Chapter 1: Introduction
1.1 Microparticles (MP) generation
MP were first described in 1967 by Wolf as platelet membrane fragments in human plasma and they were initially called ‘platelet dust’. At the beginning MP have been considered simply markers of cellular activation or damage but recent data show that MP can mediate long-range signaling, acting on cells different from their cells of origin. MP are small membrane vesicles (0.05 – 1 μM) shed from cells in response to activation, injury and/or apoptosis and their composition reflects the state of the membrane of the originating cells [5]. These vesicles can be released by all eukaryotic cells and in general they derive from circulating cells such as platelets, leukocytes and erythrocytes, and cells that compose the vessel wall like endothelial cells, macrophages and smooth muscle cells [6].
MP circulate in the bloodstream of healthy individuals and patients, and their number, cellular origin and composition can change accordingly to the different diseases and different states of the disease. The impact of these changes on their in vivo effects is still unknown. The mechanisms leading to the generation of MP have not been fully elucidated; it is known that there are two different cellular processes that can lead to the formation of MP: chemical and physical cell activation induced by agonists or shear stress respectively, and apoptosis induced both by deprivation of growth factors or by apoptotic inducers [7] (fig.1.1). Currently it is still not clear if cell activation and apoptosis lead to the formation of similar MP in terms of size, lipid and protein composition and (patho-)physiological effects. There are however differences in the mechanisms leading to their generation.
Cells can release MP after activation by many different agonists. Monocytes, endothelial cells, hepatocytes and arterial smooth muscle cells can generate MP after stimulation by bacterial lypopolysaccharide (LPS), cytokines such as Tumor Necrosis Factor – α (TNF-α) or Interleukin-1 (IL-1), the C5b-9 complex
or hydroperoxide [2], [8], [9], [10], [11]. The release of MP following cell activation is generally time- and calcium-dependent and the shedding starts within few minutes after stimulation by an agonist [12], [13]. There are many evidences that the process of vesiculation depends on the rapid increase of the cytosolic calcium concentration [14], [15], [16]. The chelation of extracellular calcium ions by EGTA blocks the increase in cytosolic calcium as well as the release of MP [15]. The increased level of intracellular calcium activates kinases, inhibits phosphatases and activates calpain leading to the collapse of membrane asymmetry. Calpain is a calcium-dependent cytosolic protease, which can be inhibited by calpeptin. However, inhibition of this protease can not block the generation of MP as efficiently as does calcium chelation and this finding suggests that the calpain pathway may not be the sole calcium-dependent mechanism for MP release [17]. The asymmetric distribution of phospholipids of the cell membrane is lost after cell stimulation and this step is crucial for the generation of MP. In the resting membrane the aminophospholipids, like phosphatidylserine (PS) and phosphatidylethanolamine, are sequestered in the inner side of the membrane by specific transporters that regulate their inward and outward phospholipid translocation. After stimulation the aminophospolipids translocate to the outer leaflet and this process is controlled by the activation of specific phospholipid transporters called “floppases” and the inhibition of other transporters including the aminophospholipid translocase. The main change in the lipid distribution is the exposure of PS on the surface of the cell membrane, and the PS externalization also represents one of the principal characteristics of MP [7]. The remodeling of the membrane is a fundamental step for the generation of MP and it has been shown that are involved also genes linked to the cytoskeletal reorganization, such as Rho kinase-II in the formation of MP by endothelial cells through a mechanism involving caspase-2 [18]. Other authors have shown the involvement of caspase-3 and Rho kinase-I in MP formation [19].
MP can be released also by apoptotic cells. Apoptosis is a process of programmed cell death characterized by cell contraction, DNA fragmentation,
and dynamic membrane blebbing. The apoptotic bodies differ from MP released by cells in size, lipid and protein composition and (patho-) physiological effects. The apoptotic membrane blebbing depends on activation of the Rho-associated kinase ROCK I. During apoptosis ROCK I is cleaved by activated caspases and becomes activated. This kinase promotes the increasing of actin-myosin force generation and couples actin-myosin filaments to the plasma membrane [20]. ROCK activity is fundamental for membrane blebbing, thus MP formation during apoptosis results from ROCK activity and the resulting disruption of the membrane skeleton structure. MP derived from apoptotic cells can contain fragmented DNA [5].
Fig.1.1 Schematic representation of the general mechanisms involved in MP formation during cell activation (left panel) and apoptosis (right panel) [5].
