Endothelial Progenitor Cell-derived Exosomes Research Articles

Exercise improves cardiac fibrosis by stimulating the release of endothelial progenitor cell-derived exosomes and upregulating miR-126 expression.

Cardiac fibrosis is an important pathological manifestation of various cardiac diseases such as hypertension, coronary heart disease, and cardiomyopathy, and it is also a key link in heart failure. Previous studies have confirmed that exercise can enhance cardiac function and improve cardiac fibrosis, but the molecular target is still unclear. In this review, we introduce the important role of miR-126 in cardiac protection, and find that it can regulate TGF-β/Smad3 signaling pathway, inhibit cardiac fibroblasts transdifferentiation, and reduce the production of collagen fibers. Recent studies have shown that exosomes secreted by cells can play a specific role through intercellular communication through the microRNAs carried by exosomes. Cardiac endothelial progenitor cell-derived exosomes (EPC-Exos) carry miR-126, and exercise training can not only enhance the release of exosomes, but also up-regulate the expression of miR-126. Therefore, through derivation and analysis, it is believed that exercise can inhibit TGF-β/Smad3 signaling pathway by up-regulating the expression of miR-126 in EPC-Exos, thereby weakening the transdifferentiation of cardiac fibroblasts into myofibroblasts. This review summarizes the specific pathways of exercise to improve cardiac fibrosis by regulating exosomes, which provides new ideas for exercise to promote cardiovascular health.

Exosomes endow photocurable 3D printing 45S5 ceramic scaffolds to enhance angiogenesis-osteogenesis coupling for accelerated bone regeneration

The reconstruction of the vascular network is crucial step in bone regeneration. Therefore, effectively modulating angiogenesis-osteogenesis coupling in bone tissue engineering scaffolds is currently an urgent need. In this study, we employed silane coupling agents containing double bonds to modify tetrahedral silicate, resulting in the preparation of a photocurable precursor of 45S5 bioactive glass (PG). PG was utilized as a binding agent for tricalcium phosphate (TCP) powder, and we employed a one-step photocuring 3D printing approach to fabricate PG/TCP (PT) scaffolds. Furthermore, the endothelial progenitor cell-derived exosomes (EPC-exos) was encapsulated by GelMA and anchored onto the PT scaffolds to create exosome-functionalized PT/G@Exos composite scaffolds. In summary, the PT/G@Exos composite scaffold effectively orchestrates the creation of a vascularized bone regeneration microenvironment by releasing EPC-exos, as well as calcium, silicon (Si), and phosphorus (P) elements. This enables an efficient modulation of the angiogenesis-osteogenesis coupling of bioactive scaffolds and accelerates bone regeneration.

Endothelial progenitor cell-derived exosomes promote anti-inflammatory macrophages via SOCS3/JAK2/STAT3 axis and improve the outcome of spinal cord injury

BackgroundMacrophage in the spinal cord injury (SCI) area imparts a chronic pro-inflammation effect that challenges the recovery of SCI. Previously, endothelial progenitor cell-produced exosomes (EPC-EXOs) have been noticed to facilitate revascularization and inflammation control after SCI. However, their effects on macrophage polarization remained unclear. This study aimed to investigate the EPC-EXOs' role in macrophage polarization and reveal its underlying mechanism.MethodsWe extracted the macrophages and EPC from the bone marrow suspension of C57BL/L mice by centrifugation. After cell identification, the EPC-EXOs were collected by ultra-high-speed centrifugation and exosome extraction kits and identified by transmission electron microscopy and nanoparticle tracking analysis. Then, macrophages were cultured with EPC-EXOs in different concentrations. We labeled the exosome to confirm its internalization by macrophage and detected the macrophage polarization marker level both in vitro and in vivo. We further estimated EPC-EXOs' protective effects on SCI by mice spinal cord tissue H&E staining and motor behavior evaluation. Finally, we performed RT-qPCR to identify the upregulated miRNA in EPC-EXOs and manipulate its expression to estimate its role in macrophage polarization, SOCS3/JAK2/STAT3 pathway activation, and motor behavior improvement.ResultsWe found that EPC-EXOs decreased the macrophages’ pro-inflammatory marker expression and increased their anti-inflammatory marker expression on the 7 and 14 days after SCI. The spinal cord H&E staining results showed that EPC-EXOs raised the tissue-sparing area rate significantly after 28 days of SCI and the motor behavior evaluation indicated an increased BMS score and motor-evoked potential by EPC-EXOs treatment after SCI. The RT-qPCR assay identified that miR-222-3P upregulated in EPC-EXOs and its miRNA-mimic also decreased the pro-inflammatory macrophages and increased the anti-inflammatory macrophages. Additionally, miR-222-3P mimic activated the SOCS3/JAK2/STAT3 pathway, and SOCS3/JAK2/STAT3 pathway inhibition blocked miR-2223P’s effects on macrophage polarization and mouse motor behavior.ConclusionComprehensively, we discovered that EPC-EXOs-derived miR-222-3p affected macrophage polarization via SOCS3/JAK2/STAT3 pathway and promoted mouse functional repair after SCI, which reveals EPC-EXOs’ role in modulation of macrophage phenotype and will provide a novel interventional strategy to induce post-SCI recovery.

