Microvesicles as Novel Biomarkers and Therapeutic Targets in Transplantation Medicine

Authors

  • F. Fleissner,

    Corresponding author
    1. Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany
    2. Division of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany
      Felix Fleissner, Fleissner.Felix@mh-hannover.de or Thomas Thum, thum.thomas@mh-hannover.de
    Search for more papers by this author
  • Y. Goerzig,

    1. Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany
    Search for more papers by this author
  • A. Haverich,

    1. Division of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany
    Search for more papers by this author
  • T. Thum

    Corresponding author
    1. Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany
    2. Centre for Clinical and Basic Research, IRCCS San Raffaele, Rome, Italy
      Felix Fleissner, Fleissner.Felix@mh-hannover.de or Thomas Thum, thum.thomas@mh-hannover.de
    Search for more papers by this author

Felix Fleissner, Fleissner.Felix@mh-hannover.de or Thomas Thum, thum.thomas@mh-hannover.de

Abstract

Microvesicles (MVs) including exosomes are emerging new biomarkers and potential regulators of inflammation and immunological processes. Such particles contain proteins and genetic information including DNA and microRNAs that may be of importance for cell/cell communication. However, their role during and after organ transplantation and immunomodulatory effects is only in its beginning of understanding. We here, in brief, introduce generation and biological importance of MVs, describe their (patho)physiological roles and their potential use as future biomarkers and therapeutic agents in transplantation medicine. Circulating MVs may have a great potential to detect possible immune rejections and MV modulation may emerge as a therapeutic approach in organ rejection therapy.

Abbreviations: 
AKI

acute kidney injury

CXCR4

CXC-motive-chemokine-receptor 4

CYP

cytochrome P450

DC

dendritic cells

eNOS

endothelial nitric oxide synthase

EPC

endothelial progenitor cells

FasL

Fas ligand

GFP

green fluorescent protein

GPCR

G protein-coupled receptor

GST

Glutathione S-transferase

miRNA

microRNA

MSC

mesenchymal stem cells

MV

microvesicles

P2X(7)R

P2X(7) receptor subtype

ROS

reactive oxygen species

STAT3

signal transducer and activator of transcription 3

TCS

trichosanthin

TCTP

translationally controlled tumor protein

TF

tissue factor

TFPI

tissue factor pathway inhibitor

TM

thrombomodulin

TNF

tumor necrosis factor

TRAP

thrombin-receptor-activating-peptide-6

TRAIL

TNF-related apoptosis-inducing ligand

Treg

regulatory T cells

UGT

uridinediphosphate-glucuronosyltransferase

Introduction

This review article focuses on the emerging field of circulating microvesicles (MVs) and their potential importance for transplantation medicine. In the first section, we shortly describe the formation, content and different mode of action of circulating MVs including exosomes. The second section describes the role of MVs in physiology and pathophysiology, as well as potential functions of MVs in inflammation and immunosuppression. In the third section, this review describes the interesting fact that MVs contain genetic molecules including microRNAs (miRNAs) and the potential underlying importance for cell/cell communication. Sections fourth and fifth focus on MVs and their relation to drug metabolism/resistance and describe potential future use of MVs as drugs and/or drug carriers in transplantation related diseases.

What are MVs?

There are two types of particles that can be detected in blood or other body fluids conferred to as MVs, exosomes and bigger so-called shedding vesicles (1). Other authors suggest to distinguish between exosomes and MVs, whereas they confer to MVs as shedding vesicles (2).

Exosomes are usually small particles of about 30–120 nm in size and are of endosomal origin. In brief, endosome compartmentalization leads to accumulation of multivesicular bodies. After fusing with the plasma membrane exocytic multivesicular bodies are released as exosomes. This release is dependent on cytoskeletal activation (3). However, the exact mechanisms of MV formation and their release remain still poorly understood. The translationally controlled tumor protein (TCTP), a p53 controlled protein related to guanine-nucleotide free chaperones, has been shown to be involved in MV formation (4). In addition, TCTP plays an important role in histamine production and inflammatory diseases (5).