MP are surrounded by a phospholipid bilayer with a symmetrical phospholipid distribution characterized by the exposition of negatively charged phospholipids such as PS and phosphatidylethanolamine, which can
efficiently bind coagulation factors. Huber and coworkers recently demonstrated the presence of oxidized phospholipids in MP from endothelial cells exposed to an oxidative stress stimulus, whereas such phospholipids were absent in MP released by endothelial cells stimulated with calcium ionophore [21]. Thus, the phospholipid composition and their oxidation status differ among MP.
MP expose membrane antigens that are specific for the “parent cell” they originate from (fig.1.2). Some of these identification antigens are always present on the cell surface, independently of the activation or apoptosis status of the parent cell, and they can be used to determine their cellular source, e.g. CD4 for MP from T-helper cells. MP can also contain molecules that have been upregulated or translocated by cell activation or apoptosis, e.g. MP derived from platelets expose molecules like P-selectin and glycoprotein 53 that both originate from intracellular granule membrane [5]. The MP composition is also agonist dependent, e.g. T-cells produce MP that are enriched in CD3ε- and ζ-chains only upon activation of the T-cell antigen receptor and not upon activation by ionomycin plus p-methoxyamphetamine hydrochloride [22]. They can also contain transcription factors and mRNA derived from their parents cells.
In conclusion, MP vary in size, phospholipid and protein composition and therefore in their functional capacity and activity. The shedding seems also a well regulated process able to create specific MP characteristics under various (patho-)physiological conditions [5].
Fig.1.2 The plasma membrane response to the cell stimulation and cellular MP as storage pool of bioactive effectors [23].
To better define MP, it is also important to distinguish them from other vesicular structures released from cell upon activation or apoptosis.
Ectosomes is a term used as synonymous of microparticles. Ectocytosis was coined from Stein and Luzio [24] to define the shedding of right -side-out membrane vesicles from the surface of eukaryotic cells and to distinguish this process from endocytosis and exocytosis. Like MP, ectosomes are vesicles generated upon budding out and pinching off from the surface membrane. Exosomes are stored intracellularly in preformed multivesicular bodies. They are secreted by the cells when multivesicular bodies fuse with cell membrane (“exocytosis”) upon stimulation. They are structures with a size range from 50 to 100 nm surrounded by a lipid bilayer. Thus, they differ from MP in both size and composition [25].
Endosomes are important elements in the endocitic pathway and are not released in the extracellular space. In response to external stimuli, certain specialized endosomes however may function to present and remove receptors at the cell surface, altering cellular responsiveness [26].
Also originated during apoptosis, the apoptotic bodies differ from MP. In contrast to MP, which are generated during activation of viable cells or during
early apoptosis, apoptotic bodies are released at later stages of programmed cell death and have a larger diameter (1-4 µm) and volume. Indeed, they are usually phagocytized by macrophages [17].
1.2 Methods to measure MP
The conventional method to measure MP is flow cytometry (FACS) which measures the number, size and properties of MP. By definition MP are small structures which display characteristic forward and side scatter patterns. Through this method it is possible to use antibodies to cell surface molecules and identify the specific subpopulation of MP and their original cell from which they derive. In addition to proteins, the presence of PS on their outer surface allows binding of Annexin V which can also be used to identify and quantify MP. The main limitation of FACS concerns the detection of the smallest MP, e.g. < 300 nm in diameter. This method is normally used to evaluate cells that are 10- to 100-fold greater in diameter than MP and these very small structures are sometimes registered as “noise” of the instrument. An alternative method to identify MP involves binding assay in a solid phase or microtiter plate format. In this approach, antibodies against cell surface molecules can capture particles for subsequent detection by another antibody or a functional assay for coagulation enzyme activation, for example. This assay can assess in a specific manner the total amount of MP and their biological origin but it does not give information about their number and size.
MP analysis can be impacted not only by type of assay but also by the manner in which are collected and processed the samples of blood, including the centrifugation and freeze-thaw methods. Currently, the measure of MP needs still to be standardized and improved [27].
1.3 MP functions
1.3.1 Antigen transfer and intercellular cross-talk
Thanks to their small size, MP can rapidly circulate in the vasculature and they can participate in both local and long-range signaling. In their interaction with cells, MP bind the surface ligands of different cells, representing a kind of direct long distance cell-cell communication for cells normally far away from each other. Indeed, MP can transfer surface molecules to other cells as well exchange membrane and cytoplasmic proteins, representing a new mechanism for intercellular interactions [28]. This transfer can also predispose to the infiltration by an organism (e.g. HIV) requiring a particular cell surface molecule to entry the cell [29].
