Asthma

Asthma is a complex respiratory disease characterized by chronic inflammatory responses that contribute to airflow obstruction and airway hyperresponsiveness, and remodelling (Ref. Reference Holgate, Wenzel, Postma, Weiss, Renz and Sly47). Over 260 million people worldwide are affected by asthma with many individuals remaining undiagnosed and untreated (Ref. Reference Momtazmanesh, Moghaddam, Ghamari, Rad, Rezaei, Shobeiri, Aali, Abbasi-Kangevari, Abbasi-Kangevari, Abdelmasseh, Abdoun, Abdulah, Md Abdullah, Abedi, Abolhassani, Abrehdari-Tafreshi, Achappa, Adane Adane, Adane, Addo, Adnan, Sakilah Adnani, Ahmad, Ahmadi, Ahmadi, Ahmed, Ahmed, Rashid, al Hamad, Alahdab, Alemayehu, Alif, Aljunid, Almustanyir, Altirkawi, Alvis-Guzman, Dehkordi, Amir-Behghadami, Ancuceanu, Andrei, Andrei, Antony, Anyasodor, Arabloo, Arulappan, Ashraf, Athari, Attia, Ayele, Azadnajafabad, Babu, Bagherieh, Baltatu, Banach, Bardhan, Barone-Adesi, Barrow, Basu, Bayileyegn, Bensenor, Bhardwaj, Bhardwaj, Bhat, Bhattacharyya, Bouaoud, Braithwaite, Brauer, Butt, Butt, Calina, Cámera, Chanie, Charalampous, Chattu, Chimed-Ochir, Chu, Cohen, Cruz-Martins, Dadras, Darwesh, das, Debela, Delgado-Ortiz, Dereje, Dianatinasab, Diao, Diaz, Digesa, Dirirsa, Doku, Dongarwar, Douiri, Dsouza, Eini, Ekholuenetale, Ekundayo, Mustafa Elagali, Elhadi, Enyew, Erkhembayar, Etaee, Fagbamigbe, Faro, Fatehizadeh, Fekadu, Filip, Fischer, Foroutan, Franklin, Gaal, Gaihre, Gaipov, Gebrehiwot, Gerema, Getachew, Getachew, Ghafourifard, Ghanbari, Ghashghaee, Gholami, Gil, Golechha, Goleij, Golinelli, Guadie, Gupta, Gupta, Gupta, Gupta, Hadei, Halwani, Hanif, Hargono, Harorani, Hartono, Hasani, Hashi, Hay, Heidari, Hellemons, Herteliu, Holla, Horita, Hoseini, Hosseinzadeh, Huang, Hussain, Hwang, Iavicoli, Ibitoye, Ibrahim, Ilesanmi, Ilic, Ilic, Immurana, Ismail, Merin J, Jakovljevic, Jamshidi, Janodia, Javaheri, Jayapal, Jayaram, Jha, Johnson, Joo, Joseph, Jozwiak, K, Kaambwa, Kabir, Kalankesh, Kalhor, Kandel, Karanth, Karaye, Kassa, Kassie, Keikavoosi-Arani, Keykhaei, Khajuria, Khan, Khan, Khan, Khreis, Kim, Kisa, Kisa, Knibbs, Kolkhir, Komaki, Kompani, Koohestani, Koolivand, Korzh, Koyanagi, Krishan, Krohn, Kumar, Kumar, Kurmi, Kuttikkattu, la Vecchia, Lám, Lan, Lasrado, Latief, Lauriola, Lee, Lee, Legesse, Lenzi, Li, Lin, Liu, Liu, Lo, Lorenzovici, Lu, Mahalingam, Mahmoudi, Mahotra, Malekpour, Malik, Mallhi, Malta, Mansouri, Mathews, Maulud, Mechili, Nasab, Menezes, Mengistu, Mentis, Meshkat, Mestrovic, Micheletti Gomide Nogueira de Sá, Mirrakhimov, Misganaw, Mithra, Moghadasi, Mohammadi, Mohammadi, Mohammadshahi, Mohammed, Mohan, Moka, Monasta, Moni, Moniruzzaman, Montazeri, Moradi, Mostafavi, Mpundu-Kaambwa, Murillo-Zamora, Murray, Nair, Nangia, Swamy, Narayana, Natto, Nayak, Negash, Nena, Kandel, Niazi, Nogueira de Sá, Nowroozi, Nzoputam, Nzoputam, Oancea, Obaidur, Odukoya, Okati-Aliabad, Okekunle, Okonji, Olagunju, Bali, Ostojic, A, Padron-Monedero, Padubidri, Pahlevan Fallahy, Palicz, Pana, Park, Patel, Paudel, Paudel, Pedersini, Pereira, Pereira, Petcu, Pirestani, Postma, Prashant, Rabiee, Radfar, Rafiei, Rahim, Ur Rahman, Rahman, Rahman, Rahmani, Rahmani, Rahmanian, Rajput, Rana, Rao, Rao, Rashedi, Rashidi, Ratan, Rawaf, Rawaf, Rawal, Rawassizadeh, Razeghinia, Mohamed Redwan, Rezaei, Rezaei, Rezaei, Rezaeian, Rodrigues, Buendia Rodriguez, Roever, Rojas-Rueda, Rudd, Saad, Sabour, Saddik, Sadeghi, Sadeghi, Saeed, Sahebazzamani, Sahebkar, Sahoo, Sajid, Sakhamuri, Salehi, Samy, Santric-Milicevic, Sao Jose, Sathian, Satpathy, Saya, Senthilkumaran, Seylani, Shahabi, Shaikh, Shanawaz, Shannawaz, Sheikhi, Shekhar, Sibhat, Simpson, Singh, Singh, Singh, Skryabin, Skryabina, Soltani-Zangbar, Song, Soyiri, Steiropoulos, Stockfelt, Sun, Takahashi, Talaat, Tan, Tat, Tat, Taye, Thangaraju, Thapar, Thienemann, Tiyuri, Ngoc Tran, Tripathy, Car, Tusa, Ullah, Ullah, Vacante, Valdez, Valizadeh, van Boven, Vasankari, Vaziri, Violante, Vo, Wang, Wei, Westerman, Wickramasinghe, Xu, Xu, Yadav, Yismaw, Yon, Yonemoto, Yu, Yu, Yunusa, Zahir, Zangiabadian, Zareshahrabadi, Zarrintan, Zastrozhin, Zegeye, Zhang, Naghavi, Larijani and Farzadfar1). Asthma has the highest prevalence among chronic respiratory diseases with variable incidence across different regions depending on several factors, including socioeconomic status and genetic predisposition (Ref. Reference Song, Adeloye, Salim, dos Santos, Campbell, Sheikh and Rudan48). EVs can be released by epithelial cells on both apical and basolateral sides (Refs. Reference Bartel, La Grutta and Cilluffo49, Reference Mwase, Phung, O’Sullivan, Mitchel, de Marzio, Kılıç, Weiss, Fredberg and Park50), and these contribute to the pathogenesis and progression of asthma by influencing pro-inflammatory responses (Refs. Reference Abreu, Lopes-Pacheco, Weiss and Rocco13, Reference Kanannejad, Arab, Soleimanian, Mazare and Kheshtchin51). For instance, house dust mite (HDM) extract significantly altered the content of BALF-derived EVs in experimental asthma (Ref. Reference Gon, Maruoka, Inoue, Kuroda, Yamagishi, Kozu, Shikano, Soda, Lötvall and Hashimoto52), corroborating findings of EVs from asthmatic patients (Ref. Reference Francisco-Garcia, Garrido-Martín, Rupani, Lau, Martinez-Nunez, Howarth and Sanchez-Elsner53). BALF- and nasal lavage fluid-derived EVs from asthmatic patients exhibited increased concentrations of interleukin (IL)-4, leukotriene C4, and other chemoattractant factors (Refs. Reference Torregrosa Paredes, Esser, Admyre, Nord, Rahman, Lukic, Rådmark, Grönneberg, Grunewald, Eklund, Scheynius and Gabrielsson54, Reference Lässer, O’Neil, Shelke, Sihlbom, Hansson, Gho, Lundbäck and Lötvall55). Likewise, an increase in EV release was observed in human bronchial epithelial cells treated with Th2 (IL-4 and IL-13) and Th17 cytokines (IL-17A and tumour necrosis factor (TNF)-α) (Ref. Reference Ax, Jevnikar, Cvjetkovic, Malmhäll, Olsson, Rådinger and Lässer56). Eosinophil-derived EVs from asthmatic patients were able to delay wound repair, promote in vitro apoptosis of small airway epithelial cells, and stimulate proliferation of smooth muscle cells (Refs. Reference Cañas, Sastre, Mazzeo, Fernández-Nieto, Rodrigo-Muñoz, González-Guerra, Izquierdo, Barranco, Quirce, Sastre and del Pozo57, Reference Cañas, Sastre, Rodrigo-Muñoz, Fernández-Nieto, Barranco, Quirce, Sastre and del Pozo58). Treatment with EV-production inhibitor GW4869 ameliorated asthma symptoms in an animal model by reducing the release of lung cell-derived EVs (Ref. Reference Gon, Maruoka, Inoue, Kuroda, Yamagishi, Kozu, Shikano, Soda, Lötvall and Hashimoto52). Such reduction was also accompanied by a reduction in the number of inflammatory cells, bronchial hyperresponsiveness, and IgE levels (Ref. Reference Kulshreshtha, Ahmad, Agrawal and Ghosh59). In BALF samples, positive correlations were established for EV counts versus eosinophilia and EV counts versus IgE titer (Ref. Reference Hough, Wilson, Trevor, Strenkowski, Maina, Kim, Spell, Wang, Chanda, Dager, Sharma, Curtiss, Antony, Dransfield, Chaplin, Steele, Barnes, Duncan, Prasain, Thannickal and Deshane60). Profiling studies have been performed to further understand EV involvement in asthma-mediated inflammatory responses. In BALF-derived EVs, proteomic and lipidomic analyses revealed increased levels of toll-like receptor (TLR) signalling-related proteins (Ref. Reference Rollet-Cohen, Bourderioux, Lipecka, Chhuon, Jung, Mesbahi, Nguyen-Khoa, Guérin-Pfyffer, Schmitt, Edelman, Sermet-Gaudelus and Guerrera43) and altered lipid mediator composition of EVs from asthmatic patients compared to healthy controls (Ref. Reference Hough, Wilson, Trevor, Strenkowski, Maina, Kim, Spell, Wang, Chanda, Dager, Sharma, Curtiss, Antony, Dransfield, Chaplin, Steele, Barnes, Duncan, Prasain, Thannickal and Deshane60).

