Extracellular vesicles and chronic obstructive pulmonary disease (COPD): a systematic review

Background Chronic Obstructive Pulmonary Disease (COPD) is a common inflammatory disease of the airways characterized by irreversible airflow limitation, ranking the third highest cause of death worldwide. Extracellular vesicles (EVs) are important intercellular communication mediators released by cells into their extracellular environment with the capacity to transfer biological signals. EVs involved in COPD hold great potential to understand disease pathogenesis and identify important biomarkers. This systematic review aims to examine all available research on EVs in the pathogenesis and diagnosis of COPD to identify existing knowledge and support further research within the field. Methods Publications were searched using PubMed and EMBASE with the search terms (Exosomes or extracellular vesicles or microvesicles or microparticles or ectosomes) AND (chronic obstructive pulmonary disease or COPD or emphysema or bronchitis). Results Initial search yielded 512 papers of which 142 were manually selected for review and 43 were eligible for analyses. The studies were divided into groups according to the role of EVs in pathogenesis, EV origin and cargo, their role in COPD exacerbations and their diagnostic utility. EVs were found to be involved in the mechanism of pathogenesis of COPD, derived from various cell types, as well as containing modified levels of miRNAs. EVs also varied according to the pathophysiological status of disease, therefore presenting a possible method for COPD diagnosis and progress monitoring. Conclusion The current findings show the limited but good quality research looking at the role of EVs in COPD, demonstrating the need for more studies to better define and provide further insight into the functional characteristics of EV in COPD pathogenesis. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-022-01984-0.


Introduction
Chronic Obstructive Pulmonary Disease (COPD) is a common inflammatory airway disease, affecting the airways, lung parenchyma and vasculature, and is characterized by irreversible airflow limitation. COPD has been found to affect about 10% of the population above 40 years of age and ranked the third highest cause of death worldwide [1]. It is estimated that 90% of all deaths from COPD can be linked to cigarette smoking [2]. Other risk factors include chronic exposure to biomass smoke, household indoor smoke and outdoor air pollution. Indeed, biomass fuels' smoke has been identified as an independent risk factor leading to the development of COPD, particularly in low-and middle-income countries highly dependent on the use of biomass fuels [3]. COPD comprises of two diseases, emphysema and chronic bronchitis, whereby an abnormal inflammatory response in the lungs occurs after exposure to noxious particles or gases, leading to airway obstruction and emphysematous changes [4]. Symptoms of COPD are commonly chronic cough, excessive mucus production, air trapping, dynamic hyperinflation and shortness of breath upon physical exertion [1,3,5]. Indeed, COPD exhibits symptoms beyond the lung, with systemic manifestations, such as inflammation, and is often associated with other diseases, such as cardiovascular diseases and metabolic syndrome [6]. COPD may manifest frequent periods of exacerbation which is linked to increased airway and systemic inflammation and presents symptoms of breathlessness and sputum production that worsen acutely [7,8]. COPD exacerbation is mainly common in patients with advanced COPD and has also been associated with viral or bacterial infections [9].
Extracellular vesicles (EVs) are important intercellular communication mediators released by cells into the extracellular environment [10]. EVs have the capacity to transfer biological signals between cells and as such, influence recipient cell function [11]. These signals are transmitted by various biomolecules including proteins, lipids, nucleic acids and sugars in phospholipid-enclosed vesicles that provide protection and allow for delivery to distal sites [11]. One of the main contents of EVs are microRNAs (miRNAs), small nucleic acids which play a significant role in the transmission of genetic information and in modulating protein synthesis, therefore affecting cellular functions. EVs can act in both an autocrine and paracrine manner, influencing a range of physiological and pathological functions of recipient cells [10][11][12]. EVs can be derived from most cell types and have been isolated from biological fluids like saliva, urine, nasal and bronchoalveolar lavage (BAL) fluid, amniotic fluid, breast milk, plasma, serum and seminal fluid [11]. They are classified into three groups based on size, biogenesis, and secretory component: (a) exosomes, (b) cellular microvesicles (microparticles/ectosomes), and (c) apoptotic bodies [11]. Exosomes are distinguished from other EV classes by their small size (approximately 50-100 nm), morphology, and their endosomal origin [11]. Exosomes are generated in multivesicular bodies (MVBs) in the form of intraluminal vesicles (ILVs), and once formed, the MVB can fuse with the plasma membrane to release its contents as exosomes [13]. Microvesicles (MVs) are larger in size (> 100 nm) and are produced by outward budding and fission of the cell membrane [11]. Apoptotic bodies (ApoBDs) are the largest of the EVs with a diameter of 1-5 μm and are generated from cells undergoing apoptosis [14]. In addition, both gramnegative and some gram-positive bacteria produce EVs, and as such may also play a role in pathogenesis of COPD as bacterial infections are linked to COPD and Acute Exacerbation of COPD (AECOPD) [9]. Bacterial EVs can also be found in indoor dust [15]. These EVs contain LPS on their surface and additional markers from their bacteria of origin and as such, inhalation of these EVs results in pulmonary inflammation, that when frequent, leads to emphysema [9].
EVs involved in COPD hold great potential to understand disease pathogenesis and identify important biomarkers. Here, a systematic review of all available research on EVs in the pathogenesis and diagnosis of COPD is presented to identify existing knowledge and support further research within the field.

