Roles of extracellular vesicles in the aging microenvironment and age‐related diseases

Cellular senescence is a physiologically stable state of cell cycle arrest that strictly regulates cell proliferation and lifespan. The discovery of this phenomenon can be traced to the 1960s. Hayflick and Moorhead cultivated human fibroblasts and observed that they underwent a limited number of cell divisions, which is now called the “Hayflick limitation” (Hayflick & Moorhead, 1961 ). Cellular senescence is a transitional state of cell death that precisely regulates survival and death at the cellular level. The expected cellular senescence trajectory is cell elimination, which plays an essential role in promoting tissue repair and regulating organ growth and body homeostasis (Mosteiro et al., 2016 ; Ritschka et al., 2017 ). However, in senescence‐associated diseases, excessive accumulation of senescent cells during aging becomes burdensome, disrupting the trajectory of programmed cell death and leading to a decline in regeneration (Masaldan et al., 2018 ). These findings indicate that short‐term accumulation of senescent cells in tissues is beneficial to the renewal of the internal environment and the maintenance of organs but that long‐term accumulation of senescent cells in tissues triggers an imbalance in the internal environment and organ disability. HIGHLIGHTS The aging microenvironment can be unstable with aging and transform into a pathological state. The SASP, with the involvement of EVs, amplifies senescent signals in autocrine, paracrine, and endocrine ways. Senescent EVs alter the levels of immunity, inflammation, gene expression and metabolism of target cells. Therapeutic strategies incorporating EVs from various materials and donors are feasible for age‐related diseases.

Among ageotypes, various soluble molecules marking the presence of senescent cells, which together are termed the SASP, have attracted our attention because of their heterogeneity and availability for clinical application. High levels of immune modulators, proinflammatory cytokines, growth factors, chemokines, and proteases are involved in the SASP in a cell non‐autonomous manner (Calcinotto et al., 2019 ). Proteomic analysis results obtained from human fibroblasts, renal epithelial cells, bone marrow, and adipose mesenchymal stromal cells have suggested that the SASP can be categorized into different groups depending on the senescence inducers and cell derivations (Basisty et al., 2020 ; Ozcan et al., 2016 ). In contrast to genotoxic and oncogenic senescence, the work by Wiley et al. presented a distinct senescent phenotype of mitochondrial dysfunction in progeroid mice with POLG D257A mitochondrial DNA (mtDNA) mutation, termed mitochondrial dysfunction‐associated senescence (MiDAS), which had a specific secretion profile without IL‐1‐dependent factors through an NADH/AMPK/p53‐dependent pathway (Wiley et al., 2016 ). Additionally, the SASP Atlas website ( www.SASPAtlas.com ), constructed by Basisty's team, can aid in a more comprehensive understanding of the complex and dynamic SASP network (Basisty et al., 2020 ). Paradoxically, the SASP can be beneficial to organismal homeostasis or detrimental in different biological contexts (Oubaha et al., 2016 ; Watanabe et al., 2017 ). When primary mouse keratinocytes are transiently subjected to SASP conditions, the levels of stem cell markers and regenerative capacity increase in vivo. In contrast, under prolonged exposure, cell plasticity and stemness are attenuated to some extent (Ritschka et al., 2017 ). Therefore, it can be assumed that the roles of the SASP can autonomously switch depending on the concentration and duration of treatment. Moreover, high levels of SASP proteins are positively correlated with the age and physical conditions of patients. Among these SASP factors, the levels of 7 SASP factors (GDF15, FAS, OPN, TNFR1, ACTIVIN A, CCL3 and IL‐15) may predict the postsurgery outcomes of patients with severe aortic stenosis and ovarian cancer (Schafer et al., 2020 ). When the SASP is stimulated, several signaling pathways are activated, and two downstream transcription factors, NF‐κB and C/EBPβ, are consistently recruited to regulate SASP‐related gene expression (Figure 1 and Table 1 ). With regard to DNA damage‐induced senescence, mounting evidence has indicated that the phosphatidylinositol 3‐kinase‐related kinase (PIKK) superfamily (ATM, ATR and DNA‐PKcs protein kinases included) is crucial for the onset of senescence and transcription of SASP genes upstream of NF‐κB signaling, responding to the DDR (Strzeszewska et al., 2018 ). Pharmacologic inhibition and genetic depletion of ATM can suppress NF‐κB activation, diminish inflammatory cytokine levels, and reverse senescence (Zhao et al., 2020 ). ATM and ATR also inhibit p62‐dependent autophagic degradation to stabilize the GATA4 protein, which evokes the NF‐κB signaling pathway, thereby contributing to the SASP (Kang et al., 2015 ). In addition, the transcription‐associated methyltransferase and oncoprotein MLL1 also mobilizes the ATM‐NF‐κB axis to control SASP genes in the context of the DDR (Capell et al., 2016 ). With a prolonged DDR, the accumulated histone variant H2A.J colocalizes with 53BP1 to be incorporated into SAHF formation and competes with H1 to increase the expression of repeated DNA sequences while activating STAT/IRF transcription factors and inducing the interferon‐related SASP (Isermann et al., 2020 ; Mangelinck et al., 2020 ). TABLE 1 Signaling pathways that are associated with SASP‐related secretion Trigger Signaling pathway Transcription factor(s) Secretory factors Ref. DNA damage response H2A.J accumulation Incorporation into SAHFs; H1 decrease STAT, IRF IL‐6, IL‐8, CXCL8, CSF2, CM‐CSF, MCP1, CCL2, IFN (Isermann et al., 2020 ); (Mangelinck et al., 2020 ) MLL1 increase ATM NF‐κB IL‐8, IL‐1B (Capell et al., 2016 ) Autophagy decrease GATA4 NF‐κB IL‐6, IL‐8, CXCL1/2/3, CM‐CSF, ICAM1, CCL2, TNF (Kang et al., 2015 ) Mitochondrial dysfunction NAD+/NADH ratio decrease NADH/AMPK/p53 / IL‐10, TNFα, CCL27 (Wiley et al., 2016 ) ROS and CCF increases cGAS/STING NF‐κB, IRF3 IL‐6, CXCL10 (Vizioli et al., 2020 ) Cell‐free mtDNA cGAS/STING; TLR9‐DC activation IRF3; T‐bet, RORγt IFN; IL‐17, IFN‐γ (Aarreberg et al., 2019 ); (Iske et al., 2020 ) Chromatin remodeling LBR decrease CCFs/cGAS/STING / IL‐6, IL‐8, MMP1 (En et al., 2020 ) LINE1 transcription increase cGAS/STING NF‐κB, IRF3 IFN, IL‐6, IL‐8, IL‐1, MMP3, ISG, MCP1 (Bi et al., 2020 ) HDAC inhibition ATM/MRN NF‐κB IL‐6, IL‐8, CM‐CSF, IL‐1 (Malaquin et al., 2020 ) BRD4 increase Acetylation of superenhancers of the SASP / IL‐6, IL‐8, CM‐CSF, IL‐1, CXCL1, G‐CSF (Tasdemir et al., 2016 ) Other triggers Oncogenic Ras increase MAPK/PEA3/SCCA; P38 MA

With aging or senescence‐associated stress, the dynamic cargos of EVs reach their targets through paracrine or endocrine patterns and exhibit destructive or supportive behaviours that affect aging signal transmission. This transmission refers to the passing of information via cell‐to‐cell or organ‐to‐organ communication through EV trafficking (Figure 3 ). Borghesan et al. confirmed that IFITM3, a protein messenger linked with the IFN pathway, was selectively encapsulated in small EVs during oncogene‐induced senescence (OIS). IFITM3 is critical for early genotoxic damage and paracrine senescence in neighbouring fibroblasts (Borghesan et al., 2019 ). Moreover, shuttling of EVs between the liver and vasculature in a non‐alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) model has revealed the distant interorgan communication of EVs. Steatotic hepatocyte‐derived EVs containing miR‐1 suppress KLF4 expression but activate the NF‐κB pathway to facilitate endothelial inflammation and senescence and participate in the development of atherosclerosis (Jiang et al., 2020 ). In these ways, transmission through EVs from senescent cells has negative impacts on the aging microenvironment, promoting chronic low‐grade inflammation and cellular senescence in recipient cells and organs and progressively inducing senescence‐associated diseases. EVs isolated from senescent cells can transform recipient cells’ immune state to mediate cellular viability through information delivery (Figure 3 ). When treated with oxidative agents, retinal pigment epithelial cells, a type of phagocytic cell, can produce more CD36‐associated microparticles, impeding phagocytosis and diminishing the viability of neighbourhood cells, as indicated by increased SA‐β‐gal staining and p21 and p15 expression (Yang et al., 2020 ). A cell‐free form of telomeric repeat‐containing RNA (cfTERRA) is abundant in EVs and is related to telomere dysfunction and DNA damage of parent cells. It is regarded as a telomere‐associated molecular pattern (TAMP) or DAMP and is detected in the blood plasma of control subjects and cancer patients, directly revealing that exosomal cfTERRA is a potential marker for telomere metabolism in senescence (Azzalin & Lingner, 2015 ; Cusanelli et al., 2013 ; Porro et al., 2014 ). It can also induce