1.3.2 MP in inflammation
The physiological and pathophysiological roles of MP are unclear; their role can be beneficial or deleterious depending on situations and composition. Inflammation is a process characterized by interactions among platelets, leukocytes and endothelial cells. MP have potent proinflammatory activities and are potentially important mediators of rheumatologic and other inflammatory diseases. Several pro-inflammatory enzymes and their metabolites are up-regulated by the direct action of MP. Recent studies of the effects of platelet-derived MP on endothelial cells showed that arachidonic acid transported by MP leads to an increase in cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression, promoting the adhesion of monocytes [30], [31]. MP can also facilitate interactions between leukocytes and endothelium, enhancing leukocyte rolling. Platelet-derived MP (PMP) allow neutrophils to aggregate and to adhere to pre-bound cells via interaction between P-selectin localized on MP and P-selectin-glycoprotein-ligand-1 (PSGL-1) on neutrophils and an induction of adhesion molecules [32]. PMP or MP of leukocytic origin (LMP) participate in the release of several cytokines from endothelial cells (such as 1, 6, IL-8, and monocyte chemoattractant protein-1 (MCP-1)) and from monocytes
(IL-1, TNF-α and IL-8). MP can also stimulate inflammation by activation of the complement cascade. They can bind C1q, the main unit of the classical complement pathway, and activate the classical complement pathway leading to the deposition of C3 and C4 on the MP surface. In this way, MP may therefore trigger the typical pro-inflammatory effects of complement activation [33]. Circulating MP from pre-eclamptic patients with endothelial dysfunction can act on lymphocyte stimulating the activation of iNOS and COX-2, as well as the NF-kB pathway, indicating that MP can act as pro-inflammatory effectors [7].
1.3.3.MP in immunity
MP seem to support a sort of physiological immune escape and an example could be in pregnancy. Abrahams et al. propose that the secretion of MP containing FasL on their surface may represent a mechanism by which trophoblast cells promote a state of immune privilege and, therefore, protect themselves from maternal immune recognition. In the first-trimester trophoblast cells lack membrane-associated FasL but constitutively secrete FasL through the release of MP, which are able to induce Fas-presenting T cell death by apoptosis. Cancer is a pathological situation in which MP are implicated in escape from the immune system. Recent studies show that epithelial ovarian cancer cells were shown to secrete functional FasL via the release of MP. In contrast, normal ovarian cells epithelial cells express but do not secrete FasL. These evidences suggest a mechanism by which tumors might neutralize Fas-bearing immune cells, facilitating escape and promoting survival [5].
1.3.4 MP and cancer
Cancer is a situation in which MP are involved at different levels. Monocyte-derived MP (MMP) were considered as a sign of vascular complication in
patients with lung cancer [34]. Clinical evidences show high levels of circulating platelet-derived MP in gastric cancer, suggesting a possible role of MP as metastasis predictors. Moreover, experimental evidences show that doxorubicin was accumulated in MP shed by cancer cells, supporting the hypothesis that they can release MP to rid themselves of toxic drugs. In this way, MP can take part in the resistance to chemotherapy [35].