Sources and searches
This review was conducted with the implementation of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Publications were searched using PubMed and EMBASE for results up to 02 February 2022 with the search terms (exosomes or extracellular vesicles or microvesicles or microparticles or ectosomes) AND (chronic obstructive pulmonary disease or COPD or emphysema or bronchitis).

Study selection 1
This systematic review was done to examine evidence on the role and function of EVs in the pathogenesis of COPD, with the hypothesis that EVs from various origins contribute to the pathogenesis of COPD. For this review, publications included were only primary research literature based on in vivo and in vitro human and animal studies that have been peer reviewed. Publications were screened by reviewing the full text of the articles, using predefined inclusion and exclusion criteria to first determine whether to include or exclude them (Table 1).

Study quality assessment
To assess the quality of the publications reporting on EVs and COPD, the studies were screened for components concerning the study of EVs and the diagnosis of COPD in study populations. The evaluation was done on publications pertaining to human studies only, allowing for evaluation on the study population based on size (score: n/3), small (n < 20) or large (n > 20), and number of control groups (score: n/2). Studies were scored according to size (n/3) using the following scale: small study populations = 1, a large study population with a small control group = 2, large study populations for all groups = 3. They were then scored for control groups (n/2), where no control group = 0, one control group = 1, and two or more control groups = 2. Studies were also assessed on the method of diagnosis of COPD (n/2), where diagnosis with spirometry only = 1 and diagnosis with GOLD criteria = 2.
Additionally, EV research is a relatively new and developing field of study, and as such criteria was set for experimental approach and techniques relating to EV studies [16]. Isolation of EVs (n/3) was scored as either 1-less preferable, 2-mid range, or 3-preferred techniques. Characterisation of EVs was also evaluated and ranked out of 4 marks. Together the total was 14 marks for all components added. This allowed for ranking from a score out of 1.0, where 1.0 indicated a high-quality study.

Results of PRISMA statement evidence search and selection
The EMBASE search identified 222 publications and the PubMed search identified 290 papers. Duplicates were removed (n = 144), leaving 368 papers whose titles and abstracts were screened for relevance to research topic. Articles were then screened on the basis of title and abstract to assess whether they reported EVs in COPD including bronchitis and emphysema, excluding 266 articles. The remaining 142 articles were fully reviewed for eligibility, and 43 papers fulfilled the criteria and were included in the present review. A detailed diagram of the review process can be seen below in Fig. 1. The first study was published in 2011 with the number of publications increasing each year and peaking with 11 publications in 2021 ( Fig. 2A). The most common cell origin of the EVs studied were from endothelial cells, although some studies did not specify the origin (Fig. 2B). Additionally, the most common biological fluids from which EVs were studied were plasma, followed by cell lines (Fig. 2C).

