- Email: [email protected]
Revealing the Mechanism of Tissue Damage Due to Tobacco Use
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The American Journal of Pathology, Vol. 182, No. 5, May 2013
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See related article on page 1552.
COMMENTARY Revealing the Mechanism of Tissue Damage Due to Tobacco Use Finally, a Smoking Gun? Philip Furmanski From the Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy; the Department of Cell Biology and Neuroscience, School of Arts and Sciences; and the Department of Biochemistry and Molecular Biology and Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey; Rutgers University, Piscataway, New Jersey CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
We have known for more than half a century that tobacco use causes cancers, emphysema, atherosclerosis, and a number of other debilitating and deadly diseases. Some gains have been made in the United States and other countries in reducing tobacco use but it remains a serious worldwide health problem with millions of new cases of tobacco-related diseases diagnosed each year. Remarkably, despite its importance and much study, we still do not have a full picture of the molecular mechanisms by which tobacco, its constituents, and pyrolytic products cause the tissue damage that underlies the wellrecognized tobacco-associated pathologies. In this issue of The American Journal of Pathology, Li et al1 provide a compelling case for a mechanism by which tobacco smoke extract (TSE) induces damage to the extracellular matrix, a key element in the pathogenesis of tobacco-related disease. They find that exposure of human macrophages to TSE initiates a progressive series of events that culminate in apoptosis and the release of small membrane-bound vesicles that carry on their surface a potent collagenolytic and gelatinolytic activity which then degrades the local matrix. Significantly, among the large number of possible proteases that might be involved, they find that only one, matrix metalloproteinase-14 (MMP-14), is responsible for practically all of the activity. Li et al1 also identify the intracellular signaling pathway that initiates the process after TSE Copyright ª 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2013.02.004
exposure, involving Jun N-terminal kinase (JNK) and p38 MAP kinases. This study offers important insights and implications regarding the mechanism of tobacco-induced tissue damage and also focuses attention on just a couple of central players in what is likely a complex pathogenetic process: a single transmembrane protease from the MMP family; and a packaging, intercellular communication, and cargo delivery system, the microvesicle (MV), that has gained in visibility, importance, and notoriety over the past few years. So, what is the nature of these TSE-induced MVs, and why do they seem to display only MMP-14? Also, what does all this imply for our understanding and management of tobacco-related disease?
From Cell Debris to Sophisticated Intercellular Communication and Cargo Delivery System Small, membrane-bound, extracellular vesicles have long been observed in biological fluids and conditioned media. Until Accepted for publication February 14, 2013. Address correspondence to Philip Furmanski, Ph.D., Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Rd., Piscataway, NJ 08854. E-mail: [email protected]
Furmanski relatively recently, most observers have considered them uninteresting debris, the dull refuse of cell death, or simply artifact. It is now apparent, however, that vesicles, or at least some of them, are key features of a ubiquitous and highly organized system of cell communication and intercellular material transport.2e5 Vesicles move cargo from cell to cell that can include enzymes, informational (mRNAs) and regulatory molecules (miRNAs and transcription factors), antigens, membrane receptors, metabolites, and other small molecules. When examined carefully, virtually all cells, including eukaryotic and prokaryotic cells, can be seen to release vesicles. The numbers released constitutively are usually small and are greatly increased by stimulation, activation, or stress. There seem to be several classes of vesicular structures that differ in size and origin. They have been observed and rediscovered many times in association with a broad array of normal physiological processes, and frequently, as is the case with mediators of powerful activities, if produced in a disorderly or uncontrolled fashion, tissue destruction or disease can result. As discussed below, they can even be usurped by malicious cells to advance their own devices. As often occurs when a cellular component or structure emerges again and again in association with a broad array of positive and negative processes, they initially acquire a wide assortment of names and attributed properties, defying classification and muddying the relationships among them. This certainly has been the case for these small cell vesicles. However, a clarifying order is being brought to what became a menagerie of little particles, helping to advance our understanding of their true functions, origins, and applications. We now recognize three general categories of these vesicles: i) the ectosomes, ii) the exosomes, and iii) apoptotic bodies.6 The ectosomes have been variously called microparticles, cell vesicles, nanovesicles, argosomes, prostasomes, prominosomes, apoptotic blebs, and microvesicles, among others. They characteristically arise by budding from the plasma membrane through the process of exocytosis, and generally are 100 to 1000 nm in diameter. Membrane asymmetry is lost during their formation, exposing phosphatidylserine from the inner membrane leaflet on their surface, meaning that they will tightly bind annexin V. The exosomes have also been given many names, including shedding microvesicles, dexosomes, and exovesicles, among others. They arise in the late endosomal system, where they are assembled into multivesicular bodies, which are carried to the cell surface to fuse with the plasma membrane and release their exosomes into the extracellular space. They are typically 40 to 100 nm in diameter, and because of their origin in the endosome compartment they have a different membrane composition from ectosomes, including very low exposure of phosphatidylserine on their surface. The third category, the apoptotic bodies are generally quite large (>1000 nm diameter) and are formed during the late stages of programmed cell death, and also contain many different cellular components, which can include organelles and nuclear fragments, indiscriminately taken up during the dismantling of the dying cell.