Shedding vesicles are bigger than exosomes and about 100 nm to 1 mm in size. Their formation is calcium- and cytoskeleton-dependent and is achieved directly from budding of small cytoplasmatic protrusions and detachment from the cell surface in response to cell stimulation (6). Cytoplasmatic changes play a vital role in the formation of MVs. Treatment of cells with colchicine, vinblastine and low temperatures leads to increased formation of shedding vesicles. This effect is most likely mediated via a disruption of the plasma membrane from the underlying cytoskeleton (7).

MVs have been identified in the vascular circulation and can be released by many different cell types (8–10). Probably the largest fraction of MVs found in the plasma originates from endothelial and circulating blood cells (11), although the exact contribution of individual cells to circulating MVs is unknown and may be affected by different stimuli and diseases.

MVs and especially exosomes have emerged as important players in cellular crosstalk, suggesting exosomes as interesting targets for new therapeutic approaches. There is growing evidence that interaction of MVs with other cells is mediated via specific receptor ligands. Interestingly, MVs are able to home specific target structures. For example, platelet-derived MVs transfer tissue factor (TF) selectively to monocytes but not to neutrophils (12). MVs can stimulate target cells either directly or via binding to specific surface receptors (13,14). After recognition of the target cell, either internalization as a result of direct fusion or endocytic uptake may occur (6).

How do MVs effect other cell types?

There are several ways how MVs may influence biology of other cells. First, MVs are able to transfer receptor ligands to target cells. For example, tumor cells can transfer the Fas ligand (FasL) via MVs to T cells, thereby inducing apoptosis and subsequent immune escape mechanisms of the tumor cell (15; see also Table 1 for MV-transported proteins). Also, platelets can transfer the adhesion receptor CD41 to endothelial cells resulting in enhanced platelet adhesion (16). Such examples highlight the roles of MVs as communication systems between various cells.

Table 1.  Proteins delivered by microvesicels/exosomes
Protein transported by MVs/exosomesFrom /target cellsFunctionReference
Fas ligandTumor cells /smooth muscle cellsApoptosis and subsequent immune escape(15)
Adhesion receptor CD41Platelets/endothelial cellsEnhanced adhesion(16)
Caspase-1Monocytes/ smooth muscle cellsInduced apoptosis(17)
Fas ligand/ Apo2 ligand/TNF-related apoptosis-inducing ligand (TRAIL)T blasts/ lymphocytesInduced apoptosis(24)
IL-1 beta, caspase-1 and -3 and cathepsin DDCs/severalInflammation(30)
Tumor cells/ monocytesInduced HLA-DR expression, induced [ROS, increased TNF, (IL)-10, IL-12p40](27,28)
Fas ligandActivated CD8(+)Induced apoptosis(31)
NKG2D ligandsPlacenta/NK, CD8+, gammadelta T cellsDown-regulation of the NKG2D; reduction of their in vitro cytotoxicity(32)
Fas ligandTrophoplast/ T cellsInduced apoptosis(34)
CD18, CD14 and tissue factorStimulated monocytes/endotheliumIncreased endothelial TF; increase endothelial thrombogenicity(39)
CYP, UGT and GST protein familiesPrimary hepatocytes/diverseDrug resistance(57)
P-GlycoproteinCancer cell line drug resistant/cancer cell lineDrug resistance(58)
Cisplatin transportersCisplatin resistant cell lineDrug resistance(59)
HedgehogLiver cells/ hepatic sinusoidal endothelial cellsAngiogenic responses in endothelial cells(56)

Next to ligand delivery, MVs may modulate target cells via transfer of other intracellular proteins. For example, monocytes, stimulated with endotoxin, are able to deliver the caspase-1 via MVs to smooth muscle cells to induce apoptosis (17). MVs can also directly stimulate target cells. For instance, MVs derived from activated platelets provide a surface for binding and assembling clotting factors. An inborn defect in platelet-derived MV formation leads to ineffective blood clotting known as “Scott syndrome” (18,19).

