Invited Review Article Microparticles and Acute Lung Injury
Transcription
Invited Review Article Microparticles and Acute Lung Injury
Articles in PresS. Am J Physiol Lung Cell Mol Physiol (June 22, 2012). doi:10.1152/ajplung.00354.2011 1 Invited Review Article 2 3 Microparticles and Acute Lung Injury 4 5 Mark McVey1,2, Arata Tabuchi1, Wolfgang M. Kuebler1,3-6 6 7 1 The Keenan Research Centre at the Li Ka Shing Knowledge Institute of St. Michael´s; 8 Departments of 2Anesthesia, 3Surgery and 4Physiology, University of Toronto, ON; 5Institute of 9 Physiology, Charité – Universitätsmedizin Berlin and 6German Heart Institute Berlin, Germany; 10 11 Author contributions: All three authors contributed towards the writing and editing of the paper. 12 Declaration of Conflicts of Interest: None 13 Running head: Microparticles and acute lung injury 14 Body of the manuscript: 8616 words 15 Address for Correspondence: 16 Wolfgang M. Kuebler, MD Dr. med. Email: [email protected] 17 The Keenan Research Centre Phone: 416 864 5924 18 Li Ka Shing Knowledge Institute Fax: 416 864 5958 19 St. Michael´s Hospital 209 Victoria Street 20 M5B 1W8 Toronto, Ontario Canada 21 Key words: Acute lung injury, ARDS, microparticles, permeability, coagulation, inflammation, 22 endothelial function Copyright © 2012 by the American Physiological Society. 23 Abstract 24 The pathophysiology of acute lung injury and its most severe form, ARDS, is characterized by 25 increased vascular and epithelial permeability, hypercoagulation and hypofibrinolysis, 26 inflammation and immune modulation. These detrimental changes are orchestrated by crosstalk 27 between a complex network of cells, mediators and signaling pathways. A rapidly growing 28 number of studies have reported the appearance of distinct populations of microparticles (MPs) 29 in both the vascular and alveolar compartment in animal models of ALI/ARDS or respective 30 patient populations, where they may serve as diagnostic and prognostic biomarkers. MPs are 31 small cytosolic vesicles with an intact lipid bilayer that can be released by a variety of vascular, 32 parenchymal or blood cells and that contain membrane and cytosolic proteins, organelles, lipids 33 and RNA supplied from and characteristic for their respective parental cells. Due to this 34 endowment, MPs can effectively interact with other cell types via fusion, receptor-mediated 35 interaction, uptake, or mediator release, thereby acting as intrinsic stimulators, modulators or 36 even attenuators in a variety of disease processes. 37 This review summarizes current knowledge on the formation and potential functional role of 38 different MPs in inflammatory diseases with a specific focus on ALI/ARDS. ALI has been 39 associated with the formation of MPs from such diverse cellular origins as platelets, neutrophils, 40 monocytes, lymphocytes, red blood cells, endothelial and epithelial cells. Due to their 41 considerable heterogeneity in terms of origin and functional properties, MPs may contribute via 42 both harmful and beneficial effects to the characteristic pathologic features of ALI/ARDS. A 43 better understanding of the formation, function and relevance of MPs may give rise to new 44 promising therapeutic strategies to modulate coagulation, inflammation, endothelial function and 45 permeability either through removal or inhibition of "detrimental" MPs, or through 46 administration or stimulation of "favorable" MPs. 47 1 48 INTRODUCTION 49 ALI/ARDS. Acute lung injury (ALI) as a result from either direct or indirect mechanical, toxic, 50 infectious, or inflammatory challenges to the lung, presents initially as dyspnea and hypoxemia 51 which can rapidly progress to respiratory failure. ALI and its most severe form, the acute 52 respiratory distress syndrome (ARDS) are characterized by bilateral exudative chest infiltrates 53 visible by roentgenograms with no evidence of left heart failure and a PaO2/FiO2 ratio < 300 54 (ALI) or even < 200 (ARDS) (151, 171). Within days, ALI progresses from an initial 55 inflammatory exudative phase with a leaky edematous lung to a proliferative phase involving 56 fibrin deposition and a concomitant decrease in respiratory compliance that finally, if the patient 57 survives, proceeds to a fibrotic phase where the lung is burdened with scarring as it remodels 58 while attempting to heal. Despite the well-documented benefit of protective ventilatory strategies 59 and increasing knowledge of the etiological mechanisms of ARDS, mortality rates remain 60 relatively stagnant around 40% (133), with incidence estimates of up to 75 per 100 000 (171). 61 A broad spectrum of different cell types contribute to the pathogenesis of ARDS, including 62 alveolar epithelial and vascular endothelial cells which undergo changes in permeability leading 63 to edema formation and alveolo-capillary injuries, as well as platelets and immune cells such as 64 polymorphonuclear neutrophils (PMNs), alveolar macrophages and monocytes which are 65 critically involved in inflammatory responses (104, 108, 170, 171). In contrast to the 66 characteristic anticoagulant and profibrinolytic basal state of the lung, ALI is associated with a 67 prothrombotic and antifibrinolytic shift which favours deposition of fibrin and accelerates and 68 potentiates inflammation (19). Considerable research has focused on the direct interaction and 69 communication between different cell types both in vivo and in vitro, and revealed important 70 pathophysiological aspects of ALI. However a rapidly growing number of studies have recently 71 started to recognize the critical role of microparticles (MPs) as biomarkers, communication 72 shuttles between these different cells and cell types, and independent functional effectors in a 73 large variety of diseases ranging from endemic pathologies such as hypertension, diabetes and 74 atherosclerosis to frequently lethal syndromes such ALI (7, 16, 27, 28, 31, 36, 44, 60, 65, 79, 99, 75 118, 129, 135, 138, 155, 172). Here we provide a brief overview on the main characteristics of 76 MPs, review the current knowledge on their role in inflammatory disease with a particular focus 2 77 on ALI, and oppose their proposed detrimental and beneficial effects in lung injury, thus 78 providing a rationale for their future use as both therapeutics targets and tools. 79 80 MICROPARTICLES. The first accounts of MPs date back to 1967 at which time they were 81 considered to be nothing more than cellular dust (177). In fact, MPs are tiny cell-derived, intact 82 vesicles that are formed by partitioning off from activated or apoptotic eukaryotic cells (Figure 83 1). MPs are defined by their size, which ranges between 50 nm and 1 µm, and their distinctive 84 lipid layer composition. MPs have intact lipid bilayers, yet their outer layer has a 85 characteristically high level of negatively charged phospholipids, particularly phosphatidylserine 86 (PS). MPs may contain membrane and cytosolic proteins, transcription factors, genetic material 87 such as ribosomal RNA (rRNA), messenger RNA (mRNA), and microRNAs (miRNA) as well 88 as lipids or even organelles supplied from parent cells (22, 32, 109, 116). Occasionally, 89 membrane vesicles of less than 0.1 µm are sub-categorized as nanoparticles, and have been 90 proposed to originate from lipid rafts on parent cell membranes; accordingly, they may display 91 distinct protein endowments and functions (reviewed in 77, 134, 155). 92 93 MICROPARTICLES SUBSERVE INTERCELLULAR INFORMATION EXCHANGE. The 94 distinct cellular elements retained from precursor cells allow MPs to function as transcellular 95 delivery systems for the exchange of biological signals. Notably, information exchange between 96 MPs and target cells can occur in a bidirectional manner, thus generating a continuous and 97 dynamic exchange of antigens, cell signaling molecules and genetic material that opens an entire 98 new spectrum of opportunities for tightly regulated (or dysregulated) signal propagation. 