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Decreased Macrophage Number and Activation Lead to Reduced Lymphatic Vessel Formation and Contribute to Impaired Diabetic Wound Healing

  • Kazuichi Maruyama
    Affiliations
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan

    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts
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  • Jun Asai
    Affiliations
    Division of Cardiovascular Research, Caritas, St. Elizabeth Medical Center, Tufts University of Medicine, Boston, Massachusetts

    Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan
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  • Masaaki Ii
    Affiliations
    Division of Cardiovascular Research, Caritas, St. Elizabeth Medical Center, Tufts University of Medicine, Boston, Massachusetts
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  • Tina Thorne
    Affiliations
    Division of Cardiovascular Research, Caritas, St. Elizabeth Medical Center, Tufts University of Medicine, Boston, Massachusetts
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  • Douglas W. Losordo
    Affiliations
    Division of Cardiovascular Research, Caritas, St. Elizabeth Medical Center, Tufts University of Medicine, Boston, Massachusetts
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  • Patricia A. D'Amore
    Correspondence
    Address reprint requests to Patricia A. D'Amore, Ph.D., Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114
    Affiliations
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts

    Departments of Ophthalmology and Pathology, Harvard Medical School, Boston, Massachusetts
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      Impaired wound healing is a common complication of diabetes. Although it is well known that both macrophages and blood vessels are critical to wound repair, the role of wound-associated lymphatic vessels has not been well investigated. We report that both the presence of activated macrophages and the formation of lymphatic vessels are rate-limiting to the healing of diabetic wounds. We have previously shown that macrophages contribute to the lymphatic vessels that form during the acute phase of corneal wound healing. We now demonstrate that this is a general phenomenon; cells that co-stain for the macrophage marker F4/80 and the lymphatic markers LYVE-1 (lymphatic vascular endothelium hyaluronate receptor) and podoplanin contribute to lymphatic vessels in full-thickness wounds. LYVE-1-positive lymphatic vessels and CD31-positive blood vessels were significantly reduced in corneal wound healing in diabetic mice (db/db) (P < 0.02) compared with control (db/+) mice. Glucose treatment of control macrophages led to the down-regulation of the lymphatic-specific receptor VEGFR3 and its ligands, vascular endothelial growth factor-C and -D (VEGF-C, -D). Interleukin-1β stimulation rescued diabetic macrophage function; application of interleukin-1β-treated db/db-derived macrophages to wounds in db/db mice induced lymphatic vessel formation and accelerated wound healing. These observations suggest a potential therapeutic approach for healing wounds in diabetic patients.
      Wound healing involves a complex interplay among cells, growth factors, and cytokines. The cascade of events that leads to wound healing begins with clotting and the recruitment of inflammatory cells, such as neutrophils and macrophages. The role of macrophages in wound healing-associated inflammation is particularly well documented. Within the first few days after injury, circulating monocytes are recruited into the wound site where they differentiate into macrophages,
      • Ross R
      • Odland G
      Human wound repair. II. Inflammatory cells, epithelial-mesenchymal interrelations, and fibrogenesis.
      • Odland G
      • Ross R
      Human wound repair. I. Epidermal regeneration.
      become activated, phagocytose debris, and produce factors such as vascular endothelial growth factor (VEGF)-A and -C that regulate tissue repair and induce the formation of lymphatic and blood vessels
      • Polverini PJ
      • Cotran PS
      • Gimbrone Jr, MA
      • Unanue ER
      Activated macrophages induce vascular proliferation.
      • Hong YK
      • Lange-Asschenfeldt B
      • Velasco P
      • Hirakawa S
      • Kunstfeld R
      • Brown LF
      • Bohlen P
      • Senger DR
      • Detmar M
      VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.
      The critical role of macrophages is demonstrated by the fact that their depletion in wounds leads to a delay in wound healing.
      • Leibovich SJ
      • Ross R
      The role of the macrophage in wound repair: a study with hydrocortisone and antimacrophage serum.
      • DiPietro LA
      Wound healing: the role of the macrophage and other immune cells.
      Lymphatic vessels in wounds function to maintain normal tissue pressure by draining the protein-rich lymph from the interstitial space, as well as by facilitating the delivery of cells that mediate the immune response.
      • Oliver G
      • Detmar M
      The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature.
      • Witte MH
      • Bernas MJ
      • Martin CP
      • Witte CL
      Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology.
      Delayed wound healing, such as that seen in infections, seems to be due, at least in part, to reduced lymphatic development that leads to persistent edema and delayed removal of debris and inflammatory cells.
      • Ji RC
      Characteristics of lymphatic endothelial cells in physiological and pathological conditions.
      However, the origin of the cells involved in the formation of lymphatic vessels in wounds has not been investigated.
      We and others have previously shown that monocyte/macrophages contribute to the formation of lymphatic vessels during inflammation.
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      • Kerjaschki D
      • Huttary N
      • Raab I
      • Regele H
      • Bojarski-Nagy K
      • Bartel G
      • Krober SM
      • Greinix H
      • Rosenmaier A
      • Karlhofer F
      • Wick N
      • Mazal PR
      Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants.
      • Schledzewski K
      • Falkowski M
      • Moldenhauer G
      • Metharom P
      • Kzhyshkowska J
      • Ganss R
      • Demory A
      • Falkowska-Hansen B
      • Kurzen H
      • Ugurel S
      • Geginat G
      • Arnold B
      • Goerdt S
      Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1(+), F4/80(+), CD11b(+) macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis.
      In addition, we demonstrated that macrophages express VEGFR3 (Flt4) and secrete VEGF-C, a ligand for VEGFR3,
      • Cursiefen C
      • Chen L
      • Borges LP
      • Jackson D
      • Cao J
      • Radziejewski C
      • D'Amore PA
      • Dana MR
      • Wiegand SJ
      • Streilein JW
      VEGF-A stimu-lates lymphangiogenesis and hemangiogenesis in inflammatory neo-vascularization via macrophage recruitment.
      which induces lymphatic vessel formation
      • Cursiefen C
      • Chen L
      • Borges LP
      • Jackson D
      • Cao J
      • Radziejewski C
      • D'Amore PA
      • Dana MR
      • Wiegand SJ
      • Streilein JW
      VEGF-A stimu-lates lymphangiogenesis and hemangiogenesis in inflammatory neo-vascularization via macrophage recruitment.
      and has been shown to be essential for reducing tissue edema in wound healing.
      • Ji RC
      Characteristics of lymphatic endothelial cells in physiological and pathological conditions.
      Diabetic patients frequently have serious problems with wound repair, and the etiology of this impaired healing process is poorly understood. We have used a genetic model of murine diabetes (db/db) to investigate diabetes-associated wound healing. db/db mice have an inactivating mutation of the gene encoding the leptin receptor ObR
      • Coleman DL
      Diabetes-obesity syndromes in mice.
      • Chen H
      • Charlat O
      • Tartaglia LA
      • Woolf EA
      • Weng X
      • Ellis SJ
      • Lakey ND
      • Culpepper J
      • Moore KJ
      • Breitbart RE
      • Duyk GM
      • Tepper RI
      • Morgenstern JP
      Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice.
      and develop obesity, insulin resistance, and severe diabetes with marked hyperglycemia, resembling adult-onset diabetes mellitus.
      • Coleman DL
      Diabetes-obesity syndromes in mice.
      Similar to human diabetics, wound healing in these animals is markedly delayed.
      • Leibovich SJ
      • Ross R
      The role of the macrophage in wound repair: a study with hydrocortisone and antimacrophage serum.
      • Wetzler C
      • Kampfer H
      • Stallmeyer B
      • Pfeilschifter J
      • Frank S
      Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair.
      • Greenhalgh DG
      • Sprugel KH
      • Murray MJ
      • Ross R
      PDGF and FGF stimulate wound healing in the genetically diabetic mouse.
      In this study we show in wild-type mice that, as in corneal inflammation,
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      lymphatic vessels that form during the acute phases of excisional wounds are comprised largely of cells that co-stain for the macrophage marker F4/80 and the lymphatic markers LYVE-1 and podoplanin. We document that macrophages contribute to lymphatic vessels in wild-type or heterozygous (+/db) spontaneously diabetic mice but not in homozygous db/db mice. Exogenous activation of db/db macrophages restores their functions and rescues their ability to contribute to wound healing.