Several studies have investigated EV-transported miRNA for potential biomarkers of asthma. Along these lines, EV miRNA content was significantly distinct in samples from asthmatic patients compared to healthy controls (Refs. Reference Duarte, Taveira-Gomes, Sokhatska, Palmares, Costa, Negrão, Guimarães, Delgado, Soares and Moreira42, Reference Levänen, Bhakta, Torregrosa Paredes, Barbeau, Hiltbrunner, Pollack, Sköld, Svartengren, Grunewald, Gabrielsson, Eklund, Larsson, Woodruff, Erle and Wheelock61). In mice, HDM exposure altered the expression of >100 miRNAs in EV content, including miR-346 and miR-574-5p (Ref. Reference Gon, Maruoka, Inoue, Kuroda, Yamagishi, Kozu, Shikano, Soda, Lötvall and Hashimoto52). Most BALF-derived EVs are released from epithelial cells in experimental asthma; however, a significant increase in EV number from immune cells was evidenced following allergen exposure. Increased levels of miR-142a and miR-223 were found in immune cell-derived EVs from this animal model (Ref. Reference Pua, Happ, Gray, Mar, Chiou, Hesse and Ansel62). In plasma-derived EVs, miR-21 and miR-223 levels were increased in patients with moderate asthma compared to healthy controls (Refs. Reference Rostami Hir, Alizadeh, Mazinani, Mahlooji Rad, Fazlollahi, Kazemnejad, Hosseini and Moin63, Reference Soccio, Moriondo, Lacedonia, Tondo, Pescatore, Quarato, Carone, Foschino Barbaro and Scioscia64). A promising miRNA to identify mildly asymptomatic asthma is let-7, which was demonstrated to be downregulated in BALF-derived EVs from patients with asthma (Ref. Reference Levänen, Bhakta, Torregrosa Paredes, Barbeau, Hiltbrunner, Pollack, Sköld, Svartengren, Grunewald, Gabrielsson, Eklund, Larsson, Woodruff, Erle and Wheelock61). On the contrary, PM2.5 led to asthma exacerbation by increasing levels of EV-packaged let-7i-5p in human bronchial epithelial cells (Ref. Reference Zheng, Du and Tian65). PM2.5 also increased levels of EV-packaged miR-129-2-30 that led to increased secretion of inflammatory mediators (IL-6, IL-8, and TNF-α) by epithelial cells (Ref. Reference Zheng, Du and Tian65). Levels of miR-126 were elevated in serum-derived EVs from patients with asthma and lung tissue of asthmatic mice (Ref. Reference Zhao, Li, Geng, Zhao, Ma, Yang, Deng, Luo and Pan66). Blood eosinophil counts positively correlated with miR-122-5a expression in plasma- and sputum-derived EVs with the potential to differentiate endotypes of asthma (Ref. Reference Bahmer, Krauss-Etschmann, Buschmann, Behrends, Watz, Kirsten, Pedersen, Waschki, Fuchs, Pfaffl, von Mutius, Rabe, Hansen, Kopp, König and Bartel67). Compared to healthy controls, EVs from asthmatic epithelial cells demonstrated increased levels of miR-9, which has been associated with steroid-resistant neutrophilic asthma (Ref. Reference Li, Tay, Maltby, Xiang, Eyers, Hatchwell, Zhou, Toop, Morris, Nair, Mattes, Foster and Yang68).

Bronchopulmonary dysplasia

BPD is a chronic lung disorder characterized by inflammation and disruption in airways and lung parenchymal vasculature, primarily affecting preterm infants or neonates requiring oxygen therapy. Although progress has been made in improving the survival of preterm infants, BPD remains a major cause of morbidity among survivors (Ref. Reference Stoll, Hansen, Bell, Walsh, Carlo, Shankaran, Laptook, Sánchez, van Meurs, Wyckoff, das, Hale, Ball, Newman, Schibler, Poindexter, Kennedy, Cotten, Watterberg, D’Angio, DeMauro, Truog, Devaskar and Higgins69). EVs have been isolated from various biofluids to investigate their role in BPD; however, studies have demonstrated that tracheal aspirate fluid is a feasible source for EV isolation and may represent the lung development stage in neonates or preterm infants (Refs. Reference Lal, Olave, Travers, Rezonzew, Dolma, Simpson, Halloran, Aghai, das, Sharma, Xu, Genschmer, Russell, Szul, Yi, Blalock, Gaggar, Bhandari and Ambalavanan70, Reference Ransom, Bunn, Negretti, Jetter, Bressman, Sucre and Pua71). Indeed, gestational age was demonstrated to be directly and negatively correlated to EV concentration and size, respectively (Refs. Reference Ransom, Bunn, Negretti, Jetter, Bressman, Sucre and Pua71, Reference Barnes, Pantazi and Holder72). Likewise, experimental hyperoxia-induced bronchial epithelial cells release more EVs of smaller size compared to cells under normoxia (Ref. Reference Lal, Olave, Travers, Rezonzew, Dolma, Simpson, Halloran, Aghai, das, Sharma, Xu, Genschmer, Russell, Szul, Yi, Blalock, Gaggar, Bhandari and Ambalavanan70). Umbilical cord-derived EVs from neonates who developed BPD reduced cell proliferation and capillary tube formation in cultured endothelial cells (Ref. Reference Zhong, Yan, Chen, Jia, Li, Liang, Gu, Wei, Lian, Zheng and Cui73). Differences in EV-surface markers have also been documented in neonates with BPD compared with those without it (Refs. Reference Lal, Olave, Travers, Rezonzew, Dolma, Simpson, Halloran, Aghai, das, Sharma, Xu, Genschmer, Russell, Szul, Yi, Blalock, Gaggar, Bhandari and Ambalavanan70, Reference Ransom, Bunn, Negretti, Jetter, Bressman, Sucre and Pua71). Tracheal aspirate-derived EVs exhibited increased levels of CD14 and CD24 (usually expressed by immune and epithelial cells, respectively) when obtained from infants with BPD (Ref. Reference Ransom, Bunn, Negretti, Jetter, Bressman, Sucre and Pua71). In experimental hyperoxia-induced BPD, rats demonstrated an increase in EV-packaged surfactant protein C in plasma. When these EVs were administered in control animals, lung and brain damage occurred, as well as morphological alterations consistent with BPD (Ref. Reference Ali, Zambrano, Duncan, Chen, Luo, Yuan, Chen, Benny, Schmidt, Young, Kerr, de Rivero Vaccari, Keane, Dietrich and Wu74).

Analysis of EV miRNAs for BPD has gained traction in recent years. Over 400 EV-derived miRNAs were differentially expressed in the umbilical cord of neonates with BPD compared to those without it (Ref. Reference Zhong, Yan, Chen, Jia, Li, Liang, Gu, Wei, Lian, Zheng and Cui73). Among these, the most significant reduction was observed for miRNA-103a-3p and miRNA-185-5p, while miRNA-200a-3p had increased expression (Ref. Reference Zhong, Yan, Chen, Jia, Li, Liang, Gu, Wei, Lian, Zheng and Cui73). An increase in endothelial cell proliferation and migration, and tube formation was observed by miR-103a-3p and miR-185-5p overexpression. On the contrary, miR-200a-3p overexpression prevented cellular angiogenic responses (Ref. Reference Zhong, Yan, Chen, Jia, Li, Liang, Gu, Wei, Lian, Zheng and Cui73). In another study, miRNA profiling revealed 40 miRNAs differentially expressed in EVs from infants with severe BPD compared to age-matched neonates without BPD (Ref. Reference Lal, Olave, Travers, Rezonzew, Dolma, Simpson, Halloran, Aghai, das, Sharma, Xu, Genschmer, Russell, Szul, Yi, Blalock, Gaggar, Bhandari and Ambalavanan70). The most sensitive alteration to predict BPD severity was the reduction of miR-876-3p. When exogenous miR-876-3p was administered in experimental hyperoxia-induced BPD, animals exhibited reduced inflammation and improved alveologenesis (Ref. Reference Lal, Olave, Travers, Rezonzew, Dolma, Simpson, Halloran, Aghai, das, Sharma, Xu, Genschmer, Russell, Szul, Yi, Blalock, Gaggar, Bhandari and Ambalavanan70). EV miR-21 has been implicated in adults with lung diseases and was upregulated in serum-derived EVs of preterm infants (≤32 weeks gestation) with versus without lung disorder (Ref. Reference Go, Maeda, Miyazaki, Maeda, Kume, Namba, Momoi, Hashimoto, Otsuru, Kawasaki, Hosoya and Dennery75).

