Biological significance of exosomes and microparticles

Classification of microvesicles based on diameter size (adapted from Nagy et al. [7])

Knowing the above division, in cases where it is not specifically one group or another, but microvesicles in general, I will use the names exosomes and summary microvesicles in the following.

Another possibility for categorization is the morphological classification based on electron microscopy, which Yang and Tsai use in their article [5]. They differentiate:

- cup-shaped and

- saucer-shaped exosomes.

Schorey et al. [8] categorize exosomes based on the mother cell rather than the mechanism of production; for hemopoietic and non-hemopoietic origins.

Edit Buzas also deals with measurement and detection issues in her lecture [9] and proposes the use of differential detergent lysis for the separation of impurities caused by protein complexes.

Now let's briefly review the biogenesis of exosomes! All eukaryotic cells produce similar microvesicles [10]. Their formation usually takes place during cell activation, although they are also continuously produced in certain cells (epithelial cells, immature dendritic cells). They can originate in two ways [10] (Fig. 1).

Those between 30-100 nm (exosomes) are multivesicular bodies branching from the endosome-lysosome pathway (Fig. 2) and leave the cell by exocytosis (this pathway was first described by Pan and Johnstone in reticulocytes in 1983).

Those between 100-1000 nm (microparticles/ectosomes) are created by reverse splicing as a result of an activation signal or an increase in the intracellular Ca ²⁺ concentration.

Although there are still areas to be described in the exploration of biogenesis, primarily the mechanisms of the selection of substances entering the vesicles.

In contrast to generation, the description of the composition and functions of exosomes is quite detailed (see the ExoCarta topic later). Exosomes also carry proteins, lipids and nucleic acids (Fig. 3).

a) b)

Fig. 1. The two ways of the formation of exosomes (Sources: a) van Niel et al, b) Pap and tsai [10])

Fig. 5. Known and suspected exosomal functions related to tumours (Source: Noerholm [25])

- they affect anti-tumour immune processes (!),

- they play a role in the development of tumour cell chemoresistance (!).

Yang and Robbins (5) summarize the role of tumour cell-derived exosomes: Antitumor effects

- Immunogenic effect - as a result of carried tumour antigens.

- Induction of tumour cell apoptosis - Bcl-2 and Bax genes trigger the metabolic cascades that lead to tutor cell death.

- Neoplasmogenic role (Fig. 6).

Tumour cells produce exosomes, the composition of which is characteristic of the cell that secretes them, and they play the following role in the progression of the neoplasm [26]:

Immunosuppressive effect - inhibition of T cells, by influencing myeloid cell differentiation and through their antigens;

Enhancement of tumour invasion and metastasis formation - with angiogenic factors (tetraspanin);

Stroma-enhancing effect (on fibroblasts) - with metalloproteinases antiapoptotic and oncogenic effect - with RNA and protein transport.

Development of chemoresistance.

Valadi and tsai [22] showed that RNA in exosomes remains functional even after delivery. This may play a role in chemotherapy resistance of tumors.

Although the exact “result” of the processes has not yet been described, it seems that the tumor stage and the patient's immune status determine whether the neoplasmogenic or the opposite processes dominate.

Fig. 6. Neoplasmogenic effects of tumor cell exosomes (Source: Yang and Robbins [5])

Regarding immunomodulation, Karlsson et al developed the so-called tolerosome theory [27]. Tolerosomes are approx. Exosome-like vesicles with a diameter of 40 nm, which carry MHC-II protein complexes. Intestinal epithelial cells produce and secrete them into the intestinal lumen. After oral antigen administration, they can also be detected in the plasma soon after. Based on their structure, they are highly effective antigen presenters, so they play an important role in tolerance to orally introduced antigens, i.e. in inhibiting intense immune reactions against such antigens. Researchers assume that exosomes play a similar role in the immunosuppressive effect of tumours.