In addition, MVs transfer genetic information between cells. MVs from endothelial progenitor cells are able to recognize endothelial cells via alpha4 and beta1 integrins and lead to an upregulation of the phosphatidylinositol 3-kinase and endothelial nitric oxide synthase expression resulting in enhanced angiogenesis (20).

MVs derived from embryonic stem cells lead to a phosphorylation of mitogen activated protein kinase p42/44 and Akt in hematopoetic stem cells. Subsequently, hematopoetic stem cells in cocultivation with MVs show markedly improved functions (19). The intravenous administration of mesenchymal stem cells (MSC) improves recovery from acute kidney injury (AKI). Bruno et al. found that these effects are in part mediated via MSC-derived MVs. The biological action of MVs required their CD44- and beta1-integrin-dependent incorporation into tubular cells within the kidney. MVs seemed to transduce miRNAs associated with changes of the mesenchymal phenotype and altered control of transcription, proliferation and immunoregulation that subsequently resulted in an improved kidney function after injury (Ref. 21; also refer to Figure 1).

Figure 1.

Different effects of secreted exosomes/microvesicles (MV) on target cells. Exosomes and MV can interact with target cells via protein, ligand and mRNA/miRNA delivery or direct stimulation.

Role of MVs in Physiology and Pathophysiology

MVs in inflammation and immunosuppression

MVs and T cells/dendritic cells (DC):  MVs have been previously described to interact with inflammatory and immunosuppressive processes. MVs derived from DC are able to induce the inflammatory mediator NF-kappaB in microglia (22). This effect can be enhanced by MV-mediated MHC class II-associated invariant chain recruitment in microglia. Thus, DC-mediated activation of microglia may contribute to the initiation of autoimmune diseases. T-cell blasts show the ability to secrete FasL and Apo2 ligand/TNF-related apoptosis-inducing ligand (TRAIL) containing MVs (23). These FasL containing MVs induce lymphocyte apoptosis subsequently attenuating antitumor activity (24). Tumor-derived MVs also influence human regulatory T cells (Treg), resulting in enhanced conversion of CD4(+)CD25(neg) T cells into CD4(+)CD25(high)FOXP3(+) Treg subsequently leading to immunosupression. Tumor-derived MVs also result in increased FasL, Il-10 TG-beta1, granzyme B and perforin expression (Figure 2). Accordingly, proliferation of responder cells can be reduced (25). Tumor-derived MV inhibit signaling and proliferation of activated CD8(+) T cells and induce apoptosis of CD8(+) T cells, including tumor-reactive, tetramer(+)CD8(+) T cells (26). Tumor-mediated MVs also induce HLA-DR expression, induce production of reactive oxygen species (ROS) and lead to tumor necrosis factor (TNF), interleukin (IL)-10 and IL-12p40 accumulation and increased secretion. The effects on TNF secretion is abolished by blockade of CD44 on the monocytes (27,28). Tumor-derived MVs may also inhibit CD8(+) proliferation and induce apoptosis. Unlike DC-derived MV, tumor-derived MV induce expansion of CD4(+)CD25(+)FOXP3(+) Tregs and enhances their suppressor activity resulting in enhanced tumor evasion. On the other hand, shedded MVs transfer antigenic components to recipient DCs leading to increase DC immunogenicity (29). These “artificial” MVs might represent a new therapeutic approach to antitumor immunotherapy. DCs can be stimulated to produce MV by the P2X(7) receptor subtype (P2X[7]R). P2X(7)R stimulation can cause fast MV shedding from DC plasma membrane. These MVs contain IL-1 beta, caspase-1 and caspase-3 and cathepsin D (30). The fact that MVs are important regulators of tumor invasion is strengthened by findings that MV from colorectal cancer cells can induce FasL-mediated and TNF-related apoptosis of activated CD8(+) cells (31).

