Am J Physiol Lung Cell Mol Physiol 303: L364–L381, 2012.

Review First published June 22, 2012; doi:10.1152/ajplung.00354.2011.

Microparticles and acute lung injury

Mark McVey,1,2 Arata Tabuchi,1 and Wolfgang M. Kuebler1,3,4,5,6
1
The Keenan Research Centre at the Li Ka Shing Knowledge Institute of St. Michael’s, University of Toronto, Toronto,
Ontario, Canada; 2Department of Anesthesia, University of Toronto, Toronto, Ontario, Canada; 3Department of Surgery,
University of Toronto, Toronto, Ontario, Canada; 4Department of Physiology, University of Toronto, Toronto, Ontario,
Canada; 5Institute of Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany; and 6German Heart Institute, Berlin,
Germany
Submitted 4 November 2011; accepted in final form 18 June 2012

McVey M, Tabuchi A, Kuebler WM. Microparticles and acute lung injury. Am

J Physiol Lung Cell Mol Physiol 303: L364 –L381, 2012. First published June 22,
2012; doi:10.1152/ajplung.00354.2011.—The pathophysiology of acute lung injury
(ALI) and its most severe form, acute respiratory distress syndrome (ARDS), is
characterized by increased vascular and epithelial permeability, hypercoagulation
and hypofibrinolysis, inflammation, and immune modulation. These detrimental
changes are orchestrated by cross talk between a complex network of cells,
mediators, and signaling pathways. A rapidly growing number of studies have
reported the appearance of distinct populations of microparticles (MPs) in both the
vascular and alveolar compartments in animal models of ALI/ARDS or respective
patient populations, where they may serve as diagnostic and prognostic biomarkers.
MPs are small cytosolic vesicles with an intact lipid bilayer that can be released by
a variety of vascular, parenchymal, or blood cells and that contain membrane and
cytosolic proteins, organelles, lipids, and RNA supplied from and characteristic for
their respective parental cells. Owing to this endowment, MPs can effectively
interact with other cell types via fusion, receptor-mediated interaction, uptake, or
mediator release, thereby acting as intrinsic stimulators, modulators, or even
attenuators in a variety of disease processes. This review summarizes current
knowledge on the formation and potential functional role of different MPs in
inflammatory diseases with a specific focus on ALI/ARDS. ALI has been associ-
ated with the formation of MPs from such diverse cellular origins as platelets,
neutrophils, monocytes, lymphocytes, red blood cells, and endothelial and epithe-
lial cells. Because of their considerable heterogeneity in terms of origin and
functional properties, MPs may contribute via both harmful and beneficial effects
to the characteristic pathological features of ALI/ARDS. A better understanding of
the formation, function, and relevance of MPs may give rise to new promising
therapeutic strategies to modulate coagulation, inflammation, endothelial function,
and permeability either through removal or inhibition of “detrimental” MPs or
through administration or stimulation of “favorable” MPs.
acute respiratory distress syndrome; permeability; coagulation; inflammation; en-
dothelial function

ACUTE LUNG INJURY (ALI) as a result from either direct or
indirect mechanical, toxic, infectious, or inflammatory chal-
lenges to the lung presents initially as dyspnea and hypoxemia,
which can rapidly progress to respiratory failure. ALI and its
most severe form, the acute respiratory distress syndrome
(ARDS), are characterized by bilateral exudative chest infiltrates
visible by roentgenograms with no evidence of left heart failure
and a PaO2/FIO2 ratio ⬍300 (ALI) or even ⬍200 (ARDS) (151,
171). Within days, ALI progresses from an initial inflammatory
exudative phase with a leaky edematous lung to a proliferative
phase involving fibrin deposition and a concomitant decrease in
respiratory compliance that finally, if the patient survives, pro-
Address for reprint requests and other correspondence: W. M. Kuebler, The
Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s
Hospital, 209 Victoria St., M5B 1W8 Toronto, Ontario, Canada (e-mail:
kueblerw@smh.ca).
L364 1040-0605/12 Copyright © 2012 the American Physiological Society
ceeds to a fibrotic phase in which the lung is burdened with
scarring as it remodels while attempting to heal. Despite the
well-documented benefit of protective ventilatory strategies and
increasing knowledge of the etiological mechanisms of ARDS,
mortality rates remain relatively stagnant around 40% (133), with
incidence estimates of up to 75 per 100,000 (171).
A broad spectrum of different cell types contribute to the
pathogenesis of ARDS, including alveolar epithelial and vas-
cular endothelial cells that undergo changes in permeability
leading to edema formation and alveolocapillary injuries, as
well as platelets and immune cells such as polymorphonuclear
neutrophils (PMNs), alveolar macrophages, and monocytes
that are critically involved in inflammatory responses (104,
108, 170, 171). In contrast to the characteristic anticoagulant
and profibrinolytic basal state of the lung, ALI is associated
with a prothrombotic and antifibrinolytic shift that favors
deposition of fibrin and accelerates and potentiates inflamma-
http://www.ajplung.org