Endothelial progenitor cell-derived exosomes inhibit pulmonary artery smooth muscle cell in vitro proliferation and resistance to apoptosis by modulating the Mitofusin-2 and Ras-Raf-ERK1/2 signaling pathway

Pulmonary arterial hypertension (PAH) mainly occurs as a result of abnormal proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Endothelial progenitor cell (EPC)-derived exosomes (Exos) (EPC-Exos) relieve PAH. However, there is still insufficient knowledge of whether EPC-Exos contribute to the pathological process of PAH, especially for PASMC repair. This study aimed to determine the effects of EPC-Exos on the proliferation, migration, and apoptosis of PASMCs and explore the possible underlying molecular mechanisms through bioinformatics analysis and in vitro testing. Bioinformatics analysis showed that the Ras signaling pathway and Exos were crucial in PAH. The PAH differential microRNAs (miRNAs) and miRNAs identified in EPC-Exos were intersected to obtain miR-21-5p. A target gene prediction program predicted mitofusin-2 (Mfn2) as a potential target of miR-21-5p. Cellular experiments demonstrated that EPC-Exos attenuated the viability, proliferation, migration, and apoptosis resistance of PASMCs under hypoxia. Mechanistically, EPC-Exos significantly upregulated Mfn2 expression and attenuated Ras-Raf-ERK1/2 signaling pathway activity. In conclusion, EPC-Exos suppress cell viability, proliferation, and migration and promote apoptosis in PASMCs under hypoxic conditions. It is possible to transport miR-21-5p to improve the expression of Mfn2 and inhibit the Ras-Raf-ERK1/2 signaling pathway directly or by targeting the expression of Mfn2. EPC-Exos are a potential therapeutic candidate for the treatment of PAH.

MiR-17-5p Mediates the Effects of ACE2-Enriched Endothelial Progenitor Cell-Derived Exosomes on Ameliorating Cerebral Ischemic Injury in Aged Mice.