Mechanism of EVs in the pathogenesis of COPD Endothelial EVs in COPD
Cigarette smoking (CS) causes stress and damage on the endothelial layer and induces the release of EVs from endothelial cells, therefore it is important to note the relationship of these EVs on the development of lung damage that may lead to COPD. In total 8 original research articles were identified that investigated endothelial EV (EEVs) levels in COPD (Table 2). Overall, the 8 studies [17][18][19][20][21][22][23][24] noted an elevation in CD31+/ CD42b− or CD31+CD62E+ endothelial microparticles (EMPs) in their investigations. Six [18-21, 23, 24] of these studies were human studies, observing EMP levels in COPD patients and of the four studies, three studies compared EMPs in COPD patients with healthy smokers and non-smokers. One study was in vitro [17] and one was an animal study [22].
The increased levels of CD31+/42b− EMPs suggested for all 8 studies that there was active endothelial apoptosis and endothelial damage. However, four studies [18,20,22,23] made additional observations. Strulovici-Barel [18] noted that upon smoking cessation, healthy smokers had a significant decrease in total EMP levels after 12 months, compared to healthy smokers who continued smoking, but that for COPD patients, the EMP levels had no significant change for patients who quit smoking compared to those that continued to smoke. Garcia-Lucio et al. [20] observed a significantly elevated EMP level Articles published in a language other than English for COPD patients and healthy smokers when compared to healthy non-smokers, noting also a reduced number of progenitor cells (PCs) for the COPD patients which would reflect an imbalance between endothelial damage and a reduced repair capacity. Liu et al. [22] observed, in an animal model of COPD, that the elevated levels of CD31+/42b− EMPs increased with an increased time of CS exposure. Nieri et al. [23] observed a direct relationship between EEVs and IL-6, suggesting there is release of EEVs upon an inflammatory stimulus. Six [18-21, 23, 24] of the studies were assessed for quality, given as they were human studies. The articles ranged from a score of 0.57 for the lowest quality to 0.86 for two studies with higher quality. All the studies included for assessment lost marks in the EV isolation method, but scored high for EV characterisation. The study that scored the lowest had a large study population but did not include any control group (Additional file 1: Table S1).

EVs from other cell types in COPD
Other cell types that play a role in the pathogenesis of COPD are also known to release EVs. Eleven studies [23,[25][26][27][28][29][30][31][32][33][34] observed EVs from other cell types involved in COPD ( Table 3). Seven of the studies [25,26,28,[30][31][32][33] investigated EVs from epithelial cells, two studies [27,34] observed EVs from neutrophils, one study [29] examined microparticles from T lymphocytes and one study [30] did not specify the cells types EVs were derived from. Six studies investigating EVs from epithelial cells carried out in vitro studies, and three of the studies carried out further analysis in vivo in mice only [26] or in vivo mice and ex vivo human [28]. The six studies noted that CS triggered and affected the release of EVs from epithelial cells, and of these studies, four [26,28,32,33] made additional observations in terms of the cargo in the EVs. Genschemer et al. [27] observed EVs from neutrophils and carried out in vitro studies using cell lines and in vivo mice and ex vivo human studies. The study concluded that exosomes released from neutrophils carried neutrophil elastase (NE) and were found to bind to the extracellular matrix (ECM), leading to emphysema. Margaroli et al. [34] also studied neutrophil-derived EVs and EV-bound NE in mediating emphysema. Qiu et al. [29] observed that T lymphocytes microparticles (TLMPs) were significantly upregulated in COPD patients compared to healthy volunteers and further noted that CD4+ and CD8+ TLMPs reduced cell viability and induced production of inflammatory cytokines. Nieri et al. [23] observed an increase in monocyte-derived EVs with COPD severity. Zou et al. [30] noted significantly increased levels of IL-1B-containing exosomes in the bronchoalveolar lavage from mice with emphysema, but did not specify the cells the exosomes were derived from.
Three studies were assessed, where two [27,28] studies scored 0.64 and one study scored 0.79. The two studies scored poorly in terms of study population, where both had small group sizes and only one control, and one study [28] did not include how COPD patients were diagnosed with the condition (Additional file 1: Table S2). The third study scored [29] had good population size and EV characterisation techniques.  Circulating endothelial microparticles involved in lung function decline in a rat exposed in cigarette smoke maybe from apoptotic pulmonary capillary endothelial cells Investigate if the number of EMPs is elevated in rats exposed in cigarette smoke, and whether the elevated EMPs are derived from pulmonary capillaries In vivo Mice Exposure of rats to CS resulted in high levels of circulating CD42b/CD31+ EMPs (cEMPs), which increased with an increase in time of exposure.
High levels of CD42b/CD31+ cEMPs reflected the decline of small airway function indirectly in early COPD and would be useful for evaluating the degree of COPD progression