MICROPARTICLES AND ACUTE LUNG INJURY
tion (19). Considerable research has focused on the direct
interaction and communication between different cell types
both in vivo and in vitro and has revealed important patho-
physiological aspects of ALI. However, a rapidly growing
number of studies have recently started to recognize the critical
role of microparticles (MPs) as biomarkers, communication
shuttles between these different cells and cell types, and
independent functional effectors in a large variety of diseases
ranging from endemic pathologies such as hypertension, dia-
betes, and atherosclerosis to frequently lethal syndromes such
as ALI (7, 16, 27, 28, 31, 36, 44, 60, 65, 79, 99, 118, 129, 135,
138, 155, 172). Here we provide a brief overview on the main
characteristics of MPs, review the current knowledge on their
role in inflammatory disease with a particular focus on ALI,
and oppose their proposed detrimental and beneficial effects in
lung injury, thus providing a rationale for their future use as
both therapeutic targets and tools.
Microparticles
The first accounts of MPs date back to 1967 at which time
they were considered to be nothing more than cellular dust
(177). In fact, MPs are tiny cell-derived, intact vesicles that are
formed by partitioning off from activated or apoptotic eukary-
otic cells (Fig. 1). MPs are defined by their size, which ranges
between 50 nm and 1 ␮m, and their distinctive lipid layer
composition. MPs have intact lipid bilayers, yet their outer
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00354.2011 • www.ajplung.org
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L365
layer has a characteristically high level of negatively charged
phospholipids, particularly phosphatidylserine (PS). MPs may
contain membrane and cytosolic proteins, transcription factors,
genetic material such as ribosomal RNA (rRNA), messenger
RNA (mRNA), and microRNAs (miRNA) as well as lipids or
even organelles supplied from parent cells (22, 32, 109, 116).
Occasionally, membrane vesicles of less than 0.1 ␮m are
subcategorized as nanoparticles and have been proposed to
originate from lipid rafts on parent cell membranes; accord-
ingly, they may display distinct protein endowments and func-
tions (reviewed in Refs. 77, 134, 155).
Microparticles subserve intercellular information exchange.
The distinct cellular elements retained from precursor cells
allow MPs to function as transcellular delivery systems for the
exchange of biological signals. Notably, information exchange
between MPs and target cells can occur in a bidirectional
manner, thus generating a continuous and dynamic exchange
of antigens, cell signaling molecules, and genetic material that
opens an entire new spectrum of opportunities for tightly
regulated (or dysregulated) signal propagation.
In principle, MPs can transfer information from the MP-
generating donor cell to a wide range of target cells either by
direct cell-cell contact for delivery of genetic material, pro-
teins, and lipids or alternatively by remote interaction through
secretion of soluble mediators and effectors by MPs. Currently,
we distinguish at least four distinct mechanisms how MPs may
Fig. 1. Release of microparticles (MPs) from
the surface of a polymorphonuclear neutro-
phil (PMN). Scanning electron micrographs
show a fixed resting PMN (scale bar: 1.5
␮m) (A) and a fixed PMN stimulated with
0.1 ␮M N-formylmethionine leucyl-phenyl-
alanine for 5 min (scale bar: 1.5 ␮m) dis-
playing large pseudopodia and, in addition,
budding of small vesicles of 70 –300 nm in
size from the PMN cell membrane (B; inset,
arrowhead; scale bar 400 nm). C: transmis-
sion electron micrographs following immu-
nogold labeling show expression of cell sur-
face antigens such as complement receptor 1
on isolated MPs (scale bar: 150 nm). D: thin
sections of MPs precipitated with Dyna-
beads prove vesicles to be bilamellar struc-
tures. Reproduced from Hess et al. (69);
Copyright 1999. The American Association
of Immunologists, Inc.
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L366 MICROPARTICLES AND ACUTE LUNG INJURY
interact with their target cells that are illustrated schematically
in Fig. 2. First, MPs are able to stimulate outside-in signaling
in target cells via presentation of membrane-associated ligands
that directly bind to their respective surface receptors, or via
release of secreted factors such as for example cytokines that
act on the target cell via receptor-mediated or receptor-inde-
pendent mechanisms (102). Second, MPs can be internalized
into recipient cells, a process that has also been proposed to
underlie the rapid clearance of circulating MPs from the blood
(41). For example, fluorescently tagged platelet MPs (PMPs)
can be endocytosed by brain microvascular endothelial cells
and are subsequently detectable in endosomes and lysosomes
(50). Third, MPs may fully or partially fuse with the target cell,
allowing for complete or selective transfer of contents includ-
ing membrane and cytosolic proteins, bioactive lipids (109,
139), or even whole cell organelles, as recently demonstrated
for the microvesicular delivery of mitochondria by mesenchy-
mal stem cells (73). Last, MPs may deliver genetic material in
form of mRNA and miRNA. The encoding mRNAs can be
directly translated to alter the target cell proteome (6, 45),
whereas miRNAs are small noncoding RNAs that can modu-
late gene expression in target cells at the posttranscriptional
level, in that they bind to complementary sequences on target
mRNAs, commonly resulting in translational repression and
gene silencing. The export of miRNA from donor cells is
largely regulated by ceramide (166), a notion that gains rele-
vance in light of the parallel implication of ceramide in MP
generation (155), which may thus facilitate miRNA egress with
MPs in a coordinated fashion. At the level of the target cell,
subsequent miRNA entry is facilitated either by endocytosis of
MPs or by fusion of their lipid bilayer with the plasma
membrane (166). Lately, miRNAs have been detected in a
wide variety of different MPs as generated from peripheral
blood cells in humans (70) or mesenchymal (39) or embryonic
stem cells (180), respectively, and miRNA transfer by MPs has
been demonstrated to critically modulate target cell responses
Fig. 2. Mechanisms of interactions between
MPs and target cells. Interaction between MPs
and their target cells can occur via essentially
4 different mechanisms: outside-in signaling
via ligand-receptor interaction or secreted fac-
tors (top right); internalization and lysosomal
processing of MPs facilitates for example sub-
sequent presentation of MP antigens on the
surface of target cells (bottom right); fusion-
mediated transfer of surface receptors, pro-
teins, and lipids (bottom left); and delivery of
genetic material such as mRNA or miRNA
(top left). MPs may fuse either completely or
temporarily with target cells resulting in either
complete or selective transfer of MP contents.
Redrawn and modified from a figure origi-
nally published in Beyer et al. (22).
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00354.2011 • www.ajplung.org
such as for example angiogenesis in endothelial cells (64).
Because miRNAs have recently been recognized to contribute
to injurious mechanisms in ALI (183), transfer of miRNA from
MPs to inflammatory or lung parenchymal cells may present an
exciting novel pathomechanism in this disease that clearly
deserves further exploration.
Origin of microparticles. Parent cells of MPs include but are
not limited to platelets, megakaryocytes, monocytes, epithelial
cells, lymphocytes, PMNs, endothelial cells, red blood cells,
reticulocytes, mast cells, stem cells, astrocytes, glial cells,
smooth muscle cells, and cancer cells (16, 20, 23, 139). Similar
to the wide range of parent cells, MP formation can be
triggered by diverse physiological, pathophysiological, or ex-
perimental stimuli as discussed in greater detail below.
Unstimulated, resting cells actively maintain their mem-
