Nitric oxide as mediator, marker and modulator of microvascular damage in ARDS.

Abstract

In this issue, Stuart-Smith and Jeremy join an interesting series of international debates regarding mechanisms of microvessel damage in acute respiratory distress syndrome (ARDS), in particular the role of endothelium derived relaxing factors (EDRF) in the pathophysiology and treatment of this disease.1Stuart-Smith K Jeremy JY Microvessel damage in acute respiratory distress syndrome: the answer may not be NO.Br J Anaesth. 2001; 87: 272-279Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Their review seems to promote the idea of endothelium-derived hyperpolarizing factor playing a primary role in ARDS whereas nitric oxide (NO), a prominent endothelium derived relaxing factor and regulator of intercellular communication, is attributed only as a minor component. The major claim of the authors, as reflected in the title, is that NO may not be the answer to microvessel damage in ARDS. This claim should certainly be investigated in the context of specific questions and in the context of our success or failure in understanding this life-threatening condition. The main characteristics of ARDS is non-cardiogenic pulmonary oedema and a mild degree of hypertension that results from acute alveolar injury causing respiratory insufficiency, decreased compliance, tissue hypoxaemia and multi-organ failure.2Anonymous Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network.N Engl J Med. 2000; 342: 1301-1308Crossref PubMed Scopus (10091) Google Scholar, 3Wyncoll DL Evans TW Acute respiratory distress syndrome.Lancet. 1999; 354: 497-501Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 4Ware LB Matthay MA The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4477) Google Scholar Although the exact cellular and molecular basis for ARDS remains uncertain more than 30 years after the original description of the syndrome, an intense and dynamic inflammatory response as discussed in the review of Stuart-Smith and Jeremy has been implicated in the microvascular and alveolar damage. A considerable body of experimental work appears to suggest that endotoxin, enhanced leukocyte and endothelial activation, and interaction with upregulation of a pro-inflammatory cytokine network all culminating in extensive oxidative stress are important components of the pathology underlying ARDS.5Hasleton PS Roberts TE Adult respiratory distress syndrome – an update.Histopathology. 1999; 34: 285-294Crossref PubMed Scopus (56) Google Scholar Complementing the observational and descriptive studies identifying these mechanisms, there is enormous research showing the functional and survival benefit from sophisticated treatments that efficiently neutralize some of these components including anti-endotoxin molecules, neutralizing antibodies against pro-inflammatory cytokines, neutrophil adhesion molecules, and recombinant and engineered antioxidants in a wide range of animal studies.6Conner BD Bernard GR Acute respiratory distress syndrome. Potential pharmacologic interventions.Clin Chest Med. 2000; 21: 563-587Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar These studies clearly demonstrate our ability to successfully prevent alveolar damage in controlled laboratory situations with one or more intelligent therapeutic strategies. As it is pointed out in many recent editorials and reviews on ARDS, none of these strategies, however, decreased mortality among patients with ARDS.2Anonymous Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network.N Engl J Med. 2000; 342: 1301-1308Crossref PubMed Scopus (10091) Google Scholar 3Wyncoll DL Evans TW Acute respiratory distress syndrome.Lancet. 1999; 354: 497-501Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar This important discrepancy between the success in the animal studies and failure in the clinical situation can be explained by a number of variables. These include: the dynamic nature of the condition and the already established dysfunction at the time of admission and treatment; great heterogeneity of the patient population; and lack of biochemical predictors of endothelial and epithelial injury to identify patients who may benefit from a given treatment strategy.3Wyncoll DL Evans TW Acute respiratory distress syndrome.Lancet. 1999; 354: 497-501Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Another, more pessimistic opinion could be that similarly to septic shock, the therapeutic premises in ARDS may be flawed.7Natanson C Hoffman WD Suffredini AF Eichacker PQ Danner RL Selected treatment strategies for septic shock based on proposed mechanisms of pathogenesis.Ann Intern Med. 1994; 120: 771-783Crossref PubMed Scopus (413) Google Scholar Targeting endotoxin or inhibiting the host inflammatory response in humans may not be beneficial. Studies of pro-inflammatory cytokines have shown that no single cytokine consistently predicts either the onset or the outcome of ARDS.8Martin TR Lung cytokines and ARDS: Roger S. Mitchell Lecture.Chest. 