Extracellular Vesicles as Emerging Regulators in Ischemic and Hypertrophic Cardiovascular Diseases: A Review of Pathogenesis and Therapeutics

Introduction

Cardiovascular diseases (CVDs) continue to be the leading cause of death worldwide, with projections estimating 23 million global deaths annually by 2030 [1]. CVDs encompass a broad range of diseases, including myocardial infarction, ischemia-reperfusion injury, coronary artery disease, atherosclerosis, cardiac hypertrophy, and heart failure. Despite a decline in overall mortality rates in recent decades, the overall burden of CVD remains unchanged due to the increased number of patients surviving into older age with serious cardiovascular conditions. Consequently, there is an increased focus on understanding the underlying pathophysiology of various CVDs and manipulating key cellular processes to mitigate the disease burden [2].

In recent years, there has been increasing interest in understanding the molecular and cellular mechanisms underlying specific forms of CVD, particularly ischemic and hypertrophic cardiovascular diseases. Among emerging regulators, extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, have attracted significant attention. These lipid bilayer-bound vesicles facilitate intercellular communication by transporting proteins, lipids, DNA, and various types of RNA. Through this cargo, EVs modulate key processes such as inflammation, apoptosis, angiogenesis, fibrosis, and immune regulation [3].

This article reviews the emerging roles of extracellular vesicles in atherosclerosis, myocardial ischemia, infarction, and reperfusion injury, and cardiac hypertrophy. We summarized emerging insights into the mechanistic roles of EVs in disease pathogenesis, highlight preclinical and clinical studies of EV-based therapies, and discuss the potential of EVs as diagnostic and therapeutic tools.

Extracellular Vesicles

MICROVESICLES:

Microvesicles are a heterogenous group of membrane-enclosed vesicles, with a size ranging from 50 to 1000 nm [10]. They are produced via the direct shedding of the vesicles from the cell membrane. This involves the outwards blebbing of small cytoplasmic protrusions on the plasma membrane, which are driven by changes in cytoskeletal reorganization and membrane lipid composition. These protrusions grow and elongate and subsequently detach by fission of their stalk [11]. Microvesicles do not have distinctive, universally recognized markers, as their surface markers primarily reflect the composition of the membrane from which they originate. This can serve as a powerful marker indicating the state of the original cell, as they can carry a snapshot of the cellular conditions, including stress, activation, or apoptosis, at the time of their formation [10]. Their versatility and the information they carry are increasingly being explored in cardiovascular research, where they may provide insights into disease progression and potential therapeutic targets [12].

Microvesicle biogenesis is notably regulated by small GTPases, particularly ADP ribosylation factor 6 (ARF6), which orchestrates membrane trafficking, actin cytoskeletal remodeling, and vesicle release [13,14]. ARF6 signaling promotes the recruitment of extracellular signal-regulated kinase (ERK) to the plasma membrane, resulting in myosin light chain kinase (MLCK)-dependent actomyosin contraction necessary for vesicle budding and scission from the cell surface [13]. Microvesicles exhibit significant heterogeneity, including specialized subpopulations such as arrestin domain-containing protein 1-mediated microvesicles (ARMMs) and large oncosomes [15]. ARMM formation involves recruitment of ESCRT-1 complex component TSG101 by arrestin domain-containing 1 (ARRDC1) at the plasma membrane [16], while large oncosomes are markedly larger vesicles released during cellular stress or oncogenic signaling, facilitated by mechanisms involving ARF6 and Rho-associated kinase (ROCK) signaling pathways [17].

The selective sorting and incorporation of bioactive cargo, including proteins and miRNAs, into microvesicles are tightly regulated. ARF6-mediated trafficking endosomes serve as crucial hubs for sorting cargo proteins, such as membrane type-1 matrix metalloprotease (MT1-MMP), which are subsequently delivered to nascent microvesicles via interactions with vesicle-associated membrane protein 3 (VAMP3) [13]. Pathological conditions, particularly hypoxia and oncogenic transformation, significantly enhance microvesicle release. Under hypoxic conditions, transcription factors such as hypoxia-inducible factor-1 (HIF-1) drive increased microvesicle production through the upregulation of proteins like RAB22A, influencing both vesicle number and cargo composition [18]. Similarly, malignant cells frequently demonstrate elevated microvesicle shedding associated with oncogenic mutations or loss of tumor-suppressor genes, underscoring their potential as biomarkers for disease monitoring [19].

APOPTOTIC BODIES:

Apoptotic bodies are membrane-bound vesicles that are considered the largest type of extracellular vesicle and display large variation in their size (50–5000 nm). Apoptotic bodies are released by dying cells during the final stage of apoptosis, unlike microvesicles and exosomes [20].

