1. Please check and comment entries here.
Table of Contents

    Topic review

    Extracellular Vesicles and Their Relationship with the Uremia

    Subjects: Pathology
    View times: 5
    Submitted by: Carolina Azevedo

    Definition

    Extracellular vesicles (EVs) have been widely investigated for their role in intercellular communication and as potential biomarkers; and could be a promising tool to improve the quality of care in kidney disease patients. Our research group previously demonstrated that the EVs can be related to endothelial dysfunction and are formed when UTs are in contact with the endothelial monolayer. Thus, this review addresses the relationship between these vesicles, cardiorenal syndrome, and uremia.

    1. Introduction

    Extracellular vesicles (EVs) have been widely investigated for their role in intercellular communication and as potential biomarkers, particularly in inflammatory pathological conditions. Cardiorenal syndrome results from interrelated heart and kidney injuries, which leads to an accumulation of uremic toxins in the body, especially with the progression of chronic kidney disease (CKD) [1][2][3]. However, the participation of EVs in CRS has not been fully elucidated. Clinical and in vitro studies have shown that uremic toxins induce the formation of EVs [4][5][6][7][8]. In this review, we address the role of EVs in CRS, especially their relationship with uremic toxins and kidney dysfunction. We also discuss the classification of EVs and the main methods for isolating and characterizing EVs, including electron microscopy, proteomics, lipidomics, transcriptome and metabolomics analyses, Fourier transform infrared (FTIR), and Raman spectroscopies, as well as possible use of EVs as biomarkers of cell injury.

     

    2. Cardiorenal Syndrome and Uremic Toxins (UTs)

    CRS is a set of diseases with clinical and metabolic consequences triggered by acute and/or chronic heart failure (CRS I and II) or acute and/or chronic kidney disease (CRS III and IV) resulting in injury to the other organs. Failure of both organs can also occur simultaneously as a consequence of a systemic disease (CRS V). Despite the current categorization of the CRS into these five groups, substantial overlap is observed between the different types [9].

    Type 2 CRS is characterized by chronic heart failure causing chronic kidney disease (CKD). The underlying mechanisms involve chronic kidney hypoperfusion, increased renal vascular resistance, overactivation of the SNS and RAAS, increased venous pressure, volume overload, endothelial dysfunction and inflammation [10]. This subtype of CRS is very common and has been described in up to 63% of patients with CRS in some reports [11]. The most common mechanisms believed to be involved in the development of type 2 CRS are neurohormonal activation, renal hypoperfusion, venous congestion, inflammation and oxidative stress [10].

    UT accumulation can be observed in all types of CRS [3][10][12][13]. Following renal injury, the structural damage in the kidney compromises renal function resulting in a reduction in GFR and/or subsequent increased proteinuria [2], this can cause an increase in UT accumulation in the blood further compounding the functional and structural deterioration of the kidneys and other organs [3]. Although it has been pointed out that this accumulation of toxins causes a primary injury to the kidney, some studies have suggest (i.e., Di Lullo et al.) that it could be considered a type 5 CRS since the uremic compounds can also directly cause damage to the cardiac tissue, featuring a systemic disease [3][10].

    The accumulation of medium-sized UT also contributes to renal structural damage, reducing the GFR and cardiac function [14]. Patients with advanced CKD present with low GFR and 1000-fold elevated FGF23 levels [15]. This has adverse effects on the heart via an independent mechanism, promoting cardiac hypertrophy and contractile dysfunction [16][17]. The Pi and FGF23 levels appear to be inter-related by their mechanisms. The Pi levels increase as soon as the renal function decreases, and plasma FGF23 concentration increases due to significant changes in phosphate or serum PTH concentration. One study considered FGF23 a secondary UT, since it only increases after phosphate accumulation [18]. There are relevant findings regarding Pi and medium-sized UT inducing hypertrophy of the myocardium, hyperplasia of cardiomyocytes, and interstitial fibrosis and vessels [2].