1999; 116: 2S-8SAbstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar In contrast, it is becoming increasingly recognized that immune cells operate in a very complex network and they play both pathogenic and protective roles. Similarly, it is becoming clear that the balance of proinflammatory and anti-inflammatory factors, oxidants and antioxidants, and cytotoxic and protective factors determine the net inflammatory response and dysfunction in the lungs. Rather than targeting one single mediator or cytokine or immune cells, current efforts are directed at increasing our scientific knowledge of the complex timing of mediator release and the net pro-inflammatory, pro-oxidant and cytotoxic imbalance during early ARDS. Our inability to treat established cellular injury might be related to deeper problems in our understanding of the biology of disease. While no one can doubt the significant progress in medical science related to molecular biology and chemistry, several great thinkers of cell biology voiced a critical opinion regarding the lack of our understanding of the determinants of normal ‘living state’, which is likely related to the dynamic and intimate supramolecular organisation especially of the cellular interior and physico-chemical properties of cytosol. As part of his introduction to electronic biology, the Nobel Prized Szent-Gyorgyi suggested that there were major ‘gaps’ in our understanding of the electronic and perhaps magnetic properties of intracellular structural proteins which he believed were the critical elements of the normal living state.9Szent-Gyorgyi A Bioelectronics and cancer.J Bioenerg. 1973; 4: 533-562Crossref PubMed Scopus (31) Google Scholar Similarly, Ling argued that a total and lasting cure of deadly diseases requires a re-evaluation of the physical basis of life and living matter.10Ling GN A physical theory of the living state: application to water and solute distribution.Scanning Microsc. 1988; 2: 899-913PubMed Google Scholar Finally, Bulkley has offered the ‘Life-as-Physics’ paradigm for consideration since ‘Life-as-Chemistry’ as a singular theory to understand life has not solved any of the really basic mysteries of life.11Bulkley DH An electromagnetic theory of life–II: Testing.Med Hypotheses. 1992; 38: 305-310Abstract Full Text PDF PubMed Scopus (3) Google Scholar Regarding ARDS, a better understanding of the physical basis of human endothelial and epithelial cell biology and survival might be needed to provide secure foundations to combat cellular injury in the future. While we are currently not successful in treating acute lung injury there are indications that some of our efforts are successful in preventing further injury and might promote healing after the original insult. Recent improvements in supporting care of ARDS patients including aggressive treatment of underlying conditions such as infections, judicious fluid management and haemodynamic support and early enteral nutrition appear to slightly reduce mortality in individual centres when compared to historical controls.3Wyncoll DL Evans TW Acute respiratory distress syndrome.Lancet. 1999; 354: 497-501Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar It has also become apparent that improper mechanical ventilation might promote further injury on an already damaged lung. The use of inappropriate ventilation pressures and tidal volumes causing alveolar overdistension or repeated collapse and reopening has been shown to produce not only barotrauma but also an inflammation related lung injury. One of the most significant progresses in this field is the recent report by the ARDS Network demonstrating a 22% decrease in mortality in a randomized multicentre trial by a protective low tidal volume ventilation strategy in 861 patients with acute lung injury.2Anonymous Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network.N Engl J Med. 2000; 342: 1301-1308Crossref PubMed Scopus (10091) Google Scholar These recent data provide a framework and study structure, where additional protective strategies could successfully be tested. It might suggest that minimizing further injury and promoting lung healing might be achievable, and could be an important aspect of our efforts. From this introduction it is obvious that no single molecule has been identified as the likely successful target of a potential magic bullet of cure for ARDS. Thus NO is not the only mediator which does not provide the full answer, but the real question is whether or not NO is involved in the complicated events underlying lung injury. In this setting, pulmonary vascular reactivity is only one of the determinants of the problem, which involves in addition changes in permeability, endothelial and epithelial physiology, cell injury and survival. We believe that there are ample theoretical considerations, experimental