Apoptotic bodies are generated through apoptotic cell disassembly – a process defined by a series of precisely regulated morphological changes, including membrane blebbing, the formation of apoptotic membrane protrusions, and fragmentation into apoptotic bodies [21]. The first step involves apoptotic membrane blebbing, which is the emergence of crescent-shaped spaces around the nucleus, which occurs due to rupture of the cytoskeletal plasma membrane and the loss of phospholipid asymmetry. Hydrostatic pressure within the cell drives membrane blebbing, while the actin cortex plays a local role in facilitating its formation. Next, apoptotic membrane protrusions are formed, such as beaded apoptopodia, microtubule spikes and apoptopodia. The final stage involves the fragmentation of nuclear blebs, where nuclear fragments condense into half-moon shapes before forming apoptotic bodies [22]. As remnants of the cell, apoptotic bodies carry cellular components and surface markers from the parent cells.

Apoptotic bodies carry molecular cargo representative of the dying parent cell, including fragmented genomic DNA, RNA, histones, mitochondria, and organelle fragments. Their surface often displays classical ‘eat-me’ signals, such as phosphatidylserine (PS), thrombospondin (TSP), and calreticulin (CRT), which promote recognition and clearance by professional phagocytes like macrophages and dendritic cells. Additional markers, including leukocyte common antigen (LCA), leukocyte common antigen protein tyrosine phosphatase receptor C (PTPRC), integrin lymphocyte function-associated antigen 1 (LFA-1), and complement proteins C3b and C1q and histone H2AX, facilitate immunological crosstalk and clearance signalling [23].

Although historically regarded as cellular debris, apoptotic bodies are increasingly recognized as bioactive structures involved in intercellular communication. They can transfer bioactive macromolecules to neighboring cells, influencing immune responses, inflammation, angiogenesis, and tissue remodeling. For instance, apoptotic bodies released by apoptotic endothelial cells have been shown to regulate vascular homeostasis and contribute to atherosclerotic plaque development through modulation of macrophage activation and cytokine production [24]. In cardiovascular pathology, improper clearance of apoptotic bodies can exacerbate disease progression. Their accumulation can lead to secondary necrosis and the release of damage-associated molecular patterns (DAMPs), promoting chronic inflammation, a process implicated in atherosclerosis, myocardial infarction, and heart failure [25].

Despite growing interest, standardized methods for apoptotic body isolation and characterization remain limited, and their heterogeneity complicates functional analysis. Future research is needed to elucidate apoptotic body subtype-specific roles, optimize isolation protocols, and explore their translational potential in cardiovascular and systemic diseases.

EXOSOMES:

In contrast to microvesicles, exosomes are a homogenous group of extracellular vesicles, typically ranging from 30 to 100 nm in diameter. Exosome formation begins with inward invagination of the plasma membrane, leading to formation of early endosomes [26]. These cup-shaped structures possess cell-surface proteins and soluble proteins associated with the extracellular milieu. As they mature into late endosomes, intraluminal vesicles (ILVs) form by inward budding within the endosomal membrane, resulting in development of multivesicular bodies (MVBs). Cargo – including proteins, RNAs (mRNA, miRNA, lncRNA), DNA fragments, lipids, and metabolites – selectively sorted into ILVs via multiple tightly regulated mechanisms. These include ESCRT (Endosomal Sorting Complex Required for Transport)-dependent pathways, in which the ESCRT-0, -I, -II, and -III complexes coordinate the budding and scission of membrane vesicles. In addition, ESCRT-independent pathways, such as those involving tetraspanins (eg, CD63, CD81, CD9), lipid raft microdomains, and ceramide-enriched domains, also contribute to cargo selection and vesicle formation [27]. The endoplasmic reticulum and Golgi apparatus play a key role in packaging the bioactive molecules into the ILVs. Each MVB can contain multiple ILVs, and most MVBs fuse with the plasma membrane to release these ILVs into the extracellular space, now referred to as exosomes. However, some MVBs fuse with lysosomes or autophagosomes for degradation and recycling [23]. Rab GTPases, particularly Rab27a/b, Rab11, and Rab35, are key regulators of MVB trafficking and exosome secretion, modulating vesicle docking and fusion with the plasma membrane. A number of exosome specific markers have been identified, including, tetraspanins (eg, CD63, CD81), tumor susceptibility gene 101 (TSG101), heat shock protein 70 (HSP70), ALG-2-interacting protein X (Alix), and actin [26]. Importantly, exosomes exhibit a characteristic cup-shaped morphology under transmission electron microscopy, although this shape may be artefactual due to sample preparation.

