UvA-DARE (Digital Academic Repository)
Microparticles: mediators of cellular and environmental homeostasis
Böing, A.N.
Publication date
2011
Link to publication
Citation for published version (APA):
Böing, A. N. (2011). Microparticles: mediators of cellular and environmental homeostasis.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
Chapter 10
Chapter 10
Aim of the thesis
The aim of this thesis was to investigate why cells release microparticles and whether microparticles are involved in maintaining cellular- and environmental homeostasis.
Microparticles and cell survival
Why do cells release microparticles? To answer this question, we inhibited the release of microparticles from endothelial cells and studied the effects thereof in Chapter 2. Inhibition of microparticle release resulted in the intracellular accumulation of caspase-3, especially in the presence of the apoptosis inducer staurosporin, which was paralleled by increased apoptosis and detachment of the endothelial cells. Therefore, we hypothesized that the release of caspase-3 containing microparticles indeed contributes to endothelial survival.
To further investigate the hypothesis that microparticles can be used by cells to remove the potentially dangerous caspase-3, we used MCF-7 cells, a human cancer cell line which is known to be deficient of functional caspase-3 and lacks “membrane blebbing”1.
Although “membrane blebbing” and “microparticle release” are often used as synonyms in the literature, it is unknown whether blebbing is a prerequisite for, or leads to, the release of microparticles. Moreover, from the literature it is unknown whether MCF-7 cells actually release any microparticles. In Chapter 3, we transfected MCF-7 cells with the 29 kDa form of caspase-3, since this form has the ability of autocleavage and self activation2. The
expression of caspase-3 by MCF-7 cells was paralleled by the release of microparticles, whereas untransfected cells did not or hardly released any microparticles, indicating that expression of caspase-3 is indeed strongly associated with the release of microparticles. Furthermore, we showed that the released microparticles themselves contain caspase-3 and are enriched in caspase-3 activity compared to the releasing cells. These data further
10
presence of caspase-3 in microparticles is a common phenomenon that is not necessarily restricted to nucleated cells.
The sorting of potentially dangerous or redundant compounds by cells into vesicles seems to be a common mechanism used for cellular survival. Earlier, de Gassart et al. showed that exosomes in conditioned culture media from several types of healthy and viable cells contain a caspase-3 like activity. He postulated that the observed packaging of caspase-3 activity into exosomes is a mechanism to ensure cell survival3. Similarly, the
release of vesicles enriched in e.g. the complement C5b-9 complex4 or cytostatics5;6 are
presumed to protect the cells against accumulation of dangerous or lethal concentrations of these compounds, and thus to promote cellular survival. Taken together, the release of microparticles containing caspase-3 and other dangerous compounds contributes to cellular survival, and, by packaging these compounds into microparticles, also the environment may be protected from exposure to such harmful compounds.
Microparticles: more than cellular homeostasis alone
Many previous studies have shown that microparticles can initiate or facilitate processes such as coagulation, inflammation and angiogenesis. Although these processes are often associated with pathological conditions, these processes are also of fundamental relevance to ensure survival by protecting an organism against threats from its environment under normal and abnormal conditions. In many diseases, however, the aforementioned processes are in a disbalance. One of those diseases is preeclampsia, a disease characterized by a pro-coagulant, pro-inflammatory and anti-angiogenic phenotype when compared to normal pregnant women. Although the exact underlying mechanisms of the development of preeclampsia are unknown, it is assumed that placental hypoxia leads to increased shedding of placental material into the maternal circulation7. In turn, this placental material, also
known as syncythiotrophoblast-derived microparticles or STBM, is thought to be an essential step in the development of preeclampsia, leading to endothelial dysfunction, coagulation, inflammation and inhibition of angiogenesis8.