EVs containing microRNA in COPD
EVs carry various biomolecules and of particular interest are nucleic acids including mRNAs, miRNAs and noncoding RNAs and DNA sequences. Interestingly, some EVs have the ability to export miRNA outside cells and affect gene expression in distance cells, thereby inducing phenotypic changes [10,35]. Four studies [36][37][38][39] investigated whether EVs containing miRNA could influence manifestations of COPD (Table 4). These studies all conducted in vitro studies, with three of the studies [36,37,39] carrying out additional human and animal studies. All four studies observed that CS caused changes in the miRNA levels in EVs. Two studies [36,37] observed that CS modified the levels of miR-21 carried by EVs. He et al. [36] noted that CS reduced levels of miR-21 of EVs derived from BEAS-2B cells but that EVs obtained from the serum of COPD patients carried significantly higher levels of miR-21. Xu et al. [37] concluded that CS exposure increased miR-21 levels in exosomes from human bronchial epithelial cells (HBECs). Two studies [38,39] observed that CS modified the miRNA components in EVs. Fujita et al. [38] noted that EVs from HBECs treated with CS had distinct and varying levels of 8 miRNAs, which had either increased levels or decreased levels when compared to the non-treated group. Together, the studies suggest that the CS-induced changes in miRNA cargo of EVs result in myofibroblast differentiation [37,38], inference of efferocytosis [39] or polarization of macrophages to M2 [36], which are all characteristic of the pathogenesis in COPD.
Three [36,37,39] of the four studies were assessed for quality and scores ranged from 0.64 to 0.93 for the highest quality paper (Additional file 1: Table S3). The study with the highest score had population groups above n = 20 and two additional control groups (non-smokers without COPD and smokers without COPD), and diagnosed COPD patients according to GOLD criteria. Additionally, EV isolation methods included centrifugation plus an exosome precipitation solution kit. Finally, characterisation of EVs for this study was done via nanoparticle tracking analysis, TEM and western blotting.

Bacterial EVs and COPD
EVs are known to also be released from all gram-negative and some gram-positive bacteria, which carry a wide variety of molecules such as proteins, lipids, DNA, and RNA, but additionally harbour various virulence factors, such as LPS, and as a result, may have several physiological and pathological functions in bacteria-host interactions [9,40]. Additionally, studies have demonstrated the presence of bacteria-derived EVs in indoor dust that has been collected from beds in apartments [15].
Four studies [41][42][43][44] investigated whether bacteriaderived EVs were related to COPD (Table 5). Three research articles [42][43][44] identified carried out ex vivo human studies and one study [41] was done in vivo using a mice model and additionally in vitro assays. Kim et al. [41] determined that EVs derived from bacteria resulted in emphysema due to neutrophilic inflammation. Kim et al. [42] showed that bacterial EVs differed in the lungs of COPD patients when compared to non-smokers and healthy smokers. The third study [43] observed that IgG levels against bacterial EVs collected from dust were highest in COPD patients than in control subject. Yang et al. [44] followed from this study to determine the specific bacterial EVs affecting COPD in indoor dust, such as S. aureus, A. baumannii, E. cloacae and P. aeruginosa. As a result, all together the studies show that bacteriaderived EVs whether found in lungs or inhaled from the environment were distinct in COPD patients than in control groups and could result in pathogenesis of COPD.
Three [42][43][44] of the studies were assessed and scores ranged from 0.50 to 0.64. Breakdown of their assessment can be noted in Additional file 1: Table S4. The studies missed marks in all categories of assessment, leading to a lower score.

EVs and exacerbation of COPD
COPD is often marked by periods of exacerbation, whereby there is increased airway and systemic inflammation, and symptoms such as breathlessness and sputum production worsen and could lead to hospitalization [8]. Four studies [45][46][47][48] investigated whether EVs were involved in occurrences of COPD exacerbation (Table 6). Three studies [45,47,48] compared EV levels in stable and exacerbated COPD patients. All three studies noted elevated levels of EVs in patients with exacerbated COPD, and each carried out additional observations in their studies. Tan et al. [47] primarily focused on levels of exosomes (CD9 exosomes) and noted the highest levels in patients with acute exacerbation of COPD compared to stable COPD patients and healthy controls. The other two studies [45,48] observed significantly elevated levels of CD62E+ (E-selectin) microparticles (MPs) in exacerbated COPD patients, with one [48] of the studies additionally observing CD41+, CD42a+ and CD14+ MPs levels in their study.
The fourth study [46] identified carried out in vitro, in vivo mice and ex vivo human studies, focusing on the cargo of EVs that may result in COPD exacerbation. This study observed that EVs released during respiratory infections carried and released IL-1b and IL-18, suggesting a mechanism that results in disease exacerbation.
Three [45,47,48] of the studies were assessed for quality and scores ranged from 0.79 to 0.86 (Additional file 1:

EVs in COPD diagnosis
According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), the current diagnosis of COPD is based on three features including spirometry, observation of symptoms and a significant exposure to toxic stimuli [49]. Twelve studies [50][51][52][53][54][55][56][57][58][59][60][61] investigated the use of EVs as a more effective and accurate diagnostic tool for COPD ( Table 7). All twelve studies involved human studies, with one study [53] carrying out additional investigation in vitro. Seven studies observed EV levels and five studies observed the cargo in the EVs. Six studies [50,52,54,56,58] noted elevated levels of EVs in COPD patients, observing specific microparticles. One study [50] observed significantly elevated levels of CD31+ MPs in the sputum of COPD patients that negatively correlated with forced expiratory volume in 1 s (FEV1). Another study [52] showed that elevated levels of CD62E+ MPs in plasma of COPD patients had significant negative correlations with FEV1 changes. The third study [54] identified a series of plasma EVs (CD45, CD28, CTLA4, TNF-R-II, and CD16) highly expressed in patients with COPD. Another study [51] noted elevated levels of EMPs with apoptotic characteristics in smokers with reduced DL CO , indicative of early lung destruction. Soni et al. [56] noted that BALF neutrophil MVs significantly correlated with clinically relevant disease severity indexes. Bazzan et al. [58] observed increased levels of EVs from alveolar macrophages in BAL of smokers with COPD compared to smokers without COPD and nonsmokers, which correlated with the pack-years and disease severity according to FEV1. Luccheti et al. [57] observed that EVs are detectable in exhaled breathe condensate (EBC) and sputum of COPD patients but did not identify the cell source of these EVs. Of the studies that observed EV cargo in COPD patients, four studies observed miRNA profiles in EVs and one study observed proteins. Sundar et al. [53] and Kaur et al. [61] observed distinct miRNA profiles in EVs of COPD patients when compared to smokers and/ or non-smokers. Carpi et al. [59] noted that miR-206, miR-133a-5p and miR-133a-3p levels can discriminate between COPD patients. Shen et al. [60] observed that expression levels of three exosomal miRNAs were negatively associated with FEV1. Koba et al. [55] study observed that expression of fibulin-3 correlated with lung function and emphysema. Together these studies indicate that EVs can lead to the development of more accurate biomarkers to diagnose COPD and monitor disease progression.
All twelve studies were assessed with scores ranging from 0.50 to 0.86 (Additional file 1: Table S6). Two studies scored 0.86 due to sample population and their techniques for EV isolation and characterisation. Other studies scored low in the areas of study populations, for either size or number of control groups or both, and in their EV isolation method. The least scoring study also received a lower score for the characterisation of EVs, due to no tracking to determine particle size or no visual characterisation of EVs.

