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The Peripheral Lymphatic System Is Impaired by the Loss of Neuronal Control Associated with Chronic Spinal Cord Injury

Open AccessPublished:July 14, 2022DOI:https://doi.org/10.1016/j.ajpath.2022.06.012
      Spinal cord injury (SCI) is associated with venous vascular dysfunction below the level of injury, resulting in dysregulation of tissue fluid homeostasis in afflicted skin. The purpose of this study was to determine whether loss of neuronal control in chronic SCI also affects the skin lymphatic system. Morphology of lymphatics was characterized by immunohistochemistry and lymphatic gene expression profiles determined by DNA microarray analysis. In SCI, skin lymphatic function appeared to be impaired, because the ratio of functionally dilated versus collapsed lymphatic vessels was 10-fold decreased compared with control. Consequently, the average lumen area of lymphatic vessels was almost halved, possibly due to the known impaired connective tissue integrity of SCI skin. In fact, collagenases were found to be overexpressed in SCI skin, and dermal collagen structure was impaired. Molecular profiling also suggested an SCI-specific phenotype of increased connective tissue turnover and decreased lymphatic contractility. The total number of lymphatic vessels in SCI skin, however, was doubled, pointing to enhanced lymphangiogenesis. In conclusion, these data show, for the first time, that lymphatic function and development in human skin are under neuronal control. Because peripheral venous and lymphatic vascular defects are associated with disturbed fluid homeostasis, inappropriate wound-healing reactions, and impaired skin immunity, they might contribute to the predisposition of afflicted individuals to pressure ulcer formation and wound-healing disorders.
      Spinal cord injury (SCI) results not only in motor and sensory deficits but also in a broad range of autonomic dysfunctions persisting in the chronic phase of SCI.
      • Karlsson A.K.
      Autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and signs.
      As a consequence, the function of several tissues and organs is impaired in chronic SCI, having a severe, life-long impact on the quality of life of afflicted individuals.
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Ditunno Jr., J.F.
      • Formal C.S.
      Chronic spinal cord injury.
      One of the peripheral tissues commonly affected is the skin, resulting in frequently occurring and recurring pressure ulcers. Thus, it is estimated that the prevalence of pressure ulcers among SCI individuals is 17%,
      • Whiteneck C.G.
      • Charlifue S.W.
      • Frankel H.L.
      • Fraser M.H.
      • Gardner B.P.
      • Gerhart K.A.
      • Krishnan K.R.
      • Menter R.R.
      • Nuseibeh I.
      • Short D.J.
      • Silver J.R.
      Mortality, morbidity, and psychosocial outcomes of persons spinal cord injured more than 20 years ago.
      and the lifetime risk of developing an ulcer ranges from 62% to 86%.
      • Salzberg C.A.
      • Byrne D.W.
      • Cayten C.G.
      • Kabir R.
      • van Niewerburgh P.
      • Viehbeck M.
      • Long H.
      • Jones E.C.
      Predicting and preventing pressure ulcers in adults with paralysis.
      ,
      • Sumiya T.
      • Kawamura K.
      • Tokuhiro A.
      • Takechi H.
      • Ogata H.
      A survey of wheelchair use by paraplegic individuals in Japan, part 2: prevalence of pressure sores.
      Apart from sociodemographic, neurologic, behavioral, or medical care–related risk factors for pressure ulcer development in SCI,
      • Marin J.
      • Nixon J.
      • Gorecki C.
      A systematic review of risk factors for the development and recurrence of pressure ulcers in people with spinal cord injuries.
      intrinsic molecular and cellular characteristics of chronic SCI skin, associated with aberrant nerve activity, are only beginning to be characterized in more detail.
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      We have recently reported that molecular and cellular homeostasis is disturbed in chronic SCI skin.
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      This was associated with increased microvascular permeability and extravasation of plasma components, such as platelets, into the dermal tissue. The consequences were wound-healing specific molecular reactions, which are inappropriate to noninjured, intact skin and potentially predispose chronic SCI skin to the development of pressure ulcers.
      Disturbance of tissue fluid homeostasis in SCI skin is also suggested by observations such as skin thickening and edema formation.
      • Stover S.L.
      • Omura E.F.
      • Buell A.B.
      Clinical skin thickening following spinal cord injury studied by histopathology.
      ,
      • Guihan M.
      • Bates-Jenson B.M.
      • Chun S.
      • Parachuri R.
      • Chin A.S.
      • McCreath H.
      Assessing the feasibility of subepidermal moisture to predict erythema and stage 1 pressure ulcers in persons with spinal cord injury: a pilot study.
      Skin fluid homeostasis, however, is maintained by the peripheral microvascular system of both blood vessels and lymphatics, with the latter draining excess fluid, particulates, and cells from the tissue.
      • Alitalo K.
      The lymphatic vasculature in disease.
      Whether aberrant nerve activity, in addition to impairing blood vessel function, may also affect skin lymphatic function; and how this potentially contributes, in addition to increased venous vascular permeability, to the disturbed tissue fluid homeostasis in chronic SCI is not known.
      To address this question, a histologic analysis of the lymphatic system in chronic SCI skin was performed, and the molecular effects of aberrant nerve activity were examined by defining SCI-specific molecular profiles for lymphatic function, connective tissue integrity, and lymphangiogenesis.

      Materials and Methods

      To identify SCI-specific alterations in the morphology of the lymphatic system, we quantified, using immunohistochemistry combined with image analysis, number and functionality of lymph vessels in SCI skin from areas afflicted by loss of neuronal control in comparison to skin from healthy individuals. In addition, we identified, at the molecular level, SCI-specific lymphatic gene regulation by whole-genome gene expression analysis. This was confirmed, by (immuno)histochemistry specific for collagenases and collagen.

      Tissue Samples

      Following informed consent, tissue samples of intact skin or of the wound edge of pressure ulcers, both from skin areas afflicted by loss of neuronal control, were collected during routine surgery of chronic SCI patients at the Werner Wicker Klinik (Bad Wildungen, Germany). Patients (n = 31; paraplegia n = 15; tetraplegia n = 16) were classified on the basis of the impairment scale (grades A through E) developed by the American Spinal Injury Association (Table 1). All patients were classified to American Spinal Injury Association grade A or B [ie, were afflicted by a complete loss of both motor and sensory function (grade A) or only motor function (grade B) below the level of injury]. Tissue samples of pressure ulcers were taken from the ischium (70%), the coccyx (13%), or the sacrum (9%) (8% from undefined body locations). During debridement and coverage of pressure ulcers, intact SCI skin samples were collected from areas approximately 10 cm distant to the wound and to high-risk weight-bearing skin areas, respectively.
      Table 1Patient Characteristics
      CharacteristicsSCI patients (n = 31)Able-bodied controls (n = 22)
      Sex
       Male27 (87)5 (23)
       Female4 (13)17 (77)
      Age, years50 (14.4)38 (11.4)
      SCI level
       Paraplegia15 (48)
       Tetraplegia16 (52)
      ASIA Impairment Scale
       A21 (68)
       B10 (32)
      SCI duration, years14.7 (11.5)
      The patient cohort is identical to the cohort analyzed by Brunner et al.
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      Data are given as number (percentage) or mean (SD).
      ASIA, American Spinal Injury Association; SCI, spinal cord injury.
      Following informed consent, control tissue samples of normal skin were collected, during routine surgical procedures, from various body locations of able-bodied (AB) patients (n = 22) at the Fachklinik Hornheide (Münster, Germany).
      Tissue samples were stored fresh-frozen at –80°C or formalin fixed and paraffin embedded at room temperature. Procedures of tissue sample collection were approved by the local ethical committee (Ärztekammer Westfalen-Lippe, Münster, Germany).

