key: cord-270213-ygb64yxc authors: Williams, Alexander T.; Muller, Cynthia R.; Govender, Krianthan; Navati, Mahantesh S.; Friedman, Adam J.; Friedman, Joel M.; Cabrales, Pedro title: Control of systemic inflammation throughearly nitric oxide supplementation with nitric oxide releasing nanoparticles date: 2020-10-02 journal: Free Radic Biol Med DOI: 10.1016/j.freeradbiomed.2020.09.025 sha: doc_id: 270213 cord_uid: ygb64yxc Amelioration of immune overactivity during sepsis is key to restoring hemodynamics, microvascular blood flow, and tissue oxygenation, and in preventing multi-organ dysfunction syndrome. The systemic inflammatory response syndrome that results from sepsis ultimately leads to degradation of the endothelial glycocalyx and subsequently increased vascular leakage. Current fluid resuscitation techniques only transiently improve outcomes in sepsis, and can cause edema. Nitric oxide (NO) treatment for sepsis has shown promise in the past, but implementation is difficult due to the challenges associated with delivery and the transient nature of NO. To address this, we tested the anti-inflammatory efficacy of sustained delivery of exogenous NO using IV infused NO releasing nanoparticles (NO-np). The impact of NO-np on microhemodynamics and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model was evaluated. NO-np treatment significantly attenuated the pro-inflammatory response by promoting M2 macrophage repolarization, which reduced the presence of pro-inflammatory cytokines in the serum and slowed vascular extravasation. Combined, this resulted in significantly improved microvascular blood flow and 72-hour survival of animals treated with NO-np. The results from this study suggest that sustained supplementation of endogenous NO ameliorates and may prevent the morbidities of acute systemic inflammatory conditions. Given that endothelial dysfunction is a common denominator in many acute inflammatory conditions, it is likely that NO enhancement strategies may be useful for the treatment of sepsis and other acute inflammatory insults that trigger severe systemic pro-inflammatory responses and often result in a cytokine storm, as seen in COVID-19. Sepsis represents a dynamic progression of host-pathogen interactions that progressively 64 causes a systemic inflammatory response syndrome (SIRS) and ultimately leads to multi-65 organ dysfunction syndrome (MODS) even after the initial insult has been controlled. 1 The 66 complications associated with sepsis are the most common cause of death in non-coronary 67 intensive care units (ICUs) worldwide. Medical care costs related to sepsis treatment add up to 68 approximately $20 billion in the United States. 2,3 However, the development of a 69 comprehensive study to target key aspects of sepsis development and progression has been 70 challenging and most of the results obtained from bench top experiments are hard to translate 71 to the bedside. The difficulty to generate translatable data in experimental studies comes 72 mainly from two reasons. First, the large number of variables that play a role in the 73 deterioration of cell and tissue function during SIRS and sepsis makes it almost impossible to 74 pinpoint a single therapeutic target. 3,4 Second, the complexity and diverse sources of human 75 sepsis makes the development of an animal model that is reproducibly translatable, in which 76 different theories could be tested, almost impossible. Thus, a middle ground should be set in 77 which individual components of sepsis can be used to understand the response of the insulted 78 organism. 79 80 A well-described hallmark of sepsis is endothelial dysfunction in response to a cytokine 81 'storm', which is associated with an increase in a series of negative consequences arising from 82 overproduction of reactive oxygen species (ROS), disruption of the glycocalyx, and endothelial 83 nitric oxide synthase (eNOS) uncoupling, all contributing to increased adhesion of red blood 84 cells (RBCs), white blood cells (WBCs), and platelets to the endothelium lining, enhanced 85 platelet activation, blood stagnation, decreased tissue perfusion and increased vascular 86 permeability. 1, 5, 6 Experimental therapies designed to target this aspect have been promising 87 and represent pathways that could be targeted to increase survival. 5,6 From a clinical 88 standpoint, several studies have highlighted that microvascular function is drastically impaired 89 in patients in different stages of sepsis. 7 The evidence indicates that the gold standard, fluid 90 replacement therapies, fail to recover microvascular blood flow causing poor perfusion, which 91 has been associated with increased mortality. 8, 9 Routinely monitored clinical variables poorly 92 reflect the true state of the microcirculation in central and peripheral tissues. 10 Recently it has 93 been proposed that in order to properly restore cardiovascular homeostasis, the 94 microcirculation should be targeted as a functional unit. Intravital microscopy and microvascular measurements. The window chamber was 169 studied using transillumination on a custom intravital microscope. Briefly, the animals were 170 restrained on a plexiglass tube with a longitudinal opening from which the window protruded 171 and then they were fixed to the stage of an upright microscope (BX51WI, Olympus, New Hyde 172 Park, NY) as described and depicted elsewhere. 