Utilizing engineered extracellular vesicles as delivery vectors in the management of ischemic stroke:a special outlook on mitochondrial delivery

Ischemic stroke is a secondary cause of mortality worldwide,imposing considerable medical and economic burdens on society.Extracellular vesicles,serving as natural nanocarriers for drug delivery,exhibit excellent biocompatibility in vivo and have significant advantages in the management of ischemic stroke.However,the uncertain distribution and rapid clearance of extracellular vesicles impede their delivery efficiency.By utilizing membrane decoration or by encapsulating therapeutic cargo within extracellular vesicles,their delivery efficacy may be greatly improved.Furthermore,previous studies have indicated that microvesicles,a subset of large-sized extracellular vesicles,can transport mitochondria to neighboring cells,thereby aiding in the restoration of mitochondrial function post-ischemic stroke.Small extracellular vesicles have also demonstrated the capability to transfer mitochondrial components,such as proteins or deoxyribonucleic acid,or their sub-components,for extracellular vesicle-based ischemic stroke therapy.In this review,we undertake a comparative analysis of the isolation techniques employed for extracellular vesicles and present an overview of the current dominant extracellular vesicle modification methodologies.Given the complex facets of treating ischemic stroke,we also delineate various extracellular vesicle modification approaches which are suited to different facets of the treatment process.Moreover,given the burgeoning interest in mitochondrial delivery,we delved into the feasibility and existing research findings on the transportation of mitochondrial fractions or intact mitochondria through small extracellular vesicles and microvesicles to offer a fresh perspective on ischemic stroke therapy.

Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery

Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery.Our previous in vitro study demonstrated that exosomes/small extracellular vesicles(sEVs)isolated from cerebral endothelial cells(CEC-sEVs)of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a(miR-27a)is an elevated miRNA in ischemic CEC-sEVs.In the present study,we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a(27a-sEVs)further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs.27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector.Small EVs isolated from CECs transfected with a scramble vector(Scra-sEVs)were used as a control.Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs.An array of behavior assays was used to measure neurological function.Compared with treatment of ischemic stroke with Scra-sEVs,treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side,and significantly improved neurological outcomes.In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth.Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone,while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a,and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone.Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs.Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes.Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

Several studies have found that transplantation of neural progenitor cells(NPCs)promotes the survival of injured neurons.However,a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application.Small extracellular vesicles(sEVs)contain bioactive molecules for neuronal protection and regeneration.Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases.In this study,we intravitreally transplanted sEVs derived from human induced pluripotent stem cells(hiPSCs)and hiPSCs-differentiated NPCs(hiPSC-NPC)in a mouse model of optic nerve crush.Our results show that these intravitreally injected sEVs were ingested by retinal cells,especially those localized in the ganglion cell layer.Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration,and regulated the retinal microenvironment by inhibiting excessive activation of microglia.Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells,which had protective effects on RGCs after optic nerve injury.These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

Targeting capabilities of engineered extracellular vesicles for the treatment of neurological diseases

Recent advances in research on extracellular vesicles have significantly enhanced their potential as therapeutic agents for neurological diseases.Owing to their therapeutic properties and ability to cross the blood–brain barrier,extracellular vesicles are recognized as promising drug delivery vehicles for various neurological conditions,including ischemic stroke,traumatic brain injury,neurodegenerative diseases,glioma,and psychosis.However,the clinical application of natural extracellular vesicles is hindered by their limited targeting ability and short clearance from the body.To address these limitations,multiple engineering strategies have been developed to enhance the targeting capabilities of extracellular vesicles,thereby enabling the delivery of therapeutic contents to specific tissues or cells.Therefore,this review aims to highlight the latest advancements in natural and targeting-engineered extracellular vesicles,exploring their applications in treating traumatic brain injury,ischemic stroke,Parkinson's disease,Alzheimer's disease,amyotrophic lateral sclerosis,glioma,and psychosis.Additionally,we summarized recent clinical trials involving extracellular vesicles and discussed the challenges and future prospects of using targeting-engineered extracellular vesicles for drug delivery in treating neurological diseases.This review offers new insights for developing highly targeted therapies in this field.

The emerging role of mesenchymal stem cell-derived extracellular vesicles to ameliorate hippocampal NLRP3 inflammation induced by binge-like ethanol treatment in adolescence

Our previous studies have reported that activation of the NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)-inflammasome complex in ethanol-treated astrocytes and chronic alcohol-fed mice could be associated with neuroinflammation and brain damage.Mesenchymal stem cell-derived extracellular vesicles(MSC-EVs)have been shown to restore the neuroinflammatory response,along with myelin and synaptic structural alterations in the prefrontal cortex,and alleviate cognitive and memory dysfunctions induced by binge-like ethanol treatment in adolescent mice.Considering the therapeutic role of the molecules contained in mesenchymal stem cell-derived extracellular vesicles,the present study analyzed whether the administration of mesenchymal stem cell-derived extracellular vesicles isolated from adipose tissue,which inhibited the activation of the NLRP3 inflammasome,was capable of reducing hippocampal neuroinflammation in adolescent mice treated with binge drinking.We demonstrated that the administration of mesenchymal stem cell-derived extracellular vesicles ameliorated the activation of the hippocampal NLRP3 inflammasome complex and other NLRs inflammasomes(e.g.,pyrin domain-containing 1,caspase recruitment domain-containing 4,and absent in melanoma 2,as well as the alterations in inflammatory genes(interleukin-1β,interleukin-18,inducible nitric oxide synthase,nuclear factor-kappa B,monocyte chemoattractant protein-1,and C–X3–C motif chemokine ligand 1)and miRNAs(miR-21a-5p,miR-146a-5p,and miR-141-5p)induced by binge-like ethanol treatment in adolescent mice.Bioinformatic analysis further revealed the involvement of miR-21a-5p and miR-146a-5p with inflammatory target genes and NOD-like receptor signaling pathways.Taken together,these findings provide novel evidence of the therapeutic potential of MSC-derived EVs to ameliorate the hippocampal neuroinflammatory response associated with NLRP3 inflammasome activation induced by binge drinking in adolescence.

Human neural stem cell-derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

Human neural stem cell-derived extracellular vesicles exhibit analogous functions to their parental cells,and can thus be used as substitutes for stem cells in stem cell therapy,thereby mitigating the risks of stem cell therapy and advancing the frontiers of stem cell-derived treatments.This lays a foundation for the development of potentially potent new treatment modalities for ischemic stroke.However,the precise mechanisms underlying the efficacy and safety of human neural stem cell-derived extracellular vesicles remain unclear,presenting challenges for clinical translation.To promote the translation of therapy based on human neural stem cell-derived extracellular vesicles from the bench to the bedside,we conducted a comprehensive preclinical study to evaluate the efficacy and safety of human neural stem cell-derived extracellular vesicles in the treatment of ischemic stroke.We found that administration of human neural stem cell-derived extracellular vesicles to an ischemic stroke rat model reduced the volume of cerebral infarction and promoted functional recovery by alleviating neuronal apoptosis.The human neural stem cell-derived extracellular vesicles reduced neuronal apoptosis by enhancing phosphorylation of phosphoinositide 3-kinase,mammalian target of rapamycin,and protein kinase B,and these effects were reversed by treatment with a phosphoinositide 3-kinase inhibitor.These findings suggest that human neural stem cell-derived extracellular vesicles play a neuroprotective role in ischemic stroke through activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.Finally,we showed that human neural stem cell-derived extracellular vesicles have a good in vivo safety profile.Therefore,human neural stem cell-derived extracellular vesicles are a promising potential agent for the treatment of ischemic stroke.

