Mesenchymal Stem/Stromal Cell-Derived Exosomes Part 2

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4.2.T Cell Regulation

MSC-exosomes also modulate the functions or activities of T cells (Table 3). BM-MSC-exosomes were reported to convert Th1 to Th2, and reduce Th17 differentiation in PBMCs [97]. More importantly, BM-MSC-exosomes increased the level of Tregs in PBMCs. These effects might be mediated by the suppression of pro-inflammatory cytokines such as TNF-α and IL-1β, and an increase of anti-inflammatory cytokine TGF-β [9]. Another report also revealed that BM-MSC-exosomes modulate immune reactions in PBMCs from asthmatic patients [98]. The proliferation and immune-suppression capacity of Tregs was promoted by BM-MSC-exosomes through upregulation of IL-10 and TGF-β1 in PBMCs. Tregs were also induced by exosomes derived from TGF-β/IFN-γ-stimulated UC-MSCs 【99】. cynomorium benefits The proposed mechanism of this Treg regulation is an antigen-presenting cell (APC)-but not a CD4+T cell-dependent pathway [97]. A previous report demonstrated that differentiation of Tregs is mediated by activated APCs, which is induced by ESC-MSC-exosomes in a myeloid differentiation primary response 88 (MYD88)-dependent manner [100]. It has been also reported that mouse ASC-exosomes induce the increase of Tregs population in the splenic mononuclear cells of mice with streptozotocin-induced autoimmune type 1 diabetes mellitus [100]. Upregulation of Tregs has been also reported in the multiple sclerosis(MS) mouse experimental autoimmune encephalomyelitis model by human BM-MSC-exosomes [101], and a concanavalin A(Con A)-induced mouse liver injury model by mouse BM-MSC-exosomes [102]. Downregulation of proliferation of activated T and B lymphocytes by BM-MSC-exosomes has been also reported [103]. Of note, studies by Del Fattore et al. and Di Trapani et al. have shown that EVs from BM-MSC suppress Tcell proliferation indirectly by induction of Treg differentiation, unlike MSCs, which directly suppress T cell proliferation [103,104]. In addition, UC-MSC-EVs purified by size exclusion chromatography only showed an inhibitory effect on T cell proliferation and did not induce cytokine response and monocyte polarization [105]. Further studies are needed to elucidate the molecular mechanism of these regulations by MSC-exosomes.

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4.3. Inflammation in Skin

It was reported that human BM-MSC-exosomes reduce photoaging and inflammation in mice, which might be helpful to prevent and treat cutaneous aging [107]. Human ASC-exosomes were reported to enhance neovascularization and the survival of the skin flap in a rat IR injury of the flap transplantation model by reducing inflammation and apoptosis [108]. In this experimental setting, ASC-exosomes derived from H, O2-preconditioned ASCs had better outcomes compared to those from unconditioned ASCs. Regulation of inflammation is also important to treat atopic dermatitis (AD), a representative skin inflammatory disease. It has been demonstrated that human ASC-exosomes can ameliorate AD in two distinct mouse models via reducing pathological symptoms and expression of multiple cytokines such as IL-4, IL-5, IL-13, IL-17, IL-23, IL-31, TNF-α, IFNy, and thymic stromal lymphopoietin (TSLP)[20,109]. Th2 cytokines, such as IL-4, IL-5, I-13, and IL-31, mainly produced by activated Th2 cells, are crucial contributing factors in the development of allergic inflammation in the skin [158,159]. desert hyacinth Notably, Th2 cytokines including IL-4, IL-13, and I-31 are therapeutic targets for AD[160]. Additionally, ASC-exosomes also reduced the infiltration of inflammatory dendritic epidermal cells(IDECs, CD86+, and CD206+), which led to the release of pro-inflammatory cytokines in lesional skin of AD[20]. Taken together, MSC-exosomes are key players in skin regeneration by promoting macrophage M2 polarization with anti-inflammatory properties and reducing pro-inflammatory cytokine-releasing cells such as M1 macrophages and IDECs.

