1. Vascular Endothelial Growth Factor-d Modulates Caliber and Function of Initial Lymphatics in the Dermis 
Sophie Paquet-Fifield, Sidney M. Levy, Teruhiko Sato, Ramin Shayan, Tara Karnezis, Natalia Davydova, Cameron J. Nowell, Sally Roufail, Gerry Zhi-Ming Ma, You-Fang Zhang, Steven A. Stacker, Marc G. Achen 
Journal of Investigative Dermatology 
Volume 133, Issue 8, Pages (August 2013)
DOI: /jid
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Initial 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.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Compromised 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.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Altered 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.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Analysis 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.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Initial 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.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions