A New Therapeutic Strategy in Regenerative Medicine
Abstract: Exosomes (EXOs), derived from endocytic membranes, are nanoscale vesicles containing biomol-ecules such as nucleic acids, proteins and lipids. They work as effective carriers of intercellular communication in prokaryotic and eukaryotic organisms, and play significant roles in the regulation of both physiological and pathological processes. Current studies suggest that stem cell-derived exosomes can promote cell proliferation, migration and immune regulation. They can be utilized as cell-free therapeutic agents in the field of regenerative medicine, including acute/chronic renal injury, ischemia reperfusion injury, liver regeneration, skin regeneration, corneal regeneration. This review focuses on the ability of exosomes in promoting cell proliferation and summarizes their possible future application directions.
Extracellular vesicles (EVs) are membranous vesicles released by cells through endocytosis, exocytosis, etc., and their discovery dates back to the 1940s, first reported by Chargaff and West in 1946 [1] , widely found in prokaryotes and eukaryotes. These EVs contain lipids, proteins, RNA, and DNA, which can be transported to distant sites through body fluids, play the role of intercellular communication, and affect the metabolism and proliferation of target cells. According to the release mechanism and size of EVs, they can be divided into three categories: 1) apoptotic bodies larger than 1000 nm formed by membrane blistering during apoptosis; 2) 100~1000 nm microvesicles formed by cell budding; 3) exosomes less than 150 nm released after fusion of polyvesicles with plasma membrane [2].
In recent years, stem cell therapy has flourished due to its pluripotency, self-renewal, and ability to promote regenerative cytokine secretion [3], and several studies have proposed that the therapeutic effects of stem cells are mainly mediated by the secretion of soluble factors in the form of paracrine action, in which exosomes play a major role in paracrine action in part of the secretion group of stem cells [4]. Exosomes are stable, easy to store, can be sterilized by filtration and produced as off-the-shelf products, and have no immune rejection, have a homing effect [5], and the dose is easy to control, avoiding many of the disadvantages of stem cells, therefore, in fact, mesenchymal stem cells (MSCs) exosomes have been used as substitutes for MSCs for new strategies for cell-free therapy in various disease models, including neurological, cardiovascular, immunological, renal, Musculoskeletal, hepatic, respiratory, ophthalmological, dermatological diseases, and cancer [6] – [12].
2. Biological properties of exosomes
(1) Biogenesis and release
The biogenesis and release mechanisms of exosomes are as follows: 1) Early endosomes produced by phagosomes and plasma membranes undergo a series of maturation steps to form late endosomes, namely multivesicular bodies (MVBs), during which intraluminal vesicles (ILVs) accumulate in their lumen, and cargo sorting into ILVs is carried out by endosomal sorting complexes required for transport, ESCRT) dependent on the endosome sorting complex mediated by the ESCRT-independent mechanism [13]; 2) The endosome-derived MVB partially enters the lysosomes and is degraded, and some fuse with the cell membrane; 3) MVB releases exosomes through exocytosis and enters the extracellular environment [14]. In addition, studies have shown that exosomes can also be released directly from the plasma membrane or by delayed budding in intracellular plasma membrane-connected compartments (IPMCs) [15], in the latter case, these IPMCs are connected to the extracellular environment by the neck, where vesicles can be stored and released in pulses [ 16]。
The fusion of MVB transport to the cell membrane is regulated by Rab guanosine triphosphatase (GTPase) proteins such as Rab27, Rab11, Rab2B, Rab5A, Rab9A, Rab27A, and Rab27B [17], and at the same time coordinate with the movement of the cytoskeleton. Recent studies have found that the actin cytoskeletal regulatory protein cortactin plays an important role in regulating exosome secretion. It was found that cortactin, Rab27a, and coronin 1b synergistically control the stability of cortical actin docking sites in multivesicular advanced endosomes, thereby promoting exosome secretion [18]。 In addition, the binding of vacuolar protein sorting factor 4 (Vps4) and ESCRT required for transport to ubiquitinated proteins can promote the release of exosomes, and the Ral family of small GTPases also plays a regulatory role in exosome biogenesis. UV radiation, oxygen radical stimulation, and changes in calcium levels or cholesterol levels can all lead to changes in exosome secretion [15].
(2) Composition
Exosomes contain many molecules, including proteins, lipids, metabolites, mRNA, mitochondrial DNA, miRNAs, and many other non-coding RNAs, among others. Currently, exosomes have been confirmed to be secreted from a variety of cells, including B cells [19], T cells [20], dendritic cells [21], platelets [22], Schwann cells [23], tumor cells [24], cardiomyocytes [25], endothelial cells [ 26], stem cells [27], etc. The size and cargo of exosomes are heterogeneous, even from the same cell. But among the different exosomes, there are some common goods.
Exosomes are rich in tetratransmembrane proteins such as CD9, CD81, CD82, CD37, and CD63 [28], which are essential for cell targeting and adhesion. In addition, Rab GTPase, annexin and raft protein are important for membrane fusion, heat shock protein (HSP) 70 and HSP90 are chaperones, and tumor susceptibility gene (TSG101) protein is involved in MVB biogenesis. Exosomes also contain cytokines, transcription factor receptors, growth factor receptors, and other bioactive molecules [13].
MicroRNAs, ribosomal RNAs, and long non-coding RNAs are present in exosomes, which are actively sorted into exosomes. A widely accepted hypothesis is that microRNAs can be delivered directly to target cells through exosomes, thereby functionally modulating their mRNA targets. However, there is still a lack of direct evidence of extracellular vesicle-mediated miRNA transfer function [29]. It has been reported that 3’UTR mRNA fragments are enriched in EVs instead of intact mRNA molecules. Since the 3’UTR contains multiple regulatory miRNA binding sites, this suggests that the RNA of EVs may compete with cellular RNA to bind miRNAs or RNA-binding proteins in recipient cells, thereby regulating stability and translation [30]. mRNA-containing EVs have also been shown to enhance cell viability and tissue repair under various stressful conditions, and human mesenchymal stem cell-derived EVs have been found to contain 239 mRNAs, most of which are involved in cell differentiation, transcription, cell proliferation, and immune regulation [31]. At the same time, its mRNA content is regulated by cellular physiological states and stress conditions, and may play a role in maintaining tissue homeostasis and synchronizing cell functional states [32].
(3) Mechanism of action
EVs promote cell proliferation and reduce apoptosis mainly by regulating signaling pathways within cells, and their therapeutic effects are mainly mediated by therapeutic proteins and miRNAs.
1) Based on the mechanism of action of proteins
Zhang et al. [33] found that in a rat skin burn model, EV-carrying Wnt4 promotes β-catenin nuclear translocation and activity to enhance skin cell proliferation and migration. Katsuda et al. [34] found that adipose-derived mesenchymal stem cells (ADSC)-derived EVs contain enzyme activity neutral lysozyme (also known as CD10), which is a rate-limiting amyloid β (Aβ) degrading enzyme in the brain. ADSC-EVs were transferred to Neuro-2a cells that overexpressed amyloid precursor proteins, thereby reducing extracellular and intracellular Aβ levels. Another study [35] also found that bone marrow (BM)-MSC-derived EVs carry the enzyme-functioning CD73 (also known as ecto-5′-nucleotidase) that metabolizes AMP to adenosine, and through a series of signaling, A2AR-expressing type 1 helper T (Th1) cells lead to apoptosis.
2) RNA-based mechanism of action
miRNAs are also considered to be key molecules that mediate the therapeutic potential of MSC-EVs. MSC-derived exosomal miRNAs not only reduce apoptosis of damaged nerve cells, but also enhance brain function by improving neuroplasticity [36] [37]. Different authors have reported that different exosomal miRNAs, such as miR-22, miR-19a, miR-223, and miR-132, exert cardioprotective effects, and they propose various related genes and signaling pathways as mechanistic explanations [38].
Exosomes are involved in various physiological and pathological processes such as immune regulation, signaling between nerve cells, reproduction and development, tumors, neurodegenerative diseases, and infections [13]. Among them, most of the exosomes with functions related to enhancing cell proliferation are derived from stem cells [39], and their functions are mainly mediated by miRNAs. Therefore, exosomes can act as alternative mediators for stem cells.
The following mainly discusses the proliferative and regenerative effects of stem cell-derived exosomes in different diseases.
(1) Kidney disease
Exosomes have certain effects on acute kidney injury (AKI), kidney transplantation conditioning, and chronic kidney disease (CKD).
Some preclinical studies have shown that stem cell-derived EVs have the function of promoting tissue repair and reducing inflammation in different AKI models, which have been summarized in other reviews. AKI is marked by a rapid decline in renal function accompanied by the loss of renal tubular cells, resulting in elevated blood urea nitrogen (BUN) and plasma creatinine, and in 2009, Bruno et al. [31] demonstrated that in a model of AKI induced by glycerol injection, BMMSC-EVs accelerate the recovery of damaged tubular cells, promote cell proliferation and protect cells from apoptosis, and BM MSC-EVs carry specific mRNA, This in turn stimulates the damaged cells of the receptor to re-enter the cell cycle.
In kidney transplant patients, pretreatment of the kidney with mesenchymal stem cells (MSCs) and MSC-EVs limits tissue damage due to ischemia-reperfusion injury and chronic allograft kidney disease [40]. MSCs and MSC-EVs were tested in a rat model of kidney with organ donation after cardiac death (DCD). DCD kidneys treated with MSC-EV during organ cold perfusion (4 hours) showed significantly reduced signs of kidney damage [41].
