key: cord-0700034-w61uawz5 authors: Okamoto, Haruka; Muraki, Isamu; Okada, Hideshi; Tomita, Hiroyuki; Suzuki, Kodai; Takada, Chihiro; Wakayama, Yugo; Kuroda, Ayumi; Fukuda, Hirotsugu; Kawasaki, Yuki; Nishio, Ayane; Matsuo, Maho; Tamaoki, Yuto; Inagawa, Risa; Takashima, Shigeo; Taniguchi, Toshiaki; Suzuki, Akio; Suzuki, Keiko; Miyazaki, Nagisa; Kakino, Yoshinori; Yasuda, Ryu; Fukuta, Tetsuya; Kitagawa, Yuichiro; Miyake, Takahito; Doi, Tomoaki; Yoshida, Takahiro; Yoshida, Shozo; Ogura, Shinji title: Recombinant Antithrombin Attenuates Acute Respiratory Distress Syndrome in Experimental Endotoxemia date: 2021-06-08 journal: Am J Pathol DOI: 10.1016/j.ajpath.2021.05.015 sha: d08b537a320720e3c6d0051474a81f0f40a71508 doc_id: 700034 cord_uid: w61uawz5 Sepsis-induced endothelial acute respiratory distress syndrome is related to microvascular endothelial dysfunction caused by endothelial glycocalyx disruption. Recently, recombinant antithrombin (rAT) was reported to protect the endothelial glycocalyx from septic vasculitis; however, the underlying mechanism remains unknown. Here, we investigated the effect of rAT administration on vascular endothelial injury under endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 10-week-old male C57BL/6 mice, and saline or rAT was administered intraperitoneally at 3 and 24 hours after LPS administration. Subsequently, serum and/or pulmonary tissues were examined for inflammation and cell proliferation and differentiation by histologic, ultrastructural, and microarray analyses. The survival rate was significantly higher in rAT-treated mice than in control mice 48 hours after LPS injection (75% versus 20%; P < 0.05). Serum interleukin-1β was increased but to a lesser extent in response to LPS injection in rAT-treated mice than in control mice. Lectin staining and ultrastructural studies showed a notable attenuation of injury to the endothelial glycocalyx after rAT treatment. Microarray analysis further showed an up-regulation of gene sets corresponding to DNA repair, such as genes involved in DNA helicase activity, regulation of telomere maintenance, DNA-dependent ATPase activity, and ciliary plasm, after rAT treatment. Thus, rAT treatment may promote DNA repair, attenuate inflammation, and promote ciliogenesis, thereby attenuating the acute respiratory distress syndrome caused by endothelial injury. The Q6 sepsis diagnostic criteria include organ failure caused by several factors, which includes endothelial cell injury. 1 Endothelial cells are coated by the vascular endothelial glycocalyx, which is present on the surface of endothelial cells and plays a pivotal role in the maintenance of vascular homeostasis. 2 In the healthy state, the glycocalyx is a critical determinant of vascular permeability. 3, 4 Endothelial glycocalyx disruption causes injury to the microcirculation system, which functions to maintain blood flow and tissue perfusion, and, thus, affects blood pressure and responses to inflammation. Disruption of the endothelial glycocalyx also is associated with lung injury and neutrophil adhesion during sepsis-induced acute respiratory distress syndrome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 associated with microvascular endothelial dysfunction. 6 Because an intact glycocalyx potentially protects against endothelial disorders, 7e10 protection of the endothelial glycocalyx could represent a novel therapeutic strategy to prevent the endothelial injuryeinduced organ failure that occurs during sepsis. However, thus far, no clinical strategies have been established for treating sepsis through endothelial glycocalyx protection. Although corticosteroids 11 and heparinoids 12 have been suggested to protect the endothelial glycocalyx, definitive evidence of the clinical utility of these compounds for the treatment of sepsis is lacking. Antithrombin (AT)da small protein molecule that inactivates several enzymes of the coagulation systemdis a physiological serine-protease inhibitor that plays a crucial role in blood coagulation. 13 AT inhibits the interaction between thrombin and activated coagulation factors and has been reported to account for 75% to 80% of the inhibitory activity directed toward thrombin. 