Another mechanism of the anti-immune effect is the so-called Fas tumour counterattack theory from Andreola et al. [28]. The Fas (also known as FasL) ligand is a type II transmembrane protein belonging to the tumour necrosis factor (TNF) family. Its trimerized form acts most actively. By binding to its T-cell receptor, it weakens the immune system through the apoptosis of the target cell and helps the tumour to hide from the immune system and to progress. It reaches the extracellular space in the form of an exosome. In particular, many active aquired Fas ligands are expressed by certain types of tumours (e.g. melanoma, breast cancer, colon cancer, esophageal cancer), as reported by O'Connell et al. in their article [29]. It has also been shown that the degree of Fas expression observed in ovarian, breast, liver and breast cancer as well as melanoma is inversely proportional to a good prognosis (Fig. 7).

Fig. 7. FasL expression in human melanoma cells detected by immunocytochemistry (Source: Andreola et al [28])

In connection with the research results related to exosomes, new directions for tumour therapy are being outlined. One direction is the idea of an anti-cancer vaccine, which has already been raised by several research groups. Results, considerations and issues related to this are described in Clayton and Mason's 2009 article [30]. Raposo and tsai [16] and then Zitvogel and tsai [31] showed that exosomal markers from antigen-presenting cells, e.g. heat shock protein number 70 (Hsp70) increases the migration and cytolytic activity of natural killer T cells.

The 2012 article by Hartmann et al also deals with the issue of tumor vaccination [32]. One of the reasons for the low effectiveness of the traditional vaccination method is that many non-cell surface antigens (exosomes) also play an important role in the case of tumours, AIDS and other diseases. The adenovirus vector transduction procedure proposed by the authors facilitates the presentation of exosomal antigens. The experiment was carried out with tumor cells, but the results seem to be applicable to viral and parasitic diseases, so research continues in all directions: T. gondii, M. bovis, S. pneumoniae [33], SARS and adenoviruses [34] are also research areas, such as autoimmune arthritis and so-called late hypersensitivity [35]. We can also be diagnostic and therapeutic helpers in many other diseases (heart attack, arteriosclerosis). Their use in relation to infarction is described in the article by Lai Ruenn Chai et al [36].

Another direction of research is aimed at the more specific use of existing drugs with fewer side effects through the artificial production and modification of microparticles (nanomedicine) [37], which, through its specificity, enhances the effect of cytostatics (cytostatics “packaged” in exosomes can be delivered to tumor cells in a targeted manner) and enables makes chemotherapy-resistant cancer cells end their resistance. In this way, the dose of cytostatics (e.g. cisplatin, doxorubicin, paclitaxel) can be reduced, thus reducing their systemic toxicity. With the possibility of specific treatment, the basis of personalized medicine (theranostics) was born. The essence of the trend is that it does not use epidemiological data and statistics based on the examination of a large number of patients, but rather examines the individual effect of the applicable drugs in the given patient and selects the ideal therapy based on this. The use of personalized medicine has already started in our country: on July 22, 2010, the Hungarian Society of Personalized Medicine (www.mszmt.hu) was established.

After tumours, let's move on to the topic of circulatory diseases [36]. Cardiovascular diseases are important targets for experimental stem cell treatment. Although, in principle, the transplanted stem cells differentiate and replace the damaged tissues. But they seem to reduce tissue damage and stimulate regeneration through the secretions they produce. The authors investigate the role of exosomes in the treatment of cardiovascular diseases (mainly myocardial infarction). Mesenchymal stem cells secrete in two ways (paracrine secretion - Fig. 8):

- traditional way: delivering soluble proteins to the extracellular space, which exert their effect by binding to the receptors of the target cell;

- with exosomes: their “packaged” proteins and nucleic acids are much more stable and protected; and they exert their effect not only on cell surface receptors, but also by reaching the target cell. Due to their complex “inner content”, they are also capable of complex effects. Parolini et al showed that the exosome assembly activity and efficiency of cells depends on several microenvironmental conditions, including tissue pH value (20). The therapeutic efficiency of stem cell secretions therefore correlates well with the fact already described by Schrader in 1985 that ischemic heart cells are characterized by acidic conditions (38). Exosomes are ideal candidates for therapeutic use. They are mainly photogenic because, paradoxically, the artificial enhancement of blood and oxygen supply to ischemic myocardial tissue leads to the exacerbation of ischemia-induced cell insults. Among exosomal proteins, the role of enzymes is particularly important in this case, as they break down ischemic metabolic products, thus correcting the cell-damaging cascade processes. The enthusiasm caused by the positive results of the animal experiments was unfortunately dampened by the fact that the stem cells often do not integrate into the heart muscle tissue or are not able to survive there. These problems can be solved by subcellular exosomes, which are capable of exerting a complex local effect without reactions occurring against the cells.

