Neutrophil Elastase Damages the Pulmonary Endothelial Glycocalyx in Lipopolysaccharide-Induced Experimental Endotoxemia
2019; Elsevier BV; Volume: 189; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2019.05.002
ISSN1525-2191
AutoresKodai Suzuki, Hideshi Okada, Genzou Takemura, Chihiro Takada, Ayumi Kuroda, Hirohisa Yano, Ryogen Zaikokuji, Kentaro Morishita, Hiroyuki Tomita, Kazumasa Oda, Saori Matsuo, Akihiro Uchida, Tetsuya Fukuta, So Sampei, Nagisa Miyazaki, Tomonori Kawaguchi, Takatomo Watanabe, Takahiro Yoshida, Hiroaki Ushikoshi, Shozo Yoshida, Yoichi Maekawa, Shinji Ogura,
Tópico(s)Traumatic Brain Injury and Neurovascular Disturbances
ResumoNeutrophil elastase (NE) is necessary for effective sterilization of phagocytosed bacterial and fungal pathogens; however, NE increases alveolocapillary permeability and induces proinflammatory cytokine production in sepsis-induced acute respiratory distress syndrome. Under septic conditions, the pulmonary endothelial glycocalyx covering on the healthy endothelium surface is injured, but the contribution of NE to this injury remains unknown. Our aim was to examine whether NE-induced pulmonary endothelial injury is associated with endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 9- to 12-week–old granulocyte colony-stimulating factor knockout (G-CSFKO) mice, which harbor few neutrophils, and littermate control mice; in a second assay, mice were injected with the NE-inhibitor sivelestat (0.2 mg/kg) at 3, 6, 9, and 12 hours after LPS administration. Subsequently, vascular endothelial injury was evaluated through ultrastructural analysis. At 48 hours after LPS injection, survival rate was more than threefold higher among G-CSFKO than control mice, and degradation of both thrombomodulin and syndecan-1 was markedly attenuated in G-CSFKO compared with control mice. Ultrastructural analysis revealed attenuated vascular endothelial injury and clear preservation of the endothelial glycocalyx in G-CSFKO mice. Moreover, after LPS exposure, survival rate was approximately ninefold higher among sivelestat-injected mice than control mice, and sivelestat treatment potently preserved vascular endothelial structures and the endothelial glycocalyx. In conclusion, NE is associated with pulmonary endothelial injury under LPS-induced endotoxemic conditions. Neutrophil elastase (NE) is necessary for effective sterilization of phagocytosed bacterial and fungal pathogens; however, NE increases alveolocapillary permeability and induces proinflammatory cytokine production in sepsis-induced acute respiratory distress syndrome. Under septic conditions, the pulmonary endothelial glycocalyx covering on the healthy endothelium surface is injured, but the contribution of NE to this injury remains unknown. Our aim was to examine whether NE-induced pulmonary endothelial injury is associated with endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 9- to 12-week–old granulocyte colony-stimulating factor knockout (G-CSFKO) mice, which harbor few neutrophils, and littermate control mice; in a second assay, mice were injected with the NE-inhibitor sivelestat (0.2 mg/kg) at 3, 6, 9, and 12 hours after LPS administration. Subsequently, vascular endothelial injury was evaluated through ultrastructural analysis. At 48 hours after LPS injection, survival rate was more than threefold higher among G-CSFKO than control mice, and degradation of both thrombomodulin and syndecan-1 was markedly attenuated in G-CSFKO compared with control mice. Ultrastructural analysis revealed attenuated vascular endothelial injury and clear preservation of the endothelial glycocalyx in G-CSFKO mice. Moreover, after LPS exposure, survival rate was approximately ninefold higher among sivelestat-injected mice than control mice, and sivelestat treatment potently preserved vascular endothelial structures and the endothelial glycocalyx. In conclusion, NE is associated with pulmonary endothelial injury under LPS-induced endotoxemic conditions. Sepsis is defined as serious organ dysfunction caused by an uncontrollable host response to infection.1Singer M. Deutschman C.S. Seymour C.W. Shankar-Hari M. Annane D. Bauer M. Bellomo R. Bernard G.R. Chiche J.D. Coopersmith C.M. Hotchkiss R.S. Levy M.M. Marshall J.C. Martin G.S. Opal S.M. Rubenfeld G.D. van der Poll T. Vincent J.L. Angus D.C. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).JAMA. 2016; 315: 801-810Crossref PubMed Scopus (11873) Google Scholar Among the multiple organ dysfunctions that occur, the development of acute respiratory distress syndrome (ARDS) is associated with short- and long-term morbidity, mortality, prolonged hospitalization, and high costs.2Iscimen R. Cartin-Ceba R. Yilmaz M. Khan H. Hubmayr R.D. Afessa B. Gajic O. Risk factors for the development of acute lung injury in patients with septic shock: an observational cohort study.Crit Care Med. 2008; 36: 1518-1522Crossref PubMed Scopus (178) Google Scholar ARDS pathogenesis was previously reported to involve the degradation of the endothelial glycocalyx, which serves as an epithelial–cell barrier.3Schmidt E.P. Yang Y. Janssen W.J. Gandjeva A. Perez M.J. Barthel L. Zemans R.L. Bowman J.C. Koyanagi D.E. Yunt Z.X. Smith L.P. Cheng S.S. Overdier K.H. Thompson K.R. Geraci M.W. Douglas I.S. Pearse D.B. Tuder R.M. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.Nat Med. 2012; 18: 1217-1223Crossref PubMed Scopus (499) Google Scholar, 4Schmidt E.P. Li G. Li L. Fu L. Yang Y. Overdier K.H. Douglas I.S. Linhardt R.J. The circulating glycosaminoglycan signature of respiratory failure in critically ill adults.J Biol Chem. 2014; 289: 8194-8202Crossref PubMed Scopus (96) Google Scholar Furthermore, we recently reported that glycocalyx disruption participated in microvascular endothelial injury that is distinctive of sepsis-induced ARDS; we found that the endothelial glycocalyx in the lung was obviously disrupted by lipopolysaccharide (LPS).5Inagawa R. Okada H. Takemura G. Suzuki K. Takada C. Yano H. Ando Y. Usui T. Hotta Y. Miyazaki N. Tsujimoto A. Zaikokuji R. Matsumoto A. Kawaguchi T. Doi T. Yoshida T. Yoshida S. Kumada K. Ushikoshi H. Toyoda I. Ogura S. Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia.Chest. 2018; 154: 317-325Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar The endothelial glycocalyx plays an important role in regulating microvascular homeostasis by maintaining endothelial permeability and microvascular tone, preserving an oncotic gradient across the endothelial barrier, and modulating adhesion/migration of leukocytes.6Chelazzi C. Villa G. Mancinelli P. De Gaudio A.R. Adembri C. Glycocalyx and sepsis-induced alterations in vascular permeability.Crit Care. 2015; 19: 26Crossref PubMed Scopus (221) Google Scholar Degradation of the endothelial glycocalyx facilitates the inflow of fluid, including lots of protein and macromolecules into the alveolar space,7Manicone A.M. Role of the pulmonary epithelium and inflammatory signals in acute lung injury.Expert Rev Clin Immunol. 2009; 5: 63-75Crossref PubMed Scopus (93) Google Scholar and exacerbated pulmonary vascular permeability is associated with endothelial glycocalyx degradation under septic conditions.5Inagawa R. Okada H. Takemura G. Suzuki K. Takada C. Yano H. Ando Y. Usui T. Hotta Y. Miyazaki N. Tsujimoto A. Zaikokuji R. Matsumoto A. Kawaguchi T. Doi T. Yoshida T. Yoshida S. Kumada K. Ushikoshi H. Toyoda I. Ogura S. Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia.Chest. 2018; 154: 317-325Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Pulmonary endothelial glycocalyx injury increases the availability of endothelial surface adhesion molecules to circulating microspheres and contributes to neutrophil adhesion under septic conditions.3Schmidt E.P. Yang Y. Janssen W.J. Gandjeva A. Perez M.J. Barthel L. Zemans R.L. Bowman J.C. Koyanagi D.E. Yunt Z.X. Smith L.P. Cheng S.S. Overdier K.H. Thompson K.R. Geraci M.W. Douglas I.S. Pearse D.B. Tuder R.M. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.Nat Med. 2012; 18: 1217-1223Crossref PubMed Scopus (499) Google Scholar Neutrophils are regarded to play a crucial role in ARDS aggravation; neutrophil activation and transmigration constitute a hallmark event in the progression of this disease.8Abraham E. Neutrophils and acute lung injury.Crit Care Med. 2003; 31: S195-S199Crossref PubMed Google Scholar Moreover, neutrophil elastase (NE), which is necessary for effective sterilization of phagocytosed bacterial and fungal pathogens,9Hagiwara S. Iwasaka H. Togo K. Noguchi T. A neutrophil elastase inhibitor, sivelestat, reduces lung injury following endotoxin-induced shock in rats by inhibiting HMGB1.Inflammation. 2008; 31: 227-234Crossref PubMed Scopus (41) Google Scholar, 10Vender R.L. Therapeutic potential of neutrophil-elastase inhibition in pulmonary disease.J Investig Med. 1996; 44: 531-539PubMed Google Scholar, 11Weiss S.J. Tissue destruction by neutrophils.N Engl J Med. 1989; 320: 365-376Crossref PubMed Scopus (3847) Google Scholar is known to increase alveolocapillary permeability, induces the production of proinflammatory cytokines, and enhances neutrophil migration.12Lee J.M. Yeo C.D. Lee H.Y. Rhee C.K. Kim I.K. Lee D.G. Lee S.H. Kim J.W. Inhibition of neutrophil elastase contributes to attenuation of lipopolysaccharide-induced acute lung injury during neutropenia recovery in mice.J Anesth. 