Chronic obstructive pulmonary disease

COPD is a heterogeneous group of respiratory diseases characterized by progressive and partially irreversible restriction of airflow due to small airway damage (chronic bronchitis) or extensive alveolar wall disruption with airspace enlargement (emphysema) (Ref. Reference Antunes, Lopes-Pacheco and Rocco76). COPD has the highest mortality rate among chronic respiratory diseases (Ref. Reference Momtazmanesh, Moghaddam, Ghamari, Rad, Rezaei, Shobeiri, Aali, Abbasi-Kangevari, Abbasi-Kangevari, Abdelmasseh, Abdoun, Abdulah, Md Abdullah, Abedi, Abolhassani, Abrehdari-Tafreshi, Achappa, Adane Adane, Adane, Addo, Adnan, Sakilah Adnani, Ahmad, Ahmadi, Ahmadi, Ahmed, Ahmed, Rashid, al Hamad, Alahdab, Alemayehu, Alif, Aljunid, Almustanyir, Altirkawi, Alvis-Guzman, Dehkordi, Amir-Behghadami, Ancuceanu, Andrei, Andrei, Antony, Anyasodor, Arabloo, Arulappan, Ashraf, Athari, Attia, Ayele, Azadnajafabad, Babu, Bagherieh, Baltatu, Banach, Bardhan, Barone-Adesi, Barrow, Basu, Bayileyegn, Bensenor, Bhardwaj, Bhardwaj, Bhat, Bhattacharyya, Bouaoud, Braithwaite, Brauer, Butt, Butt, Calina, Cámera, Chanie, Charalampous, Chattu, Chimed-Ochir, Chu, Cohen, Cruz-Martins, Dadras, Darwesh, das, Debela, Delgado-Ortiz, Dereje, Dianatinasab, Diao, Diaz, Digesa, Dirirsa, Doku, Dongarwar, Douiri, Dsouza, Eini, Ekholuenetale, Ekundayo, Mustafa Elagali, Elhadi, Enyew, Erkhembayar, Etaee, Fagbamigbe, Faro, Fatehizadeh, Fekadu, Filip, Fischer, Foroutan, Franklin, Gaal, Gaihre, Gaipov, Gebrehiwot, Gerema, Getachew, Getachew, Ghafourifard, Ghanbari, Ghashghaee, Gholami, Gil, Golechha, Goleij, Golinelli, Guadie, Gupta, Gupta, Gupta, Gupta, Hadei, Halwani, Hanif, Hargono, Harorani, Hartono, Hasani, Hashi, Hay, Heidari, Hellemons, Herteliu, Holla, Horita, Hoseini, Hosseinzadeh, Huang, Hussain, Hwang, Iavicoli, Ibitoye, Ibrahim, Ilesanmi, Ilic, Ilic, Immurana, Ismail, Merin J, Jakovljevic, Jamshidi, Janodia, Javaheri, Jayapal, Jayaram, Jha, Johnson, Joo, Joseph, Jozwiak, K, Kaambwa, Kabir, Kalankesh, Kalhor, Kandel, Karanth, Karaye, Kassa, Kassie, Keikavoosi-Arani, Keykhaei, Khajuria, Khan, Khan, Khan, Khreis, Kim, Kisa, Kisa, Knibbs, Kolkhir, Komaki, Kompani, Koohestani, Koolivand, Korzh, Koyanagi, Krishan, Krohn, Kumar, Kumar, Kurmi, Kuttikkattu, la Vecchia, Lám, Lan, Lasrado, Latief, Lauriola, Lee, Lee, Legesse, Lenzi, Li, Lin, Liu, Liu, Lo, Lorenzovici, Lu, Mahalingam, Mahmoudi, Mahotra, Malekpour, Malik, Mallhi, Malta, Mansouri, Mathews, Maulud, Mechili, Nasab, Menezes, Mengistu, Mentis, Meshkat, Mestrovic, Micheletti Gomide Nogueira de Sá, Mirrakhimov, Misganaw, Mithra, Moghadasi, Mohammadi, Mohammadi, Mohammadshahi, Mohammed, Mohan, Moka, Monasta, Moni, Moniruzzaman, Montazeri, Moradi, Mostafavi, Mpundu-Kaambwa, Murillo-Zamora, Murray, Nair, Nangia, Swamy, Narayana, Natto, Nayak, Negash, Nena, Kandel, Niazi, Nogueira de Sá, Nowroozi, Nzoputam, Nzoputam, Oancea, Obaidur, Odukoya, Okati-Aliabad, Okekunle, Okonji, Olagunju, Bali, Ostojic, A, Padron-Monedero, Padubidri, Pahlevan Fallahy, Palicz, Pana, Park, Patel, Paudel, Paudel, Pedersini, Pereira, Pereira, Petcu, Pirestani, Postma, Prashant, Rabiee, Radfar, Rafiei, Rahim, Ur Rahman, Rahman, Rahman, Rahmani, Rahmani, Rahmanian, Rajput, Rana, Rao, Rao, Rashedi, Rashidi, Ratan, Rawaf, Rawaf, Rawal, Rawassizadeh, Razeghinia, Mohamed Redwan, Rezaei, Rezaei, Rezaei, Rezaeian, Rodrigues, Buendia Rodriguez, Roever, Rojas-Rueda, Rudd, Saad, Sabour, Saddik, Sadeghi, Sadeghi, Saeed, Sahebazzamani, Sahebkar, Sahoo, Sajid, Sakhamuri, Salehi, Samy, Santric-Milicevic, Sao Jose, Sathian, Satpathy, Saya, Senthilkumaran, Seylani, Shahabi, Shaikh, Shanawaz, Shannawaz, Sheikhi, Shekhar, Sibhat, Simpson, Singh, Singh, Singh, Skryabin, Skryabina, Soltani-Zangbar, Song, Soyiri, Steiropoulos, Stockfelt, Sun, Takahashi, Talaat, Tan, Tat, Tat, Taye, Thangaraju, Thapar, Thienemann, Tiyuri, Ngoc Tran, Tripathy, Car, Tusa, Ullah, Ullah, Vacante, Valdez, Valizadeh, van Boven, Vasankari, Vaziri, Violante, Vo, Wang, Wei, Westerman, Wickramasinghe, Xu, Xu, Yadav, Yismaw, Yon, Yonemoto, Yu, Yu, Yunusa, Zahir, Zangiabadian, Zareshahrabadi, Zarrintan, Zastrozhin, Zegeye, Zhang, Naghavi, Larijani and Farzadfar1), with cigarette smoke (CS) representing the major risk factor for COPD development (Ref. Reference Osei, Mirbolouk, Orimoloye, Dzaye, Uddin, Benjamin, Hall, DeFilippis, Bhatnagar, Biswal and Blaha77). Alterations in lung structural and immune cell-derived EVs have been documented in COPD. Harmful gases or particles were demonstrated to stimulate the release of stressed lung cell-derived EVs that contribute to COPD-associated tissue damage (Ref. Reference Tan, Armitage, Teo, Ong, Shin and Moodley78). Activated neutrophils participate in alveolar deterioration by enhancing the release of EV-transported elastase, which causes significant tissue damage by degrading the lung extracellular matrix (Ref. Reference Genschmer, Russell, Lal, Szul, Bratcher, Noerager, Abdul Roda, Xu, Rezonzew, Viera, Dobosh, Margaroli, Abdalla, King, McNicholas, Wells, Dransfield, Tirouvanziam, Gaggar and Blalock79). An increase in the production of endothelial cell-derived EVs was also found in patients with exacerbated COPD (Ref. Reference Takahashi, Kobayashi, Fujino, Suzuki, Ota, He, Yamada, Suzuki, Yanai, Kurosawa, Yamaya and Kubo44). Compared to patients with stable disease, exacerbated ones exhibited a greater number of serum-derived EVs, which correlated with IL-6, C-reactive protein, and soluble TNF-R1 values (Ref. Reference Tan, Armitage, Teo, Ong, Shin and Moodley78). In an early study, a reduction in lung function (FEV₁/FVC ratio) was correlated with increased release of endothelial cell-derived EVs in COPD (Ref. Reference Takahashi, Kobayashi, Fujino, Suzuki, Ota, He, Yamada, Suzuki, Yanai, Kurosawa, Yamaya and Kubo44), while a subsequent study evidenced a negative correlation between FEV₁ and the number of circulating EVs (Ref. Reference Takahashi, Kobayashi, Fujino, Suzuki, Ota, Tando, Yamada, Yanai, Yamaya, Kurosawa, Yamauchi and Kubo80). Because pathogens also release EVs, infected patients with COPD may present worse symptoms. For instance, E. coli-derived EVs could accelerate alveolar disruption by intensifying neutrophilic inflammation via IL-17A-dependent signalling (Ref. Reference Kim, Lee, Choi, Choi, Heo, Gho, Jee, Oh and Kim81).