1.3.5 MP and the vascular system
Endothelial dysfunction is the primary event leading to the failure of the vasoactive, anticoagulant and anti-inflammatory effects of the healthy endothelium. Because endothelial dysfunction and arterial stiffness are major determinants of cardiovascular risk, several groups have investigated the effects of circulating MP on vascular function. MP can have an effect on endothelial cell and smooth muscle cells responses and, hence, vasoreactivity as well as angiogenesis [36]. Endothelial responses can be acute, resulting from the release of several factors, or prolonged, implying changes in expression of genes involved in structural and functional regulation of the vascular wall. The endothelium is also a primary target for cardiovascular risk factor and MP can constitute an adaptive phenomenon or contribute to the aggravation of diseases. The type of effect that MP have on the regulatory function of the blood vessel depends on their cellular origin. PMP stimulate platelets and endothelial cells through modifications of arachidonic acid metabolism and generation of thromboxane A2 (TxA2). Indeed, PMP induce
the expression of the inducible isoform of COX-2 and the generation and release of prostacyclin (PGI2) [37]. Therefore PMP seem to act as a cellular
source of TxA2 that can regulate the vascular tone. An increased number of
circulating endothelial MP (EMP) has been observed in several pathologic conditions reflecting endothelial cell damage and dysfunction. EMP impairs endothelium-dependent relaxation and nitric oxide (NO) production in the rat aorta. It has been shown that the effect induced by EMP is linked to an increase in superoxide anion production, that might reduce the bioavailability
of NO [38]. So far, little information is available about MP shed from smooth muscle cells (SMCs). Schecter et al. suggested that the active extracellular tissue factor (TF) found in the injured arterial wall and atherosclerotic plaques derives in part from smooth muscle cell MP. These TF-rich MP (TF+-MP) released by SMCs might have a role in the generation and maintenance of atherosclerotic plaques [39]. Interaction between lymphocytes and endothelial cells is a prerequisite for the recruitment of immune cells from blood at sites of inflammation. Under vascular damage the level of MP released by leukocytes is strongly enhanced. LPS-stimulated monocytes release TF+-MP and active adhesion complexes, disseminating a procoagulant potential [2]. Moreover, MP shed by activated monocytes represent the major secretory pathway for the rapid release of the proinflammatory cytokine IL-1β [40]. LMP behave like inflammatory mediators activating signal transduction in human vein endothelial cells (HUVEC). Among the active pathways, LMP can stimulate the secretion of IL-6 in HUVEC through the phosphorilation of JNK1 without the involvement of NF-kB or the ERK pathway [41]. It has been shown that T lymphocytes-derived MP impair endothelium-dependent relaxation in conductance and small resistance arteries in response to agonist and shear stress, respectively [42]. Indeed, T-lymphocyte-derived MP can affect vascular contraction by acting directly on smooth muscle cells [42]. The mechanism involves an interaction of MP with the smooth muscle cells through the action of Fas-Fas ligand pathway responsible for the activation of NF-kB, that in turn upregulates inducible NO synthase and COX-2 expression. These data can explain the paracrine role of MP as vectors of transcellular exchange of message in promoting vascular dysfunction during inflammatory diseases (fig.1.3).
Fig.1.3 MP effects on endothelial cells. COX-2, NO, ROS, reactive oxygen species; TF, [7].
1.3.6 MP and angiogenesis
MP are involved in the induction of angiogenesis in the vascular system. It has been shown that PMP released by healthy individuals promote proliferation, migration and tube formation in cultured endothelial cells. MP can induce angiogenesis by activation of ERK and phosphoinositide 3-kinase pathways [43]. The latter effect of PMP are mediated by their lipid components, like sphingosine 1-phosphate. Also EMP are involved in angiogenesis. They expose on their surface metalloproteinases that can regulate the proteolitic activity essential for invasion during neovascular structure formation [44].
1.4 MP and vascular diseases
Detectable in the peripheral blood in physiological conditions, circulating MP originating from blood and vascular cells are elevated in many different kind of diseases by comparing patients with matched healthy volunteers. A review by FD George summarizes the alteration of MP levels and their main clinical significance in vascular disorders (Tab 1.1).
Tab 1.1 MP in vascular diseases [45].
1.4.1 The coagulation cascade
The coagulation cascade is a multi-step series of events that lead to the activation of pro-thrombin to thrombin, which in turn converts fibrinogen to fibrin with the subsequent formation of a fibrin clot. There are two different pathways that can induce the coagulation cascade: the intrinsic and the extrinsic pathway (1.4).
The intrinsic pathway is initiated by the activation of Factor XII to Factor XIIa, the active enzyme form of the zymogen Factor XII. The extrinsic pathway is triggered by the formation of a complex that includes Factor VIIa, TF, membrane phospholipids and calcium ions. The activation of this pathway catalyses the conversion of Factor IX and Factor X to Factor IXa and Factor Xa, respectively [46].
