermis. Initial lymphatics were compared at distances of (a) 1mm or (b) 3mm from Matrigel plugs. An abundance of small lymphatic sprouts is seen arising from many initial lymphatics in response to Vegf-d in a. (c) The region of the ear 1mm from the Matrigel plug was analyzed for blood vessels (red) by PECAM-1 staining. (d) Width of initial lymphatics (n=11) and (e) size (top) and density (bottom) of blood vessels (n=5) are compared (Cn denotes treatment with control cells; Vd denotes treatment with cells producing Vegf-d). Graphs show mean±SEM, and statistical analysis was by Student’s t-test; *P<0.05; **P<0.01.

      Discussion

      The data presented here show that Vegf-d deficiency can lead to significantly smaller initial lymphatic vessels in the dermis of adult skin, an effect that was specific in terms of lymphatic vessel subtypes given that collecting lymphatic vessels were not altered in size or abundance. The reduced size of the initial lymphatics in Vegf-d-deficient mice was owing to a reduction in the size of the endothelial cells lining these vessels—the molecular mechanisms by which Vegf-d signaling modulates the size of LECs is yet to be fully determined. The altered initial lymphatics in the dermis of Vegf-d-deficient mice were less efficient in the uptake and/or transport of dextran than in wild-type controls. This may be owing to the reduced size of these lymphatics in Vegf-d-deficient mice; however, other explanations, including effects on lymphatic pump function, such as have been reported for VEGF-C (
      • Breslin J.W.
      • Gaudreault N.
      • Watson K.D.
      • et al.
      Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism.
      ), or on the ultrastructure of endothelial cells lining initial lymphatic vessels, require exploration. The dysfunction of these vessels was not sufficiently profound to give rise to detectable edema or lymphedema under nonpathological conditions. Vegf-d deficiency did not lead to significant alteration in the size or abundance of blood vessels in the dermis. Our finding that Vegf-d in wild-type mice enlarges lymphatic vessels in skin is consistent with the observation that tumor-derived VEGF-D promotes enlargement of lymphatics in the setting of cancer (
      • Karnezis T.
      • Shayan R.
      • Caesar C.
      • et al.
      VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium.
      ).
      The finding that the initial lymphatics in the dermis are altered in adult Vegf-d-deficient mice but blood vessels are not is consistent with transgenic mice in which full-length human VEGF-D (
      • Veikkola T.
      • Jussila L.
      • Makinen T.
      • et al.
      Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.
      ) or mouse Vegf-d (
      • Haiko P.
      • Makinen T.
      • Keskitalo S.
      • et al.
      Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos.
      ;
      • Huggenberger R.
      • Siddiqui S.S.
      • Brander D.
      • et al.
      An important role of lymphatic vessel activation in limiting acute inflammation.
      ) was expressed in epidermal keratinocytes under the control of the cytokeratin 14 (K14) promoter. These adult transgenic mice exhibited hyperplasia of dermal lymphatic vessels but no alterations to blood vessel architecture in the dermis. Our findings that initial lymphatics and cutaneous wound healing are altered in Vegf-d-deficient mice on pure C57Bl/6 genetic background are in contrast to the previous work with Vegf-d-deficient mice on mixed genetic backgrounds (
      • Baldwin M.E.
      • Halford M.M.
      • Roufail S.
      • et al.
      Vascular endothelial growth factor d is dispensable for development of the lymphatic system.
      ;
      • Koch M.
      • Dettori D.
      • Van Nuffelen A.
      • et al.
      VEGF-D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis.
      ) in which dermal lymphatics and wound healing were normal. The different genetic backgrounds of these Vegf-d-deficient mouse strains likely explain the different phenotypes observed. There is precedence for the genetic backgrounds of mice influencing the degree of the lymphatic vascular phenotype resulting from the targeting of a specific gene. For example, the phenotype of mice in which the gene for the Sox18 transcription factor had been inactivated varied markedly depending on the genetic background, with an extremely strong phenotype involving a complete blockade of LEC differentiation leading to gross subcutaneous edema and fetal lethality for homozygotes on pure C57BL/6 background (
      • Francois M.
      • Caprini A.
      • Hosking B.
      • et al.
      Sox18 induces development of the lymphatic vasculature in mice.
      ). In stark contrast, homozygotes on a mixed genetic background were viable, fertile, and displayed only a mild coat defect (
      • Pennisi D.
      • Bowles J.
      • Nagy A.
      • et al.