4.4. Immunomodulation in Other Inflammatory Diseases

Immunomodulation by MSC-exosomes was also reported in various inflammatory disease models. Examples are as follows:(1)Exosomes from melatonin-preconditioned rat BM-MSCs reduced the kidney injury in a rat renal IR injury model by decreasing oxidative stress and apoptosis, increasing anti-oxidant, and anti-apoptotic proteins, and enhancing angiogenesis [110]. In addition, mouse BM-MSC-exosomes reduced renal injury in a CCR2-dependent manner [111]. Human UC-MSC-exosomes have been also reported to reduce cisplatin-induced acute kidney injury(AKI) in rats in an autophagy-dependent manner [112];(2)Human UC-MSC-exosomes reduced the experimental autoimmune uveitis in rats [113];(3)Human placenta-derived MSC (PL-MSC)-exosomes reduced the tissue fibrosis and inflammation in a mouse Duchenne muscular dystrophy(DMD) model partly through the delivery of miR-29c[114];(4)Human UC-MSC-exosomes improved the pathology of lung, cardiac, and brain in neonatal mice with BPD by reducing the pulmonary inflammation and alveolar-capillary leak potentially through the delivery of TSG-6 [115] or macrophage M2 polarization [86];(5) Targeted delivery of mouse BM-MSC-exosomes by rabies viral glycoprotein (RVG) peptide improved the cognitive function of transgenic APP/PS1 mice by reducing plaque deposition, the level of Aβ, activation of astrocytes, and the expression of pro-inflammatory cytokines TNF-α, IL-β, and IL-6, while increasing the levels of IL-10, IL4, and IL-13 [116];(6) Human BM-MSC-EVs improved the neurological impairment and long-term neuroprotection in stoke mice by attenuating the post-ischemic immunosuppression and lymphopenia,and as well as stimulating neurogenesis and angiogenesis [117];and (7) Mouse BM-MSC-exosomes decreased the threshold for thermal and mechanical stimuli in a mouse diabetic peripheral neuropathy model by regulating multiple factors involved in macrophage polarization through the delivery of miRNAs targeting the TLR4/NF-kB signaling pathway [118]. flavonoid extraction method pdf Other inflammatory diseases, which can be modulated by MSC-exosomes or MSC-EVs, include OA [119,120], intervertebral disc degeneration (IVDD)[123], spinal cord injury [124-126], myocardial infarction [127,128], acute lung injury(ALI)[129-131],idiopathic pulmonary fibrosis(IPF)[132], hepatic IR injury [133],liver fibrosis [134],acute liver failure [135], IBD [92,136], necrotizing enterocolitis [137], abdominal aortic aneurysm [1391.brain injuries [139-143l, urethral stricture [144l,status epilepticus (SE)[145,146], retinal injuries [147,148], sepsis [150], and graft-versus-host disease(GvHD)[150]. The immunomodulation of MSC-exosomes was highlighted in their first clinical application in an allogeneic setting to a patient suffering from steroid-refractory GvHD[151]. In this study, MSC-exosomes modulated the status of the patient's immune cells. The differentiation of Tregs by MSC-exosome-mediated APC activation might contribute to the suppression of GvHD [99].

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In summary, MSC-exosomes or MSC-EVs suppress inflammatory responses in diverse disease settings by inducing polarization and differentiation of M2 macrophages and Tregs. Although exact cargo compositions and MoA of exosomes need to be further studied, mounting evidence suggests that MSC-exosomes have similar anti-inflammatory and immunomodulatory properties to MSCs, which could be beneficial for the treatment of inflammatory and autoimmune diseases, as well as for skin regeneration. However, MSC-exosomes may also possess distinct immunomodulatory mechanisms from those of MSCs, which needs to be further elucidated to facilitate application in clinical settings.