Functionally, exosomes play critical roles in cardiovascular homeostasis and pathology by mediating cell-to-cell communication. They influence angiogenesis, inflammation, fibrosis, and apoptosis in cardiac and vascular cells. For example, cardiomyocyte-derived exosomes can transfer stress-induced miRNAs to endothelial cells, promoting angiogenic signaling, while exosomes from cardiac fibroblasts have been shown to influence cardiomyocyte hypertrophy and survival. In cardiovascular diseases, dysregulation of exosome biogenesis, cargo composition, or uptake can contribute to disease progression, such as promoting endothelial dysfunction, inflammatory responses, and plaque instability in atherosclerosis, or maladaptive remodeling in heart failure. As such, exosomes are increasingly investigated as biomarkers of cardiovascular disease and as therapeutic vectors for RNA and drug delivery due to their endogenous origin and biocompatibility.

Roles of Extracellular Vesicles in Cardiovascular Disease

EXTRACELLULAR VESICLES IN MYOCARDIAL INFARCTION:

Extracellular vesicles play a critical role in the pathophysiology of myocardial infarction, particularly in modulating post-injury cardiac remodeling. One major mechanism involves their contribution to myocardial fibrosis. Yang et al demonstrated that in response to myocardial infarction, cardiomyocytes release exosomes containing miR-208a which is taken up by cardiac fibroblasts promoting the development of myocardial fibrosis [30]. Similarly, miR-92a-containing exosomes enhance fibroblast activation by attenuating SMAD7-mediated inhibition of α-smooth-muscle actin (α-SMA), facilitating myofibroblast conversion. Conversely, exosomal lncRNA AK139128, also derived from hypoxic cardiomyocytes, has been shown to promote apoptosis and suppress fibroblast proliferation, further highlighting the complex and context-dependent roles of exosomal content in cardiac fibrosis.

EVs also regulate cardiomyocyte survival through modulation of autophagy and apoptosis. Under hypoxic conditions, cardiomyocytes release exosomes enriched with miR-30a, which downregulates autophagy-related genes such as ATG12 and Beclin-1, thereby influencing autophagic flux and cell fate during ischemic injury [31].

The inflammatory response following MI is also strongly influenced by EVs. Loyer et al investigated the role of extracellular vesicles in regulating inflammation after myocardial infarction. Following coronary artery ligation in mice, the researchers observed a transient increase in extracellular vesicles levels in the infarcted heart compared to sham control animals. These extracellular vesicles were found to express endothelial cell and cardiomyocyte-specific markers and were classified as large vesicles. Small vesicles containing exosomal markers like CD63 and CD9 were also detected. Unlike extracellular vesicles derived from sham mice and small extracellular vesicles, large extracellular vesicles from the hearts of mice subjected to MI stimulated inflammatory monocytes to release cytokines such as IL-6, CCL2, and CCL7. Similar extracellular vesicles were also identified in human heart tissue, reinforcing the clinical relevance of the findings. The study concluded that MI triggered local extracellular vesicles production, which was rapidly taken up by infiltrating monocytes, influencing cardiac inflammation, and potentially shaping the post-MI healing processes [32].

Beyond their endogenous effects, exosomes also hold therapeutic promise. The cardioprotective effects of extracellular vesicles derived from normal heart tissue (cEVs) and kidney tissue (nEVs) in myocardial ischemic injury have been explored. After subjecting mice to myocardial infarction, they received intramyocardial injections of cEVs or nEVs. Researchers found that both cEVs and nEVs inhibited cardiomyocyte apoptosis, alleviated inflammation, enhanced angiogenesis, reduced scar size, and significantly improved cardiac function. Of note, cEVs demonstrated superior protective effects compared to nEVs. Transcriptomic and proteomic profiling identified protective mRNA clusters in both extracellular vesicle types, implicating multiple signaling pathways in mitigating post-MI damage. These findings suggest that EVs from normal heart tissue could serve as a therapeutic strategy for ischemic heart diseases [33].

Collectively, these findings underscore the multifaceted roles of exosomes in post-infarction remodeling, balancing injury and repair, and highlight their translational potential as a novel, cell-free strategy for treating myocardial infarction.

EXTRACELLULAR VESICLES IN ISCHEMIA-REPERFUSION INJURY:

The role of extracellular vesicles in mediating cardiac injury or recovery following ischemia–reperfusion injury remains a subject of debate, with both detrimental and protective functions reported. Clinical and experimental data suggest that the effects of EVs depend heavily on their origin, molecular cargo, and signaling context.

