Liquan Wang,Zhujun Li,Yunzhu Li,Jiuzuo Huang,Nanze Yu,Xiao Long

Department of Plastic and Reconstructive Surgery,State Key Laboratory of Complex Severe and Rare Diseases,Peking Union Medical College Hospital of Peking Union Medical College and Chinese Academy of Medical Sciences,Beijing 100730,China

Keywords:Mesenchymal stem cell Adipose-derived stem cell Exosome Fibrosis Animal experiment Therapeutic application

ABSTRACT Fibrosis is a condition in which connective tissue replaces normal parenchymal tissue,resulting in significant tissue remodeling.Fibrosis can affect several organs and pose a serious threat to human health and life.Adiposederived stem cells (ASCs) have been suggested as promising candidates for antifibrotic therapies.Paracrine secretion is one of the key processes in stem cell therapy due to its critical function in cellular communication.ASC-derived exosomes (ASC-exos) are used as tools for restoring and regenerating damaged tissue,and they are now thought to orchestrate antifibrosis-related events.In this review,we summarize the recent findings and present an extensive view of the therapeutic applications of ASC-exos in fibrotic diseases.

1.Introduction

Fibrosis is a condition in which connective tissue replaces normal parenchymal tissue,resulting in significant tissue remodeling and the development of permanent scar tissue.Fibrosis is caused by excessive accumulation of extracellular matrix (ECM) components due to repeated injuries,chronic inflammation,and repair.1It is a physiological response to tissue damage designed to keep tissues and organs relatively intact;however,if this type of repair is repetitive and uncontrolled,pathological changes will occur.Fibrosis can affect several organs,and the key pathological change in the organ tissue is an increase in fibrous connective tissue.Continuous pathological interference can lead to structural and functional deterioration of the organ,posing a serious threat to human health and life.2

Adipose-derived stem cells (ASCs) are multipotent stromal cells that possess considerable therapeutic potential.ASCs have several distinct characteristics,including ease of access,high proliferation capacity,self-renewal,and secretion of trophic factors and exosomes.3,4Growth factors and cytokines,which are secreted by ASCs,send out paracrine signals that have chemoattractant,angiogenic,and pro-survival effects that are essential for tissue regeneration.Moreover,ASCs have been suggested as a promising candidate for tissue engineering and have gradually become applicable as a potential cell source for cell-based therapies in antifibrosis.3,5

Paracrine secretion is one of the key therapeutic processes in stem cells,and it has garnered considerable interest because of its critical function in cellular communication and regenerative medicine.Exosomes are thought to mediate this kind of cell-to-cell connectivity.6Exosomes are lipid bilayer vesicles with a diameter of 30-200 nm that are secreted by cells grown in culture or body fluids and contain a variety of molecules,including proteins,lipids,and nucleic acids.7Exosomes are molecules that are transported to recipient cells.After they are internalized by these cells,they impair their physiological activity,serving as intercellular signaling vehicles and mediating multiple physiological and pathological processes.8

When multipotent stromal cells from various sources were compared,ASCs were found to have the ability to secrete important soluble factors.ASCs could be a promising option for mass exosome processing due to their ease of availability and lack of ethical concerns.9ASC-derived exosomes (ASC-exos) have been implicated in immune dysregulation,oxidative stress,and inflammation in previous research.10ASC-exos are used as tools for restoring and regenerating damaged tissue,and they are now thought to orchestrate antifibrosis-related events.11In this review,we summarize the recent findings and present an extensive view of the therapeutic applications of ASC-exos in fibrotic diseases.

2.Superiority of ASC-exos

ASCs have paracrine antifibrotic effects on fibroblast activity and have been used to treat fibrosis in several trials.Similarly,ASC-exos can imitate the therapeutic capabilities of ASCs for cell-free therapy,despite their lack of differentiation ability.Furthermore,given the drawbacks of using ASCs directly,such as low survival rates,immunological rejection,concerns related to protection and ethics,research has increasingly shifted to exosomes,which have unique biological characteristics and are capable of modulating the basic behavior of recipient target cells.12The superior abilities of ASC-exos compared to ASCs are listed in the following table(Table 1).

Table 1 The superior abilities of ASC-exos compared to ASCs.