      Mice null for sox18 are viable and display a mild coat defect.
      ).
      Mouse Vegf-d binds Vegfr-3 but has been reported to be a poor ligand for Vegfr-2 (
      • Baldwin M.E.
      • Catimel B.
      • Nice E.C.
      • et al.
      The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man.
      ). Vegfr-3 signaling is known to be sufficient to drive the enlargement of lymphatics in skin as illustrated by transgenic mice expressing a Vegfr-3-specific mutant of VEGF-C (
      • Veikkola T.
      • Jussila L.
      • Makinen T.
      • et al.
      Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.
      ). Hence, the smaller initial lymphatics in the dermis of Vegf-d-deficient mice likely arise owing to inadequate Vegfr-3 signaling. In contrast, it is not clear why expression of full-length mouse Vegf-d or human VEGF-D in the skin of the K14 transgenic mice, or Vegf-d deficiency in the mice studied here, did not alter the blood vessel size or abundance in the dermis, given that delivery of mature or full-length human VEGF-D in a range of tissues and tumor models promoted angiogenesis (
      • Marconcini L.
      • Marchio S.
      • Morbidelli L.
      • et al.
      c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro.
      ;
      • Stacker S.A.
      • Caesar C.
      • Baldwin M.E.
      • et al.
      VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.
      ;
      • Byzova T.V.
      • Goldman C.K.
      • Jankau J.
      • et al.
      Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo.
      ;
      • Bhardwaj S.
      • Roy H.
      • Gruchala M.
      • et al.
      Angiogenic responses of vascular endothelial growth factors in periadventitial tissue.
      ;
      • Rissanen T.T.
      • Markkanen J.E.
      • Gruchala M.
      • et al.
      VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses.
      ;
      • Rutanen J.
      • Rissanen T.T.
      • Markkanen J.E.
      • et al.
      Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart.
      ). Possible explanations include insufficient concentrations or proteolytic processing of mouse Vegf-d (or human VEGF-D) in the skin to drive angiogenesis or blood vessel enlargement, or functional redundancy with other angiogenic growth factors, such as Vegf-a.
      The exact timing at which initial lymphatics in the dermis of wild-type mice became larger than in Vegf-d-deficient mice was difficult to pinpoint given that the difference in vessel size in the adults was relatively subtle. An indication of the time at which the dermal lymphatic vasculature becomes sensitive to Vegf-d is provided by transgenic mice expressing the K14-VEGF-D transgene. The hyperplasia in the dermal lymphatics of these transgenic mice developed soon after birth despite the transgene being expressed already by embryonic day 14.5, suggesting that early postnatal lymphatic maturation involves the acquisition of sensitivity to Vegf-d (
      • Karpanen T.
      • Wirzenius M.
      • Makinen T.
      • et al.
      Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation.
      ). The molecular mechanisms underlying this observation are currently unknown, but could reflect the timing or levels of expression of receptors for Vegf-d, including Vegfr-3 (
      • Achen M.G.
      • Jeltsch M.
      • Kukk E.
      • et al.
      Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).
      ), neuropilins (
      • Karpanen T.
      • Heckman C.A.
      • Keskitalo S.
      • et al.
      Functional interaction of VEGF-C and VEGF-D with neuropilin receptors.
      ;
      • Harris N.C.
      • Paavonen K.
      • Davydova N.
      • et al.
      Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer.
      ), or integrin α9β1 (
      • Vlahakis N.E.
      • Young B.A.
      • Atakilit A.
      • et al.
      The lymphangiogenic vascular endothelial growth factors VEGF-C and -D are ligands for the integrin alpha9beta1.
      ), on dermal LEC.
      We assessed the capacity of initial lymphatics in the dermis of adult SCID/NOD mice to respond to mature mouse Vegf-d, which demonstrated that at high concentrations (i.e., proximal to the source of protein) mouse Vegf-d promoted both the enlargement of initial lymphatics as well as sprouting to form new lymphatics, whereas at lower concentrations (i.e., more distant from the source of Vegf-d) only lymphatic vessel enlargement was observed. The result observed more distant from the source of Vegf-d (i.e., that caliber of initial lymphatics, but not abundance, was altered) is consistent with our observations in Vegf-d-deficient mice in that the absence of Vegf-d led to reduced size, but not density, of initial lymphatics in the dermis. Further, our study shows that initial lymphatics in the adult are sensitive to mouse Vegf-d signaling, as was shown to be the case during the postnatal period in K14-VEGF-D transgenic mice expressing human VEGF-D (
      • Karpanen T.
