*3.3. Epithelial Recovery*

Apart from their effects on immunomodulation and angiogenesis, a growing body of evidence has revealed the therapeutic effects of MSC-exosomes on epithelial injuries. In summary, MSC-exosomes increase the proliferation and survival of epithelial cells.

MSC-exosomes accelerate epithelial recovery in wounded tissues via their miRNA cargo. By using different disease models, recent studies have reported some specific roles of MSC-exosomal miRNAs in mediating epithelial recovery, such as that of miRNA-135a in increasing epithelial cell migration by suppressing expression of the gene encoding LATS2 during cutaneous wound healing [81] and that of miRNA-126 in activating the PI3K-AKT and MAPK pathways during cutaneous healing in a rat model of diabetes [82]. Moreover, exosomes may carry specific cargo such as foreign miRNA products. For example, in a study of MSCs genetically modified to overexpress a variety of miRNAs, including miRNA-100, miRNA-146a, miRNA-21, miRNA221 and miRNA-143, it was found that these exosomes enhance DNA synthesis, thus promoting the proliferation of vaginal epithelial cells [83]. In an acute lung injury model, exosomes from miRNA-30b-3p-overexpressing

MSCs protected type II alveolar epithelial cells against apoptosis by downregulating serum amyloid A3 (SAA3) [36].

In fact, increasing cell proliferation and survival by activating PI3K/Akt and MAPK are typical effects of both MSCs and their exosomes. Other effects of MSC-exosomes during epithelial recovery should be mentioned, including antioxidation. To our knowledge, oxidation is a harmful occurrence that impairs cell survival. In a renal injury model, MSCexosomes inhibited apoptosis in tubular epithelial cells by reducing the level of reactive oxygen species (ROS) [84]. As documented, mitochondrial dysfunction is an important biological event that is closely associated with lung disease pathogenesis and/or progression [85]. Mechanistically, MSC-exosomes improve the mitochondrial function of lung epithelial cells by targeting division/fusion-related genes such as *rhot1*, *mfn1* and *opa1* [86]. Simultaneously, exosomes have been shown to carry functional mitochondria and promote mitochondrial transfer events [87], further demonstrating that MSC-exosomes have the potential to alleviate mitochondrial damage and control the progression of tissue damage.

MSC-exosomes also inhibit the epithelial-mesenchymal transition (EMT) [88–90], which is critical in inducing tissue fibrosis, resulting in pathological rather than functional restoration of damaged tissue. Although the underlying mechanisms by which MSCexosomes restrict fibrotic development are not clear, MSC-exosomes inhibit activation of the TGF-β1/Smad pathway [91] while enhancing expression of zona occludens protein-1 in epithelial cells, which is related to cellular tight junctions [92]. Therefore, MSCexosomes at least reduce epithelial depletion due to transformation, thereby maintaining the integrity of the epithelium and suppressing tissue fibrosis. Collectively, MSC-exosomes promote epithelial recovery by facilitating regeneration, inhibiting apoptosis and reducing EMT depletion.

## **4. Role of MSC-Exosomes in Repairing Radiation Damage**

Despite the use of advanced treatment techniques, radiation damage is common and often unavoidable in cancer patients during or after receiving radiotherapy. The actions of ionizing radiation on biological molecules can be segmented into direct and indirect effects. DNA damage in cells can be induced by the direct effects of ionizing radiation, and it can also be caused by the oxidative stress reaction mediated by reactive oxygen species (ROS) generated by indirect effects. Ionizing irradiation-induced DNA double-strand breaks, oxidative stress, vascular damage, and subsequent inflammation are typical events in the acute phase of the pathogenesis of radiation damage, and if these events are not well managed, fibrosis occurs as a pathogenic feature in the chronic phase [55].The potential use of MSCs in repairing radiation-induced acute damage in the hematopoietic system, liver, lung, gastrointestinal tract, or skin has been explored [88,93–95], and the results indicate that MSCs have several therapeutic features including increased proliferation and survival of tissue/organ-specific stem/progenitor cells, the promotion of angiogenesis, anti-inflammation and oxidation, and the reduction of fibrotic pathogenesis [96]. The above findings indicate that MSC-exosomes have similar potencies to those of MSCs in repairing tissue or organ damage due to disease. Moreover, recent advances have demonstrated the repair of radiation damage by MSC-exosomes. In the following sections, we elaborate on the therapeutic effects of MSC-exosomes on radiation damage in the hematopoietic system and nonhematopoietic system (Figure 2).