Fig. 8. Paracrine mechanisms of mesenchymal stem cells

microvesicles exosome intercellular communication detoxification regeneration

In addition to infarction, the role of exosomes in atherosclerosis, diabetes or hypertension has been suggested. The white blood cell-endothelial adhesion effect of microparticles is a key initial step in the development of atherosclerosis [39]. An important new result is the 2011 discovery by Vickers et al. of the relationship between the presence of HDL-miRNA complexes and familial hypercholesterolemia [40].

The relationship between neurodegenerative diseases and exosomes can be summarized as follows: I mentioned earlier that the role of exosomal sphingolipids in the development of Alzheimer's disease was described this year by Yuyama and tsai [13]. Exosomes related to Alzheimer's disease were also investigated by Street and his research group [41]. They investigated whether the cerebrospinal fluid also contains exosomes and, if so, the variability of exosomal proteins related to individuals. With their method based on ultracentrifugation, they managed to detect two characteristic exosome markers (Flotillin-1 and TSG101), which was also confirmed by transmission electron microscopy. Since the tests were carried out on a more concentrated sample than the physiological one, the goal was to develop a method for analyzing samples with a physiological concentration. Another goal is the development of detection methods, because with the current ones it is not possible to extract exosomes from biological fluids without contamination with non-exosomal proteins. One of the hallmarks of Alzheimer's disease is the extracellular accumulation of tau protein. Its role is similarly neurodegradative as that of alpha-synuclein in Parkinson's disease or prions in Kreutzfeld-Jacobs disease. The presence and accumulation of all three groups of substances indicates the presence and role of exosomes in the development of neurodegenerative pathologies.

The relationship between kidney diseases and exosomes deserves attention because the damage to the kidneys is often irreparable and the patient eventually needs dialysis or a kidney transplant. Balkom et al. [42] investigated the exosomal relationship between kidney physiology and diseases. They managed to show that the absence or mutation of the Tamm-Horsfall protein (uromodulin), which is also found in exosomes, can be the cause of kidney diseases. Unfortunately, the diagnostic rapid test is still waiting to be developed, but encouraging initial steps are reported in the differential diagnosis of kidney diseases (polycystic kidney, Nieman-Pick disease, cystinosis, Gitelman and Bartter syndrome). Urinary-derived exosomes can also be used to detect liver damage, as these are also associated with secondary renal manifestations. They also play a role in diagnosing hypertension and monitoring drug therapy, as well as comparing the effectiveness of drugs for a given patient (personalized medicine).

Finally, a few words about another topic: common upper respiratory tract infections. Lasser and his research team, already mentioned in relation to pregnancy [19], examined exosomes from nasal secretions in another experiment [43]. They were the first to reveal the RNA content of the exosomes of this secretion and its significance in the communication taking place in the nose, as well as the role of esRNA biomarker in upper respiratory diseases.

Based on the research so far, development continues at an ever-faster pace, so in 2011 many new results were achieved. Let's start with exosomal RNA research! Kosaka and Ochiya summarized the knowledge gathered on microRNA secretion [43]. MicroRNA is a biological fine-tuner, its deregulation leads to diseases, including cancer. It can be found in the circulation of both healthy and sick people, namely - since the circulation also contains RNase enzyme - in microvesicles, apoptotic bodies or linked to nucleic acid-binding proteins, this was demonstrated by Kwak et al. in 2011 (44). Because of the intervention in gene regulation, it became the focus of research on the development and diagnosis of cancer. In 1978, Stroun et al. described the in vitro modifying effect of extracellular RNA on DNA synthesis, and the possibility of this in vivo also arose [45]. The relationship between serum RNA, tumor mass and therapeutic sensitivity was described by Wieczorek et al. in 1987 [46], and Garzon et al. in 2010 that deregulation of miRNA leads to the development of cancer and changes in normal tissues, thus the miRNA diagnostic came into being, prognostic and therapeutic use in oncology [47]. The authors proved the communication significance of secreted miRNA in the transfer of information between cancer cells and with their microenvironment (Fig. 9).