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Ectosomes contain an array of molecules collected from the cytoplasm and proteins picked up from the plasma membrane that are transferred horizontally to other cells, or, as highlighted here, that are involved in surface or contact-mediated effects on other cells or the cell matrix. Exosomes contain mainly proteins destined for export. Apoptotic bodies are mainly involved in packaging the detritus of cell destruction for uptake by scavenger cells and recycling or elimination. Vesicles have been assigned to these categories on the basis of size, vesicle content, or association with a physiological stimulus or process (eg, apoptosis). Surface properties, however, such as binding of annexin V, appear to be better markers.6 Which vesicle is implicated in mediating the TSE effects? Li et al1 refer to their vesicles as MVs and also as apoptotic microvesicles or blebs (presumably to be distinguished from apoptotic bodies, which as previously noted are very different structures). The sizes they report for their MVs are consistent with an ectosomal identity, and visualization of their budding from the cell membrane distinguishes them from exosomes. Li et al1 show that release of the MVs after TSE exposure, but not induction of MMP-14 expression, is associated with the initiation of apoptosis, and is caspase and mitogen-activated protein kinase-dependent. This would place their MVs squarely in the ectosome class, although the authors note that they exhibited only a partial loss of membrane asymmetry, a defining characteristic of ectosomes. Given the indistinct identification and classification of vesicles in many prior studies, further characterization of the TSE-induced MVs using surface markers is warranted, but for now we can accept that they are probably true ectosome-derived MVs.
More on Ectosome MVs Only small numbers of ectosome MVs (hereafter referred to simply as MVs) are found in the circulation of healthy individuals and their half-life is quite short.7 The numbers of MVs and their persistence increases greatly with thrombosis, inflammation, sepsis, metabolic disorders, autoimmune diseases, trauma, as well as malaria, sickle cell disease, cancer, and others.3 They alter endothelial cell function, promote angiogenesis, participate in antigen presentation, and facilitate the development of an immune response.8e10 MVs deliver MMPs (including MMP-2 and MMP-9), as well as vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor to sites of angiogenesis.2,9 They also have a treacherous side and a great deal of attention has been focused in this regard on MVs released by cancer cells, sometimes called oncosomes, which appear to play a role in tumor progression and resistance to therapy. For example, ovarian cancer cells release MVs expressing the Fas ligand, which causes T-cell apoptosis, and could contribute to the escape of tumor cells from immunosurveillance or immunodestruction.4,11 MVs released from platelets can transfer CD41 to tumor cells, enhancing their
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Commentary adhesiveness to endothelium and their propensity to metastasize.11 They have been reported to deliver K-Ras, epidermal growth factor receptor, and PKB/Akt from cancer to surrounding normal cells, a phenomenon possibly related to the field effect of tumors.2 MVs have been implicated in horizontal transfer of P-glycoprotein, the efflux pump that mediates multidrug resistance, from drug resistant to sensitive cells, and may even play a direct role in drug resistance by packaging and eliminating chemotherapeutic drugs from treated cells.12,13 It is thus clear that the MVs are an important class of structures involved in a wide spectrum of normal and pathophysiological processes. The consequences of knowing that ectosome MVs are the mediators of matrix damage caused by TSE-stimulated macrophages are potentially quite significant. Obviously, interfering with the release of MVs might be useful therapeutically. Some have suggested that the MVs released in association with various pathogenetic processes could have characteristic biosignatures.7 This might be exploited for screening, diagnostic, or patient monitoring purposes. Indeed, it has already been shown that endothelial microparticles with apoptotic characteristics (a type of ectosome MV) are elevated in plasma from cigarette smokers and might be useful in monitoring early onset of emphysema.14 Also, because MVs can apparently exhibit homing ability, ones that possess properties enabling them to localize in areas of tissue damage, destruction, or abnormality might be useful vehicles for targeted delivery of therapeutic agents (especially ones requiring protective enclosures, such as inhibitory or interfering RNAs); this has been proposed for use in atherosclerosis, transplantation, and cancer.7,15,16 Some clinical trials are already underway using MV encapsulated drugs, and the European Medicines Agency, which evaluates and regulates medicinal products for the European Union, recently classified microvesicles as advanced therapy products (ie, drugs).5
Many Suspects But Only One Protease Found Guilty The other key finding by Li et al1 is that the TSE-induced MVs contained as their only active protease MMP-14, an anchored or membrane type 1 member of the large MMP family. The MMPs are responsible for digesting components of the extracellular matrix and basement membrane during development, tissue maintenance and repair, and remodeling.17 In addition to their effects on the physical structure of the matrix, digestion of matrix components by MMPs releases many potent bioactives, such as cytokines and growth factors, that are stored or sequestered in the matrix. Thus, activation of MMPs has major structural as well as physiological consequences. There are 24 MMPs in humans, with similar structures and closely overlapping substrate specificities.17 They are tightly regulated at the transcriptional, post-transcriptional, locational, and proenzyme activation levels, and their activities