      Materials and Methods

      Animals

      Male BKS.Cg-m Leprdb/+ +/J, BKS.Cg-m Leprdb/+ −/J, and wild-type C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME; 8 to 10 weeks) were used. All animal protocols were approved by Schepens Animal Care and Use Committee, consistent with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and St. Elizabeth's Institutional Animal Care and Use Committee, consistent with the Guide for the Care and Use of Laboratory Animals.

      Corneal Inflammation Model

      Corneal inflammation was created by suture placement. Before suture placement on the cornea, each animal was deeply anesthetized with an intraperitoneal injection of ketamine (3 to 4 mg/mouse) and xylazine (0.007 mg/mouse). Using stromal incisions that encompassed more than 120° of the corneal circumference, three 11-0 nylon (MANI, Tochigi, Japan) sutures were placed intrastromally. To obtain standardized angiogenic and lymphangiogenic responses, the outer edge of the suture was placed halfway between the limbus and the line outlined by a 2-mm trephine; the inner edge was equidistant from the 2-mm trephine (Figure 1).
      Figure thumbnail gr1
      Figure 1Contribution of macrophages to lymphatic vessel formation in normal wound healing. Sections of granulation tissue 5, 10, and 14 days after wounding stained for F4/80 (green), a macrophage marker; LYVE-1 (red), a marker of lymphatic endothelium; and TOPRO3 (blue), a nuclear marker. Results reveal vessel-like structures composed of cells that are double positive for markers of macrophages and lymphatic cells. Scale bar = 40 μm. The small window in D14/OVERLAY image shows a higher magnification of a F4/80 and LYVE-1 double-positive structure (white arrowhead). Scale bar = 16 μm.