      Gene Expression Analysis

      Gene expression analysis was performed as described previously.
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      Briefly, total RNA was prepared from fresh-frozen tissue (intact SCI skin, n = 17; pressure ulcers, n = 15; and AB control skin, n = 16) using RNeasy Fibrous Tissue Mini Kits (Qiagen, Hilden, Germany) and cyanine-3 labeled by RT-IVT. Whole human genome gene expression profiles were obtained for each sample group in blinded triplicates using G3 Human Gene Expression 8 × 60K Microarrays (Agilent, Waldbronn, Germany). Gene expression data were normalized to the mean expression of the housekeeping genes, PUM1, GUSB, and HPRT1.

      Immunohistochemistry of Lymphatic Vessels

      Formalin-fixed, paraffin-embedded tissue sections (5 μm thick) were dewaxed in xylene and rehydrated in decreasing ethanol concentrations. Following proteinase K treatment (Qiagen; 20 μg/mL) for 20 minutes at room temperature, endogenous peroxidase was inactivated using NOVADetect Peroxid–Block (Dianova, Hamburg, Germany) for 15 minutes at room temperature, and free protein binding sites were blocked with 5% human serum for 30 minutes at room temperature. Tissue sections were incubated overnight at 4°C with primary antibodies to the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1; polyclonal goat IgG at 5 μg/mL; AF2089; R&D Systems, Abingdon, UK) or nonimmune goat IgG as negative control. Antibody binding was detected by incubation for 45 minutes at room temperature with secondary anti–goat IgG horseradish peroxidase. Diaminobenzidine (Dianova) was used as a chromogenic substrate for peroxidase. Tissue sections were counterstained with Papanicolaou (Merck, Darmstadt, Germany).

      Image Analysis of Lymphatic Vessels

      Microphotographs of LYVE-1–immunostained skin tissue sections were taken using a Diaplan microscope (Leitz, Wetzlar, Germany), equipped with a DFC320 digital camera and FireCam 3.4 software (Leica Microsystems, Wetzlar, Germany), and imported into Photoshop software (Adobe, San Jose, CA). Lymph vessels were identified on the basis of LYVE-1 staining and counted, and the lumen area of the vessels as well as the length of the epidermis in the tissue section were quantified in Photoshop using the respective selection tools.

      Statistical Analysis

      All graphs show mean values and SEM. Statistical significance was determined using the Kruskal-Wallis test or the t-test, with Bonferroni correction for multiple comparisons.

      Results

      Characterization of the Lymphatic Vessel System in SCI Skin

      To analyze the skin for potential morphologic and functional alterations caused by the loss of neuronal control in chronic SCI, dermal lymphatic vessels were identified and characterized by immunostaining for the lymphatic marker, LYVE-1 (Figure 1A).
      • Podgrabinska S.
      • Braun P.
      • Velasco P.
      • Kloos B.
      • Pepper M.S.
      • Jackson D.G.
      • Skobe M.
      Molecular characterization of lymphatic endothelial cells.
      The shape of the lumen of lymphatic vessels varied, and three categories of vessels were defined on the basis of the appearance of their lumen (open, intermediate, or collapsed) (Figure 1C).
      Figure thumbnail gr1
      Figure 1Identification of lymphatic vessels in the dermis. A and C: Immunostaining for the lymphatic endothelial hyaluronan receptor-1. B: Control staining with nonimmune IgG. C: On the basis of the shape of the lumen, three types of lymphatic vessels were defined: open, intermediate, and collapsed. Scale bar = 20 μm (AC).
      To quantify lymphatic vessel number and lumen area, we developed an image analysis procedure, depicted in Figure 2. Microphotographs covering the entire area of a skin tissue section, immunostained for LYVE-1, were rearranged to visualize the section (Figure 2A). Lymphatic vessel density in each of the three categories (open, intermediate, or collapsed) (Figure 2B), lumen area of the vessels (Figure 2C), as well as the length of the epidermis in the tissue section (Figure 2D) were determined.
      Figure thumbnail gr2
      Figure 2Quantitative analysis of lymphatic vessels. A: Micrograph exemplary of a skin tissue section immunostained for the lymphatic endothelial hyaluronan receptor-1 (LYVE-1), imported into Photoshop software, and rearranged to cover and visualize the entire area of the section; immunostaining was digitally enhanced to facilitate subsequent image analysis. B: Lymphatic vessels were numbered and categorized into the three vessel types, defined in C (open, green; intermediate, yellow; and collapsed, red). C: Following conversion of LYVE-1 immunostaining to black and white, the lumen areas of the lymphatic vessels in the section were determined. D: Following conversion of the Papanicolaou staining to black and white, the length of the epidermis in the section was measured.

      Loss of Neuronal Control in Chronic SCI Skin Induces Collagenase Expression and Impairs Collagen Fibril Structure as Well as Functional Lymphatic Morphology