      • Wirzenius M.
      • Makinen T.
      • et al.
      Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation.
      ), and has been reported for adult skin in a rat study involving adenoviral delivery of human VEGF-D (
      • Byzova T.V.
      • Goldman C.K.
      • Jankau J.
      • et al.
      Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo.
      ). We also observed enlargement of blood vessels in response to mature mouse Vegf-d showing that, although this protein has been reported to be a poor ligand for Vegfr-2 (
      • Baldwin M.E.
      • Catimel B.
      • Nice E.C.
      • et al.
      The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man.
      ), it can elicit effects on blood vessels, potentially via Vegfr-3 signaling (
      • Tammela T.
      • Zarkada G.
      • Wallgard E.
      • et al.
      Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation.
      ,
      • Tammela T.
      • Zarkada G.
      • Nurmi H.
      • et al.
      VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling.
      ;
      • Benedito R.
      • Rocha S.F.
      • Woeste M.
      • et al.
      Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling.
      ).
      We monitored the effect of Vegf-d deficiency, and the abnormality of initial lymphatics arising from it, in a full-thickness cutaneous wound healing model designed to challenge lymphatic function. Wound healing in Vegf-d-deficient mice was characterized by pronounced and acute edema in the epithelium, which caused significant thickening of this tissue layer. The epithelial edema likely resulted from compromised capacity of the initial lymphatics in normal unwounded dermis immediately surrounding the wound to absorb fluid (
      • Shimamura K.
      • Nakatani T.
      • Ueda A.
      • et al.
      Relationship between lymphangiogenesis and exudates during the wound-healing process of mouse skin full-thickness wound.
      ). We also observed an increase in the proportion of lymphatics that were small in the granulation tissue of Vegf-d-deficient wounds, although there was no difference in the overall density of lymphatics in this tissue, which is consistent with our observation that Vegf-d promotes the enlargement of these vessels. The altered lymphatics in the granulation tissue may also have contributed to the edema in the epithelium. These findings indicate that although Vegf-d deficiency (and the resulting lymphatic dysfunction in fluid uptake/transport) did not lead to dermal edema in nonpathological conditions, it was sufficient to do so during cutaneous wound healing.
      Unexpectedly, myofibroblasts were far more abundant in the granulation tissue of Vegf-d-deficient wounds, which likely explained the faster wound closure from days 1 to 10 pw, given that these cells enhance contractility of wound beds (
      • Grinnell F.
      Fibroblasts, myofibroblasts, and wound contraction.
      ). Although the molecular mechanisms leading to more abundant myofibroblasts in Vegf-d-deficient wounds are unknown, it is recognized in a clinical setting that lymph stasis can lead to more fibroblasts in skin (
      • Tabibiazar R.
      • Cheung L.
      • Han J.
      • et al.
      Inflammatory manifestations of experimental lymphatic insufficiency.
      ). Hence, it is conceivable that the edema in Vegf-d-deficient wounds promoted myofibroblast recruitment. We also observed altered remodeling of granulation tissue in Vegf-d-deficient wounds suggesting a compromised quality of healing. The altered remodeling was not owing to a difference in the abundance of macrophages given that F4/80-positive macrophages were equally abundant in the granulation tissue of Vegf-d-deficient and wild-type wounds at day 10 pw (data not shown). It is not known whether the effects of Vegf-d-deficiency on fibroblast abundance or remodeling of the granulation tissue are a consequence of altered lymphatic vessel function or owing to direct effects of Vegf-d on other components of healing wounds. These aspects require further investigation involving systems biology approaches to broadly monitor the effects of Vegf-d on the variety of cell types involved in wound healing. Our observations in cutaneous wound healing suggest that Vegf-d deficiency leads to faster wound closure but a compromised quality of healing. Given the poorer quality of healing, this scenario is likely to be detrimental, but further work is required to assess the significance of Vegf-d-deficiency for different types of wounds.
      In conclusion, our study is the first to show that Vegf-d is a determinant of the caliber of initial lymphatic vessels in the dermis and the capacity of these vessels to absorb and/or transport macromolecules. Although not required for the formation of the lymphatic vasculature, Vegf-d is important for “fine-tuning” the structure and function of initial lymphatic vessels in the dermis.