      Whole-Mount Corneal Staining of Blood and Lymphatic Vessels

      Mice were euthanized 7 days after suture placement, and the corneas were excised, rinsed three times in phosphate-buffered saline (PBS), and fixed in acetone for 1 hour. They were then rinsed in PBS, blocked with 2% bovine serum albumin (BSA)-PBS with 0.03% Triton X-100, and incubated overnight at 4°C with rabbit anti-mouse LYVE-1 antibody (1:500; generously provided by Dr. D.G. Jackson, University of Oxford, Oxford, UK).
      • Cursiefen C
      • Schlotzer-Schrehardt U
      • Kuchle M
      • Sorokin L
      • Breiteneder-Geleff S
      • Alitalo K
      • Jackson D
      Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin.
      • Chang L
      • Kaipainen A
      • Folkman J
      Lymphangiogenesis: new mechanisms.
      The tissues were then washed, blocked, and stained overnight with rat anti-mouse fluorescein isothiocyanate (FITC)-conjugated CD31 (PECAM-1) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), then washed, blocked, and stained with anti-rabbit Cy3-conjugated secondary antibody (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA). Double-stained, whole-mount corneas were visualized under a Zeiss Axiophot microscope (Carl Zeiss Inc., Thornwood, NY), a Leica TSC-SP2 inverted (Leica, Wetzlar, Germany), and an upright confocal laser-scanning microscope. Digital pictures of the flat mounts were taken using a spot image analysis system (Spot Advanced; Diagnostic Instruments, Inc., Sterling Heights, MI), and the area covered by blood and lymphatic vessels positive for CD31 or LYVE-1,
      • Cursiefen C
      • Schlotzer-Schrehardt U
      • Kuchle M
      • Sorokin L
      • Breiteneder-Geleff S
      • Alitalo K
      • Jackson D
      Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin.
      • Chang L
      • Kaipainen A
      • Folkman J
      Lymphangiogenesis: new mechanisms.
      respectively, was quantified using NIH Image software (Image J, http://rsb.info.nih.gov/ij/). The total corneal area was calculated using the innermost vessel of the limbal arcade as the border; the areas of blood and lymphatic vessels within the cornea were calculated and normalized to the total corneal area (expressed as a percentage of the cornea covered by vessels). Five mice were used in each experiment, which was conducted three times with similar results. A representative experiment is shown.

      Collection and Culture of Peritoneal Macrophages

      Thioglycollate-induced macrophages were collected from the peritoneal cavity (peritoneal exudate cells; PECs) of normal 8-week-old male db/db and db/+ mice using previously described methods.
      • Takeuchi M
      • Alard P
      • Streilein JW
      TGF-beta promotes immune deviation by altering accessory signals of antigen-presenting cells.
      PECs were washed, resuspended, and cultured for 24 hours at 37°C in a 5% CO2 (106 cells/35-mm plate) in RPMI 1640 medium (Cambrex, Baltimore, MD) containing 10% BSA (Sigma Chemical Co., St. Louis, MO), 1 × 10−5 mol/L 2-mercaptoethanol (Sigma-Aldrich Corp.), 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.1 mmol/L nonessential amino acid, 1 mmol/L sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (BioWhittaker). Twenty-four hours later, adherent cells were harvested. For incubation in high glucose, macrophages were cultured for 3 days in the same medium with the addition of 30 mmol/L glucose. For stimulation with interleukin (IL)-1β, macrophages were cultured for 24 hours in the same medium with the addition of 20 ng/ml IL-1β following culture in high glucose (BD PharMingen, San Diego, CA). Five mice were used in each experiment, which were each conducted three times with similar results. A representative experiment is shown.

      Collection of F4/80- and CD11b-Positive Macrophages from Bone Marrow

      Macrophages were collected from bone marrow as previously described
      • Herembert T
      • Gogusev J
      • Zhu DL
      • Drueke TB
      • Marche P
      Control ofvascular smooth-muscle cell growth by macrophage-colony-stimulating factor.
      and were cultured for 7 days at 37°C in a 5% CO2 in RPMI 1640 containing 10% horse serum, 10% controlled processed serum replacement factor 1(CSPR-1), 10% L929 cell-conditioned medium (L929 cells provided by Dr. Joan Stein-Streilein, Harvard Medical School, Boston, MA), 1% minimal essential medium vitamins, 1% sodium pyruvate, 1% nonessential amino acids, 1% l-glutamine, and 1% penicillin and streptomycin. Nonadherent cells were then removed, and adherent cells were harvested from the culture dish with a cell scraper, rinsed in PBS, blocked by incubation for 10 minutes on ice with CD16/32 Fc-block (BD PharMingen) in 1% BSA-PBS, and labeled for 30 minutes on ice with rat anti-mouse phycoerythrin (PE)-conjugated CD11b, rat anti-mouse FITC-conjugated F4/80, or an isotype control antisera (FITC and PE-conjugated nonspecific rat IgG2bκ, 1:100; BD PharMingen). Labeled cells were analyzed on an EPIC XL flow cytometer (Beckman Coulter, New York, NY). For stimulation with IL-1β, macrophages were cultured for 24 hours in the same medium with the addition of 20 ng/ml IL-1β. For labeling with DiIAcLDL (Biomedical Technologies, Inc., Stoughton, MA), macrophages were cultured for 2 hours in DiIAcLDL according to the manufacturer's instructions (Invitrogen).

      Skin Wound Model

      Wounds were created in mice 8 to 10 weeks of age as described previously.
      • Greenhalgh DG
      • Sprugel KH
      • Murray MJ
      • Ross R
      PDGF and FGF stimulate wound healing in the genetically diabetic mouse.
      In brief, after induction of deep anesthesia by intraperitoneal injection of sodium pentobarbital (160 mg/kg), full-thickness and excisional skin wounds (one per mouse) were created on the backs of the mice using 6-mm skin biopsy punches and were covered with the semipermeable polyurethane dressing OpSite (Smith & Nephew, Massillon, OH). Macrophages (1 × 106 cells/0.2 μl) or saline was injected with a 27-gauge needle into the wound bed around the wound at four sites (total 4 × 106 cells) through the OpSite (n = 5). Wound closure was documented with a digital camera (Nikon Coolpix 995; Nikon, Tokyo, Japan) on days 0, 7, and 14. Images were analyzed using the NIH Image J analyzer by tracing the wound margin with a fine resolution computer mouse and calculating pixel area. The areas of the wounds were compared using Fisher's t-test. This experiment was conducted twice with similar results. A representative experiment is shown.