Figure 2.

Role of exosomes/microvesicles in autoimmune disease and cancer. Exosomes and MV are able to influence immunity via NF kappa B upregulation, influence antitumor activity either by increase of ROS and TNF alpha levels or by induction of apoptosis by FasL. MVs and exosomes can also regulate antitumor activity via interleukins, granzymes and perforines.

MVs and placenta physiology:  Escape mechanisms against immunity are also of relevance in placenta physiology. The placenta serves as a barrier between the fetus' and the mother's immunity. Lessons learned from the physiological immunology of the placenta may be also of importance to understand immunologic mechanisms in solid organ transplantation. Indeed, placenta-derived exosomes have been described as new immune regulators in the maternal immune tolerance. Exosomes bearing killer cell lectin-like ligands are released by human placenta and isolated placental exosomes carry UL16-binding protein 15 and MHC class I chain-related proteins A and B on their surface. Subsequently, placental exosomes can induce downregulation of the NKG2D receptor on NK, CD8(+) and gammadelta T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathways (32). Placental trophoblast- and maternal thrombocyte-derived MVs bind to circulating peripheral T and MV-lymphocyte interactions induce signal transducer and activator of transcription 3 phosphorylation of T cells. This effect is mediated via a P-selectin (CD62P)-PSGL-1 (CD162) interaction (33). Accordingly, trophoblasts can secrete MVs containing FasL that can induce T-cell death by induction of apoptosis (34).

MVs and the cardiovascular system:  MVs secreted from platelets play an important role in both coagulation and development of atherosclerosis. Increased levels of MVs derived from platelets can be found in patients with acute coronary syndrome (35), stroke (36) and peripheral arterial disease (37). Lactadherin, a breast epithelial cell antigen promotes the phagocytosis of phosphatidylserine-expressing lymphocytes and red blood cells. Lactadherin-deficient mice show increased levels of thrombin. Accordingly, Lactadherin-deficient mice show a decreased capacity to phagocytose platelet-derived MVs. This results in a shorter occlusion time in a model of endothelial damage (38). The authors conclude that a defective clearance of platelet-derived MV can induce a hypercoagulable state.

Stimulation of monocytes by starvation or by endotoxin and calcium ionophores results in the release of MVs expressing monocyte markers (CD18, CD14) and active TF.These MVs lead to increased endothelial TF expression as well as depression of anticoagulant TF pathway inhibitor and thrombomodulin resulting in an increased endothelial thrombogenicity and apoptosis (39). The shedding of MVs from endothelial cells leads to an increase in CXCL12 in residual vascular cells causing a recruitment of progenitor cells and counteraction of apoptosis, an effect that is mediated via MV delivery of miR-126. miR-126 represses the function of regulator G protein (heterotrimeric guanosine triphosphate-binding protein) signaling 16, an inhibitor of G protein-coupled receptor (GPCR) signaling. This enables CXC-motive-chemokine-receptor 4, a GPCR, to increase the production of CXCL12 (40). It is likely, that circulating miRNAs in body fluids such as plasma or urine are packed in exosomes (41–44). We recently were able to show that one of such miRNAs, miR-210, may be a mediator of acute T-cell mediated rejection in renal allograft recipients. During rejection, miR-210 was down-regulated and differed between patients with acute rejection when compared to stable transplant patients with urinary tract infection or transplant patients before/after rejection (44). Thus, the genetic content of circulating MVs maybe of great use for diagnostic and prognostic purposes after organ transplantation.

Is there a role for MVs in organ transplantation?