99 In principle, MPs can transfer information from the MP-generating donor cell to a wide range of 100 target cells by either direct cell-cell contact for delivery of genetic material, proteins and lipids or 101 alternatively by remote interaction through secretion of soluble mediators and effectors by MPs. 102 Currently, we distinguish at least four distinct mechanisms how MPs may interact with their 103 target cells which are illustrated schematically in Figure 2. First, MPs are able to stimulate 104 outside-in-signaling in target cells via presentation of membrane-associated ligands that directly 3 105 bind to their respective surface receptors, or via release of secreted factors such as for example 106 cytokines which act on the target cell via receptor-mediated or receptor-independent mechanisms 107 (102). Second, MPs can be internalized into recipient cells, a process that has also been proposed 108 to underlie the rapid clearance of circulating MPs from the blood (41). For example, 109 fluorescently tagged PMPs can be endocytosed by brain microvascular endothelial cells, and are 110 subsequently detectable in endosomes and lysosomes (50). Third, MPs may fully or partially 111 fuse with the target cell, allowing for complete or selective transfer of contents including 112 membrane and cytosolic proteins, bioactive lipids (109, 139) or even whole cell organelles as 113 recently demonstrated for the microvesicular delivery of mitochondria by mesenchymal stem 114 cells (73). Last, MPs may deliver genetic material in form of mRNA and miRNA. While the 115 encoding mRNAs can be directly translated to alter the target cell proteome (6, 45), miRNAs are 116 small non-coding RNAs that can modulate gene expression in target cells at the post- 117 transcriptional level, in that they bind to complementary sequences on target mRNAs, commonly 118 resulting in translational repression and gene silencing. The export of miRNA from donor cells is 119 largely regulated by ceramide (166), a notion that gains relevance in light of the parallel 120 implication of ceramide in MP generation (155) which may thus facilitate miRNA egress with 121 MPs in a coordinated fashion. At the level of the target cell, subsequent miRNA entry is 122 facilitated by either endocytosis of MPs, or by fusion of their lipid bilayer with the plasma 123 membrane (166). Lately, miRNAs have been detected in a wide variety of different MPs as 124 generated from peripheral blood cells in humans (70), mesenchymal (39) or embryonic stem 125 cells (180), respectively, and miRNA transfer by MPs has been demonstrated to critically 126 modulate target cell responses such as for example angiogenesis in endothelial cells (64). As 127 miRNAs have recently been recognized to contribute to injurious mechanisms in ALI (183), 128 transfer of miRNA from MPs to inflammatory or lung parenchymal cells may present an exciting 129 novel pathomechanisms in this disease which clearly deserves further exploration. 130 131 ORIGIN OF MICROPARTICLES. Parent cells of MPs include but are not limited to platelets, 132 megakaryocytes, monocytes, epithelial cells, lymphocytes, PMNs, endothelial cells, red blood 133 cells, reticulocytes, mast cells, stem cells, astrocytes, glial cells, smooth muscle cells and cancer 4 134 cells (16, 20, 23, 139). Similar to the wide range of parent cells, MP formation can be triggered 135 by diverse physiological, pathophysiological or experimental stimuli as discussed in greater 136 detail below. 137 Unstimulated, resting cells actively maintain their membrane asymmetry via phospholipid 138 transporter enzymes which localize negatively charged phospholipids on the inner leaflet, facing 139 into the cell. When cellular homeostasis is disturbed or lost such as in apoptosis, the activities of 140 these phospholipid flippases, floppases and scramblases are dysregulated leading to hastened 141 changes in membrane composition of the inner and outer membrane leaflets. This process is a 142 characteristic feature of MP formation, as it allows for the relocation of the PS rich layer of the 143 inner leaflet to the exterior leaflet (116, 155). The resulting changes in membrane composition 144 and regular asymmetry in conjunction with processes such as intracellular calcium accumulation 145 and calpain activation reviewed in detail elsewhere (116, 119) cause membrane blebbing and 146 ultimately the formation of MPs (Figure 3). 147 Externalization of PS is a precursor step in MP formation allowing for a common shared surface 148 marker for identification of many MPs which can be assayed by annexin V binding. Interestingly 149 however, it appears that there are MPs which do not stain appreciably for annexin V, which adds 150 controversy to the identification and strict definition of what constitutes an MP (116, 153). 151 Further it seems that some MPs can also be formed before regular membrane asymmetry is 152 disrupted, such as in cultured endothelial cells (155). Specifically, Davizon and colleagues have 153 linked MP formation to lipid rafts in cell membranes (42, 155), raising the possibility that lipid 154 rafts may be actively involved in MP formation under some conditions, and that MPs may 155 maintain part of the unique functional and signaling properties of membrane lipid rafts (Figure 156 3). Currently, the cellular machinery implicated in MP formation constitutes an active area of 157 research (reviewed in 134), that is expected to yield important insights not only into the 158 mechanisms of MP formation, but likewise into its regulation and function. 159 In healthy humans, circulating MPs are largely derived from platelets with some contributions 160 from leukocytes and minimally from endothelial cells (34, 36). Size and composition of this 161 population of basal MPs depends on physiological variables including exercise, menstrual cycle, 5 162 age, or exposure to extreme environments such as SCUBA diving, in addition to countless 163 pathologic conditions some of which are discussed in later sections of this review (34, 55, 163). 164 Platelet MPs have a short circulating half life of less than 10 minutes in rabbits (141), but were 165 shown to circulate for approximately 30 minutes in mice (53). MP clearance from the circulation 166 occurs predominantly by endothelial cell internalization (41) or splenic removal, and the half live 167 of platelet MPs is markedly prolonged in splenectomized mice as compared to controls (129). 168 Moreover, a recent study in thrombocytopenic humans reported a markedly longer half life for 169 circulating MPs that was in the order of 5 to 6 hours (142, 143). The considerable variation in 170 half lives in these studies suggests a major influence of both host and MP variables such as 171 species, blood cell levels and mode of activation, as well as potential methodological variability 172 with respect to isolation and assay for MPs between these different studies. 173 Tracking the origin of MPs is complicated not only by the wide variety of possible parent cell 174 sources and triggering stimuli, but in addition by the fact that these cell-derived vesicles can be 175 and presumably are constantly phenotypically modified by contact with different cell types at 176 different steps in their production. Different mechanisms of fusion between cells and MPs which 177 could lead to phenotypic modulation of MPs and/or other cells have been recently reviewed in 178 detail by Quesenberry and colleagues (139). A particularly striking example in this respect is the 179 provocative hypothesis that endothelial progenitor cells may origin from the fusion of platelet 180 microparticles (PMPs) with mononuclear cells as suggested by proteomic analyses (137). 181 Repetitive fusion events with tissue factor (TF) expressing cells may also account for the 182 expression of TF on various types of cells and MPs which would not otherwise express this 183 marker. For example, platelets can become TF positive after contact with TF expressing MPs 184 shed 185 (lipopolysaacharide; LPS) or protein kinase C activation by phorbol myristate acetate (PMA), 186 platelets and subsequent PMPs may obtain their TF antigens from either fusion with monocytes 187 or monocyte MPs (MMPs) (28). Beyond mixing of distinct lineage markers between cells and 188 MPs there are also challenging distinctions between MPs of similar parent cell origins. Subtle 189 variations in parent cells can be difficult to detect due to phenotypic similarities of their 190 respective MPs, as in the case of platelet versus megykaryocyte-derived MPs (54, 143). Hence, a from PMNs and monocytes (145). 6 Following stimulation with endotoxin 191 series of elaborate experimental techniques have been developed to allow for the exact 192 identification of the origin of MPs and the functional significance of these cell-derived vesicles. 193 194 CHARACTERIZATION OF MICROPARTICLES. In the stepwise process of experimentally 195 isolating MPs there are considerable technical variables which can impact on the recovery and 196 phenotype of MPs obtained. Some of these procedural differences include the actual phlebotomy, 197 the use of anticoagulants, storage conditions, length and number of freeze-thaw cycles, 198 centrifugation, washing, pelletting or filtration of MPs, and specific recommendations (Table 1) 199 have been developed to optimize each of these steps (186). A series of review articles have 200 scrutinized the impact of these variables which underline the sensitive and precarious nature of 201 MP isolation and stress how caution is required to avoid undesired differences in MP isolates 202 obtained from slight variations in handling and processing procedures (91, 134, 153, 155). The 203 rapidly growing number of publications pertaining to MPs showcases a diverse array of 204 techniques for preparation and isolation of MPs which is bound to generate considerable 205 variability in terms of the final products assayed (78, 134). In recent years, this dilemma has 206 stimulated workshops and meetings specifically dedicated to the attempt to limit variability and 207 standardize the study of MPs, and namely the International Society on Thrombosis and 208 Haemostatis has made strides to centralize methodologic approaches for MP isolation (90). 