The potential roles of cigarette smoke-induced extracellular vesicles in oral leukoplakia

A robust body of evidence have accumulated documenting that cigarette smoke (CS) is an essential risk factor for multiple human diseases worldwide including cancers, cardiovascular disease, chronic lung diseases, asthma and inflammatory skin disorders [1‐4]. The prevalent use of CS and the series of diseases induced by CS result in serious economic and health burden to the world, especially for the undeveloped countries [5]. A cross-over study performed by Kerr et al. found that the heart rate of healthy individuals increased only after 4 h use of tobacco smoking/electronic cigarettes [6]. Moreover, the peak expiratory flow remarkably down-regulated in electronic cigarettes group [6]. These data suggested the acute exposure to CS influenced vascular and respiratory function. It is reported that CS is a complicated mixture of chemicals that contains about 10^15–17 oxidants/free radicals and up to 4500–4700 different compounds, such as nicotine, tobacco-specific nitrosamines, reactive aldehydes and quinones, per puff [7, 8]. Most of these components manifest great cellular toxicity pathogenesis by modulating the cellular survival, apoptosis, ferroptosis, autophagy, proliferation and angiogenesis [9‐11]. They also act as mutagen and carcinogen factors promoting tumorigenesis [12, 13].

The tissues of oral cavity are always inevitably to suffer damage, because it is the first part stimulated by CS. In fact, CS is one of the most established major risk factors for many oral diseases, including periodontal disease, dental caries, aphthous ulcers, primary Sjögren’s syndrome and peri-implantitis [14‐16]. According to a meta-analysis performed by Leite et al., smoking increased the risk of periodontitis by 85% and this risk could be decreased by about 14% if smoking factor is eliminated in this population, which well-demonstrated the detrimental effect of smoking on periodontitis [17]. CS also represents the most important etiological factor that promotes oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) [12, 18, 19]. Among the OPMDs, oral leukoplakia (OLK) is the most common potentially premalignant lesion that showing a primary white change of oral mucosa [20, 21]. The prevalence for OLK is between 1.5% and 2.6%, and the risk malignant transformation is up to 5–36% [22, 23]. Strong evidence have been found that the incidence of OLK was closely associated with the addiction of tobacco [24, 25]. In addition, the correlation between CS and OLK is underlined by a dose-dependent manner [26]. The complicated pathogenic impacts of CS are considered as the attribution that leads to OLK and even its malignant transformation, In a prior study by Ye et al., they found that the immortalized human oral mucosa epithelial cell line leuk1 could significantly accumulated reactive oxygen species (ROS) upon cigarette smoke extract (CSE) exposure compared to the unexposed co-cultured cells, indicating the destructive effect of oxidative stress on the structure and function of oral epithelium [27].

Recent evidence identify that the oxidative stress contributes to the release of extracellular vesicles (EVs) [28‐30]. EVs are a heterogeneous group of cell-derived membranous structures that are categories as exosomes and ectosomes according to their different mechanism of formation and approximate size [31] (Fig. 1). Exosomes are EVs in the size range of 40–160 nm in diameter with an endosome origin, termed a multivesicular body biogenesis [31, 32]. Different from exosomes, the ectosomes, including microvesicles and microparticles, are ubiquitous vesicles directly released from the plasma membrane through outward budding with a size range of 50 nm–1 μm in diameter [31, 32] (Fig. 1). EVs are derived from almost all types of cells and can be found in diverse biological fluids, including plasma, serum, lymph, saliva, urine, bile, cerebrospinal fluid and human milk [33, 34] (Fig. 2). EVs have emerged as important modulators in intercellular information transmission by delivering their biological cargo, such as proteins, lipids, RNAs and DNAs [35, 36] (Fig. 2).

Currently, emerging evidence have attempted to explain how CS affects the generation and release of EVs, and what is the specific molecular mechanism or pathways they interfered that cause the disease occurrence, especially for lung, cardiovascular and cancer-related diseases. However, little is known about how CS-induced EVs regulate the pathophysiological processes of OLK, even though the oral mucosa is the first-line site affected by CS. Thus, attempts to understand the roles of CS-induced EVs in the microenvironment of OLK are of great importance, which not only helps to clarify the immunopathogenesis of OLK in-depth, but also provides novel strategies for the diagnosis and treatment of OLK, even inhibition of its malignant transformation.