In a clinical investigation, D’Ascenzo et al assessed the role of extracellular vesicles in ischemia–reperfusion injury (IRI) and their therapeutic potential in patients undergoing percutaneous coronary intervention (PCI) [34]. Patients were randomized to receive either a sham procedure or remote ischemic preconditioning (RIPC) prior to PCI. Interestingly, EVs isolated from sham-treated patients (EV-naive) conferred robust cardioprotective effects in both ex vivo rat heart and in vitro cardiomyocyte models, whereas EVs from RIPC-treated patients (EV-RIPC) lacked this effect. Mechanistically, EVs from EV-naive patients exerted their protective action via activation of the signal transducer and activator of transcription 3 (STAT-3) pathway, and this was mediated by dual-specificity phosphatase 6 (DUSP6) mRNA cargo, while silencing DUSP6 abolished STAT-3 phosphorylation and eliminated the protective phenotype. In contrast, EV-RIPC activated the ERK1/2 pathway but did not afford significant protection. These findings suggest that not all ischemic stimuli enhance EV-mediated cardioprotection, and the cargo composition, particularly the DUSP6–STAT-3 axis, plays a critical role.

Preclinical studies further support the therapeutic potential of EVs in IRI. Zhang et al demonstrated the protective effects of ischemic preconditioning-induced serum exosomes (IPC-Exo) in a rat model of myocardial IRI [35]. PC-Exo was injected into the infarcted myocardium, and its effects on cardiac function, apoptosis, inflammatory markers, and infarct size were assessed. The results demonstrated that IPC-Exo treatment improved cardiac function, mitigated cardiomyocyte apoptosis, reduced inflammatory cytokine levels, and minimized myocardial infarct size. These effects were associated with increased expression of pPI3K, pAKT, and BCL2 and decreased expression of Bax and caspase-3. However, blocking the PI3K/AKT pathway with LY294002 reversed these protective effects. This study reinforces the idea that manipulating EV cargo or origin may be a viable strategy for therapeutic intervention.

A novel mechanism of EV-mediated protection was reported by Crewe et al, who discovered that obesity-induced mitochondrial stress in adipocytes leads to the release of small extracellular vesicles containing respiration-competent but oxidatively-damaged mitochondrial particles [36]. These small extracellular vesicles were found to circulate in the blood and were up taken by cardiomyocytes, triggering the generation of vast amounts of reactive oxygen species (ROS) that activates compensatory antioxidant signaling, preconditioning the heart against oxidative stress. Injection of these small extracellular vesicles in mice reduced cardiac IRI, highlighting a novel form of inter-organ mitohormesis. This study suggested that adipocyte-derived small extracellular vesicles, despite carrying damaged mitochondria, serve as a physiological mechanism for cardioprotection in obesity-related stress conditions.

In addition, exosomes isolated from induced pluripotent stem cells provided cardioprotection via miR-210 and miR-21 signaling in the myocardium following IRI. This was achieved by inhibiting caspase 3/7 activation, preventing H2O2-induced oxidative stress, thus protecting cardiomyocytes from apoptosis [37]. Taken together, these findings show the multifaceted roles of EVs in IRI. Depending on their cellular origin, molecular cargo, and physiological context, EVs can exacerbate or alleviate myocardial injury. Harnessing the protective features, such as the DUSP6–STAT3 axis, PI3K/AKT signaling, or mitochondrial hormetic triggers, could inform the development of EV-based therapy for patients with ACS or undergoing revascularization procedures.

EXTRACELLULAR VESICLES IN CORONARY ARTERY DISEASE:

EVs have been increasingly recognized as key players in the pathophysiology of coronary artery disease, not only as biomarkers but also as functional mediators of vascular injury and thrombosis. Clinical evidence suggests that the pathogenic impact of EVs lies more in their functional activity than in their overall abundance. In a study by Soyama et al, arterial and venous blood samples were collected from individuals with and without coronary artery disease, before extracellular vesicles were isolated and characterized. While the total number of extracellular vesicles did not differ between groups, coronary artery disease patients exhibited significantly higher extracellular vesicles-associated pro-coagulant activity [38]. Endothelial-derived extracellular vesicles were more abundant in arterial blood compared to venous blood. Linear regression models identified that age and the level of plasma triacylglycerol independently predicted extracellular vesicles numbers in coronary artery disease patients. Proteomic analysis revealed an upregulation of coagulation-associated proteins in extracellular vesicles isolated from coronary artery disease patients. These findings suggest that, rather than quantity, the functional activity of extracellular vesicles, particularly their pro-coagulant properties, play a key role in coronary artery disease pathogenesis.

