Extracellular vesicles and pancreatitis: mechanisms, status and perspectives.

Autoimmune pancreatitis (AIP) is a special form of CP that has a pivotal role in inducing fibroinflammatory disorders of the pancreas 124 , 125 . The diagnosis and treatment of AIP have not achieved satisfactory clinical effects. Nakamaru et al. reported that the expression of miR-21 was significantly upregulated in extracellular vesicles derived from the serum of patients with type 1 autoimmune pancreatitis 126 . This study included 27 patients with type 1 AIP, 23 patients with chronic pancreatitis and 23 healthy controls (HCs). Microarray analysis showed 165 differentially expressed miRNAs in patients with type 1 AIP. Furthermore, 132 miRNAs were upregulated and 33 were downregulated in type 1 AIP patients compared with HCs. Among these results, the expression levels of miR-659-3p, miR-27a-3p, miR-99a-5p, miR-21-5p, miR-205-5p, miR-100-5p, miR-29c-3p, and miR-126b-1-3p were significantly upregulated, concurrent with a significant decrease in miR-4252 and miR-5004-1-5p expression relative to that of the HCs. Quantitative evaluation of EV miRNA expression levels by RT-PCR showed that only the expression level of miR-21-5p was significantly higher in type 1 AIP patients than in HCs. Furthermore, the results of in situ hybridization (ISH) of resected specimens of type 1 AIP patients showed that the expression of miR-21 in pancreatic duct epithelium was similar between type 1 AIP patients and HCs. However, miR-21 was highly expressed in pancreatic acinar cells in type 1 AIP patients compared to HCs. Therefore, this study demonstrated that miR-21 in EVs derived from AIP patients' serum could be used as a diagnostic marker to distinguish AIP from healthy people.

Pancreatitis refers to an inflammatory disorder of the pancreas, in which pancreatic enzymes damage pancreatic tissue, leading to acinar cell death, as well as local and systemic inflammation 1 . Previous studies have shown that acute pancreatitis, recurrent acute pancreatitis, and chronic pancreatitis represent a continuum of disease progression. Per 100,000 people in the general population, the global incidence of acute pancreatitis is 33.74 cases per year and that of chronic pancreatitis is 9.62 cases per year 2 . Similar to acute pancreatitis, chronic pancreatitis is most prevalent in middle-aged and older patients 3 , 4 . However, the incidence of chronic pancreatitis was higher among men than women, although there was no significant difference between sexes for acute pancreatitis. The global transition rate data indicated that the transition from the first episode of acute pancreatitis to recurrent acute pancreatitis occurs in approximately 21% of cases and that from recurrent acute pancreatitis to chronic pancreatitis occurs in approximately 36% of cases 5 . The global mortality rates of acute pancreatitis and chronic pancreatitis were 1.60 and 0.09 per 100,000 persons per year, respectively 2 . Recently, clinical and experimental data have shed light on the pathophysiology of pancreatitis, indicating that premature intrapancreatic activation of digestive proteases is critical in the pathogenesis of pancreatitis 6 . Furthermore, the progression and severity of pancreatitis may be influenced by dysregulated autophagy, which promotes the inflammatory response in the pancreas, leading to local and systemic inflammatory responses and multiorgan failure 7 . Unfortunately, the sequelae and mortality of pancreatitis remain substantial. Concerted efforts by not only surgeons but also researchers should strive to reduce the incidence of pancreatitis and effectively improve the treatment of its sequelae 8 - 10 . EVs are cell-derived membranous structures that are present in biological fluids and are involved in physiological and pathological processes of inflammatory disease or cancer 11 - 15 . EVs were initially regarded as membrane debris with no biological function 16 . However, in 2007, exosomes were shown to transfer mRNAs and microRNAs to recipient cells, remained functional and changed the behavior of target cells 17 . EVs exert their effects on fundamental biological processes by directly merging with the recipient cell plasma membrane and delivering their contents, including transcription factors, oncogenes, microRNAs and mRNAs, into recipient cells 18 - 20 . In this manner, EVs participate in the pathophysiological process of disease, for example, stem cell therapy 21 , tissue repair 22 , immune surveillance 23 , and tumor progression and metastasis 24 , 25 . In addition, several studies have reported the potential applications of EVs in the diagnosis and treatment of disease based on their own characteristics. Here, we report a comprehensive overview of the relationship between EVs and pancreatitis, with a special focus on their roles in pathogenesis and their potential clinical application as diagnostic biomarkers and therapeutic targets in pancreatitis. We also discuss the advantages and limitations among current studies and the need for further research. Finally, we discuss the prospects and applications of EVs in pancreatitis.

