Exosome biogenesis involves invagination of the plasma membrane to form early sorting endosomes, which mature into late endosomes and MVBs (Ka et al., 2020). The sorting of cargo into intraluminal vesicles occurs through both ESCRT-dependent and independent mechanisms (van Niel et al., 2018). MVBs then fuse with the plasma membrane to release exosomes. Once secreted, exosomes interact with recipient cells via endocytosis, receptor-mediated uptake, or direct membrane fusion. These processes are regulated by proteins such as Rab GTPases, tetraspanins, and various cytoskeletal elements (Thery et al., 2018). The biogenesis, release, and interactions with target cells of exosomes are showed in Figure 1.

Exosomes contain a lipid bilayer that protects their internal cargo. They are enriched with proteins such as CD9, CD63, and TSG101; lipids such as ceramide and cholesterol; and nucleic acids including miRNAs, lncRNAs, and circRNAs (Skog et al., 2008; Figure 2). These molecules mediate their functional effects. For example, miR-21, miR-146a, and miR-145 have been implicated in inflammation and vascular remodeling. lncRNAs and circRNAs contribute to transcriptional and post-transcriptional regulation (Valadi et al., 2007; Jiang N. et al., 2019).

After exosomes are secreted, they are taken up by target cells through several distinct mechanisms. Endocytosis is the primary method of exosome uptake and is mediated by various substances including lipid rafts, clathrin, and caveolin. For example, exosomes derived from Schwann cells enter target cells via lipid raft-mediated endocytosis and regulate macrophage polarization via the SOCS3/STAT3 pathway to alleviate inflammatory responses (Yu et al., 2019). Additionally, the primary mechanism for exosome-like vesicle uptake from Opisthorchis felineus by human cholangiocytes is clathrin-mediated endocytosis, highlighting the significance of different endocytic pathways in various cell types (Wang et al., 2019). Myriam et al. reported a unique pathway for exosome uptake in hypoxic environments. They discovered that under hypoxic conditions, heparan sulfate proteoglycan-dependent endocytosis promotes exosome uptake, leading to lipid droplet formation in glioma cells (Ren et al., 2023). Exosomes are preferentially taken up by homologous cells through receptor-ligand interactions, making them highly likely to be enclosed by lysosomes. Shabirul et al. designed exosomes to express CD19 chimeric antigen receptors, enabling them to target CD19-positive leukemia B cells through receptor-ligand interactions and induce cell death without affecting CD19-negative cells (Pakharukova et al., 2023). Membrane fusion is a common mechanism for heterologous cell uptake. Exosomal contents are delivered to the target cell when the exosomal membrane fuses with the plasma membrane, and the exosomal contents are then delivered into the target cell (Cerezo-Magana et al., 2021). Exosomes secreted by dendritic cells can fuse with target cells, creating fusion pores that release their contents (Haque et al., 2021). The uptake of exosomes by target cells is influenced by the specific surface proteins of both the exosomes and target cells, as well as the external environment. Plasma proteins attached to the surfaces of exosomes can alter their functional properties, provide new functionalities, and affect their uptake by target cells (Montecalvo et al., 2012). Lower pH levels in the environment significantly affect EV secretion and increase EV uptake (Chernomordik and Kozlov, 2008).

Common exosome isolation techniques include ultracentrifugation (Figure 3), PEG precipitation, and size-exclusion chromatography (Konoshenko et al., 2018). Immunoaffinity capture and commercial kits like ExoQuick are also used. Characterization methods include nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and flow cytometry (Table 1). While each technique has advantages and drawbacks, combining approaches improves yield and purity (Zhang et al., 2016). Detailed reviews on methodology are available for further reference (Mateescu et al., 2017).