20 Measurements were carried out using a 40ˣ 173 (LUMPFL-WIR, numerical aperture 0.8, Olympus) water immersion objective. The microscope 174 was equipped with a high-speed video camera (Fastcam 1024 PCI, Photron USA), which was 175 used to record videos of the microvascular blood flow at 1,000 frames per second (fps). 176 Briefly Survival. In addition to measuring 297 functional parameters, we also assessed 298 survival of mice dosed with LPS over 72 299 hours (Figure 4) CD206, indicating an M1-like macrophage phenotype, which is considered to be inflammatory. 323 In fact, over 72% of macrophages tested via FACS presented an M1-like phenotype for 324 animals treated with control-np, but only 16% of macrophages represented an M1-like 325 phenotype for animals treated with NO-np. However, animals treated with NO-np showed a 326 larger population of M2-like macrophages (representing 33% of macrophages harvested), 327 which are typically considered to be anti-inflammatory, and are associated with tissue repair. 328 To supplement these macrophage phenotypes, we also measured the cytokine profile of 329 treated mice after LPS injection. Cytokine profile. Cytokines were measured 24 and 48 hours after LPS injection (Figure 6) . 339 After 24 hours, animals treated with NO-np showed significantly higher levels of cytokines 340 traditionally considered to be anti-inflammatory (interleukin [IL]-10, TGF-beta), and significantly 341 lower levels of cytokines associated with a proinflammatory response (IL-1, 6, 12, MCP, and 342 TNF alpha) than animals treated with Control-np. However, after 48 hours, all cytokines 343 measured were elevated in animals dosed with Control-np compared to NO-np, but not all of 344 these comparisons were statistically significant. The principle finding of this study is that sustained delivery of exogenous NO using NO 350 releasing nanoparticles improved microvascular flow and capillary transit compared to animals 351 treated with control nanoparticles during LPS-induced endotoxemia. Additionally, this study 352 demonstrates that the adverse microcirculatory changes from LPS-induced endotoxemia There are a number of mechanisms that may be responsible for the improved survival and 372 maintenance of microvascular perfusion seen with NO-np treatment in this study, but the exact 373 mechanism or set of mechanisms is unclear. As we saw, and as others have demonstrated, 22 374 NO treatment decreased M1, and increased M2 polarization of macrophages. This can have a 375 multitude of downstream effects as M1 macrophages produce proinflammatory cytokines, 376 upregulate inducible nitric oxide synthase (iNOS), and enhance production of ROS and 377 reactive nitrogen species, which disrupt the glycocalyx, expose the endothelial layer, decrease 378 NO production from eNOS, and destroy endothelial cells. 23,24 379 380 Disruption of the glycocalyx undermines vascular integrity and eliminates the shear stress-381 based trigger for production of NO from eNOS. Mice treated with Control-np in our study 382 experienced significantly increased vascular permeability, as shown in Figure 4 , suggesting 383 endothelial cell and glycocalyx disruption in these animals. The glycocalyx has a number of 384 vital physiological roles, including acting as a barrier to preserve intravascular oncotic 385 pressure, 23 promoting RBC marginalization and the presence of a red cell-free layer, 25,26 386 reducing leukocyte adhesion and infiltration, 26 and preventing thrombus formation and 387 attachment, 27 so its disruption is likely a primary determinant for many of the clinical 388 presentations of sepsis, such as edema. As such, it is logical that the glycocalyx has been a 389 common drug target in improving outcomes from sepsis. Others have explored individual 390 mechanisms as a method to prevent glycocalyx degradation during sepsis, but these 391 J o u r n a l P r e -p r o o f strategies have not shown much clinical success. 23 However, NO-np treatment seems 392 particularly promising as it attempts to treat a more upstream target than previous therapies by 393 preventing the initial insult to the glycocalyx, and thus preserving endogenous NO signaling. 394 Endogenous NO may function to upregulate and activate nuclear factor erythroid 2-related 395 factor 2 (Nfr2) which represents a potent signaling pathway to counter an LPS-induced 396 inflammatory cascade, and to down regulate pro-inflammatory pathways and thus decrease 397 ROS production. 