     

    3. The Importance of Extracellular Vesicles in Heart/Kidney Axis and Uremia

    The EVs are vesicular nano-sized membrane-enclosed structures composed of a lipid bilayer (such as the cell plasmatic membrane) which transport body cargo such as DNA, RNA, and proteins from their cell of origin and have the ability to physiologically and pathologically influence their cell of origin and other cells [19][20][21]. They can be detected in plasma, urine, and other body fluids of healthy people [22], and their levels are increased in various diseases, mainly reflecting the injury suffered in these tissues [23]. As their composition depends on the pathophysiological and functional state of their cell of origin, they have been studied as potential biomarkers in several diseases, especially cardiovascular [24][25][26], immune [27][28][29][30], cancer [31][32][33][34][35], viral infection, including COVID-19 [36][37], CKD [4][38][39][40][41], and recently in peritoneal dialysis [42][43][44][45][46].

    According to the International Society of Extracellular Vesicles (ISEV), EVs are all vesicles released from a cell which can be classified based on their mechanism of formation, mode of release from the cells and size [47][48]. Studies have broadly divided EVs into three main groups: microvesicles (MVs) (also called microparticles), exosomes, and apoptotic bodies.

    The role of the UTs in the release of EVs, have mostly been studied in the setting of endothelial dysfunction leading to a release of endothelial-derived microvesicles (EMVs). In vitro and clinical studies observed that PCS was able to induce spreading of EMVs in cell culture as well as increase the levels of EMVs in hemodialysis patients [49]. EMVs also have an important role in patients with ESRD, mainly modulating vasorelaxation, and decreasing endothelial nitric oxide ( eNO) release [50]. Favretto et al. observed the formation of different-sized EVs from endothelial cells generated by PCS, IS, and Pi treatments in vitro. In addition, it stimulated cell adhesion markers in PCS and Pi-induced EVs and VCAM-1 expression in PCS and IS-induced EVs [4].

    When exposed to an increased hemodynamic load due to physiological stress such as CRS, the heart responds adapting to new operating conditions; however, prior to this adaptation, it responds with a particular communication using EVs to mediate the various cell populations [51][52]. This interaction was previously described by Waldenström et al. when internalization of cardiomyocyte exosomes was observed in fibroblasts and endothelial cells [53]. This study showed the presence of genetic material of cardiomyocytes inside the cytoplasm of other cells types, promoting modification of gene expression. In this context, the EV interaction between cardiomyocytes and fibroblasts was important in the progression of chronic heart failure and is given by the transport of the miR-217 from cardiomyocytes to fibroblasts promoting its proliferation and consequent fibrosis [54]. Cardiomyocytes have also been show to release exosomes internalized by endothelial cells containing miRNAs in order to increase angiogenesis after stress (miR-17,19a,19b,20a,30c,126) [55].

     

    4. Isolation and Characterization of Extracellular Vesicles (EVs)

    One of the most used methods for isolating EVs is differential ultracentrifugation (DUC), in which the solution containing the EVs is subjected to several centrifugation steps with increasing speed to pellet the EVs. The isolation of EVs by DUC usually consists of a step with low-speed centrifugation (1000 RCF) to remove cells and larger particles, followed by another intermediate speed spin (20,000 RCF) to collect larger EVs, and finally high-speed ultracentrifugation (100,000 RCF) to isolate smaller EVs [56]. However, it is worth noting that several parameters can affect the type, purity, and yield of EVs, such as rotor type, g-force, centrifugation time, sedimentation rotor angle, and viscosity of the sample [57][58][59][60]. Considering these parameters, possible adaptations to the centrifugation protocol, such as changing the centrifugation time, can improve the separation of the required EVs [58]. For instance, viscous samples need more time and greater ultracentrifugation speed [58].

    Mass spectrometry techniques are extensively used in order to determine the proteomic profile of EVs. In summary, EVs are lysed and the proteins are subjected to enzymatic digestion with subsequent separation of the peptides in the mass spectrometer [61][62][63]. The proteomic study of EVs has an important role in the research of biomarkers in EVs that would normally be masked by abundant soluble proteins [43][64].

    More recently, the metabolomic study of the content of EVs has gained ground [65]. Some studies have identified significant metabolites in EVs in pathological conditions, such as cancer [66][67]. However, it is necessary to use metabolite extraction protocols and define the analytical platform to study the metabolome of EVs using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) [68][66][69].