2017; 31: 397-404Crossref PubMed Scopus (19) Google Scholar, 13Miyazaki Y. Inoue T. Kyi M. Sawada M. Miyake S. Yoshizawa Y. Effects of a neutrophil elastase inhibitor (ONO-5046) on acute pulmonary injury induced by tumor necrosis factor alpha (TNFalpha) and activated neutrophils in isolated perfused rabbit lungs.Am J Respir Crit Care Med. 1998; 157: 89-94Crossref PubMed Scopus (61) Google Scholar, 14Passi A. Negrini D. Albertini R. De Luca G. Miserocchi G. Involvement of lung interstitial proteoglycans in development of hydraulic- and elastase-induced edema.Am J Physiol. 1998; 275: L631-L635PubMed Google Scholar However, to our knowledge, no study has investigated whether neutrophils and NE injure the pulmonary endothelial glycocalyx under septic conditions. Therefore, we hypothesized that neutrophils and NE injure the pulmonary endothelial glycocalyx during sepsis, and to test this we evaluated the condition of the pulmonary endothelial glycocalyx after LPS administration in granulocyte colony-stimulating factor knockout (G-CSFKO) mice and in wild-type (WT) mice treated with the NE inhibitor sivelestat. This study conformed to the Guide for the Care and Use of Laboratory Animals15Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Google Scholar and was approved by the Institutional Animal Research Committee of Gifu University (Gifu, Japan). Male G-CSFKO mice (B6; 129P2-Csf3tm1Ard/J) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred and genotyped as described16Morishita K. Takemura G. Tsujimoto A. Kanamori H. Okada H. Chousa M. Ushimaru S. Mikami A. Kawamura I. Takeyama T. Kawaguchi T. Watanabe T. Goto K. Morishita M. Ushikoshi H. Kawasaki M. Ogura S. Minatoguchi S. Postinfarction cardiac remodeling proceeds normally in granulocyte colony-stimulating factor knockout mice.Am J Pathol. 2015; 185: 1899-1911Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar; 9- to 12-week–old G-CSFKO and littermate control mice were used in this study. After 16 hours of starvation, mice were intraperitoneally administered LPS (20 mg/kg; MilliporeSigma, Burlington, MA), and in certain assays the NE-inhibitor sivelestat (gifted from Ono Pharmaceutical Co., Ltd., Osaka, Japan) was intraperitoneally administrated at 3, 6, 9, and 12 hours after LPS injection. Survival rate was determined at 12, 24, 36, and 48 hours after LPS administration, and surviving mice were sacrificed, and lung specimens were collected. Blood samples were collected from the maxillary artery, allowed to clot at room temperature for 2 hours, and centrifuged at 2000 × g for 4°C for 20 minutes. The supernatant was collected as the serum for measuring NE levels by using enzyme-linked immunosorbent assay quantitation kits for mouse NE (E-EL-M0444; Elabscience, Houston, TX). Before and at 6 hours after LPS administration, blood samples were collected in the same manner. Samples were prepared as previously described.17Suzuki K. Inoue S. Kametani Y. Komori Y. Chiba S. Sato T. Inokuchi S. Ogura S. Reduced immunocompetent B cells and increased secondary infection in elderly patients with severe sepsis.Shock. 2016; 46: 270-278Crossref PubMed Scopus (30) Google Scholar Cells were stained with the following antibodies (BioLegend, San Diego, CA) for 5 minutes at room temperature: anti-mouse CD45–peridinin chlorophyll protein complex (clone: 30-F11), anti-mouse CD11b-phycoerythrin (clone: M1/70), anti-mouse Ly6G-fluorescein isothiocyanate (clone: 1A8), and anti-mouse F4/80-Alexa Fluor 647 (clone: BM8). Neutrophils were defined as the CD45+CD11b+Ly6G+F4/80− white blood cell population. Flow cytometry was performed on a BD FACSCalibur instrument (Becton and Dickinson Company, Franklin Lakes, NJ), and data were analyzed with FlowJo software version 10.5.3 (TreeStar LLC, Ashland, OR). After deparaffinization, sections were cut (4 μm thick), lung tissues were counterstained with hematoxylin and eosin and were scored by a certificated pathologist as follows for neutrophilic infiltration: 1, absent to rare solitary neutrophils; 2, detectable extravasated neutrophils observed as small loose cellular accumulates in one to a few airways and/or alveoli; 3, detectable extravasated neutrophils observed as loose