Exosomes are a focal point of research in the fields of medicine and biomedicine, and optimizing their preservation is a prerequisite for further study. Research indicates that the storage methods of exosomes significantly impact their concentration, physical state, and biological functionality (Jeyaram and Jay, 2017). Therefore, optimizing storage conditions is crucial for the reliability of fundamental research results on exosomes, the efficacy of clinical applications, and the quality and safety of commercialized products. Currently, storage at −80°C is a widely adopted method. Stefano et al. systematically evaluated eight different strategies, including simple −80°C freezing and the addition of seven different protective agents, as well as the effects of freeze-thaw cycles on exosomes. The results indicated a negative correlation between storage time and the concentration and purity of exosomes, with particle size and variability changing over time. They recommended that, in most cases, exosomes should be sourced from fresh samples. For strict storage requirements, biological samples may be stored short-term, while storing isolated exosomes is not advised (Gelibter et al., 2022). Yuan et al. proposed that storage at 4°C can be utilized for short-term preservation of exosomes. However, for long-term retention of functionality and extended shelf life, freeze-drying presents a promising method (Yuan et al., 2021). André et al. found that storing exosomes in phosphate-buffered saline (PBS) leads to a significant decrease in recovery rates, particularly for pure samples. They applied a novel buffer, which is a PBS formulation supplemented with human albumin and trehalose (PBS-HAT), and discovered that it significantly enhances both the short-term and long-term preservation of exosomes, especially under −80°C conditions (Gorgens et al., 2022). Further research is essential to explore optimal methods for the long-term or short-term preservation of exosomes while maintaining their inherent properties and functionalities.

Almost all human cells secrete exosomes that fuse with target cells to transfer functional molecules or activate receptors or effectors. Exosomes released through various pathways can act as signaling messengers, regulating the behavior of nearby, distant, or even originating cells. For instance, hepatocyte-derived exosomes mediate increased synthesis of sphingosine-1-phosphate by transferring neutral ceramidase and sphingosine kinase 2, thereby promoting hepatocyte proliferation and regeneration following ischemia-reperfusion injury or partial hepatectomy (Wu and He, 2023). Exosomes released by ECs under oxidative stress conditions can enhance EC proliferation and migration and transport miR-92a-3p to VSMCs, promoting their proliferation and migration (Guo and Zhang, 2023). Using a rat model, Qu et al. demonstrated that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) enhanced endothelial function, inhibited neointimal hyperplasia in vein grafts, and accelerated reendothelialization (Nojima et al., 2016). These exosomes promoted EC proliferation and migration, thereby contributing to vascular repair and regeneration.

Exosomes play crucial roles in cellular immune responses. Certain exosomes secreted by macrophages can induce proinflammatory reactions. For instance, exosomes containing glycopeptidolipids from Mycoplasma gallisepticum-infected macrophages can stimulate resting cells to exert proinflammatory responses (Yao et al., 2022). Under stress conditions, heat shock proteins are embedded in the plasma membrane and then released into the environment, inducing tumor necrosis factor alpha (TNF-α) release and activating macrophages (Qu et al., 2020). Neutrophil-derived exosomes transfer miR-30d-5p, inducing M1 macrophage polarization and activating the nuclear factor kappa B (NF-κB) signaling pathway, leading to an inflammatory response in macrophages (Bhatnagar and Schorey, 2007). Zhang et al. studied the effects of exosomes derived from Treponema pallidum-stimulated dendritic cells on EC inflammation. These exosomes activated the LR4/MyD88/NF-κB signaling pathway, increasing the expression of interleukin (IL)-1β, IL-6, and TNF-α in ECs, thereby promoting inflammation. Plasma exosomes from patients with acute myocardial infarction can induce NF-κB signaling activation, contributing to proinflammatory immune responses and EC injury (Vega et al., 2008). Moreover, exosomes can modulate the tumor microenvironment and enhance or reduce the responsiveness of cancer cells to therapies. They transport non-coding RNAs and regulate molecular signaling pathways such as PTEN and PI3K/Akt, promoting chronic inflammation, immune evasion, and tumor progression (Hazrati et al., 2022).

Exosomes play a significant role in neuronal signal transmission, promoting neuronal survival and synaptic assembly. They can cross the blood–brain barrier and are involved in myelination. Exosomes are associated with the pathological processes of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington’s disease (Giannotta et al., 2013). Aging is regulated by the hypothalamus. A study involving mice revealed that exosomes secreted by normal hypothalamic stem cells can slow aging, suggesting that the hypothalamus partially controls this process through the release of exosomes (Gong et al., 2023).

Surgical procedures and dialysis punctures can cause tissue injury, and EC damage is particularly significant. Under physiological conditions, ECs form a continuous endothelium that lines the inner walls of blood vessels. This endothelium not only facilitates the metabolic exchange of plasma and tissue fluid but also secretes various bioactive substances that maintain vascular tone, regulate blood pressure, and balance antiinflammatory and proinflammatory responses. Compromised integrity of the endothelium leads to the invasion of inflammatory cells, platelet aggregation, and thrombosis (Galardi et al., 2023). Oxidative stress is a crucial mechanism underlying endothelial damage. During oxidative stress, the balance between oxidation and antioxidation is disrupted owing to the excessive production of reactive oxygen species (ROS). Hypoxia and substances, such as angiotensin II, can induce ROS production, leading to endothelial tissue damage (Lee and Roy-Chaudhury, 2009). ROS reduces the bioavailability of nitric oxide (NO) through redox reactions, causing vascular dysfunction. NO plays a crucial role in the cardiovascular system by relaxing vascular smooth muscle, promoting vasodilation, and enhancing blood flow. Additionally, it possesses anti-inflammatory properties, supports endothelial cell function, and inhibits platelet aggregation. Zhu et al. designed a patch that covalently binds a nitrite compound to a biodegradable polymer for localized NO release. Their study, conducted in animal models, demonstrated that the released NO improves cardiac function, aids in cardiac tissue repair, and alleviates cardiac remodeling and fibrosis (Zhu et al., 2021). In another study, researchers developed a precise system for delivering nitric oxide (NO) to tissues, which demonstrated the critical role of NO in vascular regulation and tissue repair in both rat hind limb ischemia models and mouse acute kidney injury models (Hou et al., 2019). Furthermore, ROS can trigger endothelial inflammation, disrupt mitochondrial function, and lead to abnormal energy metabolism, exacerbating EC damage (Wettschureck et al., 2019). ROS can induce EC apoptosis by increasing the expression of p38MAPK and caspases and by inhibiting the expression of Bcl-2 while promoting the expression of Bax and Fas. It can also induce apoptosis in VSMCs (Shaw et al., 2014). ROS have been recognized as a significant target in ischemia-reperfusion injury. Li et al. developed a ROS-sensitive hydrogel for the delivery of basic fibroblast growth factor (bFGF) aimed at repairing damaged cardiomyocytes and enhancing angiogenesis (Zhenhua Li et al., 2021). The NLRP3 inflammasome activation plays a significant role in endothelial dysfunction (Shaito et al., 2022). The NLRP3 inflammasome can activate and promote the maturation of IL-1β and IL-18 and mediate the formation of the pore protein, N-GSDMD, leading to cell swelling, rupture, and death (Higashi, 2022). It can also disrupt tight junction proteins between ECs, thereby increasing vascular permeability. As an upstream signal of NLRP3, ROS initiates and activates the NLRP3 inflammasome, thereby triggering pyroptosis.

Blood flow in vessels can be categorized into laminar and turbulent flows based on different flow patterns, which are closely related to the vascular configuration. In smooth and straight sections of the lumen, blood flow is typically laminar, with a Reynolds number (Re) of less than 2000. Conversely, in irregular sections, including the inner sides of curved vessels and upstream stenotic regions, the flow becomes unstable and disturbed, manifesting as a turbulent flow with an Re greater than 2000. The original vascular anatomy is altered following AVF creation, resulting in different angles between the arteries and veins. This alters the type of shear stress acting on the endothelium, shifting it from laminar shear stress to low oscillatory shear stress (Bai B. et al., 2020). The EC membrane contains numerous receptors that detect and transmit mechanical stimuli, including receptor tyrosine kinases, G protein-coupled receptors, and various ion channels (Zhang Y. et al., 2019). This mechanosensory capability helps differentiate blood flow characteristics and make physiological adjustments.