Mechanistic studies have provided novel insights into the onset and progression of COPD by investigating EV miRNA content. In human bronchial epithelial cells, CS extract exposure upregulated miR-21 and miR-210, increasing the production of α-smooth muscle actin and collagen type I and inducing lung fibroblast differentiation into myofibroblasts (Refs. Reference Fujita, Araya, Ito, Kobayashi, Kosaka, Yoshioka, Kadota, Hara, Kuwano and Ochiya82, Reference Xu, Ling, Xue, Dai, Sun, Chen, Liu, Zhou, Liu, Luo, Bian and Liu83). These alterations facilitated tissue remodelling that was prevented by administering a miR-21 inhibitor (Ref. Reference Xu, Ling, Xue, Dai, Sun, Chen, Liu, Zhou, Liu, Luo, Bian and Liu83). CS-exposed bronchial epithelial cells also demonstrated upregulation of miR-500a-5p, miR-574-5p, miR-656-5p, miR-3180-5p, and miR-3913-5p, and downregulation of miR-130b-5p, miR-222-5p, and miR-618 (Ref. Reference Corsello, Kudlicki, Garofalo and Casola84). Furthermore, EVs from CS-exposed endothelial cells were enriched with specific miRNAs (let-7d, miR-125a, miR-126, and miR-191) that, once engulfed by macrophages, inhibited their clearance abilities (Ref. Reference Serban, Rezania, Petrusca, Poirier, Cao, Justice, Patel, Tsvetkova, Kamocki, Mikosz, Schweitzer, Jacobson, Cardoso, Carlesso, Hubbard, Kechris, Dragnea, Berdyshev, McClintock and Petrache85). CS-exposed airway epithelial cells released EVs containing miR-7, miR-21-3p, miR-27b-3p, miR-125a-5p, and miR-221-3p that induced macrophage polarization toward pro-inflammatory M1 phenotype (Refs. Reference Chen, Wu, Shi, Fan, Zhang, Su, Wang and Li86–Reference Jiang, Wang, Zhang, Min and Gu89). In another study using CS-induced emphysema, airway epithelial cells exhibited increased levels of EV miR-93, which increased levels of macrophage-produced metalloproteinases 9 and 12 and promoted lung remodelling by excessively degrading elastin (Ref. Reference Xia, Wu, Zhao, Li, Lu, Ma, Cheng, Sun, Xiang, Bian and Liu90). CS-exposed airway epithelial cells can also contribute to lung remodelling by reducing EV-packaged miR-422a, leading to greater secretion of phosphoprotein-1 and production of collagen type I and smooth muscle actin by fibroblasts (Refs. Reference Xu, Ling, Xue, Dai, Sun, Chen, Liu, Zhou, Liu, Luo, Bian and Liu83, Reference Dai, Lin, Xu, Hu, Gou and Xu91).

Several studies have investigated alterations in EV miRNA expression to distinguish patients with distinct severity of COPD or from other diseases. Levels of miR-199a-5p were upregulated in plasma-derived EVs from patients with COPD compared to non-smokers (Ref. Reference Sundar, Li and Rahman92). A negative correlation between this miRNA and FEV₁ was observed (Ref. Reference Mizuno, Bogaard, Gomez-Arroyo, Alhussaini, Kraskauskas, Cool and Voelkel93). In plasma-derived EVs, a set of four miRNAs (miR-92b-3p, miR-106b-3p, miR-223-3p, and miR-374a-5p) demonstrated high diagnostic accuracy in segregating stable and exacerbated patients (Ref. Reference O’Farrell, Bowman and Fong94), while neutrophil count combined with serum-derived EV miR-1258 also demonstrated high accuracy for such (Ref. Reference Wang, Yang, Qiao, Bai, Li, Sun, Liu, Yang and Cui95). Muscle dysfunction in COPD patients was detected by upregulation of three plasma-derived EV miRNAs (miR-133a-3p, miR-133a-5p, miR-206) (Ref. Reference Carpi, Polini, Nieri, Dubbini, Celi, Nieri and Neri96). Patients with lung cancer or stable COPD differed in the expression of 14 plasma-derived EV miRNAs, of which the highest diagnostic accuracy was found by the expression of miR-27a-3p combined with miR-106b-3p and miR-361-5p (Ref. Reference O’Farrell, Bowman, Fong and Yang97). Inflammatory endotypes of COPD (neutrophilic versus eosinophilic) were also segregated by differentially expressed EV miRNAs in the BALF (Ref. Reference Burke, Cellura, Freeman, Hicks, Ostridge, Watson, Williams, Spalluto, Staples and Wilkinson98).

Cystic fibrosis

CF is a life-limiting genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene and affects over 100,000 individuals worldwide. Despite the multiorgan involvement, CF morbidity and mortality primarily result from end-stage lung disease due to impaired mucociliary clearance, chronic inflammation, and recurrent infection (Ref. Reference Lopes-Pacheco99). Emerging evidence suggests that EVs participate in inflammation and immune responses in CF (Ref. Reference Bacalhau, Camargo and Lopes-Pacheco41). In this context, higher EV counts were observed in CF cell lines (CFBE41o⁻ and CuFi-5) compared to control cells (16HBE14o⁻ and Nuli-1) (Ref. Reference Useckaite, Ward, Trappe, Reilly, Lennon, Davage, Matallanas, Cassidy, Dillon, Brennan, Doyle, Carter, Donnelly, Linnane, McKone, McNally and Coppinger27). Differences in EV cargo from CF and non-CF cell lines were also evidenced in response to Pseudomonas aeruginosa (Ref. Reference Lozano-Iturbe, Blanco-Agudín and Vázquez-Espinosa100), which is the most prevalent pathogen found in CF lungs. Interestingly, primary bronchial epithelial cell-derived EVs inhibited P. aeruginosa biofilm formation by mitigating levels of biofilm-related proteins in a CF mouse model (Ref. Reference Sarkar, Barnaby, Nymon, Taatjes, Kelley and Stanton101). On the contrary, both WT and CF airway epithelial cell-derived EVs exposed to P. aeruginosa exhibited greater content of pro-inflammatory mediators; however, responses from CF macrophages were mitigated in comparison to WT macrophages (Ref. Reference Koeppen, Nymon, Barnaby, Li, Hampton, Ashare and Stanton102). BALF-derived EVs revealed an enrichment of protein signatures related to macrophage activation and inflammation in a murine model of CF-like muco-inflammatory lung disease (Ref. Reference Mao, Suryawanshi, Patial and Saini103).

In sputum from CF patients, a high number of EVs – predominantly from granulocytes – was evidenced, and when these EVs were intratracheally administered in mice, there was strong neutrophilia in their lungs (Ref. Reference Porro, Di Gioia and Trotta104). Likewise, CF BALF-derived EVs contained higher levels of leukocyte chemotaxis-related proteins and drove neutrophil recruitment (Refs. Reference Useckaite, Ward, Trappe, Reilly, Lennon, Davage, Matallanas, Cassidy, Dillon, Brennan, Doyle, Carter, Donnelly, Linnane, McKone, McNally and Coppinger27, Reference Rollet-Cohen, Bourderioux, Lipecka, Chhuon, Jung, Mesbahi, Nguyen-Khoa, Guérin-Pfyffer, Schmitt, Edelman, Sermet-Gaudelus and Guerrera43). By analysing BALF-derived EVs, unique protein signatures were identified to differentiate CF stable and exacerbated versus controls (Ref. Reference Useckaite, Ward, Trappe, Reilly, Lennon, Davage, Matallanas, Cassidy, Dillon, Brennan, Doyle, Carter, Donnelly, Linnane, McKone, McNally and Coppinger27). BALF-derived EVs from paediatric CF patients (similar to those from asthmatic patients) were found to enhance epithelial sodium channel activity in small airway epithelia (Ref. Reference Al-Humiari, Yu and Liu105). Compared to controls, CF EVs exhibited alterations in lipid profile with increased ceramide production, which might accelerate the release of EV-packaged pro-inflammatory ceramides and perpetuate the inflammatory state in CF (Ref. Reference Zulueta, Peli, Dei Cas, Colombo, Paroni, Falleni, Baisi, Bollati, Chiaramonte, del Favero, Ghidoni and Caretti106). CF airway fluid-derived EVs demonstrated an enrichment with IL-1α, IL-1β, IL-18, and active caspase-1, which stimulate a hyperinflammatory state by inducing inflammasome activation of surrounding cells (namely, epithelial cells and newly recruited neutrophils) (Ref. Reference Forrest, Dobosh, Ingersoll, Rao, Rojas, Laval, Alvarez, Brown, Tangpricha and Tirouvanziam107). In sputum-derived EVs from CF patients treated for pulmonary exacerbation, levels of EV-transported neutrophil elastase and myeloperoxidase were decreased and demonstrated good correlation with improvement in lung function (Ref. Reference Ben-Meir, Antounians, Eisha, Ratjen, Zani and Grasemann108).

While BALF and sputum have been utilized as the main sources of EVs in CF studies, recent evidence demonstrated that urine-derived EVs can be a suitable source to distinguish CF patients and healthy controls (Ref. Reference Gauthier, Pranke, Jung, Martignetti, Stoven, Nguyen-Khoa, Semeraro, Hinzpeter, Edelman, Guerrera and Sermet-Gaudelus109). Indeed, CF EVs exhibited increased expression of epidermal growth factor receptor (EGFR) and decreased expression of klotho and matrisome, providing insights into CFTR-related kidney dysfunction (Ref. Reference Gauthier, Pranke, Jung, Martignetti, Stoven, Nguyen-Khoa, Semeraro, Hinzpeter, Edelman, Guerrera and Sermet-Gaudelus109).

Recent studies have assessed the impact of clinically approved CFTR modulators on CF EVs (Refs. Reference Useckaite, Ward, Trappe, Reilly, Lennon, Davage, Matallanas, Cassidy, Dillon, Brennan, Doyle, Carter, Donnelly, Linnane, McKone, McNally and Coppinger27, Reference Trappe, Lakkappa, Carter, Dillon, Wynne, McKone, McNally and Coppinger110) since these drugs can correct the basic defects of the mutant CFTR protein (Refs. Reference Lopes-Pacheco99, Reference Oliver, Carlon, Pedemonte and Lopes-Pacheco111). In CFBE41o⁻ cells, treatment with lumacaftor/ivacaftor or tezacaftor/ivacaftor led to a significant reduction of EV release (Ref. Reference Useckaite, Ward, Trappe, Reilly, Lennon, Davage, Matallanas, Cassidy, Dillon, Brennan, Doyle, Carter, Donnelly, Linnane, McKone, McNally and Coppinger27). Although children with CF and healthy controls demonstrated equivalent EV counts, treatment with elexacaftor/tezacaftor/ivacaftor altered EV protein content in serum-derived EVs of patients with CF from different age groups (Ref. Reference Trappe, Lakkappa, Carter, Dillon, Wynne, McKone, McNally and Coppinger110).

