Autogenous tissue lymphangiogenesis is associated with spontaneous reconnection of local lymphatics and infiltration of new lymphatic capillaries.

<p><b>A.</b> Skin grafts harvested from GFP transgenic mice express GFP at high levels and for a long period of time. A representative fluorescent picture of a mouse (top) with higher magnification of the tail (bottom) is shown 6 weeks after surgery. The skin-grafted GFP portion of the tail can easily be seen. <b>B.</b> Gross photographs of mouse tails treated with skin grafts harvested from GFP transgenic mice. Note the rapid incorporation and ingrowth of hair follicles at the 6-week time point. <b>C.</b> Microlymphangiography performed 2 (left) or 6 (right) weeks following skin graft. Distal portion of the tail is shown at the bottom. Note the flow of fluorescent dye by interstitial flow after 2 weeks. In contrast, lymphatic flow can be seen in the skin-grafted area at the 6-week time point. Representative figures of triplicate experiments are shown. <b>D.</b> Higher power photograph of microlymphangiography 2 and 6 weeks after surgery demonstrating a few honeycomb-like dermal lymphatics (white arrows) in the skin graft at the 6-week time point. <b>E.</b> Representative LYVE-1 (pink) and GFP (green) co-localization in skin-grafted mouse tails 6 weeks after surgery. Low power (5x; left) and high power (20x; right) views are shown. Note connection between GFP^- (recipient) and GFP⁺ (donor) lymphatic vessel. <b>F.</b> Representative photomicrograph (2x) demonstrating GFP (left), LYVE-1 (middle), and co-localization (right) of skin-grafted mouse tails 6 weeks after surgery. Note ingrowth of GFP^-/LYVE-1⁺ vessels (yellow circle) from the distal (yellow dotted line) portion of the wound. <b>G.</b> High power (20x) view of section shown in <b>F</b>. Note the presence of both recipient (GFP^-/LYVE-1⁺) and donor (GFP⁺/LYVE-1⁺) lymphatics at the distal margin of the wound.</p

Lymphatic regeneration after tissue transfer is associated with expression of VEGF-C.

<p><b>A.</b> VEGF-C expression in skin-grafted tails 2 weeks after surgery. Representative low power (2x; upper panel) photomicrographs encompassing the skin-grafted area and distal/proximal portions of the recipient mouse-tail are shown. High power (20x) views of the distal and proximal junctions between recipient tissues and skin grafts are shown below. Black arrow shows large number of VEGF-C⁺ cells in the distal junction. Dashed box delineates skin-grafted area. <b>B.</b> VEGF-C expression in skin-grafted tails 6 weeks after surgery. Representative low power (2x; upper panel) and high power (20x) photomicrographs encompassing the skin-grafted area and distal/middle/proximal portions of the recipient mouse tails are shown. Dashed box delineates skin-grafted area. Note small amount of wound/skin graft contracture after repair. <b>C.</b> Cell counts per high power field of VEGF-C⁺ cells in the various tail regions (D = distal, M = middle, P = proximal) 2 and 6 weeks after surgery. Cell counts are means ± SD of at least 4 high power fields/mouse/time point. At least 6 mice were analyzed in each group (*<i>p</i><0.05; <i>#</i><0.01).</p

Lymphatic regeneration after tissue transfer is associated with infiltration of macrophages.

<p><b>A.</b> F4/80 localization in skin-grafted tails 2 weeks after surgery. Representative low power (2x; upper panel) photomicrographs encompassing the skin-grafted area and distal/proximal portions of the recipient mouse-tail are shown. High power (20x) views of the distal and proximal junctions between recipient tissues and skin grafts are shown below. Dashed box delineates skin-grafted area. <b>B.</b> F4/80 localization in skin-grafted tails 6 weeks after surgery. Representative low power (2x; upper panel) and high power (20x) photomicrographs encompassing the skin-grafted area and distal/proximal portions of the recipient mouse tails are shown. Dashed box delineates skin-grafted area. Note small amount of wound/skin graft contracture after repair. <b>C.</b> Cell counts per high power field of F4/80⁺ cells in various regions of the tail 2 and 6 weeks after surgery. Cell counts are means ± SD of at least 4 high power fields/mouse/time point. At least 6 mice were analyzed in each group (*<i>p</i><0.05).</p

Spontaneous regeneration of lymphatics after tissue transfer can be used to bypass damaged lymphatics.