The main cause of renal insufficiency in patients with end-stage CKD is glomerular and tubular fibrosis. Recently, EVs isolated from BM MSCs and liver MSCs have been shown to be effective in reversing renal fibrosis in established diabetic nephropathy models [42]. MSC-EV and HLSC-EV contain a series of anti-fibrotic miRNAs that downregulate pro-fibrotic genes and restore normal kidney function.
Currently, Nassar et al. [10] published the results of their phase II/III clinical trial using umbilical cord tissue MSC-derived EVs to improve CKD progression, in which 20 patients diagnosed with CKD (eGFR 15~60 mg/ml) for more than 6 months received two doses (1 week apart) of MSC-EV (100 μg/kg/dose). After 1 year, the patients showed improvements in eGFR and urinary albumin-creatinine ratios, as well as significant reductions in BUN and creatinine, along with significantly higher levels of TGF-β and IL-10 in plasma, while TNF-α continued significant decreases.
(2) Liver disease
MSCs improve disease progression in patients with cirrhosis, and MSC-derived exosomes have similar effects. Li et al. [43] used carbon tetrachloride (CCl.) in Kunming mice4) induced liver injury model. Exosomes derived from human umbilical cord mesenchymal stem cells have been found to improve liver fibrosis by inhibiting epithelial-mesenchymal transition and collagen production in hepatocytes. In addition, studies [44] showed that DC-derived exosomes from mice receiving immunosuppressive therapy or modified to express immunosuppressive cytokines promoted a tolerant immune response, and mRNA-155 and miRNA-125b-enriched exosomes promoted differentiation of M1 macrophages over M2 macrophages, thereby improving the inflammatory response in mice. Human amniotic epithelial cell-derived exosomes significantly reduce the number of macrophages and macrophage infiltration during liver fibrosis [45]. Hepatocyte-derived exosomes can transfer sphingosine kinase 2 to form sphingosine 1-phosphate in target hepatocytes, thereby causing cell proliferation and liver regeneration [46].
To date, only a few groups have studied the therapeutic role of MSC exosomes in acute liver injury. Some studies have shown that MSC-EVs can inhibit the proliferation and activation of pro-inflammatory macrophages, thereby reducing the secretion of cytokines such as interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor α (TNF-α α), thereby significantly improving acute liver failure (ALF). This mechanism may be related to MSC-EV inhibition of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway [47] [48] [49]. Lou et al. [8] found that adipose tissue-derived MSCs significantly reduced elevated serum alanine aminotransferase and aspartate aminotransferase levels, as well as serum pro-inflammatory factor levels. Therefore, it is reasonable to believe that transplantation of MSC-derived exosomes may be a new treatment for various types of acute liver injury.
(3) Cardiovascular disease
The beneficial effects of exosomes on the heart include anti-apoptotic, anti-inflammatory, anti-cardiac remodeling, and cardiac regeneration [50]. miR-22 and miR-221 in exosomes target methyl CpG-binding protein 2 (Mecp2) [51] and PUMA [52], respectively (a subclass of the Bcl-2 protein family), which reduces its expression, thereby exerting an anti-apoptotic effect and improving cardiomyocyte loss in the area of myocardial infarction. Direct injection of MSC exosomes into the border region of myocardial infarction reduces fibrosis and inflammation in animal models. Analysis of target genes and pathways suggests that the PI3k-Akt-mTOR pathway may be the main mechanism responsible for these phenomena, as miR-29 and miR-24 expressions are upregulated, while miR-34, miR-130, and miR-378 expressions are expressed [53]. Upregulation of miR-24 limits aortic vascular inflammation. Importantly, in vivo expression of miR-24 in mouse myocardial infarction models inhibits apoptosis in cardiomyocytes, reduces infarct area, and reduces cardiac death [54]. Cardiac progenitor cell (CPC)-derived exosomes promote endothelial cell migration and vascular endothelial growth factor secretion [55] to induce cardiac regeneration and improve cardiac function.
Exosomes can also promote the formation of new blood vessels, which are related to the proliferation, migration, and differentiation of endothelial cells and vascular smooth muscle cells [50]. At the same time, MSCs-derived exosomes have recently been shown to upregulate wnt5a, which plays an important role in endothelial cell migration by stabilizing neutins at cell junctions to help cells move together, thereby enhancing vascularization and migration [56].