Idiopathic pulmonary fibrosis

IPF is a progressive disorder characterized by abnormal deposition of extracellular matrix in diverse regions of the lung parenchyma in response to chronic epithelial cell damage. The disease affects over three million people worldwide and presents a median survival rate of 2–5 years with only 20–25% of patients living beyond 10 years (Ref. Reference Nathan, Shlobin, Weir, Ahmad, Kaldjob, Battle, Sheridan and du Bois112). Under normal conditions, lung fibroblasts communicate with surrounding cells by EVs enriched with prostaglandins to inhibit myofibroblast differentiation; however, a reduction in EV-transported prostaglandins could trigger pulmonary fibrosis (Refs. Reference Lacy, Woeller, Thatcher, Pollock, Small, Sime and Phipps36, Reference Kadota, Yoshioka, Fujita, Araya, Minagawa, Hara, Miyamoto, Suzuki, Fujimori, Kohno, Fujii, Kishi, Kuwano and Ochiya113). Several studies have demonstrated the detrimental influence of EV-transported cargo on cell senescence and EMT during IPF development and progression. For instance, decreased reparative properties were observed for senescent cell-derived EVs (Refs. Reference Kadota, Fujita, Yoshioka, Araya, Kuwano and Ochiya114, Reference Antunes, Braga, Oliveira, Kitoko, Castro, Xisto, Coelho, Rocha, Silva-Aguiar, Caruso-Neves, Martins, Carvalho, Galina, Weiss, Lapa e Silva, Lopes-Pacheco, Cruz and Rocco115). Administration of syndecan-1-positive-EVs into mice lungs led to epithelial reprogramming by upregulating TGF-β and WNT signalling (Ref. Reference Parimon, Yao, Habiel, Ge, Bora, Brauer, Evans, Xie, Alonso-Valenteen, Medina-Kauwe, Jiang, Noble, Hogaboam, Deng, Burgy, Antes, Königshoff, Stripp, Gharib and Chen116). WNT signalling plays a role in IPF pathogenesis by promoting fibroblast proliferation, and an elevated number of WNT5A-positive EVs was found in BALF from IPF patients compared to non-IPF controls (Ref. Reference Martin-Medina, Lehmann, Burgy, Hermann, Baarsma, Wagner, de Santis, Ciolek, Hofer, Frankenberger, Aichler, Lindner, Gesierich, Guenther, Walch, Coughlan, Wolters, Lee, Behr and Königshoff117). Fibroblast-derived EVs contain high levels of fibronectin on their surface that, once bound to other fibroblasts, stimulate invasion by focal adhesion kinase- and steroid receptor coactivator kinase-mediated mechanisms (Ref. Reference Chanda, Otoupalova, Hough, Locy, Bernard, Deshane, Sanderson, Mobley and Thannickal118).

Various studies have investigated the influence of EV-transferred miRNAs on aberrant EMT and fibrosis development. For instance, bronchial epithelial cells from healthy subjects release much less EVs than those cells from IPF patients, which also contain increased levels of miR-7, miR-137, miR-195, and miR-411. These EVs can also induce senescence in naïve epithelial cells (Ref. Reference Asghar, Monkley, Smith, Hewitt, Grime, Murray, Overed-Sayer and Molyneaux119). In IPF sputum-derived EVs, seven miRNAs were upregulated, and there was a negative correlation between miR-142-3p levels and lung diffusing capacity (Ref. Reference Njock, Guiot, Henket, Nivelles, Thiry, Dequiedt, Corhay, Louis and Struman120). Interestingly, increased expression of miR-142-3p was suggested to come from macrophages and was able to reduce TGFβ-R1 expression in lung fibroblasts and alveolar epithelial cells (Ref. Reference Guiot, Cambier, Boeckx, Henket, Nivelles, Gester, Louis, Malaise, Dequiedt, Louis, Struman and Njock121). Compared to non-IPF cells, lung fibroblasts from IPF patients release more EVs that can induce increased senescence and mitochondrial dysfunction in airway epithelial cells (Refs. Reference Kadota, Yoshioka, Fujita, Araya, Minagawa, Hara, Miyamoto, Suzuki, Fujimori, Kohno, Fujii, Kishi, Kuwano and Ochiya113, Reference Chanda, Otoupalova, Hough, Locy, Bernard, Deshane, Sanderson, Mobley and Thannickal118). IPF fibroblast-derived EVs carry high levels of miR-23b-3p and miR-494-3p, which lead to epithelial phenotypic alterations by inhibiting sirtuin-3. Such mechanisms may be involved in mitochondrial dysfunction and increased levels of reactive oxygen species that result in DNA damage and epithelial cell senescence (Ref. Reference Kadota, Yoshioka, Fujita, Araya, Minagawa, Hara, Miyamoto, Suzuki, Fujimori, Kohno, Fujii, Kishi, Kuwano and Ochiya113). Upregulation of miR-21-5 was found in serum-derived EVs of IPF patients with a good prediction of mortality in the 30-month follow-up (Ref. Reference Makiguchi, Yamada, Yoshioka, Sugiura, Koarai, Chiba, Fujino, Tojo, Ota, Kubo, Kobayashi, Yanai, Shimura, Ochiya and Ichinose122). Levels of EV-transferred miR-21-5p and the rate of decline of spirometry FVC also demonstrated good correlation (Ref. Reference Makiguchi, Yamada, Yoshioka, Sugiura, Koarai, Chiba, Fujino, Tojo, Ota, Kubo, Kobayashi, Yanai, Shimura, Ochiya and Ichinose122).

Lung cancer

Lung cancer is the leading cause of malignant neoplasms and accounts for ~25% of all global cancer deaths (Ref. Reference Siegel, Miller, Wagle and Jemal123). Extensive research has been performed to investigate EVs and their cargo for early cancer detection. For instance, increased expression of EGFR has been detected in lung cancer cells with an even higher level of EGFR in EVs from patients with lung cancer compared to healthy controls (Ref. Reference Yamashita, Kamada, Kanasaki, Maeda, Nagano, Abe, Inoue, Yoshioka, Tsutsumi, Katayama, Inoue and Tsunoda124). Lung cancer cell-derived EVs contain miR-21 and miR-29a that bind to TLR8 on immune cells within the tumour niche, leading to a pro-inflammatory state via NF-κB activation that results in tumour progression and metastasis (Ref. Reference Fabbri, Paone, Calore, Galli, Gaudio, Santhanam, Lovat, Fadda, Mao, Nuovo, Zanesi, Crawford, Ozer, Wernicke, Alder, Caligiuri, Nana-Sinkam, Perrotti and Croce125). Early tumour formation is usually marked by hypoxia with increased levels of EV hypoxic signature proteins associated with EMT (Ref. Reference Lobb, Visan, Wu, Norris, Hastie, Everitt, Yang, Bowman, Siva, Larsen, Gorman, MacManus, Leimgruber, Fong and Möller126). Under hypoxic conditions, lung cancer cell-derived EVs express high levels of EGFR, TGF-β, miR-23a, and miR-619-5p that create an immunosuppressive state by polarizing macrophages (Ref. Reference Kim, Park, Kim, Choi, Kim, Sung, Sung, Choi, Yun, Yi, Lee, Kim, Lee and Rho127) and eliminating NK cells (Ref. Reference Cohnen, Chiang, Stojanovic, Schmidt, Claus, Saftig, Janßen, Cerwenka, Bryceson and Watzl128), while inducing angiogenesis (Ref. Reference Fabbri, Paone, Calore, Galli, Gaudio, Santhanam, Lovat, Fadda, Mao, Nuovo, Zanesi, Crawford, Ozer, Wernicke, Alder, Caligiuri, Nana-Sinkam, Perrotti and Croce125), thus facilitating the dissemination of abnormal cells.

The cargo of lung cancer-derived EVs contributes to drug resistance (Ref. Reference Liu, Gao, Ma, Huang, Chen, Zeng, Ashby, Zou and Chen129). Analysis of lung cancer-derived EVs indicated cisplatin response linked to high expression of miR-146a-5p and cisplatin resistance associated with miR-96 and miR-425-3p (Refs. Reference Yamashita, Kamada, Kanasaki, Maeda, Nagano, Abe, Inoue, Yoshioka, Tsutsumi, Katayama, Inoue and Tsunoda124, Reference Zhang and Xu130). Furthermore, poor prognosis was observed by miR-378 upregulation of serum-derived EVs from patients with non-small cell lung cancer (NSCLC) (Ref. Reference Zhang and Xu130). In patients with lung adenocarcinoma, plasma EVs exhibited elevated expression of Ras homolog family member V. By EV array, CD151/tetraspanin-24, CD171/L1CAM, and tetraspanin-8 were found to be abnormally expressed in histological slices of lung tumours (Ref. Reference Kato, Vykoukal, Fahrmann and Hanash131). Prospective miRNAs for early diagnosis and prognosis have been identified for lung cancer. In transcriptomic analysis, miR-30a-3p, miR-30e-3p, miR-181-5p, and miR-361-5p were identified for the diagnosis of lung adenocarcinoma, while miR-10b-5p, miR-15b-5p, and miR-320b were suggested for lung squamous cell carcinoma (Ref. Reference Jin, Chen, Chen, Fei, Chen, Cai, Liu, Lin, Su, Zhao, Su, Pan, Shen, Xie and Xie132). Prediction of survival rate by lung cancer was also suggested by analysis of let-7, miR-137, miR-182, miR-221, and miR-372 expressed in lung cancer cell-derived EVs (Ref. Reference Jin, Chen, Chen, Fei, Chen, Cai, Liu, Lin, Su, Zhao, Su, Pan, Shen, Xie and Xie132).