brane asymmetry via phospholipid transporter enzymes, which
localize negatively charged phospholipids on the inner leaflet,
facing into the cell. When cellular homeostasis is disturbed or
lost such as in apoptosis, the activities of these phospholipid
flippases, floppases, and scramblases are dysregulated, leading
to hastened changes in membrane composition of the inner and
outer membrane leaflets. This process is a characteristic feature
of MP formation, since it allows for the relocation of the
PS-rich layer of the inner leaflet to the exterior leaflet (116,
155). The resulting changes in membrane composition and
regular asymmetry in conjunction with processes such as
intracellular calcium accumulation and calpain activation re-
viewed in detail elsewhere (116, 119) cause membrane bleb-
bing and ultimately the formation of MPs (Fig. 3).
Externalization of PS is a precursor step in MP formation,
allowing for a common shared surface marker for identification
of many MPs that can be assayed by annexin V binding.
Interestingly, however, it appears that there are MPs that do not
stain appreciably for annexin V, which adds controversy to the
identification and strict definition of what constitutes an MP
(116, 153). Furthermore, it seems that some MPs can also be

MICROPARTICLES AND ACUTE LUNG INJURY
Fig. 3. Pathways of MP release from parent cells. Cell activation and/or
stimulation of proapoptotic signaling cascades cause a rapid loss in lipid
membrane asymmetry due to dysregulation of flippases, floppases, and scram-
blases via sudden increases in intracellular calcium, resulting in externalization
of phosphatidylserine (PS). Concurrent cytoskeletal reorganization and con-
traction cause blebbing and subsequent shedding of MPs into the extracellular
space. MP formation from membrane lipid rafts may lead to release of MPs
that maintain part of the unique signaling characteristics of these platforms.
Redrawn and modified from a figure originally published in Chironi et al. (36).
formed before regular membrane asymmetry is disrupted, such
as in cultured endothelial cells (155). Specifically, Davizon and
colleagues (42) have linked MP formation to lipid rafts in cell
membranes, raising the possibility that lipid rafts may be
actively involved in MP formation under some conditions, and
that MPs may maintain part of the unique functional and
signaling properties of membrane lipid rafts (Fig. 3). Currently,
the cellular machinery implicated in MP formation constitutes
an active area of research (reviewed in Ref. 134) that is
expected to yield important insights not only into the mecha-
nisms of MP formation but likewise into its regulation and
function.
In healthy humans, circulating MPs are largely derived from
platelets with some contributions from leukocytes and mini-
mally from endothelial cells (34, 36). Size and composition of
this population of basal MPs depend on physiological variables
including exercise, menstrual cycle, age, or exposure to ex-
treme environments such as SCUBA diving, in addition to
countless pathological conditions, some of which are discussed
in later sections of this review (34, 55, 163).
PMPs have a short circulating half-life of less than 10 min in
rabbits (141) but were shown to circulate for ⬃30 min in mice
(53). MP clearance from the circulation occurs predominantly
by endothelial cell internalization (41) or splenic removal, and
the half-life of PMPs is markedly prolonged in splenectomized
mice compared with controls (129). Moreover, a recent study
in thrombocytopenic humans reported a markedly longer half-
life for circulating MPs that was on the order of 5– 6 h (142,
143). The considerable variation in half-lives in these studies
suggests a major influence of both host and MP variables such
as species, blood cell levels, and mode of activation, as well as
potential methodological variability with respect to isolation
and assay for MPs between these different studies.
Tracking the origin of MPs is complicated not only by the
wide variety of possible parent cell sources and triggering
stimuli, but in addition by the fact that these cell-derived
vesicles can be and presumably are constantly phenotypically
modified by contact with different cell types at different steps
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L367
in their production. Different mechanisms of fusion between
cells and MPs that could lead to phenotypic modulation of MPs
and/or other cells have been recently reviewed in detail by
Quesenberry and colleagues (139). A particularly striking ex-
ample in this respect is the provocative hypothesis that endo-
thelial progenitor cells may origin from the fusion of PMPs
with mononuclear cells as suggested by proteomic analyses
(137). Repetitive fusion events with tissue factor (TF)-express-
ing cells may also account for the expression of TF on various
types of cells and MPs that would not otherwise express this
marker. For example, platelets can become TF positive after
contact with TF-expressing MPs shed from PMNs and mono-
cytes (145). Following stimulation with endotoxin [lipopoly-
saccharide (LPS)] or protein kinase C activation by phorbol
myristate acetate (PMA), platelets, and subsequent PMPs may
obtain their TF antigens from fusion with either monocytes or
monocyte microparticles (MMPs) (28). Beyond mixing of
distinct lineage markers between cells and MPs there are also
challenging distinctions between MPs of similar parent cell
origins. Subtle variations in parent cells can be difficult to
detect because of phenotypic similarities of their respective
MPs, as in the case of platelet vs. megakaryocyte-derived MPs
(54, 143). Hence, a series of elaborate experimental techniques
have been developed to allow for the exact identification of the
origin of MPs and the functional significance of these cell-
derived vesicles.
Characterization of microparticles. In the stepwise process
of experimentally isolating MPs there are considerable techni-
cal variables that can have impact on the recovery and pheno-
type of MPs obtained. Some of these procedural differences
include the actual phlebotomy, the use of anticoagulants, stor-
age conditions, length and number of freeze-thaw cycles,
centrifugation, washing, and pelleting or filtration of MPs; and
specific recommendations (Table 1) have been developed to
optimize each of these steps (186). A series of review articles
have scrutinized the impact of these variables that underline the
sensitive and precarious nature of MP isolation and stress how
caution is required to avoid undesired differences in MP
isolates obtained from slight variations in handling and pro-
cessing procedures (91, 134, 153, 155). The rapidly growing
number of publications pertaining to MPs showcases a diverse
array of techniques for preparation and isolation of MPs that is
bound to generate considerable variability in terms of the final
products assayed (78, 134). In recent years, this dilemma has
stimulated workshops and meetings specifically dedicated to
the attempt to limit variability and standardize the study of
MPs; namely, the International Society on Thrombosis and
Haemostasis has made strides to centralize methodological
approaches for MP isolation (90).
Detection and characterization of MPs has involved strate-
gies such as antibody-capture ELISA assays, flow cytometry,
functional coagulation assays, electron microscopy (Fig. 1),
atomic force or confocal microscopy, HPLC, capillary electro-
phoresis, and mass spectrometry (11, 24, 37, 43, 75, 77–79, 89,
91, 115, 129, 137, 153, 155, 162, 174, 181, 185). Each of these
techniques comes with its specific advantages and limitations,
as summarized in Table 2. Because of its general availability
and versatility, flow cytometry has become the most commonly
used technique for characterization of MPs and discerning their
parent cell of origin depending on the expression of character-
istic surface markers (Fig. 4). Most strategies with flow cy-
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L368 MICROPARTICLES AND ACUTE LUNG INJURY