Fig. 9. The effect of miRNA secretion of healthy and pathological cells on the recipient cell (Source: Kosaka and Ochiya [43])

A new discovery (Lim and tsai, 2011) is that the intercellular transport of miRNA can also take place via gap junctions [48]. The mechanism that selects the miRNA entering the exosomes and binds it to proteins still needs to be described and clarified, but there are many indications that the Ago2 protein plays an important role in the process [49].

Hood et al (2011) describe that miRNA plays a role in melanoma's ability to break through the immune barrier of lymph nodes and metastasize to distant organs through genetic modification of the target organ [50].

It has been known for nearly a decade that RNA transport takes place in plants and nematodes as well, through transmembrane channels formed by SID-1 proteins; and also, that the membrane of human cells also contains proteins similar to SID-1. From the report of Molnar et al. [51], we can conclude that the intercellular transport of RNA is characteristic of all multicellular organisms.

It was a real surprise, however, when Zhang and Tsai reported in 2011 that exogenous plant-derived miRNA was detected in animal tissues and serum [52]. MIR168a from rice, which has a very strong regulatory effect, probably entered the animal body through nutrition, since esRNA can withstand the acidic pH of the alimentary canal, as Lasser et al. showed at about the same time [19]. In addition, MIR168a has already been found in the serum of the healthy Chinese population. In the examined sera, Zhang and tsai detected nearly 30 similar miRNAs. They also managed to prove that the materials of the exosomes are taken up by the cells of the small intestine and passed on to the cells of the body. Exogenous MIR168a inhibits hepatocyte LDL uptake, thereby increasing serum LDL levels. The authors came to the conclusion that the exosomal RNAs taken up with plant food - with their regulatory effects - are just as essential nutrients as e.g. the vitamins. The article is the first report that there can be a direct interaction between plant and human cells at the molecular level.

Gan and Gould research the process of development of retroviruses, with particular reference to the HIV virus [53]. The relationship between viral development and exosomes is being investigated. They found that neither the HIV protease nor the lack of p6 glycosaminoglycan sequences characteristic of the virus inhibits viral development, as previously assumed. However, surprisingly, the virus's own Gag-Pol protein has a developmental inhibitory effect. The inhibitory signal for the development of HIV is therefore found in the virus itself. Nabhan and tsai report [54] that some developing viruses take advantage of the cell's TSG101 protein and the endosomal sorting mechanism (ESCRT) so that virus particles are transferred to exosomes instead of physiological particles, thus spreading from cell to cell.

One of the latest immunological publications discusses the role of exosomes in the development of atypical allergic rhinitis [55]. The pathological role of CD8+ type T cells, which are activated by exosomes produced in mucosal cells and containing microbial substances indicative of previous infection, has been confirmed.

In their article published this year, Choi and Tsai report on the protein networks created by exosomes [56] and Figure 10. Exosomes have been shown to possess a number of cluster-forming proteins that are similar to proteins in other subcellular networks.

Fig. 10. Network of exosomal proteins (left) and the cell covered by them (right) (Source: choi et al. [56])

It was experimentally proven that the protein interactions in the cell have an influence on the biogenesis of exosomes and the sorting of their proteins.

Of course, new discoveries are also due to new techniques. Let's look at some of them!

On April 7, 2011, Global Newswire reported on the new technology developed by Caris Life Sciences [57]. The procedure is a so-called a bloodbased test that is based on the role of circulating microvesicles and is just as effective in the diagnosis of colon, breast, lung and prostate cancer as other, much more complicated procedures. The essence of the test is the “capture” of circulating microvesicles with the help of specific antibodies, followed by the analysis, examination, and classification of the captured vesicles (Fig. 11, 12) [58]. The exact details of the technology are not known as they are under patent protection.

The test fits perfectly into the toolset of Theragnostic, a new trend that uses nano-diagnostic and nanotherapeutic procedures primarily used in tumour treatment [37].

Fig. 11. Logo of Carisome technology (Source: Caris Life Science [58])

Fig. 12. The essence of the Carisome process (Source: Caris Life Science [58])

The procedure allows for diagnostic, therapeutic and theranostic conclusions based on the different miRNA profiles of microvesicle populations detectable in the blood circulation. In this case, the researchers used the biomarker function of exosomes.