Pulmonary arterial hypertension

PAH is a rare, progressive disease characterized by pulmonary vascular inflammation and remodelling that leads to right ventricular failure. In PAH patients, the number of circulating EVs was demonstrated to be significantly increased (Refs. Reference Bakouboula, Morel, Faure, Zobairi, Jesel, Trinh, Zupan, Canuet, Grunebaum, Brunette, Desprez, Chabot, Weitzenblum, Freyssinet, Chaouat and Toti133–Reference Khandagale, Åberg, Wikström, Bergström Lind, Shevchenko, Björklund, Siegbahn and Christersson135) and directly correlated with functional impairment (Ref. Reference Bakouboula, Morel, Faure, Zobairi, Jesel, Trinh, Zupan, Canuet, Grunebaum, Brunette, Desprez, Chabot, Weitzenblum, Freyssinet, Chaouat and Toti133) and mortality rate (Ref. Reference Amabile, Heiss, Chang, Angeli, Damon, Rame, McGlothlin, Grossman, de Marco and Yeghiazarians136). Proteomic analysis revealed 13 proteins associated with coagulation and oxidative stress that were differentially expressed in blood-derived EVs from PAH patients compared to healthy controls (Ref. Reference Khandagale, Åberg, Wikström, Bergström Lind, Shevchenko, Björklund, Siegbahn and Christersson135). Endothelial cell-derived EV counts and levels of tricuspid annular plane systolic excursion (prognostic predictor for hypertension) were also higher in urine samples from PAH patients compared to controls (Ref. Reference Rose, Wanner, Cheong, Queisser, Barrett, Park, Hite, Naga Prasad, Erzurum and Asosingh137). Treatment with prostacyclin analogues reduced platelet- and leukocyte-derived EV counts and inhibited platelet reactivity and thrombus formation in PAH patients (Ref. Reference Gąsecka, Banaszkiewicz, Nieuwland, van der Pol, Hajji, Mutwil, Rogula, Rutkowska, Pluta, Eyileten, Postuła, Darocha, Huczek, Opolski, Filipiak, Torbicki and Kurzyna138).

EV content has been profiled to identify miRNAs and other factors that may be useful for PAH diagnosis and prognosis. For instance, miR-26a-5p was downregulated and miR-486-5p was upregulated in plasma-derived EVs of PAH patients (Ref. Reference Khandagale, Corcoran, Nikpour, Isaksson, Wikström, Siegbahn and Christersson139). A high level of miR-596 in plasma-derived EVs was associated with severe PAH and poor prognosis (Ref. Reference Huang, Wang, Tang, Gong, Sun, Wang, Jiang, Wu, Luo, Zhang, Yang, Li, Yuan, Zhao and Yuan140). Translationally controlled tumour protein has increased expression in lung tissue sections of PAH patients, and its transfer by EVs led to proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells that resulted in vascular remodelling (Ref. Reference Ferrer, Dunmore, Hassan, Ormiston, Moore, Deighton, Long, Yang, Stewart and Morrell141). In experimental monocrotaline-induced PAH, EV-packaged miR-211 induced proliferation of pulmonary arterial smooth muscle cells by inhibiting CaMK1/PPAR-γ signalling. Downregulation of miR-211 mitigated PAH in rats (Ref. Reference Zhang, Liu, Zheng, Chen, Sun, Yao, Sun, Lin, Lin and Yuan142).

Other respiratory diseases

Recent evidence indicates that cilia-derived EVs possess distinct mechanisms of release and molecular features compared to cytosolic-derived EVs (Ref. Reference Mohieldin, Pala, Beuttler, Moresco, Yates and Nauli143). In primary ciliary dyskinesia, a rare genetic disease caused by mutations in genes related to cilia development and function, proteomic analysis demonstrated high levels of proteins involved in leukocyte chemotaxis and antioxidant activity (Ref. Reference Rollet-Cohen, Bourderioux, Lipecka, Chhuon, Jung, Mesbahi, Nguyen-Khoa, Guérin-Pfyffer, Schmitt, Edelman, Sermet-Gaudelus and Guerrera43).

Both silicosis and pulmonary sarcoidosis exhibit granuloma formation and extensive lung parenchyma fibrosis resulting from different aetiologies. While silicosis is caused by silica inhalation mainly in work environments, systemic inflammation with unknown causes is responsible for sarcoidosis. In experimental silicosis, macrophage-derived EVs contributed to silica-induced lung fibrosis by activating fibroblasts in a mechanism dependent on endoplasmic reticulum stress. A significant decrease in lung fibrosis and levels of pro-inflammatory mediators (TNF-α, IL-1β, and IL-6) in BALF was observed when animals were pre-treated with the EV-production inhibitor GW4869 (Ref. Reference Qin, Lin, Liu, Li, Li, Deng, Chen, Chen, Niu, Li and Hu144). Serum-derived EVs from an animal model of silicosis demonstrated increased levels of miR-107 and miR-125-5p, which can promote fibroblast differentiation and contribute to lung fibrosis (Refs. Reference Xia, Wang, Guo, Pei, Zhang, Bao, Li, Qu, Zhao, Hao and Yao145,Reference Ding, Pei, Zhang, Qi, Xia, Hao and Yao146). In addition, levels of miR-223-3p were significantly altered in EVs and tissue from silicosis patients, thus being considered a promising biomarker (Ref. Reference Hou, Zhu, Jiang, Zhao, Jia, Jiang, Wang, Xue, Wang and Tian147). On the contrary, sarcoidosis patients demonstrated altered levels of CD14 and lipopolysaccharide-binding protein on serum-derived EVs by proteomics analysis (Ref. Reference Futami, Takeda, Koba, Narumi, Nojima, Ito, Nakayama, Ishida, Yoshimura, Naito, Fukushima, Takimoto, Edahiro, Matsuki, Nojima, Hirata, Koyama, Iwahori, Nagatomo, Shirai, Suga, Satoh, Futami, Miyake, Shiroyama, Inoue, Adachi, Tomonaga, Ueda and Kumanogoh148). Treatment with methotrexate also reduced levels of EV-transported serpin C1 in sarcoidosis patients, suggesting that it can be a good predictor for therapy monitoring (Ref. Reference Kraaijvanger, Janssen Bonás, Grutters, Paspali, Veltkamp, de Kleijn and van Moorsel149).

Asthma

Therapeutic effects of MSC-derived EVs have been investigated in several experimental models of asthma, including Aspergillus hyphal extract, HDM, and ovalbumin. In an early study, MSC-derived conditioned medium prevented peribronchial inflammation and airway smooth muscle thickening in experimental asthma by an adiponectin-promoted mechanism (Ref. Reference Ionescu, Alphonse, Arizmendi, Morgan, Abel, Eaton, Duszyk, Vliagoftis, Aprahamian, Walsh and Thébaud151). Subsequent studies demonstrated that EVs from both murine and human MSCs reduce airway hyperresponsiveness and lung inflammation by mitigating BALF neutrophil and eosinophil counts and levels of IL-4, IL-5, and IL-17 in Aspergillus hyphal extract-induced asthma (Ref. Reference Cruz, Borg, Goodwin, Sokocevic, Wagner, Coffey, Antunes, Robinson, Mitsialis, Kourembanas, Thane, Hoffman, McKenna, Rocco and Weiss152). MSC-derived EVs also stimulated T regulatory cell proliferation and immunosuppressive properties by enhancing IL-10 and TGF-β levels in peripheral blood mononuclear cells of asthmatic patients (Ref. Reference Du, Zhuansun and Chen153).

In experimental ovalbumin-induced asthma, MSC-derived EVs were efficient at reducing lung eosinophilia, fibrosis, and IgE and TGF-β levels (Refs. Reference de Castro, Xisto, Kitoko, Cruz, Olsen, Redondo, Ferreira, Weiss, Martins, Morales and Rocco154, Reference Mun, Kang, Park, Yu, Cho and Roh155), decreasing the number of mucus-producing goblet cells (Ref. Reference Song, Zhu and Wei156) and modulating macrophage polarization (Ref. Reference Fang, Zhang, Meng, Wang, He, Peng, Xu, Fan, Wu, Wu, Zheng and Fu157). MSC-derived EVs mitigated airway remodelling by promoting M2-like macrophage activation via FoxO1 signalling (Ref. Reference Shang, Sun, Xu, Ge, Hu, Xiao, Ning, Dong and Bai158). TNF receptor-associated factor 1 was also involved in macrophage polarization by MSC-derived EVs by regulating NF-κB and AKT signalling pathways (Ref. Reference Dong, Wang, Zhang, Zhang, Gu, Guo, Zuo, Pan, Hsu, Wang and Wang159). Furthermore, EMT and airway remodelling in asthma are influenced by WNT/β-catenin signalling activation, and MSC-derived EVs prevented tissue remodelling by inhibiting WNT/β-catenin signalling pathway-related factors. Such effects were reversed by the administration of BML-284, a WNT agonist (Ref. Reference Song, Zhu and Wei156). Likewise, the downregulation of miR-188, an EV-enriched miRNA, mitigated the protective effects of MSC-derived EVs in experimental ovalbumin-induced asthma (Ref. Reference Shan, Liu, Zhang, Zhou and Shang160). EV-transferred miR-221-3p inhibited FGF2 expression and ERK1/2 signalling, leading to mitigated lung inflammation and remodelling in experimental asthma (Ref. Reference Liu, Lin, Nie, Wan, Jiang and Zhang161). In samples from asthmatic patients, STAT3 and miR-301a-3p levels were demonstrated to be negatively correlated. MSC-derived EV miR-301a-3p was efficiently internalized by airway smooth muscle cells, which inhibits their proliferation and migration by targeting STAT3 (Ref. Reference Feng, Bai, Li, Zhao, Sun, Bao, Ren and Su162). miR-223-3p is highly expressed in MSC-derived EVs and has a major protective action on asthma-induced airway remodelling by regulating NLRP3/caspase-1/gasdermin D-mediated inflammasome activation and pyroptosis (Ref. Reference Li and Yang163).