28 Limitations: LPS induced endotoxemia does not fully replicate the cascade of events that 464 occurs during septic shock, but no study can fully replicate the morbidity of septic shock due to 465 its distinct etiologies. However, this study did demonstrate that this mouse model replicates 466 many of the early changes that occur in septic shock, including increased vascular 467 permeability, and impaired systemic oxygen transport, and allowed us to observe these 468 changes in an awake, unanesthetized model. Since anesthetics have a poorly characterized 469 influence on inflammation, the awake unanesthetized state is most representative of changes 470 that may occur during sepsis. Unfortunately, this study did not measure any parameters 471 examining the status of the vascular endothelial glycocalyx, since sepsis damages the 472 glycocalyx, and exogenous NO could help protect the glycocalyx. Future studies should 473 examine changes in the glycocalyx by measuring its breakdown molecules, such as heparan 474 sulfate and sydecan-1, in the plasma, or measure glycocalyx integrity and thickness via 475 fluorescent lectin binding, in order to directly study glycocalyx disruption during LPS-induced 476 endotoxemia, and its protection by NO-nps. 477 478 Conclusion 479 Pathogenetic Mechanisms of Septic Shock 506 Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and 507 associated costs of care Epidemiology of severe sepsis Severe Sepsis and Septic Shock Sepsis and endothelial permeability Novel therapies for microvascular permeability in 515 sepsis The microcirculation is the motor of sepsis Severe abnormalities in microvascular perfused vessel density are associated 520 to organ dysfunctions and mortality and can be predicted by hyperlactatemia and 521 norepinephrine requirements in septic shock patients Microvascular resuscitation as a therapeutic goal in 523 severe sepsis 525 Early difference in tissue pH and microvascular hemodynamics in hemorrhagic shock 526 resuscitation using polyethylene glycol-albumin-and hydroxyethyl starch-based plasma 527 expanders B. & Intaglietta, M. Microcirculation: Its Significance in Clinical and Molecular Microcirculation: Physiology, Pathophysiology, and 532 Clinical Application Clinical review: Clinical imaging of the 534 sublingual microcirculation in the critically ill -where do we stand? LPS/TLR4 signal transduction pathway Animal Models of sepsis: setting the stage Sustained release nitric oxide releasing nanoparticles: characterization of a novel 542 delivery platform based on nitrite containing hydrogel/glass composites A nanoparticle delivery vehicle for S-nitroso-N-acetyl cysteine: 546 Sustained vascular response 548 Sustained release nitric oxide from long-lived circulating nanoparticles. Free radical 549 biology & medicine 49 Technical report-a new chamber 551 technique for microvascular studies in unanesthetized hamsters A. & Leunig, M. Dorsal Skinfold Chamber Preparation in Mice: Studying 554 Microhemodynamic parameters quantification 556 from intravital microscopy videos Macrophage Polarization Mediates Anti-inflammatory Effects of Endothelial Nitric Oxide 560 Signaling Glycocalyx and 562 sepsis-induced alterations in vascular permeability The M1 and M2 paradigm of macrophage activation: time 564 for reassessment Implications Enzymatic Degradation 566 of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red 567 Cell Free Layer of the Rat Cremaster Muscle Modulates Immobilization of Leukocytes at the Endothelial Surface The glycocalyx: a novel diagnostic and 572 therapeutic target in sepsis Activation of 574 endothelial nitric oxide synthase by dietary isoflavones: Role of NO in Nrf2-mediated 575 antioxidant gene expression The Coagulopathy of Acute Sepsis The role of microvascular thrombosis in sepsis Endogenous and exogenous nitric oxide protect against intracoronary 582 thrombosis and reocclusion after thrombolysis Nitric Oxide Regulates Exocytosis by S-Nitrosylation of N-586 ethylmaleimide-Sensitive Factor Nitric oxide and its relationship to thrombotic disorders 590 Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot 591 properties The role of 593 the microcirculation in multiple organ dysfunction syndrome (MODS): a review and 594 perspective Nitroglycerin in septic shock after intravascular volume resuscitation Nitrite protects against morbidity and mortality associated with TNF-or LPS-induced 600 shock in a soluble guanylate cyclase-dependent manner Reducing Ischemia/Reperfusion Injury by the Targeted Delivery of Nitric 604 Oxide from Magnetic-Field-Induced Localization of S-Nitrosothiol-Coated Paramagnetic 605 Nanoparticles 607 COVID-19: consider cytokine storm syndromes and immunosuppression Interactions of coronaviruses with ACE2, angiotensin II, and RAS 610 inhibitors-lessons from available evidence and insights into COVID-19 CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked 615 by a Clinically Proven Protease Inhibitor Antigenicity of the SARS-CoV-2 Spike Glycoprotein A 622 crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced 623 lung injury ACE2-angiotensin-(1-7)-Mas axis and oxidative 625 stress in cardiovascular disease Harnessing nitric oxide 627 for preventing, limiting and treating the severe pulmonary consequences of COVID-19 Inflammatory response was reduced with nitric oxide releasing nanoparticles (NO-nps) Endotoxemia-induced microvascular deficits were alleviated by NO-nps NO-nps promoted M2 macrophage repolarization, and reduced M1 macrophage population NO-nps could improve outcomes of insults that cause a cytokine storm