Under laminar shear stress, the expression of the cyclin-dependent kinase inhibitor p21 increases, slowing EC DNA synthesis and inhibiting proliferation. Laminar shear stress can suppress apoptosis induced by serum starvation, oxidative stress, and TNFα, potentially through increased expression of nitric oxide synthase and superoxide dismutase (Pfenniger et al., 2015). NO produced by ECs can elevate cGMP and PKG levels, reducing Ca2+ concentration and promoting vasodilation (He et al., 2022). Under physiological conditions, laminar shear stress also reduces the expression of the adhesion molecules, vascular cell adhesion molecule one and intercellular adhesion molecule 1. This in turn decreases leukocyte and platelet aggregation, reduces ROS levels, activates anticoagulant genes, and inhibits VSMC proliferation and migration. These actions help maintain EC vitality and functional integrity (Rennier and Ji, 2013; Cirino et al., 2017). In contrast, under low shear stress, cell proliferation is accelerated, ROS levels are increased, and IL-6 and IL-8 secretion is significantly increased, activating neutrophils and promoting their adhesion to ECs, thereby fostering inflammation and apoptosis.

Inflammation is a key factor in IH development. The inflammatory response involves various miRNAs that target and regulate multiple proteins to promote inflammation; these include miR-125, miR-155, and miR-125. Following tissue injury, leukocytes aggregate at the injury site under the influence of chemokines (Zheng et al., 2013). Macrophages and neutrophils release a plethora of inflammatory cytokines such as interleukins, C-reactive protein, and TNF-α during the inflammatory response. These pro-inflammatory cytokines, in conjunction with growth factors, further promote the aggregation of inflammatory cells and initiate the phenotypic transformation of VSMCs, leading to their proliferation and migration (Wang et al., 2014). Chemokines that promote leukocyte aggregation, such as platelet-derived growth factor (PDGF) and tumor growth factor-β, are secreted by VSMCs and activated by ECs. Local inflammation also triggers vascular wall remodeling and neointimal formation, leading to vascular intima stenosis. Yi et al. discovered that Nik-related kinase expression in VSMCs affects IH and vascular inflammation by regulating the expression of matrix metalloproteinases and inflammatory cytokines/chemokines (Charo and Taubman, 2004). The epigenetic factor, PCAF, is also involved in regulating vascular inflammation by promoting NF-κB-mediated inflammation (Carracedo et al., 2019). Further, reduced PCAF levels significantly decreased IH, the intima/media ratio, and luminal stenosis. Xu et al. revealed that Sox10 is a key regulator of vascular inflammation (Lu et al., 2020). It activates the PI3K/AKT signaling pathway and drives the transdifferentiation of VSMCs into macrophages through lactylation and phosphorylation modifications, thereby promoting pyroptosis and exacerbating the inflammatory response.

Upon stimulation, ECs contract, leading to the disruption of adhesive junctions. Adhesion between ECs is primarily maintained by vascular endothelial cadherin (VE-cadherin), which interacts with cytoskeletal molecules to regulate EC adhesion, vascular regeneration, and migration (de Jong et al., 2017). VE-cadherin connects with β-catenin, plakoglobin, and p120-catenin to stabilize EC junctions (Xu et al., 2023). The barrier function of ECs is dynamic and maintained by adhesive junctions. Endothelial permeability increases under the influence of autocrine substances such as vascular endothelial growth factor (VEGF) and thrombin. This dynamic change during vascular injury promotes the accumulation of inflammatory mediators, leukocyte migration, and Ca2+ elevation and activates Ca2+-driven proteins such as myosin light chain kinase and membrane-associated protein A2. These processes promote actin contraction and the degradation of endothelial cadherin, leading to EC detachment and barrier dysfunction (Giannotta et al., 2013; Oas et al., 2013). Additionally, talin-dependent activation of EC β1-integrin weakens adhesion and reduces tyrosine phosphorylation of the VE-cadherin-catenin complex, and VEGF activation of FAK kinase opens endothelial junction structures (Dalal et al., 2020). Collectively, these processes contribute to the loss of endothelial function.