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are counteracted by a series of physiological inhibitors, particularly the tissue inhibitors of metalloproteinases. Not surprisingly, the dynamic balance between matrix deposition and degradation is closely regulated to assure tissue integrity and to enable proper growth, repair, and remodeling. When the balance is disrupted, serious damage and destruction ensue. Thus, dysregulation of the MMP system contributes to a broad spectrum of diseases, including cancer, atherosclerosis, venous thrombosis, and emphysema, to name a few relevant just to tobacco use. What do we know about MMP-14 that might make it special in the context of TSE exposure? As with other MMPs, MMP-14 digests collagen during soft tissue remodeling and degrades the basement membrane in the initial stages of angiogenesis, among other activities.18 MMP-14 also participates in the activation of other MMPs from their proenzyme forms, including MMP-2 (progelatinase A) and MMP-13, which suggests a possible cascading effect that would enhance the overall amount of enzymatic activity and broaden the range of substrates degraded at the point of MMP-14 action. Interestingly, mice in which members of the MMP family have been knocked out mostly have no overt phenotype (although many do exhibit altered responses to various physiological stimuli and/or physiological insults). The exception is the MMP-14 knock-out, as these animals appear normal at birth but then develop multiple abnormalities in connective tissue and bone, and die starting at 3 weeks of age. A simple interpretation of this finding is that loss of function of any of the other MMPs can be offset by fellow members of the family, but that there is something particular regarding MMP-14 activity that cannot be compensated for by another MMP. Whether or not these characteristics relate to TSE-mediated tissue damage, and how, is not yet clear. The fact that most or all of the TSE-treated macrophageinduced matrix degradation might be attributable to one enzyme suggests opportunities for specific therapeutic intervention to mitigate the effects of TSE. Members of the MMP family have long been seen as potential targets for drug development because of their pervasive involvement in a variety of diseases. A number of MMP inhibitors have been developed. However, because of the strong similarity in their active sites, selective inhibitors of individual MMPs have been elusive, and clinical trials with the more promiscuous inhibitors, not unexpectedly, have been disappointing. If MMP-14 does have special attributes or a substrate specificity that can be differentiated from its other family members, there may be opportunities for selective inhibition useful in treating the effects of smoking.
It’s Always More Complicated Than It Seems Li et al1 have importantly reduced TSE-induced macrophagemediated matrix degradation to a core process: TSE causes apoptosis of macrophages accompanied by release of potent MMP-14 on MVs, which degrades the local matrix. But there is likely more to it than that.
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Furmanski Other investigators have shown that cigarette smoke extracts cause degradation of the extracellular matrix by inducing MMP expression in lung epithelial cells. In one case, MMP-1 (an interstitial collagenase active at neutral pH) was the enzyme responsible.19 A TSE responsive element was identified in the MMP-1 promoter region of the MMP1 gene. Activation of the promoter was prevented by inhibitors of ERK 1/2 phosphorylation. Another study showed that TSE increased MMP-1 expression (and decreased expression of a potent inhibitor of its activity, tissue inhibitor of metalloproteinase 3) by lung epithelial cells in vitro and rabbit aortic tissue in vivo.20 The TSE effects appeared to be mediated through inhibition of the mTOR/p70S6K pathway. Inhibitors of ERK decreased constitutive MMP-1 expression but did not prevent the induction by TSE. Li et al1 have themselves also made the story more complex. The current article shows that TSE treatment of human THP-1 macrophages [derived from THP-1 monocytes transiently exposed to phorbol 12-myristate 13-acetate (PMA)] and primary human monocyte-derived macrophages (produced by isolating adherent monocytes from Buffy coats and differentiating them into macrophages) increased phosphorylation of JNK, p38, and ERK. Inhibitors of JNK or p38 completely blocked induction of MMP-14 expression, but an inhibitor of ERK did not. Similarly, the JNK and p38 inhibitors, but not the ERK inhibitor, prevented TSE-induced apoptosis and MV release. However, the authors have published prior work that implicated ERK signaling (also MEK, Ras, and caspase 3), but not JNK or p38, in TSE induced apoptosis and apoptotic bleb (MV) formation in human monocyte-derived macrophages and THP-1 monocytes not treated with PMA.21 Obviously, there are differences among all these experiments in cell types studied (epithelial, endothelial, monocytic, and macrophage-like), species of cell origin (human, rabbit), MV cargo (MMPs, tissue factor), and experimental conditions (with or without PMA treatment) that may explain the divergent results regarding the MMPs and signaling pathways involved. The more important message, however, is that it is very likely that multiple signaling pathways, effector cells, and enzymes contribute to the full expression of tobacco-induced matrix degradation and tissue damage in vivo. Monocyte/macrophages and other inflammatory responders are certain to play a key role but so are epithelial and endothelial cells (and probably others) exposed to TSE and other noxious stimuli, and the mechanics of their responses will probably be different. The full interplay of all these elements in vivo in causing the pathologies associated with tobacco use clearly will require further investigation.