Although the traditional tissue biopsies remains the gold standard for disease diagnosis, the procedure is invasive and commonly constrained by its repeatability and brings economic and mental burden to patients [37, 38]. These limitations of traditional tissue biopsy promote the application of biofluids/liquid biopsy technique in clinical practice. Interestingly, EVs display great superiority in liquid biopsy with the following reasons (Fig. 2): first, the prevalence of EVs that exist in almost all body fluids makes it possible to implement less invasive operation and to apply in different types of diseases [33, 34]. Second, it is time saving, sample repeatable and cost effective due to the rapid advancement in EVs-related purification and identification techniques [39, 40]. Third, EVs are secreted continuously by living cells, providing exciting opportunities for real time monitoring of lesions, disease diagnosis, prediction and surveillance [41]. Fourth, the EVs contain differentially expressed cargos and specific surface markers from their parental cells, which could accurately reflect the pathological factors of diseases on both molecular and cellular levels [42, 43]. This might largely contribute to the progression of targeted therapy in future. Finally, the membranous shell of EVs show high stability to guarantee the long-term existence of their contents [44], which would not be affected by the different processing time of samples, ensuring the stable and reliable detection results.

The exposure of CS could increase the production of EVs from diverse types of cells, including endothelial cells, epithelial cells and fibroblasts, which might rely on the redox modifications of protein thiols [45, 46]. Serban et al. showed that CS-induced endothelial microparticles (EMPs) contained predominantly exosomes that were largely enriched in let-7d, miR-191, miR-126 and miR-125a [47], which may be important in predicting potential function of exosomes as paracrine effectors in patients with chronic obstructive pulmonary disease (COPD). In addition, these EMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for microparticle release [47]. The aSMase is an enzyme that is classically associated with stress-induced apoptosis and shown higher activity in plasma of patients with COPD or of CS-exposed mice. These reveal that CS-induced EMPs could be a promising indicator of the functional state of the endothelium in smokers. Stassen et al. identified that CSE promoted the secretion of CD63⁺CD81⁺ extracellular vesicles (exosomes) by bronchial epithelial cells [48]. Moreover, these exosomes were enriched in tissue factor (TF) and exerted a TF-dependent procoagulant activity compared with exosomes by unexposed cells [49], suggesting that TF⁺ exosomes might contribute to the elevated cardiovascular risk in smokers and consider as biomarkers of exposure to CSE.

The changes of circulating EVs induced by CS are in essence due to the changes of EVs secreted by various cells under the stimulation of CS, and finally released into the peripheral blood or local tissues. The altered composition in EVs upon CS exposure ultimately leads to their dysregulated functions after being engulfed by recipient cells. Researches on the production of EVs, the specific changes of contents, and the mechanism of modulating cells upon CS stimulation could contribute to our deeper understanding in the field of EVs, which will benefits for future use of EVs as diagnostic biomarkers and therapeutic targets of smoke-induced-related diseases.

The health of oral mucosal epithelial is prone to be damaged, because it is the first affected site by CS. Exposure to CS is identified as a major risk factor for OLK and its malignant transformation to OSCC. However, evidence done on the role of EVs in CS-induced OLK is still in its infancy. Based on the prior literature, upon CS stimulation, the biogenesis of EVs is significantly elevated, the composition of EVs and the signaling transduction via EVs are obviously changed, which is closely associated with the occurrence and progression of disease. Exposure to cigarette smoke contributes to the production of EVs from different types of cells including epithelial cells, macrophages, T cells, and monocytes. The bioactive cargo (proteins, cytokines, RNAs, and DNAs) of these EVs induced by CS stimulation are significantly changed, which acts as mediators for intra/inter-cellular communication. When these EVs are engulfed, they can change the biological behavior of the recipient/target cells and finally lead to the onset of OLK pathogenesis or even carcinogenesis (Fig. 3). Further researches should be performed to identify these assumptions. Elucidating the functions of CS-induced EVs in the pathophysiological processes of OLK is of great significance for the development of diagnostic biomarkers, prognostic evaluation, and therapeutic targets of OLK.