    Recent studies have also shown that the IR spectroscopy–based protein quantification can be successfully adapted to experimental practice to analyze EVs. In contrast, vibrational spectroscopy presents a reagent-free alternative to traditional colorimetric protein determination assays and demands no special sample preparation to explore EVs [70]. According to results obtained by Paolini et al., FTIR also has the potential to promptly characterize EV subpopulations [71], suggesting it as an attractive complement or alternative method for understanding EVs in healthy and pathological situations.

    This entry is adapted from 10.3390/toxins13110778

    References

    1. Kumar, U.; Wettersten, N.; Garimella, P.S. Cardiorenal Syndrome: Pathophysiology. Cardiol. Clin. 2019, 37, 251–265.
    2. Barreto, F.C.; Stinghen, A.E.M.; de Oliveira, R.B.; Franco, A.T.B.; Moreno, A.N.; Barreto, D.V.; Pecoits-Filho, R.; Drüeke, T.B.; Massy, Z.A. The quest for a better understanding of chronic kidney disease complications: An update on uremic toxins. J. Bras. Nefrol. 2014, 36, 221–235.
    3. Falconi, C.A.; da Cruz Junho, C.V.; Fogaça-Ruiz, F.; Vernier, I.C.S.; da Cunha, R.S.; Stinghen, A.E.M.; Carneiro-Ramos, M.S. Uremic Toxins: An Alarming Danger Concerning the Cardiovascular System. Front. Physiol. 2021, 12, 667.
    4. Favretto, G.; da Cunha, R.S.; Flores Santos, A.; Leitolis, A.; Schiefer, E.M.; Gregório, P.C.; Franco, C.R.C.; Massy, Z.; Dalboni, M.A.; Stinghen, A.E.M. Uremic endothelial-derived extracellular vesicles: Mechanisms of formation and their role in cell adhesion, cell migration, inflammation, and oxidative stress. Toxicol. Lett. 2021, 347, 12–22.
    5. Meijers, B.K.I.; De Loor, H.; Bammens, B.; Verbeke, K.; Vanrenterghem, Y.; Evenepoel, P. p-cresyl sulfate and indoxyl sulfate in hemodialysis patients. Clin. J. Am. Soc. Nephrol. 2009, 4, 1932–1938.
    6. Abbasian, N.; Goodall, A.H.; Burton, J.O.; Bursnall, D.; Bevington, A.; Brunskill, N.J. Hyperphosphatemia Drives Procoagulant Microvesicle Generation in the Rat Partial Nephrectomy Model of CKD. J. Clin. Med. 2020, 9, 3534.
    7. Soriano, S.; Carmona, A.; Triviño, F.; Rodriguez, M.; Alvarez-Benito, M.; Martín-Malo, A.; Alvarez-Lara, M.A.; Ramírez, R.; Aljama, P.; Carracedo, J. Endothelial damage and vascular calcification in patients with chronic kidney disease. Am. J. Physiol.-Ren. Physiol. 2014, 307, 1302–1311.
    8. Faure, V.; Dou, L.; Sabatier, F.; Cerini, C.; Sampol, J.; Berland, Y.; Brunet, P.; Ddignat-George, F. Elevation of circulating endothelial microparticles in patients with chronic renal failure. J. Thromb. Haemost. 2006, 4, 566–573.
    9. Mavrakanas, T.A.; Khattak, A.; Singh, K.; Charytan, D.M. Epidemiology and natural history of the cardiorenal syndromes in a cohort with echocardiography. Clin. J. Am. Soc. Nephrol. 2017, 12, 1624–1633.
    10. Di Lullo, L.; Bellasi, A.; Barbera, V.; Russo, D.; Russo, L.; Di Iorio, B.; Cozzolino, M.; Ronco, C. Pathophysiology of the cardio-renal syndromes types 1–5: An uptodate. Indian Heart J. 2017, 69, 255–265.