Table 1. Technical variables and recommendations for MP isolation

Variables Current Recommendation

Venipuncture needle & tourniquet ⬎21-gauge needle, light or no tourniquet to avoid platelet activation
2
Blood collection anticoagulant citrate, CTAD (as EDTA can activate platelets)
2 transportation & time to centrifuge avoid agitation, ⬍2 h to centrifuge (as PMPs increase over time)
PFP preparation speed & time of centrifuge wide variety of speeds and times
platelets may persist in PFP after a single centrifugation step
2
(Storage) number of freeze & thaw cycles ⫺80°C, single freeze & thaw cycle (multiple cycles reduce number of MPs)
2
(Further purification of MPs) speed & time of centrifuge wide variety of speeds and times
2 further purification can reduce background signal from plasma at the risk of losing MPs
Analysis
Flow chart of variables and current recommendations for the isolation, storage, and purification of microparticles (MPs) as proposed for platelet microparticles
(PMPs) by Zwicker and colleagues (186). CTAD, citrate theophylline adenosine and diphyridamole; PFP, platelet-free plasma.

tometry involve identification of MPs based on forward and
side scatter characteristics as well as annexin V staining for PS
in conjunction with analysis of markers characteristic for
specific parent cells to test for their contribution to the indi-
vidual MPs being assayed. The use of flow cytometry to this
end bears a series of strengths, but also some limitations (77,
78, 89, 147, 153) that have been discussed in detail in recent
literature. Proteomics presents an important alternative tool for
particularly small particles not amenable to flow cytometry
(117) since it can yield comprehensive information on charac-
teristic protein expression patterns that may be utilized to
identify and/or characterize MPs. This approach has been used
in a series of investigations to assay different aspects of the
human MP proteome (75, 96).
Heterogeneity of microparticles. Our current understanding
of the conditions under which MPs are formed, and their
individual actions, is still incomplete. Little is known about
exact parameters that dictate when, how, from where, why,
which, and how many MPs are formed. What is becoming
evident is the vast variation in terms of genetic material and
antigens MPs can express from their parent cells of origin or
other cells or MPs they contact during formation or during
circulation, which can be incorporated or eventually redistrib-
uted to other MPs or cells. The literature has likely not
captured the full extent of variations possible since the char-
acterization of MPs under different conditions is incomplete
and we currently lack the tools to accurately assay antigens
present at very low abundances on the surface of MPs. MPs
come in a variety of sizes ranging from smaller nanoparticles
such as those often produced by red blood cells up to MPs large
enough to rival platelets (134). Similar parent cells are able to
produce a spectrum of different-sized MPs depending upon
whether MP formation occurs spontaneously at baseline or
following stimulation (25, 74). Even cell lines such as Jurkat T
cells yield a spectrum of different-sized MPs (164). Notably,
this heterogeneity in size can directly impact MP surface
marker expression and function as shown for platelet MPs that
contain different antigens such as growth factors, chemokines,
and receptors depending on their size (74).
Similar to their heterogeneity in terms of origin, size, and
endowment with antigens, MPs also differ widely with regard
to their functional effects. MPs can shuttle information from
remote cells to similar or phenotypically different cells that
might never be in direct proximity. Different MPs can affect
certain cells in different ways, as for example diabetics’ T
cell-derived MPs can decrease endothelial nitric oxide (NO)
synthase (eNOS) and increase caveolin-1 expression causing
endothelial dysfunction as opposed to MPs formed by in vitro

Table 2. Advantages and limitations of techniques commonly applied in MP analysis

Enumeration Sizing Origin Throughput Cost Availability

FCM (79,89,155,185) ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫺ ⫹⫹ limited sensitivity for small sized particles
(⬍500 nm)
new technologies (e.g., impedance FCM)
are being proposed to address this
limitation
ELISA (78,153,155) ⫺ ⫺ ⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ high sensitivity
Functional assays (78,129,153,155) ⫺ ⫺ ⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ no direct enumeration but able to assay
functional parameters (e.g., coagulation)
EM (11,37) ⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ can visualize organelles
CLM (162) ⫹⫹ ⫹ ⫹⫹ ⫺ ⫺ ⫹⫹ can investigate cell-MP interactions (e.g.,
internalization)
AFM (181) ⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫺ ⫺ high sensitivity
HPLC (24,43,174) ⫺ ⫹* ⫹ ⫺ ⫺ ⫹ can analyze phospholipid composition
MS (43,75,115,137) ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫺ can perform proteome and lipidome
analyses
Commonly applied techniques for the analysis of MPs and their relative strengths and weaknesses in terms of MP enumeration, MP sizing, determining the
parent cell of origin, throughput, cost, and availability. AFM, atomic force microscopy; CLM, confocal laser microscopy; ELISA, enzyme-linked immune sorbent
assay; EM, electron microscopy; FCM, flow cytometry; HPLC, high-performance liquid chromatography; MS, mass spectrometry. *Size fractionation.