5. Anti-Aging Effects of MSC-Exosomes

Aging, defined as the irreversible deterioration of physiological processes of organisms over time, is characterized by nine hallmarks: cellular senescence, mitochondrial dysfunction, deregulated nutrient sensing, epigenetic alterations, telomere attrition, genomic instability, altered intercellular communication, and stem cell exhaustion [161,162]. Among these, cellular senescence has recently been focused on as one of the key factors in the complex aging process as it is interlinked with other hallmarks [163]. Senescent cells are accumulated in the tissues of vertebrates with age. flavonoids Interestingly, the removal of senescent cells in animals results in the delayed onset of age-associated diseases [164-168]. Senescence is characterized by a stable cell-cycle arrest in the Gl phase and an inflammatory response called senescence-associated secretory phenotype (SASP), which modifies the microenvironment around senescent cells [161]. Senescence is induced by intracellular and extracellular stresses, including replicative stress, DNA damage, oncogene activation, telomere damage or shortening, inflammation, mitochondrial dysfunction, oxidative stress, and drug insults, to eliminate damaged cells, and prevent potential malignant cell transformation [161,169]. Components of the SASP include growth factors, pro-inflammatory cytokines, chemokines, and extracellular matrix remodeling enzymes [170-172]. SASP contributes to inflammaging, a term coined by Franceschi et al. in 2000, which describes low-grade, controlled, asymptomatic, chronic, and systemic inflammation associated with aging processes [173]. Indeed, many pieces of evidence point out that inflammation may ultimately lead to age-related diseases [174-176]. Thus, interventions that suppress SASP and inflammation processes may hold the potential to alleviate various chronic diseases [177]. In addition, senescent cells display the expression of senescence-associated β-galactosidase (SA-β-gal), increases in mRNAs/proteins including p53， p21， p16， and γ-H2AX， and a decrease in cell proliferation 【161】.

5.1. EVs in Senescence

EVs or exosomes have a role in both transferring the senescence phenotype and alleviating or even rejuvenating senescence cells, depending on their originating cells. Studies suggest that EVs or exosomes act as new components of the SASP and age-related disease markers [169-171]. Age-related changes in EVs or exosomes have been reported to result in the following:(1) an increase in the number of EVs or exosomes released during senescence of fibroblast, epithelial cells, and cancer cells [178,179];(2) a decrease in the levels of circulating EVs with age, at least from the 30s to 60s in humans, as well as in mice and rats [180-182]; and (3) changes of EV or exosome composition (miRNAs, proteins, or lipids) associated with aging or senescence [171,183-189]. In fact, EVs or exosomes mediate paracrine senescence, transmitting senescence from senescent or diseased cells to normal cells, in both normal and disease conditions [169,190-195]. This paracrine senescence is positively correlated with the uptake of exosomes by target cells and is prevented by inhibition of exosome generation [169].

It has also been reported that various long noncoding RNAs (ncRNAs) are enriched in exosomes from senescent cells and accumulating evidence shows that these RNAs may contribute to the progression of age-related diseases such as atherosclerosis, type 2 diabetes, osteoporosis, OA, rheumatoid arthritis, Parkinson's disease, and multiple sclerosis [196]. It has also been reported that various long noncoding RNAs (ncRNAs) are enriched in exosomes from senescent cells, and accumulating evidence shows that these RNAs may contribute to the progression of age-related diseases such as atherosclerosis, type 2 diabetes, osteoporosis, OA, rheumatoid arthritis, Parkinson's disease, and multiple sclerosis. For instance, in atherosclerosis, monocytes exposed to oxidized low-density lipoprotein (oxLDL) drive the progression of the disease. A study by Chen et al. has shown that THP-1, a monocyte cell line, treated with oxLDL shows significant upregulation of exosomal lncRNA GAS5, and these exosomes cause apoptosis of endothelial cells [197]. The role of exosomal lncRNA was also highlighted by Ruan et al. In this study, it was found that exosomal lncRNA-p3134 contents in diabetic patients were higher than those in non-diabetic subjects [198]. Senescent cells also exert effects by transferring protein cargo. For instance, exosomes from drug-induced senescent multiple myeloma cells promote activation and proliferation of NK cells by transferring IL-15RA and IL-15 [199]. Taken together, EVs from senescent cells may serve as disease markers.