209 Detection and characterization of MPs has involved strategies such as antibody capture ELISA 210 assays, flow cytometry, functional coagulation assays, electron (Figure 1), atomic force or 211 confocal microscopy, HPLC, capillary electrophoresis and mass spectrophotometry (11, 24, 37, 212 43, 75, 77-79, 89, 91, 115, 129, 137, 153, 155, 162, 174, 181, 185). Each of these techniques 213 comes with its specific advantages and limitations, as summarized in table 2. Due to its general 214 availability and versatility, flow cytometry has become the most commonly used technique for 215 characterization of MPs and discerning their parent cell of origin depending on the expression of 216 characteristic surface markers (Table 3). Most strategies with flow cytometry involve 217 identification of MPs based on forward and side scatter characteristics as well as annexin V 218 staining for PS in conjunction with analysis of markers characteristic for specific parent cells to 219 test for their contribution to the individual MPs being assayed. The use of flow cytometry to this 7 220 end bears a series of strengths, but also some limitations (77, 78, 89, 147, 153) which have been 221 discussed in detail in recent literature. Proteomics presents an important alternative tool for 222 particularly small particles not amenable to flow cytometry (117) as it can yield comprehensive 223 information on characteristic protein expression patterns which may be utilized to identify and/or 224 characterize MPs. This approach has been used in a series of investigations to assay different 225 aspects of the human MP proteome (75, 96). 226 227 HETEROGENEITY OF MICROPARTICLES. Our current understanding of the conditions 228 under which MPs are formed, and their individual actions is still incomplete. Little is known 229 about exact parameters which dictate when, how, from where, why, which and how many MPs 230 are formed. What is becoming evident is the vast variation in terms of genetic material and 231 antigens MPs can express from their parent cells of origin or other cells or MPs they contact 232 during formation or during circulation, which can be incorporated or eventually redistributed to 233 other MPs or cells. The literature has likely not captured the full extent of variations possible as 234 the characterization of MPs under different conditions is incomplete and we currently lack the 235 tools to accurately assay antigens present at very low abundances on the surface of MPs. MPs 236 come in a variety of sizes ranging from smaller nanoparticles such as those often produced by 237 red blood cells up to microparticles large enough to rival platelets (134). Similar parent cells are 238 able to produce a spectrum of different sized MPs depending upon whether MP formation occurs 239 spontaneously at baseline or following stimulation (25, 74). Even cell lines such as Jurkat T cells 240 yield a spectrum of different sized MPs (164). Notably, this heterogeneity in size can directly 241 impact on MP surface marker expression and function as shown for platelet MPs which contain 242 different antigens such as growth factors, chemokines and receptors depending on their size (74). 243 Similar to their heterogeneity in terms of origin, size, and endowment with antigens, MPs also 244 differ widely with regard to their functional effects. MPs can shuttle information from remote 245 cells to similar or phenotypically different cells which might never be in direct proximity. 246 Different MPs can affect certain cells in different ways as for example diabetics’ T cell-derived 247 MPs can decrease endothelial NO synthase (eNOS) and increase caveolin-1 expression causing 248 endothelial dysfunction as opposed to MPs formed by in vitro activation of human T cells which 8 249 express sonic hedge hog on their membranes and can stimulate NO production as evidenced by 250 the improved endothelial function in a cardiac ischemia reperfusion injury model (106, 114). 251 Alternatively, a single MP subtype such as PMPs can interact with and recruit numerous target 252 cells such as monocytes, NK cell, B and T cells (114). 253 MP generation is triggered by a spectrum of stimuli ranging from physiological to experimental 254 stimuli (Table 4). MPs can be formed constitutively by various parent cells under physiological 255 conditions but in differing amounts, resulting in a higher abundance of circulating PMPs as 256 compared to red blood cell or lymphocyte derived MPs (RMP and LMP, respectively) in healthy 257 patients (116). Additional physiological triggers for MP formation involve stresses such as heavy 258 exercise or cyclic changes such as menstruation. MP formation in response to pathophysiological 259 stimuli adds another level of complexity, as different triggers will stimulate MP formation from 260 different parent cell types. Multiple simultaneous or sequential triggers can further enhance MP 261 diversity and therefore potentially expand their range of function. For example, platelet storage 262 conditions including platelet rich plasma, apheresis blood bank derived human platelets, or cold 263 storage of platelets with and without contact with propyl gallate will produce distinct PMP 264 variants (33, 144, 168, 178). Addition of platelet activation inhibitors such as prostaglandin E1 265 (PGE1), theophylline and aprotinin during storage reduce the number of large but not small 266 PMPs formed, and modify their function in terms of platelet factor 3 activity (25). Under 267 experimental conditions, a diverse array of potent cell stimulants such as calcium ionophores, 268 phytohemagglutinins, LPS, fMLP and PMA can be utilized to induce MP formation with a high 269 efficiency and reproducibility (Table 4). 270 271 MICROPARTICLES IN INFLAMMATORY DISEASE 272 Some of the MPs characterized to date have been identified as biomarkers for a wide variety of 273 inflammatory conditions. Elevated levels of circulating PMP, LMP, MMP and endothelial MPs 274 (EMPs) have been shown to accompany pathologic conditions such as atherosclerosis, type 2 275 diabetes, vasculitis, pulmonary hypertension, coronary disease, cardiopulmonary resuscitation, 276 lupus, Crohn´s disease and rheumatoid arthritis (7, 27, 28, 36, 52, 118, 129, 135, 138, 155). 9 277 Likewise, systemic inflammatory states in pregnancy such as preeclampsia are associated with 278 elevated plasma levels of MPs (146). Infectious diseases are either directly associated with 279 increased levels of MPs as seen in malaria (61), or can give rise to disproportionate inflammatory 280 responses such as the systemic inflammatory response syndrome (SIRS), sepsis or the multi- 281 organ dysfunction syndrome (MODS), which are again associated with elevated levels of MPs 282 (129, 155). MPs have also been implicated mechanistically on several levels in the 283 pathophysiology of sepsis, including proinflammatory signaling pathways such as target cell 284 activation through reactive oxygen species production and coagulopathy in terms of MP 285 mediated TF presentation (113). 286 A survey comparing 36 patients with sepsis with 18 healthier patients without sepsis showed 287 elevated blood levels of total MPs, specifically PMPs and EMPs, whereas CD45+ LMPs were 288 lower in patients with sepsis as compared to controls (122). Interestingly, MPs from septic 289 patients enhanced aortic contraction when transfused intravenously to mice. This effect was 290 independent of NO modulation or COX enzyme expression but blocked by the specific 291 thromboxane A2 (TXA2) antagonist SQ-29548, indicating that circulating MPs may prevent 292 vascular hyporeactivity accounting for systemic hypotension in septic shock through a TXA2- 293 mediated mechanism (122). Furthermore, patients with SIRS have elevated levels of circulating 294 EMPs, increased binding of MPs to PMNs, higher procoagulant activity and higher circulating 295 levels of triggers for MP formation such as plasminogen activator inhibitor 1 (PAI-1) compared 296 with healthy controls (126). As we discuss later in greater detail, the effects of MPs are 297 frequently considered to aggravate the pathology in inflammatory and cardiovascular diseases, 298 and increased formation of MPs as seen in systemic inflammatory disorders is therefore 299 generally considered to constitute not only a biomarker, but an important pathophysiological 300 mechanism driving the disease. However, inflammation and the associated increase in circulating 301 MPs may also have protective effects, as pointed out by Soriano and coworkers who 302 demonstrated that increased inflammation and the presence of EMPs, PMPs and platelet 303 leukocyte conjugates are significantly associated with survival in septic patients (158). Hence, 304 more detailed and comprehensive experimental and clinical investigations are in dire need to 10 305 discern which MPs under what conditions exert beneficial or detrimental effects in inflammatory 306 diseases. 