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MICROPARTICLES AND ACUTE LUNG INJURY
activation of human T cells that express sonic hedgehog on
their membranes and can stimulate NO production as evi-
denced by the improved endothelial function in a cardiac
ischemia-reperfusion injury model (106, 114). Alternatively, a
single MP subtype such as PMPs can interact with and recruit
numerous target cells such as monocytes, NK cells, and B and
T cells (114).
MP generation is triggered by a spectrum of stimuli ranging
from physiological to experimental stimuli (Fig. 5). MPs can
be formed constitutively by various parent cells under physi-
ological conditions but in differing amounts, resulting in a
higher abundance of circulating PMPs compared with red
blood cell- or lymphocyte-derived MPs (RMP and LMP, re-
spectively) in healthy patients (116). Additional physiological
triggers for MP formation involve stresses such as heavy
exercise or cyclic changes such as menstruation. MP formation
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L369
Fig. 4. Characteristic surface markers indic-
ative of MP origin. Characteristic surface
markers used to detect MP populations
(right) that originate from specific parent
cells (left). CD, cluster of differentiation;
EMP, endothelial microparticle; IgG, immu-
noglobin; LMP, lymphocyte microparticle;
MegMP, megakaryocyte microparticle; MP,
microparticle; MMP, monocyte micropar-
ticle; NMP, neutrophil microparticle; PMP,
platelet microparticle; RMP, red blood cell
microparticle; vWF, von Willebrand factor.
in response to pathophysiological stimuli adds another level of
complexity, since different triggers will stimulate MP forma-
tion from different parent cell types. Multiple simultaneous or
sequential triggers can further enhance MP diversity and there-
fore potentially expand their range of function. For example,
platelet storage conditions including platelet-rich plasma,
apheresis blood bank-derived human platelets, or cold storage
of platelets with and without contact with propyl gallate will
produce distinct PMP variants (33, 144, 168, 178). Addition of
platelet activation inhibitors such as prostaglandin E1 (PGE1),
theophylline, and aprotinin during storage reduce the number
of large but not small PMPs formed and modify their function
in terms of platelet factor 3 activity (25). Under experimental
conditions, a diverse array of potent cell stimulants such as
calcium ionophores, phytohemagglutinins, LPS, N-formylme-
thionyl-leucyl-phenylalanine, and PMA can be utilized to in-
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L370 MICROPARTICLES AND ACUTE LUNG INJURY

Fig. 5. Triggers for MP formation from differ-

ent parent cells of origin. Physiological, patho-
physiological, and experimental triggers of MP
formation (right) from platelets, megakaryo-
cytes, lymphocytes, monocytes, red blood
cells, polymorphonuclear neutrophils, and en-
dothelial cells (left). APC, activated protein C;
ATII, angiotensin II; autoAb, autoantibodies;
C5a, complement factor C5a; C5B-9, comple-
ment factor C5B-9; CD40L, CD40 ligand;
CHX, cyclohexamide; EC, endothelial cell;
fMLP, N-formylmethionyl-leucyl-phenylala-
nine; IL-1, interleukin-1; L-NAME, L-NG-ni-
troarginine methyl ester; LPS, lipopolysaccha-
ride; PAF, platelet activating factor; PHA,
phytohemagglutinin; PAI-I, plasminogen acti-
vator inhibitor 1; PMA, phorbol myristate ac-
etate; PtdIns(4,5)P2, phosphatidylinositol-4,5-
bisphosphate; ROS, reactive oxygen species;
STS, staurosporine; TNF-␣, tumor necrosis
factor-␣; TRAP, thrombin receptor agonist
peptide SFLLRN.

duce MP formation with a high efficiency and reproducibility
(Fig. 5).
Microparticles in Inflammatory Disease
Some of the MPs characterized to date have been identified
as biomarkers for a wide variety of inflammatory conditions.
Elevated levels of circulating PMP, LMP, MMP, and endothe-
lial MPs (EMPs) have been shown to accompany pathological
conditions such as atherosclerosis, Type 2 diabetes, vasculitis,
pulmonary hypertension, coronary disease, cardiopulmonary
resuscitation, lupus, Crohn’s disease, and rheumatoid arthritis
(7, 27, 28, 36, 52, 118, 129, 135, 138, 155). Likewise, systemic
inflammatory states in pregnancy such as preeclampsia are
associated with elevated plasma levels of MPs (146). Infec-
tious diseases are either directly associated with increased
levels of MPs as seen in malaria (61) or can give rise to
disproportionate inflammatory responses such as the systemic
inflammatory response syndrome (SIRS), sepsis, or the multi-
organ dysfunction syndrome, that are again associated with
elevated levels of MPs (129, 155). MPs have also been impli-
cated mechanistically on several levels in the pathophysiology
of sepsis, including proinflammatory signaling pathways such
as target cell activation through reactive oxygen species pro-
duction (ROS) and coagulopathy in terms of MP-mediated TF
presentation (113).
A survey comparing 36 patients with sepsis with 18 healthier
patients without sepsis showed elevated blood levels of total
MPs, specifically PMPs and EMPs, whereas CD45⫹ LMPs
were lower in patients with sepsis compared with controls
(122). Interestingly, MPs from septic patients enhanced aortic
contraction when transfused intravenously to mice. This effect
was independent of NO modulation or cyclooxygenase enzyme
expression but blocked by the specific thromboxane A2
(TXA2) antagonist SQ-29548, indicating that circulating MPs
may prevent vascular hyporeactivity accounting for systemic
hypotension in septic shock through a TXA2-mediated mech-