Pathological and therapeutic effects of extracellular vesicles in neurological and neurodegenerative diseases

Extracellular vesicles are released by all cell types and contain proteins,microRNAs,mRNAs,and other bioactive molecules.Extracellular vesicles play an important role in intercellular communication and in the modulation of the immune system and neuroinflammation.The cargo of extra cellular vesicles(e.g.,proteins and microRNAs)is altered in pathological situations.Extracellular vesicles contribute to the pathogenesis of many pathologies associated with sustained inflammation and neuroinflammation,including cance r,diabetes,hype rammonemia and hepatic encephalopathy,and other neurological and neurodegenerative diseases.Extracellular vesicles may cross the blood-brain barrier and transfer pathological signals from the periphery to the brain.This contributes to inducing neuroinflammation and cognitive and motor impairment in hyperammonemia and hepatic encephalopathy and in neurodegenerative diseases.The mechanisms involved are beginning to be unde rstood.For example,increased tumor necrosis factor a in extracellular vesicles from plasma of hype rammonemic rats induces neuroinflammation and motor impairment when injected into normal rats.Identifying the mechanisms by which extracellular vesicles contribute to the pathogenesis of these diseases will help to develop new treatments and diagnostic tools for their easy and early detection.In contrast,extra cellular vesicles from mesenchymal stem cells have therapeutic utility in many of the above pathologies,by reducing inflammation and neuroinflammation and improving cognitive and motor function.These extra cellular vesicles recapitulate the beneficial effects of mesenchymal stem cells and have advantages as therapeutic tools:they are less immunoge nic,may not diffe rentiate to malignant cells,cross the blood-brain barrier,and may reach more easily target organs.Extracellular vesicles from mesenchymal stem cells have beneficial effects in models of ischemic brain injury,Alzheimer's and Parkinson's diseases,hyperammonemia,and hepatic encephalopathy.Extracellular vesicles from mesenchymal stem cells modulate the immune system,promoting the shift from a pro-inflammato ry to an anti-inflammatory state.For example,extracellular vesicles from mesenchymal stem cells modulate the Th17/Treg balance,promoting the anti-inflammatory Treg.Extracellular vesicles from mesenchymal stem cells may also act directly in the brain to modulate microglia activation,promoting a shift from a pro-inflammatory to an anti-inflammatory state.This reduces neuroinflammation and improves cognitive and motor function.Two main components of extracellular vesicles from mesenchymal stem cells which contribute to these beneficial effects are transforming growth factor-βand miR-124.Identifying the mechanisms by which extracellular vesicles from mesenchymal stem cells induce the beneficial effects and the main molecules(e.g.,proteins and mRNAs)involved may help to improve their therapeutic utility.The aims of this review are to summarize the knowledge of the pathological effects of extracellular vesicles in different pathologies,the therapeutic potential of extra cellular vesicles from mesenchymal stem cells to recover cognitive and motor function and the molecular mechanisms for these beneficial effects on neurological function.

Mesenchymal stem cell-derived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration

Traumatic brain injury is a serious and complex neurological condition that affects millions of people worldwide.Despite significant advancements in the field of medicine,effective treatments for traumatic brain injury remain limited.Recently,extracellular vesicles released from mesenchymal stem/stromal cells have emerged as a promising novel therapy for traumatic brain injury.Extracellular vesicles are small membrane-bound vesicles that are naturally released by cells,including those in the brain,and can be engineered to contain therapeutic cargo,such as anti-inflammatory molecules,growth factors,and microRNAs.When administered intravenously,extra cellular vesicles can cross the blood-brain barrier and deliver their cargos to the site of injury,where they can be taken up by recipient cells and modulate the inflammatory response,promote neuroregeneration,and improve functional outcomes.In preclinical studies,extracellular vesicle-based therapies have shown promising results in promoting recove ry after traumatic brain injury,including reducing neuronal damage,improving cognitive function,and enhancing motor recovery.While further research is needed to establish the safety and efficacy of extra cellular vesicle-based therapies in humans,extra cellular vesicles represent a promising novel approach for the treatment of traumatic brain injury.In this review,we summarize mesenchymal ste m/stromal cell-de rived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration and brainderived extracellular vesicles as potential biofluid biomarkers in small and large animal models of traumatic brain injury.

