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

2022; Elsevier BV; Volume: 192; Issue: 10 Linguagem: Inglês

10.1016/j.ajpath.2022.06.012

ISSN

1525-2191

Autores

Georg Brunner, Meike S. Roux, Thomas Falk, Martina Bresch, Volker Böhm, Norbert Blödorn‐Schlicht, Thomas Meiners,

Tópico(s)

Pressure Ulcer Prevention and Management

Resumo

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 decreased 10-fold 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) 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 decreased 10-fold 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.1Karlsson A.K. Autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and signs.Prog Brain Res. 2006; 152: 1-8Crossref PubMed Scopus (118) Google Scholar 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.2Rappl L. Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.Int Wound J. 2008; 5: 435-444Crossref PubMed Scopus (41) Google Scholar,3Ditunno Jr., J.F. Formal C.S. Chronic spinal cord injury.N Engl J Med. 1994; 330: 550-556Crossref PubMed Scopus (136) Google Scholar One of the peripheral tissues commonly affected is the skin, resulting in frequently occurring and recurring pressure ulcers. It is estimated that in individuals with SCI, the prevalence of pressure ulcers is 17%,4Whiteneck 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.Paraplegia. 1992; 3: 617-630Google Scholar and the lifetime risk of developing an ulcer ranges from 62% to 86%.5Salzberg 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.Adv Wound Care. 1998; 11: 237-246PubMed Google Scholar,6Sumiya 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.Spinal Cord. 1997; 35: 595-598Crossref PubMed Scopus (44) Google Scholar Apart from sociodemographic, neurologic, behavioral, or medical care–related risk factors for pressure ulcer development in SCI,7Marin 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.Spinal Cord. 2013; 51: 522-527Crossref PubMed Scopus (63) Google Scholar intrinsic molecular and cellular characteristics of chronic SCI skin, associated with aberrant nerve activity, are only beginning to be characterized in more detail.2Rappl L. Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.Int Wound J. 2008; 5: 435-444Crossref PubMed Scopus (41) Google Scholar Molecular and cellular homeostasis is disturbed in chronic SCI skin.8Brunner G. Roux M. Böhm V. Meiners T. Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.Int Wound J. 2021; 18: 728-737Crossref PubMed Scopus (3) Google Scholar This is associated with increased microvascular permeability and extravasation of plasma components, such as platelets, into the dermal tissue. The consequences are 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.9Stover S.L. Omura E.F. Buell A.B. Clinical skin thickening following spinal cord injury studied by histopathology.J Am Paraplegia Soc. 1994; 17: 44-49Crossref PubMed Scopus (13) Google Scholar,10Guihan 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.J Spinal Cord Med. 2012; 35: 46-52Crossref PubMed Scopus (50) Google Scholar 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.11Alitalo K. The lymphatic vasculature in disease.Nat Med. 2011; 17: 1371-1380Crossref PubMed Scopus (701) Google Scholar 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. To identify SCI-specific alterations in the morphology of the lymphatic system, immunohistochemistry combined with image analysis was used to quantify number and functionality of lymph vessels in SCI skin from areas afflicted by loss of neuronal control in comparison to skin from able-bodied (AB) individuals. In addition, SCI-specific lymphatic gene regulation was identified at the molecular level by whole-genome gene expression analysis. This was confirmed by (immuno)histochemistry specific for collagenases and collagen. 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 as grade A or B [ie, 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%), coccyx (13%), sacrum (9%), or from undefined body locations (8%). 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 CharacteristicsCharacteristicsSCI 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.8Brunner G. Roux M. Böhm V. Meiners T. Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.Int Wound J. 2021; 18: 728-737Crossref PubMed Scopus (3) Google Scholar Data are given as number (percentage) or mean (SD).—, not applicable; ASIA, American Spinal Injury Association; SCI, spinal cord injury. Open table in a new tab The patient cohort is identical to the cohort analyzed by Brunner et al.8Brunner G. Roux M. Böhm V. Meiners T. Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.Int Wound J. 2021; 18: 728-737Crossref PubMed Scopus (3) Google Scholar Data are given as number (percentage) or mean (SD). —, not applicable; 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 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 was performed as described previously.8Brunner G. Roux M. Böhm V. Meiners T. Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.Int Wound J. 2021; 18: 728-737Crossref PubMed Scopus (3) Google Scholar 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 reverse transcription–in vitro transcription. 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. 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). 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 (version 12.0.5; 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. 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. 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 and B).12Podgrabinska S. Braun P. Velasco P. Kloos B. Pepper M.S. Jackson D.G. Skobe M. Molecular characterization of lymphatic endothelial cells.Proc Natl Acad Sci U S A. 2002; 99: 16069-16074Crossref PubMed Scopus (396) Google Scholar The shape of the lumen of lymphatic vessels varied, and three categories of vessels (open, intermediate, or collapsed) were defined on the basis of the appearance of their lumen (Figure 1C). To quantify lymphatic vessel number and lumen area, an image analysis procedure was developed, as 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. 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. To support these morphologic observations, differential gene expression analysis of functional lymphatic markers in SCI (n = 17) versus AB (n = 16) skin was performed. As a reference to wound healing, the analysis also comprised the wound edge of pressure ulcers (n = 15). An SCI-specific molecular signature of nine known functional lymphatic markers [CACNG1, ACTA1, LYVE1, ESR1, PF4, CCL21, MYLK2, TLR4, and transforming growth factor (TGF)-β1] was identified, SCI-specific differential regulation of which may result in impaired lymphatic contractility and immune cell trafficking (Table 2),13Tsai M.-K. Lai C.-H. Chen L.-M. Jong G.-P. Calcium channel blocker-related chylous ascites: a systematic review and meta-analysis.J Clin Med. 2019; 8: 466Crossref PubMed Scopus (12) Google Scholar, 14Imtiaz 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.Biophys J. 2007; 92: 3843-3861Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 15Muthuchamy M. Gashev A. Boswell N. Dawson N. Zawieja D. Molecular and functional analyses of the contractile apparatus in lymphatic muscle.FASEB J. 2003; 17: 920-922Crossref PubMed Scopus (133) Google Scholar, 16Jackson D.G. Hyaluronan in the lymphatics: the key role of the hyaluronan receptor LYVE-1 in leucocyte trafficking.Matrix Biol. 2019; 78-79: 219-235Crossref PubMed Scopus (69) Google Scholar, 17Morfoisse 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.Arterioscler Thromb Vasc Biol. 2018; 38: 1346-1357Crossref PubMed Scopus (36) Google Scholar, 18Ma 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.JCI Insight. 2020; 5: e135109Crossref PubMed Scopus (24) Google Scholar, 19Furtado 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.Proc Natl Acad Sci U S A. 2007; 104: 5026-5031Crossref PubMed Scopus (88) Google Scholar, 20Wang 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.Am J Physiol Heart Circ Physiol. 2009; 29: H726-H734Crossref Scopus (55) Google Scholar, 21Zampell 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.Am J Physiol Cell Physiol. 2012; 302: C709-C719Crossref PubMed Scopus (46) Google Scholar, 22Avraham 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.Am J Physiol Cell Physiol. 2010; 299: C589-C605Crossref PubMed Scopus (109) Google Scholar supporting the morphologic findings. Differential regulation of four of the nine signature markers (ACTA1, LYVE1, ESR1, and TGF-β1) was further enhanced (P < 0.001) in pressure ulcers as compared to SCI skin.Table 2SCI-Specific Molecular Signature of Lymphatic Function in the SkinGene/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 vs ABP valuePressure ulcer vs ABP valueCACNG1Calcium channel γ subunit13Tsai M.-K. Lai C.-H. Chen L.-M. Jong G.