<p><b>A.</b> Gross photographs comparing nude mice that had undergone tail excision with (right) and without (left) skin grafting are shown 6 weeks after surgery. Note obvious difference in tail swelling. <b>B.</b> Tail volume measurements in nude mice that had undergone tail excision with or without skin grafting. Data are presented as percent change from baseline (i.e. preoperatively) with mean ± SD (*<i>p</i><0.05). <b>C, D.</b> Representative lymphoscintigraphy of nude mice that had undergone tail excision with or without skin grafting. <b>E.</b> Representative photomicrograph (5x) of H&E stained tails sections from nude mice treated with (left) or without (right) 6 weeks after surgery. Dashed box delineates area of skin graft. Note decreased inflammation (cellularity) and dermal thickness in skin-grafted mice distal (to the left) of the wound. <b>F.</b> High power (40x) photomicrographs of tail skin harvested 5 mm distal to the excision site. Note decreased cellularity in skin-grafted section (left) as compared with excision section (right; arrow). Also note decreased dermal thickness. <b>G.</b> Dermal thickness measurements and representative figures (40x) in nude mice that had undergone tail excision with or without skin grafting 6 weeks following surgery (*<i>p</i><0.05). <b>H.</b> Scar index measurements in tail tissues localized just distal to the site of lymphatic injury 6 weeks after treatment with excision with or without skin grafting. Representative Sirius red birefringence images are shown to the right. Orange-red is indicative of scar; yellow-green is consistent with normal (i.e. non-fibrosed) tissue (*<i>p</i><0.01).</p

CD4 cell depletion reduces lymphedema. A.

<p>Flow cytometry analysis of splenic single cell suspensions from mice treated with isotype control antibodies or depleted of CD4+ cells (upper panel) or CD8+ cells (lower panel) using neutralizing antibodies (n = 5–7/group). Representative dot plots are shown to the right. <b>B.</b> Representative photograph of control, CD8+ cell depleted, or CD4+ cell depleted mice 6 weeks after tail superficial and deep lymphatic excision. Note near complete resolution of edema in CD4+ treated animals and loss of fixed tail contracture (“J” shape seen in control or CD8+ treated animals). <b>C.</b> Representative cross sectional histology and quantification of subcutaneous tissue thickness (brackets) in control, CD8+, and CD4+ depleted animals. <b>D.</b> Tail volumes in control, CD8+, or CD4+ depleted animals over the course of the experiment. CD4+ or CD8+ cell depletion was begun 2 weeks after surgery (arrow). <b>E.</b> Analysis of lymphatic vessel diameter (podoplanin+ vessels) in control, CD8+, or CD4+ depleted animals (left) and representative photomicrographs (right). Lymphatic vessel diameter is shown in brackets.</p

Chronic lymphedema results in a mixed inflammatory cell response.

<p><b>A.</b> Photograph of mouse-tails 6 weeks after skin/lymphatic excision (lymphedema; left) or skin incision (control; right). <b>B.</b> Representative cross sectional histology of mouse tails comparing lymphedema (left) and control (right) mice 6 weeks after surgery. Cross sections were obtained 2 centimeters distal to the tail wound (arrow in figure A). Note subcutaneous fat deposition (brackets), dilated lymphatics, and inflammation in lymphedema section. <b>C.</b> Flow cytometry analysis for CD45+ cells in single cell suspensions prepared from tail tissue 2 cm distal to the wound of lymphedema or control mice (n = 5–7/group) 6 weeks after surgery. The percentage of CD45+ cells as a function of total cell population is shown. A representative histogram is shown to the right. <b>D., E.</b> Flow cytometry analysis of T-helper, T-cytotoxic, natural killer T cells (NKT), B cell (Figure D) and neutrophils, monocytes, macrophage, and dendritic cells (Figure E) in single cell suspensions of lymphedematous or control mice (n = 5–7/group) 6 weeks after surgery. Representative dot plots are shown to the right. Oval gates indicate double positive cell populations.</p

CD4⁺ cell depletion reduces lymphedema induced chronic inflammation. A, B, C.