Although MVs play important role in immunology and inflammation, their role in organ transplantation remains less investigated. Schuerholz et al. investigated the influence of graft ischemia in kidney transplants on MV formation. Here, prolonged graft ischemia time resulted in decreased MV formation in platelets after ex vivo thrombin-receptor-activating-peptide-6 stimulation. The authors concluded that attenuated platelet MV formation may be a sign of former activation of platelets that could influence graft function and survival (45). Concerning kidney function, experiments revealed that the beneficial effect of MSC on recovery from AKI is in part mediated via MVs. MV function in this setting was CD-44 and beta-1 integrin dependent. Because miRNAs treatment of MSC-derived MVs abolished the beneficial effects, the authors concluded that this effect is most likely mediated via horizontal gene transfer (21). Subcutaneous injection of exosomes derived from donor bone marrow derived DC leads to a prolonged survival of bone marrow transplants in rats. The allograft of rats treated with exosomes before transplantation showed a markedly decrease in interferon gamma mRNA and leucocyte infiltration (46). In a model of rat heart transplantation, presentation of donor-MHC antigens from exosomes was able to induce regulatory responses modulating allograft rejection and inducing donor-specific allograft tolerance (47).

Importance of MVs, mRNA and miRNA Content

In a study transferring mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells. This important experiment indicates that transferred exosomal mRNA can be translated into proteins after entering another cell leading to altered cellular functions (41,48). Pegtel et al. showed mature EBV-encoded miRNAs to be secreted by EBV-infected B cells through exosomes (see also Table 2). The functionality of these miRNAs delivered through exosomes was confirmed by a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene downregulated in primary EBV-associated lymphomas (49). In a study identifying miRNAs in exosomes, the majority of the miRNAs expressed in the MVs from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways as well as modulators of immune function (50). In patients with ovarian benign and malign tumors, a distinct exosomal miRNA profile from ovarian cancer patients was exhibited that was significantly distinct from profiles observed in benign disease (51). Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum MVs of glioblastoma patients (52). In patients with nasopharyngeal carcinoma, miRNAs with abundant transcription of a family of viral miRNAs called BART miRNAs, are to a large extent associated with exosomes. This leads to the conclusion that miRNAs in exosomes have a potential to serve as a source of novel tumor biomarkers and may play a possible role in the communication between malignant and nonmalignant cells (53). In vitro data suggest a stable production of exosomes by a gastric cancer line. Especially miRNAs of the let 7 family play a tumor-suppressive role because of their targeting of oncogenes, such as RAS and HMGA2 (54). Circulating miRNAs in the urine of patients with kidney rejection may also be of causative and/or predictive importance (44).

Table 2.  mRNA and miRNA delivered by microvesicles/exosomes
miRNA/mRNA transported by MVs/exosomesFrom /target cellsFunctionReference
BART1–5p, BART2-p and BART3EBV-infected B cells/several lymphomasCXCL11/ITAC, immunoregulatory gene(49)
miR-223, -484, -191, -146a, -16, -26a, -222, -24, -126, and -32 miR-223, -484, -191, -146a, -16, -26a and othersBlood circulating MVsDiverse; cellular differentiation of blood cells and metabolic pathways(50)
EGFR, diverse miRNAsGlioblastoma/glioma cell lineImproved proliferation(52)
let 7 and othersGastric cancer line/diverseOncogenes, such as RAS and HMGA2, tumor proliferation(54)
miR-126Endothelial cells/endothelial cellsGPCR, CXCR4, increasing CXCL12, increasing apoptosis(40)
mRNA associated with (PI3K)/AKT signaling pathwayEndothelial progenitor cell/endothelial cellsActivation of angiogenic program(20)