307 Over the past decade, it has become evident that inflammatory diseases are closely associated 308 with impaired coagulation and fibrinolysis, processes that can be regulated through MPs. Many 309 but not all MPs express tissue factor (TF) which interacts with factor VIIa facilitating activation 310 of factors IX and X, thus leading to generation of thrombin (103). TF MPs are characteristically 311 found in diseases where thrombosis is common such as cancer, sepsis, unstable angina, ALI, 312 sickle cell disease and thromboembolism (128, 129, 138, 165), giving rise to the speculation that 313 they may contribute critically to the disease process. TF is a key player in the activation of the 314 coagulation pathway, yet its action is not only determined by its expression levels, but also by its 315 accessibility in terms of conformational state which is controversial for different cells types or 316 conditions (128). Much of the circulating TF is in fact in an encrypted and therefore, inactive 317 state, while MPs represent a source of decrypted, active TF (127). LPS stimulates monocytes and 318 endothelial cells to produce TF which can lead to a coagulopathy that can escalate into DIC 319 (103). Co-incubation of platelets with monocytes and PMNs which produce high amounts of TF 320 following stimulation with pro-inflammatory agents such as LPS leads to enhanced presentation 321 of decrypted TF in the form of MPs, thus generating a prothrombotic milieu (127). Even MPs not 322 expressing TF can still possess considerable procoagulant properties as they contain anionic 323 phospholipids, including PS which acts as a surface to facilitate assembly of the clotting factor 324 machinery (103, 129, 138). Hence, MPs can be involved in coagulopathies associated with 325 inflammatory diseases such as sepsis as they possess prothrombotic characteristics with exposed 326 TF and PS, but conversely may also activate fibrinolysis via changes in plasminogen and 327 metalloproteases (113), or exert anticoagulant properties mediated through protein C and tissue 328 factor pathway inhibitor (TFPI) (117). In line with the notion that MPs may have both pro- and 329 anticoagulant effects, thrombus weight was shown to correlate with plasma levels of PMPs and 330 older MPs in a murine thrombus model, while LMPs showed an inverse correlation (140). 331 332 MICROPARTICLES AND ACUTE LUNG INJURY. Several studies have shown associations 333 of MPs with key structures and cells in ALI, giving rise to the notion that similar to systemic 11 334 inflammatory diseases, MPs may be a biomarker of the disease and play important roles by either 335 aggravating or counteracting (or potentially both) the ongoing disease process. For example, 336 interactions between cells such as platelets, PMNs and endothelial cells are central to the 337 pathology of ALI/ARDS, and MPs have been demonstrated to critically regulate these 338 interactions, and/or modify the phenotypes of the contributing cells by fusion and subsequent 339 transfer of cellular components. In the following, we will summarize recent data in support of an 340 association and possible functional role of MPs in ALI/ARDS and discuss potential novel 341 interactions between MPs and lung parenchyma or circulating blood cells that are likely pertinent 342 to lung injury. 343 344 POTENTIAL DETRIMENTAL EFFECTS OF MICROPARTICLES IN ALI 345 Excessive inflammation, changes in coagulation and fibrin deposition and increased permeability 346 of the alveolocapillary barrier with resulting protein extravasation and lung edema formation are 347 characteristic hallmarks of ALI (Figure 4). In the following, we highlight how MPs may impact 348 either directly or indirectly on the pathophysiology underlying these symptoms. 349 350 INFLAMMATION. Inflammatory changes in ALI/ARDS involve an abundance of 351 proinflammatory intra- and intercellular signaling pathways which include, but are in no way 352 confined to signaling via arachadonic acid (AA), TXA2, plasminogen activator inhibitor-1 (PAI- 353 1), nitric oxide (NO), reactive oxygen species (ROS), cytokines and protease activated receptors 354 (PARs). In this section, we highlight data demonstrating that MPs can modulate and/or replicate 355 these pathways (114, 120), and thus act as proinflammatory agents promoting ALI/ARDS. 356 Over past years, the tight interplay between platelets and PMNs has emerged as important aspect 357 in the pathogenesis of ALI (100, 160, 182). Similar to platelets, circulating PMPs can interact 358 with PMNs via linkages including glycoprotein Ib/Mac-1, β2-integins (CD11b)/Mac-1, or P- 359 selectin/PSGL-1 on PMPs/PMNs, thus causing PMN activation involving secretion of 360 interleukin (IL)-8 secretion, oxidative burst, degranulation, and enhanced leukocyte rolling (79, 12 361 99). Likewise, PMPs generated from LPS-stimulated platelets promote endothelial activation as 362 exemplified by the finding that they further increase the IL-1β induced expression of vascular 363 cell adhesion molecule (VCAM)-1 (30). Hence, circulating PMPs are not only a biomarker, but 364 more importantly, an active promoter of inflammatory disease processes in that they stimulate 365 endothelial cells, PMNs and notably also platelets. The latter was demonstrated in experiments 366 where PMPs were stimulated by soluble phospholipase A2; the newly formed AA is then rapidly 367 converted by PMPs to TXA2, a classic activator of platelets (14). In line with this view, ExoU, a 368 Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, increases both, PMP release 369 and TXA2 formation, and accordingly, resulted in lung thrombus formation and increased 370 vascular permeability in a murine model of pneumosepsis (101). Overall, a series of 371 proinflammatory effects of PMPs has emerged that is based on the activation of platelets, 372 monocytes, and vascular endothelial cells either directly by MP-derived AA, or by its 373 metabolism to bioactive prostanoids (12, 13, 15). This recognition substantiates a critical role of 374 PMPs in ALI, as TXA2 contributes pivotally to lung vascular leakage and inflammation in 375 experimental models of the disease (72). 376 Platelet-PMN and endothelial-PMN co-stimulation may not only be promoted by PMPs, but 377 likewise by PMN microparticles (NMPs), as endotoxin (LPS) can stimulate adherent PMNs to 378 release NMPs that generate platelet-activating factor (PAF), thus causing activation of platelets 379 (172), endothelium (149) and other inflammatory cells. In the airspaces, NMPs may also trigger 380 inflammatory responses and epithelial cell damage, as suggested from their abundance in sputum 381 from patients with cystic fibrosis (136). NMPs can furthermore directly activate endothelial cells 382 to generate proinflammatory cytokines such as interleukin-6, thereby further promoting vascular 383 inflammation (8, 111, 112). Endothelial cells may in turn likewise release MPs (EMPs) which 384 modulate inflammation and coagulation, and most prominently vascular barrier function, for 385 which reason they are discussed in greater detail in the section Permeability. 386 Lastly, inflammatory diseases such as ALI and ARDS are often associated with immune 387 modulation and its complex functional consequences including inflammatory damage to 388 bystander cells (123, 158, 173). Such immune modulation also involves platelets which can 389 actively alter innate and adaptive immune function (94, 152). For example, PMPs can stimulate 13 390 endothelial cells to generate a range of proinflammatory cytokines including IL-1, IL-6, IL-8 and 391 monocyte chemoattractant protein-1 (MCP-1) as well as monocyte derived interleukin (IL)-1, 392 tumor necrosis factor alpha (TNFα) and IL-8 (reviewed in 20, 120). MPs have also been directly 393 associated with immunomodulatory effector molecules such as RMP and PMP derived CD40L, 394 IgG and complement found in stored blood products (80). Immunomodulatory and 395 proinflammatory effects of MPs may be particularly relevant in the context of transfusion-related 396 ALI (TRALI), as storage of blood products will propel formation of MPs (25, 33, 144) that will 397 subsequently activate PMNs (80). Taken together, MPs of diverse cellular origins including 398 LMPs, PMPs, RMPs, NMPs and EMPs directly or indirectly promote inflammatory responses 399 and may thus propel ALI, in that they stimulate cytokine release and promote PMN activation, 400 migration and PMN-platelet interaction. 401 402 COAGULATION. Severe infections lead to activation of the coagulation cascade and altered 403 fibrinolysis, with the lung being a key recipient of fibrin and clots during ALI and sepsis (165). 404 The hypercoagulable and hypofibrinolytic changes seen in ALI/ARDS contribute pivotally to the 405 pathology of the disease (170, 171) and involve characteristic changes in mediators of 406 coagulation and fibrinolysis, including TF, thrombomodulin, TFPI, plasminogen activator 407 inhibitor 1 (PAI-1), PS, and von Willebrand factor (vWF). 