to compact cellular accumulates in multiple to coalescing airway and/or alveoli with some effacement of lung architecture; and 4, detectable extravasated neutrophils observed as compact cellular accumulates effacing most adjacent pulmonary structure. Pulmonary edema was scored as 1, absent; 2, detectable seroproteinaceous fluid in one to a few alveoli; and 3, seroproteinaceous fluid filling alveoli in a multifocal to coalescing pattern in lung.18Langlois R.A. Meyerholz D.K. Coleman R.A. Cook R.T. Waldschmidt T.J. Legge K.L. Oseltamivir treatment prevents the increased influenza virus disease severity and lethality occurring in chronic ethanol consuming mice.Alcohol Clin Exp Res. 2010; 34: 1425-1431PubMed Google Scholar Lung sections incubated with primary antibodies against the neutrophil surface marker Gr-1 (ab8592; Abcam, Cambridge, UK) and the endothelial-injury surface marker thrombomodulin (ab6980; Abcam). Sections were immunostained with the Vectastain Elite ABC system (Vector Laboratories, Burlingame, CA) as described.5Inagawa R. Okada H. Takemura G. Suzuki K. Takada C. Yano H. Ando Y. Usui T. Hotta Y. Miyazaki N. Tsujimoto A. Zaikokuji R. Matsumoto A. Kawaguchi T. Doi T. Yoshida T. Yoshida S. Kumada K. Ushikoshi H. Toyoda I. Ogura S. Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia.Chest. 2018; 154: 317-325Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Okada H. Takemura G. Suzuki K. Oda K. Takada C. Hotta Y. Miyazaki N. Tsujimoto A. Muraki I. Ando Y. Zaikokuji R. Matsumoto A. Kitagaki H. Tamaoki Y. Usui T. Doi T. Yoshida T. Yoshida S. Ushikoshi H. Toyoda I. Ogura S. Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions.Crit Care. 2017; 21: 261Crossref PubMed Scopus (72) Google Scholar A trained pathologist counted the incidence of thrombomodulin-positive cells in the total endothelial cells. The cell count was performed on five randomly chosen high-power field (HPF) in each section. The number of endothelial cells evaluated was 253 ± 21 cells per lung. Tissue lysate preparation and electrophoresing were performed as previously described.5Inagawa R. Okada H. Takemura G. Suzuki K. Takada C. Yano H. Ando Y. Usui T. Hotta Y. Miyazaki N. Tsujimoto A. Zaikokuji R. Matsumoto A. Kawaguchi T. Doi T. Yoshida T. Yoshida S. Kumada K. Ushikoshi H. Toyoda I. Ogura S. Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia.Chest. 2018; 154: 317-325Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar The membranes were probed with antibodies against thrombomodulin (Abcam), syndecan-1 (ab34164; Abcam), or α-tubulin (sc-5546; Santa Cruz Biotechnology, Dallas, TX), and then immunoreactive bands were visualized with enhanced chemiluminescence (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK), and signal intensities were quantified (as arbitrary units) with the use of ImageJ software version 1.51j8 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Western blot analysis was performed on four samples from each group. Electron microscopy analysis of the endothelial glycocalyx was performed as previously described.5Inagawa R. Okada H. Takemura G. Suzuki K. Takada C. Yano H. Ando Y. Usui T. Hotta Y. Miyazaki N. Tsujimoto A. Zaikokuji R. Matsumoto A. Kawaguchi T. Doi T. Yoshida T. Yoshida S. Kumada K. Ushikoshi H. Toyoda I. Ogura S. Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia.Chest. 2018; 154: 317-325Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Okada H. Takemura G. Suzuki K. Oda K. Takada C. Hotta Y. Miyazaki N. Tsujimoto A. Muraki I. Ando Y. Zaikokuji R. Matsumoto A. Kitagaki H. Tamaoki Y. Usui T. Doi T. Yoshida T. Yoshida S. Ushikoshi H. Toyoda I. Ogura S. Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions.Crit Care. 2017; 21: 261Crossref PubMed Scopus (72) Google Scholar Briefly, mice were anesthetized and then perfused with a solution composed of 2% glutaraldehyde, 2% sucrose, 0.1 mol/L sodium cacodylate buffer (pH 7.3), and 2% lanthanum nitrate, at a steady flow-rate of 1 mL/min, through a cannula placed in the left ventricle. After the mice were sacrificed, lung samples were fixed in a solution without glutaraldehyde and then washed in alkaline (0.03 mol/L NaOH) 2% sucrose solution. The freeze-fracture method was used to prepare samples for scanning electron microscopy (S-4800; Hitachi High-Technologies Global, Tokyo, Japan). To prepare samples for transmission electron microscopy (TEM), specimens were embedded in epoxy resin and then ultrathin (90-nm) sections were generated, stained with uranyl acetate and lead citrate, and