MVs, Liver Biology and Drug Metabolism/Resistance

Next to their role in immunology, inflammation and coagulation, there is evidence that MVs are involved in drug metabolism and development of drug resistance. MVs originating from the liver contain RNA molecules of Hepatitis C virus in infected patients that leads to the conclusion, that also liver cells excrete MVs (55). In response to liver injury and stimulation with platelet-derived growth factor, MVs containing active Hedgehog can be found in the serum. This leads to an altered angiogenic response in endothelial cells representing a novel pathway for cirrhotic liver development (56). In a study using a proteomic-based approach, Jin et al. revealed a significant percentage of proteins involved in drug metabolism in exosomes derived from plasma. For instance, these proteins include glutathione transferase and albumin. These findings are verified by the fact that also primary hepatocytes secrete MVs containing proteins of the cytochrome P450, uridinediphosphate-glucuronosyltransferase and Glutathione S-transferase protein families (57). P-Glycoprotein, which is involved in multidrug resistance as an ATP binding cassette transporter, has a role in the cell-to-cell communication via MVs between drug resistant cancer cells and drug sensitive cells (58). A possible role in drug resistance is indicated by the fact that cisplatin as an antitumoral drug is secreted by an exosome-dependent mechanism along with cisplatin transporters in a cisplatin resistant cell line (59–61).

MVs as Novel Drugs and/or Drug Carriers

As MVs play diverse roles in both physiologic as well as pathophysiologic processes and are able to transport many different proteins as well as genetic information, usage of MVs as drugs and/or drug carriers seems eligible. See also Figure 3 for possible therapeutic options using MVs.

Figure 3.

Diagnostic and therapeutic use of exosomes/micro-vesicles in transplantation medicine. Future medical approaches in organ transplantation might involve using exosomes for both therapeutic as well as diagnostic approaches.

Artificially produced exosomes are traceable both in vitro and in vivo via fluorescent and magnetic resonance imaging and lead to the activation and expandation of functional antigen specific T cells at sufficient levels (62). Liposomal constructs can be synthesized by mixing phosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy [polyethylene glycol]-2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide [polyethylene glycol]-2000). To label such artificial liposomes, super paramagnetic iron oxide nano particles have been used. The liposomes were then coated with MHC peptide complexes and Fab regions. In a rat model of rheumatoid arthritis, liposomal-bound APO2L/TRAIL proved as an effective antiinflammatory treatment (63).

The findings of Zhang et al. reveal that trichosanthin (TCS), a plant toxin with ribosome-inactivating activity with abortifacient, antitumor and anti-HIV effects, is able to penetrate host cells. Endocytosed TCS is incorporated into intraluminal vesicles of the multivesicular body and is then secreted via MVs. The secreted TCS loaded MVs target syngenic and specific allogeneic cells and lead to subsequent internalization, delivery of the toxin into the cytosol and finally resulting in ribosomal inactivation and cell death (64). This shows how a plant toxin can be used as a novel drug approach by using exosome-related pathways. A further exciting approach is to use exosomes for oligonucleotide-based therapies. Here, purified exosomes were shown to be loadable with exogenous miRNA and subsequently used to target BACE1, a therapeutic target in Alzheimer's disease, in mice by intravenous injection (65).

Whereas detection of exosomes and MVs for diagnostic use is already feasible, an understanding of the cellular production of MVs or exosomes and the potential use of MVs and exosomes as drugs is still in its infancy. Specific hurdles that need to be overcome include design of synthetic targeted endogenous microparticles that may home to specific cells and/or organs, to optimize the “loading” of exosomes with specific proteins or genetic contents and to isolate and/or produce the needed amount for targeted therapies. However, the “delivery” to specific targets as discussed above may be of great clinical relevance for many diseases.

Conclusion

MVs are of importance for immunology and inflammatory processes and subsequently play a role in transplantation medicine. However, their role and future therapeutic relevance in transplantation medicine needs to be further investigated. The newly discovered involvement of miRNA gene transfer in MV mediated cell/cell communication opens a new perspective in future treatment options. MVs usage as drugs is highly eligible, because their use represents a way to use endogenous carriers for drug treatment. Because MVs can be (a) directed at certain target cells and (b) “loaded” with both genetic information as well as proteins, use of MVs seems to be a very elegant way for future target specific therapies, especially in transplantation medicine.

Acknowledgment

The research support of the BMBF (IFB-Transplantation; 01EO0802 to TT) is acknowledged.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Ancillary