      Lymphatic function requires opening of the lumen as well as contractile properties of lymphatic vessels. In chronic SCI skin, the proportion of open lymphatic vessels was drastically reduced (by 4.9-fold compared with AB control skin) (Figure 3A). Correspondingly, the proportion of collapsed vessels was almost doubled. Thus, the ratio of functionally dilated versus collapsed lymphatic vessels decreased by almost 10-fold compared with AB control skin. These alterations were reflected in a 1.7-fold reduction in the average lumen area of individual lymphatic vessels in SCI skin compared with control (Figure 3B). These findings suggested impairment of lymphatic function.
      Figure thumbnail gr3
      Figure 3Number of functionally dilated skin lymphatic vessels and vessel lumen area were decreased in chronic spinal cord injury (SCI). A: Lymphatic vessels in each of the three categories (open, intermediate, and collapsed) (C) were identified in tissue sections of SCI skin or able-bodied (AB) control skin using lymphatic endothelial hyaluronan receptor-1 immunostaining and counted following image analysis (B). The difference in the vessel category intermediate was not significant. B: The average lumen area of lymphatic vessels was determined by image analysis (C). Data are mean values + SEM (A and B). n = 11 tissue sections of SCI skin (A); n = 9 tissue sections of AB control skin (A). ∗P < 0.05, ∗∗P < 0.01 by the Kruskal-Wallis test.
      To support the above morphologic observations, we performed differential gene expression analysis of functional lymphatic markers in SCI (n = 17) versus AB (n = 16) skin. As a reference to wound healing, the analysis also comprised the wound edge of pressure ulcers (n = 15). We identified an SCI-specific molecular signature of eight known functional lymphatic markers [CACNG1, ACTA1, LYVE1, ESR, PF4, MYLK2, TLR4, and transforming growth factor (TGF)-β1], SCI-specific differential regulation of which may result in impaired lymphatic contractility and immune cell trafficking (Table 2),
      • Tsai M.-K.
      • Lai C.-H.
      • Chen L.-M.
      • Jong G.-P.
      Calcium channel blocker-related chylous ascites: a systematic review and meta-analysis.
      • Imtiaz M.S.
      • Zhao J.
      • Hosaka K.
      • von der Weid P.-Y.
      • Crowe M.
      • van Helden D.F.
      Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.
      • Muthuchamy M.
      • Gashev A.
      • Boswell N.
      • Dawson N.
      • Zawieja D.
      Molecular and functional analyses of the contractile apparatus in lymphatic muscle.
      • Jackson D.G.
      Hyaluronan in the lymphatics: the key role of the hyaluronan receptor LYVE-1 in leucocyte trafficking.
      • Morfoisse F.
      • Tatin F.
      • Chaput B.
      • Therville N.
      • Vaysse C.
      • Métivier R.
      • Malloizel-Delaunay J.
      • Pujol F.
      • Godet A.-C.
      • De Toni F.
      • Boudou F.
      • Grenier K.
      • Dubuc D.
      • Lacazette E.
      • Prats A.-C.
      • Guillermet-Guibert J.
      • Lenfant F.
      • Garmy-Susini B.
      Lymphatic vasculature requires estrogen receptor-alpha signaling to protect from lymphedema.
      • Ma W.
      • Gil H.J.
      • Escobedo N.
      • Benito-Martin A.
      • Ximénez-Embún P.
      • Munoz J.
      • Peinado H.
      • Rockson S.G.
      • Oliver G.
      Platelet factor 4 is a biomarker for lymphatic-promoted disorders.
      • Furtado G.C.
      • Marinkovic T.
      • Martin A.P.
      • Garin A.
      • Hoch B.
      • Hubner W.
      • Chen B.K.
      • Genden E.
      • Skobe M.
      • Lira S.A.
      Lymphotoxin β receptor signaling is required for inflammatory lymphangiogenesis in the thyroid.
      • Wang W.
      • Nepiyushchikh Z.V.
      • Zawieja D.C.
      • Chakraborty S.
      • Zawieja S.D.
      • Gashev A.A.
      • Davis M.J.
      • Muthuchamy M.
      Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.
      • Zampell J.C.
      • Elhadad S.
      • Avraham T.
      • Weitman E.
      • Aschen S.
      • Yan A.
      • Mehrara B.J.
      Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury.
      • Avraham T.
      • Yan A.
      • Zampbell J.C.
      • Daluvoy S.V.
      • Haimovitz-Friedman A.
      • Cordeiro A.P.
      • Mehrara B.J.
      Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-β1-mediated tissue fibrosis.
      supporting the above morphologic findings. Differential regulation of four of the eight signature markers (ACTA1, LYVE1, ESR, and TGF-β1) was further enhanced (P < 0.001) in pressure ulcers of SCI individuals.
      Table 2SCI-Specific Molecular Signature of Lymphatic Function in the Skin
      Gene/protein symbolGene/proteinDifferential gene expression
      Determined by DNA microarray analysis (1 to 10 probes/gene, assayed on triplicate microarrays).
      /growth factor activity
      Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al8).
      (x-fold)
      SCI versus ABP valuePressure ulcer versus ABP value
      CACNG1Calcium channel γ subunit
      • Tsai M.-K.
      • Lai C.-H.
      • Chen L.-M.
      • Jong G.-P.
      Calcium channel blocker-related chylous ascites: a systematic review and meta-analysis.
      ,
      • Imtiaz M.S.
      • Zhao J.
      • Hosaka K.
      • von der Weid P.-Y.
      • Crowe M.
      • van Helden D.F.
      Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.
      >34 ↓
      Data are x-fold differences of mean expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15).
      <0.00121.7 ↓<0.001
      ACTA1Actin α 1
      • Muthuchamy M.
      • Gashev A.
      • Boswell N.
      • Dawson N.
      • Zawieja D.
      Molecular and functional analyses of the contractile apparatus in lymphatic muscle.
      8.7 ↓<0.00178.7 ↓<0.001
      LYVE1Lymphatic vessel endothelial hyaluronan receptor-1
      • Jackson D.G.
      Hyaluronan in the lymphatics: the key role of the hyaluronan receptor LYVE-1 in leucocyte trafficking.
      5.4 ↓<0.0018.5 ↓<0.001
      ESREstrogen receptor
      • Morfoisse F.
      • Tatin F.
      • Chaput B.
      • Therville N.
      • Vaysse C.
      • Métivier R.
      • Malloizel-Delaunay J.
      • Pujol F.
      • Godet A.-C.
      • De Toni F.
      • Boudou F.
      • Grenier K.
      • Dubuc D.
      • Lacazette E.
      • Prats A.-C.
      • Guillermet-Guibert J.
      • Lenfant F.
      • Garmy-Susini B.
      Lymphatic vasculature requires estrogen receptor-alpha signaling to protect from lymphedema.
      4.3 ↓<0.0017.7 ↓<0.001