14 Similarly, AT binds to heparin sulfate on endothelial cells and to syndecan-4 on neutrophils to subsequently attenuate inflammation by more than 120% of the level activated by LPS. 15 AT also has been reported to attenuate endothelial glycocalyx injury caused by LPS administration, 16 and a nonfucosylated recombinant AT (rAT) was found to maintain vascular structure. 17 However, the mechanisms underlying these effects are unknown. Therefore, our aim was to assess the state of the pulmonary endothelial glycocalyx after LPS injection in rAT-treated mice. The present study conformed to the NIH's Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Research Committee of Gifu University , Gifu, Japan) . 18 Ten-week-old, male C57BL/6 mice were obtained from Chubu Kagaku Shizai, Co., Ltd. (Nagoya, Japan). After a 16-hour starvation period, the mice were injected intraperitoneally with LPS (20 mg/kg; MilliporeSigma, Burlington, MA) and with rAT (750 IU/kg; Kyowa Kirin, Co., Ltd., Tokyo, Japan) for certain assays at 3 and 24 hours after LPS injection. The survival rate was evaluated every 12 hours after LPS administration; the mice that survived were euthanized, and lung specimens were obtained. We have confirmed that there was no significant difference in the survival rate between male and female mice in a preliminary study. Therefore, we used male mice for this experiment. Blood samples were prepared as described previously. 19 Serum was collected to quantify IL-1b and IL-6 concentrations using Enzyme-Linked Immunosorbent Assay Quantitation Kits for mouse IL-1b (MLB00C; R&D Systems, Minneapolis, MN), and mouse IL-6 (M6000B; R&D Systems), respectively. The clinical scoring of lung was performed as described previously. 19, 20 These experiments were performed in a blinded manner to avoid bias (H.O ., H.T, and T.T.). For the quantitative analysis of glycocalyx injury, scoring of wheat germ agglutinin (WGA) (B-1025-5; Vector Laboratories, Burlingame, CA) staining intensity was performed using a confocal fluorescence microscope (BZ-X810; Keyence, Osaka, Japan) and ImageJ software version 1.51j8 (NIH, Bethesda, MD; https://imagej.nih.gov) as described previously. 19 WGA (100 mL), which allows good visualization of the endothelial glycocalyx, 21 was injected into the jugular vein 10 minutes before sacrifice. The lung of each mouse was embedded in ornithine carbamoyltransferase Q10 compound and frozen with liquid nitrogen. The frozen blocks were stored at À80 C. Sections of frozen tissues (5to 7-mm thick) were prepared with a cryostat. The intensity of WGA was scored manually in 10 high-power fields Q11 per sample (n Z 6 per sample) in the focal plane. Deparaffinized sections (4-mm thick) were incubated with primary antibodies against Ki-67 (ab16667; Abcam, Cambridge, UK), the endothelial cell marker thrombomodulin (ab6980; Abcam), proliferating cell nuclear antigen (PCNA) (M0879; Dako, Santa Clara, CA), phosphorylated g-H2A.X (S139. ab11174; Abcam), and ARL13B (17711-1-AP; Proteintech, Rosemond, IL). The localization of the target proteins was visualized using the VECTASTAIN Elite ABC system (Vector Laboratories) or Alexa Fluor 488e/ 568econjugated secondary antibodies (Invitrogen, Carlsbad, CA); Hoechst staining was used to visualize nuclei. Electron microscopic analysis of the endothelial glycocalyx was performed as described previously. 22 Because it is difficult to distinguish between the glycocalyx and nonspecific staining by osmium when the glycocalyx is visualized using lanthanum nitrate, osmium was not used in transmission Q12 electron microscopy imaging without lanthanum nitrate during glycocalyx visualization. Lung tissues used for microarray analysis were obtained from saline-or rAT-treated mice at 30 hours after LPS administration, and total RNA was extracted as described previously. 19 Gene expression analysis was performed by the Life Science Research Center, Gifu University (Gifu, Japan), using Agilent Expression Arrays (SurePrint G3 Mouse GE 8 Â 60K microarray). The obtained data were analyzed and visualized as described previously. 