2.1.Intercellular communication

Exosomes,as paracrine signaling mediators,have been found to influence the progression of fibrotic diseases by delivering biogenic molecules to target cells,as well as affecting pathological fibrogenesis.They have a higher level of tissue penetrability than ASCs and shuttle between cells via a complex intercellular communication mechanism.The advantages of small size and easy shuttling further contribute to mediating intercellular communication and regulating biological activity.13

2.2.Protection

Exosomes contain cell-specific combinations of proteins,nucleic acids,lipids,and enzymes,which are similar to a cartage truck that efficiently delivers cargoes to target cells.These cargoes are enclosed in stable membranes to shield them from decay and are transported to the surrounding cells.14For example,the high activity of plasma ribonucleases was one of the major challenges for synthesizing and transporting microRNAs (miRNAs),but exosomes could shield miRNAs from degradation due to a stable membrane structure.15

2.3.Modification

Exosomes are cell-specific biogenic transport systems that are gaining increasing popularity as either drug or gene delivery tools.Moreover,exosomes have a membranous structure similar to liposomes,which prompts researchers to reassemble exosomes using their previous knowledge of drug loading into liposomes.They are thought to deliver drug molecules in a targeted manner while also performing their natural functions.16ASCs are the best candidates for exosome-wrapped miRNA production.Human organs affected by various chronic diseases can benefit from exosome-delivered miRNA cocktails.17Exosomes are remodeled for therapeutic needs and used in novel ways for selective drug delivery and as gene carriers in regenerative medicine.

2.4.Management

Maintaining cellular activity is essential for stem cells in order to function efficiently;therefore,the transport and storage processes need to be carefully designed.Exosomes,unlike ASCs,are microscopic substances that can be sterilized and frozen without the use of cryopreservatives.Generally,there is no need to preserve cell viability during therapeutic applications.ASC-exos have certain advantages over ASCs with respect to processing,packaging,shelf life,and shipping and are potentially ready-to-use biological products.18

2.5.Immunogenicity

Considering the issue of immune rejection,ASC-exos may be a better therapeutic agent than ASCs.While questions about immune rejection remain unanswered,syngeneic stem cell transplantation is performed more frequently than allogeneic transplantation.Exosomes,as a noncellular material,have fewer immune-recognition molecules than normal cells while retaining the same homing function as ASCs.19Exosomes are believed to be able to resist immune system detection and protect the integrity of the cell membrane,thereby preventing degradation.The low immunogenicity of exosomes makes them a good material for allogeneic transplantation.20

2.6.Biosafety

ASC-exos,as opposed to ASCs,can prevent cell therapy-related issues such as genetic instability,unfavorable differentiation,and the risk of tumor formation.21Although the use of ASCs has been proposed as a strategy to stabilize autologous fat grafts for regenerative therapy,their safety in the context of reconstructive surgery following mastectomy is unknown.Based on these findings,it has been suggested that ASCs promote the growth of active tumor cells but not of dormant tumor cells.22In contrast to other gene delivery vehicles,exosomes may be a potential therapeutic strategy since they are non-cytotoxic and non-mutagenic to the recipient.Maji et al.23examined the genotoxic,hematological,and immunological effects,as well as the amount of endotoxin in mesenchymal stem cell (MSC)-derived exosomes at two doses,and presented anin vitrosafety profile of MSC-exosomes.Data from recent toxicological testing and assessment of exosomes revealed that,while the use of exosomes is beneficial,concerns regarding the side effects of MSC-exosomes include tumorigenesis from cell transplantation and occlusion of the distal vasculature due to intravascular administration.24

3.Therapeutic applications in fibrotic diseases

Exosomes play a role in fibrosis-related diseases in a variety of organs,and in the purified form they may act either by artificially editing and modifying their internal substances or by being borne on various carriers(Fig.1).In the following sections we review the recent research regarding the current applications of ASC-exos in fibrotic diseases.

3.1.Skin

Scar formation in cutaneous healing is an intractable medical problem,which is characterized by morphological and histopathological changes in the skin and soft tissue,affecting esthetic appearance and impairing organ function.25Emerging evidence has demonstrated that ASC-exos alleviate skin fibrosis.By controlling fibroblast differentiation and gene expression,ASC-exos can promote ECM regeneration,facilitate wound healing,and prevent scar proliferation.26

Fig.1.Different forms of exosomes treat fibrosis in different organs.

The role of ASC-exos in cutaneous wound healing was investigated by Hu et al.19They discovered that ASC-exos could be picked up and internalized by fibroblasts in a dose-dependent manner to control cell migration,proliferation,and collagen synthesis.Exosomes increased collagen I and III synthesis during the early stages of wound healing,while they suppressed collagen expression in the late stages to decrease scar development,according to histological observations.This phenomenon is consistent with the histological differences found during normal healing of soft tissue wounds,in which collagen deposition is more significant in the early stages of healing,and collagen rearrangement is more crucial in the later stages.Collectively,their findings confirmed that ASC-exos can facilitate scarless wound healing by optimizing the characteristics of fibroblasts.