Aging is one of the key mechanisms of vascular dysfunction and contributes to the initiation and progression of ischemic stroke (IS). Our previous study demonstrated that ACE2 priming enhanced the protective effects of exosomes derived from endothelial progenitor cells (EPC-EXs) on hypoxia-induced injury in aging endothelial cells (ECs). Here, we aimed to investigate whether ACE2-enriched EPC-EXs (ACE2-EPC-EXs) could attenuate brain ischemic injury by inhibiting cerebral EC damage through their carried miR-17-5p and the underlying molecular mechanisms. The enriched miRs in ACE2-EPC-EXs were screened using the miR sequencing method. EPC-EXs, ACE2-EPC-EXs, and ACE2-EPC-EXs with miR-17-5p deficiency (ACE2-EPC-EXsantagomiR-17-5p) were administered to transient middle cerebral artery occlusion (tMCAO)-operated aged mice or coincubated with hypoxia/reoxygenation (H/R)-treated aging ECs. The results showed that (1) the level of brain EPC-EXs and their carried ACE2 were significantly decreased in aged mice compared to in young mice, and (2) after tMCAO, aged mice displayed increases in brain cell senescence, infarct volume, and neurological deficit score (NDS) and a decrease in cerebral blood flow (CBF). (3) Compared with EPC-EXs, ACE2-EPC-EXs were enriched with miR-17-5p and more effective in increasing ACE2 and miR-17-5p expression in cerebral microvessels, accompanied by obvious increases in cerebral microvascular density (cMVD) and cerebral blood flow (CBF) and decreases in brain cell senescence, infarct volume, neurological deficit score (NDS), cerebral EC ROS production, and apoptosis in tMCAO-operated aged mice. Moreover, silencing of miR-17-5p partially abolished the beneficial effects of ACE2-EPC-EXs. (4) In H/R-treated aging ECs, ACE2-EPC-EXs were more effective than EPC-EXs in decreasing cell senescence, ROS production, and apoptosis and increasing cell viability and tube formation. In a mechanistic study, ACE2-EPC-EXs more effectively inhibited PTEN protein expression and increased the phosphorylation of PI3K and Akt, which were partially abolished by miR-17-5p knockdown. Altogether, our data suggest that ACE-EPC-EXs have better protective effects on ameliorating aged IS mouse brain neurovascular injury by inhibiting cell senescence, EC oxidative stress, apoptosis, and dysfunction by activating the miR-17-5p/PTEN/PI3K/Akt signaling pathway.

Manipulating bioactivities of endothelial progenitor cell-derived exosomes for promoting angiogenesis in ischemic vascular diseases

Exosomes paracrine-secreted by EPC (EPC-EXO) may exert strong pro-angiogenic effects, which offers a potential means to treat ischemic diseases. However, the mechanism of EPC-EXO-mediated angiogenesis remains unknown, and EPC-EXO shows poor homing to ischemic sites, which greatly limits the pro-angiogenic effect in vivo. We found that EPC-EXO from patients with coronary heart disease (C-EPC-EXO) was much less effective than EPC-EXO from healthy volunteers (H-EPC-EXO) in improving angiogenesis in murine hindlimb ischemia model. The miRNA sequencing and RT-PCR analyses detected abundant microRNA-199a-3p (miRNA-199a-3p) in C-EPC-EXO, and computational and luciferase reporter gene analyses further found that miR-199a-3p targeted the seed sequence of 4856–4862 in the 3′-untranslated region of RNA-binding protein Quaking isoform 5 (QKI-5) mRNA in 293T cells. Increasing the intracellular miR-199a-3p level of human umbilical vein endothelial cells (HUVECs) downregulated the QKI-5 gene expression and decreased angiogenesis, whereas inhibiting miR-199a-3p led to opposite results. Therefore, the miR-199a-3p-inhibited C-EPC-EXO promoted ECs-mediated angiogenesis more effectively than the original C-EPC-EXO. Besides, decorating C-EPC-EXO with cRGD enhanced targeting delivery to ischemic sites, further strengthening the pro-angiogenic effect of exosomes. Overall, manipulating the bioactivities of C-EPC-EXO through miR-199a-3p inhibition and cRGD decoration remarkably promotes ECs-mediated angiogenesis for treating ischemic vascular diseases.

Endothelial progenitor cell derived exosomes mediated miR-182-5p delivery accelerate diabetic wound healing via down-regulating PPARG.