What’s Next? Aside from more precisely specifying the type of vesicle produced by TSE-treated macrophages, and clarifying the signaling pathways involved, the most important next step is to determine whether the macrophage response to TSE
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reported by Li et al1 occurs in vivo, in both experimental models and in humans. It would be quite valuable to have long-lived conditional knock-outs of MMP-14 to more closely evaluate its specific role in TSE-induced tissue destruction, as well as many other processes in which it has been implicated. It would also be of interest to determine whether the promoter region of MMP14 contains a TSE responsive element, as does MMP1.19 Many observations support a strong role for activated, or in this case apoptotic, macrophages in the tissue destruction caused by smoking. Li et al1 have made a significant contribution to our understanding of the mechanisms involved. Translation into new and improved strategies for disease management is the ultimate goal and these findings offer some good leads to pursue. Meanwhile, there are more than 45 million smokers in the United States and probably hundreds of millions more globally, which results in an enormous health burden. It is extremely important that we continue to define the pathophysiological events that lead to tobacco-use related diseases. Without taking anything away from the significance of the results summarized here, however, it is worth noting that all this morbidity and mortality is entirely preventable, and effective strategies for smoking cessation should be further pursued as well.
References 1. Li C, Liu Y, Chen Y, Yu D, Williams KJ, Liu M-L: Novel proteolytic microvesicles released from human macrophages after exposure to tobacco smoke. Am J Pathol 2013, 182:1552e1562 2. Lee TH, D’Asti E, Magnus N, Al-Nedawi K, Meehan B, Rak J: Microvesicles as mediators of intercellular communication in cancer e the emerging science of cellular ‘debris.’ Semin Immunopathol 2011, 33:455e467 3. Morel O, Morel N, Jesel L, Freyssinet J-M, Toti F: Microparticles: a critical component in the nexus between inflammation, immunity and thrombosis. Semin Immunopathol 2011, 33:469e486 4. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S: Microvesicles in Health and Disease. Arch Immunol Ther Exp 2012, 60:107e121 5. Tetta C, Bruno S, Fonsanto V, Deregibus MC, Camussi G: The role of microvesicles in tissue repair. Organogenesis 2011, 7:105e115 6. Sadallah S, Eken C, Schifferli JA: Ectosomes as modulators of inflammation and immunity. Clin Exptl Immunol 2010, 163:26e32 7. Owens AP, Mackman N, Weber C, Mause S: Microparticles in hemostasis and thrombosis. Circulation Res 2011, 108:1284e1297 8. Thery C, Ostrowski M, Segura E: Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 2009, 9:581e593 9. Taraboletti G, D’Ascenzo S, Borsotti P: Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MTI-MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol 2002, 160:673e680 10. Sidhu SS, Mengistab AT, Tauscher AN, LaVail J, Basbaum C: The microvesicle as a vehicle for EMMPRIN in tumor-stromal interactions. Oncogene 2004, 23:956e963 11. Pap E: The role of microvesicles in malignancies. Adv Exptl Med and Biol 2011, 714:183e199 12. Bebawy M, Combes V, Lee E: Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cells. Leukemia 2009, 23: 1643e1649
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Commentary 13. 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: 4331e4337 14. Gordon C, Gudi K, Krause A, Sackrowitz R, Harvey B-G, StruloviciBarel Y, Mezey JG, Crystal RG: Circulating endothelial microparticles as a measure of early lung destruction in cigarette smokers. Am J Respir Crit Care Med 2011, 184:224e232 15. Fleissner F, Goerzig Y, Haverich A, Thum T: Microvesicles as novel biomarkers and therapeutic targets in transplantation medicine. Am J Transplant 2012, 12:289e297 16. Vlassov AV, Magdaleno S, Setterquist R, Conrad R: Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 2012, 1820:940e948
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17. Loffek S, Schilling O, Franzke C-W: Biological role of matrix metalloproteinases: a critical balance. Europ Resp J 2011, 38:191e208 18. Schneider F, Sukhova GK, Aikawa M, Canner J, Gerdes N, Tang S-MT, Shi G-P, Apte SS, Libby P: Matrix metalloproteinase-14 deficiency in bone marrow-derived cells promotes collagen accumulation in mouse atherosclerotic plaques. Circulation 2008, 117:931e939 19. Mercer BA, Wallace AM, Brinckerhoff CE, D’Armiento JM: Identification of a cigarette smoke-responsive region in the distal MMP-1 promoter. Am J Resp Cell Molec Biol 2008, 40:4e12 20. Lemaitre V, Dabo AJ, D’Armiento JD: Cigarette smoke components induce matrix metalloproteinase-1 in aortic endothelial cells through inhibition of mTOR signaling. Toxicol Sci 2011, 123:542e549 21. Li M, Yu D, Williams KJ, Liu M-L: Tobacco smoke induces the generation of procoagulant microvesicles from human monocyte/macrophages. Arterioscler Thromb Vasc Biol 2010, 30:1818e1824
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The American Journal of Pathology, Vol. 182, No. 5, May 2013
ajp.amjpathol.org
See related article on page 1552.