Endothelial–mesenchymal transition (EndoMT) refers to a pathological process in which endothelial cells lose their specific markers (e.g., CD31 and VE-cadherin) and acquire a mesenchymal phenotype characterized by the expression of α-SMA, vimentin, and other mesenchymal proteins. This transition is commonly induced by inflammatory cytokines, oxidative stress, or transforming growth factor-beta (TGF-β), and has been shown to play a pivotal role in vascular remodeling and neointimal hyperplasia, particularly following vascular injury (Qian et al., 2025). Emerging studies have indicated that exosomes also play critical roles in modulating EndoMT. Exosomes derived from diseased or inflamed tissues may promote EndoMT, whereas those from mesenchymal stem cells or other regenerative sources can inhibit EndoMT and preserve endothelial identity, offering potential therapeutic avenues for limiting intimal hyperplasia in AVF (Sishuai et al., 2024). Additionally, under oxidative stress conditions, exosomes derived from adipose-derived stem cells (ADSC-Exo) have been shown to alleviate hydrogen peroxide-induced EndoMT in human umbilical vein endothelial cells (HUVECs) by inhibiting the miR-486-3p/Sirt6/Smad signaling axis, suggesting their antifibrotic potential in endothelial dysfunction (Li et al., 2024).

During neointimal formation following vascular injury, ECs play a crucial role, with some cells undergoing competitive selection to become tip cells that rearrange dynamically to form new blood vessels (Pulous et al., 2019). In this process, the interaction between the Notch and YAP/TAZ signaling pathways regulate the selection and phenotype of tip cells (Chen et al., 2012). Chen et al. found that the transcription factor Foxp1 in ECs can regulate VSMC proliferation and migration by targeting the matrix metalloproteinase (MMP)-9 and Cdkn1b signaling pathways (Blanco and Gerhardt, 2013). When Foxp1 is absent, VSMC proliferation and migration increase, while endothelial cell proliferation decreases.

VSMCs are abundant in the vasculature and primarily comprise the medial layer of blood vessels. The active contraction and passive recoil of elastin fibers contribute to the elasticity of the blood vessels, thereby regulating blood pressure and flow distribution. VSMCs exhibit high plasticity, phenotypic switching capabilities, and proliferative potential, which enable adaptive responses to environmental changes. They are regulated by many miRNAs (Table 2). Under normal physiological conditions, VSMCs express many contractile proteins such as myosin heavy chain 11, SM22α, and α smooth muscle actin, which confer a contractile phenotype (Guo et al., 2024). Their contraction activity is regulated by calcium ions, and calmodulin activates myosin light chain phosphorylation, leading to myofibril contraction. However, under various pathological conditions, VSMCs can switch from a contractile phenotype to a synthetic phenotype, aiding in injury repair. In the synthetic phenotype, the cells change from a spindle shape to an irregular shape, with a significant reduction in contractile markers. Their proliferation and migration abilities are enhanced, and they secrete large amounts of extracellular matrix, including collagen, elastin, and matrix metalloproteinases, in response to injury (Chen et al., 2022; Chakraborty et al., 2021). Phenotypic switching of VSMCs is typically regulated by inflammatory mediators, growth factors, transcription factors, and noncoding RNAs. For instance, growth factors can stimulate VSMC dedifferentiation through the MAPK pathway, whereas transcription factors such as KLF4 can regulate phenotypic switching by modulating epithelial adhesion complexes and actin cytoskeleton dynamics (Lynch et al., 2016; Jain et al., 2020). Synthetic VSMCs migrate from the medial layer to the intima, forming a new intimal layer that causes venous neointimal hyperplasia.