      Histological Analysis

      Wound tissues were harvested at 7 and 14 days after macrophage application. The tissues were fixed in 4% paraformaldehyde for 24 hours and were incubated overnight in 15% and then 30% sucrose before being placed in 100% acetone for 3 hours and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin and then observed by masked observers who assigned histological scores. In brief, each specimen was given a score of 1 to 12 as previously described
      • Greenhalgh DG
      • Sprugel KH
      • Murray MJ
      • Ross R
      PDGF and FGF stimulate wound healing in the genetically diabetic mouse.
      : 1 to 3, none to minimal cell accumulation and granulation tissue or epithelial migration; 4 to 6, thin, immature granulation tissue dominated by inflammatory cells but with few fibroblasts, capillaries, or collagen deposition and minimal epithelial migration; 7 to 9, moderately thick granulation tissue, dominated by inflammatory cells and more fibroblasts and collagen deposition; and 10 to 12, thick, vascular granulation tissue dominated by fibroblasts and extensive collagen deposition.

      Evaluation of Wound Lymphatic Vessel-Like Structures

      Sections of wounds were stained with rabbit anti-LYVE-1 antibody (1:500; Angiobio, Del Mar, CA), hamster anti-mouse podoplanin antibody (Angiobio), rat anti-mouse F4/80, and rat anti-mouse CD31 (1:250; BD PharMingen). Labeling with F4/80 and CD31 was visualized with FITC streptavidin (Vector Laboratories, Burlingame, CA) and biotinylated anti-rat antibody (Vector Laboratories). Lymphatic vessel-like structures were defined as three or more cells associated to form a cord-like structure (see below). Lymphatic vessels were normalized per 1 mm2 using percentage of fluorescence area, as described previously.
      • Jacobi J
      • Jang JJ
      • Sundram U
      • Dayoub H
      • Fajardo LF
      • Cooke JP
      Nicotine accelerates angiogenesis and wound healing in genetically diabetic mice.

      Reverse Transcriptase (RT)-PCR and Real-Time PCR Analysis

      For RT-PCR analysis, RNA was extracted using RNA-Stat (Tel-Test Inc., Friendswood, TX), according to the manufacturer's instructions. Total RNA was reverse-transcribed using TaqMan Mutiscribe RT Kit (Applied Biosystems, Foster City, CA), and amplification was performed on the ABI 7300 Real Time PCR System (Applied Biosystems). The following primers and probes were designed using PrimerExpress software (Applied Biosystems): VEGF-A (all isoforms): forward, 5′-CATCTTCAAGCCGTCCTGTGT-3′; reverse, 5′-CAGGGCTTCATCGTTACAGCA-3′; and 5-carboxyfluorescein (FAM)-CCGCTGATGCGCTGTGCAGG-BHQ. VEGFR3: forward, 5′-TGCTGAAAGAGGGCGCTACT-3′; reverse, 5′-TGCCGATGTGAATTAGGATCTTG-3′; and FAM-AGCACCGTGCCCTGATGTCGGA-TAMRA.
      The relative expression levels of the target gene mRNAs were calculated by the comparative CT method. All target sequences were normalized to 18S mRNA (VIC-labeled TaqMan reagents; Applied Biosystems) in multiplexed reactions performed in duplicate. Differences in CT values were calculated for each target mRNA after subtracting the mean value of 18S rRNA (relative expression = 2−ΔCT). Results were analyzed using analysis of variance.

      Tube Formation Assay

      Adherent cells were used in the tube formation assay as previously described.
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      An aliquot (100 μl) of growth factor-depleted Matrigel (Becton Dickinson, Bedford, MA) with 100 μl of EBM-2 (endothelial cell culture medium) (Cambrex Corporation, East Rutherford, NJ) was added to four-chamber slides (Lab-Tek; Nunc Inc., Naperville, IL) and allowed to gel for 30 minutes at 37°C. PECs were preincubated with high glucose (2 × 106 cells/ml) in 500 μl of EBM-2 containing 3% fetal bovine serum (normal glucose). Tube formation was monitored over 5 days. Digital pictures were taken using a Spot image analysis system. The areas covered by cell aggregates and tube-like structures were measured at 48 hours using NIH Image software. The total area covered by the Matrigel was outlined (18 mm2); the area of the aggregates and the tube-like structures was calculated and expressed as a percentage of the Matrigel area.