<p>Representative photomicrographs of CD45 (figure A), F4/80 (figure B), and CD4 (figure C) immunohistochemical staining in tail tissues of control, CD8+, or CD4+ cell depleted animals 6 weeks after tail superficial and deep lymphatic excision. Quantification of cell numbers per high-powered field (hpf) are shown below for each cell type. <b>D.</b> Representative (of triplicate experiments) western blots from tail tissues for Th1 (IFN-y, Tbet), T-reg (FoxP3), and Th2 (Gata-3) markers in control, CD8+, and CD4+ depleted animals 6 weeks after tail superficial and deep lymphatic excision. Quantification of band density relative to controls (arbitrarily set at 1 and represented by dotted line) is shown to the right.</p

CD25⁺ cell depletion does not improve lymphedema, decrease fibrosis, or augment lymphatic function. A.

<p>Representative photograph of control or CD25+ depleted mice 6 weeks after tail superficial and deep lymphatic excision. <b>B.</b> Representative cross sectional histology and quantification of subcutaneous tissue thickness (brackets) in control and CD25+ cell depleted animals. <b>C.</b> Tail volumes in control and CD25+ cell depleted animals over the course of the experiment. CD25+ cell depletion was begun 2 weeks after surgery (arrow). <b>D.</b> Representative (of triplicate experiments) western blots from tail tissues for Th1 (IFN-y, Tbet), and Th2 (Gata-3, IL4) markers in control and CD25+ cell depleted animals 6 weeks after tail superficial and deep lymphatic excision. Quantification of band density relative to controls (fold change) is shown to the right. <b>E.</b> Lymphoscintigraphy and sacral lymph node uptake in control and CD25+ cell depleted mice 6 weeks after surgery. Representative heat map is shown to the right (white arrow = injection site; red circle = sacral lymph nodes). <b>F.</b> Scar index analysis (below) and representative photomicrographs of polarized light microscopic views (above) in control and CD25+ cell depleted animals (n = 5–7 per group) 6 weeks after surgery. <b>G.</b> Representative photomicrographs of type I collagen immunohistochemistry (above) and calculation of type I collagen staining in the dermis (positive pixels/mm²; below) in control and CD25+ cell depleted animals 6 weeks after surgery. <b>H.</b> Calculation of type I:type III collagen staining ratio in tail tissue sections from control and CD25+ cell depleted mice 6 weeks after surgery. <b>I.</b> Representative (of triplicate experiments) western blot analysis of a-sma, E-cadherin, type III collagen, pSMAD, and TGF-B1 in protein lysates obtained from tail tissues of control and CD25+ cell depleted animals 6 weeks after surgery. Quantification of band density relative to controls (fold change) is shown to the right.</p

Cell surface markers for identification of leukocyte cell types.

<p>Cell surface markers for identification of leukocyte cell types.</p

CD4+ cells regulate inflammatory lymphangiogenesis.

<p><b>A.</b> LYVE-1+ vessel density in popliteal lymph nodes 7 days after CFA/OVA induced lymph node lymphangiogenesis in control, CD4+ cell depleted, or CD4 knockout (CD4KO) mice. Representative cross sectional histology of the lymph node (blue DAPI stain, red LYVE-1 stain) are shown to the right. <b>B., C.</b> Expression of VEGF-A (A) and VEGF-C (B) protein by ELISA in popliteal lymph nodes harvested 7 days after CFA/OVA induced lymph node lymphangiogenesis in control or CD4 depleted mice.</p