19 All microarray data were deposited in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo; accession number GSE160929). Data are presented as the means AE SEM. A two-tailed t-test was used for comparing two groups, and survival data were analyzed using the log-rank test; P < 0.05 was considered significant. All calculations were performed using GraphPad Prism (La Jolla, CA). To produce an experimental endotoxemia model, LPS (20 mg/kg) was injected intraperitoneally into 10-week-old C57BL/6 male mice. At 48 hours after LPS administration, the survival rate of rAT-treated mice (75%; 24 of 32) was significantly higher than that of control mice (20%; 10 of The American Journal of Pathologyajp.amjpathol. org 3 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 50) ( Figure 1 ½F1 ½F1 A). To confirm the adverse effects of rAT, we injected it into the mice without LPS (n Z 20). The survival rate of the rAT-injected mice was 100% at 48 hours after injection. However, adverse effects of rAT, including hemorrhage and infusion site reaction, were not tested Q14 . In the serum of control mice, the inflammatory cytokine IL-1b reached concentrations of 290.0 AE 31.6 ng/mL and 211.8 AE 29.8 ng/mL at 6 and 12 hours after LPS injection, respectively, and then decreased to 37.1 AE 10.7 ng/mL at 48 hours after injection ( Figure 1B ). However, in rAT-treated mice, the IL-1b concentration was significantly lower at 6 hours after LPS administration (186.3 AE 32.8 ng/mL). Next, pulmonary injury was assessed at 48 hours after LPS injection using a clinical scoring system. 20, 23 Pulmonary edema and neutrophil infiltration were caused by LPS injection (Figure 1 , CeF). However, at 48 hours after LPS administration, pulmonary edema was attenuated in the rAT-treated mice relative to that in the saline-injected mice (Figure 1 , C and D), and neutrophil infiltration also was decreased significantly in the rAT-treated mice compared with that in the saline-injected mice (Figure 1 , E and F). These results su Q15 ggest that rAT treatment attenuated the pulmonary injury that occurs after LPS administration. To quantify endothelial glycocalyx injury, we performed WGA staining, which enables the visualization of the endothelial glycocalyx, 21 and measured the staining intensity. WGA staining was weaker in saline-injected mice after LPS administration than in mice not injected with LPS (sham mice) (Figure 2 , A and D). After LPS administration, granulocytes were detected in the pulmonary capillary lumen along with induced vasculitis, which subsequently led to injury of the inner surface of the vascular endothelium ( Figure 3 , B and E). However, rAT administration ameliorated this injury (Figure 3 , C and F). Next, to visualize the endothelial glycocalyx, SEM analysis was performed after lanthanum nitrate staining. The endothelial glycocalyx structure appeared thin and formed a continuous structure, and it covered the inner surface of the vascular endothelium in sham mice (Figure 3 , G and J). After LPS injection, the endothelial glycocalyx was injured and its continuous structure was disrupted in saline-treated mice (Figure 3 , H and K), whereas the continuous structure was retained in rAT-treated mice (Figure 3 , I and L). In conventional transmission electron microscopy imaging, the inner surface of pulmonary capillaries appeared smooth and the endothelial capillary wall was thin in sham mice (Figure 4 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 whereas LPS-induced endothelial edema was attenuated in rAT-treated mice (Figure 4 , C and F). Transmission electron microscopy analyses further indicated that the endothelial glycocalyx forms a continuous structure and covers the inner surface of the vascular endothelium (Figure 4 , G and J), as also observed in SEM imaging. After LPS injection, the endothelial glycocalyx was degraded, and, thus, the continuous structure was damaged in saline-treated mice (Figure 4, H and K) . However, in rAT-treated mice, the continuous structure was retained and the endothelial glycocalyx injury was ameliorated ( Figure 4, I and L) . These results show that rAT treatment ameliorated endothelial glycocalyx injury under endotoxemia. Gene set enrichment analysis was performed on salinetreated (control) and rAT-treated mice. In gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of gene set enrichment analysis Q17 , we observed significant upregulation of gene sets related to DNA helicase activity ( Figure 5 A), regulation of telomere maintenance ( Figure 5B ), DNA-dependent ATPase activity ( Figure 5C ), and ciliary plasm ( Figure 5D ) in rAT-treated