      Results

      Macrophages Contribute to Lymphatics in Normal Wound Healing

      To determine whether the involvement of macrophages in lymphatic vessel formation that we observed during corneal inflammation
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      is common to other forms of wound healing, we investigated the healing of excisional skin wounds. Examination of granulation tissue in wild-type C57BL/6 mice 5 days after wounding revealed lymphatic structures consisting of cells that were double-positive for F4/80 and LYVE-1 (Figure 1; day 5). Cells expressing both F4/80 and LYVE-1 were not detectable in the pre-existing lymphatics in the nonwounded tissue at the wound edge. Double positive cells persisted 10 to 14 days after wounding, though in lower numbers (Figure 1; white arrowhead), and lymphatic structures that remained in the granulation tissue stained for LYVE-1 only. We could not definitively determine whether these LYVE-1-only-positive structures had sprouted from local existing lymphatic vessels or if they were formed in situ by F4/80-positive cells that had down-regulated F4/80 expression. However, the fact that lymphatic vessels in the wound at day 14 expressed low levels of F4/80 suggests that new lymphatic vessels were formed from F4/80-positive cells. These observations indicate that, as for corneal inflammation, the lymphatic vessels that assemble transiently during the acute phase (days 5 to 7) of wound healing are derived largely from F4/80 macrophages and suggest an important role for macrophages in the formation of lymphatic vessels during the early stages of wound healing.

      Lymphangiogenesis in db/db Mouse Cornea

      Although reduced wound healing-associated angiogenesis has been documented in diabetic patients and in animal models,
      • Coleman DL
      Diabetes-obesity syndromes in mice.
      • Bohlen HG
      • Niggl BA
      Adult microvascular disturbances as a result of juvenile onset diabetes in Db/Db mice.
      the status of lymphangiogenesis in diabetic wounds has not been well investigated. We used the corneal suture model of inflammation
      • Cursiefen C
      • Chen L
      • Borges LP
      • Jackson D
      • Cao J
      • Radziejewski C
      • D'Amore PA
      • Dana MR
      • Wiegand SJ
      • Streilein JW
      VEGF-A stimu-lates lymphangiogenesis and hemangiogenesis in inflammatory neo-vascularization via macrophage recruitment.
      to assess lymphangiogenesis in the db/db mouse. Lymphangiogenesis and hemangiogenesis in db/+ mouse cornea were equivalent to that in wild-type mice (Figure 2b). Thus, db/+ littermate mice were used as controls in subsequent studies. Quantification of LYVE-1-positive lymphatic vessels and CD31-positive blood vessels in the cornea 7 days after suture placement revealed that both lymphangiogenesis and hemangiogenesis were significantly suppressed in the diabetic mice (P < 0.02) as compared with control (db/+) mice (Figure 2, b and c).
      Figure thumbnail gr2
      Figure 2Angiogenesis and lymphangiogenesis in the corneal suture model assay in db/db mice. a: A schematic illustrating the method of the suture placement on cornea. b: Quantification of lymphangiogenesis and hemangiogenesis in the corneal suture model assay in wild-type C57BL/6 mice. c: Hemangiogenesis (CD31; green) and lymphangiogenesis (LYVE-1; orange) in the db/db and db/+ (control) mouse corneal 7 days after suture placement. Graphs display quantification of lymphatic and blood vessel area.

      Macrophage Function in db/db Mice

      Using the corneal suture model of inflammation and wound healing, we have previously shown that macrophages play a critical role in the formation of lymphatic vessels.
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      As described above, macrophages contribute to lymphatics in normal skin wound healing. Based on these observations, we hypothesized that the reduced lymphatic vessels observed in diabetic mice might be due, at least in part, to an altered ability of macrophages to participate in lymphatic vessel formation. To test this possibility, peritoneal macrophages, collected as previously described
      • Takeuchi M
      • Alard P
      • Streilein JW
      TGF-beta promotes immune deviation by altering accessory signals of antigen-presenting cells.
      from db/db mice and db/+ controls, were quantified and assessed for their ability to form lymphatic vessels in vitro. Significantly fewer macrophages were recruited by thioglycolate stimulation in db/db mice than in control (db/+) mice (P = 0.02) (Figure 3a). Moreover, mRNA levels for VEGFR3 and its ligands VEGF-C and VEGF-A were significantly reduced in db/db-derived macrophages compared with macrophages from control (db/+) mice (P < 0.05) (Figure 3b). Stimulation of db/db-derived macrophages with an inflammatory cytokine, such as IL-1β (20 ng/ml), led to a marked increase in VEGFR3 mRNA and a modest increase in VEGF-C expression (Figure 3c).
      Figure thumbnail gr3
      Figure 3Characterization of PECs from db/db and db/+ mice. a: Thioglycollate-induced macrophages were collected from the peritoneal cavity of normal 8-week-old male db/db and db/+ mice and cell numbers compared (n = 4). b: Real-time RT-PCR analysis of VEGFR3, VEGF-C, and VEGF-A relative expression (RE) = 2DCT; by PECs from db/db and db/+ mice. c: RT-PCR analysis of VEGFR3 and VEGF-C after IL-1β (20 ng/ml) stimulation on PECs from db/db mice.