5.2.Anti-Aging Effects

It has been elusive that circulating mediators are responsible for rejuvenating multiple tissues of old organisms by parabiosis of young organisms[200]. Very recently, it was demonstrated that EVs from young mice plasma extend the lifespan of old mice by delaying aging through exosomal nicotinamide phosphoribosyltransferase (en amp)[201]. Another study also reported that exosomes from young mice could transfer miR-126b-5p to the tissue of old mice, and reverse the expression of aging-associated molecules such as p16, mTOR, IGF-1R, and telomerase-related genes including Men1, Mre11a, Tep1, Terf2, Tert, and Tnks, in aged mice [202]. Another report revealed that EVs derived from the serum of young mice attenuated inflammation in old mice by partially rejuvenating aged T-cell immunotolerance [203]. Implantation of hypothalamic stem/progenitor cells, which were genetically engineered to survive aging-related hypothalamic inflammation, was reported to induce retardation of aging and extension of lifespan in mid-aged mice [204].

More importantly, growing evidence suggests that cellular senescence can be alleviated or reversed by EVs or exosomes derived from stem cells (Table 4) [205-214]. Human ASC-exosomes reduced the high glucose-induced premature senescence of endothelial progenitor cells (EPCs) and enhanced wound healing in diabetic rats [205]. In the same study, overexpression of nuclear factor erythroid 2-related factor 2 (NRF2)in human ASC-exosomes further reduced premature senescence of EPCs and promoted wound healing in diabetic rats by modulating the expression of various proteins [205]. Since high glucose in diabetic patients induces reactive oxygen species (ROS) and inflammation, which promotes senescence and impairs the function of EPCs, reduced senescence of EPCs by ASC-exosomes may be beneficial for the treatment of diabetic foot ulcers [205]. It has also been reported that human ASC-exosomes contain lncRNA MALAT1 and recover the function of motor behavior with a reduction of cortical brain injury in a rat traumatic brain injury model [142]. Regarding this, a study revealed that the MALATl expression is reduced in aged mice and that treatment of human UC-MSC-exosomes containing MALAT1 prevents aging in D-galactose (gal)-treated mice and senescence in H, O-treated H9C2 cardiomyocytes [206]. MALATl is one of the candidates for anti-aging effects in stemcell-derived exosomes since MALAT1-knockdown in UC-MSCs abolished these effects of UM-MSC-exosomes. Similarly, exosomal miR-146a was known to negatively regulate the senescence of MSCs by targeting the NF-B signaling [191]. Recently, miR-146a in AF-MSC-exosomes was reported to reduce LPS-induced inflammation in the human trophoblast cells [215]. The miR-146a is also known to be enriched in human UC-MSC-exosomes by TNF-α-preconditioning, and mediate anti-inflammatory effects in a rat urethral stricture model [145]. Antioxidant enzymes peroxiredoxins (PRDXs) were reported as being highly enriched in iPSC-EVs and BM-MSC-EVs [208]. Transferring of PRDXs by these EVs resulted in alleviation of cellular aging phenotypes such as increases of SA-β-gal, p21, p53, IL-1α, IL-6, and γ-H2AX in both replicative and genetically induced senescent MSCs 【209】. hesperidin uses Interestingly， proteomic analysis revealed that ASC-exosomes also contain PRDXs such as PRDX1, PRDX4, and PRDX6 [109]. Human ASC-exosomes was also reported to reduce IL-1β-induced senescence in osteoblasts from OA patients 【209】.In this study， ASC-exosomes reduced not only the levels of SA-β-gal，γ-H2AX， and IL-6 protein, but also the levels of prostaglandin E2, oxidative stress, and mitochondrial membrane potential. It has been reported that miR-214 in exosomes prevents senescence of endothelial cells by repressing the expression of ataxia telangiectasia mutated (ATM) protein by targeting the 3'-untranslated region (UTR) of its mRNA [216]. Interestingly, the next generation sequencing (NGS)analysis revealed that ASC-exosomes also contain miR-214 (Ha et al. unpublished observation).