    11. Cruz, D.N.; Schmidt-Ott, K.M.; Vescovo, G.; House, A.A.; Kellum, J.A.; Ronco, C.; McCullough, P.A. Pathophysiology of cardiorenal syndrome type 2 in stable chronic heart failure: Workgroup statements from the eleventh consensus conference of the acute dialysis quality initiative (ADQI). In Contributions to Nephrology; Karger: Basel, Switzerland, 2013; Volume 182, pp. 117–136. ISBN 9783318024067.
    12. Chaudhary, K.; Malhotra, K.; Sowers, J.; Aroor, A. Uric acid-key ingredient in the recipe for cardiorenal metabolic syndrome. Cardiorenal Med. 2013, 3, 208–220.
    13. Tamariz, L.; Hernandez, F.; Bush, A.; Palacio, A.; Hare, J.M. Association between serum uric acid and atrial fibrillation: A systematic review and meta-analysis. Heart Rhythm 2014, 11, 1102–1108.
    14. Fujii, H.; Goto, S.; Fukagawa, M. Role of uremic toxins for kidney, cardiovascular, and bone dysfunction. Toxins 2018, 10, 202.
    15. Edmonston, D.; Wolf, M. FGF23 at the crossroads of phosphate, iron economy and erythropoiesis. Nat. Rev. Nephrol. 2020, 16, 7–19.
    16. Faul, C.; Amaral, A.P.; Oskouei, B.; Hu, M.-C.; Sloan, A.; Isakova, T.; Gutiérrez, O.M.; Aguillon-Prada, R.; Lincoln, J.; Hare, J.M.; et al. FGF23 induces left ventricular hypertrophy. J. Clin. Investig. 2011, 121, 4393–4408.
    17. Navarro-García, J.A.; Delgado, C.; Fernández-Velasco, M.; Val-Blasco, A.; Rodríguez-Sánchez, E.; Aceves-Ripoll, J.; Gómez-Hurtado, N.; Bada-Bosch, T.; Mérida-Herrero, E.; Hernández, E.; et al. Fibroblast growth factor-23 promotes rhythm alterations and contractile dysfunction in adult ventricular cardiomyocytes. Nephrol. Dial. Transplant. 2019, 34, 1864–1875.
    18. Kuczera, P.; Adamczak, M.; Wiecek, A. Fibroblast growth factor-23—A potential uremic toxin. Toxins 2016, 8, 369.
    19. Borges, F.T.; Reis, L.A.; Schor, N. Extracellular vesicles: Structure, function, and potential clinical uses in renal diseases. Braz. J. Med. Biol. Res. 2013, 46, 824–830.
    20. Yáñez-Mó, M.; Siljander, P.R.M.; Andreu, Z.; Zavec, A.B.; Borràs, F.E.; Buzas, E.I.; Buzas, K.; Casal, E.; Cappello, F.; Carvalho, J.; et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 2015, 4, 1–60.
    21. Nederveen, J.P.; Warnier, G.; Di Carlo, A.; Nilsson, M.I.; Tarnopolsky, M.A. Extracellular Vesicles and Exosomes: Insights From Exercise Science. Front. Physiol. 2021, 11, 1757.
    22. Burger, D.; Schock, S.; Thompson, C.S.; Montezano, A.C.; Hakim, A.M.; Touyz, R.M. Microparticles: Biomarkers and beyond. Clin. Sci. 2013, 124, 423–441.
    23. Akbari, S.; Abou-Arkoub, R.; Sun, S.; Hiremath, S.; Reunov, A.; McCormick, B.B.; Ruzicka, M.; Burger, D. Microparticle formation in peritoneal dialysis: A proof of concept study. Can. J. Kidney Health Dis. 2017, 4, 1–8.
    24. Ridger, V.C.; Boulanger, C.M.; Angelillo-Scherrer, A.; Badimon, L.; Blanc-Brude, O.; Bochaton-Piallat, M.L.; Boilard, E.; Buzas, E.I.; Caporali, A.; Dignat-George, F.; et al. Microvesicles in vascular homeostasis and diseases position paper of the european society of cardiology (ESC) working group on atherosclerosis and vascular biology. Thromb. Haemost. 2017, 117, 1296–1316.
    25. Pironti, G.; Strachan, R.T.; Abraham, D.; Mon-Wei Yu, S.; Chen, M.; Chen, W.; Hanada, K.; Mao, L.; Watson, L.J.; Rockman, H.A. Circulating Exosomes Induced by Cardiac Pressure Overload Contain Functional Angiotensin II Type 1 Receptors. Circulation 2015, 131, 2120–2130.