408 TF is considered to play an important role in the pathophysiology of lung injury, as it can 409 activate PAR1 and PAR2 which are involved in inflammatory and procoagulatory responses 410 causing fibrin deposition in the lung (82, 165). Importantly, TF is not only expressed on 411 activated mononuclear cells, endothelial cells, alveolar epithelium, or respiratory macrophages, 412 but likewise on many MPs (11, 165). The procoagulant activity of PMP membranes exceeds that 413 of their parent platelet membranes by up to 100-fold (116, 156). In vascular homeostasis, the 414 effects of TF are counteracted by TFPI which blocks the TF-induced stimulation of the 415 coagulation cascade by reversibly inhibiting factor Xa and thrombin. Severe infections such as 416 sepsis which constitute typical predispositions for ALI are characterized by an imbalance 417 between elevated TF and lowered TFPI levels causing enhanced coagulation and inflammation 418 (165). The relevance of this imbalance is highlighted by observations from humans (56) and 14 419 experimental models of ALI in which blockade of TF by antibodies (175) or TFPI (48) reduced 420 lung damage and improved pulmonary function. Monocytic MPs (MMPs) can attenuate the 421 expression of TFPI and thrombomodulin, an endothelium-derived anti-coagulant factor, thus 422 increasing thrombogenicity in vitro (4), whereas TF is abundantly expressed on MPs under 423 inflammatory conditions (91). In healthy human volunteers, MP-borne TF procoagulant activity 424 increased 8-fold within 4 hours after infusion of LPS (11). Hence, infectious or inflammatory 425 stressors may contribute to and/or aggravate ALI in part by the formation and release of TF 426 studded MPs which in the presence of an attenuated anticoagulatory response will generate the 427 characteristic prothrombotic milieu associated with ALI, especially in consideration of the 428 previously discussed fact that TF on MPs is often decrypted and active. Notably, TF activity on 429 MPs in ALI/ARDS is not necessarily confined to the vascular compartment (16), but – in line 430 with the clinical presentation of fibrin clots in the distal airspaces – may similarly apply to the 431 alveolar compartment where increased TF production by alveolar epithelial cells has been 432 documented (18, 165). In pulmonary edema fluid from mechanically ventilated (<7 ml/kg tidal 433 volume) patients with ARDS, Bastarache and colleagues detected higher concentrations of 434 epithelial cell marker receptor for advanced glycation end products (RAGE) and TF enriched 435 MPs in BALF as compared to patients with hydrostatic lung edema whose MPs also expressed 436 less TF and RAGE (16). It is possible that ARDS leads to either larger MPs that express more 437 RAGE receptors or perhaps a numeric increase in RAGE positive MPs compared with ARDS- 438 negative hydrostatic edema controls. Patients who died had a trend towards a higher MP 439 concentration in lung edema fluid compared with those who survived (p=0.073), attesting to a 440 detrimental effect of MPs in ALI/ARDS. Similar MPs could be generated in vitro by stimulation 441 of cultured alveolar epithelial cells with a mix of the proinflammatory mediators TNF-α, IL-1β, 442 and interferon-γ (16, 17). While both ARDS and non-ARDS controls in Bastarache’s study were 443 severely sick, BALF analyses from healthy pigs with volume controlled ventilation set to 444 maintain normocapnia (37.5-41 mmHg PaCO2) with 5 cmH2O PEEP revealed PMPs that were 445 positive for fibrinogen, vWF and P-selectin (124). This finding gains relevance in light of the 446 fact that mechanical ventilation, in particular at tidal volumes > 6 mL/kg body weight, will 447 exacerbate ALI and promote ventilator induced lung injury (VILI). PMPs in BALF were evident 15 448 after only 1 h of mechanical ventilation, yet unfortunately, actual tidal volumes applied were not 449 reported in this study so that it is unclear whether this effect was attributable to overventilation or 450 occurs even under conditions of protective ventilation. Notably, presence of PMPs was likewise 451 detected in human tracheal mucus obtained from five humans at extubation after surgery, 452 suggesting that this finding may reflect a more general effect of mechanical ventilation and/or 453 surgical trauma. 454 Similar to TF expressing MPs, a pro-coagulant environment in ALI may be likewise promoted 455 by PMPs. However, the degree to which circulating PMPs are actually elevated in patients with 456 ALI or ARDS remains to be elucidated. Earlier work by George and coworkers did not detect an 457 increase in blood PMPs in patients with ARDS from various causes, while post cardiac bypass 458 patients exhibited an increased plasma concentration of PMPs (60). Yet, it is conceivable that the 459 radiolabelled GPIIb antibody technique used to assay PMPs in this study did not screen for the 460 entire population of PMPs in the ARDS cohort. Beyond TF expression, MPs expose large 461 amounts of PS on their outer membranes thus creating an ideal surface for activation of the 462 coagulation pathway (148). In addition, MPs generate and release other prothrombotic factors 463 including thrombin (80) or vWF multimers which can promote and stabilize platelet aggregation 464 (20). In summary, there is ample evidence from in vitro, animal and human studies 465 demonstrating the formation of MPs with procoagulant activity under inflammatory conditions 466 and in ALI in both the vascular and alveolar compartment, where they are likely to contribute 467 pivotally to the hypercoagulable and hypofibrinolytic state characteristic for ALI/ARDS. 468 469 PERMEABILITY. The increased permeability of capillary and alveolar barriers in ALI leads to 470 the extravasation of high molecular weight protein and inflammatory cells into the airspaces of 471 the lung, thus impairing respiratory mechanics, attenuating gas exchange, causing increased 472 shunting and ventilation perfusion mismatch and contributing to the consolidation of lung tissue 473 during the subsequent fibroproliferative phase of ALI/ARDS. Several biolipid mediators such as 474 TXA2, phospho- and sphingolipids have been implicated in the pathogenesis of permeability- 475 type lung edema, and are notably also linked directly or indirectly to the formation and function 476 of MPs (62, 104). 16 477 As discussed before, PMPs can promote the formation of TXA2 from AA (14). This finding 478 gains relevance in the context of permeability-type edema as TXA2 inhibitors can reduce lung 479 vascular permeability in animal models of ALI (72, 182). In line with this view, increased levels 480 of circulating PMPs were associated with elevated TXA2 plasma concentrations and lung 481 vascular permeability in a mouse model of bacterial pneumosepsis with histological evidence of 482 thrombus formation in the lungs (101). In sepsis, the increased MP associated formation of TXA2 483 may serve to maintain a basal vasopressor effect (120, 122), yet this beneficial effect does not 484 translate to the low pressure system of the lung where increased TXA2 formation will primarily 485 promote lung edema. 486 In addition to eicosanoids, phospho- and sphingolipid mediators such as PAF and ceramide have 487 been proposed to contribute critically to vascular hyperpermeability in ALI (62). PAF can be 488 expressed on the surface of NMPs and promote platelet aggregation and association with PMNs 489 (172) while ceramide has been implicated in MP formation due to its involvement in lipid rafts 490 (155). Acid sphingomyelinase (ASM), which catalyzes the conversion of sphingomyelin to 491 ceramide, triggers the release of brain glial MPs, an effect that is blocked by ASM inhibitors or 492 in astrocytes obtained from ASM deficient (Smpd1-/-) mice (23). While it remains to be shown 493 whether a similar ASM-mediated mechanism contributes to MP formation in other non-glial cell 494 types, this finding raises the intriguing notion that the demonstrated detrimental effects of 495 ASM/ceramide signaling in ALI (87) relate to the increased formation of MPs. 496 497 ENDOTHELIAL FUNCTION. Apart from biolipid mediators, various MPs may directly impact 498 on endothelial function, thereby critically regulating vascular barrier properties as well as 499 promoting the hyper-inflammatory and hyper-coagulatory responses discussed in previous 500 sections. EMPs can attenuate NO release from cultured endothelial cells, and induce a 501 corresponding decrease in eNOS phosphorylation at Ser1179 and a decrease in hsp90 association 502 consistent with the notion of EMP-induced endothelial dysfunction (44). In line with this view, 503 EMP treatment impaired endothelium-mediated vasodilation in both mouse and human ex vivo 504 vessel preparations (44). Likewise, EMPs attenuate vasorelaxation in rat aortas in a dose 505 dependent fashion involving impaired NO signaling and endothelial function (29). These finding 17 506 gain relevance in the context of ALI in view of the frequently barrier-protective (85, 179), anti- 507 inflammatory and anti-coagulatory effects of basal endothelial NO production in the lung. 508 Endothelial dysfunction and impaired aortic vasorelaxation was likewise inducible in mice by 509 LMPs which caused overexpression of caveolin-1 and concomitant inhibition of endothelial 510 nitric oxide synthase (eNOS) (106). Endothelial NO production is also inhibited by smooth 511 muscle cell MPs via a β3 integrin mediated mechanism (49). The relevance of impaired 512 endothelial NO formation in the context of ALI and inflammation is highlighted by the fact that 513 IL-8 dependent PMN migration and endothelial transmigration in vitro are enhanced by the NO 514 synthase inhibitor L-NAME, and likewise by NMPs generated by L-NAME treatment, but not by 515 D-NAME or untreated PMNs (125). Conversely, NO may also regulate MP formation although 516 in different ways depending on cell type and/or experimental conditions, as highlighted by the 517 finding that L-NAME induced NMP formation from PMNs (125), while macrophage MP 518 formation in response to toll-like receptor agonists such as LPS is reduced by inhibition of 519 inducible nitric oxide synthase (iNOS) (59). Taken together, MP formation can be modulated by 520 the bioavailability of NO, and once formed MPs can attenuate local NO production, in particular 521 with respect to endothelial cells (20), which in turn may promote ALI and barrier permeability. 