COMMENTARY Revealing the Mechanism of Tissue Damage Due to Tobacco Use Finally, a Smoking Gun? Philip Furmanski From the Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy; the Department of Cell Biology and Neuroscience, School of Arts and Sciences; and the Department of Biochemistry and Molecular Biology and Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey; Rutgers University, Piscataway, New Jersey CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
We have known for more than half a century that tobacco use causes cancers, emphysema, atherosclerosis, and a number of other debilitating and deadly diseases. Some gains have been made in the United States and other countries in reducing tobacco use but it remains a serious worldwide health problem with millions of new cases of tobacco-related diseases diagnosed each year. Remarkably, despite its importance and much study, we still do not have a full picture of the molecular mechanisms by which tobacco, its constituents, and pyrolytic products cause the tissue damage that underlies the wellrecognized tobacco-associated pathologies. In this issue of The American Journal of Pathology, Li et al1 provide a compelling case for a mechanism by which tobacco smoke extract (TSE) induces damage to the extracellular matrix, a key element in the pathogenesis of tobacco-related disease. They find that exposure of human macrophages to TSE initiates a progressive series of events that culminate in apoptosis and the release of small membrane-bound vesicles that carry on their surface a potent collagenolytic and gelatinolytic activity which then degrades the local matrix. Significantly, among the large number of possible proteases that might be involved, they find that only one, matrix metalloproteinase-14 (MMP-14), is responsible for practically all of the activity. Li et al1 also identify the intracellular signaling pathway that initiates the process after TSE Copyright ª 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2013.02.004
exposure, involving Jun N-terminal kinase (JNK) and p38 MAP kinases. This study offers important insights and implications regarding the mechanism of tobacco-induced tissue damage and also focuses attention on just a couple of central players in what is likely a complex pathogenetic process: a single transmembrane protease from the MMP family; and a packaging, intercellular communication, and cargo delivery system, the microvesicle (MV), that has gained in visibility, importance, and notoriety over the past few years. So, what is the nature of these TSE-induced MVs, and why do they seem to display only MMP-14? Also, what does all this imply for our understanding and management of tobacco-related disease?
From Cell Debris to Sophisticated Intercellular Communication and Cargo Delivery System Small, membrane-bound, extracellular vesicles have long been observed in biological fluids and conditioned media. Until Accepted for publication February 14, 2013. Address correspondence to Philip Furmanski, Ph.D., Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Rd., Piscataway, NJ 08854. E-mail: [email protected]
Furmanski relatively recently, most observers have considered them uninteresting debris, the dull refuse of cell death, or simply artifact. It is now apparent, however, that vesicles, or at least some of them, are key features of a ubiquitous and highly organized system of cell communication and intercellular material transport.2e5 Vesicles move cargo from cell to cell that can include enzymes, informational (mRNAs) and regulatory molecules (miRNAs and transcription factors), antigens, membrane receptors, metabolites, and other small molecules. When examined carefully, virtually all cells, including eukaryotic and prokaryotic cells, can be seen to release vesicles. The numbers released constitutively are usually small and are greatly increased by stimulation, activation, or stress. There seem to be several classes of vesicular structures that differ in size and origin. They have been observed and rediscovered many times in association with a broad array of normal physiological processes, and frequently, as is the case with mediators of powerful activities, if produced in a disorderly or uncontrolled fashion, tissue destruction or disease can result. As discussed below, they can even be usurped by malicious cells to advance their own devices. As often occurs when a cellular component or structure emerges again and again in association with a broad array of positive and negative processes, they initially acquire a wide assortment of names and attributed properties, defying classification and muddying the relationships among them. This certainly has been the case for these small cell vesicles. However, a clarifying order is being brought to what became a menagerie of little particles, helping to advance our understanding of their true functions, origins, and applications. We now recognize three general categories of these vesicles: i) the ectosomes, ii) the exosomes, and iii) apoptotic bodies.6 The ectosomes have been variously called microparticles, cell vesicles, nanovesicles, argosomes, prostasomes, prominosomes, apoptotic blebs, and microvesicles, among others. They characteristically arise by budding from the plasma membrane through the process of exocytosis, and generally are 100 to 1000 nm in diameter. Membrane asymmetry is lost during their formation, exposing phosphatidylserine from the inner membrane leaflet on their surface, meaning that they will tightly bind annexin V. The exosomes have also been given many names, including shedding microvesicles, dexosomes, and exovesicles, among others. They arise in the late endosomal system, where they are assembled into multivesicular bodies, which are carried to the cell surface to fuse with the plasma membrane and release their exosomes into the extracellular space. They are typically 40 to 100 nm in diameter, and because of their origin in the endosome compartment they have a different membrane composition from ectosomes, including very low exposure of phosphatidylserine on their surface. The third category, the apoptotic bodies are generally quite large (>1000 nm diameter) and are formed during the late stages of programmed cell death, and also contain many different cellular components, which can include organelles and nuclear fragments, indiscriminately taken up during the dismantling of the dying cell.