Diabetic wound is one of the most common and serious complications of diabetes, which is characterized by abnormal number and quality of wound repair related cells. Previous studies have shown that human endothelial progenitor cells derived exosomes (EPCs-EXO) can promote diabetic wound healing through modulating vascular endothelial cell function. The purpose of this study was to investigate the biological effects and molecular mechanisms of EPCs-EXO on diabetic wound healing. The regulation of EPCs-EXO on human immortalized epidermal cell line HaCaT in high glucose (HG) environment was evaluated. Our data showed that EPCs-EXO promoted the proliferation, migration, while inhibited apoptosis of HaCaTs challenged by HG via elevating miR-182-5p expression level in vitro. Skin wound healing was significantly enhanced by EPCs-EXO in diabetic mice. Moreover, bioinformatics analyses and luciferase reporter assay indicated that exosomal miR-182-5p was bound to PPARG 3' UTR sequence and inhibited the expression of PPARG. Collectively, our findings provided a new role of EPCs-EXO in the clinical treatment of diabetic skin wounds. Diabetic wound is one of the most common and serious complications of diabetes, which is characterized by abnormal number and quality of wound repair related cells. Previous studies have shown that human endothelial progenitor cells derived exosomes (EPCs-EXO) can promote diabetic wound healing through modulating vascular endothelial cell function. The purpose of this study was to investigate the biological effects and molecular mechanisms of EPCs-EXO on diabetic wound healing. The regulation of EPCs-EXO on human immortalized epidermal cell line HaCaT in high glucose (HG) environment was evaluated. Our data showed that EPCs-EXO promoted the proliferation, migration, while inhibited apoptosis of HaCaTs challenged by HG via elevating miR-182-5p expression level in vitro. Skin wound healing was significantly enhanced by EPCs-EXO in diabetic mice. Moreover, bioinformatics analyses and luciferase reporter assay indicated that exosomal miR-182-5p was bound to PPARG 3' UTR sequence and inhibited the expression of PPARG. Collectively, our findings provided a new role of EPCs-EXO in the clinical treatment of diabetic skin wounds.

Compromised endothelial progenitor cell exosomal communication with endothelial cells in hypertension ischemia conditions

We have previously demonstrated that endothelial progenitor cell (EPC) derived exosomes (EPC-EXs) can protect endothelial cells (ECs) against hypoxia injury. Given that EX function varies upon the cellular status and EPC function is declined in hypertension, we speculate the function of EPC-EXs is altered in hypertension-ischemia conditions. Here, we studied the EPC-EX mediated communications of EPCs with ECs in hypertension-ischemia conditions. EPC-EXs were prepared from the bone marrow EPCs of wild-type (WT) and hypertensive renin transgene (R+) mice (WT-EPC-EXs and R-EPC-EXs, respectively). To mimic hypertension-ischemia injury, ECs were challenged with angiotensin II (Ang II; 10−6 M) plus hypoxia (1% O2 for 6 h) and reoxygenation (21% O2 for 24 h). To determine the function of EPC-EXs, ECs were co-cultured with EXs during the reoxygenation period. EX uptake efficiency, EC viability, and angiogenic function were assessed. We found that: (1) The incorporation efficiency of R-EPC-EXs by ECs was significantly decreased compared to the WT-EPC-EXs. (2) Ang II plus hypoxia reoxygenation-injured ECs displayed decreased cell viability, increased cell apoptosis, and compromised angiogenic ability, which were alleviated by R-EPC-EXs. (3) WT-EPC-EXs elicited better effects than R-EPC-EXs on protecting ECs from hypertension plus hypoxia injury. In conclusion, our data have demonstrated that EPC-EXs mediated communication of EPCs and ECs is compromised in hypertension-ischemia conditions, suggesting that impairment of EPC exosomal communication might contribute to the exaggerated cerebral ischemia injury in hypertension-associated ischemic stroke.

MiR-221-3p targets HIPK2 to promote diabetic wound healing.

Diabetic foot ulcer is a severe complication of diabetes and is prone to being a chronic non-healing wound. We previously demonstrated that endothelial progenitor cell-derived exosomes, which contain miR-221-3p, alleviate diabetic ulcers. Here, to explore the mechanisms underlying this wound healing, we investigated the potential angiogenic effects of miR-221-3p in vitro using cultured human umbilical vein endothelial cells (HUVECs) and in vivo using a streptozotocin-induced mouse model of diabetes. We found that miR-221-3p promoted HUVEC viability, migration, and capillary-like tube formation. HUVECs cultured in high glucose showed up-regulated expression of homeodomain-interacting protein kinase 2 (HIPK2), a predicted target of miR-221-3p that may decrease angiogenesis. Knockdown of HIPK2 enhanced high glucose-suppressed HUVEC viability, migration, and tube formation, counteracting the effects of high glucose. Using a dual luciferase reporter assay, we found that HIPK2 was indeed a direct target of miR-221-3p. Subcutaneous injection of miR-221-3p agomir into diabetic mice promoted wound healing and suppressed HIPK2 expression in wound margin tissue. These findings indicate that HIPK2, as a direct target of miR-221-3p, contributes to the regulatory role of miR-221-3p in diabetic wound healing and may be a novel therapeutic target for diabetic foot ulcer.