Ferroptosis, a research hotspot in recent years, is a form of programmed cell death that is dependent on iron and lipid peroxidation. It is primarily regulated by three major mechanisms—iron metabolism, lipid metabolism, and the glutathione peroxidase 4 (GPX4) pathway activation. Other pathways involved include FSP1-CoQ10-NAD(P)H, BH4-DHFR, and P53 signaling (Yoshida et al., 2012). (Figure 5.) Ferroptosis is closely associated with intimal vascular stenosis. Using differentially expressed gene analysis, Zhang et al. identified 34 and 31 significantly differentially expressed ferroptosis-related genes at 2 days and 14 days after carotid artery ligation in mice, respectively (Han et al., 2021). Exposure to ZnO nanoparticles (ZnONP) induces endothelial dysfunction via ferroptosis. When ZnONP exposure triggers vascular inflammation, ferroptosis markers increase, and the inhibition of ferroptosis reduces vascular damage (Zhang et al., 2021). Chen et al. found that the phospholipid oxidation product, PGPC, increases ferrous ion content and peroxide production, inducing EC ferroptosis via the CD36 receptor, thereby impairing endothelial function by increasing FABP3 expression (Zhang L. et al., 2022). Luo et al. discovered that ferroptosis is closely related to EC death, activating the p53-xCT-GSH axis and causing endothelial dysfunction (Qin et al., 2021). Zhang et al. found that BaP/BPDE exposure upregulates free iron, accelerates lipid peroxidation, and reduces GPX4 protein levels, thereby inducing ferroptosis, inhibiting EC function, and affecting angiogenesis (Chen et al., 2024).

Ferroptosis also induces VSMC phenotypic switching. In vivo activation of ferroptosis by RSL3 promotes VSMC phenotypic switching and exacerbates IH. A previous study showed that the ferroptosis inhibitor, Fer-1, counteracted the effects of RSL3 by reducing IH (Luo et al., 2021). Sun et al. discovered that ferroptosis promotes VSMC aging and vascular aging (Zhang et al., 2023). Activation of ferroptosis signaling led to a decrease in NAD + levels and drove VSMC aging via NCOA4-mediated ferritinophagy. Using a specific inhibitor of lysine-specific demethylase 1 has been found to reduce intracellular iron levels and decrease VSMC ferroptosis (Zhang S. et al., 2022). Targeted inhibition of the endogenous histone acetyltransferase P300 activates the hypoxia-inducible factor 1-alpha (HIF1-α)/HMOX1(Heme Oxygenase 1) axis, promoting CD and IKE-induced VSMC ferroptosis, thus contributing to vascular stenosis-related diseases. Ferroptosis is closely related to inflammation, which influences vascular intimal stenosis by modulating inflammation-related pathways. Chen et al. concluded that ferroptosis regulate the activity of five inflammatory signaling pathways—JAK-STAT, NF-κB, inflammasome, cGAS-STING, and MAPK, thereby affecting cellular functions (Sun et al., 2024).

Exosomes, found in various body fluids, can reflect the state of their parent cells. Their significant advantages include the capability for rapid detection and noninvasive collection of body fluids. Notably, miRNAs within exosomes offer superior sensitivity and specificity compared to circulating miRNAs in blood, as they can be purified to ensure high sensitivity and specificity. A study utilizing microarray analysis compared miRNA expression profiles between stenotic AVF sites and control veins. The results showed that hsa-miR-214-3p, hsa-miR-374B-3p, and hsa-miR30e-3p were upregulated, whereas hsa-miR-4708-3p, hsa-miR-371b-5p, and hsa-miR-3960 were downregulated in primary AVF (Li et al., 2023). These findings provide valuable guidance for the use of exosomes as biomarkers to detect AVF stenosis. Zhao et al. induced VSMC proliferation using angiotensin II and observed that the expressions of KLF5 and cyclin D1 also increased with increased VSMC proliferation, along with miR-146a upregulation (Li and Chen, 2023). They used salvianolic acid B to downregulate miR-146a expression, thereby inhibiting neointimal hyperplasia. miR-146a has been proposed as a biomarker for inflammation and fibrosis (Lv et al., 2016). A study on patients with periodontitis revealed that serum exosomal miR-146a levels were significantly elevated and exhibited a positive correlation with inflammatory factor levels and disease severity. Inflammation is critical in acute coronary syndrome. Serum miR-146a levels are significantly higher in patients with this condition than in healthy controls. miR-21 is also crucial for vascular intimal stenosis. Overexpression studies have shown that miR-21 downregulation reduces intimal formation in rat carotid arteries after angioplasty. miR-21 can inhibit VSMC proliferation and promote apoptosis. Clinically, samples from stenotic AVF sites show high miR-21 expression, which is predominantly localized in the intimal region where fibroblasts are situated (Zhao et al., 2019). Exosomes containing miR-155 induce EC inflammation, leading to endothelial dysfunction. They increase the production of IL-6 and TNF-α and exert their effects through SHIP1 and SOCS1 (Liao et al., 2023).