Mouse miR-29la-3p was identified to target the TGF-β2 receptor and as a cargo of mouse ESC-exosomes [211]. Treatment of mouse ESC-exosomes reduced the SA-β-gal expression and promoted cell proliferation and migration of replicative or adriamycin-induced senescent HDFs [211]. It was reported that human ESC-exosomes inhibited D-gal-induced senescence of human vascular endothelial cells(HUVECs)[212]. Treatment of ESC-exosomes resulted in a decrease in SA-β-gal activity, p16 and p21 protein levels, and ROS in HUVECs, and an increase in cell proliferation, migration, and tube formation of HUVECs. The miR-200a in ESC-exosomes reduced the level of Kelch-like ECH-associated protein 1 (KEAP1)by targeting the 3'-UTR of KEAP1 mRNA.As a result, the level of NRF2, a master regulator of anti-oxidative responses [217], was increased to induce the expression of its downstream targets such as heme oxygenase 1(HO1), superoxide dismutase (SOD), and catalase (CAT) [213]. ESC-exosomes promoted pressure ulcer healing in D-gal-induced aged mice by reducing endothelial senescence and increasing angiogenesis [212]. Human iPSC-exosomes were reported to protect HDFs from UVB damage, reduce the senescence-associated MMP-1/3 expression, and induce synthesis of collagen type I in both UVB-damaged and senescent HDFs [214]. Human iPSC-exosomes were also reported to reduce SA-β-gal and increase cell viability and tube formation of high glucose-injured HUVECs with unknown mechanisms [214]. Exosomes from various cells are also useful as a delivery vehicle of biomolecules to suppress senescence. The miR-675 was discovered as a candidate marker for aging [207]. Delivery of miR-675 through UC-MSC-exosomes reduced the SA-β-gal expression, and the levels of p21 and TGF-β1 proteins in H2O2-induced senescent H9C2 cells by targeted downregulation of TGF-β1. Additionally, miR-675-UC-MCS-exosomes promoted perfusion in ischemic hindlimb by inhibiting the expression of both mRNAs and proteins of p21 and TGF-β1[207]. Another study reported that exosomes derived from Wnt4-overexpressed mouse thymic epithelial cells (TECs) inhibited dexamethasone-induced aging phenotypes in TECs [218].

Taken together, MSC-exosomes confer anti-senescence effects through their unique miRNA, lncRNA, and enzyme contents. By inducing proliferation and reducing SASP in senescent cells, they hold great potential to reduce senescent cells in tissues. Since the removal of senescent cells from tissues was reported to create a pro-regenerative environment [168]and tissue homeostasis [166], application of MSC-exosomes to remove the senescent cells may be a preferable approach to induce the regeneration or rejuvenation of tissues.

6. Cutaneous Wound Healing by MSC-Exosomes

A wound is a type of injury to the skin. An open wound is caused by a tear, cut, or puncture, and a closed wound is caused by blunt trauma [219]. Cutaneous wounds can be classified into acute and chronic wounds [220]. Acute wounds are highly prevalent from a loss of dermis and epidermis caused by mechanical, chemical, biological, or thermal injuries. Chronic wounds, on the other hand, are common comorbidities of complex diseases such as obesity, diabetes, and vascular disorders. Four categories of chronic wounds include pressure ulcers, diabetic ulcers, venous ulcers, and arterial insufficiency ulcers according to the Wound Healing Society [221]. Since chronic wounds do not heal within three months, they are considered as non-healing wounds [222,223]. Another major medical issue is pathological wound healing and scar formation, which cause both physiological and psychological challenges [224]. The annual Medicare cost for the treatment of acute and chronic wounds was estimated from $28.1 to $96.8 billion [225]. In addition, the annual product market for wound care is estimated to reach $15 to $22 billion by 2024[225].