the transport of alarmin to elicit a recipient inflammatory response (Wang & Lieberman, 2016 ). Incubation of peripheral blood mononuclear cells with exosomes containing cfTERRA stimulates innate immune signaling and upregulates inflammatory cytokines, such as TNFα, IL‐6, and CXCL10 (Wang et al., 2015 ). In addition to cfTERRA, other exosomal DAMPs modulate innate immunity and cell conditions in the aging microenvironment. CD63 + EVs from irradiated cancer cells loaded with DNA:RNA hybrids and LINE 1 retrotransposons trigger the senescence of adjacent cells and induce innate immune signaling to polarize M1 macrophages (Tesei et al., 2021 ). EVs from senescent oral keratinocytes can carry mtDNA and nuclear DNA to activate the cGAS‐STING pathway and interferon expression in monocytes (Schwartz et al., 2021 ). Hence, these DAMPs in EVs consequently contribute to chronic inflammation and age‐related diseases through the innate immune signaling pathway, which can be considered a positive feedback loop of SASP modulation. In genotoxic aging models with either irradiated breast cancer cells or irradiated mice, EVs can directly induce telomere attrition and DDR in recipients, causing genomic instability and aging. However, this effect is reduced by RNase treatment or thermal denaturation by boiling, which suggests that protein‐ and RNA‐containing EVs indeed have effects on telomere shortening and DNA damage (Al‐Mayah et al., 2017 ; Hargitai et al., 2021 ). In addition, EVs can mediate genomic instability through senescence‐associated genome‐wide hypomethylation to spread senescence characteristics to neighbouring cells. In a model consisting of replicative senescent human umbilical vein endothelial cells (HUVECs), EVs carried miR‐21‐5p, and miR‐217 downregulated DNA methyltransferase 1 (DNMT1) and Sirtuin 1 (SIRT1) expression in young cells, leading to high levels of SASP molecules and cell cycle inhibitors (Mensa et al., 2020 ). In nature, DNMT1 cooperates with SIRT1 (a well‐known longevity‐related gene) to maintain genetic methylation patterns in order to ensure genome integrity through mitosis (Lee et al., 2019 ; Zheng et al., 2015 ). Therefore, EVs transmit aging signals at the genetic and epigenetic levels (Figure 3 ). Cargos and their corresponding EVs can co‐destroy energetic and proteinic networks to upset the intracellular balance of the targeted cells, promoting aging (Figure 3 ). In a model of recipient mitochondrial dysfunction, lung fibroblast‐derived EVs from idiopathic pulmonary fibrosis (IPF) patients carried elevated levels of miR‐23b‐3p and miR‐494‐3p to suppress SIRT3 expression. These EVs are critical for

As the frequencies of neurodegenerative diseases exponentially increase with age, postmitotic cells in the brain show extreme sensitivity to systemic and cellular senescence. Alzheimer's disease (AD) and Parkinson's disease (PD) are the most extensively studied neuronal senescence‐associated conditions to date, and both are mainly attributed to insoluble misfolded protein aggregation in specific regions of the brain. These neuropathic proteins are closely linked with senescence‐associated autophagy and lysosomal dysfunction, which lead to their stable presence in specific EVs (Chung et al., 2019 ; Kapogiannis et al., 2019 ; Lamontagne‐Proulx et al., 2019 ; Minakaki et al., 2018 ). Additionally, evidence has shown that injection of EVs isolated from AD patients into the hippocampi of wild‐type mice is neurotoxic in vivo, leading to increased tau phosphorylation (Aulston et al., 2019 ). After such EVs are intravenously injected from the serum of PD patients into mice, protein aggregation, dopamine neuron degeneration, microglial activation, and mouse movement defects are also observed (Han et al., 2019a ). This directly indicates that EVs packaged with a pathogenic pattern of molecules may play vital roles in neural aging phenotype transmission. To precisely evaluate and diagnose age‐associated neurodegeneration, secreted EVs in various body fluids may be the optimal biomarkers. Taking advantage of blood‐brain barrier permeability to EVs, brain‐derived exosomes can be detected in the peripheral circulation. Neuron‐derived EVs in serum, sorted by L1CAM and neural cell adhesion molecule expression, were found to exhibit elevated phosphorylated tau‐181 and 231, Aβ42 and insulin receptor substrate 1 levels in an AD group (Kapogiannis et al., 2019 ). Compared to exosome‐unbound Aβ, exosome‐bound Aβ can better reflect amyloid plaques of the brain and be used to differentiate