    26. Vasina, E.; Heemskerk, J.W.M.; Weber, C.; Koenen, R.R. Platelets and platelet-derived microparticles in vascular inflammatory disease. Inflamm. Allergy Drug Targets 2010, 9, 346–354.
    27. Vajen, T.; Mause, S.F.; Koenen, R.R. Microvesicles from platelets: Novel drivers of vascular inflammation. Thromb. Haemost. 2015, 114, 228–236.
    28. Burbano, C.; Villar-Vesga, J.; Orejuela, J.; Muñoz, C.; Vanegas, A.; Vásquez, G.; Rojas, M.; Castaño, D. Potential Involvement of Platelet-Derived Microparticles and Microparticles Forming Immune Complexes during Monocyte Activation in Patients with Systemic Lupus Erythematosus. Front. Immunol. 2018, 9, 322.
    29. Mobarrez, F.; Svenungsson, E.; Pisetsky, D.S. Microparticles as autoantigens in systemic lupus erythematosus. Eur. J. Clin. Investig. 2018, 48, e13010.
    30. Ullal, A.J.; Reich, C.F.; Clowse, M.; Criscione-Schreiber, L.G.; Tochacek, M.; Monestier, M.; Pisetsky, D.S. Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus. J. Autoimmun. 2011, 36, 173–180.
    31. Crow, J.; Atay, S.; Banskota, S.; Artale, B.; Schmitt, S.; Godwin, A.K. Exosomes as mediators of platinum resistance in ovarian cancer. Oncotarget 2017, 8, 11917–11936.
    32. Choi, D.Y.; You, S.; Jung, J.H.; Lee, J.C.; Rho, J.K.; Lee, K.Y.; Freeman, M.R.; Kim, K.P.; Kim, J. Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment. Proteomics 2014, 14, 1845–1856.
    33. Challagundla, K.B.; Wise, P.M.; Neviani, P.; Chava, H.; Murtadha, M.; Xu, T.; Kennedy, R.; Ivan, C.; Zhang, X.; Vannini, I.; et al. Exosome-Mediated Transfer of microRNAs Within the Tumor Microenvironment and Neuroblastoma Resistance to Chemotherapy. J. Natl. Cancer Inst. 2015, 107, djv135.
    34. Caivano, A.; Laurenzana, I.; De Luca, L.; La Rocca, F.; Simeon, V.; Trino, S.; D’Auria, F.; Traficante, A.; Maietti, M.; Izzo, T.; et al. High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders. Tumor Biol. 2015, 36, 9739–9752.
    35. Bouvy, C.; Wannez, A.; Laloy, J.; Chatelain, C.; Dogné, J.M. Transfer of multidrug resistance among acute myeloid leukemia cells via extracellular vesicles and their microRNA cargo. Leuk. Res. 2017, 62, 70–76.
    36. Bello-Morales, R.; Ripa, I.; López-Guerrero, J.A. Extracellular vesicles in viral spread and antiviral response. Viruses 2020, 12, 623.
    37. Hassanpour, M.; Rezaie, J.; Nouri, M.; Panahi, Y. The role of extracellular vesicles in COVID-19 virus infection. Infect. Genet. Evol. 2020, 85, 104422.
    38. Burton, J.O.; Hamali, H.A.; Singh, R.; Abbasian, N.; Parsons, R.; Patel, A.K.; Goodall, A.H.; Brunskill, N.J. Elevated Levels of Procoagulant Plasma Microvesicles in Dialysis Patients. PLoS ONE 2013, 8, e72663.
    39. Buendía, P.; De Oca, A.M.; Madueño, J.A.; Merino, A.; Martín-Malo, A.; Aljama, P.; Ramírez, R.; Rodríguez, M.; Carracedo, J. Endothelial microparticles mediate inflammation-induced vascular calcification. FASEB J. 2015, 29, 173–181.
    40. Amabile, N.; Guérin, A.P.; Leroyer, A.; Mallat, Z.; Nguyen, C.; Boddaert, J.; London, G.M.; Tedgui, A.; Boulanger, C.M. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J. Am. Soc. Nephrol. 2005, 16, 3381–3388.
    41. Lau, Y.C.; Xiong, Q.; Blann, A.D.; Lip, G.Y.H. Relationship between renal function and circulating microparticles, soluble P-selectin and E-selectin levels in atrial fibrillation. J. Thromb. Thrombolysis 2017, 43, 18–23.