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Ectosomes contain an array of molecules collected from the cytoplasm and proteins picked up from the plasma membrane that are transferred horizontally to other cells, or, as highlighted here, that are involved in surface or contact-mediated effects on other cells or the cell matrix. Exosomes contain mainly proteins destined for export. Apoptotic bodies are mainly involved in packaging the detritus of cell destruction for uptake by scavenger cells and recycling or elimination. Vesicles have been assigned to these categories on the basis of size, vesicle content, or association with a physiological stimulus or process (eg, apoptosis). Surface properties, however, such as binding of annexin V, appear to be better markers.6 Which vesicle is implicated in mediating the TSE effects? Li et al1 refer to their vesicles as MVs and also as apoptotic microvesicles or blebs (presumably to be distinguished from apoptotic bodies, which as previously noted are very different structures). The sizes they report for their MVs are consistent with an ectosomal identity, and visualization of their budding from the cell membrane distinguishes them from exosomes. Li et al1 show that release of the MVs after TSE exposure, but not induction of MMP-14 expression, is associated with the initiation of apoptosis, and is caspase and mitogen-activated protein kinase-dependent. This would place their MVs squarely in the ectosome class, although the authors note that they exhibited only a partial loss of membrane asymmetry, a defining characteristic of ectosomes. Given the indistinct identification and classification of vesicles in many prior studies, further characterization of the TSE-induced MVs using surface markers is warranted, but for now we can accept that they are probably true ectosome-derived MVs.
More on Ectosome MVs Only small numbers of ectosome MVs (hereafter referred to simply as MVs) are found in the circulation of healthy individuals and their half-life is quite short.7 The numbers of MVs and their persistence increases greatly with thrombosis, inflammation, sepsis, metabolic disorders, autoimmune diseases, trauma, as well as malaria, sickle cell disease, cancer, and others.3 They alter endothelial cell function, promote angiogenesis, participate in antigen presentation, and facilitate the development of an immune response.8e10 MVs deliver MMPs (including MMP-2 and MMP-9), as well as vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor to sites of angiogenesis.2,9 They also have a treacherous side and a great deal of attention has been focused in this regard on MVs released by cancer cells, sometimes called oncosomes, which appear to play a role in tumor progression and resistance to therapy. For example, ovarian cancer cells release MVs expressing the Fas ligand, which causes T-cell apoptosis, and could contribute to the escape of tumor cells from immunosurveillance or immunodestruction.4,11 MVs released from platelets can transfer CD41 to tumor cells, enhancing their
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Commentary adhesiveness to endothelium and their propensity to metastasize.11 They have been reported to deliver K-Ras, epidermal growth factor receptor, and PKB/Akt from cancer to surrounding normal cells, a phenomenon possibly related to the field effect of tumors.2 MVs have been implicated in horizontal transfer of P-glycoprotein, the efflux pump that mediates multidrug resistance, from drug resistant to sensitive cells, and may even play a direct role in drug resistance by packaging and eliminating chemotherapeutic drugs from treated cells.12,13 It is thus clear that the MVs are an important class of structures involved in a wide spectrum of normal and pathophysiological processes. The consequences of knowing that ectosome MVs are the mediators of matrix damage caused by TSE-stimulated macrophages are potentially quite significant. Obviously, interfering with the release of MVs might be useful therapeutically. Some have suggested that the MVs released in association with various pathogenetic processes could have characteristic biosignatures.7 This might be exploited for screening, diagnostic, or patient monitoring purposes. Indeed, it has already been shown that endothelial microparticles with apoptotic characteristics (a type of ectosome MV) are elevated in plasma from cigarette smokers and might be useful in monitoring early onset of emphysema.14 Also, because MVs can apparently exhibit homing ability, ones that possess properties enabling them to localize in areas of tissue damage, destruction, or abnormality might be useful vehicles for targeted delivery of therapeutic agents (especially ones requiring protective enclosures, such as inhibitory or interfering RNAs); this has been proposed for use in atherosclerosis, transplantation, and cancer.7,15,16 Some clinical trials are already underway using MV encapsulated drugs, and the European Medicines Agency, which evaluates and regulates medicinal products for the European Union, recently classified microvesicles as advanced therapy products (ie, drugs).5