High-mobility group box 1 (HMGB1) present in exosomes, and they play a pivotal role in neointimal hyperplasia. HMGB1 promotes inflammatory responses through damage-associated molecular patterns and activates immune cells, leading to the generation of IL-1β (Kilari et al., 2019). HMGB1 and IL-1β together can upregulate MMP-1, MMP-3, and MMP-9 expression, which exacerbate vascular wall degradation and remodeling (Jiang K. et al., 2019). Following vascular injury, VSMCs migrate from the intima to the site of injury. During this process, HMGB1 interacts with receptors for advanced glycation end products and TLRs, thereby promoting VSMC proliferation and migration (Yang and Tracey, 2010). This nuclear protein, which circulates via EVs, can be used as a biomarker, offering significant insights into vascular pathology.

Currently, many nanometer-scale particles are used as platforms for the delivery of therapeutic drugs. However, artificially prepared carriers often face issues such as immune rejection and inability to penetrate specific biological barriers. In contrast, exosomes exhibit excellent biocompatibility and possess membrane structures on their surfaces, making them ideal drug carriers. In addition, proteins, nucleic acids, and other drug molecules encapsulated within exosomes demonstrate greater stability. Exosomes can be artificially engineered to encapsulate various types of drugs and are designed with surface antigens that target specific cells or tissues. Common antiproliferative drugs, such as paclitaxel, can be delivered in a targeted manner using exosomes. Exosomes contain a plethora of proangiogenic and antiinflammatory factors that promote angiogenesis and mitigate inflammatory responses following vascular injury. Exosome-releasing stents have been developed to gradually release exosomes fixed to the stent surface, thereby promoting EC regeneration and vascular wall repair. These stents significantly enhance EC migration and proliferation while reducing smooth muscle cell proliferation in animal models. This technology effectively reduces the restenosis rate after stent implantation and accelerates vascular healing (Wang and Khalil, 2018). Researchers have initiated mesenchymal stem cells release with paclitaxel, reporting that the released microvesicles exhibit strong antiproliferative activity (Wang and Khalil, 2018; Inoue et al., 2007).

The therapeutic applications of exosomes encapsulating miRNAs, lncRNAs, and circRNAs have been extensively studied. VSMC-derived exosomes are rich in miR-145, which regulates VSMC function by inhibiting autophagy, thereby reducing VSMC proliferation and migration (Hu et al., 2021). Animal studies on miR-145 transduction have demonstrated its role in phenotypic modulation, converting proliferative VSMCs into a contractile state. This conversion alleviates neointimal hyperplasia and has shown promising results in the treatment of venous graft diseases (Wang et al., 2020a). Zhao et al. loaded an miR-145-5p agomir (miR-145) into injectable and in situ self-assembling RAD hydrogels, allowing for the slow and controlled release of miR-145. This method reduced smooth muscle cell migration, promoted the contractile phenotype, and facilitated EC regeneration. Studies using rat vascular injury models revealed that miR-451 upregulation improved intimal thickening by mitigating VSMC damage caused by PDGF-BB and inhibited VSMC proliferation and migration (Nishio et al., 2020). Researchers using the Shexiang Baoxin pill to influence miR-451 expression and regulate PDGF-BB found that miR-451 can also regulate VSMC phenotypic transformation and promote apoptosis to reduce neointimal hyperplasia after vascular injury (Zhang W. et al., 2019). In another study using a mouse tissue-engineered vascular graft model, miR-451 expression was found to be downregulated in graft stenosis (Li et al., 2022). miR-451 downregulation leads to acute proliferation of macrophages and VSMCs, highlighting a direct regulatory relationship between miR-451 and macrophage migration inhibitory factor, suggesting miR-451 as a potential therapeutic target for intimal stenosis. Exosomes derived from hucMSCs are rich in miR-148a, which targets the 3′untranslated region to inhibit SERPINE1, reducing VSMC phenotypic transformation and migration (Hibino et al., 2016). SERPINE1, also known as plasminogen activator inhibitor-1, is a critical factor contributing to IH.