Discussion
Investigating the role of EVs in COPD holds great potential to understand disease pathogenesis and identify biomarkers. The aim of this systematic review was to interpret and present all available research on EVs in the pathogenesis and diagnosis of COPD to identify existing knowledge and support further research within the field.
The studies identified for this review consistently reported significantly elevated levels of EVs in patients with COPD when compared to their control groups, being either healthy smokers or non-smokers. Many of the studies identified the EVs to be from endothelial cells, suggesting damage in the endothelial layer in patients with COPD. Studies have described an association between endothelial dysfunction and COPD and an increase in levels of apoptotic endothelial cells in the lungs of patients with COPD, with further studies demonstrating in animal models the resulting development of emphysema [62,63]. In addition, endothelial dysfunction has been identified as a possible key mechanism in airflow obstruction and is associated with increased risk of mortality in COPD patients [64]. The studies identified for this review support the hypothesis that endothelial apoptosis is involved in the early developments of emphysema and that endothelial cells increase the release of EVs upon exposure to CS.
Other cell types also release increased levels of EVs during exposure to cigarette smoke. Elevated levels of EVs originated from lung epithelial cells, neutrophils and T lymphocytes, all cells which have previously been identified in the response to exposure of CS and thus play a role in the pathogenesis of COPD [65]. Of particular interest in these studies was the cargo of the EVs that triggered inflammation and degradation of the extracellular matrix (ECM). Epithelial cells were found to release EVs containing proteins and pro-inflammatory cytokines that drive local and systemic inflammation and that resulted in further recruitment of inflammatory cells, particularly neutrophils. Indeed, neutrophilia is a key feature of COPD where neutrophils have previously been  in several independent cohorts to identify potential biomarkers to determine the extent of any pulmonary damage at an early stage Ex vivo Human Next generation sequencing results identified three differentially expressed miRNAs in the BALF and one in the lung-derived exosomes from COPD patients, compared to healthy non-smokers. Of these, miR-122-5p was three-or fivefold downregulated among the lung-tissue-derived exosomes of COPD patients compared to healthy non-smokers and smokers, respectively. The identified lung-specific miRNAs associated with COPD can serve as potential biomarkers or therapeutic targets observed to secrete proteases that cause destruction of lung tissue and release mediators that further promote inflammation [66]. The studies included described that neutrophils released EVs containing neutrophil elastase, a serine protease known to degrade the ECM, contributing to tissue destruction and emphysema in COPD [67]. T lymphocytes released EVs that reduced cell viability and induced significant production of inflammatory cytokines IL-6, MCP-1, MCP-9 and TNF-a in human bronchial epithelial cells (HBEs), with decreased levels of anti-inflammatory cytokine IL-10 [29]. IL-6 and TNF-a both have a role in the acute phase response of COPD, and TNF-a particularly is significantly associated with disease progression [68]. Together the studies showed that upon CS exposure, epithelial cells, T lymphocytes and neutrophils release increased levels of EVs containing biomolecules that further enhance the inflammatory response and degrade the ECM, causing lung tissue damage. This mechanism in turn drives the development of emphysema. Furthermore, other cells are important in COPD pathogenesis, however, to date there are limited studies on EVs in COPD and current literature only examines EVs produced by endothelial cells, epithelial cells, T lymphocytes, neutrophils and monocytes. Furthermore, some studies identified investigated the role of EVs containing microRNAs (miRNAs, mi-R) in the mechanism of COPD. miRNAs are small non-coding RNAs essential to key biological functions with the capacity to regulate tens to hundreds of genes simultaneously [69]. As a result, identifying expressions of miRNAs in COPD will enable better understanding of the mechanism of COPD. The studies reported EVs containing significantly increased levels of miRNAs from cells after exposure to CS and in particular, noted this difference in levels for COPD patients when compared to healthy people. One particular miRNA identified in the studies included is miR-21, a microRNA expressed at increased levels in patients with COPD, and when upregulated, drives excessive autophagy in COPD [70]. In addition, the studies noted that upon uptake of the EVs carrying miR-21, macrophages polarised into M2 phenotype and bronchial fibroblast cells differentiated into myofibroblasts. An enhanced polarisation level of M2 phenotype macrophages has been observed in the lungs of smokers, with even higher levels in COPD patients, linking these cells to the pathogenesis of COPD [71]. EVs from cells exposed to CS were also found to carry increased levels of miR-210 among other microRNAs compared to non-exposed cells. These miRNAs caused increased collagen type I, myofibroblast differentiation, and reduced clearance of dead cells. Further to this, myofibroblasts differentiation results in the reduced lung function observed in COPD patients [72]. As a result, investigation into biomolecules or cargo of EVs is essential as these may affect the recipient cells which in turn play a role in the development of COPD.