Many Suspects But Only One Protease Found Guilty The other key finding by Li et al1 is that the TSE-induced MVs contained as their only active protease MMP-14, an anchored or membrane type 1 member of the large MMP family. The MMPs are responsible for digesting components of the extracellular matrix and basement membrane during development, tissue maintenance and repair, and remodeling.17 In addition to their effects on the physical structure of the matrix, digestion of matrix components by MMPs releases many potent bioactives, such as cytokines and growth factors, that are stored or sequestered in the matrix. Thus, activation of MMPs has major structural as well as physiological consequences. There are 24 MMPs in humans, with similar structures and closely overlapping substrate specificities.17 They are tightly regulated at the transcriptional, post-transcriptional, locational, and proenzyme activation levels, and their activities
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are counteracted by a series of physiological inhibitors, particularly the tissue inhibitors of metalloproteinases. Not surprisingly, the dynamic balance between matrix deposition and degradation is closely regulated to assure tissue integrity and to enable proper growth, repair, and remodeling. When the balance is disrupted, serious damage and destruction ensue. Thus, dysregulation of the MMP system contributes to a broad spectrum of diseases, including cancer, atherosclerosis, venous thrombosis, and emphysema, to name a few relevant just to tobacco use. What do we know about MMP-14 that might make it special in the context of TSE exposure? As with other MMPs, MMP-14 digests collagen during soft tissue remodeling and degrades the basement membrane in the initial stages of angiogenesis, among other activities.18 MMP-14 also participates in the activation of other MMPs from their proenzyme forms, including MMP-2 (progelatinase A) and MMP-13, which suggests a possible cascading effect that would enhance the overall amount of enzymatic activity and broaden the range of substrates degraded at the point of MMP-14 action. Interestingly, mice in which members of the MMP family have been knocked out mostly have no overt phenotype (although many do exhibit altered responses to various physiological stimuli and/or physiological insults). The exception is the MMP-14 knock-out, as these animals appear normal at birth but then develop multiple abnormalities in connective tissue and bone, and die starting at 3 weeks of age. A simple interpretation of this finding is that loss of function of any of the other MMPs can be offset by fellow members of the family, but that there is something particular regarding MMP-14 activity that cannot be compensated for by another MMP. Whether or not these characteristics relate to TSE-mediated tissue damage, and how, is not yet clear. The fact that most or all of the TSE-treated macrophageinduced matrix degradation might be attributable to one enzyme suggests opportunities for specific therapeutic intervention to mitigate the effects of TSE. Members of the MMP family have long been seen as potential targets for drug development because of their pervasive involvement in a variety of diseases. A number of MMP inhibitors have been developed. However, because of the strong similarity in their active sites, selective inhibitors of individual MMPs have been elusive, and clinical trials with the more promiscuous inhibitors, not unexpectedly, have been disappointing. If MMP-14 does have special attributes or a substrate specificity that can be differentiated from its other family members, there may be opportunities for selective inhibition useful in treating the effects of smoking.
It’s Always More Complicated Than It Seems Li et al1 have importantly reduced TSE-induced macrophagemediated matrix degradation to a core process: TSE causes apoptosis of macrophages accompanied by release of potent MMP-14 on MVs, which degrades the local matrix. But there is likely more to it than that.