Cardioprotective Roles of Endothelial Progenitor Cell-Derived Exosomes.

With the globally increasing prevalence, cardiovascular diseases (CVDs) have become the leading cause of mortality. The transplantation of endothelial progenitor cells (EPCs) holds a great promise due to their potential for vasculogenesis, angiogenesis, and protective cytokine release, whose mechanisms are essential for CVD therapies. In reality, many investigations have attributed the therapeutic effects of EPC transplantation to the secretion of paracrine factors rather than the differentiation function. Of note, previous studies have suggested that EPCs could also release exosomes (diameter range of 30–150 nm), which carry various lipids and proteins and are abundant in microRNAs. The EPC-derived exosomes (EPC-EXs) were reported to act on the heart and blood vessels and were implicated in anti-inflammation, anti-oxidation, anti-apoptosis, the inhibition of endothelial-to-mesenchymal transition (EndMT), and cardiac fibrosis, as well as anti-vascular remodeling and angiogenesis, which were considered as protective effects against CVDs. In this review, we summarize the current knowledge on using EPC-EXs as therapeutic agents and provide a detailed description of their identified mechanisms of action to promote the prognosis of CVDs.

Exosomal miR-218-5p/miR-363-3p from Endothelial Progenitor Cells Ameliorate Myocardial Infarction by Targeting the p53/JMY Signaling Pathway.

Accumulating evidence has shown that endothelial progenitor cell-derived exosomes (EPC-Exos) can ameliorate myocardial fibrosis. The purpose of the present study was to investigate the effects of EPC-Exos-derived microRNAs (miRNAs) on myocardial infarction (MI). A miRNA-Seq dataset of miRNAs differentially expressed between EPCs and exosomes was collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the miRNA expression indicated by miRNA-Seq. Immunofluorescence, cell proliferation, and angiogenesis assays were employed to investigate the effects of miRNAs on cardiac fibroblasts (CFs) in vitro. Interactions between miRNAs and their respective targets were examined via immunoblotting, qRT-PCR, and luciferase reporter assays. An MI rat model was constructed, and various staining and immunohistochemical assays were performed to explore the mechanisms underlying the miRNA-mediated effects on MI. miR-363-3p and miR-218-5p were enriched in EPC-Exos, and miR-218-5p and miR-363-3p mimic or inhibitor enhanced or suppressed CF proliferation and angiogenesis, respectively. miR-218-5p and miR-363-3p regulated p53 and junction-mediating and regulatory protein (JMY) by binding to the promoter region of p53 and the 3′ untranslated region of JMY. Additionally, treatment of CFs with Exo-miR-218-5p or Exo-miR-363-3p upregulated p53 and downregulated JMY expression, promoted mesenchymal-endothelial transition, and inhibited myocardial fibrosis. Administration of exosomes containing miR-218-5p mimic or miR-363-3p mimic ameliorated left coronary artery ligation-induced MI and restored myocardial tissue integrity in the MI model rats. In summary, these results show that the protective ability of EPC-Exos against MI was mediated by the shuttled miR-218-5p or miR-363-3p via targeting of the p53/JMY signaling pathway.

MiR-137 boosts the neuroprotective effect of endothelial progenitor cell-derived exosomes in oxyhemoglobin-treated SH-SY5Y cells partially via COX2/PGE2 pathway