In Chapters 5 and 6, we studied the association between circulating microparticles, angiogenesis and inflammation in preeclampsia. In Chapter 5, we showed that plasma samples from preeclamptic women contain increased numbers of microparticles exposing
Chapter 10
the vascular endothelial growth factor (VEGF)-receptor 1, FMS-like tyrosine kinase (Flt-1), compared to normal pregnant women. These Flt-1-exposing microparticles originated from the placenta and platelets. We hypothesize that the capturing of VEGF by microparticles exposing Flt-1 will reduce the plasma level of VEGF, leading to less VEGF available for binding to its cellular receptors, and thus resulting in the inhibition of angiogenesis. Further evidence supporting a role of microparticles in inhibition of angiogenesis in preeclampsia comes from a recent study, in which a perfusate of human placenta was shown to contain high numbers of microparticles exposing endoglin9. Endoglin is a receptor for transforming
growth factor beta (TGF-β), which, like VEGF, is an angiogenesis-promoting growth factor. Thus, similar to microparticles exposing Flt-1, also endoglin-exposing microparticles may be involved in capturing a pro-angiogenic factor before this factor can bind to its cellular receptor as a prerequisite for the induction of angiogenesis9.
In Chapter 6, we investigated the possible involvement of microparticles in inflammation in preeclampsia. We studied the expression of inflammation-related genes in endothelial cells exposed to microparticles from plasma of preeclamptic patients and healthy pregnant control individuals. Overall, our data showed no upregulation of inflammation-related genes in the presence of preeclamptic microparticles. In contrast, incubation of monocytes with STBM resulted in the upregulation of the inflammatory mediators including interleukin (IL)-12p70, tumor necrosis factor (TNF)-α, IL-8 and IL-6 in vitro10;11. Since STBM bind to monocytes in vivo10, one may speculate that in experiments where microparticles are isolated from plasma samples, the biologically most active and interesting microparticles are absent due to their interaction with, or binding to, blood cells and endothelial cells.
10
anionic phospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the membrane environment of TF13-17, and / or a conformation change in the TF molecule
induced by protein disulfide isomerase (PDI)18.
In Chapter 7, we investigated the phospholipid composition and the ability of endothelial microparticles to initiate thrombus formation in vivo in a rat venous stasis model. We showed that microparticles released from IL-1α-activated endothelial cells, but not microparticles released from non-activated cells, expose TF and trigger TF-dependent thrombus formation in vivo. The microparticles from activated endothelial cells were enriched in PS and PE compared to microparticles from non-activated endothelial cells. Since both PS and PE promote the coagulant activity of TF13-17, these data suggest that
activated cells release microparticles with a fully equipped coagulant profile. Microparticles from resting endothelial cells also expose PS and PE, albeit to a lesser extent than microparticles from activated endothelial cells, but these microparticles did not trigger thrombus formation in vivo, suggesting that the presence of these phospholipids alone is insufficient to initiate thrombus formation.
In Chapter 8 we showed that plasma membranes and microparticles simultaneously contain a non-coagulant form and a coagulant active form of TF. Furthermore, we showed that PDI and the non-coagulant form of TF are present in rafts of plasma membranes and microparticles, whereas the coagulant form of TF is present outside the rafts. We hypothesized that, if PDI plays any role in of the regulation of the coagulant activity of TF, there may be a two-step activation mechanism, in which PDI first converts TF from a non-coagulant form into a non-coagulant form within the rafts, but where the non-coagulant activity of TF remains functionally dormant. In turn, TF is then immediately translocated to the non-raft domains of the membrane, which are enriched in PS and PE compared to the non-rafts, and this enables the coagulant activity of TF to be decrypted. However, since PDI can also reduce the coagulant activity of TF via a conformation change18 and can also reduce the
presence of PS exposed by membranes19, additional studies are essential to unravel this
complex interrelationship.
In Chapter 9, we investigated whether the alternatively spliced (as) form of TF is associated with microparticles, and whether this form of TF is coagulant. In contrast to full length TF, however, microparticles from cells expressing asTF did not contain detectable
Chapter 10
amounts of this form of TF, and, in contrast to earlier studies20;21, we found that asTF is not
secreted and lacks coagulant activity. Thus, asTF is unlikely to play any role in microparticle-initiated and TF-dependent coagulation. This does not rule out a role for asTF, however, in other TF-dependent processes such as angiogenesis22-24.