1.4.2 TF
TF is an integral membrane protein with a molecular weight of 47,000 KDa constitutively expressed by non vascular cells and therefore considered extrinsic to blood [47]. A small amount of Factor VII circulates in its active form as Factor VIIa but is kept physically segregated from TF by the integrity of the endothelium and has no significant biologic activity. After vascular damage Factor VIIa comes in contact with TF expressed by the underlying non-vascular tissues when these tissues are exposed to flowing blood, and Factor IXa and Factor Xa are generated. This means that TF is not constitutively synthesized by cells normally in contact with the blood stream. However, the expression of TF is not restricted to the subendothelium; TF can also be generated by monocytes and endothelial cells upon their activation. Different agonists can induce synthesis of TF by endothelial cells and/or monocytes, such as LPS, IL-1, TNF-α, immune complexes and platelets. The expression of tissue factor by monocytes and endothelial cells involves de novo protein synthesis and requires several hours. For this reason the monocytes and endothelial cell-derived TF cannot trigger the blood coagulation upon tissue injury but rather it is linked to wound repair and remodeling, thrombus formation and atherosclerosis.
Fig.1.4 The two different ways of coagulation cascade. TF as the initiator of the extrinsic pathway of the coagulation cascade [46].
1.4.3 MP and the Coagulation system
One of the first roles described for circulating MP is the initiation and amplification of the coagulation cascade in both physiological and pathological conditions, such as thrombosis. They provide a procoagulant phospholipid surface for the assembly of the enzymatic complexes of the blood coagulation cascade. Their catalytic properties rely to the anionic aminophospholipid PS translocated to the exoplasmic leaflet after membrane remodeling. PS enables local concentrations necessary to achieve the kinetics required for optimal thrombin generation and efficient homeostasis. Moreover, in PMP and activated monocytes the procoagulant activity is enhanced by the presence on their surface of TF. Elevated PMP associated with calpain activity have been documented in plasma of patients with thrombotic thrombocytopenic purpura [4]. MP are involved in several vascular diseases including acute myocardial infarction, antiphospholipid syndrome, preeclampsia, rheumatoid arthritis, hemolytic uremic syndrome, vasculitis, heparin-induced thrombocytopenia and paroxysmal nocturnal hemoglobinuria [48]. EMP express von Willebrand factor (vWF) multimers, with promote platelet aggregation and increase their stability. It has been shown that vWF bound to EMP can bind more readily the platelet vWF receptors than soluble vWF [49]. PMP can express on their surface P-selectin, an adhesion molecule expressed at the platelet and endothelial cell surface, necessary for TF accumulation and leukocyte incorporation into the thrombus after endothelial injury. The interaction between P-selectin and PSGL-1 is necessary to concentrate TF activity at the thrombus edge. Indeed, MP generated by activated monocytes express TF and PSGL-1. These MP circulate at a relatively low concentration and TF is too dilute to initiate blood coagulation. Upon vessel injury, the endothelium becomes activated, expressing P-selectin. The circulating PSGL-1-bearing MP bind to the activated endothelial cell, delivering TF to the developing thrombus. Once concentrated within the thrombus, TF reaches a critical concentration and induce the beginning of blood coagulation [3]. The TF+-MP recruited through P-selectin interactions may stabilize the thrombus by inducing fibrin formation. Because P-selectin-PSGL-1 interactions usually mediate unstable rolling, additional cytoadhesins such as MAC-1, a β-integrin on leukocyte MP, could contribute to thrombus stabilization. Recently, Del Conde et al. postulated that TF on MMP plays a key role in normal coagulation physiology.
Since 1999 when Giesen demonstrated in a in vitro system that the so called blood-borne TF is functionally active and potentially thrombogenic [50], several new further experimental evidences supported this hypothesis challenging the dogma that TF is only a constitutive protein. TF+-MP represent a source of “functional circulating TF” and they can act as important mediators in the process of coagulation and therefore in all diseases wherein thrombotic events are involved. Several studies point at MP of various origins as effectors of vascular wall inflammation [5]. MP can modulate cytokine expression in monocytes and endothelium, promote leukocyte-leukocyte aggregation and recruitment through P-selectin. LMP would stimulate interleukin-6 IL-6 and MCP-1 endothelial release and TF expression trough a JNK1 signaling pathway.
1.5 Pharmacological modulation of circulating MP
Several therapies known to be beneficial in cardiovascular disorders seem to reduce both MP concentration and procoagulant activity. These observations support the hypothesis that part of the beneficial effect is linked to decreased MP pathogenicity at quantitative or qualitative level. It has been shown that Statins like fluvastatin can inhibit EMP shedding, partly through Rho small GTPase, key regulators in cytoskeleton remodeling. Indeed, different anti-platelet treatments, such as GIIbIIIa antagonists and thienopyridines can decrease circulating PMP levels [51].