BackgroundWe have previously verified the beneficial effects of exosomes from endothelial progenitor cells (EPC-EXs) in ischemic stroke. However, the effects of EPC-EXs in hemorrhagic stroke have not been investigated. Additionally, miR-137 is reported to regulate ferroptosis and to be involved in the neuroprotection against ischemic stroke. Hence, the present work explored the effects of miR-137-overexpressing EPC-EXs on apoptosis, mitochondrial dysfunction, and ferroptosis in oxyhemoglobin (oxyHb)-injured SH-SY5Y cells.MethodsThe lentiviral miR-137 was transfected into EPCs and then the EPC-EXs were collected. RT-PCR was used to detect the miR-137 level in EPCs, EXs, and neurons. The uptake mechanisms of EPC-EXs in SH-SY5Y cells were explored by the co-incubation of Dynasore, Pitstop 2, Ly294002, and Genistein. After the transfection of different types of EPC-EXs, flow cytometry and expression of cytochrome c and cleaved caspase-3 were used to detect the apoptosis of oxyHb-injured neurons. Neuronal mitochondrial function was assessed by reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) depolarization, and cellular ATP content. Cell ferroptosis was measured by lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4. Additionally, recombinational PGE2 was used to detect if activation of COX2/PGE2 pathway could reverse the protection of miR-137 overexpression.ResultsThe present work showed (1) EPC-EXs could be taken in by SH-SY5Y cells via caveolin-/clathrin-mediated pathways and macropinocytosis; (2) miR-137 was decreased in neurons after oxyHb treatment, and EXsmiR-137 could restore the miR-137 levels; (3) EXsmiR-137 worked better than EXs in reducing the number of apoptotic neurons and pro-apoptotic protein expression after oxyHb treatment; (4) EXsmiR-137 are more effective in improving the cellular MMP, ROS, and ATP level; (5) EXsmiR-137, but not EXs, protected oxyHb-treated SH-SY5Y cells against lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4; and (6) EXsmiR-137 suppressed the expression of the COX2/PGE2 pathway, and activation of the pathway could partially reverse the neuroprotective effects of EXsmiR-137.ConclusionmiR-137 overexpression boosts the neuroprotective effects of EPC-EXs against apoptosis and mitochondrial dysfunction in oxyHb-treated SH-SY5Y cells. Furthermore, EXsmiR-137 rather than EXs can restore the decrease in miR-137 levels and inhibit ferroptosis, and the protection mechanism might involve the miR-137-COX2/PGE2 signaling pathway.

Angiotensin-converting enzyme 2 augments the effects of endothelial progenitor cells-exosomes on vascular smooth muscle cell phenotype transition.

Phenotype transition of vascular smooth muscle cells (VSMCs) is implicated in vascular diseases. Angiotensin-converting enzyme 2 (ACE2) is a perspective cardiovascular target due to its ability of converting angiotensin (Ang II) to Ang (1-7). Our group recently showed that ACE2 can regulate the function of endothelial progenitor cell-derived exosomes (EPC-EXs). Here, we investigate whether ACE2 could affect the role of EPC-EXs on phenotype transition of VSMCs. After co-incubation with EXs released from EPC overexpressed ACE2 (EPC-EXsACE2), the ACE2 level and Ang II/Ang (1-7), proliferation/migration, phenotype gene, cytokine and NF-κB level on VSMCs were assessed. To determine the EX uptake route, VSMCs were pretreated with inhibitors. We found that (1) EPC-EXs and EPC-EXsACE2 were uptaken by VSMCs dominantly through caveolin-dependent endocytosis. (2) EPC-EXsACE2 remarkably increased the ACE2 level and decreased Ang II/Ang (1-7) in VSMCs activated by Ang II, whereas EPC-EXsACE2 pretreated by proteinase A blocked this effect. (3) EPC-EXsACE2 had better effects than EPC-EXs on reducing proliferation/migration activities and cytokine (MCP-1, TNF-α) secretion of Ang II-activated VSMCs. (4) EPC-EXs attenuated Ang II-induced VSMC synthetic phenotype change as evidenced by upregulated expressions of calponin and a-SMA and downregulated expressions of CRBP-1 and MYH10, associated with a decreased NF-κB level. EPC-EXsACE2 augmented these effects, which were attenuated by ACE2 inhibitor (DX600). In conclusion, EPC-EXsACE2 reduced Ang II-induced VSMC phenotype change by conveying functional ACE2 to downregulate the activated NF-κB pathway.