Clinical relevance and future directions
At present, there is a growing interest in the biological relevance of microparticles and other types of cell-derived vesicles. In the first chapters of this thesis data are presented that provide additional evidence to support the hypothesis that microparticles indeed play a role in the maintenance of cellular homeostasis. Concurrently, by packaging unwanted compounds into vesicles the environment may be protected as well. Combined with efficient clearance mechanisms, this packaging seems an elegant mechanism of cells and the organism to remain healthy. When studying the “health state” of a cell, it may be worthwhile to focus not only on the cells themselves but also on the (composition of the) vesicles that are released by those cells. In this way, an improved insight into the “health state” can be obtained. For instance, when endothelial cells were incubated in vitro with clinically relevant concentrations of simvastatin, a widely used cholesterol-lowering drug that significantly reduces the risks of cardiovascular disease, no apparent changes in the cell cultures were observed. The cells remained seemingly healthy and viable in the presence of simvastatine, which is in line with the overall beneficial affects of statins in vivo. Nevertheless, there was a 2-3 fold increase in the release of caspase-3 containing microparticles by endothelial cells in the presence of simvastatin25, suggesting that, for
unknown reasons, the cells seem to consider simvastatin as a form of extracellular stress and thus release more caspase-3 containing microparticles to overcome the effects of simvastatin. Given the fact that other in vitro studies, usually using higher concentrations of
10
The fact that microparticles maintain cellular homeostasis and facilitate protective mechanisms that support survival of the organism at the same time, including coagulation, inflammation and angiogenesis, is a beautiful convergence of functions. Obviously, this will be a very delicate balance and a shift may impose changes that contribute to the development or aggregation of diseases. For instance, the presence of microparticles exposing coagulant TF within the blood can be potentially lethal and lead to development of disseminated intravascular coagulation or venous thromboembolism. As shown in
Chapter 8, cells as well as microparticles can expose two different conformational forms
of TF. Most of this TF seems to be in a non-coagulant form, but the function(s) of this form of TF is unknown. These findings make the research on TF even more complex, leading to a multitude of intriguing questions, e.g. do both forms of TF occur on microparticles from cancer cells, what is the function of the non-coagulant form of TF, are both forms of TF transferred to recipient cells, etc.
Due to their small size and heterogeneity, the detection of vesicles is still cumbersome. Although flow cytometry is widely used, this technology detects only the larger vesicles and more than 98% of the vesicles in body fluids is thought to be below the detection limit of flow cytometry26;27. Improvement in reliable detection techniques is an
essential prerequisite for future vesicle research. The use of vesicles as clinically relevant diagnostic and prognostic tools may then come within better reach, and may also help to answer the question whether microparticles are cause or consequence of disease, or both. Also the underlying mechanisms of microparticle release are only fragmentary understood. More insight into the underlying mechanisms of microparticle release and how these mechanisms are affected by drugs will help to keep cellular and environmental homeostasis in a healthy balance.
Finally, despite a wealth of literature, more thorough investigations and comparisons of vesicle subpopulations well divided into e.g. microparticles and exosomes, will be essential to improve our understanding of their functions and the underlying mechanisms of release. Given the fact that also prokaryotes use vesicles for communication and protection suggests that common and conserved mechanisms may be present underlying the release of such vesicles and this knowledge may help to understand the formation and function of microvesicles in eukaryotes. At present, vesicles have become widely accepted throughout
Chapter 10
the literature as an exciting new element in the many cascades of protein and cellular systems.
References
1. Janicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J.Biol.Chem. 1998;273:9357-9360.
2. Meergans T, Hildebrandt AK, Horak D, Haenisch C, Wendel A. The short prodomain influences caspase-3 activation in HeLa cells. Biochem.J. 2000;349:135-140.