various clinical groups via amplification techniques (Lim et al., 2019 ). The levels of advanced glycation end products N‐(1‐carboxymethyl)‐L‐lysine (CML) in EVs present dynamic changes in early and moderate AD (Haddad et al., 2019 ). However, there is little correlation in the miRNA contents of EVs in brain and blood samples obtained from AD subjects (Cheng et al., 2020 ), while similar levels of exosomal Aβ42 and tau in CSF and blood have been verified (Jia et al., 2019a ). A novel tau fragment in AD, N‐224 tau, can be packaged in neuron‐specific EVs and secreted in CSF to upregulate cognitive decline (Cicognola et al., 2019 ). In PD, in addition to α‐synuclein detected in CSF, numerous noncoding RNAs are significantly upregulated in EVs, including miR‐153, miR‐409‐3p, and let‐7 g‐3p (Gui et al., 2015 ; Stuendl et al., 2016 ). Interestingly, the α‐synuclein oligomer and the total ratio of α‐synuclein in salivary EVs can be noninvasively assessed in PD patients, and α‐synuclein levels increase with disease severity (Cao et al., 2019 ). Consequently, it is valuable to classify specific EVs in biofluids as ageotypes and obtain profiles of AD and PD patients for early and safe diagnosis. In particular, EVs can contribute to the development and aggravation of these neurodegenerative diseases through the intercellular transmission of misfolded proteins or other pathological molecules. In AD, EVs with tau protein are released by primarily affected neurons after depolarization and internalized by the next neurons through synaptic transmission to cause protein folding errors in normal cells (Wang et al., 2017b ). SHswe cells (a neuroblastoma cell line transfected with the Swedish mutant of amyloid precursor protein) are capable of stimulating the microglial immune response and increasing senescence hallmark activation through EV‐containing miRNA transmission between neurons and microglia (Fernandes et al., 2018 ). Microglia, which are tissue‐resident macrophages, not only facilitate tau release in EVs but also facilitate neuroinflammation development via EVs (Asai et al., 2015 ). With age, the accumulation of internal antigen‐antibody compounds and misfolded proteins stimulates microglia to overexpress several complement components involved in the complement cascade and maladaptive neuroinflammatory effects (Spani et al., 2015 ). High complement levels can also be found in astrocytic EVs and neuronal EVs (Goetzl et al., 2018 ). These inflammatory EVs cause cellular membrane disruption and neurite density reduction in rat cortical neurons by activating membrane attack complex (MAC) formation (Nogueras‐Ortiz et al., 2020 ). Age‐associated chronic inflammatory states are similarly evident during PD progression. α‐Synuclein in EVs, whether from the brain or the periphery, can incessantly evoke innate immune responses and interfere with the clearance system, resulting in synucleinopathy pathogenesis (Grozdanov et al., 2019 ; Matsumoto et al., 2017 ; Xia et al., 2019 ). In a sense, pathological pattern transmission via EVs is equivalent to aging phenotype t

It has been confirmed that aging greatly increases susceptibility to chronic and refractory age‐related diseases. In turn, age‐related diseases, in a mechanism similar to positive feedback, exacerbate the aging microenvironment and accelerate the aging process. In particular, a diabetic microenvironment is replete with factors leading to senescence and hyperglycaemia, both of which collaborate to induce long‐standing diabetes mellitus and severe complications. In the endocrine system, message delivery via EVs is essential for realization of aging transmission. In a diabetic microenvironment, EVs from MSCs are aging factor carriers that induce renal tubular cell senescence, fibrosis and dysfunction (Li et al., 2020c ). These outcomes indicate that stem cells problems should be monitored when they are utilized in tissue repair of T2DM patients. The increased EVs from the circulation and cells induce diabetic senescence and organ injury, subsequently causing systemic inflammation and insulin resistance. Evidence has shown that mouse islets under attack from mixed cytokines and streptozotocin can produce dramatically elevated amounts of injury‐related exosomal miR‐375‐3p in serum prior to the development of blood glucose and insulin disorders (Fu et al., 2018 ). In addition, CD31 + /annexin V‐labelled EVs from damaged endothelial cells show higher sensitivity in T2DM than in metabolic syndrome, which leads to immune cytokine