      PF4Platelet factor 4
      • Ma W.
      • Gil H.J.
      • Escobedo N.
      • Benito-Martin A.
      • Ximénez-Embún P.
      • Munoz J.
      • Peinado H.
      • Rockson S.G.
      • Oliver G.
      Platelet factor 4 is a biomarker for lymphatic-promoted disorders.
      4.1 ↑<0.001<0.001
      CCL21Chemokine (C-C motif) ligand 21
      • Furtado G.C.
      • Marinkovic T.
      • Martin A.P.
      • Garin A.
      • Hoch B.
      • Hubner W.
      • Chen B.K.
      • Genden E.
      • Skobe M.
      • Lira S.A.
      Lymphotoxin β receptor signaling is required for inflammatory lymphangiogenesis in the thyroid.
      3.7 ↓<0.0013.6 ↓0.016
      MYLK2Myosin light chain kinase 2
      • Wang W.
      • Nepiyushchikh Z.V.
      • Zawieja D.C.
      • Chakraborty S.
      • Zawieja S.D.
      • Gashev A.A.
      • Davis M.J.
      • Muthuchamy M.
      Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.
      3.6 ↓<0.001<0.001
      TLR4Toll-like receptor 4
      • Zampell J.C.
      • Elhadad S.
      • Avraham T.
      • Weitman E.
      • Aschen S.
      • Yan A.
      • Mehrara B.J.
      Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury.
      3.0 ↓0.0023.2 ↓
      TGF-β1Transforming growth factor-β1
      • Avraham T.
      • Yan A.
      • Zampbell J.C.
      • Daluvoy S.V.
      • Haimovitz-Friedman A.
      • Cordeiro A.P.
      • Mehrara B.J.
      Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-β1-mediated tissue fibrosis.
      2.2 ↑
      Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al8).
      <0.0013.3 ↑
      ↓, Differentially down-regulated (threefold or greater); ↑, differentially up-regulated (threefold or greater); AB, able bodied; SCI, spinal cord injury.
      Determined by DNA microarray analysis (1 to 10 probes/gene, assayed on triplicate microarrays).
      Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      ).
      Data are x-fold differences of mean expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15).
      Opening of lymphatic vessels and proper skin lymphatic function are critically dependent on the mechanical integrity of the dermal connective tissue.
      • Skobe M.
      • Detmar M.
      Structure, function, and molecular control of the skin lymphatic system.
      Previous findings indicating increased collagen degradation in skin and bone of SCI patients
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Rodriguez G.P.
      • Claus-Walker J.
      • Kent M.C.
      • Garza H.M.
      Collagen metabolite excretion as a predictor of bone- and skin-related complications in spinal cord injury.
      ,
      • Claus-Walker J.
      Clinical implications of the disturbance in calcium and collagen metabolism in quadriplegia.
      prompted us to investigate connective tissue weakening as a potential cause contributing to skin lymphatic dysfunction. Differential gene expression profiling of tissue degradation and production identified an SCI-specific 13-gene signature of proteinases (MMP1, MMP13, KLK12, and MMP8), enzyme inhibitors (SPINK2 and HPSE2), connective tissue constituents (FNDC1, THBS4, TNN, DPT, and TNXB), and modifying enzymes (HS3ST2 and LOXL4). Differential gene expression of this signature might result in a shift of SCI connective tissue homeostasis toward enhanced degradation (in particular, of collagen) and reduced production (Table 3).
      • Ala-aho R.
      • Kähäri V.-M.
      Collagenases in cancer.
      • Kryza T.
      • Parent C.
      • Pardessus J.
      • Petit A.
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      • Iochmann S.
      • Labas V.
      • Courty Y.
      • Heuzé-Vourc’h N.
      Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix.
      • Fischer J.
      • Meyer-Hoffert U.
      Regulation of kallikrein-related peptidases in the skin – from physiology to diseases to therapeutic options.
      • Gaskin S.M.
      • Soares Da Costa T.P.
      • Hulett M.D.
      Heparanase: cloning, function and regulation.
      • Pinhal M.A.S.
      • Melo C.M.
      • Nader H.B.
      The good and bad sides of heparanase-1 and heparanase 2.
      • Bagordakis E.
      • Sawazaki-Calone I.
      • Carneiro Soares Macedo C.
      • Carnielli C.M.
      • Ervolino de Oliveira C.
      • Campioni Rodrigues P.
      • Rangel A.L.C.A.
      • Nunes Dos Santos J.
      • Risteli J.
      • Graner E.
      • Salo T.
      • Franco Paes Leme A.
      • Coletta R.D.
      Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.
      • Stenina-Adognravi O.
      • Plow E.
      Thrombospondin-4 in tissue remodeling.
      • Chiquet-Ehrismann R.
      • Tucker R.P.
      Connective tissues: signalling by tenascins.
      • Bret C.
      • Hose D.
      • Reme T.
      • Sprynski A.-C.
      • Mahtouk K.
      • Schved J.-F.
      • Quittet P.
      • Rossi J.-F.
      • Goldschmidt H.
      • Klein B.
      Expression of genes encoding for proteins involved in heparan sulphate and chondroitin sulphate chain synthesis and modification in normal and malignant plasma cells.
      • Takeda U.
      • Utani A.
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      • Adachi E.
      • Koseki H.
      • Taniguchi M.
      • Matsumoto T.
      • Ohashi T.
      • Sato M.
      • Shinkai H.
      Targeted disruption of dermatopontin causes abnormal collagen fibrillogenesis.
      • Huang M.
      • Cai G.
      • Baugh L.M.
      • Liu Z.
      • Smith A.
      • Watson M.
      • Popovich D.
      • Zhang T.
      • Stawski L.S.
      • Trojanowska M.
      • Georgakoudi I.
      • Black III, L.D.
      • Piolo P.A.
      • Whitfield M.L.
      • Garlick J.
      Systemic sclerosis dermal fibroblasts induce cutaneous fibrosis through lysyl oxidase-like 4: new evidence from three-dimensional skin-like tissues.
      Differential expression of 6 of the 13 signature genes was further enhanced (P < 0.001) in pressure ulcers (MMP1, MMP13, KLK12, HPSE2, TNXB, and LOXL4) (Table 3).
      Table 3SCI-Specific Molecular Signature of Skin Connective Tissue
      Gene/protein symbolGene/protein nameDifferential gene expression
      Determined by DNA microarray analysis (1 to 10 probes/gene, assayed on triplicate microarrays).
      (x-fold)
      SCI versus ABP valuePressure ulcer versus ABP value
      Connective tissue degradation
      MMP1Matrix metalloproteinase-1 (collagenase-1)
      • Ala-aho R.
      • Kähäri V.-M.
      Collagenases in cancer.
      16.3 ↑
      Data are x-fold differences of mean gene expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15).