subjected to TEM analysis (HT-7700, Hitachi High-Technologies Global, Tokyo, Japan). To prepare samples for conventional electron microscopy, 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) without lanthanum nitrate was used as the fixative. Data are presented as means ± SEM. Two-tailed t-test was used for comparing two groups, and survival data were analyzed with the log-rank test; P < 0.05 was considered significant. All calculations were performed with Prism software version 7.02 (GraphPad, La Jolla, CA). Analysis of the enzyme-linked immunosorbent assay revealed serum G-CSF deficiency, and flow cytometric analysis revealed neutropenia in G-CSFKO mice (Figure 1, A and B ), confirming the original report.20Lieschke G.J. Grail D. Hodgson G. Metcalf D. Stanley E. Cheers C. Fowler K.J. Basu S. Zhan Y.F. Dunn A.R. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.Blood. 1994; 84: 1737-1746Crossref PubMed Google Scholar To produce LPS-induced experimental endotoxemia model mice, 20 mg/kg LPS was injected intraperitoneally to 9- to 12-week–old G-CSFKO and littermate control male mice. At 48 hours after LPS administration, the survival rate of G-CSFKO mice (77%, 24 of 31) was markedly higher (P < 0.05) than that of control WT mice (23%, 6 of 26) (Figure 1C). To determine pulmonary injury 48 hours after LPS injection, a scoring system was used (Figure 1, D–G).18Langlois R.A. Meyerholz D.K. Coleman R.A. Cook R.T. Waldschmidt T.J. Legge K.L. Oseltamivir treatment prevents the increased influenza virus disease severity and lethality occurring in chronic ethanol consuming mice.Alcohol Clin Exp Res. 2010; 34: 1425-1431PubMed Google Scholar After LPS administration, levels of neutrophil infiltration and pulmonary edema increased compared with before LPS injection. Conversely, G-CSFKO mice had a significant decrease in neutrophil infiltration and pulmonary edema. In WT mice, serum NE concentration was increased at 6 hours after LPS administration and then the level decreased gradually; by comparison, serum NE was considerably lower before and at 6 and 12 hours after LPS injection in G-CSFKO mice (Figure 2A). Furthermore, peripheral neutrophil numbers were markedly lower in G-CSFKO mice than WT mice, both before and at 6 hours after LPS administration, the time at which serum NE concentration peaked (Figure 2B), and at 6 hours after LPS injection substantially fewer Gr-1-positive cells were present in the pulmonary tissues of G-CSFKO mice (24 ± 4 cells/HPF) than WT mice (104 ± 4 cells/HPF) (Figure 2, C and D). To investigate pulmonary endothelial injury, the localization of thrombomodulin, an endothelial-injury marker, was examined. Immunohistochemical analysis revealed that thrombomodulin was expressed on pulmonary capillaries in both WT and G-CSFKO mice under sham treatment (Figure 3, A and B), but little thrombomodulin was expressed in WT mice after LPS administration. LPS treatment markedly reduced thrombomodulin expression in WT mice but not in G-CSFKO mice (Figure 3, A–D). In accordance with these results, the expression of syndecan-1, an endothelial glycocalyx injury marker, was considerably lower in LPS-treated WT mice than in untreated WT mice (Figure 3, E and F). Conversely, syndecan-1 expression in LPS-treated G-CSFKO mice was not different from that in untreated WT mice (Figure 3, E and F). These results suggest that pulmonary endothelial injury and endothelial glycocalyx injury in LPS-treated G-CSFKO mice were diminished relative to those in LPS-treated WT mice. The ultrastructure of the endothelium and endothelial glycocalyx was analyzed with electron microscopy. Conventional scanning electron microscopy results showed that pulmonary capillaries were of the continuous type, characterized by the presence of an uninterrupted endothelium and a continuous basal lamina, in untreated WT and G-CSFKO mice (Figure 4A ). Conventional TEM revealed that after LPS injection the endothelial wall became edematous, but the extent was lesser in G-CSFKO mice than in WT mice (Figure 4B). Scanning electron microscopy and TEM analyses performed with lanthanum revealed an endothelial glycocalyx that featured a moss-like structure on the surface of the vascular endothelium in both WT and G-CSFKO mice under normal conditions; however, after LPS injection, the endothelial glycocalyx was peeled away in WT mice, and this was again attenuated in G-CSFKO mice (Figure 4, C–P). Next, to determine the contribution of NE in septic mice, 0.2 mg/kg sivelestat was injected intraperitoneally