In a study by Wang et al.,27intravenous injection of ASC-exos reduced scar size,increased the ratio of collagen III to collagen I,and controlled fibroblast differentiation and gene expression in murine incisional wounds,suggesting that ASC-exos may be a new therapy for scarless cutaneous repair.They also found that ASC-exos prevented the differentiation of fibroblasts into myofibroblasts but increased the ratio of transforming growth factor-β3 (TGF-β3) to TGF-β1in vivo.In dermal fibroblasts,ASC-exos also increased matrix metalloproteinase-3 (MMP3)expression,resulting in a high MMP3 to tissue inhibitor of MMP1 ratio.This helped in remodeling the ECM and reduced scarring.

After a burn injury or trauma,formation of a hypertrophic scar is a fibro-proliferative condition of the dermis that causes esthetic disfigurement and functional weakness in patients.28The conversion of fibroblasts to myofibroblasts is an important step in the pathogenesis of scar formation,which is characterized by alpha smooth muscle actin-positive fibroblasts that can stimulate collagen synthesis,particularly Col1 and Col3.29Li et al.30observed the effect of ASC-exos on the propagation and migration of hypertrophic scar-derived fibroblasts(HSFs).They verified that ASC-exos could inhibit the bioactivity of HSFs and collagen deposition using a special pathway,thereby acting as an anti-fibrotic substance in preventing the development of hypertrophic scars.Furthermore,they found that miR-192-5p inhibited fibrosis in HSFs when expressed in ASC-exos,and that IL17RA was a direct target of miR-192-5p.Finally,IL17RA inhibited the expression of pro-fibrotic proteins in HSFs by modulating the Smad pathway.In conclusion,their findings revealed that the miR-192-5p/IL-17RA/Smad axis is responsible for the antifibrotic properties of ASC-exos.

These findings show that ASC-exos have highin vitroandin vivopotential for therapeutic use in skin fibrosis.However,further research into the basic mechanism by which exosomes play a role in antifibrosis is required.31

3.2.Liver

Liver fibrosis is a pathophysiological condition characterized by abnormal connective tissue hyperplasia in the liver due to various pathogenic factors.32The process of liver fibrosis occurs during the repair and healing of any liver injury.If the damage factors are not removed for an extended period,fibrosis will progress to cirrhosis.Hepatic stellate cells(HSCs)become activated and ECM proteins accumulate during liver fibrosis,which is a common pathological condition.By secreting collagens that form fibrous scar tissue,proliferating HSCs contribute to ECM aggregation.Drug-induced chronic liver disease,hepatitis C virus deficiency,autoimmune imbalances,and metabolic disorders are also common causes of liver fibrosis.33

Owing to the complex histopathological changes in the liver,MSC transplantation alone may not be sufficient to cure liver fibrosis.According to the study by Zhu et al.,34exosomes,which are secreted by ASCs,can be used to deliver circular RNAs (circRNAs) to treat liver fibrosis.Exosomes derived from mmu_circ_0000623-modified ASCs were found to prevent liver fibrosis by triggering autophagy.CircRNA is a type of non-coding RNA that has a continuous loop structure that is covalently closed.The expression of mmu_circ_0000623 was shown to be downregulated in mice with carbon tetrachloride(CCl4)-induced liver injury.Exosomes from mmu_circ_0000623-modified ASCs significantly suppressed CCl4-induced liver fibrosis by fostering autophagy activationin vitroandin vivo.Moreover,exosome therapy was substantially reversed upon using an autophagy inhibitor.

Many studies have shown that some miRNAs are crucial in various stages of liver fibrosis including,HSC activation and ECM protein production.35miR-122,which is the most common miRNA expressed in a healthy liver,is decreased in advanced liver diseases such as cirrhosis and hepatocellular carcinoma.Lou et al.36demonstrated that ASC-exos mediated miR-122 connectivity between ASCs and HSCs,influencing the expression levels of miR-122 target genes,all of which are involved in HSC proliferation and collagen maturation.Furthermore,by blocking HSC activation and reducing collagen deposition,miR-122 modification improved the therapeutic effectiveness of ASCs in the treatment of CCl4-induced liver fibrosis.Their findings showed that exosomemediated miR-122 connectivity increased the therapeutic effectiveness of ASCs when miR-122 was modified.Exosomes containing miR-181-5p have also been shown to improve autophagy and decrease TGF-β1-induced liver fibrosis in HST cells and in a mouse model of CCl4-induced liver fibrosis by inhibiting the STAT3/Bcl-2/Beclin 1 pathway.37The ability of engineered ASCs to selectively deliver miR-181-5p via exosomes to weakened liver cells,as well as their antifibrotic activity,will contribute to the improvement of exosomal therapeutics.