      Effect of High Glucose on Macrophage Function

      We next investigated whether the reduced gene expression observed in db/db-derived macrophages might be due to the effects of elevated glucose that characterizes the diabetic state. Incubation of control (db/+) macrophages under glucose conditions that mimic those seen in diabetes (30 mmol/L glucose) led to the suppression of VEGFR3 and VEGF-C mRNA to a level similar to those measured in db/db-derived macrophages (Figure 4). Treatment of the glucose-incubated db/+ macrophages with IL-1β (20 ng/ml) attenuated the inhibition of VEGFR3, VEGF-C, and VEGF-A expression.
      Figure thumbnail gr4
      Figure 4Effect of glucose and IL-1β on control macrophages. Real-time RT-PCR analysis of VEGFR3, VEGF-C, and VEGF-A of db/+ mouse PECs cultured in high glucose (30 mmol/L) for 72 hours (n = 6) relative expression (RE) = 2DCT and in the presence or absence of IL-1β. The statistical analysis was assessed by analysis of variance post hoc analysis (Fisher's PLSD).
      We have previously shown that macrophages cultured in Matrigel can form vessel-like structures that express lymphatic markers.
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      Whereas culture of control (db/+) macrophages led to the formation of tube-like structures, control macrophages cultured for 72 hours in high glucose (30 mmol/L glucose) did not form tubes (Figure 5c). The glu-cose-treated macrophages associated with one another but did not assemble into tubes. However, IL-1β treatment of the glucose-incubated macrophages restored their ability to assemble into vessel-like structures (Figure 5, c and d).
      Figure thumbnail gr5
      Figure 5Effect of IL-1β on tube formation by macrophages. a: Schematic of the tube formation assay. b: Flow cytometric analysis of cultured macrophages isolated from bone marrow and cultured in L929 cell condition medium. c: Quantification of cluster and tube-like structure formation in Matrigel (18 mm2) of db/+ peritoneal macrophages (1 × 106 cell/ml) plated into Matrigel and grown for 24 hours in high glucose alone or in high glucose with IL-1β. d: Photomicrograph of Matrigel assay in c. Statistical analysis was performed using analysis of variance post hoc analysis (Fisher's PLSD).

      Effect of Macrophage Administration on Wound Healing

      We next compared the effect of db/db-derived and control macrophages on wound healing. Because the reduced number of macrophages recruited by thioglycolate in db/db mice (Figure 3a) makes it technically difficult to collect sufficient macrophages to conduct these studies, macrophages for wound healing studies were harvested from bone marrow. Bone marrow was collected from db/db mice and cultured in L929 medium for 7 days.
      • Herembert T
      • Gogusev J
      • Zhu DL
      • Drueke TB
      • Marche P
      Control ofvascular smooth-muscle cell growth by macrophage-colony-stimulating factor.
      • Rutherford MS
      • Schook LB
      Differential immunocompetence of macrophages derived using macrophage or granulocyte-macrophage colony-stimulating factor.
      • Takashima A
      • Edelbaum D
      • Kitajima T
      • Shadduck RK
      • Gilmore GL
      • Xu S
      • Taylor RS
      • Bergstresser PR
      • Ariizumi K
      Colony-stimulating factor-1 secreted by fibroblasts promotes the growth of dendritic cell lines (XS series) derived from murine epidermis.
      More than 95% of the macrophages collected from wild-type C57BL/6 bone marrow using this method express CD11b and F4/80 (Figure 5b).
      Macrophages were harvested from db/db mouse bone marrow and cultured for an additional 24 hours in normal glucose in the absence or presence of IL-1β (20 ng/ml). Skin wounds were created in db/db mice and 1 × 106 db/db-derived macrophages (activated or nonactivated) were injected into the wounds. Wounds that received IL-1β-activated macrophages exhibited significantly more wound healing at day 7 (P < 0.0001), as evidenced by the amount of granulation tissue, compared with wounds that received nonactivated macrophages or saline (Figure 6, a and b). This difference in wound healing was no longer apparent 14 days after treatment (Figure 6b), suggesting that activated macrophages are rate-limiting only during the initial phase of wound healing.
      Figure thumbnail gr6
      Figure 6Wound healing in db/db mice. a: Photomicrographs of wounds in db/db mice 7 and 14 days after administration of the indicated macrophages (1 × 106 cells), including saline (no macrophages), db/db macrophages, db/db macrophages treated with IL-1β, or untreated db/+ macrophages. b: Quantification of newly formed granulation tissue over wounds at days 7 and 14. The area of the wounds at day 7 that received the IL-1β-treated macrophages was significantly smaller than those that received nontreated db/db macrophages or db/+ macrophages. At day 14, there was a significant difference between the area of the wounds that received IL-1β-treated db/db macrophages and untreated db/db macrophages but no difference between IL-1β-treated db/db macrophages or db/+ macrophages. Statistical analysis was performed by analysis of variance post hoc analysis (Fisher's PLSD).

      Wound Histology

      There were gross differences in wound healing between db/db mice that received IL-1β-treated db/db macrophages and those that received untreated db/db macrophages. Histological analysis revealed significant granulation tissue in wounds receiving IL-1β-treated macrophages (Figure 7); numerous infiltrating cells were evident, some of which appeared to be assembling into cord-like structures (Figure 7, a and b), representing lymphatic vessel-like structures. In contrast, wounds in db/db mice treated by saline injection contained little granulation tissue. Tissue sections from each group were scored for the extent of granulation tissue and the number of inflammatory cells by previously described methods
      • Greenhalgh DG
      • Sprugel KH
      • Murray MJ
      • Ross R
      PDGF and FGF stimulate wound healing in the genetically diabetic mouse.
      at 7 and 14 days after treatment (Figure 7b). Wounds receiving IL-1β-treated macrophages had thicker granulation tissue and more infiltrating cells at both 7 and 14 days than control wounds that received untreated macrophages. These observations are consistent with the well-documented role of the activated macrophage in wound healing and suggest that insufficient macrophage number and incomplete activation may account, at least in part, for the impaired wound healing observed in diabetes.
      Figure thumbnail gr7
      Figure 7Histological assessment of wounds in db/db mice treated with macrophages. a1 through a8: Photomicrograph of H&E-stained paraffin sections. a-5: Wounds that received IL-1-treated macrophages display many inflammatory cells in their granulation. a-6: Arrows indicate cord-like structures in granulation tissue that received IL-1β-treated-macrophages. b: Histological scores at days 7 and 14 after receiving the indicated macrophages. There was more extensive granulation tissue at day 7 in wounds that had received IL-1β-treated macrophages than in wounds that had received the untreated db/db macrophages or the db/+ macrophages. At day 14, there was a significant difference between the wounds that had received IL-1β-treated db/db macrophages versus untreated db/db macrophages but no difference between IL-1β-treated db/db macrophages and db/+ macrophages. The statistical analysis was performed using analysis of variance post hoc analysis (Fisher's PLSD).