Abstract 272: Epigenetic Regulation Involved in Diabetes-induced Impairment in Myocardial Reparative Function of Endothelial Progenitor Cell-derived Exosomes

Myocardial infarction (MI) occurs frequently in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Moreover, emerging evidence supports that exosomes (Exo) mediate stem cell therapy by carrying cell-specific biological signatures and by inducing signaling via transfer of bioactive molecules to target cells. However, autologous cell-based therapies yielded modest clinical results, suggesting that cellular/Exo reparative function may be compromised on a background of disease such as diabetes. In addition, recent studies suggest epigenetic mechanisms, such as histone methylation for gene silencing, promotes diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content which compromise EPC reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic mice (Lepr db/+ ) and diabetic mice (Lepr db/db ) and examined their effect on tube formation and cardiomyocyte/endothelial cell survival in vitro as well as their reparative effects on permanent and acute ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo . Diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival compared to cells treated with WT EPC-Exo. In vivo studies revealed diabetic EPC-Exo significantly attenuated cardiac function, reduced capillary density, increased fibrosis and infarct size in permanent LAD ligation and I/R MI models. Mechanistically,H3K9Me3 was increased in mouse cardiac endothelial cells treated with diabetic EPC-exo, suggesting inhibition of angiogenic genes. Our results provide evidence that diabetic EPC-derived exosomes lose their cardiac reparative activities. Specific angiogenic genes will be examined by CHIP analysis of H3K9Me3. Reversing EPC-Exo function by manipulating H3K9Me3 expression will augment autologous therapies in regenerative medicine.

Moderate exercise has beneficial effects on mouse ischemic stroke by enhancing the functions of circulating endothelial progenitor cell-derived exosomes

Exosomes (EXs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. We have recently revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived EXs (cEPC-EXs) on protecting endothelial cells against hypoxia injury. However, the relationship between the changes of cEPC-EXs and the effects of exercise on ischemic stroke (IS) is unknown. Here, we investigated whether exercise-regulated EPC-EXs contribute to the beneficial effects of exercise on IS. C57BL/6 mice received moderate treadmill exercise (10 m/min) for 4-wks and then were subjected to middle cerebral artery occlusion (MCAO) stroke. The neurologic deficit score (NDS), infarct volume, microvessel density, cell apoptosis, angiogenesis/neurogenesis, sensorimotor functions were determined on day 2 (acute stage) and/or day 28 (chronic stage) post-stroke. The miR-126 and EPC-EX levels were analyzed by RT-PCR or nanoparticle tracking analysis combined with microbeads and used for correlation analyses. The function of EPC-EXs from exercised mice was detected in a hypoxia neuron model. Cell apoptosis, axon growth ability and gene expressions (cas-3 and Akt) were measured. Our data showed that: i) On day 2, exercised mice had decreased NDS and infarct volume, reduced cell apoptosis rate and cleaved cas-3 level, and a higher microvessel density than those in control (no-exercise) mice. The levels of EPC-EXs in plasma and brain tissue were raised and positively correlated in exercised mice. Meanwhile, the miR-126 level in cEPC-EXs and in ischemic tissue were upregulated in exercised mice. The EPC-EXs and their carried miR-126 levels negatively correlated with the infarct volume and cell apoptosis, whereas positively correlated with microvessel density. In addition, cEPC-EXs from exercised mice elicited protective effects on neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by miR-126 and PI3k inhibitors in vitro. ii) On day 28, exercised mice had less infarct volume, higher microvessel density, angiogenesis/neurogenesis and better sensorimotor functions. The levels of BDNF, p-TrkB/TrkB and p-Akt/Akt were upregulated in the brain of exercised mice. These recovery indexes correlated with the levels of cEPC-EXs and their miR-126. In conclusion, our data suggest that moderate exercise intervention has protective effects on the brain against MCAO-induced ischemic injury in both acute and chronic stages which might via the release of miR-126 enriched EPC-EXs.

MiRNA-221-3p in Endothelial Progenitor Cell-Derived Exosomes Accelerates Skin Wound Healing in Diabetic Mice.