In this review, we report a comprehensive overview of the relationship between EVs and pancreatitis, with a special focus on their roles in pathogenesis and their potential clinical applications as diagnostic biomarkers and therapeutic targets in pancreatitis. We have discussed EV isolation and application of clinical-grade EVs and the advantages and limitations of current studies, as well as the need for further research. EVs are regarded as a mechanism for intercellular communication, transferring proteins, lipids and genetic material in pancreatitis. EVs are involved in the pathogenesis of pancreatitis and are used as diagnostic markers for pancreatitis and have clear potential as treatment targets in pancreatitis. However, there are still no uniform and standardized methods available for the purification and isolation of EVs. Therefore, it remains unclear whether different isolation methods of EVs may lead to different results. Currently, there is a need to establish standardized methods of sample collection, storage, and application to minimize the influence of the complexity and heterogeneity of EVs. EVs have not been applied drug delivery in the treatment of AP. Therefore, the clinical application of EVs in the diagnosis and treatment of pancreatitis is promising, and additional extensive research is required before clinical application.

The study of EVs has the potential to identify cellular and molecular communication and value in the diagnosis and treatment of diseases 26 , 31 . Due to their diverse and unique contents, such as nucleic acids, proteins, lipids, and metabolites, EVs not only can reflect their origin cells but can also be used as a diagnostic marker for disease. In addition, EVs protect their contents through their stable membrane structure and serve as an effective carrier for drug delivery in the therapeutics of cancer and inflammatory diseases 15 , 36 , 127 . In recent years, EV research has focused on the classification of EVs, isolation methods, and their functions in disease diagnosis, progression and therapy 30 , 128 - 130 . Despite the increase in different isolation methods of EVs, there are still no uniform and standardized methods available for the purification and isolation of EVs 131 , 132 . Therefore, it remains unclear whether different isolation methods of EVs may lead to different results 26 . Currently, there is a need to establish standardized methods of sample collection, storage, and application to minimize the influence of the complexity and heterogeneity of EVs 133 . In addition, the conventional isolation methods of exosomes in blood, such as ultracentrifugation, cannot completely remove lipoprotein, which is similar in size and density to EVs 134 . However, the volume and time of blood collection, handling, storage condition and application of anticoagulants all impact the isolation of EVs 135 . For the isolation of EVs from cultured cells, it is recommended to use serum-free medium or EV-free serum in cell culture medium 136 . For clinical blood sample collection, it is important to minimize the influence of the activation and release of platelet and red blood cell-derived EVs and the contamination of cell debris. A number of studies have shown that -80°C and minimized freeze-thaw cycles are the optimum conditions for the storage of EVs based on size, composition, and functionality. An unfavorable temperature and increased freeze-thaw cycles can cause EV aggregation and lysis, leading to an increase in size, a reduction in counting and a loss of content. The current methods of EV isolation mainly include ultracentrifugation, size-exclusion chromatography, filtration, commercial reagents, microfluidics, asymmetric flow field-flow fractionation, and nanoflow cytometry 133 . Diverse methods have their advantages and disadvantages. Moreover, high-efficiency isolation of EVs is needed to eliminate protein contamination and increase purity for clinical application of EVs. In the diagnosis of AP, there are only a few studies on exosomal biomarkers for AP. Zhao et al. indicated that 30 exosomal miRNAs were upregulated in pancreatic acinar AR42J cells and could be used as biomarkers for AP. However, the results of this study are only derived from in vitro experiments and have not been verified by in vivo experiments and human samples 89 . Therefore, for an EV diagnostic biomarker of AP, large samples and multicenter clinical studies are needed. In the diagnosis of CP, no literature has indicated that EVs can be used as diagnostic biomarkers to distinguish CP patients from healthy people. However, PDAC is sometimes difficult