evidence and clinical observations to suggest that NO remains an important mediator, marker and modulator of alveolar damage in ARDS. Nitric oxide has unquestionably occupied a pivotal position in lung research during the last 15 years. With the masterful discovery of EDRF and its identification as NO, basic scientists and clinicians have embarked on an important journey to study the relevance of this molecule in the pulmonary circulation and airway in both health and disease. This early period was characterised by extreme enthusiasm; NO achieved a status of the ‘molecule of the year’ in 1992 by Science with the lead article announcing ‘NO News Is Good News’. The recent atmosphere with headlines of ‘Just say NO to inhaled nitric oxide for ARDS’, and ‘Is nitric oxide inhalation a “cosmetic” therapy in ARDS?’, and the article of Stuart-Smith and Jeremy obviously reflect a changing climate and reduced enthusiasm regarding the role of NO as a therapeutic strategy in ARDS.12Matthay MA Pittet JF Jayr C Just say NO to inhaled nitric oxide for the acute respiratory distress syndrome.Crit Care Med. 1998; 26: 1-2Crossref PubMed Scopus (36) Google Scholar 13Payen DM Is nitric oxide inhalation a “cosmetic” therapy in acute respiratory distress syndrome?.Am J Respir Crit Care Med. 1998; 157: 1361-1362Crossref PubMed Scopus (26) Google Scholar But before we investigate this problem let us look into the potential role of NO and especially endogenous NO in normal lung physiology and during injury. As in the systemic circulation, the primary function of NO in the pulmonary vasculature is vasodilation. Although this is more easily demonstrated with conduit vessels, NO also functions in small vessels and regulates pulmonary vascular resistance in both animals and in humans. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Studies utilizing NO synthase inhibitors and selective targeted deletions of eNOS show acute modulation of pulmonary vascular tone in response to hypoxia in isolated perfused lungs of the mouse.14Fagan KA Tyler RC Sato K Fouty BW Morris KGJ Huang PL McMurtry IF Rodman DM Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation.Am J Physiol. 1999; 277: L472-L478PubMed Google Scholar In chronic situations right ventricular systolic pressure was elevated in eNOS- and iNOS-deficient mice. Similarly, in humans, infusion of NO synthase inhibitor caused an increase in PVR and changes in pressure flow relationships demonstrating that tonic release of NO is an important regulator of pulmonary vascular tone.15Cremona G Wood AM Hall LW Bower EA Higenbottam T Effect of inhibitors of nitric oxide release and action on vascular tone in isolated lungs of pig, sheep, dog and man.J Physiol. 1994; 481: 185-195Crossref PubMed Scopus (96) Google Scholar These data strongly imply that contrary to the suggestion by Stuart-Smith and Jeremy, NO is an important feature of lung microvessels and is not a restricted feature of large vessels. Thus pathological conditions characterized by increased pulmonary vascular resistance may indeed be explained by attenuation of NO bioactivity. Although the constitutive expression profiles of NOS in airway epithelial cells appear to be more complicated, it is conceivable to deduce that NO also acts by modulating bronchial tone and counteracts airway hyper-reactivity in stimulated conditions. A result of the bronchodilator and vasodilator effect might be co-ordination of ventilation and perfusion to the same alveoli. In addition to regulation of vascular tone, endogenous NO has been implicated in modulating capillary permeability. Although there appears to be some species variability, there is enough evidence to conclude that under normal conditions endothelial NO acts to reduce capillary permeability and attenuation of NO release by NOS inhibitors appears to augment oedema formation in the lung.16Bernareggi M Mitchell JA Barnes PJ Belvisi MG Dual action of nitric oxide on airway plasma leakage.Am J Respir Crit Care Med. 1997; 155: 869-874Crossref PubMed Scopus (71) Google Scholar 17Sprague RS Stephenson AH McMurdo L Lonigro AJ Nitric oxide opposes phorbol ester-induced increases in pulmonary microvascular permeability in dogs.J Pharmacol Exp Ther. 1998; 284: 443-448PubMed Google Scholar While we argue strongly that the above considerations establish a potential role for endogenous NO to regulate microvascular injury in terms of vascular tone, reactivity and permeability, we agree with Stuart-Smith and Jeremy that NO appears to be a double-edged sword in the setting of lung injury. This likely relates to important characteristics of NO as a redox active molecule with great reactivity towards other reactive species and to its important ability to modulate cellular signal transduction. Depending on the experimental conditions NO might act either as antioxidant or an oxidizing species. Under normal conditions NO is involved in mediating