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Furmanski Other investigators have shown that cigarette smoke extracts cause degradation of the extracellular matrix by inducing MMP expression in lung epithelial cells. In one case, MMP-1 (an interstitial collagenase active at neutral pH) was the enzyme responsible.19 A TSE responsive element was identified in the MMP-1 promoter region of the MMP1 gene. Activation of the promoter was prevented by inhibitors of ERK 1/2 phosphorylation. Another study showed that TSE increased MMP-1 expression (and decreased expression of a potent inhibitor of its activity, tissue inhibitor of metalloproteinase 3) by lung epithelial cells in vitro and rabbit aortic tissue in vivo.20 The TSE effects appeared to be mediated through inhibition of the mTOR/p70S6K pathway. Inhibitors of ERK decreased constitutive MMP-1 expression but did not prevent the induction by TSE. Li et al1 have themselves also made the story more complex. The current article shows that TSE treatment of human THP-1 macrophages [derived from THP-1 monocytes transiently exposed to phorbol 12-myristate 13-acetate (PMA)] and primary human monocyte-derived macrophages (produced by isolating adherent monocytes from Buffy coats and differentiating them into macrophages) increased phosphorylation of JNK, p38, and ERK. Inhibitors of JNK or p38 completely blocked induction of MMP-14 expression, but an inhibitor of ERK did not. Similarly, the JNK and p38 inhibitors, but not the ERK inhibitor, prevented TSE-induced apoptosis and MV release. However, the authors have published prior work that implicated ERK signaling (also MEK, Ras, and caspase 3), but not JNK or p38, in TSE induced apoptosis and apoptotic bleb (MV) formation in human monocyte-derived macrophages and THP-1 monocytes not treated with PMA.21 Obviously, there are differences among all these experiments in cell types studied (epithelial, endothelial, monocytic, and macrophage-like), species of cell origin (human, rabbit), MV cargo (MMPs, tissue factor), and experimental conditions (with or without PMA treatment) that may explain the divergent results regarding the MMPs and signaling pathways involved. The more important message, however, is that it is very likely that multiple signaling pathways, effector cells, and enzymes contribute to the full expression of tobacco-induced matrix degradation and tissue damage in vivo. Monocyte/macrophages and other inflammatory responders are certain to play a key role but so are epithelial and endothelial cells (and probably others) exposed to TSE and other noxious stimuli, and the mechanics of their responses will probably be different. The full interplay of all these elements in vivo in causing the pathologies associated with tobacco use clearly will require further investigation.
What’s Next? Aside from more precisely specifying the type of vesicle produced by TSE-treated macrophages, and clarifying the signaling pathways involved, the most important next step is to determine whether the macrophage response to TSE
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reported by Li et al1 occurs in vivo, in both experimental models and in humans. It would be quite valuable to have long-lived conditional knock-outs of MMP-14 to more closely evaluate its specific role in TSE-induced tissue destruction, as well as many other processes in which it has been implicated. It would also be of interest to determine whether the promoter region of MMP14 contains a TSE responsive element, as does MMP1.19 Many observations support a strong role for activated, or in this case apoptotic, macrophages in the tissue destruction caused by smoking. Li et al1 have made a significant contribution to our understanding of the mechanisms involved. Translation into new and improved strategies for disease management is the ultimate goal and these findings offer some good leads to pursue. Meanwhile, there are more than 45 million smokers in the United States and probably hundreds of millions more globally, which results in an enormous health burden. It is extremely important that we continue to define the pathophysiological events that lead to tobacco-use related diseases. Without taking anything away from the significance of the results summarized here, however, it is worth noting that all this morbidity and mortality is entirely preventable, and effective strategies for smoking cessation should be further pursued as well.
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Commentary 13. 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: 4331e4337 14. Gordon C, Gudi K, Krause A, Sackrowitz R, Harvey B-G, StruloviciBarel Y, Mezey JG, Crystal RG: Circulating endothelial microparticles as a measure of early lung destruction in cigarette smokers. Am J Respir Crit Care Med 2011, 184:224e232 15. Fleissner F, Goerzig Y, Haverich A, Thum T: Microvesicles as novel biomarkers and therapeutic targets in transplantation medicine. Am J Transplant 2012, 12:289e297 16. Vlassov AV, Magdaleno S, Setterquist R, Conrad R: Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 2012, 1820:940e948
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17. Loffek S, Schilling O, Franzke C-W: Biological role of matrix metalloproteinases: a critical balance. Europ Resp J 2011, 38:191e208 18. Schneider F, Sukhova GK, Aikawa M, Canner J, Gerdes N, Tang S-MT, Shi G-P, Apte SS, Libby P: Matrix metalloproteinase-14 deficiency in bone marrow-derived cells promotes collagen accumulation in mouse atherosclerotic plaques. Circulation 2008, 117:931e939 19. Mercer BA, Wallace AM, Brinckerhoff CE, D’Armiento JM: Identification of a cigarette smoke-responsive region in the distal MMP-1 promoter. Am J Resp Cell Molec Biol 2008, 40:4e12 20. Lemaitre V, Dabo AJ, D’Armiento JD: Cigarette smoke components induce matrix metalloproteinase-1 in aortic endothelial cells through inhibition of mTOR signaling. Toxicol Sci 2011, 123:542e549 21. Li M, Yu D, Williams KJ, Liu M-L: Tobacco smoke induces the generation of procoagulant microvesicles from human monocyte/macrophages. Arterioscler Thromb Vasc Biol 2010, 30:1818e1824
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