522 Accordingly, EMPs derived from PAI-1 stimulated endothelial cells can induce direct ALI in 523 mice and stimulate the release of IL-1β and tumor necrosis factor (TNF)-α leading to increased 524 PMN recruitment to the lung (31). In brown Norway rats, intravenous infusion of EMPs at 525 pathophysiologically relevant concentrations induced characteristic signs of pulmonary edema, 526 lung PMN infiltration and increased lung vascular permeability (44). Increased lung capillary 527 permeability was likewise evident in C57BL/6 mice when EMPs were infused as either primary 528 or secondary hit (44). EMPs infusion into C57BL/6 mice likewise led to increased inflammatory 529 cytokine release and lung PMN recruitment (31) and exacerbated LPS-induced lung injury. 530 Notably, the aggravation of LPS-induced lung injury was particularly prominent when EMPs 531 were infused prior to rather than simultaneous with LPS (31), indicating that EMPs can both 532 prime the lung for subsequent injurious stimuli or exacerbate lung damage in previously 533 challenged lung tissue. 534 18 535 CLINICAL IMPLICATIONS. The recognition of a potential detrimental effect of MPs in the 536 pathophysiology of ALI/ARDS opens up new therapeutic perspectives, in that removal of MPs 537 and/or inhibition of MP functions may present promising strategies for future treatment 538 interventions (32, 184). Based on a similar rationale, removal of PMPs by a membrane plasma 539 separator was recently tested in 8 patients undergoing apheresis, yet while considerable amounts 540 of PMPs were removed by filtration total numbers of circulating PMPs could not be diminished 541 by this intervention, potentially due to a stimulation of PMP formation by the plasmapheresis 542 procedure per se (66). As compared with mechanical removal, pharmacological attenuation of 543 circulating MP levels may not only be feasible, but may have inadvertently already been 544 introduced into the treatment of inflammatory diseases, as glucocorticoid treatment has been 545 shown to significantly reduce circulating PMP levels in patients with polymyositis and 546 dermatomyositis (154). Yet at the current stage, the significance of immunosuppression or 547 immunomodulation by glucocorticoids in the treatment of ALI/ARDS remains unclear due to 548 mixed clinical results (173), and a thorough analysis of the effects of steroid administration on 549 circulating MP levels and subpopulations is yet lacking. 550 Notably, agonists of peroxisome proliferator activated receptor (PPAR)-γ such as rosiglitazone, 551 which have been introduced as anti-diabetic drugs but also show efficiency in lung diseases such 552 as pulmonary hypertension (67, 68) may present an alternative approach to target deleterious MP 553 functions in inflammatory diseases. MMPs increase macrophage and monocyte superoxide anion 554 production, cytokine release and nuclear factor kappa-light-chain-enhancer of activated B cells 555 (NF-κB) activation via a PPAR-γ regulated mechanism that can be inhibited by PPAR-γ agonists 556 (5). PPAR-γ agonists have also been demonstrated to inhibit LMP mediated vascular dysfunction 557 and increased release of proinflammatory proteins from isolated murine aortae, (120), and the 558 upregulation of proinflammatory cytokines including IL-8 and monocyte chemotactic protein-1 559 by monocyte MPs in cultured human lung epithelial cells (83). PPAR-γ expression has been 560 shown to be reduced in ALI (97) while agonists of PPAR-α or -γ can attenuate experimental ALI 561 (9, 98, 150). While PPAR agonists may thus have therapeutic potential in the context of 562 ALI/ARDS, their effects on circulating MP levels and MP function in this disease remain to be 563 determined. 19 564 Several reviews of MPs have highlighted the interesting strategy to modulate PMP formation by 565 statin or polyunsaturated fatty acid (PUFA) therapy (42, 91). While PUFAs have been pursued as 566 therapeutic interventions in ARDS with reported benefits in terms of decreased permeability and 567 PMN recruitment (157), statin treatment remains controversial yet is actively pursued as 568 potential therapy for ALI/ARDS (26, 86). As randomized clinical trials are currently underway, 569 it will be of interest to see whether potential benefits of statin therapy in ARDS are likewise 570 associated with reduced levels or altered profiles of circulating MPs. 571 Importantly, attenuation of MP formation may also provide an attractive strategy to reduce 572 complications associated with the administration of stored blood products, including TRALI. 573 RMPs typically form after 10 days of storage, while PMPs accumulate earlier within days, and 574 levels of both can be significantly reduced if packed cells are leukoreduced or in the presence of 575 storage additives such as glucose, pyruvate, inosine, adenine and phosphate (80). Storage of 576 platelets with PGE1, theophylline or aprotinin as well as pharmacological inhibition of αIIbβ3 577 integrin signaling have been proposed to reduce ex vivo PMP production (25, 33) and may thus 578 help to reduce PMN activation and sequestration in the lungs subsequent to transfusion of stored 579 blood products. Compounds such as cyclospoin A and Orai 1 inhibitors have been proposed as a 580 means of decreasing levels of EMPs and PMPs respectively (5, 10, 21, 32). Further work is 581 required to examine the effects of eliminating or altering selected MP populations generated in 582 stored blood products to test whether such interventions may positively impact on the mortality 583 and morbidity of transfused experimental animals and ultimately patients. 584 585 POTENTIAL BENEFICIAL EFFECTS OF MICROPARTICLES IN ALI 586 MPs are not necessarily always associated with progressive organ dysfunction and disease 587 outcome. As recently discussed in seminal reviews by Morel and coworkers (120) and Dignat- 588 George and Boulanger (46), MPs are by no way always detrimental, but may frequently exert 589 beneficial effects, in that they exhibit anti-inflammatory and anti-coagulant potential and can 590 improve endothelial and vascular function under pathologic conditions (Figure 5). Experimental 591 evidence for the beneficial effects of MPs comprise the promotion of myocardial tissue repair 592 post infarction by PMPs, the stimulation of matrix degradation and new blood vessel formation 20 593 by EMPs through activation of matrix metalloproteases, or the triggering of angiogenic 594 endothelial responses and the reconstitution of endothelial function and NO generation 595 associated with reduced ROS production by LMPs carrying the developmental morphogen sonic 596 hedgehog (3, 20, 114). These findings exemplify the seemingly paradoxical beneficial effects 597 which have been documented for a variety of MPs under different pathologic conditions, a 598 notion that gains particular relevance in the context of ALI by the clinical observation that higher 599 levels of circulating LMPs or EMPs, respectively, are associated with increased survival in both 600 sepsis and ARDS (65, 158). The potential role of beneficial MPs gains additional momentum in 601 light of the fact that considerable numbers of MPs can also be released by mesenchymal stem 602 cells (MSCs) and may thus – at least in part – account for the paracrine effects by which MSCs 603 have emerged as a promising new therapeutic strategy in ALI (63, 92, 93, 107). MSCs secrete 604 MPs that are enriched in pre-miRNAs (35) and – in a rat renal injury model - could inhibit 605 apoptosis and promote proliferation of kidney epithelial cells by paracrine effects that are in part 606 mediated via intercellular transfer of mRNA and miRNA (58). MSCs have also recently been 607 shown to donate mitochondria to lung parenchymal cells in ALI by a microvesicular mechanism 608 that could imply microparticles (73); yet, the relevance of mitochondrial transfer for the effects 609 of MSCs has been challenged by others (38). Further investigations