to distinguish from CP in the early clinical diagnosis, leading to unnecessary pancreatic resection 104 , 106 . Therefore, Lai et al. 117 found that high expression of exosomal miR-10b, miR-20a, miR-21, miR-30c, miR-106b and miR-181a can effectively differentiate patients with PDAC from those with CP. After resection, the high expression of these miRNAs decreased to normal values. Moreover, Nakamura et al. 118 reported that the expression of exosomal miR-21 and miR-155 from pancreatic juice was significantly higher in PDAC patients than in CP patients. Compared with those of serum CA19-9 levels, the AUC values of exosomal miR-21 and miR-155 levels were significantly higher. Therefore, exosomal miRNAs may be useful and stable biomarkers for distinguishing patients with CP from those with PDAC. Furthermore, several studies have shown that exosomal DNA can also distinguish patients with CP from those with PDAC and healthy subjects. Yang et al. 122 indicated that exosomal DNA identified KRAS mutations in 29 of 48 (39.6%) cases and TP53 mutations in 2 of 48 (4.2%) cases in PDAC patients. For CP patients, the KRAS mutation was found in 5 of 9 (55.6%) cases; however, none had the TP53 mutation. In healthy subjects, the KRAS mutation was observed in 3 of 114 (2.6%) individuals, and none had the TP53 mutation. Therefore, the study indicates that circulating exosomal KRAS and TP53 mutations can be used to distinguish patients with CP from those with PDAC and healthy subjects. In recent years, compared with exosomal miRNAs and DNA as diagnostic biomarkers of disease, the concentrations and diameters of EVs could discriminate PDAC and CP patients 123 . In bile samples, the median concentration of EVs was 4.00×10 15 vs 1.26×10 14 nanoparticles/L in the PDAC group vs the CP group. In serum samples, the median concentration of EVs was 4.64×10 13 vs 7.58×10 12 nanoparticles/L in the PDAC group vs the CP group. In addition, the average diameter of EVs in the PDAC group was 277.8 nm; however, the average diameter of EVs in the CP group was 169.9 nm. Thus, EVs derived from bile have larger sizes and contain more proteins in the PDAC vs CP group. In the diagnosis of AIP, Nakamaru et al. reported that the expression of miR-21 was significantly upregulated in extracellular vesicles derived from serum from patients with type 1 autoimmune pancreatitis 126 . This study included 27 patients with type 1 AIP, 23 patients with chronic pancreatitis and 23 healthy controls (HCs). Microarray analysis and RT-PCR showed that the expression level of miR-21-5p was significantly higher in type 1 AIP patients than in HCs. Therefore, miR-21 of EVs derived from AIP patients' serum could be used as a diagnostic marker to distinguish AIP patients from healthy people. At present, there are no reports on potential therapeutic application of EVs in the treatment of AIP. However, it has been reported that EVs have potential therapeutic effect in other autoimmune diseases, such as type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus 134 . Research has been suggested that mesenchymal stem cells-derived exosomes might protect the pancreatic islets of patients with Type 1 diabetes by immunomodulatory effect to slow disease progression 135 . Similarly, Multiple sclerosis (MS) is a T cell-mediated autoimmune disease, which underlying mechanisms are unclear. Kimura have showed that MS derived exosomal let-7i regulates MS pathogenesis by blocking the insulin like growth factor 1 receptor and transforming growth factor beta receptor 1 pathway 136 . Therefore, more studies are needed to further investigate the mechanism and treatment of EVs in AIP. In the treatment of AP, previous studies have shown that exosomes derived from MSCs can be used to reduce inflammatory responses and treat inflammatory diseases 90 - 100 . Therefore, Wang et al. reported that exosomes derived from mesenchymal stem cells that overexpress Klotho attenuated the severity of pancreatic inflammation in caerulein-stimulated AR42J cells 101 . In this study, exosomes derived from MSCs that overexpressed Klotho decreased the expression of IL-6 and TNF-α compared to the control group. In conclusion, these results showed that MSC-exo Klotho alleviated inflammation and apoptosis in AP and that Klotho could be a potential targeted therapy for clinical treatment in AP. However, EVs have not been applied for drug delivery in the treatment of AP. The application of drug-loaded EVs can effectively improve the targeting ability of drugs. In addition, compared with liposomes, EVs have an advantage in the application of drug delivery for targeted treatment.