mice (P < 0.01 versus control). Collectively, these results indicate that rAT treatment against LPS-induced vasculitis functionally influenced DNA repair. The American Journal of Pathologyajp.amjpathol. org 5 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 Considering the gene set enrichment analysis results indicating that rAT treatment could affect cell proliferation/ differentiation, we examined Ki-67 expression. Ki-67 was expressed at a higher level in rAT-treated mice after LPS injection than in saline-treated mice after LPS administration and in sham mice ( Figure 6 ½F6 ½F6 , AeD) and was detected in several types of cells such as inflammatory cells, alveolar macrophages, and endothelial cells. Therefore, for further confirmation, we performed double immunostaining for Ki-67 and thrombomodulin, a marker of endothelial cells, which showed colocalization of Ki-67 and thrombomodulin expression in rAT-treated mice ( Figure 6E ). To further confirm the DNA repair, we performed immunofluorescence assays, with staining for PCNA and phosphorylated g-H2A.X. PCNA is a protein participating in the replication and repair of DNA, and the adjustment of the cell cycle. 24 Phosphorylated g-H2A.X is related to the activation of the local DNA damage repair pathway. 25 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 Furthermore, in rAT-treated mice, phosphorylated g-H2A.Xepositive cell numbers were much larger than those in the saline group (Figure 7 , C and D). PCNA-positive cells and especially phosphorylated g-H2A.Xepositive cells include several types of cells, not only endothelial cells, which show flat nuclei (Figure 7, AeD) . Lastly, to assess the gene set related to ciliary proteins, we performed ARL13B immunostaining. Immunohistochemical staining with an ARL13B-specific antibody showed that ARL13B expression was localized to tracheal epithelial cells ( Figure 7E ). After LPS injection, in rATtreated mice, the cilia (ARL13B-positive) existed continuously on tracheal cells, whereas the structure of the cilia layer was injured and eroded ( Figure 7E ) in the salinetreated group. These results showed that rAT treatment attenuated cilial injury on tracheal cells after LPS injection. This study showed that compared with saline treatment, rAT treatment ameliorated pulmonary endothelial glycocalyx injury in mice after LPS injection. Although previous studies have shown that rAT shows anti-inflammatory effects, 16, 17 the novel insight of the present study is that rAT administration causes the acceleration of DNA repair via enhancement of DNA helicase activity, regulation of telomere maintenance, and DNA-dependent ATPase activity. The results of this study showed that rAT treatment decreased the levels of the inflammatory cytokine IL-1b and inhibited neutrophil infiltration in the lung. These findings are in agreement with those of previous work indicating that AT ameliorates inflammation by interacting with endothelial cells and neutrophils. 15 Neutrophils play a crucial role in endothelial injury. Sepsis causes modification of neutrophil entrapment and neutrophil deformability in pulmonary capillaries, and neutrophil entrapment is followed by hyperpermeability of the lung capillaries and edema formation. 26, 27 Moreover, in acute respiratory distress syndrome, a pathologic alteration print & web 4C=FPO 7 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 secondary to sepsis is the recruitment of neutrophils in the lung. 28 Conversely, neutrophil disruption ameliorates organ failure by attenuating endothelial injury under septic conditions. 29, 30 Although the endothelial glycocalyx is present on healthy endothelial cells and plays a critical role in vascular homeostasis, 2,22,31e35 neutrophils and secreted cytokines injure the endothelial glycocalyx and subsequently cause organ failure. A previous study also indicated that disruption of the endothelial glycocalyx affects the pathogenesis of acute respiratory distress syndrome. 5 Prostacyclin-2 production from endothelial cells is promoted through binding with AT and heparan sulfate on endothelial cells. Prostacyclin-2 inhibits inflammatory cytokine secretion and binding to endothelial cells and neutrophils. Furthermore, AT binds to syndecan-4 on the surface of neutrophils and subsequently inhibits neutrophil migration. 