are needed to characterize 610 the contributions of MSC derived MPs in the benefits these cells appear to offer in the setting of 611 lung injury. In the following, we will discuss mechanisms which may potentially contribute to 612 the beneficial effects of non-stem cell derived MPs in ALI focusing on the same 613 pathophysiological characteristics of the disease as previously done for the detrimental role of 614 MPs. 615 616 INFLAMMATION. While we previously emphasized the role of TXA2 in the context of the 617 delivery and release of AA and AA metabolites by MPs, it must be taken into consideration that 618 not all eicosanoids are proinflammatory and increase barrier permeability. Notably, PMPs 619 contain lipoxygenase 12 which, when transferred to mast cells promotes production of the anti- 620 inflammatory leukotriene lipoxin 4A (LXA4) (161). In a murine model of experimental colitis, 621 injection of PMPs and LXA4 attenuated inflammatory responses, whereas lipoxygenase 12 21 622 protein deficient MPs or mast cell depletion reversed this beneficial effect (161). As LXA4 623 analogs have been shown to attenuate ALI in animal models (47, 76), MP mediated generation of 624 LXA4 might prove beneficial in ALI. 625 Though certain MPs formed in ALI may have the ability to cause detrimental immune 626 modulation as described in previous sections, others may confer beneficial immune functions 627 capable of enhancing survival or reducing morbidity. NMPs can stimulate secretion of 628 transforming growth factor (TGF)-β which blocks macrophage activity, and can express annexin 629 A1 which acts as an anti-inflammatory protein blocking adhesion of PMNs to endothelial cells 630 (40, 57). In addition, MPs generated from KATO-III human gastric cancer cells can interact with 631 monocytes and change their cytokine release profile from proinflammatory mediators such as 632 TNF-α to anti-inflammatory cytokines such as IL-10 (83). While the relevance of such effects in 633 the context of ALI/ARDS remains to be elucidated, these findings underscore the anti- 634 inflammatory potential harbored by many MPs. A recent analysis of ARDS patients 635 demonstrated elevations in both LMPs and NMPs within BAL samples of ARDS patients 636 compared to spontaneously breathing controls (65), while levels of PMPs did not differ amongst 637 ARDS patients compared with simplified acute physiology score (SAPS II) and sequential organ 638 failure assessment (SOFA) score matched ventilated intensive care patient controls without 639 ARDS and spontaneously breathing patients being bronchoscopically investigated for suspected 640 chronic lung diseases. Notably, survivors of ARDS had statistically elevated levels of LMPs 641 compared to the ventilated and spontaneously breathing control groups. This unexpected finding 642 raises the possibility that LMPs may not only be a biomarker of ARDS and/or potentially 643 promote inflammatory signaling and parenchymal damage, but may at times also be protective 644 and reflect a "healthy" level of immune function. This physiological effect may be absent in 645 severe inflammatory diseases such as ARDS, causing "immune paralysis" or a relative anergy of 646 immune function which may have ramifications on the host that directly impact mortality (123). 647 648 COAGULATION. While the high expression levels of TF in MPs are generally considered to be 649 procoagulant and barrier disruptive, they may also have anti-coagulant and barrier protective 650 properties through PAR1-mediated transactivation of PAR2 signaling pathways (82). In addition, 22 651 MPs can express a variety of anti-coagulant factors including thrombomodulin, TFPI, EPCR and 652 protein S (120). Thus, the potential involvement of MPs in anticoagulant pathways may present 653 both an intrinsic protective mechanism and a possible future therapeutic target in lung injury. 654 Notably, LMPs from hepatitis C patients have been shown to cause increased fibrinolysis after 655 fusion with hepatic stellate cells, thus decreasing fibrin deposition (84). 656 Activated protein C (APC) proteolytically inactivates key coagulation factors including Factor 657 Va and Factor VIIIa. Recombinant activated protein C has been shown to reduce acute lung 658 injury and increase alveolar ventilation in experimental animal models (71, 110, 167) and 659 individual clinical case reports (81). In sepsis patients, treatment with activated protein C (APC) 660 has been reported to show a trend towards increased survival that is notably associated with MP 661 formation and concomitant anti-inflammatory effects (132). However, a recent Cochrane review 662 did not recommend its use due to a failure to improve survival and an increased bleeding risk, 663 which has led to a decrease in its use as a therapy for severe sepsis (105). Interestingly 664 nevertheless in the context of this review, APC induces the release of MP-associated endothelial 665 protein C receptor (EPCR) that can bind APC and exert anti-coagulatory (131) and potentially 666 also anti-inflammatory and barrier protective effects (120). EPCR (131) is a member of the 667 major histocompatibility complex/CD1 superfamily that promotes protein C activation at the cell 668 surface (159), yet upon metalloprotease-mediated cleavage circulates in a soluble form (sEPCR) 669 (88) which neutralizes APC so that it can no longer inactivate Factor Va (95). Importantly, APC 670 bound to EPCR in microparticulate form retains anti-coagulant activity (131), and has been 671 shown to exert both anti-apoptotic and barrier-protective effects (130). 672 673 PERMEABILITY. APC may also exert important barrier protective effects, in that EPCR 674 ligation promotes transactivation of sphingosine 1-phosphate receptor 1 (S1P1) via PI3K- 675 dependent phosphorylation (51). Hence, APC-bearing MPs may limit vascular permeability via 676 the well-documented barrier enhancing signaling cascade downstream of S1P1 (169). This notion 677 is supported by experimental data demonstrating that EPCR/APC-bearing EMPs reduce 678 endothelial permeability in vitro via a mechanism that involves PAR-1, PI3K and notably also 679 the vascular endothelial growth factor receptor 2 (130). 23 680 681 ENDOTHELIAL FUNCTION. While MPs are frequently associated with endothelial and 682 vascular dysfunction (vide supra), several studies by the group of Ramarosin Andriantsitohaina 683 and Maria Carmen Martinez have demonstrated that the endothelium may also receive beneficial 684 signals from MPs carrying sonic hedgehog resulting in correction of endothelial injury, 685 reconstitution of NO release and reduced ROS production (3, 114, 121). The role of NO in this 686 context however is complex as it can exert both attenuating and aggravating effects on 687 inflammatory, coagulatory or permeability changes depending on its location, concentrations and 688 local microenvironment (85). While a recent meta-analysis did not support the rationale for 689 inhaled NO as an effective treatment strategy in ALI/ARDS (2), reconstitution of endogenous 690 NO production and an intact endothelial and vascular function may nonetheless provide 691 important benefits which could positively impact on clinical outcome in ALI/ARDS. 692 693 CLINICAL IMPLICATIONS. Taken together, MPs possess a variety of mechanisms by which 694 they may exert anti-inflammatory, anti-coagulatory and barrier-enhancing effects under healthy 695 or diseased conditions. While our understanding of these properties of MPs in general, and in the 696 context of ALI/ARDS in particular, is still in its infancy, the potential to exploit this intrinsic 697 property for therapeutic purposes poses a challenging, yet promising path to pursue. 698 699 SYNOPSIS AND IMPLICATIONS 700 There has been little in the way of effective innovations and interventions in the treatment of 701 ALI/ARDS beyond the introduction of protective ventilation (1) and restrictive fluid 702 management strategies (176). ALI/ARDS is commonly associated with increased formation of 703 MPs from various cellular origins found in both the distal airspaces and circulating blood. These 704 MPs are small vesicles with an intact lipid bilayer that may contain membrane and cytosolic 705 proteins, lipids, organelles, and genetic material from their parent cells that can function 706 autonomously or transfer their contents to other cells. While most studies have looked at MPs as 707 potential biomarkers for disease and progression of illness, MPs bear the potential to convey 24 708 both detrimental as well as beneficial effects with respect to the excessive inflammatory, 709 coagulatory and permeability responses characteristic for ALI/ARDS (Figure 4). Further 710 investigations into MPs actions are required to better understand their role in the 711 pathophysiology of acute lung disease, and to utilize their potential for therapeutic interventions. 