Hematopoietic cells are sensitive to radiation exposure, which can lead to bone marrow failure. Several studies have shown that MSC-exosomes are capable of repairing radiationinduced hematopoietic system injury, but the exact mechanism is unclear. A few studies have suggested that the following processes may contribute to the relevant mechanism. (i) MSC-exosomes can transfer miRNAs with pro-regenerative or anti-apoptotic effects to irradiated hematopoietic cells. For example, intravenous delivery of human bone marrow MSC-derived extracellular vesicles (MSC-EVs, mainly comprising exosomes and microvesicles) swiftly normalized the counts of peripheral blood cells in mice that received whole-body irradiation because their cargo content, including miRNA-221, miRNA-451

and miRNA-654-3p, promoted the proliferation of irradiated marrow cells and miRNA210- 5p, miRNA106b-3p and miRNA155-5p prevented radiation-induced hematopoietic cell apoptosis [17]. (ii) MSC-exosomes can restore hematopoiesis by stimulating secretion of hematopoiesis-related cytokines. A previous study has suggested that human placental MSCs rescue radiation-induced hematopoiesis in mice by secreting human hematopoiesisrelated cytokines, including G-CSF, MCP-1, IL-6 and IL-8 [16], and this effect can be observed with MSC-exosomes as well [18]. In fact, the data from a recent study show that MSC-exosomes are capable of inducing production of high levels of hematopoiesis-related cytokines such as G-CSF, IL-6, IL-8 and VEGF by macrophages in vitro [18]. (iii) MSCexosomes have several other features that cause the remodeling of hematopoietic cells. For example, incubation with MSC-exosomes enhances the activity of macrophages, which are regarded as the key regulators of demand-adapted hematopoiesis [89]. MSC-exosomes are also able to directly restore irradiated bone marrow MSCs, which are considered to be potent contributors to hematopoiesis. One critical mechanisms involves the alleviation of DNA damage and oxidative stress via Wnt/β-catenin signaling pathway activation [90].

In addition to remodeling the hematopoietic system, MSC-exosomes are capable of protecting the skin, gastrointestinal system, respiratory system and other systems against radiation damage. At the micro level, radiation damage is essentially attributed to the large number of oxygen free radicals generated by ionizing radiation, which subsequently result in DNA double-strand breaks. Previous studies have reported that MSCs play a key role in alleviating DNA damage and oxidative stress damage [97]. MSC-exosomes, the functional role of which depend on their cargo derived from cells of origin, exert similar remodeling effects [98–100]. For example, in an oxidative stress-induced skin injury model, MSC-exosome treatment decreased ROS generation and subsequent DNA damage and improved the antioxidant capacities of damaged cells through NRF2 signaling [100]. Other studies have found that after MSC-exosome treatment in an ischemic renal disease model, damaged renal cells showed reduced oxidative stress marker (MDA) levels, increased anti-oxidant marker (SOD and CAT) levels, and significantly reduced DNA damage parameters [98]. However, the underlying molecular mechanism is poorly understood. Notably, studies have shown that some miRNAs that are contained in MSCexosomes such as miRNA210 are able to repair DNA double-strand breaks [17,19], which suggests that exosomes may exert remodeling functions in a noncoding RNA-mediated epigenetic manner. This may explain the decrease in the apoptosis of skin epidermal cells, lung alveolar epithelium, intestinal epithelium and various parenchymal cells after MSCexosome exposure [60,101–103]. Such noncoding RNA cargo may affect nonhomologous end-joining (NHEJ), which is common and essential in mammalian cell DSB repair [104]. Current evidence suggests that MSC-exosomes treatment is beneficial for the repair of oxidative stress-induced damage [22,105,106], although the exact functional components remain to be revealed. In addition, it has been found that intravenously injected MSC-EVs (including exosomes) are highly distributed in parenchymal organs such as the liver and spleen in a whole-body irradiation mouse model [105]. This may provide the context for the development of cures for radiation-induced parenchymal organ injury. Microvascular endothelial apoptosis has been recognized as the primary process that initiates radiation-induced injury [106]. Studies have found that local MSC-exosome treatment can facilitate the proliferation of vascular endothelial cells by activating the Wnt4/β-catenin pathway [75,103]. MSC-exosomes also have the potential to cure radiation-induced injury partly due to their potent pro-angiogenic factor cargo such as PDGF, FGF and EGF [61], which induce endothelial proliferation and differentiation in vitro and neovascularization in vivo [107,108].