At the molecular level, EV cargo can directly influence vascular remodeling through RNA-based signaling. Hosen et al identified dysregulated incorporation of long noncoding RNAs (lncRNAs) into extracellular vesicles in coronary artery disease patients [39]. lncRNAs array experiments identified significantly elevated levels of growth arrest-specific 5 (GAS5) and ArfGAP with GTPase Domain, Ankyrin Repeat, and PH Domain 2 (AGAP2) antisense RNA 1 (AGAP2-AS1). Knockdown of AGAP2-AS1 impaired extracellular vesicle-mediated endothelial cell migration, angiogenesis, and proliferation. Subsequent analysis revealed that AGAP2-AS1 regulated vascular endothelial growth factor A (VEGFA) expression and stability, promoting a pro-angiogenic response. This effect was mediated via altered expression and stabilization of vascular endothelial growth factor A (VEGFA), suggesting that EV-associated lncRNAs contribute to angiogenic remodeling in CAD.

Additional mechanisms linking EVs to CAD pathogenesis involve inflammation, oxidative stress, and endothelial dysfunction. Circulating EVs derived from platelets, endothelial cells, and leukocytes are elevated in CAD and have been shown to impair endothelial nitric oxide (NO) production, increase endothelial permeability, and promote monocyte adhesion through upregulation of adhesion molecules such as intercellular adhesion molecule 1 (ICAM1) and CD11a [40]. The pro-inflammatory and pro-thrombotic effects are mediated by EV cargo such as interleukins (IL-6, IL-8), chemokines (CCL5), microRNAs (miR-10a, miR-222), and active enzymes (caspases, MMPs) [41,42]. Endothelial-derived EVs from CAD patients also accelerate endothelial senescence and thrombogenicity via angiotensin II and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways, contributing to diffuse vascular dysfunction beyond the focal atherosclerotic lesions [43,44].

Moreover, EVs are abundant in atherosclerotic plaques and can contribute to plaque instability and thrombus formation. Microvesicles in particular are enriched with tissue factor and phosphatidylserine, key initiators of the coagulation cascade, thus amplifying thrombogenic potential at sites of plaque rupture [45,46]. While exosomes have been less extensively studied in this context, emerging data suggest that platelet-derived exosomes can counteract thrombosis by inhibiting CD36-mediated platelet aggregation.

Collectively, these findings support a paradigm in which EVs serve not only as biomarkers but also as active effectors in CAD development and progression. Their cargo composition, cellular origin, and pro-coagulant or inflammatory potential may be more critical than their total quantity in influencing disease outcomes. These insights open avenues for EV-targeted therapies and biomarker discovery in coronary artery disease.

EXTRACELLULAR VESICLES IN ATHEROSCLEROSIS:

Growing experimental evidence has revealed the multifaceted role of EVs in the initiation and progression of atherosclerosis [47]. Circulating levels of extracellular vesicles have been shown to be elevated in atherosclerosis, playing a crucial role in various stages of disease progression. These stages include smooth-muscle cell proliferation, lipid accumulation, intimal thickening, calcification, vascular media remodeling, thrombus formation, and plaque development and progression [48]. These vesicles act not only as passive biomarkers but also as active regulators of vascular cell function and inflammation.

Kestecher et al explored the relationship between low-density lipoproteins (LDL) and extracellular vesicles during atherogenesis using clinical samples and mouse models [49]. Platelet-free plasma blood samples were collected from normal and hypercholesterolemic clinical patients before extracellular vesicles were extracted and characterized. In addition, male wild-type, proprotein convertase subtilisin/kexin type 9−/− (PCSK9−/−) and LDLR−/− C57BL/6 mice were fed a high-fat diet for 12 weeks or normal diet until they reached old age (22 months). The high-fat diet led to increased extracellular vesicle levels in LDLR−/− and PCSK9−/− mice, as evidenced by annexin V and CD63 staining, whereas CD81 levels remained unchanged. PCSK9−/− mice had better cardiac function and lower cholesterol levels compared to LDLR−/− and wild-type mice, reinforcing the cardiovascular benefits of PCSK9 inhibition. In mice and in hypercholesterolemic patients, CD63+ extracellular vesicle were significantly depleted, suggesting an inverse relationship between cholesterol and extracellular vesicle. These findings highlight extracellular vesicles as potential biomarkers for CVDs and support the therapeutic benefits of PCSK9 inhibitors in managing hypercholesterolaemia.