      <0.001130.2 ↑<0.001
      MMP13Matrix metalloproteinase-13 (collagenase-3)
      • Ala-aho R.
      • Kähäri V.-M.
      Collagenases in cancer.
      >11 ↑<0.00121.7 ↑<0.001
      KLK12Kallikrein-related peptidase 12
      • Kryza T.
      • Parent C.
      • Pardessus J.
      • Petit A.
      • Burlaud-Gaillard J.
      • Reverdiau P.
      • Iochmann S.
      • Labas V.
      • Courty Y.
      • Heuzé-Vourc’h N.
      Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix.
      4.0 ↑<0.00124.3 ↑<0.001
      SPINK2Serine proteinase inhibitor Kazal type 2
      • Fischer J.
      • Meyer-Hoffert U.
      Regulation of kallikrein-related peptidases in the skin – from physiology to diseases to therapeutic options.
      4.0 ↓<0.0015.2 ↓<0.001
      MMP8Matrix metalloproteinase-8 (collagenase-2)
      • Ala-aho R.
      • Kähäri V.-M.
      Collagenases in cancer.
      >3.8 ↑0.031
      HPSE2Heparanase 2 (heparanase 1 inhibitor)
      • Gaskin S.M.
      • Soares Da Costa T.P.
      • Hulett M.D.
      Heparanase: cloning, function and regulation.
      ,
      • Pinhal M.A.S.
      • Melo C.M.
      • Nader H.B.
      The good and bad sides of heparanase-1 and heparanase 2.
      3.5 ↓<0.00110.1 ↓<0.001
      Connective tissue production
      FNDC1Fibronectin type III domain containing 1
      • Bagordakis E.
      • Sawazaki-Calone I.
      • Carneiro Soares Macedo C.
      • Carnielli C.M.
      • Ervolino de Oliveira C.
      • Campioni Rodrigues P.
      • Rangel A.L.C.A.
      • Nunes Dos Santos J.
      • Risteli J.
      • Graner E.
      • Salo T.
      • Franco Paes Leme A.
      • Coletta R.D.
      Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.
      4.7 ↓0.03
      THBS4Thrombospondin 4
      • Stenina-Adognravi O.
      • Plow E.
      Thrombospondin-4 in tissue remodeling.
      3.5 ↓<0.001
      TNNTenascin W
      • Chiquet-Ehrismann R.
      • Tucker R.P.
      Connective tissues: signalling by tenascins.
      3.4 ↓<0.0013.9 ↓<0.001
      HS3ST2Heparan sulfate 3-O-sulfotransferase 2
      • Bret C.
      • Hose D.
      • Reme T.
      • Sprynski A.-C.
      • Mahtouk K.
      • Schved J.-F.
      • Quittet P.
      • Rossi J.-F.
      • Goldschmidt H.
      • Klein B.
      Expression of genes encoding for proteins involved in heparan sulphate and chondroitin sulphate chain synthesis and modification in normal and malignant plasma cells.
      3.2 ↓0.0473.6 ↓0.049
      DPTDermatopontin
      • Takeda U.
      • Utani A.
      • Wu J.
      • Adachi E.
      • Koseki H.
      • Taniguchi M.
      • Matsumoto T.
      • Ohashi T.
      • Sato M.
      • Shinkai H.
      Targeted disruption of dermatopontin causes abnormal collagen fibrillogenesis.
      3.2 ↓<0.001
      TNXBTenascin X
      • Chiquet-Ehrismann R.
      • Tucker R.P.
      Connective tissues: signalling by tenascins.
      3.1 ↓<0.0018.1 ↓<0.001
      LOXL4Lysyl oxidase-like 4
      • Huang M.
      • Cai G.
      • Baugh L.M.
      • Liu Z.
      • Smith A.
      • Watson M.
      • Popovich D.
      • Zhang T.
      • Stawski L.S.
      • Trojanowska M.
      • Georgakoudi I.
      • Black III, L.D.
      • Piolo P.A.
      • Whitfield M.L.
      • Garlick J.
      Systemic sclerosis dermal fibroblasts induce cutaneous fibrosis through lysyl oxidase-like 4: new evidence from three-dimensional skin-like tissues.
      3.1 ↓<0.0018.0 ↓<0.001
      ↑, Differentially up-regulated (threefold or greater); ↓, differentially down-regulated (threefold or greater); AB, able bodied; SCI, spinal cord injury.
      Determined by DNA microarray analysis (1 to 10 probes/gene, assayed on triplicate microarrays).
      Data are x-fold differences of mean gene expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15).
      To provide further support for increased collagen degradation in SCI skin, as suggested previously
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Rodriguez G.P.
      • Claus-Walker J.
      • Kent M.C.
      • Garza H.M.
      Collagen metabolite excretion as a predictor of bone- and skin-related complications in spinal cord injury.
      ,
      • Claus-Walker J.
      Clinical implications of the disturbance in calcium and collagen metabolism in quadriplegia.
      and by our gene expression profiling (see above), we analyzed collagenase protein expression and dermal collagen structure by (immuno)histochemistry. Collagenase-3 [matrix metalloproteinase (MMP)-13] was strongly and collagenase-2 (MMP-8) was moderately expressed in SCI skin (Figure 4). In accordance with mRNA levels (Table 3), protein expression of both collagenases was de novo (ie, expression was undetectable in AB control skin). Expression occurred predominantly in the epidermis but also throughout the dermis. In contrast to mRNA levels, no significant MMP-1 protein was detected in SCI or AB skin. Histologic appearance of the collagen fibril structure in SCI skin was strikingly different to that in AB control skin (Figure 5). Throughout the SCI dermis, collagen appeared less dense (Figure 5C versus Figure 5D) and less fibrillar (Figure 5A versus Figure 5B). In the dermal-epidermal junction zone, however, collagen appeared to be enriched compared with control (Figure 5A versus Figure 5B).
      Figure thumbnail gr4
      Figure 4Induction of collagenase protein expression in spinal cord injury (SCI) skin. AD: Expression was analyzed in SCI skin (A and C) and able-bodied control skin (B and D) by immunohistochemistry. A and B: Collagenase-3 [monoclonal mouse anti–matrix metalloproteinase (MMP)-13; 16 μg/mL]. C and D: Collagenase-2 (monoclonal mouse anti–MMP-8; 10 μg/mL). D: Arrows indicate single cells stained for MMP-8, most likely representing neutrophils. Staining with corresponding antibody isotype controls was negative (data not shown). Scale bar = 100 μm (AD). Original magnification, ×10 (AD).
      Figure thumbnail gr5
      Figure 5Structure of the network of collagen fibrils in the dermis. Collagen was visualized in spinal cord injury dermis (A and C) and able-bodied control dermis (B and D) by Masson trichrome staining. Scale bars: 200 μm (A and B); 20 μm (C and D).
      Taken together, loss of neuronal control in chronic SCI appears to impair skin lymphatic function, as documented by a decrease in functionally dilated lymphatic vessels, by molecular signatures of functional inhibitors and connective tissue degradation, and by histopathologic evidence of impaired collagen structure.