LncRNAs have emerged as significant targets for intervention in neointimal hyperplasia. Silencing lncRNA-p21 induces proliferation while inhibiting the apoptosis of VSMCs and macrophages (Zhang X. et al., 2022). Liang et al. found that the lncRNA Xist functions in VSMCs by interacting with miR-29b-3p and inducing arterial smooth muscle cell apoptosis via the miR-29b-3p/Eln pathway (Wu et al., 2014). Song et al. demonstrated that overexpression of lncRNA SENCR regulates the miR206/myocardin axis, thereby inhibiting VSMC proliferation, migration, and phenotypic transformation (Liang et al., 2021). Cheng et al. identified that the lncRNA LLNC00281 negatively regulates ANXA2 and inhibits VSMC proliferation, migration, and dedifferentiation through the NF-κBp65 signaling pathway (Song et al., 2022). Another study found that the lncRNA RP11-531A24.3 also negatively regulates ANXA2 (Verma Nagendra and Arora, 2025). Similarly, circRNAs have been implicated in IH and are considered potential therapeutic targets. Fu et al. found that circMAPK1 regulates VSMC proliferation and migration via the miR22-3p/MECP2 axis (Cheng et al., 2022). Lin et al. reported that circ_0021155 expression increases ox-LDL-induced VSMC proliferation and migration (Wu et al., 2021). This circRNA promotes TRPM7 expression while inhibiting the expression of α-smooth muscle actin and calmodulin, thereby enhancing smooth muscle cell proliferation and migration.

Exosomes influence neointimal hyperplasia by interfering with ferroptosis. Various miRNAs within exosomes regulate iron and ROS metabolism during ferroptosis, thereby affecting cellular redox states (Lin et al., 2022). Understanding the mechanisms by which miRNAs regulate ferroptosis is crucial for diagnosing and treating IH. Exosomes derived from human umbilical cord blood stem cells can inhibit ferroptosis by suppressing DMT1 expression via miR-23a-3p (Zong et al., 2022). Jia et al. found that exosomes from endothelial progenitor cells upregulate miR-30e-5p in HUVECs, activating the AMPK pathway. miR-30e-5p directly targets and inhibits SP1, thereby preventing EC ferroptosis (Song et al., 2021). Additionally, Li et al. demonstrated that exosomes from endothelial progenitor cells transfer miR-199a-3p to ECs, reducing ROS production and iron accumulation, ultimately inhibiting EC ferroptosis and death (Xia et al., 2022).

However, the use of exosomes as drug carriers has several limitations. First, exosomes derived from stem cells are limited by the manufacturing methods, making large-scale, high-yield production challenging (Li et al., 2021). Second, the loading capacity of exosomes is relatively low, and they are easily captured by non-target organs, thereby reducing their effective dosage. Thus, surface modification of exosomes is crucial to enhance their targeting and efficacy (Pascucci et al., 2014; Colao et al., 2018).

Despite the promising roles of exosomes in regulating vascular remodeling, inflammation, and cell proliferation in AVF stenosis, several critical challenges hinder their clinical translation. First, the heterogeneity of exosome populations derived from different cell types and biofluids complicates standardization and quality control. Secondly, current isolation and purification techniques, such as ultracentrifugation and precipitation, often yield preparations with impurities or low reproducibility, limiting their scalability for clinical-grade production. Moreover, the targeting efficiency and in vivo biodistribution of exogenously administered exosomes remain suboptimal, raising concerns about their delivery to pathological sites. In addition, the mechanisms underlying exosome-mediated signaling are not fully elucidated, especially in the context of AVF-specific microenvironments. Regulatory hurdles, lack of large-scale clinical trials, and insufficient understanding of long-term safety further complicate the path to clinical application. Therefore, while exosomes hold great promise, their use in treating AVF stenosis remains at a preclinical stage and requires further technological, mechanistic, and translational advancements (Verma Nagendra and Arora, 2025).