      Lymphatic-Like Vessels in Wounds Treated with IL-1β Stimulated db/db Macrophages

      Seven days after macrophages were applied to wounds, the mice were sacrificed and the wound areas were sectioned and stained with antisera against LYVE-1, CD31, and F4/80. There were more LYVE-1-positive cord-like structures in the granulation tissue of wounds that had received IL-1β treated db/db-derived macrophages than in wounds that had received nontreated db/db macrophages (Figure 8). A majority of F4/80-positive cells in the granulation tissue co-stained with LYVE-1 (data not shown). Within the wound itself, the newly formed small lymphatic vessel structures were nearly all double-labeled with LYVE-1 and F4/80 (Figure 9a). However, outside of granulation tissue, there were only a few F4/80-positive cells in the lymphatic vessels (Figure 9b). Most of the cells that made up the lymphatic vessels of the granulation tissue double-stained with LYVE-1 and CD31, whereas none of the cells in blood vessels expressed LYVE-1 (data not shown). The small lymphatic vessel-like structures stained with antisera against both podoplanin and F4/80 (Figure 9c).
      Figure thumbnail gr8
      Figure 8Effect of IL-1β treatment on formation of cord-like structures in the granulation tissue. a: Immunofluorescence images at day 7 after treatment with untreated db/db macrophages (LYVE-1; orange) and IL-1β-stimulated db/db macrophages (LYVE-1; orange). b: Quantification of cord-like structures (three or more cells) (1 mm2). Cells were counted under the fluorescence microscope following DAPI staining (data not shown).
      Figure thumbnail gr9
      Figure 9Effect of IL-1β-treated macrophages on formation of lymphatic structures. Confocal microscopic images of wounds treated with IL-1β-stimulated db/db macrophages. a: Small vessel-like structures stained with LYVE-1 (orange) and F4/80 (green) at the edge of the granulation tissue (blue is nuclear staining with TOPRO-3). The arrow indicates the lumen that was constituted by cells that stained for both F4/80 (green) and LYVE-1 (red). Scale bar = 20 μm. b: Area outside of the granulation tissue stained with LYVE-1 (orange) and F4/80 (green) (blue is nuclear staining with TOPRO-3). V, vessel; L, lymphatic vessel. Note that some lymphatic vessels did not stain with F4/80 (arrowhead), whereas others stained for both F4/80 and LYVE-1 (arrow). Scale bar = 40 μm. c: A vessel-like structure in the granulation tissue stained with podoplanin (orange) and F4/80 (green). Scale bar = 40 μm.
      Prelabeled macrophages that had been injected also contributed to LYVE-1 and podoplanin-positive lymphatic-like structures in the wound (Figure 10). Four weeks after macrophage injection, when wound-healing inflammation had resolved, there were no lymphatic vessel-like structures that co-labeled with LYVE-1 and F4/80; only LYVE-1-positive lymphatic vessels were evident (data not shown).
      Figure thumbnail gr10
      Figure 10The contribution of exogenously administered macrophages to lymphatic vessels in wound in db/db mice. Macrophages were isolated from wild-type C57BL/6 mice, prelabeled with DiLDL, and injected into excisional wounds created in diabetic mice. Granulation tissue was examined 7 days later. Top: DiI-prelabeled macrophages stained with LYVE-1 located in granulation tissue 7 days after transplantation. Bottom: DiI-prelabeled macrophages stained with antisera against podoplanin in granulation tissue 7 days after transplantation.