overproduction and vascular aging (Berezin et al., 2015 ). Research on EVs obtained from T2DM plasma, mainly erythrocyte‐derived EVs, has revealed the immune function of these EVs, which are internalized by circulating leukocytes; this internalization alters their gene expression related to cell survival, oxidative stress and cytokine release (Freeman et al., 2018 ). Bone can be regarded as another endocrine tissue that regulates systemic hormone metabolism. BMSCs from aged mice target SIRT1 in adipocytes, myocytes and hepatocytes through exosomal miR‐29b‐3p to significantly induce age‐related insulin resistance in vitro and increase blood glucose in vivo (Su, Xiao et al., 2019 ). Moreover, EVs from obese tissue engage in age‐related DM. Excessive calorie intake in mice can induce cellular senescence in adipose tissue through accumulation of ROS and activation of p53, which have been proven to play roles in insulin resistance (Minamino et al., 2009 ). Thereinto, exosomal miR‐27a in adipocytes has been verified to participate in the insulin resistance of muscle cells through repression of downstream PPARγ (Yu et al., 2018 ). In addition to adipocytes, macrophages from adipose tissue can release EVs to modulate cellular insulin action via the same target gene (Ying et al., 2017 ). These findings suggest that controlling energy intake not only maintains glucose homeostasis but also ameliorates senescence. However, the true risk of diabetes involves its complications, especially microvascular and macrovascular diseases. Long‐term exposure to high glucose causes endothelial cells to transfer exosomal Notch3 to vascular smooth muscle cells, consequently increasing senescence and osteocalcin expression to induce vascular calcification and aging (Lin et al., 2019 ). Additionally, renal tubular epithelial cells are injured by albumin in diabetic macroalbuminuria. Exosomal miR‐4756 accelerates the aging and injury of these cells by suppressing Sestrin2 to induce endoplasmic reticulum stress and the epithelial‐to‐mesenchymal transition (Jia et al., 2019b ). Despite the multiorgan hazards and refractoriness of T2DM, metformin therapy has been confirmed as a jack‐of‐all‐trades drug to achieve antiaging, antidiabetes, and antiobesity effects (Cabreiro et al., 2013 ; Onken & Driscoll, 2010 ). A spectrum of circulating miRNAs in EVs in T2DM subjects treated with metformin has been found to be surprisingly similar to that of healthy controls (Ghai et al., 2019 ). However, caution should be exercised when considering the use of metformin as an antiaging agent for treating healthy elderly patients. A recent study warns that it will enhance mitochondrial dysfunction and metabolic disturbance in late life, shortening lifespan (Espada et al., 2020 ).

5.1 Targeting senescence through senotherapy There is a growing consensus that researching and developing therapeutic interventions for age‐related disorders is necessary to alleviate the severe economic, healthcare and humanitarian challenges of the global aging society (Zhao & Stambler, 2020 ). Our aging population must cope with increasing threats of senescence‐related dysfunction, among which mortality is the top concern and the most challenging. COVID‐19 results in markedly higher in‐hospital mortality in patients with advanced chronological age than in younger patients, possibly due to involvement of CD26 and ACE‐2 (Koff & Williams, 2020 ; Li et al., 2020a ; Radzikowska et al., 2020 ) (two host receptors that show a significant association with senescence (Li et al., 2018 ; Takeshita et al., 2018 )). In experimental animals, inhibition of cellular senescence with the transgenic suicide gene INK‐ATTAC can expand lifespan and healthspan through inducible elimination of p16 INK4a ‐positive senescent cells upon drug treatment (Baker et al., 2011 ). Using small molecules to therapeutically target exclusive markers of aging cells will likely have positive effects on the quality of life and the burden of age‐related diseases. Hence, there is a growing interest in the development of antiaging treatment, termed senotherapy (Figure 4 ). In principle, senotherapeutic strategies can be broadly classified into two categories: senolytic strategies (antiaging strategies for selective removal of senescent cells (Jeon et al., 2017 )) and senomorphic strategies (antiaging strategies that block senescent phenotypes by inhibiting the SASP without compromising cell death (Short et al., 2019 )). FIGURE 4 Approaches for exploiting senescence for therapeutics: senolytics, senomorphics