      Materials and Methods

      Mice

      Mice with targeted inactivation of the vegfd gene, on a mixed C57Bl/6 and 129SV genetic background (
      • Baldwin M.E.
      • Halford M.M.
      • Roufail S.
      • et al.
      Vascular endothelial growth factor d is dispensable for development of the lymphatic system.
      ), were backcrossed to C57Bl/6 mice for 10 generations and used for this study. SCID/NOD mice were from the Australian Resource Centre. Experiments were conducted according to ethical guidelines of the National Health and Medical Research Council of Australia.

      Lymphangiography

      Hair was removed from the tails of anesthetized mice with depilatory cream, and mice placed in a chamber at 37°C in an IX81 Olympus microscope (Mt Waverley, Victoria, Australia) connected to a Lambda XL Xenon plasma lamp (Sutter Instruments, Novato, CA). Tails were immobilized with tape and tail tips injected intradermally with 20μl of 25mgml−1 FITC–Dextran (2000KD, Sigma) using a 29 gauge syringe. The fluorescent signal within initial lymphatics located 2.5cm from the tail tip was recorded every second for 50minutes. Images were captured using an × 4 objective and data analysis was with MetaMorph software (Version 7.6.2, Molecular Devices, Middle Cove, New South Wales, Australia).

      Wound healing

      Eight to 10-week-old hemizygous vegfdo/- and wild-type male mice were anesthetized by intraperitoneal injection of xylazine (20mgkg−1) and ketamine (100mgkg−1). They were shaved on the back and treated with depilatory cream (Veet, Reckitt Benckiser, Slough, UK) for 1minute, followed by rinsing with warm water, then 70% ethanol. Two 6-mm-diameter full-thickness excisional wounds were made on the upper dorsal back, one on each side of the spine, using a biopsy punch (Kai Industries, Gifu, Japan)—the skin collected through the punch biopsies was used as a control (Day 0). All wounds analyzed were free of infection and contamination with hair. Four mice were used per time point, and each time point was performed in duplicate. Wounds were not dressed and mice were housed individually until harvesting. Wounds were measured with callipers and photographed with an Olympus SP-550UZ digital camera. At selected time points, mice were killed and wound biopsies, surrounded by a 5-mm margin of tissue, were harvested with scissors. The orientation of samples for embedding was always the same. Skin samples were fixed flat in porous holders in 4% paraformaldehyde, cut in half by transverse incision, and embedded in paraffin. Samples were sectioned vertically over the middle of the wound.

      Injection of matrigel plugs into the ear for delivery of Vegf-d

      293EBNA-1 cells stably transfected to express a mature form of mouse Vegf-d (
      • Achen M.G.
      • Roufail S.
      • Domagala T.
      • et al.
      Monoclonal antibodies to vascular endothelial growth factor-D block interactions with both VEGF receptor-2 and VEGF receptor-3.
      ;
      • Baldwin M.E.
      • Catimel B.
      • Nice E.C.
      • et al.
      The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man.
      ) or control cells stably transfected with expression vector lacking DNA sequence for Vegf-d were grown in Dulbecco’s Modified Eagle Medium containing 10% fetal calf serum. Secretion of Vegf-d was verified by western blotting as described previously (
      • Harris N.C.
      • Paavonen K.
      • Davydova N.
      • et al.
      Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer.
      ). Cells (2.5 × 105) were resuspended in 30μl of Matrigel (Matrigel Basement Membrane Matrix, BD Biosciences) and injected into the center of an ear of an 8-week old SCID/NOD mouse. Ears were harvested for processing one week later.

      Computer-assisted morphometric vessel analysis

      For flank wounds, images were taken on a Nikon Eclipse 90i microscope with a x20/NA 0.6 PlanApo objective and a digital camera (Coolsnap HQ, Photometrics). Four individual fields per section in non-wounded samples and three in wounded samples (one in the wound bed and one at each wound edge) were randomly photographed—fewer images were taken for wounded samples because they were smaller than non-wounded samples. Morphometric analysis was performed using MetaMorph Premier (Version 7.6.2) software program (Molecular Devices). Each vessel was outlined to quantify its size (assessed as number of pixels in endothelium and lumen) and the number of vessels per image. For the analysis of lymphatics in whole mounts, the “Lymphatic Vessel Analysis Protocol” plug-in was used as described (
      • Shayan R.
      • Karnezis T.
      • Tsantikos E.
      • et al.
      A system for quantifying the patterning of the lymphatic vasculature.
      ).

      Statistical analysis

      Results were evaluated using Excel 2003 (Microsoft, Los Angeles, CA) or Graph Pad Prism 4.0 (GraphPad Software, San Diego, CA) software. Data are shown as mean±SEM as indicated, and were analyzed with a two-tailed unpaired Student’s t-test. For all analyses, P<0.05 was considered significant.

      ACKNOWLEDGMENTS

      This work was supported by a Program Grant and Research Fellowships (to SA Stacker and MG Achen) from the National Health and Medical Research Council of Australia (NHMRC), and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. R Shayan is supported by the Raelene Boyle Sporting Chance Foundation and the Royal Australasian College of Surgeons (RACS) Foundation Scholarship, and the RACS Surgeon Scientist Program. S Levy is supported by a NHMRC Postgraduate Scholarship, a RACS Eric Bishop Scholarship, a RACS Foundation for Surgery Catherine Marie Enright Scholarship, and a National Breast Cancer Foundation Doctoral Scholarship.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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