Beyond systemic circulation, EVs derived directly from atherosclerotic plaques may actively propagate vascular inflammation at distant sites. Another study explored the role of extracellular vesicles derived from atherosclerotic plaques in promoting atherogenesis in remote vascular locations. The study used Ldlr knockout rats fed a high-cholesterol diet [50]. These rats were subsequently subjected to partial carotid ligation to induce local atherosclerosis, then researchers isolated extracellular vesicles from carotid artery tissues and downstream blood. qPCR and miRNA sequencing revealed that extracellular vesicles from atherosclerotic plaques contained higher levels of miR-23a-3p compared to control rats. Functional analyses demonstrated that these extracellular vesicles increased the expression of endothelial adhesion molecules VCAM-1, ICAM-1, and E-selectin, promoting endothelial inflammation [50]. Mechanistically, miR-23a-3p was found to target Dusp5 to sustain pERK1/2 expression, thereby driving inflammation. In vivo experiments demonstrated that inhibiting extracellular vesicle release reduced atherogenesis and macrophage infiltration, while intravenous administration of atherosclerosis-derived extracellular vesicles induced lumen narrowing, endothelial inflammation and arterial wall thickening. Notably, these effects were mitigated by co-administration of a miR-23a-3p antagomir. These findings suggest that atherosclerotic extracellular vesicles contribute to disease progression by transferring pro-inflammatory factors, particularly miR-23a-3p, to distant vascular sites, indicating a novel mechanism of atherosclerosis propagation.

Collectively, these findings show the dual role of EVs in atherosclerosis – as both disease amplifiers and therapeutic targets. Circulating and plaque-derived EVs contribute to vascular inflammation, remodeling, and lesion propagation, primarily via miRNA-mediated signaling pathways such as miR-23a-3p/ERK1/2. These results also suggest that inhibiting specific EV subtypes or modifying their cargo may offer a novel approach for limiting atherosclerotic progression and its systemic complications.

EXTRACELLULAR VESICLES IN CARDIAC HYPERTROPHY:

Cardiac hypertrophy is a key compensatory response to chronic pressure overload and neurohormonal stimulation but is also a major risk factor for heart failure. Recent studies have explored the therapeutic potential of EVs, particularly those derived from MSCs, in preventing or reversing maladaptive hypertrophy.

In a study by Lu et al, neonatal rat cardiomyocytes were subjected to angiotensin II stimulation before being treated with extracellular vesicles derived from human mesenchymal stem cells subjected to hypoxic conditions (Hypo-EVs) [51]. They found that Hypo-EVs were able to inhibit cardiac hypertrophy induced by angiotensin II treatment mediated by Parkinson disease protein 7 (DJ-1) activation. DJ-1 was found to directly interact with and inhibit proteasome subunit PSMB10, which in turn suppressed angiotensin II type 1 receptor-mediated signaling pathways and protein degradation. DJ-1 was also found to mitigate excess reactive oxygen species production and mitochondria dysfunction, demonstrating the potential of Hypo-EVs for managing cardiac hypertrophy.

Constantin et al investigated the effects of mesenchymal stem cell-derived extracellular vesicles from human adipose tissue-derived stem cells (EV-ADSCs) and bone marrow-derived stem cells (EV-BMMSCs) on cardiac hypertrophy [52]. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to TGF-β1 and angiotensin II, in the presence or absence of extracellular vesicles. TGF-β1 and angiotensin II treatment induced reactive oxygen species generation, increased cell surface area, elevated expression of pro-hypertrophic markers such as atrial natriuretic factor, and increased α-SMA, SMAD2, COL1A1, and NF-kBp50 expression. EV-ADSCs or EV-BMMSCs treatment mitigated this hypertrophic response, with EV-BMMSCs primarily decreasing transcription factor gene expression involved in hypertrophic signaling, and EV-ADSCs primarily decreasing expression of inflammatory and hypertrophic markers. These results suggest source-specific EV signatures and support their therapeutic potential.

The protective effects of EVs in cardiac hypertrophy are largely attributed to their microRNA cargo. In the hypertrophic heart, extracellular vesicles are released by cardiac fibroblasts containing miR-21, which was subsequently taken up by cardiomyocytes. miR-21 is known to target signaling pathways involved in cardiac hypertrophy, including PDZ and LIM domain 5 (PDLIM5), sorbin, and SH3 domain-containing 2 (SORBS2). Administration of a miR-21 antagonist protected the myocardium from angiotensin II-induced cardiac hypertrophy [53].

Together, these studies highlight the promising role of EVs as cell-free therapeutic agents in cardiac hypertrophy. By delivering regulatory miRNAs and protein effectors, EVs derived from stem cells or cardiac fibroblasts can modulate oxidative stress, inflammation, and hypertrophic gene expression, offering a potential strategy to prevent the transition to heart failure.