and EV‐mediated regeneration and rejuvenation. There is a growing interest in developing antiaging therapies, termed senotherapies. Senotherapeutic strategies can be traditionally classified into two categories: senolytic strategies (antiaging strategies for selective removal of senescent cells, including compound treatment and immune system‐mediated clearance) and senomorphic strategies (antiaging strategies that block senescent phenotypes by inhibiting the SASP without compromising cell death). Recently, EVs, as next‐generation candidates for antiaging therapy, have been well studied for their preventive, rejuvenating and anti‐inflammatory effects on senescence. There are several natural sources (from stem cells, young or centenarian donors), modified sources and artificial sources of therapeutic EVs 5.1.1 Senolytics Senolytic therapy specifically kills senescent cell populations depending on apoptotic resistance, an essential hallmark of cellular senescence. Using apoptosis‐inducing drugs, several intracellular prosurvival pathway members have been targeted to eliminate senescent cells, including Bcl‐2 family members (including Bcl‐2, Bcl‐W, and Bcl‐X L ), p53/FOXO4/p21 pathway members, PI3K/Akt pathway members, receptor tyrosine kinases, HSP90, HDACs, and glutamine metabolism pathway members (Song et al., 2020 ). The senolytic agent ABT‐737 and its orally bioavailable formulation ABT‐263 (navitoclax), which belong to the class of BH3 mimetics, can inhibit Bcl‐2, Bcl‐W, and Bcl‐X L and initiate apoptosis of senescent cells through a mitochondria‐dependent pathway (Basu, 2021 ). The highly potent and selective Bcl‐X L inhibitors A‐1155462 and A‐1331852 have also been proven to have proapoptotic ability but less hematological toxicity than navitoclax (Zhu et al., 2017 ). In multiple senescence events, the transcription factor FOXO4 is upregulated and interacts with p53 in the nucleus, preventing apoptosis. Administration of a peptide of FOXO4, FOXO4‐DRI, can perturb this interaction and cause p53 nuclear exclusion and cell‐intrinsic apoptosis in vitro and in vivo (Baar et al., 2017 ). In senescent liver cancer cells with p53 mutations, the antidepressant sertraline exerts a selective proapoptotic function via inhibition of the DNA‐replication kinase CDC7 and suppression of mTOR signaling (Wang et al., 2019 ). In addition, quercetin and fisetin, two natural flavonoids, have also been shown to have senolytic properties in the modulation of several pathways, such as the PI3K/Akt/mTOR and NF‐κB pathways (Ngoi et al., 2021 ). There exists a classic senolytic cocktail of dasatinib, a tyrosine kinase inhibitor, plus quercetin (D+Q), which has been well studied in the elimination of a wide range of senescent cells (Zhu et al., 2015 ). The combination of D+Q treatment can alleviate physical dysfunction and improve lifespan and healthspan in naturally aged mice by reducing the numbers of senescent cells and the levels of cytokines (Xu et al., 2018a ). This senolytic combination has been verified to be effective in phase I clinical trials for diabetic kidney disease ( NCT02848131 ) and idiopathic pulmonary disease ( NCT02874989 ) (Hickson et al.,

Extracellular vesicles are able to program their target cells to follow a Trojan‐horse approach, which enables them to exert pathogenic and curative effects depending on their source cells. In the aging microenvironment, EVs can transmit aging phenotypes from senescent cells in amplifying ways. However, they can also be cell‐free therapeutic materials and tools with no risk of tumour induction, and they show a low probability of immune rejection during regeneration and rejuvenation (Adamiak et al., 2018 ; Zhai et al., 2020 ). It has been determined that EVs are next‐generation candidates for use in achieving antiaging goals. 5.2.1 Natural sources of EVs There are natural, modified and artificial sources of therapeutic EVs (Table 3 ). Among natural sources, stem cells are the primary sources, and young donors are the main population group supplying these sources. EVs originating from stem cells contain diverse cargos that can execute antioxidative stress and anti‐inflammatory activities in order to induce antiaging effects (Figure 4 ). TABLE 3 Novel antiaging treatments via extracellular vesicles Treatments and sources Cargo(s) Subjects Mechanism(s) and Effect(s) Ref. Stem cell‐derived therapeutics Hypothalamic stem cells miRNAs Aged mice Blocked NF‐κB activation and restored GnRH secretion in neurons; Reduced age‐related physiological