Extracellular vesicles in anti-tumor drug resistance: Mechanisms and therapeutic prospects

Drug resistance presents a significant challenge to achieving positive clinical outcomes in anti-tumor therapy.Prior research has illuminated reasons behind drug resistance,including increased drug efflux,alterations in drug targets,and abnormal activation of oncogenic pathways.However,there's a need for deeper investigation into the impact of drug-resistant cells on parental tumor cells and intricate crosstalk between tumor cells and the malignant tumor microenvironment(TME).Recent studies on extracellular vesicles(EVs)have provided valuable insights.EVs are membrane-bound particles secreted by all cells,mediating cell-to-cell communication.They contain functional cargoes like DNA,RNA,lipids,proteins,and metabolites from mother cells,delivered to other cells.Notably,EVs are increasingly recognized as regulators in the resistance to anti-cancer drugs.This review aims to summarize the mechanisms of EV-mediated anti-tumor drug resistance,covering therapeutic approaches like chemo-therapy,targeted therapy,immunotherapy and even radiotherapy.Detecting Ev-based biomarkers to predict drug resistance assists in bypassing anti-tumor drug resistance.Additionally,targeted inhibition of EV biogenesis and secretion emerges as a promising approach to counter drug resistance.We highlight the importance of conducting in-depth mechanistic research on EVs,their cargoes,and functional ap-proaches specifically focusing on EV subpopulations.These efforts will significantly advance the devel-opment of strategies to overcome drug resistance in anti-tumor therapy.

Extracellular vesicles loaded dual-network bioactive sealant via immunoregulation and annulus fibrosus repair for intervertebral disc herniation

Intervertebral disc herniation(IVDH)is a common manifestation of intervertebral disc degeneration(IVDD)characterized by inflammation that results in the rupture of the annulus fibrosus(AF)and her-niation of the nucleus pulposus(NP).While current clinical research primarily focuses on regulating the degenerative NP,the crucial role of the AF in maintaining the mechanical stability and metabolic balance of the intervertebral disc(IVD)has been overlooked.Resolving immunoregulation and AF repair is im-perative to effectively prevent recurrent herniation.Therefore,this study introduces a bioactive sealant(OD/GM/QCS-sEVs),which combines gelatin methacryloyl(GM)and oxidized dextran(OD)with quater-nized chitosan(QCS)and incorporates small extracellular vesicles(sEVs).The developed sealant possesses injectability,self-healing capabilities,tissue adhesiveness,and mechanical stability,with an average ad-hesive strength of 109.63 kPa.In vitro experiments demonstrate that OD/GM/QCS-sEVs effectively seal AF defects while preserving mechanical properties comparable to those of a normal IVD.Additionally,the sealant releases sEVs through a pH-responsive mechanism,thereby modulating macrophage polarization to the M2 phenotype via the NF-κB signaling pathway.This mechanism facilitates immunoregulation and anti-inflammatory effects,and promotes stem cell differentiation into fibrocartilage.Animal experiments confirm the ability of OD/GM/QCS-sEVs to seal defects,prevent proteoglycan loss,inhibit IVDD develop-ment,and promote AF regeneration.Overall,OD/GM/QCS-sEVs hold promise as an innovative bioactive sealant for recurrent herniation by resolving immunoregulation and AF regeneration.