into mice at 3, 6, 9, and 12 hours after LPS administration. Sivelestat forms the acyl complex with an active region of the NE and inhibits NE activity.21Imaki K. Okada T. Nakayama Y. Nagao Y. Kobayashi K. Sakai Y. Mohri T. Amino T. Nakai H. Kawamura M. Non-peptidic inhibitors of human neutrophil elastase: the design and synthesis of sulfonanilide-containing inhibitors.Bioorg Med Chem. 1996; 4: 2115-2134Crossref PubMed Scopus (32) Google Scholar At 48 hours after LPS treatment, the survival rate was significantly higher (P < 0.05) among sivelestat-injected mice (82%, 22 of 27) than control mice (9%, 3 of 32) (Figure 5A). In sivelestat-treated mice, the levels of neutrophil infiltration and pulmonary edema were attenuated compared with untreated mice 48 hours after LPS injection (Figure 5, B–D). At 24 hours after LPS administration, serum NE concentration was markedly lower (P < 0.05) in sivelestat-treated mice than in control (saline-treated) mice (Figure 5E). Moreover, the Gr-1–positive cell numbers within pulmonary tissues were obviously increased (P < 0.01) in LPS-injected mice (108 ± 4 cells/HPF) compared with that in untreated mice (12 ± 1 cells/HPF), but there were no significant differences between the numbers of peripheral neutrophils in untreated and sivelestat-treated mice (Figure 5F) or the numbers of extravasated Gr-1–positive cells in the lung sections from these mice at 6 hours after LPS administration (Figure 5, G and H). Finally, to examine the protective effect of sivelestat treatment against pulmonary endothelial injury and endothelial glycocalyx injury under endotoxemia, thrombomodulin and syndecan-1 expression was analyzed. In sivelestat-treated mice, thrombomodulin was detected on pulmonary capillaries after LPS administration in the same manner as in untreated control mice (Figure 5, I and J); however, quantitative Western blot analyses showed that thrombomodulin and syndecan-1 expression in lung tissue after LPS administration was enhanced in sivelestat-injected mice compared with that in saline-injected mice (Figure 5, K–N). Notably, ultrastructural analysis revealed that after LPS administration, the endothelial surface was less edematous and the endothelial glycocalyx was preserved to a greater extent in sivelestat-treated WT mice than in saline-treated WT mice (Figure 6). These results suggest that pulmonary endothelial injury and endothelial glycocalyx injury were ameliorated in sivelestat-treated mice compared with that in untreated mice after LPS injection. This study revealed that G-CSFKO mice and sivelestat-treated WT mice showed attenuated pulmonary endothelial glycocalyx injury after LPS administration compared with LPS-treated WT mice. Our findings indicate that the absence of G-CSF and the inhibition of NE critically contribute to glycocalyx protection. The inner surface of vascular endothelial cells is coated with a glycocalyx of membrane-bound macromolecules, including glycoproteins, hyaluronan, sulfated proteoglycans, and plasma proteins that adhere to this surface matrix.22Weinbaum S. Tarbell J.M. Damiano E.R. The structure and function of the endothelial glycocalyx layer.Annu Rev Biomed Eng. 2007; 9: 121-167Crossref PubMed Scopus (821) Google Scholar Degradation of the endothelial glycocalyx has been suggested to contribute to ARDS pathogenesis,3Schmidt E.P. Yang Y. Janssen W.J. Gandjeva A. Perez M.J. Barthel L. Zemans R.L. Bowman J.C. Koyanagi D.E. Yunt Z.X. Smith L.P. Cheng S.S. Overdier K.H. Thompson K.R. Geraci M.W. Douglas I.S. Pearse D.B. Tuder R.M. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.Nat Med. 2012; 18: 1217-1223Crossref PubMed Scopus (499) Google Scholar, 4Schmidt E.P. Li G. Li L. Fu L. Yang Y. Overdier K.H. Douglas I.S. Linhardt R.J. The circulating glycosaminoglycan signature of respiratory failure in critically ill adults.J Biol Chem. 2014; 289: 8194-8202Crossref PubMed Scopus (96) Google Scholar and in a previous report we presented three-dimensional images that revealed exacerbated pulmonary vascular permeability and degradation of the endothelial glycocalyx on the inner surface of endothelial cells under septic conditions.5Inagawa R. Okada H. Takemura G. Suzuki K. Takada C. Yano H. Ando Y. Usui T. Hotta Y. Miyazaki N. Tsujimoto A. Zaikokuji R. Matsumoto A. Kawaguchi T. Doi T. Yoshida T. Yoshida S. Kumada K. Ushikoshi H. Toyoda I. Ogura S. Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia.Chest. 