The procedure is suitable for detecting certain cancers and some other complex diseases. Its complexity is enhanced by the fact that it uses all three size ranges of circulating microvesicles (exosomes - ectosomes - apoptotic bodies).

Almost two years before the appearance of the Carisome method, NanoSight [59] reported on an instrument (Fig. 13) and a procedure that allows for multifaceted analysis of nanoparticles (size, concentration, aggregation) with one instrument.

The fast and direct procedure (Nanoparticle Tracking Analysis - NTA) detects, measures and counts nanoparticles in a soluble state (Fig 14).

Fig. 13. Nanosight's instrument, the LM20, and its operation diagram

Fig. 14. Video frame to illustrate NTA

The smallest detectable size is 10-20 nm, which can be detected even at a concentration of 10 ⁷-10⁹ particles/ml. The procedure is based on fluorescence, which enables visual validation in a unique way: the movement of the particles, reminiscent of Brownian motion, can be recorded on video and archived for later use.

The advantage of the procedure is that it is cheap, fast, easy to use and high-performance, the disadvantage is that it always requires a diluted sample and, since it is a new procedure, the limits of its reliability range require further testing. However, it is still used today for the additional validation of already introduced measurement procedures, and the NTA can also be calibrated with their help.

A similar procedure, also based on Brownian motion, is Dynamic light scattering (DLS) from Malvern [60]. With this, even particles smaller than 1 nm can be detected. The procedure has already been calibrated and cooperates with the ISO 13321 and 21 CFR Part 11 standards. The conventional 90° instrument has been replaced by two types of Non Invasive Back Scatter: Zetasizer Nano S and Nano ZS, which already operate at an angle of 173°.

Mikkel Noerholm, director responsible for European operations of Exosome Diagnostics, also reported on his company's latest research results in the aforementioned November 2011 presentation [25]. Their technology examines the RNA content of exosomes extracted from body fluids (they call it exoRNA, but it is the same as the nucleic acid called esRNA by Valadi et al.) [22]. The Exosome Biofluid Molecular Diagnostic Procedure developed for this purpose is used to detect prostate cancer, glioblastoma multiforme and malignant melanoma. The procedure was trademarked in 2008 by Harvard/ Massachusetts General Hospital.

Cataloging of the components of exosomes is currently underway, based on published and unpublished results [61]. This catalog is ExoCarta (www. exocarta.org). During the printing time of Mathivanan et al.'s article (61), so much changed that 19.12.2011. Since version 3.2 is currently available and can be downloaded, browsed and searched with more than fourteen thousand data. The data are contained in five text files (Table 2):

Table 2

Summary

Exosomes were first described more than 30 years ago. The biological role of these particles, produced in cells and then secreted, bounded by a lipid membrane, seems to extend to almost all areas of the living world. They have been described in plants, animals and humans. It has been shown that different species and even biological countries (in taxonomy: regnum), e.g. it is also possible to transfer exosomes between plants and animals. This also increases the significant genetic variability created by mutations and recombinations through the transfer of nucleic acids. The process is practically an alternative path of evolution. It can also be seen as the “fast lane” that has existed since the time of single-celled earth history, in addition to the traditionally slow “evolution highway”. Just like on the evolutionary high street, positive (resistance) and negative (apoptosis) changes can occur here as well. Changes at the cellular level in higher organisms can result in disease or even healing.

Therefore, the importance of exosomes is increasing in several areas, e.g. in cancer research, viral (e.g. HIV, HSV) or prion infections (Jacobs-Kreutzfeld) or Alzheimer's disease. These determine the main research directions, but of course further investigations are also underway. The main practical research areas are:

- isolation and characterization methods (detection, measurement, analysis),

- normal physiological role of exosomes, communication between cells,

- infection processes (HIV, prions, Trypanosoma),

- exosomal RNA (and tumors),

- use of exosomes as a therapeutic carrier (e.g. autoimmune vaccination).

In the three decades that have passed since their discovery, exosomes have become an independent scientific subfield, whose practitioners are already specializing and researching one area of expertise (detection, diagnostic use, therapy). For years, multi-section conferences have been held to summarize exosome research, and more and more research results reach the phase of practical application. Размещено на Allbest.Ru