Mesenchymal stem cell-derived extracellular vesicles in skin wound healing:roles,opportunities and challenges

Skin wounds are characterized by injury to the skin due to trauma,tearing,cuts,or contusions.As such injuries are common to all human groups,they may at times represent a serious socioeconomic burden.Currently,increasing numbers of studies have focused on the role of mesenchymal stem cell(MSC)-derived extracellular vesicles(EVs)in skin wound repair.As a cell-free therapy,MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy.Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures,including the regeneration of vessels,nerves,and hair follicles.In addition,MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization,wound angiogenesis,cell proliferation,and cell migration,and by inhibiting excessive extracellular matrix production.Additionally,these structures can serve as a scaffold for components used in wound repair,and they can be developed into bioengineered EVs to support trauma repair.Through the formulation of standardized culture,isolation,purification,and drug delivery strategies,exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair.In conclusion,MSCderived EV-based therapies have important application prospects in wound repair.Here we provide a comprehensive overview of their current status,application potential,and associated drawbacks.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Extracellular vesicles for delivering therapeutic agents in ischemia/reperfusion injury

Ischemia/reperfusion(I/R)injury ismarked by the restriction and subsequent restoration of blood supply to an organ.This process can exacerbate the initial tissue damage,leading to further disorders,disability,and even death.Extracellular vesicles(EVs)are crucial in cell communication by releasing cargo that regulates the physiological state of recipient cells.The development of EVs presents a novel avenue for delivering therapeutic agents in I/R therapy.The therapeutic potential of EVs derived from stem cells,endothelial cells,and plasma in I/R injury has been actively investigated.Therefore,this review aims to provide an overview of the pathological process of I/R injury and the biophysical properties of EVs.We noted that EVs serve as nontoxic,flexible,and multifunctional carriers for delivering therapeutic agents capable of intervening in I/R injury progression.The therapeutic efficacy of EVs can be enhanced through various engineering strategies.Improving the tropism of EVs via surface modification and modulating their contents via preconditioning are widely investigated in preclinical studies.Finally,we summarize the challenges in the production and delivery of EV-based therapy in I/R injury and discuss how it can advance.This review will encourage further exploration in developing efficient EV-based delivery systems for I/R treatment.

tRNA^(Glu)-derived fragments from embryonic extracellular vesicles modulate bovine embryo hatching

Transfer RNA-derived small RNAs(tsRNAs)have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells.However,it is unknown whether tsRNAs also regulate embryo hatching.In this study,we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA(miRNA)cargo of preimplantation embryonic extracellular vesicles(EVs)influences embryo development.We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts.The majority of tsRNAs was identified as tRNA halves originating from the 5'ends of tRNAs.Among the 148 differentially expressed tsRNAs,the 19 nt tRNA fragment(tRF)tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts.RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group(P<0.05).Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching(P<0.05).Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation.In summary,tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions,and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching,while influencing embryo implantation-related genes and pathways.These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.

Extracellular vesicles and angiotensin-converting enzyme 2 in COVID-19 disease

Extracellular vesicles(EVs)are membranous vesicular structures released from almost all eukaryotic cell types under different physiological or pathological conditions.Growing evidence demonstrates that EVs can serve as mediators of intercellular communication between donor and recipient cells or microorganism-infected and noninfected cells.Coronavirus disease 2019(COVID-19)disease is caused by infection of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)of host cells in the respiratory system and various extra-pulmonary tissue/organs,resulting in complications of multiple organ systems.As the cell surface receptor,angiotensin-converting enzyme 2(ACE2)mediates cellular entry of SARS-CoV-2 into the host cells in patients with COVID-19.Recent studies have found that ACE2 can be released with EVs,which have been shown to interfere with the entry of the virus into host cells and thus may be involved in COVID-19 pathophysiology.In addition,ACE2,neprilysin(NEP),and thimet oligopeptidase(TOP)are the key enzymes that regulate angiotensin metabolism by converting angiotensin II or angiotensin I to angiotensin 1-7,the latter of which has protective effects in counterbalancing the harmful effects of angiotensin II in COVID-19 disease.This review summarizes the recent research progress regarding EV-associated ACE2,NEP,and TOP and the perspectives of their potential involvement in the pathophysiology of COVID-19 disease.