2018; 154: 317-325Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar We presented ultrastructural imaging of the endothelial glycocalyx in the present study. The endothelial glycocalyx forms a continuous structure that allows for vascular homeostasis. Other than confirming the location of the existence of glycocalyx with the use of a lectin staining technique, the resolution of an optical microscope is not enough to accurately detect the structure. We recently reported that the structures of the endothelial glycocalyx differ greatly among the brain, heart, and lung.23Ando Y. Okada H. Takemura G. Suzuki K. Takada C. Tomita H. Zaikokuji R. Hotta Y. Miyazaki N. Yano H. Muraki I. Kuroda A. Fukuda H. Kawasaki Y. Okamoto H. Kawaguchi T. Watanabe T. Doi T. Yoshida T. Ushikoshi H. Yoshida S. Ogura S. Brain-specific ultrastructure of capillary endothelial glycocalyx and its possible contribution for blood brain barrier.Sci Rep. 2018; 8: 17523Crossref PubMed Scopus (88) Google Scholar In this report, the pulmonary capillary glycocalyx is constructed of a thin layer; therefore, it is particularly difficult to detect its structure without ultrastructural imaging. Likewise, in a quantitative assessment of protein expression by Western blot analysis, it is impossible to confirm that the endothelial glycocalyx actually exists on the inner surface of the vascular endothelium. Therefore, we believe that the glycocalyx ultrastructural imaging is a helpful tool in the present study. Sepsis induces alterations of neutrophil deformability and neutrophil entrapment in lung capillaries; neutrophil entrapment is followed by permeability alterations and edema formation.24Wiener-Kronish J.P. Albertine K.H. Matthay M.A. Differential responses of the endothelial and epithelial barriers of the lung in sheep to Escherichia coli endotoxin.J Clin Invest. 1991; 88: 864-875Crossref PubMed Scopus (263) Google Scholar, 25Wiggs B.R. English D. Quinlan W.M. Doyle N.A. Hogg J.C. Doerschuk C.M. Contributions of capillary pathway size and neutrophil deformability to neutrophil transit through rabbit lungs.J Appl Physiol (1985). 1994; 77: 463-470Crossref PubMed Scopus (85) Google Scholar In ARDS, a histologic hallmark secondary to sepsis is the recruitment of neutrophils in the lung.26Grommes J. Soehnlein O. Contribution of neutrophils to acute lung injury.Mol Med. 2011; 17: 293-307Crossref PubMed Scopus (918) Google Scholar A previous study conducted with the use of sepsis models suggested that mortality was also diminished in granulocyte macrophage-CSFKO mice.27Spight D. Trapnell B. Zhao B. Berclaz P. Shanley T.P. Granulocyte-macrophage-colony-stimulating factor-dependent peritoneal macrophage responses determine survival in experimentally induced peritonitis and sepsis in mice.Shock. 2008; 30: 434-442Crossref PubMed Scopus (35) Google Scholar G-CSF, which is produced by monocytes, endothelial cells, and fibroblasts, stimulates neutrophil production and regulates the function and activity of developing and mature neutrophils.28Stephens D.P. Fisher D.A. Currie B.J. An audit of the use of granulocyte colony-stimulating factor in septic shock.Intern Med J. 2002; 32: 143-148Crossref PubMed Scopus (50) Google Scholar G-CSFKO mice reveal granulocyte and macrophage progenitor cell deficiencies, chronic neutropenia, and impaired neutrophil mobilization.20Lieschke G.J. Grail D. Hodgson G. Metcalf D. Stanley E. Cheers C. Fowler K.J. Basu S. Zhan Y.F. Dunn A.R. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.Blood. 1994; 84: 1737-1746Crossref PubMed Google Scholar In G-CSFKO mice, fewer neutrophils are present than in WT mice, even under endotoxemic conditions. Given that endothelial glycocalyx injury after LPS injection was markedly attenuated in G-CSFKO mice compared with that in WT mice, neutrophils might exert harmful effects on the endothelial glycocalyx under septic conditions. However, two explanations can account for the increased survival rate and attenuated endothelial glycocalyx injury in G-CSFKO mice after LPS administration: neutropenia produced a protective effect against endotoxemia in mice; and the amount of neutrophil-secreted mediators was insufficient for causing endothelial injury. NE was previously reported to play a pivotal role during the pathogenesis of acute lung injury/ARDS,26Grommes J. Soehnlein O. Contribution of neutrophils to acute lung injury.Mol Med. 2011; 17: 293-307Crossref PubMed Scopus (918) Google Scholar and elevated NE levels were correlated with the severity of lung injury.29Donnelly S.C. MacGregor I. Zamani A. Gordon M.W. Robertson C.E. Steedman