      Enhanced Lymphangiogenesis in Chronic SCI Skin

      Immunohistochemical analysis of skin lymphatics revealed that the total number of lymphatic vessels (open, intermediate, and collapsed) was almost doubled in SCI skin compared with AB skin (Figure 6), indicating enhanced lymphangiogenesis.
      Figure thumbnail gr6
      Figure 6Skin lymphangiogenesis was stimulated in chronic spinal cord injury (SCI). Total lymphatic vessels were identified in tissue sections of SCI versus normal skin using lymphatic endothelial hyaluronan receptor-1 immunostaining and counted following image analysis (B). Data are mean values + SEM. n = 11 tissue sections of SCI skin; n = 9 tissue sections of normal skin. ∗∗P < 0.01 by the Kruskal-Wallis test. AB, able bodied.
      To verify the above morphologic observation at the molecular level, lymphangiogenesis was analyzed by differential gene expression in SCI versus AB control skin. We identified an SCI-specific molecular signature of seven lymphangiogenic factors
      • Morfoisse F.
      • Tatin F.
      • Chaput B.
      • Therville N.
      • Vaysse C.
      • Métivier R.
      • Malloizel-Delaunay J.
      • Pujol F.
      • Godet A.-C.
      • De Toni F.
      • Boudou F.
      • Grenier K.
      • Dubuc D.
      • Lacazette E.
      • Prats A.-C.
      • Guillermet-Guibert J.
      • Lenfant F.
      • Garmy-Susini B.
      Lymphatic vasculature requires estrogen receptor-alpha signaling to protect from lymphedema.
      ,
      • Wu M.
      • Du Y.
      • Liu Y.
      • He Y.
      • Yang C.
      • Wang W.
      • Gao F.
      Low molecular weight hyaluronan induces lymphangiogenesis through LYVE-1-mediated signaling pathways.
      • Cueni L.N.
      • Detmar M.
      New insights into the molecular control of the lymphatic vascular system and its role in disease.
      • Jung Y.J.
      • Lee A.S.
      • Nguyen-Thanh T.
      • Kang K.P.
      • Lee S.
      • Jang K.Y.
      • Kim K.Y.
      • Kim S.H.
      • Park S.W.
      • Kim W.
      Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via toll-like receptor 4-dependent signal pathway.
      • Han L.
      • Zhang M.
      • Wang M.
      • Jia J.
      • Zhao M.
      • Fan Y.
      • Li X.
      High mobility group box-1 promotes inflammation-induced lymphangiogenesis via toll-like receptor 4-dependent signalling pathway.
      • Clavin N.W.
      • Avraham T.
      • Fernandez J.
      • Daluvoy S.V.
      • Soares M.A.
      • Chaudhry A.
      • Mehrara B.J.
      TGF-β1 is a negative regulator of lymphatic regeneration during wound repair.
      • Bauer J.
      • Rothley M.
      • Schmaus A.
      • Quagliata L.
      • Ehret M.
      • Biskup M.
      • Orian-Rousseau V.
      • Jackson D.G.
      • Pettis R.J.
      • Harvey A.
      • Bräse S.
      • Thiele W.
      • Sleeman J.P.
      : TGFβ counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.
      • Gao N.
      • Liu X.
      • Wu J.
      • Li J.
      • Dong C.
      • Wu X.
      • Xiao X.
      • Yu F.-S.X.
      CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13.
      • Iriyama S.
      • Matsunaga Y.
      • Amano S.
      Heparanase activation induces epidermal hyperplasia, angiogenesis, lymphangiogenesis and wrinkles.
      • Dicke N.
      • Pielensticker N.
      • Degen J.
      • Hecker J.
      • Tress O.
      • Bald T.
      • Gellhaus A.
      • Winterhager E.
      • Willecke K.
      Peripheral lymphangiogenesis in mice depends on ectodermal connexin-26 (Gjb2).
      (Table 4). Differential regulation of four of the seven factors (LYVE1, ESR, TLR4, and TGF-β1) results in impairment of both lymphatic function (Table 2) as well as lymphangiogenesis (Table 3). Differential expression of the remaining three signature genes (MMP13, HPSE2, and GJB2) stimulates specifically lymphangiogenesis, with MMP13 being most prominently up-regulated in SCI skin (13.3-fold compared with AB skin). Differential regulation of six of the seven signature markers was further enhanced (P < 0.001) in pressure ulcers of SCI individuals (LYVE1, ESR, TGF-β1, MMP13, HPSE2, and GJB2) (Table 4).
      Table 4SCI-Specific Molecular Signature of Lymphangiogenesis in the Skin
      Gene/protein symbolGene/protein nameDifferential gene expression
      Determined by DNA microarray analysis (one to nine probes/gene, assayed on triplicate microarrays).
      /growth factor activity
      Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al8).
      (x-fold)
      SCI versus ABP valuePressure ulcer versus ABP value
      Inhibition of lymphangiogenesis
      LYVE1Lymphatic vessel endothelial hyaluronan receptor-1
      • Wu M.
      • Du Y.
      • Liu Y.
      • He Y.
      • Yang C.
      • Wang W.
      • Gao F.
      Low molecular weight hyaluronan induces lymphangiogenesis through LYVE-1-mediated signaling pathways.
      5.4 ↓
      Data are x-fold differences of mean gene expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15).
      <0.0018.5 ↓<0.001
      ESREstrogen receptor
      • Morfoisse F.
      • Tatin F.
      • Chaput B.
      • Therville N.
      • Vaysse C.
      • Métivier R.
      • Malloizel-Delaunay J.
      • Pujol F.
      • Godet A.-C.
      • De Toni F.
      • Boudou F.
      • Grenier K.
      • Dubuc D.
      • Lacazette E.
      • Prats A.-C.
      • Guillermet-Guibert J.
      • Lenfant F.
      • Garmy-Susini B.
      Lymphatic vasculature requires estrogen receptor-alpha signaling to protect from lymphedema.
      4.3 ↓<0.0017.7 ↓<0.001
      CCL21Chemokine (C-C motif) ligand 21
      • Cueni L.N.
      • Detmar M.
      New insights into the molecular control of the lymphatic vascular system and its role in disease.
      3.7 ↓<0.0013.6 ↓<0.001
      TLR4Toll-like receptor 4
      • Jung Y.J.
      • Lee A.S.
      • Nguyen-Thanh T.
      • Kang K.P.
      • Lee S.
      • Jang K.Y.
      • Kim K.Y.
      • Kim S.H.
      • Park S.W.
      • Kim W.
      Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via toll-like receptor 4-dependent signal pathway.
      ,
      • Han L.
      • Zhang M.
      • Wang M.
      • Jia J.
      • Zhao M.
      • Fan Y.
      • Li X.
      High mobility group box-1 promotes inflammation-induced lymphangiogenesis via toll-like receptor 4-dependent signalling pathway.
      3.0 ↓0.0023.2 ↓0.016
       TGF-β1Transforming growth factor-β1
      • Clavin N.W.
      • Avraham T.
      • Fernandez J.
      • Daluvoy S.V.
      • Soares M.A.
      • Chaudhry A.
      • Mehrara B.J.
      TGF-β1 is a negative regulator of lymphatic regeneration during wound repair.
      ,
      • Bauer J.
      • Rothley M.
      • Schmaus A.
      • Quagliata L.
      • Ehret M.
      • Biskup M.
      • Orian-Rousseau V.
      • Jackson D.G.
      • Pettis R.J.
      • Harvey A.
      • Bräse S.
      • Thiele W.
      • Sleeman J.P.
      : TGFβ counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.
      2.2 ↑
      Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al8).
      <0.0013.3 ↑<0.001
      Stimulation of lymphangiogenesis
      MMP13Matrix metalloproteinase-13
      • Gao N.
      • Liu X.
      • Wu J.
      • Li J.
      • Dong C.
      • Wu X.
      • Xiao X.
      • Yu F.-S.X.
      CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13.
      >11 ↑<0.001>47 ↑<0.001
      HPSE2Heparanase 2 (heparanase 1 inhibitor)
      • Pinhal M.A.S.
      • Melo C.M.
      • Nader H.B.
      The good and bad sides of heparanase-1 and heparanase 2.
      ,
      • Iriyama S.
      • Matsunaga Y.
      • Amano S.
      Heparanase activation induces epidermal hyperplasia, angiogenesis, lymphangiogenesis and wrinkles.
      3.5 ↓<0.00110.1 ↓<0.001
      GJB2Gap junction protein β 2 (connexin-26)
      • Dicke N.
      • Pielensticker N.
      • Degen J.
      • Hecker J.
      • Tress O.
      • Bald T.
      • Gellhaus A.
      • Winterhager E.
      • Willecke K.
      Peripheral lymphangiogenesis in mice depends on ectodermal connexin-26 (Gjb2).
      3.1 ↑<0.00111.3 ↑<0.001
      ↓, Differentially down-regulated (threefold or greater); ↑, differentially up-regulated (threefold or greater); AB, able bodied; SCI, spinal cord injury.
      Determined by DNA microarray analysis (one to nine probes/gene, assayed on triplicate microarrays).
      Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      ).
      Data are x-fold differences of mean gene expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15).
      Taken together, increased lymphatic vessel numbers and strong activation of lymphangiogenic stimulators, such as MMP-13, documented enhanced lymphangiogenesis in chronic SCI skin, possibly in response to impaired lymphatic functionality.