The most prevalent liver disease is nonalcoholic fatty liver disease(NAFLD),which is linked to increased systemic inflammation and insulin tolerance induced by visceral adipose tissue(VAT),although the precise causes remain unknown.A tissue-cooperative,homeostatic model of NAFLD was proposed by Baranova et al.17During the early stages of NAFLD,intrahepatic miR-122 synthesis decreases,while adipose secretion of miRNA-containing exosomes increases.Exosomes travel through the bloodstream to the liver,where their miRNA cargo is released to monitor intrahepatic targets.Upon fibrotic decompensation of the liver and collapse of the hepatic parenchyma,the external supply of liver-supporting miRNAs steadily diminishes,leading to hepatic carcinogenesis and fibrotic decompensation.These findings pave the way for the production of exosome-delivered miRNA cocktails that can help the organs,affected from various chronic diseases,perform better.

Koeck et al.38isolated exosomes from VAT,categorized their material,and discovered one of their possible targets,the TGF-pathway,which has been related to NAFLD.In HepG2 cells,exosome exposure increased tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression and decreased MMP7 and plasminogen activator inhibitor-1 expression.In HSCs,exosome exposure increased the expression of TIMP-1,TIMP-4,Smad-3,and MMP9.In vitro,exosomes from VAT were incorporated into liver cells and caused dysregulation of the TGF-pathway participants,suggesting a possible pathogenesis for NAFLD.

As a result,delivering biomolecules to the liver using exosomes may be a new therapeutic strategy for treating liver fibrosis.39

3.3.Lungs

Exosomes,which serve as intercellular messengers,have been the subject of research over the last decade and are gaining increasing interest from pulmonologists as resources for studying the pathogenesis as well as diagnosis of the disease.40Recent research suggests that the induction of fibroblasts from capillary vessels with compromised endothelial cells,epithelial mesenchymal transition(EMT)of type II alveolar epithelial cells,and the transformation of fibroblasts to myofibroblasts are all linked to pulmonary fibrosis.41Researchers have discovered that exosomes derived from MSCs aided in wound healing and inhibited myofibroblast differentiation in patients with pulmonary fibrosis.Exosomes and their cargoes,such as miRNAs and proteins,can promote or inhibit EMT,modulate the transformation of fibroblasts into myofibroblasts,contribute to fibroblast proliferation,and promote immunoregulatory and mitochondrial damage during pulmonary fibrosis.42

MSCtherapy has shown potential in experimental models of idiopathic pulmonary fibrosis.Mansouri et al.43investigated the mechanisms of action of extracellular vesicles produced by MSCs in a bleomycin-induced pulmonary fibrosis model.Transplantation of MSCs significantly reduced pulmonary fibrosis and inflammation.According to proteomic analysis,MSC therapy promoted an immunoregulatory,anti-inflammatory monocyte phenotype.They concluded that MSCs prevented and reversed the key features of bleomycin-induced pulmonary fibrosis and that the beneficial effects of MSCs may be mediated through modulation of systemic monocyte phenotypes.In another model of idiopathic pulmonary fibrosis,MSC-derived extracellular vesicles were also found to suppress fibroblast proliferation by downregulating FZD6 expression in fibroblasts via microRNA-29b-3p.44

These studies showed that treatment with MSCs from various tissues could help patients with pulmonary disease by reducing pulmonary fibrosis through paracrine action,which provides a new perspective and therapeutic strategy for the use of stem cell-derived exosomes.43,45

3.4.Kidneys

Acute kidney injury (AKI) is a life-threatening medical condition caused by sudden kidney damage or failure,which results in patient death within hours or days.46In contrast to the traditional notion that the kidney is a static organ with limited cellular turnover and regenerative capacity,kidney cells have recently been discovered to be capable of regenerating and repairing themselves throughout their lifespan.47The regenerative capacity of renal tubules is required for recovery from AKI,and failure to replace injured tubular epithelial cells during recovery can lead to fibrosis.48New therapeutics with regenerative potential,in addition to targeting multiple pathways,may be the most promising treatment strategy for AKI in this context.Exosomes derived from MSCs of various tissues have been shown to have beneficial effects on renal injuries in a variety of experimental animal models of kidney disease,including AKI.49