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MICROPARTICLES AND ACUTE LUNG INJURY
anism (122). Furthermore, patients with SIRS have elevated
levels of circulating EMPs, increased binding of MPs to PMNs,
higher procoagulant activity and higher circulating levels of
triggers for MP formation such as plasminogen activator in-
hibitor 1 (PAI-1) compared with healthy controls (126). As we
discuss later in greater detail, the effects of MPs are frequently
considered to aggravate the pathology in inflammatory and
cardiovascular diseases, and increased formation of MPs as
seen in systemic inflammatory disorders is therefore generally
considered to constitute not only a biomarker, but an important
pathophysiological mechanism driving the disease. However,
inflammation and the associated increase in circulating MPs
may also have protective effects, as pointed out by Soriano and
coworkers (158), who demonstrated that increased inflamma-
tion and the presence of EMPs, PMPs, and platelet leukocyte
conjugates are significantly associated with survival in septic
patients. Hence, more detailed and comprehensive experimen-
tal and clinical investigations are in dire need to discern which
MPs under what conditions exert beneficial or detrimental
effects in inflammatory diseases.
Over the past decade, it has become evident that inflamma-
tory diseases are closely associated with impaired coagulation
and fibrinolysis, processes that can be regulated through MPs.
Many but not all MPs express TF, which interacts with factor
VIIa facilitating activation of factors IX and X, thus leading to
generation of thrombin (103). TF MPs are characteristically
found in diseases in which thrombosis is common such as
cancer, sepsis, unstable angina, ALI, sickle cell disease, and
thromboembolism (128, 129, 138, 165), giving rise to the
speculation that they may contribute critically to the disease
process. TF is a key player in the activation of the coagulation
pathway, yet its action is determined not only by its expression
levels, but also by its accessibility in terms of conformational
state that is controversial for different cells types or conditions
(128). Much of the circulating TF is in fact in an encrypted and,
therefore, inactive state, whereas MPs represent a source of
decrypted, active TF (127). LPS stimulates monocytes and
endothelial cells to produce TF that can lead to a coagulopathy
that can escalate into disseminated intravascular coagulation
(103). Coincubation of platelets with monocytes and PMNs
that produce high amounts of TF following stimulation with
proinflammatory agents such as LPS leads to enhanced pre-
sentation of decrypted TF in the form of MPs, thus generating
a prothrombotic milieu (127). Even MPs not expressing TF can
still possess considerable procoagulant properties because they
contain anionic phospholipids, including PS, which acts as a
surface to facilitate assembly of the clotting factor machinery
(103, 129, 138). Hence, MPs can be involved in coagulopathies
associated with inflammatory diseases such as sepsis since they
possess prothrombotic characteristics with exposed TF and PS
but conversely may also activate fibrinolysis via changes in
plasminogen and metalloproteases (113) or exert anticoagulant
properties mediated through protein C and tissue factor path-
way inhibitor (TFPI) (117). In line with the notion that MPs
may have both pro- and anticoagulant effects, thrombus weight
was shown to correlate with plasma levels of PMPs and older
MPs in a murine thrombus model, whereas LMPs showed an
inverse correlation (140).
Microparticles and acute lung injury. Several studies have
shown associations of MPs with key structures and cells in
ALI, giving rise to the notion that, similar to systemic inflam-
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L371
matory diseases, MPs may be a biomarker of the disease and
play important roles by either aggravating or counteracting (or
potentially both) the ongoing disease process. For example,
interactions between cells such as platelets, PMNs, and endo-
thelial cells are central to the pathology of ALI/ARDS, and
MPs have been demonstrated to critically regulate these inter-
actions and/or modify the phenotypes of the contributing cells
by fusion and subsequent transfer of cellular components. In
the following, we will summarize recent data in support of an
association and possible functional role of MPs in ALI/ARDS
and discuss potential novel interactions between MPs and lung
parenchyma or circulating blood cells that are likely pertinent
to lung injury.
Potential Detrimental Effects of Microparticles in ALI
Excessive inflammation, changes in coagulation and fibrin
deposition, and increased permeability of the alveolocapillary
barrier with resulting protein extravasation and lung edema
formation are characteristic hallmarks of ALI (Fig. 6). In the
following, we highlight how MPs may impact either directly or
indirectly on the pathophysiology underlying these symptoms.
Inflammation. Inflammatory changes in ALI/ARDS involve
an abundance of proinflammatory intra- and intercellular sig-
naling pathways that include, but are in no way confined to,
signaling via arachidonic acid (AA), TXA2, PAI-1, NO, ROS,
cytokines, and protease-activated receptors (PARs). In this
section, we highlight data demonstrating that MPs can modu-
late and/or replicate these pathways (114, 120) and thus act as
proinflammatory agents promoting ALI/ARDS.
Over past years, the tight interplay between platelets and
PMNs has emerged as an important aspect in the pathogenesis
of ALI (100, 160, 182). Similar to platelets, circulating PMPs
can interact with PMNs via linkages including glycoprotein
Ib/Mac-1, ␤2-integrins (CD11b)/Mac-1, or P-selectin/PSGL-1
on PMPs/PMNs, thus causing PMN activation involving se-
cretion of interleukin (IL)-8, oxidative burst, degranulation,
and enhanced leukocyte rolling (79, 99). Likewise, PMPs
generated from LPS-stimulated platelets promote endothelial
activation as exemplified by the finding that they further
increase the IL-1␤-induced expression of vascular cell adhe-
sion molecule-1 (30). Hence, circulating PMPs are not only a
biomarker, but also, more importantly, an active promoter of
inflammatory disease processes in that they stimulate endothe-
lial cells, PMNs, and notably also platelets. The latter was
demonstrated in experiments in which PMPs were stimulated
by soluble phospholipase A2; the newly formed AA is then
rapidly converted by PMPs to TXA2, a classic activator of
platelets (14). In line with this view, ExoU, a Pseudomonas
aeruginosa cytotoxin with phospholipase A2 activity, increases
both PMP release and TXA2 formation and accordingly re-
sulted in lung thrombus formation and increased vascular
permeability in a murine model of pneumosepsis (101). Over-
all, a series of proinflammatory effects of PMPs has emerged
that is based on the activation of platelets, monocytes, and
vascular endothelial cells either directly by MP-derived AA or
by its metabolism to bioactive prostanoids (12, 13, 15). This
recognition substantiates a critical role of PMPs in ALI, since
TXA2 contributes pivotally to lung vascular leakage and in-
flammation in experimental models of the disease (72).
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Fig. 6. Schematic representation of the proposed role of MPs in acute lung injury. I: depiction of normal healthy lung, interstitium and vasculature with associated
basal MP populations (color coded). Acute lung injury (II) involves characteristic pathologies including increased permeability (IIa), coagulation leading to
thrombosis and fibrin deposition (IIb), and inflammation and immunomodulation (IIc). Each of these pathologies is affected in distinct ways by specific MP
populations that are in turn activated by specific stimuli. AA, arachidonic acid; APC, activated protein C; EMP, endothelial MP (blue), EpMP, epithelial MP (light
purple); IL-1␤, interleukin 1␤; INF␥, interferon-␥; LMP, lymphocyte MP (red); LPS, lipopolysaccharide; LXA4, lipoxin A4; MacMP, macrophage MP (pink);
MMP, monocyte MP (purple); MP, microparticle; NMP, polymorphonuclear neutrophil MP (orange); PAI-1, plasminogen activator inhibitor 1; PHA,
phytohemagglutinin; PMN, polymorphonuclear neutrophil; PMP, platelet MP (light green); RMP, red blood cell MP (dark green); ROS, reactive oxygen species;
TFMP, tissue factor expressing microparticle (dark purple); TNF-␣, tumor necrosis factor-␣; TXA2, thromboxane A2.

Platelet-PMN and endothelial-PMN costimulation may not
only be promoted by PMPs, but likewise by PMN micropar-
ticles (NMPs), since endotoxin (LPS) can stimulate adherent
PMNs to release NMPs that generate platelet-activating factor
(PAF), thus causing activation of platelets (172), endothelium
(149), and other inflammatory cells. In the air spaces, NMPs
may also trigger inflammatory responses and epithelial cell
damage, as suggested from their abundance in sputum from
patients with cystic fibrosis (136). NMPs can furthermore
directly activate endothelial cells to generate proinflammatory
cytokines such as IL-6, thereby further promoting vascular
inflammation (8, 111, 112). Endothelial cells may in turn
likewise release MPs (EMPs) that modulate inflammation and
coagulation, and most prominently vascular barrier function,
for which reason they are discussed in greater detail in the
section Permeability.
Lastly, inflammatory diseases such as ALI and ARDS are
often associated with immune modulation and its complex
functional consequences including inflammatory damage to
bystander cells (123, 158, 173). Such immune modulation also
involves platelets that can actively alter innate and adaptive
immune function (94, 152). For example, PMPs can stimulate
endothelial cells to generate a range of proinflammatory cyto-
kines including IL-1, IL-6, IL-8, and monocyte chemoattrac-
tant protein-1 (MCP-1) as well as monocyte-derived IL-1,
TNF-␣, and IL-8 (reviewed in Refs. 20, 120). MPs have also
been directly associated with immunomodulatory effector mol-
ecules such as RMP- and PMP-derived CD40L, IgG, and
complement found in stored blood products (80). Immuno-
modulatory and proinflammatory effects of MPs may be par-
ticularly relevant in the context of transfusion-related ALI
(TRALI), because storage of blood products will propel for-