Mesenchymal stem cells-extracellular vesicles alleviate pulmonary fibrosis by regulating immunomodulators

BACKGROUND Pulmonary fibrosis(PF)is a chronic interstitial lung disease characterized by fibroblast proliferation and extracellular matrix formation,causing structural damage and lung failure.Stem cell therapy and mesenchymal stem cells-extracellular vesicles(MSC-EVs)offer new hope for PF treatment.AIM To investigate the therapeutic potential of MSC-EVs in alleviating fibrosis,oxidative stress,and immune inflammation in A549 cells and bleomycin(BLM)-induced mouse model.METHODS The effect of MSC-EVs on A549 cells was assessed by fibrosis markers[collagen I andα-smooth muscle actin(α-SMA),oxidative stress regulators[nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1),and inflammatory regu-lators[nuclear factor-kappaB(NF-κB)p65,interleukin(IL)-1β,and IL-2].Similarly,they were assessed in the lungs of mice where PF was induced by BLM after MSC-EV transfection.MSC-EVs ion PF mice were detected by pathological staining and western blot.Single-cell RNA sequencing was performed to investigate the effects of the MSC-EVs on gene expression profiles of macrophages after modeling in mice.RESULTS Transforming growth factor(TGF)-β1 enhanced fibrosis in A549 cells,significantly increasing collagen I andα-SMA levels.Notably,treatment with MSC-EVs demonstrated a remarkable alleviation of these effects.Similarly,the expression of oxidative stress regulators,such as Nrf2 and HO-1,along with inflammatory regulators,including NF-κB p65 and IL-1β,were mitigated by MSC-EV treatment.Furthermore,in a parallel manner,MSC-EVs exhibited a downregulatory impact on collagen deposition,oxidative stress injuries,and inflammatory-related cytokines in the lungs of mice with PF.Additionally,the mRNA sequencing results suggested that BLM may induce PF in mice by upregulating pulmonary collagen fiber deposition and triggering an immune inflammatory response.The findings collectively highlight the potential therapeutic efficacy of MSC-EVs in ameliorating fibrotic processes,oxidative stress,and inflammatory responses associated with PF.CONCLUSION MSC-EVs could ameliorate fibrosis in vitro and in vivo by downregulating collagen deposition,oxidative stress,and immune-inflammatory responses.

Gamma-aminobutyric acid enhances miR-21-5p loading into adipose-derived stem cell extracellular vesicles to alleviate myocardial ischemia-reperfusion injury via TXNIP regulation

BACKGROUND Myocardial ischemia-reperfusion injury(MIRI)poses a prevalent challenge in current reperfusion therapies,with an absence of efficacious interventions to address the underlying causes.AIM To investigate whether the extracellular vesicles(EVs)secreted by adipose mesenchymal stem cells(ADSCs)derived from subcutaneous inguinal adipose tissue(IAT)underγ-aminobutyric acid(GABA)induction(GABA-EVs^(IAT))demonstrate a more pronounced inhibitory effect on mitochondrial oxidative stress and elucidate the underlying mechanisms.METHODS We investigated the potential protective effects of EVs derived from mouse ADSCs pretreated with GABA.We assessed cardiomyocyte injury using terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/propidium iodide assays.The integrity of cardiomyocyte mitochondria morphology was assessed using electron microscopy across various intervention backgrounds.To explore the functional RNA diversity between EVs^(IAT)and GABA-EVs^(IAT),we employed microRNA(miR)sequencing.Through a dual-luciferase reporter assay,we confirmed the molecular mechanism by which EVs mediate thioredoxin-interacting protein(TXNIP).Western blotting and immunofluorescence were conducted to determine how TXNIP is involved in mediation of oxidative stress and mitochondrial dysfunction.RESULTS Our study demonstrates that,under the influence of GABA,ADSCs exhibit an increased capacity to encapsulate a higher abundance of miR-21-5p within EVs.Consequently,this leads to a more pronounced inhibitory effect on mitochondrial oxidative stress compared to EVs from ADSCs without GABA intervention,ultimately resulting in myocardial protection.On a molecular mechanism level,EVs regulate the expression of TXNIP and mitigating excessive oxidative stress in mitochondria during MIRI process to rescue cardiomyocytes.CONCLUSION Administration of GABA leads to the specific loading of miR-21-5p into EVs by ADSCs,thereby regulating the expression of TXNIP.The EVs derived from ADSCs treated with GABA effectively ameliorates mitochondrial oxidative stress and mitigates cardiomyocytes damage in the pathological process of MIRI.