3. de Gassart A., Geminard C, Fevrier B, Raposo G, Vidal M. Lipid raft-associated protein sorting in exosomes. Blood 2003;102:4336-4344.
4. Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. J.Biol.Chem. 1988;263:18205-18212.
5. Safaei R, Larson BJ, Cheng TC et al. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells. Mol.Cancer Ther. 2005;4:1595-1604.
6. Shedden K, Xie XT, Chandaroy P, Chang YT, Rosania GR. Expulsion of small molecules in vesicles shed by cancer cells: association with gene expression and chemosensitivity profiles. Cancer Res. 2003;63:4331-4337.
7. Knight M, Redman CW, Linton EA, Sargent IL. Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. Br.J.Obstet.Gynaecol. 1998;105:632-640.
8. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science 2005;308:1592-1594.
9. Guller S, Tang Z, Ma YY et al. Protein composition of microparticles shed from human placenta during placental perfusion: Potential role in angiogenesis and fibrinolysis in preeclampsia. Placenta 2010
10. Germain SJ, Sacks GP, Sooranna SR, Sargent IL, Redman CW. Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles. J.Immunol. 2007;178:5949-5956.
11. Messerli M, May K, Hansson SR et al. Feto-maternal interactions in pregnancies: placental microparticles activate peripheral blood monocytes. Placenta 2010;31:106-112.
12. Donate F, Kelly CR, Ruf W, Edgington TS. Dimerization of tissue factor supports solution-phase autoactivation of factor VII without influencing proteolytic activation of factor X. Biochemistry 2000;39:11467-11476.
13. Bach R, Rifkin DB. Expression of tissue factor procoagulant activity: regulation by cytosolic calcium. Proc.Natl.Acad.Sci.U.S.A 1990;87:6995-6999.
10
17. Wolberg AS, Monroe DM, Roberts HR, Hoffman MR. Tissue factor de-encryption: ionophore treatment induces changes in tissue factor activity by phosphatidylserinedependent and -independent mechanisms. Blood Coagul.Fibrinolysis 1999;10:201-210.
18. Ahamed J, Versteeg HH, Kerver M et al. Disulfide isomerization switches tissue factor from coagulation to cell signaling. Proc.Natl.Acad.Sci.U.S.A 2006;103:13932-13937.
19. Popescu NI, Lupu C, Lupu F. Extracellular protein disulfide isomerase regulates coagulation on endothelial cells through modulation of phosphatidylserine exposure. Blood 2010;116:993-1001.
20. Bogdanov VY, Balasubramanian V, Hathcock J et al. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat.Med. 2003;9:458-462.
21. Szotowski B, Antoniak S, Rauch U. Alternatively spliced tissue factor: a previously unknown piece in the puzzle of hemostasis. Trends Cardiovasc.Med. 2006;16:177-182.
22. Hobbs JE, Zakarija A, Cundiff DL et al. Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model. Thromb.Res. 2007;120 Suppl 2:S13-S21.
23. Signaevsky M, Hobbs J, Doll J, Liu N, Soff GA. Role of alternatively spliced tissue factor in pancreatic cancer growth and angiogenesis. Semin.Thromb.Hemost. 2008;34:161-169.
24. van den Berg YW, van den Hengel LG, Myers HR et al. Alternatively spliced tissue factor induces angiogenesis through integrin ligation. Proc.Natl.Acad.Sci.U.S.A 2009;106:19497-19502.
25. Diamant M, Tushuizen ME, Abid Hussein MN et al. Simvastatin-induced endothelial cell detachment and microparticle release are prenylation dependent. Thromb.Haemost. 2008;100:489-497.
26. Lacroix R, Robert S, Poncelet P, Dignat-George F. Overcoming limitations of microparticle measurement by flow cytometry. Semin.Thromb.Hemost. 2010;36:807-818.
27. van der Pol E, Hoekstra AG, Sturk A et al. Optical and non-optical methods for detection and characterization of microparticles and exosomes. J.Thromb.Haemost. 2010;8:2596-2607.