On the other hand, exosomal targeting of cells is mediated by members of the integrin and tetraspanin families or other associated molecules based on their expression [109,110]. An experimental study demonstrated that radiation contributes to increased formation of the integrin and tetraspanin complex CD29/CD81 on the cell surface, thus enhancing uptake of exosomes by irradiated cells [15]. This further illustrates the potential use of

MSC-exosomes in the treatment of radiation damage. In general, the repair effect of MSCexosomes on radiation damage in multiple systems is partly attributed to their bioactive cargo, which predominately consists of noncoding RNAs and functional proteins. These molecules influence the expression of target genes associated with radiation-induced damage or tissue regeneration due to epigenetic regulation. Overall, more experimental studies are required to further explore the molecular mechanisms involved.

#### **5. MSC-Exosomes in Repairing Radiation Damage: Perspective and Challenges**

With regard to radiation-induced damage, it has been revealed that MSCs play a crucial role in tissue damage treatment and prevention. Moreover, the superior properties and improved safety of MSC-exosomes make them novel candidates for curing radiation-induced damage. They exert therapeutic effects mainly by facilitating angiogenesis, promoting cellular regeneration, and probably by enhancing the repair function through immunomodulatory effects. More importantly, there are several methods that can be used to enhance the efficacy of remodeling damaged tissue. On the one hand, exosomes secreted by MSCs with genetic modifications are a promising alternative treatment, such as exosomes derived from SDF1-overexpressing MSCs for microvascular regeneration [111]. On the other hand, MSCs can be pretreated in vitro before exosomes are collected, such as with hypoxia-treated MSC-exosomes in ischemia-related disease [55]. Last, but equally important, the tropism of exosomes can be improved by increasing expression of specific receptors on the surface of the original MSCs. Current studies on the treatment of radiation-induced damage by MSC-exosomes are mostly based on the acute phase, whereas little work has been performed on the treatment of chronic radiation-induced damage by MSC-exosomes. Notably, evidence suggests that MSC-exosomes reverse EMT of endometrial epithelial cells via the TGF-β1/Smad pathway [91] and of tubular epithelial cells via enhanced tight junctions [92]. In general, sustained EMT is a critical mechanism that underlies the fibrotic pathology of tissue [112]. Thus, it can be reasonably inferred that MSC-exosome treatment has potential for preventing tissue fibrosis in the chronic phase of tissue damage. Therefore, despite limited evidence of the repair role of MSC-exosomes in chronic radiation-induced damage, it is important that researchers make further efforts to explore their therapeutic and underlying potential in chronic radiation-induced damage. This will provide a new context for the future application of MSC-exosomes to treating chronic radiation damage (Figure 2).

In fact, there are several deficiencies with regard to managing diseases by using MSC-exosomes. (i) One concern is the challenges due to the instability of contents of exosomes. For example, studies have shown that the amount of exosomal miRNA cargo is influenced by the irradiation dose and pH value of the culture medium [113,114]. The precise experimental conditions for exosomes are more difficult to control compared to MSCs. (ii) Another concern is the lack of a uniform standard for the purification and quantification of exosomes from conditioned media. Overall, it is difficult to determine the equivalent dose of exosomes in dose-dependent experimental studies, which may lead to different conclusions as results can be affected by exosome content and impurities. Therefore, it is appropriate to find an ideal method for constructing a precise equivalent dose of exosomes for experimental purpose. Although the effects of MSC-exosomes in various disease models have been clearly shown, the exact components and mechanisms of therapy are not entirely clear. miRNAs and functional proteins may play major roles, yet the role of MSC-exosomes in tumor growth and metastasis remains controversial. Previous studies have shown that MSC-exosomes can promote tumor growth in vivo [115], but a recent study revealed that MSC-exosomes enhance radiotherapy-induced tumor cell death in primary and metastatic tumor foci through synergistic and bystander effects [116]. Urgent issues for cancer patients receiving radiotherapy include the adjuvant antitumor effect and resistance to radiation damage. There is a great need for researchers to elaborate on the role of MSC-exosomes in regenerative medicine for the treatment of radiation damage.