      Discussion

      Chronic SCI is associated with prolonged dysfunction of the cardiovascular system below the level of injury, due to interruption of spinal motor pathways and impairment of the sympathetic nervous system.
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Claus-Walker J.
      • Halstead L.S.
      Metabolic and endocrine changes in spinal cord injury: II (section 1): partial decentralization of the autonomic nervous system.
      The consequences in affected body areas are loss of vascular tone, vasodilation, and venous vascular dysfunction.
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Wecht J.M.
      • de Meersman R.E.
      • Weir J.P.
      • Bauman W.A.
      • Grimm D.R.
      Effects of autonomic disruption and inactivity on venous vascular function.
      We have reported previously that this results in increased peripheral extravasation of plasma components from the microvasculature into the dermis, followed by activation of platelets and cytokines as well as wound-healing type cellular and molecular reactions, inappropriate to noninjured skin.
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      Whether loss of neuronal control in chronic SCI also affects the skin lymphatic system has not yet been studied so far.
      The lymphatic system regulates fluid homeostasis, absorption of gastrointestinal lipids, and trafficking of immune cells.
      • Aspelund A.
      • Robciuc M.R.
      • Karaman S.
      • Makinen T.
      • Alitalo K.
      Lymphatic system in cardiovascular medicine.
      Skin lymphatic vascular function critically depends on the composition, geometry, and integrity of the connective tissue.
      • Skobe M.
      • Detmar M.
      Structure, function, and molecular control of the skin lymphatic system.
      In a functional state, the lumina of lymphatic microvessels is dilated by the increased tissue fluid pressure stretching the connective tissue fibers to which the lymphatic endothelial cells are firmly attached.
      • Skobe M.
      • Detmar M.
      Structure, function, and molecular control of the skin lymphatic system.
      In SCI skin, however, the ratio of functionally dilated/collapsed lymphatic vessels was 10-fold reduced, and the average vessel lumen area was almost halved, compared with AB control skin. This suggested that lymphatic function in SCI skin was impaired, which was unexpected considering the presumably high tissue fluid pressure due to increased microvascular permeability,
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Wecht J.M.
      • de Meersman R.E.
      • Weir J.P.
      • Bauman W.A.
      • Grimm D.R.
      Effects of autonomic disruption and inactivity on venous vascular function.
      plasma leakage,
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      and predisposition to edema formation.
      • Guihan M.
      • Bates-Jenson B.M.
      • Chun S.
      • Parachuri R.
      • Chin A.S.
      • McCreath H.
      Assessing the feasibility of subepidermal moisture to predict erythema and stage 1 pressure ulcers in persons with spinal cord injury: a pilot study.
      One plausible explanation for the collapse of lymphatic vessels in SCI skin might be reduced mechanical integrity of the dermis. Structural impairment of SCI connective tissue has been suggested previously, based on increased collagen degradation in SCI skin and bone,
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Rodriguez G.P.
      • Claus-Walker J.
      • Kent M.C.
      • Garza H.M.
      Collagen metabolite excretion as a predictor of bone- and skin-related complications in spinal cord injury.
      enhanced excretion of collagen and glycosaminoglycan fragments in SCI individuals,
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Rodriguez G.P.
      • Claus-Walker J.
      • Kent M.C.
      • Garza H.M.
      Collagen metabolite excretion as a predictor of bone- and skin-related complications in spinal cord injury.
      ,
      • Claus-Walker J.
      Clinical implications of the disturbance in calcium and collagen metabolism in quadriplegia.
      ,
      • Pilonchery G.
      • Minaire P.
      • Milan J.J.
      • Revol A.
      Urinary elimination of glycosaminoglycans during the immobilization osteoporosis of spinal cord injury patients.
      and diminished collagen cross-linking by lysyl hydroxylation,
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Rodriguez G.P.
      • Claus-Walker J.
      Biochemical changes in skin composition in spinal cord injury: a possible contribution to decubitus ulcers.
      altogether apparently diminishing the quality of the collagen structure.
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Rodriguez G.P.
      • Claus-Walker J.
      Biochemical changes in skin composition in spinal cord injury: a possible contribution to decubitus ulcers.
      Increased turnover and impaired structure of SCI dermal collagen is, at least in part, consistent with our observations that gene and protein expression of two major collagen-degrading enzymes, MMP-13 (collagenase-3) and MMP-8 (collagenase-2), was significantly induced de novo in SCI skin. Gene expression of the collagen cross-linking enzyme, lysyl oxidase-like 4 (LOXL4), was down-regulated, potentially further affecting structural integrity of collagen. In addition, heparanase-2 (HPSE2), an inhibitor of the glycosaminoglycan-degrading enzyme, heparanase-1, was down-regulated, which is consistent with an increased turnover of glycosaminoglycans. Finally, the ratio of thicker collagen type I fibrils/thinner type III fibrils has been found to be decreased in SCI dermis,
      • Rodriguez G.P.
      • Claus-Walker J.
      Biochemical changes in skin composition in spinal cord injury: a possible contribution to decubitus ulcers.
      also reducing collagen integrity and possibly explaining the less dense and less fibrillar appearance of the collagen network observed herein. Thus, connective tissue in SCI dermis might be affected by a shift of homeostasis toward enhanced turnover and reduced integrity, potentially leading to the observed collapse of lymphatic microvessels.
      The functional state of lymphatics, however, cannot be determined solely based on their morphology.
      • Skobe M.
      • Detmar M.
      Structure, function, and molecular control of the skin lymphatic system.
      For example, proper fluid transport, particularly through the larger lymphatics in the deeper layer of the dermis, also critically depends on the contractile properties of lymphatic muscle cells.
      • Wang W.
      • Nepiyushchikh Z.V.
      • Zawieja D.C.
      • Chakraborty S.
      • Zawieja S.D.
      • Gashev A.A.
      • Davis M.J.
      • Muthuchamy M.
      Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.
      We defined an SCI-specific signature of eight molecular markers, which are inhibitory with regard to lymphatic function (CACNG1, ACTA1, LYVE1, ESR, PF4, MYLK2, TLR4, and TGF-β). Three of these markers (CACNG1, ACTA1, and MYLK2) are critical for smooth muscle cell contraction. The most striking lymphatic inhibitory feature of SCI skin was a >34-fold down-regulation of the voltage-dependent calcium channel γ subunit 1 (CACNG1). Because lymphatic contractility requires calcium influx into smooth muscle cells,
      • Imtiaz M.S.
      • Zhao J.
      • Hosaka K.
      • von der Weid P.-Y.