Dual role of extracellular vesicles in neurodegenerative diseases

Extracellular vesicles(EVs)are cell-to-cell interaction tools that are attracting increasing interest in the literature in two opposing areas.In addition to their role in physiological development,there is growing evidence of their involvement in healing and protective processes.However,EVs also mediate pathological conditions,particularly contributing to the progression of several chronic diseases,such as neurodegenerative diseases.On the other hand,EVs also form the core of a new therapeutic strategy for neuroprotection,which is based on the administration of EVs derived from a wide range of donor cells.In particular,the possibility of obtaining numerous EVs from stem cells of different origins,which is feasible for therapeutic aims,is now under investigation.In this review,we focused on neurodegenerative diseases,in which EVs could have a propagative detrimental effect or could also be exploited to deliver protective factors.This review explores the different hypotheses concerning the dual role of EVs,with the aim of shedding light on the following question:Can vesicles be used to fight vesicle-propagated diseases?

Extracellular Vesicles from Mesenchymal Stromal Cells (imEVs) Improve Cold Preservation of Isolated Mitochondria

Mitochondrial organelle transplantation (MOT) is an innovative strategy for the treatment of mitochondrial dysfunction such as cardiac ischemic reperfusion injuries, Parkinson’s diseases, brain and spinal cord injuries, and amyotrophic lateral sclerosis (ALS). However, one of the major challenges for widespread usage is a methodology for preservation of isolated mitochondria. Extracellular vesicles (EVs) are phospholipid bilayer-enclosed vesicles released from cells. EVs carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles such as mitochondria. Purpose: To test if EVs enhance the stability of isolated mitochondria. Methods: We mixed isolated mitochondria of fibroblasts with EVs of mesenchymal stromal cells (imEVs) (9:1 in volume) and stored the mixture at 2°C - 6°C for different time periods. We measured morphology, mitochondrial membrane potential (MMP) and mitochondrial ATP content at 0, 2, 5 days. Key findings: After 2 days of storage, the mito-chondria without imEVs lost approximate 70% MMP (RFU: 1822 ± 68), compared to the fresh mitochondria (RFU: 5458 ± 52) (p 0.05). In agreement with MMP, mitochondria without imEVs lost significant mitochondrial ATP content (p 0.05), after 2 days of cold storage, compared to fresh mitochondria. Microscopy showed that imEVs promoted aggregation of isolated mitochondria. Summary: The preliminary data showed that imEVs enhanced the stability of isolated mitochondria in cold storage.

Mesenchymal stem cell-derived extracellular vesicles:A promising therapeutic strategy in diabetic osteoporosis

Diabetic osteoporosis(DOP)is a serious complication of diabetes mellitus.It is urgent to explore efficient clinical treatment strategies for DOP.It has been found that mesenchymal stem cell-derived extracellular vesicles(MSC-EVs),as an emerging cell-free therapy,show great potential in DOP treatment.MSC-EVs can effectively promote bone formation,inhibit bone resorption,and modulate the inflammatory microenvironment by delivering cargoes of microRNAs,long non-coding RNAs,and proteins to target cells,thereby ameliorating bone loss in DOP.However,there are limited reports on the treatment of DOP with MSC-EVs.To evoke more attention to this potential strategy,this article summarised the extant literature on MSC-EVs for DOP to provide new directions for further research and to promote the application of MSC-EVs in the clinical management of DOP.