      Discussion

      We have shown that, as we observed during corneal inflammation, macrophages contribute to the formation of lymphatics in healing of excisional wounds. Furthermore, whereas the addition of macrophages isolated from db/db mouse bone marrow to wounds in db/db mice did not significantly affect wound healing, IL-1β activation of the db/db macrophages restored their ability to contribute to wound healing. Observations of the effects of glucose treatment on macrophages from nondiabetic mice lead us to speculate that high glucose suppression of macrophage function, and the resulting reduced inflammatory response may account, at least in part, for the impaired wound repair that is frequently observed in association with diabetes. Instead of the acute inflammation observed during normal healing, wound healing in the diabetic is characterized by chronic moderate inflammation that is apparently insufficient to move the healing process forward. The role of macrophages as a source of cytokines and growth factors and as a phagocyte during wound healing has been well documented.
      • Singer AJ
      • Clark RA
      Cutaneous wound healing.
      The function of macrophages has been well investigated in diabetic mice
      • Zykova SN
      • Jenssen TG
      • Berdal M
      • Olsen R
      • Myklebust R
      • Seljelid R
      Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.
      but not in humans. Macrophages isolated from diabetic mice have been shown to secrete reduced levels of inflammatory cytokines such as IL-1β and tumor necrosis factor-α.
      • Zykova SN
      • Jenssen TG
      • Berdal M
      • Olsen R
      • Myklebust R
      • Seljelid R
      Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.
      In addition, as macrophages have been shown to play a critical role in keratinocyte deposition of extracellular matrix in wound area,
      • Falanga V
      Wound healing and its impairment in the diabetic foot.
      reduced macrophages may also have a variety of indirect effects.
      Our findings of reduced macrophage number in both the corneal suture model and in peritoneal exudates in db/db mice suggest that inadequate macrophage number may be one factor contributing to impaired wound repair. We have previously shown that peritoneal monocytes/macrophages express markers of lymphatic endothelium, including LYVE-1, podoplanin, and Prox-1 and that macrophages contribute to lymphatic vessels during acute inflammation in the cornea.
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      • Kerjaschki D
      • Huttary N
      • Raab I
      • Regele H
      • Bojarski-Nagy K
      • Bartel G
      • Krober SM
      • Greinix H
      • Rosenmaier A
      • Karlhofer F
      • Wick N
      • Mazal PR
      Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants.
      • Schledzewski K
      • Falkowski M
      • Moldenhauer G
      • Metharom P
      • Kzhyshkowska J
      • Ganss R
      • Demory A
      • Falkowska-Hansen B
      • Kurzen H
      • Ugurel S
      • Geginat G
      • Arnold B
      • Goerdt S
      Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1(+), F4/80(+), CD11b(+) macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis.
      Furthermore, we observed that peritoneal macrophages could form lymphatic vessel-like tubes in vitro in a density-dependent manner.
      • Maruyama K
      • Ii M
      • Cursiefen C
      • Jackson DG
      • Keino H
      • Tomita M
      • Van Rooijen N
      • Takenaka H
      • D'Amore PA
      • Stein-Streilein J
      • Losordo DW
      • Streilein JW
      Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.
      Thus, the reduced macrophage number in db/db mice may be the cause of the significant reduction in lymphatic structures in the granulation tissue of wounds in db/db mice compared with controls, which, in turn, may result in impaired wound healing.
      Our observation of VEGF-C expression by macrophages within the inflamed corneal stroma and by bone marrow-derived macrophages
      • Cursiefen C
      • Chen L
      • Borges LP
      • Jackson D
      • Cao J
      • Radziejewski C
      • D'Amore PA
      • Dana MR
      • Wiegand SJ
      • Streilein JW
      VEGF-A stimu-lates lymphangiogenesis and hemangiogenesis in inflammatory neo-vascularization via macrophage recruitment.
      indicates that macrophages are a likely source of VEGF-C in tumors and in inflammation where they may function to induce lymphatic vessel formation.
      • Karkkainen MJ
      • Haiko P
      • Sainio K
      • Partanen J
      • Taipale J
      • Petrova TV
      • Jeltsch M
      • Jackson DG
      • Talikka M
      • Rauvala H
      • Betsholtz C
      • Alitalo K
      Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.
      Therefore, it is relevant that the db/db-derived macrophages were qualitatively different from the db/+ macrophages; db/db macrophages exhibited significantly reduced VEGFR3, VEGF-C, and VEGF-A mRNA expression relative to db/+ macrophages. The fact that incubation of macrophages from control mice in high glucose suppressed the expression of VEGFR3 and VEGF-C suggest that this suppression may be secondary to hyperglycemia. IL-1β stimulation of db/db-derived macrophages led to increased expression of lymphatic endothelial markers, including VEGFR3 and its ligand VEGF-C. Consistent with these observations, unstimulated macrophages from db/+ mice incubated in high glucose formed fewer clusters and tube-like structures in a three-dimensional assay than cells stimulated with IL-1β. Thus, in addition to the lower number of macrophages, reduced expression of VEGFR3, VEGF-C, and VEGF-A may contribute to impaired wound healing by leading to reduced lymphatic vessel formation.
      Our results indicate that activated macrophages with normal function are important for wound repair and participate in the formation of transient lymphatic vessels that seem to regress when inflammation is resolved. It has been previously suggested that lymphangiogenesis is not essential for closure of excisional skin wounds.
      • Hong YK
      • Lange-Asschenfeldt B
      • Velasco P
      • Hirakawa S
      • Kunstfeld R
      • Brown LF
      • Bohlen P
      • Senger DR
      • Detmar M
      VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.
      We found that the administration of macrophages that could contribute to the formation of lymphatic vessels accelerated and enhanced the formation of the granulation tissue. The early formation of lymphatic vessel-like structures in granulation tissue that strongly expressed LYVE-1 and podoplanin seems to be important for normal wound healing. The db/db mice share a number of similarities with human adult-onset diabetes mellitus, including impaired wound healing. Thus, our observations of wound healing rescue by exogenously activated macrophages may hold promise for human therapies.

      Acknowledgements

      We thank Dr. Eric Finkelstein, Dr. Magali Saint-Geniez, and Arindel Maharaj for helpful discussions and critical reading of the manuscript. We also thank Christine Bagley for constant support, Donald Pottle for help with confocal and immunofluorescent imaging, Marie Ortega for excellent management of the Schepens animal facility, and Randy Huang for assistance with flow cytometry.

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