712 Such concepts may include strategies to prevent the formation of MPs by altering flip flop 713 scramblase enzyme activities (116) or therapeutic manipulation of MP populations by 714 pharmacological interventions (138). Even more attractive is the potential perspective to use 715 specifically engineered "designer" MPs as vectors to move genes, proteins or specific cellular 716 functions between cells (20). Clearly, MPs add another layer of complexity to the already 717 multifaceted mechanisms involved in ALI/ARDS, but in parallel they may offer hope of 718 obtainable and specific sites for future pharmacologic manipulation. 719 720 ACKNOWLEDGEMENTS 721 Illustrations in tables 3 and 4, and figure 1 are courtesy of Stefania Spano. 722 25 723 LEGENDS 724 Table 1: Technical variables and recommendations for microparticle isolation. 725 Flow chart of variables and current recommendations for the isolation, storage and purification 726 of microparticles as proposed for platelet microparticles by Zwicker and colleagues (186). 727 CTAD, citrate theophylline adenosine and diphyridamole; MP, microparticle; PFP, platelet free 728 plasma; PMP, platelet microparticle. 729 730 Table 2: Advantages and limitations of techniques commonly applied in microparticle analysis. 731 Shown are commonly applied techniques for the analysis of microparticles and their relative 732 strengths and weaknesses in terms of microparticle enumeration, microparticle sizing, 733 determining the parent cell of origin, throughput, cost and availability. AFM, atomic force 734 microscopy; CLM, confocal laser microscopy; ELISA, enzyme-linked immune sorbent assay; 735 EM, electron microscopy; FCM, flow cytometry; HPLC, high performance liquid 736 chromatography; MS, mass spectrometry. 737 738 Table 3: Characteristic surface markers indicative of microparticle origin. 739 Given are characteristic surface markers used to detect microparticle populations (RIGHT) 740 which originate from specific parent cells (LEFT). CD, cluster of differentiation; EMP, 741 endothelial microparticle; IgG, immunoglobin; LMP, lymphocyte microparticle; MegMP, 742 megakaryocyte microparticle; MP, microparticle; MMP, monocyte microparticle; NMP, 743 neutrophil microparticle; PMP, platelet microparticle; RMP, red blood cell microparticle; vWF, 744 von Willebrand factor. 745 746 Table 4: Triggers for microparticle formation from different parent cells of origin. 747 Depicted are physiological, pathophysiological and experimental triggers of microparticle 748 formation (RIGHT) from platelets, megakaryocytes, lymphocytes, monocytes, red blood cells, 26 749 polymorphonuclear neutrophils and endothelial cells (LEFT). APC, activated protein C; ATII, 750 angiotensin II; autoAb, autoantibodies; C5a, complement factor C5a; C5B-9, complement factor 751 C5B-9; CD40L, CD40 ligand; CHX, cyclohexamide; EC, endothelial cell; fMLP, N- 752 formylmethionyl-leucyl-phenylalanine; IL-1, interleukin-1; L-NAME, L-NG-nitroarginine 753 methyl 754 phytohemagglutinin; PAI-I, plasminogen activator inhibitor 1; PMA, phorbol myristate acetate; 755 PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; ROS, reactive oxygen species; STS, 756 staurosporine; TNF-α, tumor necrosis factor-α; TRAP, thrombin receptor agonist peptide 757 SFLLRN. ester; LPS, lipopolysaccharide; PAF, platelet activating factor; PHA, 758 759 Figure 1: Release of microparticles from the surface of a polymorphonuclear neutrophil (PMN). 760 Electron micrographs of PMN and PMN derived microparticles (MPs). Scanning electron 761 micrographs show a) a fixed resting PMN (scale bar: 1.5 µm) and b) a fixed PMN stimulated 762 with 0.1 µM N-formylmethionine leucyl-phenylalanine for 5 minutes (scale bar: 1.5 µm) 763 displaying large pseudopodia and in addition, budding of small vesicles of 70-300 nm in size 764 from the PMN cell membrane (inset, arrowhead; scale bar: 400 nm). c) Transmission electron 765 micrographs following immunogold labelling show expression of cell surface antigens such as 766 complement receptor 1 on isolated MPs (scale bar – 150 nm); d) Thin sections of MPs 767 precipitated with Dynabeads prove vesicles to be bilamellar structures. Reproduced from Hess et 768 al. (69); Copyright 1999. The American Association of Immunologists, Inc. 769 770 Figure 2: Mechanisms of interactions between microparticles and target cells. 771 Interaction between microparticles (MPs) and their target cells via essentially four different 772 mechanisms: Depicted clockwise from top right to top left are a) outside-in-signaling via ligand- 773 receptor interaction or secreted factors, b) internalization and lysosomal processing of MPs 774 facilitates for example subsequent presentation of MP antigens on the surface of target cells, c) 775 fusion-mediated transfer of surface receptors, proteins and lipids, and d) delivery of genetic 776 material such as mRNA or miRNA. MPs may fuse either completely (c) or temporarily (d) with 27 777 target cells resulting in either complete or selective transfer of MP contents. Redrawn and 778 modified from a figure originally published in Beyer et al. (22). 779 780 Figure 3: Pathways of microparticle release from parent cells. 781 Cell activation and/or stimulation of pro-apoptotic signaling cascades cause a rapid loss in lipid 782 membrane asymmetry due to dysregulation of flippases, floppases, and scramblases via sudden 783 increases in intracellular calcium, resulting in externalization of phosphatidylserine (PS). 784 Concurrent cytoskeletal reorganization and contraction cause blebbing and subsequent shedding 785 of microparticles (MPs) into the extracellular space. MP formation from membrane lipid rafts 786 may lead to release of MPs that maintain part of the unique signaling characteristics of these 787 platforms. Redrawn and modified from a figure originally published in Chironi et al. (36). 788 789 Figure 4: Schematic representation of the proposed role of microparticles in acute lung injury. 790 (I) Depiction of normal healthy lung, interstitium and vasculature with associated basal 791 microparticle populations (color-coded). Acute lung injury (II) involves characteristic 792 pathologies including (IIa) increased permeability, (IIb) coagulation leading to thrombosis and 793 fibrin deposition, and (IIc) inflammation and immunomodulation. Each of these pathologies is 794 affected in distinct ways by specific microparticle populations that are in turn activated by 795 specific stimuli. AA, arachadonic acid; APC, activated protein C; EMP, endothelial MP (blue), 796 EpMP, epithelial MP (light purple); IL-1β, inteleukin 1β; INFγ, interferon-γ; LMP, lymphocyte 797 MP (red); LPS, lipopolysaccharide; LXA2, lipoxin A2; MacMP, macrophage MP (pink); MMP, 798 monocyte MP (purple); MP, microparticle; NMP, , polymorphonuclear neutrophil MP (orange); 799 PAI-1, plasminogen activator inhibitor 1; PHA, phytohemagglutinin; PMN, polymorphonuclear 800 neutrophil; PMP, platelet MP (light green); RMP red blood cell MP (dark green); ROS, reactive 801 oxygen species; TFMP, tissue factor expressing microparticle (dark purple); TNF-α, tumor 802 necrosis factor-α; TXA2, thromboxane A2. 803 28 804 Figure 5: Beneficial and detrimental potential of microparticle as highlighted by their effects on 805 endothelial cells. 806 Microparticles (MPs) influence endothelial cells in different ways (both beneficial and 807 detrimental) depending on their cell origin and the specific stimulating trigger leading to their 808 formation. 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Methods Mol Biol 788: 127-139, 2012. 41 analysis (further purification of MPs) (storage) PFP preparation blood collection venipuncture Table 1 citrate, CTAD (as EDTA can activate platelets) avoid agitation, < 2h to centrifuge (as PMPs increase over time) wide variety of speeds and times platelets may persist in PFP after a single centrifugation step -80rC, single freeze & thaw cycle (multiple cycles reduce number of MPs) wide variety of speeds and times further purification can reduce background signal from plasma at the risk of losing MPs transportation & time to centrifuge speed & time of centrifuge number of freeze & thaw cycles speed & time of centrifuge >21G needle, light or no tourniquet to avoid platelet activation current recommendation anti-coagulant needle & tourniquet variables i bl (181) (162) (11,37) ++ ++ + MS (43,75,115, 137) - (24 43 174) HPLC (24,43,174) - AFM CLM EM - +* +++ + ++ - + Functional assays (78,129, 153,155) +++ - (78,153,155) (79,89,155, 185) - ELISA FCM Table 2 ++ + + ++ ++ + + +++ - - - - - +++ +++ ++ - - - - - + + - - + - ++ - +++ +++ ++ can perform proteome and lipidome analyses can analyze phospholipid composition * size fractionation high sensitivity can investigate cell-microparticle interactions (e.g. internalization) can visualize organelles no direct enumeration but able to assay functional parameters (e.g. coagulation) high sensitivity limited sensitivity for small sized particles (<500 nm) new technologies (e.g. impedance FCM) are being proposed to address this limitation Table 3 Table 4 Figure 1 MP translation miRNA mRNA MP Receptor/protein/lipid transfer MP MP mRNA miRNA MP Nucleic acid transfer lysosome MP MP MP MP MP internalization/processing outside-in-signaling MP Receptor and soluble factor signaling Figure 3 Figure 4 Figure 5