Renal fibrosis is the gradual process of healthy kidneys becoming injured and dysfunctional.Shi et al.50demonstrated that glial cell linederived neurotrophic factor (GDNF) enhanced the therapeutic effect of ASC-exosinthe treatment ofrenalinjury.The mechanismmightinvolve the transfer of endogenous molecules to injured cells via paracrine factors,but these factors are unknown.Exosomes were isolated after GDNF was transfected into human adipose MSCs using a lentiviral transfection system.GDNF-ASC-exos could help with peritubular capillary(PTC) loss in tubulointerstitial fibrosis,and the Sirtuin 1 (SIRT1) signaling pathway was involved in this process.SIRT1 signaling was improved by GDNF-ASC-exos,which was accompanied by increased levels of phosphorylated endothelial nitric oxide synthase (eNOS).In addition,they found a link between the number of PTCs and the level of SIRT1 expression in the kidneyin vivo.Finally,their study revealed the mechanism by which exosomes ameliorated renal fibrosis.They suggested that GDNF-ASC-exos probably activated an angiogenesis program in surviving PTCs after injury by triggering the SIRT1/eNOS signaling pathway.

Considering the complex mechanism of renal fibrosis,more studies are required to develop a novel strategy for the clinical application of ASC-exos.

3.5.Uterus

Intrauterine adhesion(IUA)caused by endometrial damage may lead to endometrial fibrosis,which is one of the most common causes of infertility in women of reproductive age.Increasing data indicate that the therapeutic effects of ASCs are regulated by ASC-exos and are primarily dependent on their ability to secrete paracrine factors.Zhao et al.51identified ASC-exos and investigated their therapeutic potential in rat models of IUA.Treatment with ASC-exos maintained normal uterine structure,promoted endometrial regeneration and collagen remodeling,and increased the expression of integrin-3 and vascular endothelial growth factor in the IUA model,which facilitated endometrial regeneration and fertility repair.Topical administration of ASC-exos to the uterus may be a promising strategy for patients with extreme IUA and infertility.IUA,also known as Asherman’s syndrome,52is a gynecological disorder characterized by hypomenorrhea,amenorrhea,recurrent miscarriage,and infertility.Infection and trauma are the most common causes of IUA,especially after pregnancy.Researchers have found that administering ASC-exos to rats with IUA improved endometrial growth,increased the number of glands,and reduced endometrial fibrosis.

Lin et al.53reported that in a clinical setting,exosome-hydrogel was be able to facilitate neovascularization,endometrial regeneration,and fertility repair while reducing local tissue fibrosis,resulting in higher rates of healthy pregnancy outcomes and offspring born alive.The ASC-exos hydrogel was inserted into the uterine cavity,where it facilitated endometrial regeneration,enhanced endometrial receptivity,stimulated neovascularization,and exerted anti-infective and anti-fibrotic action by facilitating sustained ASC-exosome and Ag+secretion.Therefore,ASC-exos hydrogel appears to effectively suppress inflammation and fibrosis by enabling the sustained release of ASC-exos,resulting in robust vascular and glandular proliferation in rats with endometrial injury.Finally,they developed a microenvironment-protected exosome-hydrogel that promoted angiogenesis and endometrial regeneration,thus facilitating fertility restoration.Moreover,this hydrogel could facilitate human umbilical vein endothelial cell proliferation,migration,and tube formationin vitro,and could also induce neovascularization and tissue regenerationin vivowhile suppressing localized fibrosis.

In conclusion,these findings show that transplantation of ASC-exos in rats with IUA can promote endometrial regeneration,regulate the expression of endometrial receptivity-related molecular markers,and improve fertility,indicating that this might be a viable strategy for improving the treatment outcome of endometrial fibrosis.

4.Conclusion

This study provides sufficient evidence for the antifibrotic properties of ASC-exos.Owing to their multiple features,ASC-exos have shown therapeutic potential in many clinical fibrotic diseases related to multiple organs and have become a promising material for cell-free biotherapeutic approach in the future.Nevertheless,since research on exosomes is still in its infancy,there are limitations regarding their use in a clinical scenario.The safety and efficacy of ASC-exos cannot be guaranteed.A substantial degree of heterogeneity in the dosing regimens was observed in the reported cases.When exosomes are used as therapeutics in clinical practice,it is necessary to have an optimal and effective dose.There is also a need to resolve the lack of protocols for the collection and purification of exosomes.To create more successful ASC-exos-based drugs and allow for broader applications in antifibrosis,more research is required to better clarify their mode of action.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Long X and Wang L:Conceptualization,Methodology,Software,Data curation,Writing-Original draft.Li Z and Li Y:Visualization,Investigation.Huang J and Yu N:Supervision,Validation,Writing-Reviewing and Editing.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant no.81670444) and the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (grant no.2020-I2M-C&T-A-004).