      • Crowe M.
      • van Helden D.F.
      Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.
      the complete abolishment of muscle cell–specific CACNG1 expression in SCI skin (<1.4-fold background level) most likely has a significant impact on lymphatic function.
      Although lymphatic function appeared to be impaired in SCI skin, lymphangiogenesis was almost doubled, as determined morphologically by analyzing total vessel numbers. Enhanced lymphangiogenesis was supported, at the molecular level, by SCI-specific differential expression of a seven-marker signature of lymphangiogenesis. Although the anti-lymphangiogenic part of the signature (LYVE1, ESR, TLR4, and TGF-β) overlaps with the inhibitory functional lymphatic signature, the prolymphangiogenic part (MMP13, HPSE2, and GJB2) partially overlaps with the connective tissue signature. In particular, the dominant de novo expression of the lymphangiogenic factor, MMP13,
      • Gao N.
      • Liu X.
      • Wu J.
      • Li J.
      • Dong C.
      • Wu X.
      • Xiao X.
      • Yu F.-S.X.
      CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13.
      is consistent with a stimulation of lymphangiogenesis in SCI skin. One possible interpretation of these data is that enhanced lymphangiogenesis might represent a skin response to counteract functional lymphatic defects and restore tissue fluid homeostasis. However, because the proportion of fully dilated vessels was fivefold decreased in SCI skin, a doubling in total vessel number is most likely insufficient to compensate for the impairment of lymphatic function.
      In this study, we have extended our SCI-skin–specific gene expression profiling. Together with our previous study on venous dysfunction and inadequate wound-healing reactions in SCI skin,
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      we have defined four distinct but partially overlapping molecular signatures (relating to wound-healing reactions, lymphatic function, connective tissue integrity, and lymphangiogenesis) comprising 26 differentially regulated molecular markers. Intriguingly, differential regulation of 15 of these markers was further enhanced in pressure ulcers, corroborating our previous hypothesis that intact, noninjured SCI skin is pre-activated and has already acquired part of the molecular properties of a chronic wound.
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      However, in addition to the above 26 molecular markers, several additional genes were found to be differentially regulated in SCI skin. It must be assumed, therefore, that other skin functions and properties might be affected in a similar manner by the loss of neuronal control, requiring future, more comprehensive molecular and functional analyses.
      In conclusion, venous and lymphatic skin vasculature and, consequently, tissue fluid homeostasis as well as connective tissue integrity and wound healing appear to be affected, directly or indirectly, by the loss of neuronal control in chronic SCI. The latter comprises, apart from motor and sensory dysfunctions, also the impairment of autonomic functions of the nervous system.
      • Karlsson A.K.
      Autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and signs.
      Although the sympathetic neuronal control of blood flow in human skin has been extensively studied,
      • Johnson J.M.
      • Minson C.T.
      • Kellog Jr., D.L.
      Cutaneous vasodilator and vasoconstrictor mechanisms in temperature regulation.
      control of lymphatic function in vivo by the autonomic nervous system has only recently been shown using an animal model.
      • Bachmann S.B.
      • Gsponer D.
      • Montoya-Zegarra J.A.
      • Schneider M.
      • Scholkmann F.
      • Tacconi C.
      • Noerrelykke S.F.
      • Proulx S.T.
      • Detmar M.
      A distinct role of the autonomic nervous system in modulating the function of lymphatic vessels under physiological and tumor-draining conditions.
      To our knowledge, our studies provide first evidence that, in human skin, the lymphatic vasculature is under neuronal control. However, because our study comprised a limited number of patients, results require confirmation in an independent patient cohort. Nevertheless, our findings may help to explain the frequently observed disturbance of tissue fluid homeostasis observed in several tissues of SCI individuals, including skin.
      • Karlsson A.K.
      Autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and signs.
      ,
      • Rappl L.
      Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.
      ,
      • Brunner G.
      • Roux M.
      • Böhm V.
      • Meiners T.
      Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.
      ,
      • Guihan M.
      • Bates-Jenson B.M.
      • Chun S.
      • Parachuri R.
      • Chin A.S.
      • McCreath H.
      Assessing the feasibility of subepidermal moisture to predict erythema and stage 1 pressure ulcers in persons with spinal cord injury: a pilot study.
      Because the skin lymphatic system is not only involved in the regulation of tissue fluid homeostasis but is also essential for pathogen recognition and cellular responses in skin immunity,
      • Petrova T.V.
      • Koh G.Y.
      Organ-specific lymphatic vasculature: from development to pathophysiology.
      ,
      • Lund A.W.
      • Medler T.R.
      • Leachman S.A.
      • Coussens L.M.
      Lymphatic vessels, inflammation, and immunity in skin cancer.
      our results may have important implications with regard to the predisposition to and the treatment of wound-healing disorders of SCI individuals. Thus, molecular signatures defining the homeostatic balance of connective tissue turnover and lymphatic dysfunctionality in SCI skin, as identified herein, may have translational potential for diagnostic stratification and/or prognosis of SCI individuals with regard to the risk of pressure ulcer development. Furthermore, stimulation of contractile lymphatic function by specific receptor agonists
      • Bachmann S.B.
      • Gsponer D.
      • Montoya-Zegarra J.A.
      • Schneider M.
      • Scholkmann F.
      • Tacconi C.
      • Noerrelykke S.F.
      • Proulx S.T.
      • Detmar M.
      A distinct role of the autonomic nervous system in modulating the function of lymphatic vessels under physiological and tumor-draining conditions.
      or inhibition of inappropriate connective tissue degradation by specific MMP inhibitors
      • Fields G.B.
      The rebirth of matrix metalloproteinase inhibitors: moving beyond the dogma.
      might be evaluated, in future clinical studies, for its efficacy in preventing and/or treating pressure ulcers of individuals affected by SCI.

      Acknowledgments

      We thank Maryla Brode and Tamara Berger for technical assistance; and Nicola Tidow for help with bioinformatics.

      Author Contributions

      G.B. and T.M.: study concept and design; T.M. and V.B.: tissue samples and clinical data; M.S.R. and G.B.: development of method, experiments, statistical analysis, and writing of the article; M.S.R., T.F., N.B.-S., and M.B.: (immuno)histochemistry; G.B., T.M., and M.S.R.: analysis and interpretation of the data. All authors read and approved the final article. G.B. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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