biological responses and a subtle oxidative stress associated with normal cellular metabolism is involved only in elucidating some oxidant-dependent signal transduction. Upon excessive oxidative stress, cellular constituents including proteins, nucleic acid and lipids become targets of the oxidizing species underlying cell injury. Under these conditions NO might react with a near diffusion limited reaction with superoxide to form peroxynitrite to further augment oxidative stress.18Beckman JS Beckman TW Chen J Marshall PA Freeman BA Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.Proc Natl Acad Sci USA. 1990; 87: 1620-1624Crossref PubMed Scopus (6698) Google Scholar Equally important however is the efficiency of NO to terminate lipid peroxide propagation and to act as antioxidant in this situation.19Rubbo H Radi R Trujillo M et al.Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives.J Biol Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar These reactions are greatly modulated by the concentrations of reactants and the composition of the micro-environment in which these reactions take place. Importantly both of these reactions consume NO with the consequence of decreased availability of NO to elicit its normal bioactivity to mediate cGMP accumulation and vasodilatation. Taking these considerations into account it is not surprising to recognize that there are data suggesting increased NOS expression, increased NO concentrations, peroxynitrite formation and modulation of lung injury in various experimental models and in the human situation. These changes appear to vary greatly depending on the stimulus used. In sepsis and LPS models, overproduction of NO appears to dominate perhaps via iNOS induction. This overproduction of NO might actually contribute to oxidative damage and increased permeability through peroxynitrite formation.16Bernareggi M Mitchell JA Barnes PJ Belvisi MG Dual action of nitric oxide on airway plasma leakage.Am J Respir Crit Care Med. 1997; 155: 869-874Crossref PubMed Scopus (71) Google Scholar 20Haddad IY Pataki G Hu P Galliani C Beckman JS Matalon S Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.J Clin Invest. 1994; 94: 2407-2413Crossref PubMed Scopus (574) Google Scholar In other models of primary oxidative insult such as activated neutrophils, and ischaemia-reperfusion models, NO might reduce oxidative stress, neutrophil adhesion, pulmonary transit time and cellular injury.21Sato Y Walley KR Klut ME English D D’yachkova Y Hogg JC van Eeden SF Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression.Am J Respir Crit Care Med. 1999; 159: 1469-1476Crossref PubMed Scopus (89) Google Scholar In addition to modulation of oxidative stress and antioxidant status, NO has implications to pro-inflammatory cytokine balance and apoptotic mechanisms. Cytokine release and upregulation of inflammatory genes largely depend on intracellular activation of NF-κB-transcription factor. There are important studies to show that NO might stabilise IκB, the protein primarily responsible for controlling NF-κB-activation.22Peng HB Libby P Liao JK Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B.J Biol Chem. 1995; 270: 14214-14219Crossref PubMed Scopus (631) Google Scholar Thus NO might be important in regulating the intensity and duration of pro-inflammatory cytokine activation. Similarly, NO has been implicated in the inhibition of caspase mediated apoptosis and conferring anti-apoptotic characteristics.23Dimmeler S Haendeler J Nehls M Zeiher AM Suppression of apoptosis by nitric oxide via inhibition of interleukin-1 beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.J Exp Med. 1997; 185: 601-607Crossref PubMed Scopus (786) Google Scholar This might be an important aspect of NO mediated cytoprotection. However, in both of these respects NO can act not only as a friend but a foe activating NF-κB and causing apoptosis at higher concentrations. In any instance, these arguments do not lessen but strengthen the claim regarding the primary importance of NO as a potential mediator and modulator of microvascular damage in ARDS. If NO plays such an important role can it be used as a biochemical marker of onset and disease activity, and as a predictor of outcome? The above considerations suggest that ARDS is associated with a complicated picture of NOS expression, NO generation and NO consumption and that NO concentrations will be different accordingly to the dynamically changing cytokine environment, the nature of airway inflammation, neutrophil activation, production of reactive oxygen species, and acidity in the immediate environment of endothelial and epithelial cells. In addition there are important sampling and methodological considerations. The dynamic nature of the disease, and the spatial heterogeneity of the pathological lesions would require serial samples from affected regions. Although there are no methods to sample lung tissue repeatedly in patients with ARDS, alveolar and bronchial cells might be obtained by bronchoalveolar lavage (BAL) and brushings, which might provide information about dynamism of NOS expression and its correlation with inflammatory and cytotoxic activities. Due to its short half-life, only stable products of NO can be analysed as nitrite and nitrate in BAL and blood specimens; these, however, may not reflect tissue levels and consumption of NO. There is interesting data regarding peroxynitrite formation and nitration of BAL and plasma proteins in ARDS, which require further confirmation and analysis.20Haddad IY Pataki G Hu P Galliani C Beckman JS Matalon S Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.J Clin Invest. 