    42. Carreras-Planella, L.; Soler-Majoral, J.; Rubio-Esteve, C.; Lozano-Ramos, S.I.; Franquesa, M.; Bonet, J.; Troya-Saborido, M.I.; Borràs, F.E. Characterization and proteomic profile of extracellular vesicles from peritoneal dialysis efflux. PLoS ONE 2017, 12, e0176987.
    43. Carreras-Planella, L.; Soler-Majoral, J.; Rubio-Esteve, C.; Morón-Font, M.; Franquesa, M.; Bonal, J.; Troya-Saborido, M.I.; Borràs, F.E. Proteomic profiling of peritoneal dialysis effluent-derived extracellular vesicles: A longitudinal study. J. Nephrol. 2019, 32, 1021–1031.
    44. Corciulo, S.; Nicoletti, M.C.; Mastrofrancesco, L.; Milano, S.; Mastrodonato, M.; Carmosino, M.; Gerbino, A.; Corciulo, R.; Russo, R.; Svelto, M.; et al. AQP1-Containing Exosomes in Peritoneal Dialysis Effluent As Biomarker of Dialysis Efficiency. Cells 2019, 8, 330.
    45. Aufricht, C.; Beelen, R.; Eberl, M.; Fischbach, M.; Fraser, D.; Jörres, A.; Kratochwill, K.; LópezCabrera, M.; Rutherford, P.; Schmitt, C.P.; et al. Biomarker research to improve clinical outcomes of peritoneal dialysis: Consensus of the European Training and Research in Peritoneal Dialysis (EuTRiPD) network. Kidney Int. 2017, 92, 824–835.
    46. Pearson, L.J.; Klaharn, I.Y.; Thongsawang, B.; Manuprasert, W.; Saejew, T.; Somparn, P.; Chuengsaman, P.; Kanjanabuch, T.; Pisitkun, T. Multiple extracellular vesicle types in peritoneal dialysis effluent are prominent and contain known biomarkers. PLoS ONE 2017, 12, e0178601.
    47. Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7, 1535750.
    48. Gurunathan, S.; Kang, M.H.; Qasim, M.; Khan, K.; Kim, J.H. Biogenesis, membrane trafficking, functions, and next generation nanotherapeutics medicine of extracellular vesicles. Int. J. Nanomed. 2021, 16, 3357–3383.
    49. Meijers, B.K.I.; Van kerckhoven, S.; Verbeke, K.; Dehaen, W.; Vanrenterghem, Y.; Hoylaerts, M.F.; Evenepoel, P. The Uremic Retention Solute p-Cresyl Sulfate and Markers of Endothelial Damage. Am. J. Kidney Dis. 2009, 54, 891–901.
    50. Erdbrügger, U.; Le, T.H. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers? J. Am. Soc. Nephrol. 2016, 27, 12–26.
    51. Shimizu, I.; Minamino, T. Physiological and pathological cardiac hypertrophy. J. Mol. Cell. Cardiol. 2016, 97, 245–262.
    52. Bellin, G.; Gardin, C.; Ferroni, L.; Chachques, J.; Rogante, M.; Mitrečić, D.; Ferrari, R.; Zavan, B. Exosome in Cardiovascular Diseases: A Complex World Full of Hope. Cells 2019, 8, 166.
    53. Waldenström, A.; Gennebäck, N.; Hellman, U.; Ronquist, G. Cardiomyocyte Microvesicles Contain DNA/RNA and Convey Biological Messages to Target Cells. PLoS ONE 2012, 7, e34653.
    54. Nie, X.; Fan, J.; Li, H.; Yin, Z.; Zhao, Y.; Dai, B.; Dong, N.; Chen, C.; Wang, D.W. miR-217 Promotes Cardiac Hypertrophy and Dysfunction by Targeting PTEN. Mol. Ther. Nucleic Acids 2018, 12, 254.
    55. Garcia, N.A.; Ontoria-Oviedo, I.; González-King, H.; Diez-Juan, A.; Sepúlveda, P. Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells. PLoS ONE 2015, 10, e0138849.
    56. Veziroglu, E.M.; Mias, G.I. Characterizing Extracellular Vesicles and Their Diverse RNA Contents. Front. Genet. 2020, 11, 1–30.
    57. Taylor, D.D.; Shah, S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 2015, 87, 3–10.