13, 15 Similarly, AT was reported to inhibit the secretion of IL-6, IL-8, and p-selectin from endothelial cells by acting through protease-activated receptors. Moreover, AT was found to attenuate acute lung injury by decreasing high mobility group box 1 production. 36 Our study here showed that rAT treatment inhibited the endothelial glycocalyx injury that occurs with LPS-induced endotoxemia Q18 . Because attenuation of inflammation might protect the endothelial glycocalyx structure, 29,30 the antiinflammatory effect of rAT can be regarded as one of the beneficial effects of rAT treatment targeting LPS-induced vasculitis. Endothelial Cell Repair by rAT The occurrence of accelerated DNA repair after rAT administration was supported by the results of our gene ontology analyses, which showed the up-regulation of gene sets related to DNA helicase activity, regulation of telomere maintenance, and DNA-dependent ATPase activity in rAT-treated mice. Ki-67, which is related to the cell cycle, was expressed at higher levels in pulmonary endothelial cells in rAT-treated mice than in untreated mice. These findings suggest that DNA repair was promoted more strongly in endothelial cells in rAT-treated mice than in control mice. Endogenous cellular processes cause several types of DNA damage, including oxidation of bases, generation of DNA strand interruptions by reactive oxygen species, hydrolysis of bases, alkylation of bases, and The American Journal of Pathologyajp.amjpathol.org 9 mismatch of bases. In acute respiratory distress syndrome, neutrophils migrate to the lungs, secrete reactive oxygen species and inflammatory cytokines, and, ultimately, cause pulmonary tissue injury. 37 Thus, although the DNA of endothelial cells is injured under endotoxemia, rAT treatment might promote DNA repair. PCNA is a protein involved in the replication and repair of DNA and the adjustment of the cell cycle. 24 Phosphorylated g-H2A.X is related to the activation of the local DNA damage repair pathway. 25 rAT treatment increases the PCNA-positive cell number and g-H2A.X phosphorylation, along with increasing the levels of Ki-67epositive cells. Because these molecules are integral to DNA repair, these results suggest that rAT treatment may accelerate DNA repair in several cell types in the lungs. We found that rAT treatment attenuated endothelial glycocalyx injury caused by septic vasculitis. Because the endothelial glycocalyx exists on the surface of endothelial cells and is synthesized by endothelial cells, 38 DNA repair of endothelial cells might be closely associated with endothelial glycocalyx synthesis in septic vasculitis. We also found that rAT treatment up-regulated the gene set related to ciliary plasm. Cilia are organelles present in eukaryotic cells that are shaped like slender protuberances and are classified into two types: motile and nonmotile. Motile cilia exist on the respiratory epithelium covering the respiratory tract, where they serve in the mucociliary clearance that sweeps mucus and dirt out of the lungs. 39 Here, our results showed that cilia were present on the respiratory epithelium lining the respiratory tract and that rAT treatment might affect respiratory epithelial cells and influence the repair of cilia. Because nonmotile cilia (primary cilia) are present on almost all types of cells, excluding blood cells, the up-regulation of the gene set related to ciliary plasm also could be affected by endothelial primary cilia. However, clarification of this matter requires further investigation. Sepsis is a complex disease compared with the LPS-induced simple endotoxemia that developed in an experimental model. The focus of the current study was to investigate the direct relationship between septic vasculitis and endothelial glycocalyx injury. Thus, we used an endotoxemia model. However, this animal model does not mimic certain typical septic conditions, such as bacterial infection. This represents a limitation of our study. In addition, because alveolar epithelial cells, the basement membrane, and endothelial cells make three tightly bound layers, it is difficult to completely distinguish them using a light microscope. Furthermore, we analyzed gene expression using RNA isolated from total lung. For further confirmation Q19 , future study is required. This study also did not investigate the adverse effects of rAT in healthy mice. Because the endothelium appears normal in healthy mice, it is possible that rAT did not cause adverse effects such as hemorrhage or infusion site reactions. Treatment with rAT protects the endothelial glycocalyx (which is essential for vascular homeostasis) against injury by both inhibiting the damage caused by several factors including anti-inflammation and accelerating the restoration of endothelial cells. Because rAT already is used in clinical applications, its administration could be regarded as a novel strategy for the treatment of septic vasculitis. ajp.amjpathol.org -The American Journal of Pathology 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 References Q22 The third international consensus definitions for sepsis and septic shock (Sepsis-3) Glycocalyx and sepsis-induced alterations in vascular permeability Permeation of the luminal capillary glycocalyx is determined by hyaluronan Capillary endothelial surface layer selectively reduces plasma solute distribution volume The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis Ultrastructural alteration of pulmonary capillary endothelial glycocalyx during endotoxemia Effects of two weeks of metformin treatment on whole-body glycocalyx barrier properties in db/db mice Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo TNF-alpha induced shedding of the endothelial glycocalyx is prevented by hydrocortisone and antithrombin Therapeutic restoration of endothelial glycocalyx in sepsis The purification and mechanism of action of human antithrombin-heparin cofactor What do dysfunctional serpins tell us about molecular mobility and disease? Clinical review: molecular mechanisms underlying the role of antithrombin in sepsis Protection of the endothelial glycocalyx by antithrombin in an endotoxin-induced rat model of sepsis Protective effect of a newly developed fucose-deficient recombinant antithrombin against histoneinduced endothelial damage Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition Recombinant thrombomodulin protects against LPS-induced acute respiratory distress syndrome via preservation of pulmonary endothelial glycocalyx Oseltamivir treatment prevents the increased influenza virus disease severity and lethality occurring in chronic ethanol consuming mice Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions Chronic alcohol consumption increases the severity of murine influenza virus infections Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks Critical role of lysine 134 methylation on histone H2AX for gamma-H2AX production and DNA repair Differential responses of the endothelial and epithelial barriers of the lung in sheep to Escherichia coli endotoxin Contributions of capillary pathway size and neutrophil deformability to neutrophil transit through rabbit lungs Contribution of neutrophils to acute lung injury Neutrophil elastase inhibition ameliorates endotoxin-induced myocardial injury accompanying degradation of cardiac capillary glycocalyx Neutrophil elastase damages the pulmonary endothelial glycocalyx in lipopolysaccharide-induced experimental endotoxemia Endothelial glycocalyx and coronary vascular permeability: the fringe benefit Fine structures of capillary and endocapillary layer as revealed by ruthenium red Endothelial glycocalyx as an additional barrier determining extravasation of 6% hydroxyethyl starch or 5% albumin solutions in the coronary vascular bed The endothelial glycocalyx: composition, functions, and visualization Revised Starling equation and the glycocalyx model of transvascular fluid exchange: an improved paradigm for prescribing intravenous fluid therapy High dose antithrombin III inhibits HMGB1 and improves endotoxin-induced acute lung injury in rats The acute respiratory distress syndrome Disparate effects of catecholamines under stress conditions on endothelial glycocalyx injury: an in vitro model Epithelial sodium channels (ENaC) are uniformly distributed on motile cilia in the oviduct and the respiratory airways Antithrombin Reduces Lung Injury The American Journal of Pathology -ajp.amjpathol.org We thank Yasuko Nogaki and Shoko Kumazaki for technical assistance. Recombinant antithrombin was provided by Kyowa Kirin, Co., Ltd.