1994; 94: 2407-2413Crossref PubMed Scopus (574) Google Scholar 24Lamb NJ Quinlan GJ Westerman ST Gutteridge JM Evans TW Nitration of proteins in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome receiving inhaled nitric oxide.Am J Respir Crit Care Med. 1999; 160: 1031-1034Crossref PubMed Scopus (70) Google Scholar 25Gole MD Souza JM Choi I et al.Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome.Am J Physiol Lung Cell Mol Physiol. 2000; 278: L961-L967PubMed Google Scholar In addition to these biochemical assays, there has been an exciting development and attempt to investigate intrapulmonary levels of NO at the bedside by using analysis of exhaled gases. Many properties of NO favour partition between the fluid and gas phase and many fascinating and difficult questions remain in this rapidly developing field, the important progress allows us to formulate an hypothesis regarding the link between NO concentrations in exhaled breath and NO consumption in ARDS.26Marczin N Riedel B Gal J Polak J Yacoub M Exhaled nitric oxide during lung transplantation [letter].Lancet. 1997; 350: 1681-1682Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar 27Marczin N, Exhaled nitric oxide in acute lung injury: measurements and physiological implications. In: Matalon S and Sznajder JI, eds. Etiology and Treatment of Acute Lung Injury. EOS Press, in pressGoogle Scholar Current data suggest that similarly to experimental conditions, NO consumption reactions dominate in ARDS, since exhaled NO levels are lower in both fully developed ARDS and lung injury during transplantation.26Marczin N Riedel B Gal J Polak J Yacoub M Exhaled nitric oxide during lung transplantation [letter].Lancet. 1997; 350: 1681-1682Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar 28Brett SJ Evans TW Measurement of endogenous nitric oxide in the lungs of patients with the acute respiratory distress syndrome.Am J Respir Crit Care Med. 1998; 157: 993-997Crossref PubMed Scopus (81) Google Scholar What about replacement therapy using intravenous and inhaled NO donors? If NO is consumed in ARDS, is it not the logical therapy of choice? Before we answer this question we have to admit that NO administration has already provided medicine with great therapeutic tools to help patients. Nitroglycerin is routinely used as a vasodilator and anti-anginal agent. Similarly, inhaled NO, a very simple respiratory therapy has provided anaesthetists with a much-needed selective pulmonary vasodilator of unmatched value in managing acute right ventricular failure even in the absence of lung injury. The surprising qualities including vasodilatation in ventilated regions of the injured lung thereby improving shunt and arterial oxygenation prompted the use of inhaled NO in ARDS.29Frostell C Fratacci MD Wain JC Jones R Zapol WM Inhaled nitric oxide. A selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction.Circulation. 1991; 83: 2038-2047Crossref PubMed Scopus (968) Google Scholar As pointed out by Claude Lenfant in his introduction to the monograph of NO and the Lung by Zapol and Bloch, ‘The story of NO is almost unique in the sense that the original discovery… precipitated an urge for clinical and therapeutic application that often bypassed the need for further work on the fundamentals’.30 The discovery of peroxynitrite and the central regulatory role of NO in pulmonary oxidant reactions appear to be two of these fundamentals.18Beckman JS Beckman TW Chen J Marshall PA Freeman BA Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.Proc Natl Acad Sci USA. 1990; 87: 1620-1624Crossref PubMed Scopus (6698) Google Scholar These reactions might offset the many theoretical and potential benefits of inhaled NO in ARDS. Further studies will clarify whether these benefits can be unmasked by targeting reactive oxygen species and augmenting the efficiency of inhaled NO by specific phosphodiesterase inhibitors. We do not believe that we have an answer yet. N. Marczin1 2 D. Royston2 1National Heart and Lung Institute and Department of Anaesthesia Imperial College of Science Technology and Medicine Heart Science Centre 2Department of Anaesthetics Royal Brompton and Harefield NHS Trust Harefield Hospital, UK