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MICROPARTICLES AND ACUTE LUNG INJURY
mation of MPs (25, 33, 144) that will subsequently activate
PMNs (80). Taken together, MPs of diverse cellular origins
including LMPs, PMPs, RMPs, NMPs, and EMPs directly or
indirectly promote inflammatory responses and may thus pro-
pel ALI, in that they stimulate cytokine release and promote
PMN activation, migration, and PMN-platelet interaction.
Coagulation. Severe infections lead to activation of the
coagulation cascade and altered fibrinolysis, with the lung
being a key recipient of fibrin and clots during ALI and sepsis
(165). The hypercoagulable and hypofibrinolytic changes seen
in ALI/ARDS contribute pivotally to the pathology of the
disease (170, 171) and involve characteristic changes in medi-
ators of coagulation and fibrinolysis, including TF, thrombo-
modulin, TFPI, PAI-1, PS, and von Willebrand factor (vWF).
TF is considered to play an important role in the pathophys-
iology of lung injury, because it can activate PAR1 and PAR2,
which are involved in inflammatory and procoagulatory re-
sponses causing fibrin deposition in the lung (82, 165). Impor-
tantly, TF is expressed not only on activated mononuclear
cells, endothelial cells, alveolar epithelium, or respiratory mac-
rophages, but likewise on many MPs (11, 165). The procoagu-
lant activity of PMP membranes exceeds that of their parent
platelet membranes by up to 100-fold (116, 156). In vascular
homeostasis, the effects of TF are counteracted by TFPI, which
blocks the TF-induced stimulation of the coagulation cascade
by reversibly inhibiting factor Xa and thrombin. Severe infec-
tions such as sepsis that constitute typical predispositions for
ALI are characterized by an imbalance between elevated TF
and lowered TFPI levels causing enhanced coagulation and
inflammation (165). The relevance of this imbalance is high-
lighted by observations from humans (56) and experimental
models of ALI in which blockade of TF by antibodies (175) or
TFPI (48) reduced lung damage and improved pulmonary
function. MMPs can attenuate the expression of TFPI and
thrombomodulin, an endothelium-derived anticoagulant factor,
thus increasing thrombogenicity in vitro (4), whereas TF is
abundantly expressed on MPs under inflammatory conditions
(91). In healthy human volunteers, MP-borne TF procoagulant
activity increased eightfold within 4 h after infusion of LPS
(11). Hence, infectious or inflammatory stressors may contrib-
ute to and/or aggravate ALI in part by the formation and
release of TF studded MPs that in the presence of an attenuated
anticoagulatory response will generate the characteristic pro-
thrombotic milieu associated with ALI, especially in consider-
ation of the previously discussed fact that TF on MPs is often
decrypted and active. Notably, TF activity on MPs in ALI/
ARDS is not necessarily confined to the vascular compartment
(16), but, in line with the clinical presentation of fibrin clots in
the distal air spaces, may similarly apply to the alveolar
compartment, where increased TF production by alveolar ep-
ithelial cells has been documented (18, 165). In pulmonary
edema fluid from mechanically ventilated (⬍7 ml/kg tidal
volume) patients with ARDS, Bastarache and colleagues (16)
detected higher concentrations of epithelial cell marker recep-
tor for advanced glycation end products (RAGE) and TF-
enriched MPs in bronchoalveolar lavage fluid (BALF) com-
pared with patients with hydrostatic lung edema whose MPs
also expressed less TF and RAGE. It is possible that ARDS
leads to either larger MPs that express more RAGE receptors
or perhaps a numeric increase in RAGE-positive MPs com-
pared with ARDS-negative hydrostatic edema controls. Pa-
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L373
tients who died had a trend toward a higher MP concentration
in lung edema fluid compared with those who survived (P ⫽
0.073), attesting to a detrimental effect of MPs in ALI/ARDS.
Similar MPs could be generated in vitro by stimulation of
cultured alveolar epithelial cells with a mix of the proinflam-
matory mediators TNF-␣, IL-1␤, and interferon-␥ (16, 17).
Whereas both ARDS and non-ARDS controls in Bastarache’s
study were severely sick, BALF analyses from healthy pigs
with volume-controlled ventilation set to maintain normocap-
nia (37.5– 41 mmHg PaCO2) with 5 cmH2O positive end-
expiratory pressure revealed PMPs that were positive for fi-
brinogen, vWF, and P-selectin (124). This finding gains rele-
vance in light of the fact that mechanical ventilation, in
particular at tidal volumes ⬎6 ml/kg body wt, will exacerbate
ALI and promote ventilator-induced lung injury. PMPs in
BALF were evident after only 1 h of mechanical ventilation,
yet, unfortunately, actual tidal volumes applied were not re-
ported in this study so that it is unclear whether this effect was
attributable to overventilation or occurs even under conditions
of protective ventilation. Notably, presence of PMPs was
likewise detected in human tracheal mucus obtained from five
humans at extubation after surgery, suggesting that this finding
may reflect a more general effect of mechanical ventilation
and/or surgical trauma.
Similar to TF expressing MPs, a procoagulant environment
in ALI may be likewise promoted by PMPs. However, the
degree to which circulating PMPs are actually elevated in
patients with ALI or ARDS remains to be elucidated. Earlier
work by George and coworkers (60) did not detect an increase
in blood PMPs in patients with ARDS from various causes,
whereas postcardiac bypass patients exhibited an increased
plasma concentration of PMPs. Yet it is conceivable that the
radiolabeled GPIIb antibody technique used to assay PMPs in
this study did not screen for the entire population of PMPs in
the ARDS cohort. Beyond TF expression, MPs expose large
amounts of PS on their outer membranes, thus creating an ideal
surface for activation of the coagulation pathway (148). In
addition, MPs generate and release other prothrombotic factors
including thrombin (80) or vWF multimers that can promote
and stabilize platelet aggregation (20). In summary, there is
ample evidence from in vitro animal and human studies dem-
onstrating the formation of MPs with procoagulant activity
under inflammatory conditions and in ALI in both the vascular
and alveolar compartment, where they are likely to contribute
pivotally to the hypercoagulable and hypofibrinolytic state
characteristic for ALI/ARDS.
Permeability. The increased permeability of capillary and
alveolar barriers in ALI leads to the extravasation of high
molecular weight protein and inflammatory cells into the air
spaces of the lung, thus impairing respiratory mechanics,
attenuating gas exchange, causing increased shunting and ven-
tilation perfusion mismatch, and contributing to the consolida-
tion of lung tissue during the subsequent fibroproliferative
phase of ALI/ARDS. Several biolipid mediators such as TXA2,
phospholipids, and sphingolipids have been implicated in the
pathogenesis of permeability-type lung edema and are notably
also linked directly or indirectly to the formation and function
of MPs (62, 104).
As discussed before, PMPs can promote the formation of
TXA2 from AA (14). This finding gains relevance in the
context of permeability-type edema because TXA2 inhibitors
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L374 MICROPARTICLES AND ACUTE LUNG INJURY
can reduce lung vascular permeability in animal models of ALI
(72, 182). In line with this view, increased levels of circulating
PMPs were associated with elevated TXA2 plasma concentra-
tions and lung vascular permeability in a mouse model of
bacterial pneumosepsis with histological evidence of thrombus
formation in the lungs (101). In sepsis, the increased MP-
associated formation of TXA2 may serve to maintain a basal
vasopressor effect (120, 122), yet this beneficial effect does not
translate to the low pressure system of the lung where in-
creased TXA2 formation will primarily promote lung edema.
In addition to eicosanoids, phospho- and sphingolipid me-
diators such as PAF and ceramide have been proposed to
contribute critically to vascular hyperpermeability in ALI (62).
PAF can be expressed on the surface of NMPs and promote
platelet aggregation and association with PMNs (172), whereas
ceramide has been implicated in MP formation due to its
involvement in lipid rafts (155). Acid sphingomyelinase
(ASM), which catalyzes the conversion of sphingomyelin to
ceramide, triggers the release of brain glial MPs, an effect that
is blocked by ASM inhibitors or in astrocytes obtained from
ASM-deficient (Smpd1⫺/⫺) mice (23). Although it remains to
be shown whether a similar ASM-mediated mechanism con-
tributes to MP formation in other nonglial cell types, this
finding raises the intriguing notion that the demonstrated det-
rimental effects of ASM/ceramide signaling in ALI (87) relate
to the increased formation of MPs.
Endothelial function. Apart from biolipid mediators, various
MPs may directly impact endothelial function, thereby criti-
cally regulating vascular barrier properties as well as promot-
ing the hyperinflammatory and hypercoagulatory responses
discussed in previous sections. EMPs can attenuate NO release
from cultured endothelial cells and induce a corresponding
decrease in eNOS phosphorylation at Ser1179 and a decrease
in hsp90 association consistent with the notion of EMP-in-
duced endothelial dysfunction (44). In line with this view,
EMP treatment impaired endothelium-mediated vasodilation in
both mouse and human ex vivo vessel preparations (44).
Likewise, EMPs attenuate vasorelaxation in rat aortas in a
dose-dependent fashion involving impaired NO signaling and
endothelial function (29). These findings gain relevance in the
context of ALI in view of the frequently barrier-protective (85,
179), anti-inflammatory, and anticoagulatory effects of basal
endothelial NO production in the lung. Endothelial dysfunction
and impaired aortic vasorelaxation were likewise inducible in
mice by LMPs, which caused overexpression of caveolin-1 and
concomitant inhibition of eNOS (106). Endothelial NO pro-
duction is also inhibited by smooth muscle cell MPs via a
␤3-integrin-mediated mechanism (49). The relevance of im-
paired endothelial NO formation in the context of ALI and
inflammation is highlighted by the fact that IL-8-dependent
PMN migration and endothelial transmigration in vitro are
enhanced by the NO synthase inhibitor NG-nitro-L-arginine
methyl ester (L-NAME), and likewise by NMPs generated by
L-NAME treatment, but not by D-NAME or untreated PMNs
(125). Conversely, NO may also regulate MP formation al-
though in different ways depending on cell type and/or exper-
imental conditions, as highlighted by the finding that L-NAME
induced NMP formation from PMNs (125), whereas macro-
phage MP formation in response to Toll-like receptor agonists
such as LPS is reduced by inhibition of inducible nitric oxide
synthase (iNOS) (59). Taken together, MP formation can be
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modulated by the bioavailability of NO, and once formed MPs
can attenuate local NO production, in particular with respect to
endothelial cells (20), which in turn may promote ALI and
barrier permeability. Accordingly, EMPs derived from PAI-1-
stimulated endothelial cells can induce direct ALI in mice and
stimulate the release of IL-1␤ and TNF-␣, leading to increased
PMN recruitment to the lung (31). In Brown Norway rats,
intravenous infusion of EMPs at pathophysiologically relevant
concentrations induced characteristic signs of pulmonary
edema, lung PMN infiltration, and increased lung vascular
permeability (44). Increased lung capillary permeability was
likewise evident in C57BL/6 mice when EMPs were infused as
either primary or secondary hit (44). EMPs infusion into
C57BL/6 mice likewise led to increased inflammatory cytokine
release and lung PMN recruitment (31) and exacerbated LPS-
induced lung injury. Notably, the aggravation of LPS-induced
lung injury was particularly prominent when EMPs were in-
fused prior to rather than simultaneous with LPS (31), indicat-
ing that EMPs can both prime the lung for subsequent injurious
stimuli or exacerbate lung damage in previously challenged
lung tissue.
Clinical implications. The recognition of a potential detri-
mental effect of MPs in the pathophysiology of ALI/ARDS
opens up new therapeutic perspectives, in that removal of MPs
and/or inhibition of MP functions may present promising
strategies for future treatment interventions (32, 184). Based on
a similar rationale, removal of PMPs by a membrane plasma
separator was recently tested in eight patients undergoing
apheresis, yet although considerable amounts of PMPs were
removed by filtration total numbers of circulating PMPs could
not be diminished by this intervention, potentially because of a
stimulation of PMP formation by the plasmapheresis procedure
per se (66). Compared with mechanical removal, pharmaco-
logical attenuation of circulating MP levels may not only be
feasible but may have inadvertently already been introduced
into the treatment of inflammatory diseases, since glucocorti-
coid treatment has been shown to significantly reduce circu-
lating PMP levels in patients with polymyositis and dermato-
myositis (154). Yet at the current stage the significance of
immunosuppression or immunomodulation by glucocorticoids
in the treatment of ALI/ARDS remains unclear owing to mixed
clinical results (173), and a thorough analysis of the effects of
steroid administration on circulating MP levels and subpopu-
lations is yet lacking.
Notably, agonists of peroxisome proliferator-activated re-
ceptor (PPAR)-␥ such as rosiglitazone, which have been intro-
duced as antidiabetic drugs but also show efficiency in lung
diseases such as pulmonary hypertension (67, 68), may present
an alternative approach to target deleterious MP functions in
inflammatory diseases. MMPs increase macrophage and mono-
cyte superoxide anion production, cytokine release, and NF-␬B
activation via a PPAR-␥-regulated mechanism that can be
inhibited by PPAR-␥ agonists (5). PPAR-␥ agonists have also
been demonstrated to inhibit LMP-mediated vascular dysfunc-
tion and increased release of proinflammatory proteins from
isolated murine aortae (120) and the upregulation of proinflam-
matory cytokines including IL-8 and monocyte chemotactic
protein-1 by monocyte MPs in cultured human lung epithelial
cells (83). PPAR-␥ expression has been shown to be reduced in
ALI (97) whereas agonists of PPAR-␣ or -␥ can attenuate
experimental ALI (9, 98, 150). Although PPAR agonists may