Bacteria are known to colonise the lower airways in COPD patients and these pathogens also release EVs [9,40,73]. The studies included note that bacterial derived EVs in COPD patients had distinct characteristics when compared to healthy smokers and non-smokers. Furthermore, the exposure of bacterial EVs resulted in neutrophilia and increased inflammation, features that lead to the development of emphysema [66]. COPD patients were also found to have higher anti-dust EV IgG titres in serum when compared to control subjects. Dust EVs typically originate from microorganism and as such, may induce neutrophilic pulmonary inflammation and subsequent emphysema as seen in previous animal experiments [43]. Overall, the studies show that bacterial EVs have a key role in the development of COPD, yet not enough research has been done to date. In addition, the assessment scores of these studies were low, highlighting the need for more thorough research in this area.
Patients with COPD suffer from frequent acute exacerbations that cause significant morbidity and mortality, and thus are necessary to prevent [74]. Currently, the exacerbation of COPD is a contentious area to define, despite the fact that these periods present a significant burden on COPD patients. A broad definition of COPD exacerbation is the worsening of the patient's conditions, although this may be faulty due to no established clinical markers, signs or symptoms that can identify an exacerbation of the condition [75]. COPD exacerbations is defined clinically as periods of increasing respiratory symptoms including cough, increased sputum volume and purulence, wheezing, increased dyspnoea and/or systemic distress, and where there is a need for antibiotics [74]. The studies here observe elevated EV levels in patients with exacerbated COPD when compared to those with stable COPD. In addition, respiratory infection, whether viral or bacterial, may be responsible for periods of exacerbations, as they drive an increased release of functional EVs which contain pro-inflammatory cytokines IL-1B and IL-18 and result in exacerbated neutrophilia. The immune response to the EVs released by these infectious agents can result in increased inflammation and therefore, cause an exacerbation of COPD [65]. Also, CD62E+, CD31+ and other MPs linked to endothelial cells, monocytes and platelets were significantly elevated in AECOPD patients, indicative of endothelial damage and vascular injury in the lungs during exacerbation periods that lead to increased severity of disease. These observations are based on high quality research papers that clearly defined exacerbation of COPD as episodes of worsening symptoms and airway function beyond normal daily variation that required treatment with antibiotics and/or corticosteroids.
Current diagnosis of COPD in clinical practice depends largely on the presence of chronic airflow limitation, normally assessed by post-bronchodilator spirometry [76]. Studies have shown that EVs may present a possible method for COPD diagnosis and progress monitoring, in addition to enabling identification of exacerbation status. Levels of MPs were found to be elevated in patients with COPD which correlate with a rapid FEV1 decline. EMPs with apoptotic characteristics were also found in increased levels, indicating lung damage and endothelial apoptosis. As a result, EMP levels can be used to measure early lung destruction in healthy smokers with normal FEV1 and also allow for COPD diagnosis and monitoring of disease course. Additionally, specific MV subtype populations have been shown to correlate with Body-mass, airflow Obstruction, Dyspnea and Exercise (BODE) index as well as other markers of COPD severity [56]. Furthermore, levels of miRNAs in EVs were significantly different in COPD patients when compared to smokers and to nonsmokers, demonstrating that smoking and progression of COPD alter the miRNA levels in circulating EVs, potentially allowing for EV analysis as an added tool for disease biomarkers. Indeed, specific miRNAs were identified as possible biomarkers for diagnosis COPD and discriminating between COPD patients. As a result, the studies show that EVs would serve as a biomarker that would allow for identification of lung damage and diagnosis and monitoring of COPD. These were poor to good quality research studies, highlighting the need to further investigate EVs as a possible biomarker for COPD diagnosis.
Research in this area is limited, therefore primary studies included in the review were heterogeneous (in vivo, ex vivo, in vitro) and were limited to 43 studies with small samples sizes. Additionally, COPD comprises of emphysema and bronchitis, however, current literature did not observe EVs in bronchitis. Furthermore, methods to characterise and measure EVs are heterogeneous and until recently, guidelines for measuring and characterising EVs were not considered. Future research studies should follow the Minimal Information for Studies of Extracellular Vesicle 2018 (MISEV 2018) guidelines. Importantly, this paper highlights the importance of studying EVs in COPD pathogenesis. This review shows consistent reporting of significantly elevated levels of EVs in patients with COPD and AECOPD and that cargo of the EVs from cigarette smoking trigger mechanisms, such as inflammation, that drive pathogenesis of COPD. The studies here also highlighted EVs as possible biomarkers of lung damage for COPD diagnosis and monitoring of disease course.
In conclusion, the studies together show the limited but good quality research examining the role of EVs in COPD. Therefore, more studies are needed to help better define and provide further insight into the functional characteristics of EV in COPD pathogenesis.