deficits and extended lifespan (Zhang et al., 2017 ) Human umbilical cord MSCs LncRNA MALAT1 Cardiomyocytes with H 2 O 2 ‐induced aging; Mice with D‐galactose‐induced aging Inhibited the NF‐κB/TNFα pathway; increased TERT expression; Increased the left ventricular ejection fraction and fractional shorting in aging mice (Zhu et al., 2019 ) Human umbilical cord MSCs / UVB‐irradiated HDFs Reduced production of ROS and increased glutathione peroxidase; Inhibited UVB‐induced senescence and induced fibroblast proliferation (Deng et al., 2020 ) ADSCs / IL‐1β‐treated OA osteoblasts Reduced inflammatory mediators including IL‐6 and prostaglandin E2; Controlled mitochondrial membrane stability and reduced oxidative stress (Tofino‐Vian et al., 2017 ) ADSCs miRNAs Proteins UVB‐irradiated HDFs Induced senescent fibroblast proliferation and DNA repair and attenuated aging; Suppressed the expression levels of MMP‐1, 2, 3, and 9 and enhanced collagen; Provided protection from UVB irradiation and enhanced recovery from skin photoaging (Choi et al., 2019 ) ADSCs / Myocardial infarction mice, myocytes, fibroblasts, and macrophages Activated S1P/SK1/S1PR1 signaling and promoted M2 polarization; Suppressed cardiac dysfunction, apoptosis, fibrosis, and the inflammatory state (Deng et al., 2019 ) ADSCs / Photoaging mice and fibroblasts Attenuated macrophage infiltration and ROS production; Promoted cell proliferation and decreased skin wrinkles (Xu et al., 2020 ) Human iPSCs / Aged and UVB‐irradiated HDFs Reduced the expression levels of SA‐β‐Gal and MMP1, 3; Restored collagen type I and ameliorated UVB‐induced skin aging (Oh et al., 2018b ) Human urine‐derived stem cells CTHRC1; OPG Osteoporotic mice Enhanced osteoblastic bone formation and suppressed bone resorption (Chen et al., 2019b ) Human embryonic stem cells miR‐200a Aged mice with pressure‐induced ulcers; Senescent endothelial cells Downregulated Keap1 and activated the Nrf2 pathway; Enhanced EC proliferation and angiogenesis; Accelerated wound closure and ameliorated senescent phenotypes (Chen et al., 2019a ) Human embryonic stem cells 4122 proteins Senescent BMSCs Rescued the function of senescent BMSCs and promoted proliferation; Induced osteogenic differentiation and reduced age‐related bone loss (Gong et al., 2020 ) Embryonic stem cells Several miRNAs Vascular dementia mice Alleviated senescence and loss of hippocampal stem cells; Reduced cognitive decline by activating mTORC1 and promoting TFEB translocation and lysosome resumption (Hu et al., 2020 ) Embryonic stem cells SMAD4, 5 Aged mice Activated MYT1 and improved HIF‐2α, NAMPT and SIRT1 levels; Alleviated hippocampal stem cell senescence, reversed cognitive impairment (Hu et al., 2021 ) BMSCs Neprilysin APP/PS1 AD mice Reduced Aβ plaques and dystrophic neurites in the cortex and hippocampus (Elia et al., 2019 ) Human decidua‐derived MSCs / High glucose‐treated fibroblasts; Diabetic mice Rescued the senescent state via inhibition of RAGE and activation of Smad; Accelerated collagen deposition and enhanced diabetic wound healing (Bian et al., 2020 ) Amniotic fluid MSCs miR‐21 Mice with premature ovarian dysfunction Increased total follicular counts and AMH levels and restored regular oestrous cycles by modulating the PTEN and caspase 3 apoptotic pathway (Thabet et al., 2020 ) Amniotic fluid MSCs miR‐320a Mice with premature ovarian insufficiency; POI human granulosa cells Decreased SIRT4 and ROS levels; Improved proliferation, inhibited apoptosis and elevated ovarian function (Ding et al., 2020 ) Dental pulp stem cells miR‐302b Senescent dental pulp stem cells Improved stemness thro

The aging microenvironment, which is faced with external and internal risk factors, is equipped with considerable antioxidant machinery to eliminate ROS in endothelial EVs and HUVEC EVs, which mainly contain antioxidant enzymes and peptides as well as NADPH‐synthesizing enzymes (Bodega et al., 2017 , 2018). Given this adaptive ability, centenarian donor‐derived EVs may have more potent antioxidant agents to maintain organ and systemic balance. In fibroblasts of centenarians, EVs contain high levels of the enzyme RNAseH2C, which can restrain DNA damage‐induced inflammation and ameliorate age‐associated low‐grade chronic inflammation (Storci et al., 2019 ). This phenomenon also implies that the antioxidant machinery is another controllable node of pathological transformation in the aging microenvironment. To some degree, intervention with antioxidant elements can support microenvironment stability.