Cutaneous wound healing is the complex process of restoring the injured skin. It consists of four phases: the homeostasis, inflammatory, proliferative, and remodeling phases [226-228]. Responses in these phases are tightly coordinated to secure vital skin barrier functions [224]. However, the mechanism of cutaneous wound healing and the interplays between a variety of cells during the wound healing process have been only partly delineated [229]. Many cell types interact with each other in a highly sophisticated sequence during the cutaneous wound healing process as follows [230]:(1)the platelets initiate the formation of the blood clots, which consist of platelets, red blood cells, and extracellular matrix molecules in the first homeostasis phase;(2) neutrophils, monocytes, as well as macrophages are major players during the inflammatory phase. Chemotactic factors released by neutrophils attract monocytes and cytokines from macrophages and stimulate the migration of fibroblasts to enter the injured site from the surrounding normal tissues;(3) angiogenesis and vascularization of endothelial cells provide oxygen supply to support the proliferation of migrated cells in the wound site during the proliferative phase. Fibroblasts also differentiate into myofibroblasts to generate a tensile strength in the wound. In addition, fibroblasts secrete growth factors, which activate the migration and proliferation of keratinocytes. Reepithelialization is completed by stopping the migration of cells by contact inhibition [230]; and (4)remodeling through apoptosis of fibroblasts, myofibroblasts, and other cells, and degradation of extracellular matrix occur during the wound scar remodeling phase, which spans months to years. Adverse scarring, caused by aberrant wound healing, includes chronic non-healing wounds and pathological scarrings such as hypertrophic scars and keloids, and it affects millions of people globally since currently, no effective treatment option is available [224]. The prevention or reduction of scars is also an important issue to solve in regenerative aesthetics [231].

MSC-EVs or MSC-exosomes orchestrate all phases of skin wound healing because of their ability to modulate inflammation, activate migration and proliferation of various cells including immune cells, fibroblasts, and keratinocytes, and even ameliorate scarring (Table 5)[85,87,88,205,226,231-245]. As an example, complete reepithelialization was reported in a rabbit cutaneous wound healing model by EVs from rabbit ASCs and BM-MSCs with an unknown mechanism [232]. Human ASC-EVs were also reported to enhance cutaneous wound healing in rats [233].

6.1. Hom1eostasis Phase

During the homeostasis phase, the formation of blood clots by platelets protects the injured site. Up to now, no direct evidence has been available that shows the involvement of MSC-exosomes in blood clotting during wound healing. A recent result might suggest the potential benefit of MSC-exosomes on blood clotting in the wound healing process; human UC-MSC-EVs have been reported to induce blood coagulation in vitro [244]. However, further studies are required to analyze the effects of MSC-EVs or MSC-exosomes in blood clotting in both healthy and disease conditions.

6.2.Inflammatory Phase

Regulation of inflammation is also important in skin regeneration during the wound healing process. Although inflammation is one phase of the normal skin repair cascade, prolonged inflammation is harmful and may cause excessive scarring [245]. The prolonged inflammation happens mainly in chronic or burn wounds [226,246] and it is of important to appropriately transit from inflammatory to proliferative phases in normal wound healing [247]. Macrophages are crucial in the wound healing process, which should appropriately transition from M1 to M2 macrophages [248,249]. M2 macrophages have anti-inflammatory properties, which are promoted in order to repair wounds in the latter phases of skin wound healing [248,249]. As mentioned earlier, MSC-exosomes promote the polarization of macrophages from M1 to M2 in cutaneous wound healing models(see 4. Anti-inflammation and immunomodulation by MSC-exosomes):(1)human BM-MSC-exosomes and IM-MSC-exosomes promote cutaneous wound healing in mice by transferring miR-223[85]; (2) human UC-MSC-exosomes promoted diabetic cutaneous wound healing in rats by delivering let-7b [88]; and (3) human UC-MSC-exosomes enhanced the wound healing in rats with severe burn injury through miR-181c transfer [88].

This article is extracted from Cells 2020, 9, 1157; doi:10.3390/cells9051157 www.mdpi.com/journal/cells