MICROPARTICLES AND ACUTE LUNG INJURY
thus have therapeutic potential in the context of ALI/ARDS,
their effects on circulating MP levels and MP function in this
disease remain to be determined.
Several reviews of MPs have highlighted the interesting
strategy to modulate PMP formation by statin or polyunsatu-
rated fatty acid (PUFA) therapy (42, 91). Although PUFAs
have been pursued as therapeutic interventions in ARDS with
reported benefits in terms of decreased permeability and PMN
recruitment (157), statin treatment remains controversial yet is
actively pursued as potential therapy for ALI/ARDS (26, 86).
Because randomized clinical trials are currently underway, it
will be of interest to see whether potential benefits of statin
therapy in ARDS are likewise associated with reduced levels or
altered profiles of circulating MPs.
Importantly, attenuation of MP formation may also provide
an attractive strategy to reduce complications associated with
the administration of stored blood products, including TRALI.
RMPs typically form after 10 days of storage, whereas PMPs
accumulate earlier within days, and levels of both can be
significantly reduced if packed cells are leukoreduced or in the
presence of storage additives such as glucose, pyruvate, ino-
sine, adenine, and phosphate (80). Storage of platelets with
PGE1, theophylline, or aprotinin as well as pharmacological
inhibition of ␣IIb␤3-integrin signaling have been proposed to
reduce ex vivo PMP production (25, 33) and may thus help to
reduce PMN activation and sequestration in the lungs subse-
quent to transfusion of stored blood products. Compounds such
as cyclosporin A and Orai 1 inhibitors have been proposed as
a means of decreasing levels of EMPs and PMPs, respectively
(5, 10, 21, 32). Further work is required to examine the effects
of eliminating or altering selected MP populations generated in
stored blood products to test whether such interventions may
positively impact the mortality and morbidity of transfused
experimental animals and ultimately patients.
Potential Beneficial Effects of Microparticles in ALI
MPs are not necessarily always associated with progressive
organ dysfunction and disease outcome. As recently discussed
in seminal reviews by Morel and coworkers (120) and Dignat-
George and Boulanger (46), MPs are by no means always
detrimental but may frequently exert beneficial effects, in that
they exhibit anti-inflammatory and anticoagulant potential and
can improve endothelial and vascular function under patholog-
ical conditions (Fig. 7). Experimental evidence for the benefi-
cial effects of MPs comprise the promotion of myocardial
tissue repair postinfarction by PMPs, the stimulation of matrix
degradation and new blood vessel formation by EMPs through
activation of matrix metalloproteases, or the triggering of
angiogenic endothelial responses and the reconstitution of
endothelial function and NO generation associated with re-
duced ROS production by LMPs carrying the developmental
morphogen sonic hedgehog (3, 20, 114). These findings exem-
plify the seemingly paradoxical beneficial effects that have
been documented for a variety of MPs under different patho-
logical conditions, a notion that gains particular relevance in
the context of ALI by the clinical observation that higher levels
of circulating LMPs or EMPs, respectively, are associated with
increased survival in both sepsis and ARDS (65, 158). The
potential role of beneficial MPs gains additional momentum in
light of the fact that considerable numbers of MPs can also be
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L375
Fig. 7. Beneficial and detrimental potential of MP as highlighted by their
effects on endothelial cells. MPs influence endothelial cells in different ways
(both beneficial and detrimental) depending on their cell origin and the specific
stimulating trigger leading to their formation. Highlighted are examples of
divergent MP effects on nitric oxide (NO) release, proreactive oxidative
species (ROS) production, expression of inflammatory enzymes such as
cyclooxygenase-2 (COX-2) or procoagulant factors such as tissue factor (TF),
or angiogenic responses including proliferation and migration. Reproduced
from Benameur et al. (20) with kind permission from Pharmacological
Reports.
released by mesenchymal stem cells (MSCs) and may thus, at
least in part, account for the paracrine effects by which MSCs
have emerged as a promising new therapeutic strategy in ALI
(63, 92, 93, 107). MSCs secrete MPs that are enriched in
pre-miRNAs (35) and, in a rat renal injury model, could inhibit
apoptosis and promote proliferation of kidney epithelial cells
by paracrine effects that are in part mediated via intercellular
transfer of mRNA and miRNA (58). MSCs have also recently
been shown to donate mitochondria to lung parenchymal cells
in ALI by a microvesicular mechanism that could imply MPs
(73); yet the relevance of mitochondrial transfer for the effects
of MSCs has been challenged by others (38). Further investi-
gations are needed to characterize the contributions of MSC-
derived MPs in the benefits these cells appear to offer in the
setting of lung injury. In the following, we will discuss mech-
anisms that may potentially contribute to the beneficial effects
of non-stem-cell-derived MPs in ALI focusing on the same
pathophysiological characteristics of the disease as previously
done for the detrimental role of MPs.
Inflammation. Although we previously emphasized the role
of TXA2 in the context of the delivery and release of AA and
AA metabolites by MPs, it must be taken into consideration
that not all eicosanoids are proinflammatory and increase
barrier permeability. Notably, PMPs contain lipoxygenase 12
that, when transferred to mast cells, promotes production of the
anti-inflammatory leukotriene lipoxin A4 (LXA4) (161). In a
murine model of experimental colitis, injection of PMPs and
LXA4 attenuated inflammatory responses, whereas lipoxygen-
ase 12 protein-deficient MPs or mast cell depletion reversed
this beneficial effect (161). Since LXA4 analogs have been
shown to attenuate ALI in animal models (47, 76), MP-
mediated generation of LXA4 might prove beneficial in ALI.
Although certain MPs formed in ALI may have the ability to
cause detrimental immune modulation as described in previous
sections, others may confer beneficial immune functions capa-
ble of enhancing survival or reducing morbidity. NMPs can
stimulate secretion of transforming growth factor-␤, which
blocks macrophage activity, and can express annexin A1,
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L376 MICROPARTICLES AND ACUTE LUNG INJURY
which acts as an anti-inflammatory protein blocking adhesion
of PMNs to endothelial cells (40, 57). In addition, MPs gen-
erated from KATO-III human gastric cancer cells can interact
with monocytes and change their cytokine release profile from
proinflammatory mediators such as TNF-␣ to anti-inflamma-
tory cytokines such as IL-10 (83). Although the relevance of
such effects in the context of ALI/ARDS remains to be eluci-
dated, these findings underscore the anti-inflammatory poten-
tial harbored by many MPs. A recent analysis of ARDS
patients demonstrated elevations in both LMPs and NMPs
within BAL samples of ARDS patients compared with spon-
taneously breathing controls (65), whereas levels of PMPs did
not differ among ARDS patients compared with simplified
acute physiology score (SAPS II) and sequential organ failure
assessment (SOFA) score matched ventilated intensive care
patient controls without ARDS and spontaneously breathing
patients being bronchoscopically investigated for suspected
chronic lung diseases. Notably, survivors of ARDS had statis-
tically elevated levels of LMPs compared with the ventilated
and spontaneously breathing control groups. This unexpected
finding raises the possibility that LMPs may not only be a
biomarker of ARDS and/or potentially promote inflammatory
signaling and parenchymal damage but may at times also be
protective and reflect a “healthy” level of immune function.
This physiological effect may be absent in severe inflammatory
diseases such as ARDS, causing “immune paralysis” or a
relative anergy of immune function that may have ramifica-
tions on the host that directly impact mortality (123).
Coagulation. Whereas the high expression levels of TF in
MPs are generally considered to be procoagulant and barrier
disruptive, they may also have anticoagulant and barrier-
protective properties through PAR1-mediated transactivation
of PAR2 signaling pathways (82). In addition, MPs can express
a variety of anticoagulant factors including thrombomodulin,
TFPI, endothelial protein C receptor (EPCR), and protein S
(120). Thus the potential involvement of MPs in anticoagulant
pathways may present both an intrinsic protective mechanism
and a possible future therapeutic target in lung injury. Notably,
LMPs from hepatitis C patients have been shown to cause
increased fibrinolysis after fusion with hepatic stellate cells,
thus decreasing fibrin deposition (84).
Activated protein C (APC) proteolytically inactivates key
coagulation factors including factor Va and factor VIIIa. Re-
combinant APC has been shown to reduce ALI and increase
alveolar ventilation in experimental animal models (71, 110,
167) and individual clinical case reports (81). In sepsis pa-
tients, treatment with APC has been reported to show a trend
toward increased survival that is notably associated with MP
formation and concomitant anti-inflammatory effects (132).
However, a recent Cochrane review did not recommend its use
because of a failure to improve survival and an increased
bleeding risk, which has led to a decrease in its use as a therapy
for severe sepsis (105). Interestingly nevertheless in the con-
text of this review, APC induces the release of MP-associated
EPCR that can bind APC and exert anticoagulatory (131) and
potentially also anti-inflammatory and barrier-protective ef-
fects (120). EPCR (131) is a member of the major histocom-
patibility complex/CD1 superfamily that promotes protein C
activation at the cell surface (159), yet upon metalloprotease-
mediated cleavage circulates in a soluble form (sEPCR) (88)
that neutralizes APC so that it can no longer inactivate factor
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00354.2011 • www.ajplung.org
Va (95). Importantly, APC bound to EPCR in microparticulate
form retains anticoagulant activity (131), and has been shown
to exert both antiapoptotic and barrier-protective effects (130).
Permeability. APC may also exert important barrier-protec-
tive effects, in that EPCR ligation promotes transactivation of
sphingosine 1-phosphate receptor 1 (S1P1) via phosphatidyl-
inositol 3-kinase (PI3K)-dependent phosphorylation (51).
Hence, APC-bearing MPs may limit vascular permeability via the
well-documented barrier-enhancing signaling cascade down-
stream of S1P1 (169). This notion is supported by experimental
data demonstrating that EPCR/APC-bearing EMPs reduce endo-
thelial permeability in vitro via a mechanism that involves PAR-1,
PI3K, and notably also the vascular endothelial growth factor
receptor 2 (130).
Endothelial function. Although MPs are frequently associ-
ated with endothelial and vascular dysfunction (vide supra),
several studies by the group of Ramarosin Andriantsitohaina
and Maria Carmen Martinez (3, 114, 121) have demonstrated
that the endothelium may also receive beneficial signals from
MPs carrying sonic hedgehog, resulting in correction of endo-
thelial injury, reconstitution of NO release, and reduced ROS
production. The role of NO in this context, however, is com-
plex because it can exert both attenuating and aggravating
effects on inflammatory, coagulatory, or permeability changes
depending on its location, concentrations, and local microen-
vironment (85). Although a recent meta-analysis did not sup-
port the rationale for inhaled NO as an effective treatment
strategy in ALI/ARDS (2), reconstitution of endogenous NO
production and an intact endothelial and vascular function may
nonetheless provide important benefits that could positively
impact on clinical outcome in ALI/ARDS.
Clinical implications. Taken together, MPs possess a variety
of mechanisms by which they may exert anti-inflammatory,
anticoagulatory, and barrier-enhancing effects under healthy or
diseased conditions. Although our understanding of these prop-
erties of MPs in general, and in the context of ALI/ARDS in
particular, is still in its infancy, the potential to exploit this
intrinsic property for therapeutic purposes poses a challenging
yet promising path to pursue.
Synopsis and Implications
There has been little in the way of effective innovations and
interventions in the treatment of ALI/ARDS beyond the intro-
duction of protective ventilation (1) and restrictive fluid man-
agement strategies (176). ALI/ARDS is commonly associated
with increased formation of MPs from various cellular origins
found in both the distal air spaces and circulating blood. These
MPs are small vesicles with an intact lipid bilayer that may
contain membrane and cytosolic proteins, lipids, organelles,
and genetic material from their parent cells that can function
autonomously or transfer their contents to other cells. Although
most studies have looked at MPs as potential biomarkers for
disease and progression of illness, MPs bear the potential to
convey both detrimental as well as beneficial effects with
respect to the excessive inflammatory, coagulatory, and per-
meability responses characteristic for ALI/ARDS (Fig. 6).
Further investigations into MPs’ actions are required to better
understand their role in the pathophysiology of acute lung
disease and to utilize their potential for therapeutic interven-
tions. Such concepts may include strategies to prevent the

MICROPARTICLES AND ACUTE LUNG INJURY
formation of MPs by altering flip-flop scramblase enzyme
activities (116) or therapeutic manipulation of MP populations
by pharmacological interventions (138). Even more attractive
is the potential perspective to use specifically engineered “de-
signer” MPs as vectors to move genes, proteins, or specific
cellular functions between cells (20). Clearly, MPs add another
layer of complexity to the already multifaceted mechanisms
involved in ALI/ARDS, but in parallel they may offer hope of
obtainable and specific sites for future pharmacological manip-
ulation.
ACKNOWLEDGMENTS
Illustrations in Figs. 4, 5, and 6 are courtesy of Stefania Spano.
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