Under hypoxic conditions, a higher number of EVs was released by MSCs (Refs. Reference Dong, Wang, Zheng, Pu, Ma, Qi, Zhang, Xue, Shan, Liu, Wang and Mao164, Reference Sholihah and Barlian165). When these EVs were administered in ovalbumin-induced asthma, there was greater improvement in lung inflammation (BALF total cell and eosinophil counts and levels of IL-4 and IL-13) and remodelling (levels of α-smooth muscle actin, collagen-1, and TGF-β1) compared to EVs from normoxia-cultured MSCs (Ref. Reference Dong, Wang, Zheng, Pu, Ma, Qi, Zhang, Xue, Shan, Liu, Wang and Mao164). Nebulization of hypoxic MSC-derived EVs was also effective at reducing lung inflammation and remodelling (Ref. Reference Xu, Wang, Luo, Gao, Gu, Ma, Xu, Yu, Liu, Liu, Wang, Zheng, Mao and Dong166), levels of IgE and pro-inflammatory cytokines (Ref. Reference Luo, Wang, Mao, Xu, Gu, Li, Mao, Zheng and Dong167). Upregulation of miR-146a-5p was evidenced on these EVs, and reduction of this EV-transferred miRNA impaired lung protective effects in experimental ovalbumin-induced asthma (Refs. Reference Dong, Wang, Zheng, Pu, Ma, Qi, Zhang, Xue, Shan, Liu, Wang and Mao164, Reference Xu, Wang, Luo, Gao, Gu, Ma, Xu, Yu, Liu, Liu, Wang, Zheng, Mao and Dong166).

Bronchopulmonary dysplasia

Several studies have investigated the potential of MSC-derived EVs for BPD therapy. In an initial study, MSC-derived EVs were demonstrated to improve pulmonary hypertension and lung function by reducing vascular remodelling, alveolar simplification, and fibrosis in hyperoxia-induced BPD in newborn mice (Ref. Reference Willis, Fernandez-Gonzalez, Anastas, Vitali, Liu, Ericsson, Kwong, Mitsialis and Kourembanas168). These effects were associated with macrophage polarization to M2 anti-inflammatory phenotype instead of M1 pro-inflammatory one (Ref. Reference Willis, Fernandez-Gonzalez, Anastas, Vitali, Liu, Ericsson, Kwong, Mitsialis and Kourembanas168). Both early and late administration of MSC-derived EVs were effective at improving core features of BPD on this model, thus preventing or reversing cardiorespiratory abnormalities (Ref. Reference Willis, Fernandez-Gonzalez, Reis, Yeung, Liu, Ericsson, Andrews, Mitsialis and Kourembanas169). Biodistribution analysis revealed MSC-derived EVs in lung tissue of hyperoxia-exposed newborn mice and epigenetic and phenotypic reprogramming of myeloid cells to a non-inflammatory phenotype (Ref. Reference Willis, Reis, Gheinani, Fernandez-Gonzalez, Taglauer, Yeung, Liu, Ericsson, Haas, Mitsialis and Kourembanas170). MSC-derived EVs also restored thymocyte development/maturation profile and thymic medullary structure by enhancing the expression of antioxidant-stress-related genes (Ref. Reference Reis, Willis, Fernandez-Gonzalez, Yeung, Taglauer, Magaletta, Parsons, Derr, Liu, Maehr, Kourembanas and Mitsialis171).

In a rat model of hyperoxia-induced BPD, MSC-derived EVs reduced alveolar simplification, lung damage, and fibrosis in a dose-dependent manner (Ref. Reference Ai, Shen, Sun, Zhu, Gao, du, Yuan, Chen and Zhou172). In vitro analysis revealed that EVs delay the transdifferentiation of alveolar type II cells into type I cells by downregulating WNT5a (Ref. Reference Ai, Shen, Sun, Zhu, Gao, du, Yuan, Chen and Zhou172). Antenatal administration of MSC-derived EVs was also able to reduce NLRP3- and IL-1β-mediated inflammation and preserve distal lung growth and mechanics in chorioamnionitis-induced rat BPD (Ref. Reference Abele, Taglauer, Almeda, Wilson, Abikoye, Seedorf, Mitsialis, Kourembanas and Abman173). These therapeutic benefits were associated with increased expression of vascular endothelial growth factor (VEGF) (Ref. Reference Abele, Taglauer, Almeda, Wilson, Abikoye, Seedorf, Mitsialis, Kourembanas and Abman173), which is decreased in preterm infants (Ref. Reference Lassus, Ristimäki and Ylikorkala174). Along these lines, EV-transferred VEGF mitigated lung injury by decreasing inflammation and improving alveolarization and angiogenesis in hyperoxia-induced rat BPD. Such effects were not found by EVs from VEGF-knockdown MSCs (Ref. Reference Ahn, Park, Kim, Sung, Sung, Ahn and Chang175). Neurologic alterations are also observed in infants with BPD. MSC-derived EVs not only reduced lung damage but also improved neurodevelopmental features in an animal model (Ref. Reference Lithopoulos, Strueby, O’Reilly, Zhong, Möbius, Eaton, Fung, Hurskainen, Cyr-Depauw, Suen, Xu, Collins, Vadivel, Stewart, Burger and Thébaud176).

Chronic obstructive pulmonary disease

Initial studies assessed the effects of MSC-derived conditioned media in COPD models and demonstrated a variety of therapeutic benefits. In CS-induced emphysema, conditioned media promoted tissue repair and increased the number of small pulmonary vessels (Ref. Reference Huh, Kim, Lee, Lee, van Ta, Kim, Oh, Lee and Lee177). Hepatocyte growth factor-mediated protection was observed in lung tissue when MSC-derived conditioned media were administered at the onset of elastase-induced emphysema (Ref. Reference Kennelly, Mahon and English178).

EVs released by damaged alveolar type II cells promoted MSC migration and upregulated genes related to mitochondrial synthesis and transfer (Ref. Reference Song, Peng and Guo179). In co-culture experiments, MSCs released EVs to protect the BEAS2B lung epithelial cell line against CS-induced mitochondrial abnormalities (Ref. Reference Maremanda, Sundar and Rahman180). MSC-derived EVs also reduced CS-induced mitochondrial dysfunction, peribronchial and perivascular inflammation, alveolar septa thickening, and mucus-producing goblet cell counts in rodent lungs (Refs. Reference Maremanda, Sundar and Rahman180–Reference Zhu, Lian, Su, Wu, Zeng and Chen182). CS exposure elevated pyroptosis and inhibited phagocytic abilities in alveolar macrophages, which was reversed by MSC-derived EVs (Ref. Reference Zhu, Lian, Su, Wu, Zeng and Chen182). In experimental papain-induced emphysema, MSC-derived EVs prevented endothelial cell apoptosis by activating VEGF/VEGF receptor-2-mediated AKT and MEK/ERK signalling pathways (Ref. Reference Chen, Lin, Deng and Qian183). MSC-derived EVs were also able to improve lung function and mitigate airway inflammation and levels of pro-inflammatory cytokines in CS-exposed mice (Ref. Reference Harrell, Miloradovic, Sadikot, Fellabaum, Markovic, Miloradovic, Acovic, Djonov, Arsenijevic and Volarevic184). Interestingly, cardiorespiratory abnormalities in emphysematous mice were mitigated by MSC-derived EVs from healthy donors but not from elastase-induced emphysema (Ref. Reference Antunes, Braga, Oliveira, Kitoko, Castro, Xisto, Coelho, Rocha, Silva-Aguiar, Caruso-Neves, Martins, Carvalho, Galina, Weiss, Lapa e Silva, Lopes-Pacheco, Cruz and Rocco115). These EVs exhibited downregulation of various anti-inflammatory and anti-oxidant mediators (Ref. Reference Antunes, Braga, Oliveira, Kitoko, Castro, Xisto, Coelho, Rocha, Silva-Aguiar, Caruso-Neves, Martins, Carvalho, Galina, Weiss, Lapa e Silva, Lopes-Pacheco, Cruz and Rocco115). Artificial EVs from MSCs were generated by sequential penetration through polycarbonate membranes and demonstrated similar size, shape, and surface markers as natural MSC-derived EVs. When these EVs were administered in a model of elastase-induced emphysema, lower doses of artificial EVs promoted similar reparative actions as higher doses of natural EVs (Ref. Reference Kim, Kim, Cho, Shin, Lee and Oh185).

Lung cancer

Current research suggests MSCs as a double-edged sword in cancer as they demonstrate pro- and anti-tumorigenic actions (Refs. Reference Liang, Chen, Zhang, Fang, Chen, Xu and Chen186, Reference Slama, Ah-Pine, Khettab, Arcambal, Begue, Dutheil and Gasque187). On the contrary, most studies revealed tumour-suppression effects by MSC-derived EVs, although further research is still needed. MSC EV-overexpressing miR-30b-5p prevented tumorigenesis and promoted NSCLC cell apoptosis by regulating the EZH2/PI3K/AKT pathway (Ref. Reference Wu, Tian, Liu, Xu, Shi, Zhang, Gao, Yin, Xu and Wang188). Both miR-204 and miR-598 have reduced expression in NSCLC tissues, which was associated with poor prognosis (Refs. Reference Li and Wu189, Reference Liu, Zhang, Lin, Tang, Zhou, Wen and Li190). MSC-derived EVs transferred miR-598 into NSCLC cells and prevented proliferation and migration by inhibiting THBS2 production (Ref. Reference Li and Wu189). Inhibition of EMT and NSCLC cell migration and invasion was also evidenced by EV-transferred miR-204 by targeting the KLF7/AKT/HIF-1α pathway (Ref. Reference Liu, Zhang, Lin, Tang, Zhou, Wen and Li190). NSCLC tissues exhibited high expression of CCNE1 and CCNE2 and low expression of miR-144. MSC-derived EVs reduced NSCLC proliferation and the number of S-phase-blocked cells by transferring miR-144 in both in vitro and in vivo models (Ref. Reference Liang, Zhang, Li, Xin, Zhao, Ma and du191). Another therapeutically relevant miRNA in lung cancer is miR-320a which, once transferred by MSC-derived EVs, reduced cancer cell migration and invasion by binding to sex-determining region Y-box 4 (Ref. Reference Xie and Wang192). Let-7i has a reduced expression in lung cells, and its transfer by MSC-derived EVs inhibited lung cell proliferation by downregulating the KDM3A/DCLK1/FXYD3 signalling pathway (Ref. Reference Liu, Feng, Zeng, He, Gong and Liu193).