D.J. Little K. Haslett C. Plasma elastase levels and the development of the adult respiratory distress syndrome.Am J Respir Crit Care Med. 1995; 151: 1428-1433Crossref PubMed Scopus (149) Google Scholar, 30Zemans R.L. Colgan S.P. Downey G.P. Transepithelial migration of neutrophils: mechanisms and implications for acute lung injury.Am J Respir Cell Mol Biol. 2009; 40: 519-535Crossref PubMed Scopus (254) Google Scholar, 31Li G. Jia J. Ji K. Gong X. Wang R. Zhang X. Wang H. Zang B. The neutrophil elastase inhibitor, sivelestat, attenuates sepsis-related kidney injury in rats.Int J Mol Med. 2016; 38: 767-775Crossref PubMed Scopus (25) Google Scholar These reports support our present results. We demonstrated that the pulmonary endothelial glycocalyx injury of sivelestat-treated WT mice was attenuated compared with that of saline-treated WT mice under endotoxemia. Because sivelestat is a competitive antagonist of NE, we suspect that NE concentrations did not differ substantially between sivelestat-treated and untreated mice in this study. However, a previous clinical study indicated that intravenous sivelestat injection exerted no effect on all-cause mortality or ventilator-free days in patients with heterogeneous acute lung injury.32Zeiher B.G. Artigas A. Vincent J.L. Dmitrienko A. Jackson K. Thompson B.T. Bernard G. STRIVE Study GroupNeutrophil elastase inhibition in acute lung injury: results of the STRIVE study.Crit Care Med. 2004; 32: 1695-1702Crossref PubMed Scopus (241) Google Scholar One reason for this loss of effect of NE inhibition on neutrophil function could be related to tissue penetration. If sivelestat can be delivered selectively to injury sites by using new drug-delivery systems in the future, sivelestat administration might emerge as an effective clinical treatment against sepsis. Sepsis is an extremely complicated disease compared with simple endotoxemia in an experimental model. Because our focus here was to investigate the direct relationship between endothelial glycocalyx injury and neutrophils, we used an endotoxemia model that does not reflect certain typical septic conditions such as bacterial infection, and this represents one limitation of our study. A previous study showed that KO mice that lack neutrophil elastase have impaired host defenses to Gram-negative bacteria,33Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis.Nat Med. 1998; 4: 615-618Crossref PubMed Scopus (539) Google Scholar although the present study showed that neutrophil elastase was deleterious to the host. It is thought that the difference may be due to the presence or absence of bacteria. Neutrophils are one of the first responders of inflammatory cells and work against acute inflammation as a result of bacterial infection. Neutrophil elastase injures the endothelium through the endothelial glycocalyx while also requiring innate immunity to successfully battle a bacterial infection. Therefore, innate immunity may not be sufficient in the bacteremia model because G-CSFKO mice have depleted neutrophils. In other words, the neutrophil has two roles. In the present study, it was not confirmed whether neutrophil elastase altered endothelial glycocalyx directly. Additional studies that use a bacteremia model to further explore the role of endothelial glycocalyx in endothelial injury during sepsis are needed. Another limitation of this study is that we did not examine inflammatory cytokines such as IL-6; the serum concentration of IL-6 in particular was reported to contribute to the survival rate in sepsis.34Iwamura H. Sato M. Wakitani K. Comparative study of glucocorticoids, cyclosporine A, and JTE-607 [(-)-Ethyl-N[3,5-dichloro-2-hydroxy-4-[2-(4-methylpiperazin-1-yl)ethoxy]benzoyl]-L-phenylalaninate dihydrochloride] in a mouse septic shock model.J Pharmacol Exp Ther. 2004; 311: 1256-1263Crossref PubMed Scopus (18) Google Scholar In addition, the aforementioned report suggested that the endothelial injury was caused by several factors, including superoxides. The present study has not analyzed the influences of superoxides in endotoxemia mice.35Adachi T. Fukushima T. Usami Y. Hirano K. Binding of human xanthine oxidase to sulphated glycosaminoglycans on the endothelial-cell surface.Biochem J. 1993; 289: 523-527Crossref PubMed Scopus (170) Google Scholar NE plays a pivotal role in pulmonary endothelial glycocalyx degradation; thus, inhibition of NE might offer a potential means to protect the endothelial glycocalyx.
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