Cellular senescence can be a potential launching point for various diseases, not only because of its association with organ/systemic aging but also because of its nature. As a relatively stable intermediate state between survival and death, senescence is associated with specific markers and cell morphological changes, which is convenient for qualitative and quantitative detection and evaluation (Hernandez‐Segura et al., 2018 ). There are significant differences and hidden weaknesses in chromatin structures, gene expression, and metabolic profiles of senescent cells compared with nonsenescent cells, which can be monitored by the immune system (Liu et al., 2020 ; Wang et al., 2019 ). Moreover, for tumours, the characteristics of cell cycle arrest can be leveraged for a physiological anticancer effect. In this review, we have discussed the characteristics of the aging microenvironment for the first time, highlighting new ideas that might be developed for the treatment of age‐related diseases. On the one hand, in view of the instability of the aging microenvironment, prompt prevention and exclusion of interfering factors must be the focal points and be managed accordingly. On the other hand, considering the amplification effect of the aging microenvironment, inhibiting the secretion of harmful molecules and cutting off routes of aging transmission can buffer the adverse effects of the inflammatory microenvironment. In addition, in light of the transformative nature of the aging microenvironment, exploring the key mechanism of transformation and intervening in key events can effectively forestall and reverse the establishment and progression of pathological states. Accordingly, EVs play undeniable roles in cellular senescence and age‐related diseases and can be used in promising approaches to identify and therapeutically target senescent cells. However, there are still several challenges associated with the development of this young but emerging field of research, including a lack of optimal and verified experimental models, details on the biogenetic mechanism, a comprehensive age‐related EV atlas and a rapid capture and visualization system. Given the long aging period of humans, fast‐aging model organisms, such as Caenorhabditis elegans and yeasts, and animals, such as naturally aging mice or gene‐edited mice, are widely used in geroscience. However, they do not faithfully recapitulate human states due to the complex multicell and multiorgan networks of senescence. Better‐suited animal models (e.g., nonhuman primates) or human organoids are required for research on the topics of EVs and aging. As secretion marks the onset of EV function, clarifying the detailed molecular mechanism of EV secretion may be helpful for elucidating the aging transmission pathway and developing efficient therapeutic interventions. In addition, given the heterogeneity of senescence and EVs, creation of a comprehensive atlas of age‐related EVs from different cells under different stresses would be enormously helpful for this field. It is possible that a specific ageotype may indicate an individual physiological and pathological status as a more distinctive biomarker than others now available. Senescence‐associated EVs can now be detected in blood, urine, CSF, and saliva, which provides us with new inspiration for diagnostic and prognostic tools using liquid biopsy (Machida et al., 2015 ; Perrotte et al., 2020 ; Santelli et al., 2019 ). Due to the prion‐like behaviour of EVs, in vivo imaging techniques have been designed, such as techniques involving EV labelling combined with CT that longitudinally and quantitatively trace aberrant neurons in the brains of PD, AD, and stroke patients (Perets et al., 2019 ). However, a full understanding of which cells are senescent and which cells secrete and take up aging EVs in vivo is lacking owing to the unsuitable and inaccurate capture and visualization systems. To develop clinical antiaging therapies, safety should first be considered, followed by sensitivity and specificity. Because of the specificity of immune recognition, senolytic CAR‐T and senescent CD153 vaccines have been proven feasible and efficient (Amor et al., 2020 ; Yoshida et al., 2020 ). This finding suggests that immune‐modified EVs may be able to preferentially target senescent cells and tissues to promote senotherapy. However, before EV transplantation is performed, the routes of administration, reasonable dosages, dosing intervals, and host nutritional status should be strictly verified. EVs can be considered promising materials and tools for antiaging treatments after rigorous trials.