Extracellular vesicles in chronic pulmonary vascular diseases: novel promising biomarkers?

Abstract

MPs are classified as EVs, which represent shed-membrane structures released into the blood circulation during different cellular processes, such as apoptosis and/or cellular activation (113, 115). EVs can be categorized as exosomes (45 100 nm), MPs (100 1000 nm), and apoptotic bodies (1000 4000 nm), depending on their size and mechanisms of formation and release (113). It was reported that circulating endothelial cell-derived and procoagulant MPs, which are a type of EVs, are enhanced in patients with PH (2, 115, 129, 133, 153). The literature suggests that circulating endothelial cell-derived MPs are significantly augmented in precapillary PH and that they show a positive correlation with clinical parameters such as mPAP (115). Endothelial cell-derived EVs are elucidated to be promising biomarkers, as they fulfil many of the criteria as good biomarkers, and are also considered to play a pivotal role in the development of pulmonary vascular diseases by acting as transport vehicles for miRNAs (129). Thus, strong evidence about endothelial cellderived MPs in the field of PH, could provide knowledge about their importance in the therapeutic field. However, very little is known about the inflammatory cell-derived MPs (115). It was shown by Savai et al. that there is a massive accumulation and infiltration of inflammatory/immune cells in PH (134).Therefore, it was hypothesized that, inflammatory cell-derived EVs may play an important role in the pathogenesis of different pulmonary vascular diseases, since it is known that PH is an inflammatory disease. In this study, analysis of the endothelial cell-derived MPs in various clinical PH subgroups was done. Therefore, this study was designed to analyze circulating profiles of various inflammatory (CD3 (T cells), CD14 (monocytes), CD45 (leukocyte common antigen), CD68 (macrophages), endothelial (CD62E (E-selectin), CD31 (PECAM, monocytes, neutrophils), CD144 (VE-Cadherin), and CD209 (dendritic) cellderived EVs in different clinical forms of PH.The central blood was obtained during right heart catheterization from patients with different forms of PH (I,h PAH, associated PAH, COPD-PH, Fibrosis-PH, LHD-PH and CTEPH, and non-PH controls . The central blood from each patient during right heart catheterization was collected into citrated tubes which were further centrifuged at 500xg for 15 minutes, followed by 10.000xg for 5 minutes to obtain the platelet free plasma (PFP), as described by Amabile et al. (115). The PFP was used for flow cytometry analysis with a BD LSRFortessatm flow cytometer.Focusing on inflammatory cells-derived EVs, the flow cytometry analysis revealed that there was an augmentation of circulating CD3 (T cell) cells-derived EVs (events/mikro l) in all of the above-mentioned clinical groups of PH, compared to the non-PH controls. However, this was only statistically significant in the case of I,h PAH and CTEPH. Interestingly, only a moderate and non-significant positive correlation between circulating levels of CD3 cellsderived EVs and the relevant clinical parameters of PH, such as mPAP (mmHg) and PVR (dynscm-5) was observed. Following the analysis of endothelial cell-derived EVs, it was identified that the circulating levels of CD62E-derived-EVs were increased in associated the PAH, COPD-PH and CTEPH groups. Conversely, there was no visible alterations in other groups, as compared to non-PH donors. A statistical significance was observed in the CTEPH group. Importantly, a positive correlation between CD62E cell-derived EVs and clinical parameters like PVR, but no correlation with mPAP was observed.The present study shows for the first time that enhanced levels of circulating T cells-derived EVs are present in several forms of PH. This major finding supports the knowledge from the literature, that there is an accumulation and infiltration of inflammatory / immune cells, such as T Cells in redesigned pulmonary vasculature and the lung tissue of IPAH patients (134). Even though, the literature states a role for monocytes/macrophages in the pathogenesis of PH, a consistent alteration in the inflammatory monocytes/macrophages derived-EVs was not observed in the present study. The enhancement of endothelial cell derived-EVs in the pathophysiology of precapillary PH has been explained in the following literature (115, 126). This study also observed the augmentation of endothelial cell-derived CD62E-EVs in the associated PAH, COPD-PH and CTEPH groups.Finally, it can be concluded from this study that there is an enhancement of circulating inflammatory cell-derived EVs originating from T cells in various PH subgroups. Thus, these inflammatory T cell-derived EVs may represent novel promising biomarkers in the pathogenesis of severe pulmonary vascular diseases. Future studies should target the precise roles of T cells-derived EVs, which may act as novel players in the disease development and/or progression, since EVs are known as carriers of mediators, such as miRNAs. Mikropartikel (MPs), die als Abschnürung der Zellmembran während zellulärer Prozesse, wie der Zell aktivierung und/oder Apoptose in den Blutkreislauf freigesetzt werden, zählen zu den extrazellulären Vesikeln (EVs) (113, 115). Je nach Größe und Mechanismen der Bildung und Freisetzung, werden die extrazellulären Vesikel in Exosomen (45-100 nm), Mikropartikel (100-1000 nm) und apoptotische Körperchen (1000-4000 nm) klassifiziert (113). Es wird berichtet, dass im Blut zirkulierende endotheliale und pro-koagulierende Mikropartikeln bei Patienten mit pulmonaler Hypertonie erhöht sind (2, 115, 129, 133, 153). Es wird in der Literatur erwähnt, dass die zirkulierenden endothelialen MPs in der präkapillären PH signifikant erhöht sind und auch ihre positive Korrelation mit klinischen Parametern wie mPAP wurde gezeigt (115). Die endothelialen EVs werden als wichtige Biomarker angesehen, da sie alle Eigenschaften eines guten Biomarkers erfüllen und als Träger der Mikro RNAs eine entscheidende Rolle bei den pulmonalen vaskulären Krankheiten spielen (129). Da endotheliale MPs als wichtige Faktoren im Bereich der PH anzusehen sind, haben Sie auch eine wesentliche Bedeutung im therapeutischen Bereich. Jedoch ist sehr wenig über die inflammatorischen MPs bekannt (115). Savai et al. Wiesen nach, dass es zu einer massiven Akkumulation und Infiltration von Entzündungs- / Immunzellen in der PH kommt (134).Aufgrund dieser Erkenntnisse stellten wir die Hypothese auf, dass inflammatorische EVs eine wichtige Rolle in der Pathogenese verschiedener Lungengefäßerkrankungen spielen könnten, da der bisherige Wissenstand zeigt, dass die PH eine entzündliche Erkrankung ist. Unsere Studie analysiert zum ersten Mal die endothelialen MPs in verschiedenen klinischen PH-Untergruppen. Daher wurde unsere Studie entwickelt, um die zirkulierenden Profile von verschiedenen inflammatorischen (CD3 (T-Zellen), CD14 (Monozyten), CD45 (Leukocyte Common Antigen), CD68 (Makrophagen)) und endothelialen (CD62E (ESelectin), CD31 (PECAM, Monozyten, Neutrophile), CD144 (VE-Cadherin)) und CD209 (dendritische) EVs in verschiedenen klinischen Formen der PH zu untersuchen.