-P. Calcium channel blocker-related chylous ascites: a systematic review and meta-analysis.J Clin Med. 2019; 8: 466Crossref PubMed Scopus (12) Google Scholar,14Imtiaz 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.Biophys J. 2007; 92: 3843-3861Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar>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 ↓<0.001ACTA1Actin α 115Muthuchamy M. Gashev A. Boswell N. Dawson N. Zawieja D. Molecular and functional analyses of the contractile apparatus in lymphatic muscle.FASEB J. 2003; 17: 920-922Crossref PubMed Scopus (133) Google Scholar8.7 ↓<0.00178.7 ↓<0.001LYVE1Lymphatic vessel endothelial hyaluronan receptor-116Jackson D.G. Hyaluronan in the lymphatics: the key role of the hyaluronan receptor LYVE-1 in leucocyte trafficking.Matrix Biol. 2019; 78-79: 219-235Crossref PubMed Scopus (69) Google Scholar5.4 ↓<0.0018.5 ↓<0.001ESR1Estrogen receptor 117Morfoisse 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.Arterioscler Thromb Vasc Biol. 2018; 38: 1346-1357Crossref PubMed Scopus (36) Google Scholar4.3 ↓<0.0017.7 ↓<0.001PF4Platelet factor 418Ma 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.JCI Insight. 2020; 5: e135109Crossref PubMed Scopus (24) Google Scholar4.1 ↑<0.001—CCL21Chemokine (C-C motif) ligand 2119Furtado 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.Proc Natl Acad Sci U S A. 2007; 104: 5026-5031Crossref PubMed Scopus (88) Google Scholar3.7 ↓<0.0013.6 ↓<0.001MYLK2Myosin light chain kinase 220Wang 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.Am J Physiol Heart Circ Physiol. 2009; 29: H726-H734Crossref Scopus (55) Google Scholar3.6 ↓<0.001—TLR4Toll-like receptor 421Zampell 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.Am J Physiol Cell Physiol. 2012; 302: C709-C719Crossref PubMed Scopus (46) Google Scholar3.0 ↓0.0023.2 ↓0.016TGF-β1Transforming growth factor-β122Avraham 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.Am J Physiol Cell Physiol. 2010; 299: C589-C605Crossref PubMed Scopus (109) Google Scholar2.2 ↑†Determined by plasminogen activator inhibitor-1/luciferase bioassay (data taken from Brunner et al8).<0.0013.3 ↑<0.001—, not significant; ↓, 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 al8Brunner G. Roux M. Böhm V. Meiners T. Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.Int Wound J. 2021; 18: 728-737Crossref PubMed Scopus (3) Google Scholar).‡ Data are x-fold differences of mean expression in the respective groups (SCI, n = 17; AB control, n = 16; and pressure ulcer, n = 15). Open table in a new tab —, not significant; ↓, differentially down-regulated (threefold or greater); ↑, differentially up-regulated (threefold or greater); AB, able-bodied; SCI, spinal cord injury. Opening of lymphatic vessels and proper skin lymphatic function are critically dependent on the mechanical integrity of the dermal connective tissue.23Skobe M. Detmar M. Structure, function, and molecular control of the skin lymphatic system.J Invest Dermatol Symp Proc. 2000; 5: 14-19Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Previous findings indicating increased collagen degradation in skin and bone of SCI patients2Rappl L. 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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).26Ala-aho R. Kähäri V.-M. Collagenases in cancer.Biochimie. 2005; 87: 273-286Crossref PubMed Scopus (289) Google Scholar, 27Kryza 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.Sci Rep. 2018; 8: e6331Crossref PubMed Scopus (13) Google Scholar, 28Fischer J. Meyer-Hoffert U. 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Systemic sclerosis dermal fibroblasts induce cutaneous fibrosis through lysyl oxidase-like 4: new evidence from three-dimensional skin-like tissues.Arthritis Rheumatol. 2020; 72: 791-801Crossref PubMed Scopus (21) Google Scholar Differential expression of 6 of the 13 signature genes was further enhanced (P < 0.001) in pressure ulcers as compared to SCI skin (MMP1, MMP13, KLK12, HPSE2, TNXB, and LOXL4) (Table 3).Table 3SCI-Specific Molecular Signature of Skin Connective TissueGene/protein symbolGene/protein nameDifferential gene expression∗Determined by DNA microarray analysis (1 to 10 probes/gene, assayed on triplicate microarrays). (x-fold)SCI vs ABP valuePressure ulcer vs ABP valueConnective tissue degradation MMP1Matrix metalloproteinase-1 (collagenase-1)26Ala-aho R. Kähäri V.-M. Collagenases in cancer.Biochimie. 2005; 87: 273-286Crossref PubMed Scopus (289) Google Scholar16.3 ↑†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 ↑ 11 ↑<0.00121.7 ↑<0.001 KLK12Kallikrein-related peptidase 1227Kryza 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.Sci Rep. 2018; 8: e6331Crossref PubMed Scopus (13) Google Scholar4.0 ↑<0.00124.3 ↑<0.001 SPINK2Serine proteinase inhibitor Kazal type 228Fischer J. Meyer-Hoffert U. Regulation of kallikrein-related peptidases in the skin – from physiology to diseases to therapeutic options.Thromb Haemost. 2013; 110: 442-449Crossref PubMed Scopus (34) Google Scholar4.0 ↓<0.0015.2 ↓<0.001 M

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