    58. Momen-Heravi, F.; Balaj, L.; Alian, S.; Trachtenberg, A.J.; Hochberg, F.H.; Skog, J.; Kuo, W.P. Impact of Biofluid Viscosity on Size and Sedimentation Efficiency of the Isolated Microvesicles. Front. Physiol. 2012, 3, 1–6.
    59. Livshits, M.A.; Khomyakova, E.; Evtushenko, E.G.; Lazarev, V.N.; Kulemin, N.A.; Semina, S.E.; Generozov, E.V.; Govorun, V.M. Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci. Rep. 2015, 5, 17319.
    60. Cvjetkovic, A.; Lötvall, J.; Lässer, C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J. Extracell. Vesicles 2014, 3, 23111.
    61. Shao, H.; Im, H.; Castro, C.M.; Breakefield, X.; Weissleder, R.; Lee, H. New Technologies for Analysis of Extracellular Vesicles. Chem. Rev. 2018, 118, 1917–1950.
    62. Choi, D.S.; Kim, D.K.; Kim, Y.K.; Gho, Y.S. Proteomics of extracellular vesicles: Exosomes and ectosomes. Mass Spectrom. Rev. 2015, 34, 474–490.
    63. Subedi, P.; Schneider, M.; Philipp, J.; Azimzadeh, O.; Metzger, F.; Moertl, S.; Atkinson, M.J.; Tapio, S. Comparison of methods to isolate proteins from extracellular vesicles for mass spectrometry-based proteomic analyses. Anal. Biochem. 2019, 584, 113390.
    64. Gidlöf, O.; Evander, M.; Rezeli, M.; Marko-Varga, G.; Laurell, T.; Erlinge, D. Proteomic profiling of extracellular vesicles reveals additional diagnostic biomarkers for myocardial infarction compared to plasma alone. Sci. Rep. 2019, 9, 1–13.
    65. Williams, C.; Palviainen, M.; Reichardt, N.C.; Siljander, P.R.M.; Falcón-Pérez, J.M. Metabolomics applied to the study of extracellular vesicles. Metabolites 2019, 9, 276.
    66. Clos-Garcia, M.; Loizaga-Iriarte, A.; Zuñiga-Garcia, P.; Sánchez-Mosquera, P.; Rosa Cortazar, A.; González, E.; Torrano, V.; Alonso, C.; Pérez-Cormenzana, M.; Ugalde-Olano, A.; et al. Metabolic alterations in urine extracellular vesicles are associated to prostate cancer pathogenesis and progression. J. Extracell. Vesicles 2018, 7, 1470442.
    67. Luo, X.; An, M.; Cuneo, K.C.; Lubman, D.M.; Li, L. High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics. Anal. Chem. 2018, 90, 8314–8319.
    68. Arraud, N.; Linares, R.; Tan, S.; Gounou, C.; Pasquet, J.M.; Mornet, S.; Brisson, A.R. Extracellular vesicles from blood plasma: Determination of their morphology, size, phenotype and concentration. J. Thromb. Haemost. 2014, 12, 614–627.
    69. Dudzik, D.; Macioszek, S.; Struck-Lewicka, W.; Kordalewska, M.; Buszewska-Forajta, M.; Waszczuk-Jankowska, M.; Wawrzyniak, R.; Artymowicz, M.; Raczak-Gutknecht, J.; Siluk, D.; et al. Perspectives and challenges in extracellular vesicles untargeted metabolomics analysis. TrAC Trends Anal. Chem. 2021, 143, 116382.
    70. Szentirmai, V.; Wacha, A.; Németh, C.; Kitka, D.; Rácz, A.; Héberger, K.; Mihály, J.; Varga, Z. Reagent-free total protein quantification of intact extracellular vesicles by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Anal. Bioanal. Chem. 2020, 412, 4619–4628.
    71. Paolini, L.; Federici, S.; Consoli, G.; Arceri, D.; Radeghieri, A.; Alessandri, I.; Bergese, P. Fourier-transform Infrared (FT-IR) spectroscopy fingerprints subpopulations of extracellular vesicles of different sizes and cellular origin. J. Extracell. Vesicles 2020, 9, 1741174.
    More