Despite promising therapeutic actions, some studies demonstrated an opposite effect in which MSC-derived EVs facilitate lung cancer development. In lung adenocarcinoma cells, EV-transferred miR-410 increased their proliferation and reduced apoptosis (Ref. Reference Dong, Pu, Zhang, Qi, Xu, Li, Wei, Wang, Zhou, Zhu, Wang, Liu, Chen and Su194). Under hypoxic conditions, MSC-derived EVs express certain miRNAs (miR-21-5p, miR-193a-3p, miR-210-3p, and miR-5100) that activate STAT3-mediated EMT and accelerate cancer cell invasion (Refs. Reference Zhang, Sai, Wang, Wang, Wang, Zheng, Li, Tang and Xiang195, Reference Ren, Hou, Yang, Wang, Wu, Wu, Zhao and Lu196).

Lung fibrosis

MSC-derived EVs have demonstrated a variety of therapeutic benefits in experimental lung fibrosis. In lung fibroblasts, MSC-derived EVs inhibited TGF-β1-mediated myofibroblast differentiation by a Thy-1-dependent mechanism (Ref. Reference Shentu, Huang, Cernelc-Kohan, Chan, Wong, Espinoza, Tan, Gramaglia, van der Heyde, Chien and Hagood197). Blockage of Thy-1 reduced EV uptake and preserved myofibroblast differentiation (Ref. Reference Shentu, Huang, Cernelc-Kohan, Chan, Wong, Espinoza, Tan, Gramaglia, van der Heyde, Chien and Hagood197). MSC-derived EVs mitigated bleomycin-induced lung fibrosis by stimulating the proliferation of bronchoalveolar stem cells (Ref. Reference Tan, Lau, Leaw, Nguyen, Salamonsen, Saad, Chan, Zhu, Krause, Kim, Sievert, Wallace and Lim198). Proteomic analysis demonstrated that human MSC-derived EVs modulate macrophages to an anti-inflammatory phenotype, thus mitigating lung inflammation and fibrosis in a model of bleomycin-induced lung fibrosis (Ref. Reference Mansouri, Willis, Fernandez-Gonzalez, Reis, Nassiri, Mitsialis and Kourembanas199). In PM2.5-induced lung fibrosis, MSC-derived EVs reduced levels of reactive oxygen species and apoptosis of alveolar epithelial cells (Ref. Reference Gao, Sun, Dong, Zhao, Hu and Jin200). MSC-derived EVs were effective at mitigating macrophage counts, collagen fibre content, and granuloma size in lung parenchyma, thus improving lung function in silica-induced lung fibrosis (Ref. Reference Bandeira, Oliveira, Silva, Menna-Barreto, Takyia, Suk, Witwer, Paulaitis, Hanes, Rocco and Morales201). EVs from three-dimensional cultured MSCs also reduced collagen deposition in silica-exposed fibroblasts (Ref. Reference Xu, Zhao, Li, Hou, Wang, Li, Jiang, Zhu and Tian202).

The influence of MSC EV-transferred miRNAs in preventing or reversing lung fibrosis has been documented in several studies. For instance, miR-29b-3p demonstrated low expression in lung tissue, and its overexpression in MSC-derived EVs prevented interstitial fibroblast proliferation, differentiation, migration, and invasion by targeting frizzled 6 (Ref. Reference Wan, Chen, Fang, Zuo, Cui and Xie203). EV-transferred miR-186 also prevented fibroblast activation by downregulating SRY-related HMG box transcription factor 4 and its downstream gene Dickkopf-1, thus alleviating lung fibrosis in mice (Ref. Reference Zhou, Lin, Kang, Liu, Zhang and Xu204). TGF-β signalling pathway was inhibited by EV-transferred miR-21 and miR-23, preventing myofibroblast differentiation and lung damage in experimental bleomycin-induced lung fibrosis (Ref. Reference Shi, Ren, Li, Wang, Wang, Qin, Li, Zhang, Li and Wang205). EV-transferred let-7 mitigated lung epithelial fibrosis and remodelling in mice by reducing NLRP3-mediated inflammation activation, mitochondrial damage, and levels of reactive oxygen species in alveolar epithelial cells (Ref. Reference Sun, Zhu, Feng, Lin, Yin, Jin and Wang206). Levels of miR-30b were downregulated in the serum of IPF patients, and EV-transferred miR-30b reduced inflammation and apoptosis of TGF-β1-stimulated lung epithelial cell lines by targeting Runx1 and Spred2 (Ref. Reference Zhu, Xu, Wang, Zhang, Zhou and Wu207). EV-transferred miR-29c and miR-129 reduced lung damage and myofibroblast differentiation in experimental bleomycin-induced lung fibrosis (Ref. Reference Nataliya, Mikhail, Vladimir, Olga, Maksim, Ivan, Ekaterina, Georgy, Natalia, Pavel, Andrey, Maria, Maxim, Anastasiya, Uliana, Zhanna, Vsevolod, Natalia and Anastasiya208). In radiation-induced pulmonary fibrosis, EV-transferred miR-214-3p reduced endothelial cell damage, inflammation, and fibrosis by downregulating ataxia telangiectasia mutated/p53/p21-mediated signalling pathway (Ref. Reference Lei, He, Zhu, Zhou, Zhang, Wang, Huang, Chen, Li, Liu, Han, Guo, Han and Li209). EV-transferred miR-218 inhibited endothelial-mesenchymal transition and restored endothelial properties by downregulating the MeCP2/BMP2 pathway (Ref. Reference Zhao, Du and Sun210). Knockdown of miR-218 reduced the therapeutic effects of MSC-derived EVs on endothelial-mesenchymal transition (Ref. Reference Zhao, Du and Sun210).

In silica-induced lung fibrosis, MSCs release EV-containing miRNAs that inhibit macrophage activation by suppressing TLR signalling (Ref. Reference Phinney, Di Giuseppe and Njah211). EV-transferred miR-223-3p reduced silica-induced inflammation (IL-1β, IL-18, and cleaved caspase-1) and remodelling (collagen I and III, fibronectin, and α-smooth muscle actin) by suppressing NLRP3-mediated signalling pathway (Ref. Reference Hou, Zhu, Jiang, Zhao, Jia, Jiang, Wang, Xue, Wang and Tian147). In experimental silicosis, EV-containing let-7i-5p prevented fibroblast activation by targeting the TGF-β receptor/Smad3 pathway (Ref. Reference Xu, Hou, Zhao, Wang, Jiang, Jiang, Zhu and Tian212), while EV-transferred let-7d-5p reduced PM2.5-induced lung fibrosis (Ref. Reference Gao, Sun, Dong, Zhao, Hu and Jin200). Likewise, miR-26a-5p is downregulated in silicosis, and its transfer by MSC-derived EVs prevented EMT by targeting Adam17/Notch signalling, thus mitigating lung fibrosis in mice (Ref. Reference Zhao, Jiang, Xu, Jia, Wang, Xue, Wang, Zhu and Tian213).

Pulmonary arterial hypertension

In early studies, MSC-derived conditioned media demonstrated protective effects against PAH by preventing cardiac remodelling and improving pulmonary blood flow (Ref. Reference Rathinasabapathy, Bruce, Espejo, Horowitz, Sudhan, Nair, Guzzo, Francis, Raizada, Shenoy and Katovich214) and by downregulating calcineurin and nuclear factor of activated T-cells, thus inhibiting the inflammation-mediated over-proliferation of pulmonary artery smooth muscle cells (Ref. Reference Liu, Han and Han215). Subsequently, MSC-derived EVs reduced right ventricular hypertrophy and small pulmonary artery area index in experimental sugen/hypoxia- (Ref. Reference Klinger, Pereira and Del Tatto216) and monocrotaline-induced PAH (Ref. Reference Chen, An, Liu, Wang, Chen, Hong, Liu, Xiao and Chen217). MSC-derived EVs promoted therapeutic effects on experimental monocrotaline-induced PAH by transferring miR-34a, miR-122, miR-124, and miR-127 (Ref. Reference Aliotta, Pereira, Wen, Dooner, del Tatto, Papa, Goldberg, Baird, Ventetuolo, Quesenberry and Klinger218) and by shifting the balance from the ACE/AngII/AT1R axis toward ACE2/Ang(1–7)/Mas axis (Ref. Reference Liu, Liu, Xiao, Wang, Yao, Zeng, Yu, Guan, Wei, Peng, Zhu, Wang, Yang, Zhong and Chen219). MSC-derived EVs also reduced monocrotaline-induced lung fibrosis, right ventricular hypertrophy, and pulmonary vascular remodelling by regulating the WNT5A/BMP signalling pathway (Ref. Reference Zhang, Ge, Zhang, Wang, Jiang, Xin and Luan220). In experimental hypoxia-induced PAH, macrophages were polarized to M2 phenotype by MSC-derived EVs, leading to reduced levels of inflammatory mediators and increased levels of IL-10, inhibiting over-proliferation of pulmonary artery smooth muscle cells (Ref. Reference Liu, Zhang, Liu, Zhang, Wang, Huang, Ma and Ge221).