Therapeutic Management of CVD Using Extracellular Vesicles

The therapeutic application of EVs, particularly exosomes and microvesicles, has emerged as a promising strategy for CVDs, offering a cell-free alternative to stem cell therapy. Their endogenous origin, nanoscale size, and ability to deliver a wide range of bioactive molecules allow EVs to modulate key pathological processes such as inflammation, fibrosis, and endothelial dysfunction (Figure 2). Lai et al were among the first to demonstrate the cardioprotective effects of exosomes in a myocardial ischemia–reperfusion injury model. Exosomes derived from human MSCs exhibited canonical exosome markers (CD9, CD81, Alix) and significantly reduced infarct size upon injection into post-IRI mice. This study showed not only the regenerative potential of exosomes but also their role as paracrine mediators of repair, offering a new direction in post-infarction therapy by modulating the local microenvironment [54]. Expanding beyond myocardial infarction, Lapchak et al applied EV-based therapy in a rabbit model of embolic stroke using cardiosphere-derived cell EVs. Fluorescent tracking confirmed selective accumulation of these EVs in the ischemic brain region. Treatment led to significant functional recovery and a 245% increase in the P50 value – a neurobehavioral recovery index – without inducing hemorrhagic complications. These results illustrate the ability of EVs to cross biological barriers, such as the blood–brain barrier, and exert targeted therapeutic effects in non-cardiac vascular territories [55]. Liu et al further advanced EV therapy by integrating induced pluripotent stem cell (iPSC)-derived EVs with a slow-release hydrogel patch. In a rat model of myocardial infarction, this delivery platform improved left ventricular function, reduced infarct size, mitigated arrhythmia, and suppressed cardiomyocyte apoptosis and hypertrophy. Molecular analyses revealed enrichment of cardiac-specific microRNAs (miR-1, miR-133) within the EV cargo, suggesting that fine-tuned RNA delivery via engineered EVs could recapitulate developmental and repair pathways essential for myocardial regeneration [56].

Recent work has expanded the therapeutic landscape of EVs to heart failure with preserved ejection fraction (HFpEF), a condition with limited pharmacological treatment options. Zhang et al showed that immortalized CDC-derived EVs (imCDCevs) reversed atrial electrical remodeling in a rat model of HFpEF with atrial fibrillation (AF) [57]. ImCDCevs restored connexin 43 localization, decreased fibrosis and oxidative stress, and markedly reduced AF inducibility from 84% to 33%, without affecting blood pressure or diastolic function. These findings underscore the potential of EVs to modulate structural and electrical remodeling in the atria, providing a targeted therapy for arrhythmias in HFpEF [57]. The potential therapeutic benefits of milk-derived extracellular vesicles (EVs) were also explored on HFpEF [58]. Researchers found that oral administration of mEVs reduced body weight gain and lung weight and improved heart function, as evidenced by reduced cardiac hypertrophy, along with better echocardiographic parameters (E/A and E/E’ ratios) and lower blood pressure. Notably, these effects were not due to direct cardiac action but to gut microbiota restoration, specifically the enrichment of Lachnospiraceae and the production of butyrate, a cardioprotective short-chain fatty acid. Supplementation with butyrate alone recapitulated the benefits of mEVs, suggesting that dietary EVs could act as systemic modulators through microbiome-mediated mechanisms [59].

Several studies have identified specific miRNAs as critical mediators of EV-based cardioprotection. Ibrahim et al identified exosomes released from cardiosphere-derived cells possessing the ability to induce therapeutic regeneration of infarcted heart tissue [60]. miR-146a was particularly abundant in cardiosphere-derived cell exosomes. Injection of these exosomes into mice subjected to myocardial infarction significantly improved cardiac function, reduced the infarct size, and increased the infarct wall thickness compared to vehicle control injections. The levels of inflammatory cytokine infiltration were also significantly blunted following exosome injection. Mechanistically, miR-146a expression induced downregulation of Traf6 and Irak1 expression. Ingenuity pathway analysis revealed that miR-146a is involved in key cellular pathways such as cell cycle, cellular organization, and cell survival [60]. Prompted by this finding, Coute et al investigated the cardioprotective effects of cardiosphere-derived cell exosomes in cellular postconditioning following myocardial infarction in a rat and pig model. Infusion of cardiosphere-derived cell exosomes significantly reduced the infarct size in both in vivo models and reduced the number of CD68+ macrophages in the infarct tissue, and the exosomes were enriched in several miRNAs, including miR-146a, miR-126, and miR-181b. Reverse pathway analysis showed miR-181b is a key mediator of macrophage polarisation, with protein kinase C δ (PKCδ) as a downstream target. Consequently, exosomal transfer of miR-181b from cardiosphere-derived cells to macrophages decreased PKCδ expression levels and mediated the cardioprotective effects observed [61]. Furthermore, Khan et al found that embryonic stem cell-derived exosome administration after myocardial infarction enhanced cardiac progenitor cell proliferation and survival, angiogenesis, and cardiac function, and reduced fibrosis and inflammation [62]. A further study found pro-angiogenic effects of cardiac progenitor cell-derived extracellular vesicles in protecting the myocardium from IRI. These effects were found to be linked to PAPP-A and the IGF-R signaling pathways [63]. These findings suggest that EVs not only regenerate tissue but also reshape immune, vascular, and fibrotic responses in a concerted fashion.