(4) Skin regeneration
Adipose stem cell-derived exosomes (ADSCs-EXOs) are rich in miRNA-125a and miRNA-31, which can be transferred to vascular endothelial cells to stimulate proliferation and promote angiogenesis [57] [58] ]。 During the proliferative phase of skin healing, fibroblasts proliferate to produce an extracellular matrix (ECM), while epithelial cells proliferate and migrate to the center of the wound to promote wound healing. Therefore, the proliferation and re-epithelialization of skin cells are important for skin regeneration. ADSCs-EXO are internalized by fibroblasts and stimulate proliferation, migration, and collagen synthesis in a dose-dependent manner [59]. Finally, ADSCs-EXOs can stimulate the reconstruction of the extracellular matrix by modulating fibroblast differentiation and gene expression, thereby promoting wound healing and preventing scar proliferation. Wang et al. [60] found that ADSCs-EXOs prevent the differentiation of fibroblasts into myofibroblasts, but increase the ratio of transforming growth factor-β3 (TGF-β3) to TGF-β1 in vivo, and increase matrix metalloproteinase 3 (MMP3) in cutaneous dermal fibroblasts , resulting in a high ratio of MMP3 to matrix metalloproteinase-1 tissue inhibitor (TIMP1), which is beneficial for extracellular matrix (ECM) remodeling and reducing scarring.
(5) Corneal regeneration
The cornea covers the first 1/6 of the total surface of the eyeball, and the surface is non-keratinized stratified squamous epithelium, which is rich in innervation. Other cellular components of the cornea are corneal stromal cells and endothelial cells. Corneal damage triggers repair pathways, and scarring during healing can impair corneal transparency and potentially blind [61].
Corneal endothelial dystrophy is one of the causes of vision loss and corneal transplantation, and Buono et al. [62] found that MSC-EVs were able to induce significant downregulation of most of the ER stress-related genes in human corneal endothelial cells in an in vitro model of corneal dystrophy. At the same time, they upregulate the Akt pathway and limit caspase-3 activation and apoptosis. Shang et al. [63] found that treatment of rabbit corneal stromal cells with adipose mesenchymal stem cell-derived exosomes increased cell proliferation, decreased apoptosis, and deposition of extracellular matrix, proving that exosomes may be an important mediator of corneal regeneration.
Han et al. [64] have demonstrated that epithelial-derived exosomes mediate communication between corneal epithelial cells, corneal stromal cells, and vascular endothelial cells. In addition, limbal stromal cell-derived exosomes contribute to the proliferation and wound healing of limbal epithelial cells (LECs) [65], and human corneal mesenchymal stem cell (cMSC) exosomes can also accelerate corneal epithelial wound healing. The study by Shojaati et al. [66] demonstrated that EVs from corneal stromal stem cells can reduce inflammation, scarring, and fibrosis, thereby improving corneal clarity in a mouse model of corneal debridement.
A study by Samaeekia et al. showed that corneal mesenchymal stem cell-derived exosomes can increase wound healing in corneal injuries [67] In damaged corneas, exosomes upregulate the expression of anti-angiogenic factors such as thrombospondin 1 (TSP-1) and anti-inflammatory cytokines, including IL-10, TGF-β1, and IL-6, while down-regulating the expression of pro-inflammatory factors such as IL-2 and interferon-γ (interferon-γ, IFN-γ), macrophage inflammatory protein-1α, and vascular endothelial growth factor (VEGF) [68]. Leszczynska et al. found that limbal keratinocyte-derived exosomes activate Akt signaling and promote wound healing in limbal epithelial cells [65]. Currently, the use of EVs for the treatment of eye diseases is still in the preclinical experimental stage.
(6) Neurological diseases
Exosomes have potential therapeutic effects for neurological disorders. Xin et al. [69] found that oxygen-glucose consumption (OGD) in miR-133b-rich astrocyte-derived exosomes mediates neuronal growth and elongation after stroke. Astrocyte-derived exosomes also transport miR-190b to prevent OGD-induced autophagy and inhibit neuronal apoptosis [70]. At the same time, the miR-17-92 cluster contained in MSC exosomes mediates the activation of the signaling pathway PI3K/Akt/mTOR, leading to neuronal remodeling and neurogenesis in stroke rodent models [71].
One study [72] showed that there is an excess of phosphorylated tau protein, a biomarker of Alzheimer’s disease (AD), in astrocyte-derived exosomes exposed to β-amyloid. Another study [73] showed that, based on animal experiments, β-amyloid and tau released into serum most likely came from astrocyte-derived exosomes in the brain. The powerful role of astrocyte-derived EVs in AD patients can also show that astrocyte-derived EVs have good development prospects for brain-targeted therapies [74].
Exosomes play an important role in cell-to-cell communication by transporting nucleic acids, proteins, and lipids, and participate in a variety of physiological and pathological processes, and their main functions may be mediated by RNA transmitted. Although there has been a strong interest in exosome research since the discovery of exosomes, the mechanisms of exosome formation, protein and RNA separation, and cargo release are still unclear. However, its therapeutic effect on promoting cell proliferation and regeneration is unquestionable. In the future, exosomes will be a more promising treatment modality for cardiovascular, renal, hepatic, neurological, ophthalmic and other diseases.
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