In parallel, synthetic delivery systems such as liposomes have been explored to overcome pharmacokinetic limitations of small molecules. Allijin et al used berberine-loaded liposomes for the management of adverse ventricular remodeling in response to MI. Although berberine is known for its cardioprotective properties, it is limited by a short half-life and poor solubility [64]. To overcome these limitations, berberine was encapsulated in long-circulating liposomes before being injected in mice subjected to permanent ligation of the left anterior descending artery. Analysis 28 days after MI found that berberine-loaded liposomes significantly preserved cardiac ejection fraction as opposed to control liposomes and free berberine. The authors suggested that berberine-loaded liposomes could be a promising approach to treating adverse remodeling following MI [64]. Another study explored the use of targeted delivery of pro-angiogenic compounds to enhance myocardial repair after infarction. The researchers focused on targeting the infarcted tissue by use of upregulated endothelial cell adhesion molecules in the region. They used anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF) to selectively deliver VEGF to post-MI tissue in a rat model. Liposomes-mediated VEGF delivery significantly improved cardiac function, as evidenced by a 74% increase in perfused vessels and 21% increase in anatomical vessels in the infarcted area, but systemic VEGF did not induce any significant improvements. The study concluded that targeted delivery of pro-angiogenic compounds can effectively enhance both cardiac function and vascular structure after MI [65]. Another study used liposomes loaded with fumagillin, a selective inhibitor of endothelium cell proliferation and migration, for the treatment of atherosclerotic plaque lesions. Fumagillin-loaded liposomes were injected into ApoE-knockout mice fed an atherogenic high-fat diet. Treatment with fumagillin-loaded liposomes inhibited the growth of atherosclerotic plaques, indicating that even early intervention can substantially reduce the progression of atherosclerosis [66]. However, these studies used liposomes, which are synthetic in nature and therefore lack specificity towards target cells. Extracellular vesicles are considered an endogenous counterpart to liposomes and may be able to overcome some of these limitations.

Future Directions

Despite promising preclinical findings, the clinical application of extracellular vesicles in cardiovascular disease therapy remains in its early stages. While numerous trials have confirmed their safety in immunotherapy, challenges such as efficient isolation, purification, scalability, targeted delivery, and in vivo biodistribution must be addressed. Additionally, optimizing drug-loading techniques and improving selectivity for target tissues will be crucial for translating extracellular vesicle-based therapies into clinical practice [67]. This review explicitly highlights recent progress in EV-mediated cross-organ interactions, including adipocyte-cardiomyocyte communication during ischemic conditions, novel insights into EV-contained miRNA therapeutic mechanisms, and emerging evidence on gut microbiota-EV interactions influencing cardiovascular diseases. These innovative aspects have been relatively unexplored and are promising therapeutic avenues for future research. This review identified clear limitations in existing preclinical studies, including inadequate characterization of EV subpopulations and insufficient in vivo targeting specificity, which should be the focus of future research. Our review thus not only synthesizes existing knowledge but also distinctly outlines a roadmap for critical unresolved questions in EV-based cardiovascular therapeutics. Overcoming these hurdles could unlock their full potential as a novel, cell-free therapeutic strategy for cardiovascular diseases.

Conclusions

In summary, extracellular vesicles have emerged as key mediators of intracellular communications and influence various cellular processes involved in cardiovascular diseases, such as inflammation, tissue remodeling, apoptosis, and proliferation. As carriers of biomolecular cargo, including proteins, lipids, and microRNA, extracellular vesicles possess the potential to modulate molecular pathways underlying cardiovascular diseases. Furthermore, the therapeutic application of extracellular versicles holds promise by improving cardiac function following injury. Further research investigating the translational potential of extracellular vesicle-based therapies is warranted. Nevertheless, extracellular vesicles are considered a highly flexible platform, allowing for targeted, non-immunogenic, and efficient delivery systems for management of cardiovascular diseases.