BackgroundPatients with diabetic cutaneous ulcers experience financial burden and a lower quality of life and life expectancy. Endothelial progenitor cell (EPC)-derived exosomes facilitate skin wound healing by positively modulating vascular endothelial cell function. Exosomes play their important regulatory role through microRNA (miRNA). We explored the potential role and molecular mechanisms of miRNA in EPC-derived exosome healing of diabetic skin wounds.MethodsExosomes were isolated from the media of EPCs derived from mice bone marrow. High-throughput sequencing was used to detect the expression of exosome miRNA, and miRNA target genes were predicted using online databases. A diabetic mouse skin wound model was established, and wounds were treated with exosomes, miRNA-221-3p, or phosphate-buffered saline.ResultsExosomes from EPCs accelerated skin wound healing in both control and diabetic mice. High-throughput sequencing showed that miRNA-221-3p was highly expressed in EPC-derived exosomes. Skin wound healing in control and diabetic mice was significantly enhanced by EPC-derived exosomes and miRNA-221-3p administration. Immunohistochemical analyses showed that EPC-derived exosomes and miRNA-221-3p increased protein expression levels of the angiogenesis-related factors VEGF, CD31 and cell proliferation marker Ki67. Bioinformatics analyses indicated that miRNA-221-3p may be involved in the AGE-RAGE signaling pathway in diabetic complications, cell cycle, and the p53 signaling pathway.ConclusionWe concluded that miRNA-221-3p is one of the high-expressed miRNAs in EPC-derived exosomes and promoted skin wound healing in diabetic mice. The finding uncovers the molecular mechanism of EPC-derived exosomes and provides a potential novel approach to the clinical treatment of diabetic skin wounds.

Endothelial progenitor cell-derived exosomes facilitate vascular endothelial cell repair through shuttling miR-21-5p to modulate Thrombospondin-1 expression.

Background: Our previous studies observed that administration of exosomes from endothelial progenitor cells (EPC) facilitated vascular repair in the rat model of balloon injury. However, the molecular events underlying this process remain elusive. Here, we aim to interrogate the key miRNAs within EPC-derived exosomes (EPC-exosomes) responsible for the activation of endothelial cell (EC) repair. Methods: The efficacy of EPC-exosomes in re-endothelialization was examined by Evans Blue dye and histological examination in the rat model of balloon-induced carotid artery injury. The effects of EPC-exosomes on human vascular EC (HUVEC) were also studied by evaluating the effects on growth, migratory and tube formation. To dissect the underlying mechanism, RNA-sequencing assays were performed to determine miRNA abundance in exosomes and mRNA profiles in exosome-treated HUVECs. Meanwhile, in vitro loss of function assays identified an exosomal miRNA and its target gene in EC, which engaged in EPC-exosomes-induced EC repair. Results: Administration of EPC-exosomes potentiated re-endothelialization in the early phase after endothelial damage in the rat carotid artery. The uptake of exogenous EPC-exosomes intensified HUVEC in proliferation rate, migration and tube-forming ability. Integrative analyses of miRNA-mRNA interactions revealed that miR-21-5p was highly enriched in EPC-exosomes and specifically suppressed the expression of an angiogenesis inhibitor Thrombospondin-1 (THBS1) in the recipient EC. The following functional studies demonstrated a fundamental role of miR-21-5p in the pro-angiogenic activities of EPC-exosomes. Conclusions: The present work highlights a critical event for the regulation of EC behavior by EPC-exosomes, which EPC-exosomes may deliver miR-21-5p and inhibit THBS1 expression to promote EC repair.

Retraction Note: Endothelial progenitor cell-derived exosomes, loaded with miR-126, promoted deep vein thrombosis resolution and recanalization

The authors have retracted this article [1] because there is erroneous data in Figure 1C. Flow cytometry results of EPC cell markers CD31 and CD45 could not be replicated. Due to incorrect cell markers, the cells cultured may not have been pure EPCs. Therefore the scientific content of the article is no longer reliable. All authors agree to this retraction.

Abstract TP134: Exercise Enhanced the Function of Endothelial Progenitor Cell-derived Exosomes on Protecting Neurons Against Hypoxia/reoxygenation Insult

Our recent study has revealed that moderate exercise upregulates the levels of miR-126 in circulating endothelial progenitor cell-derived exosomes (EPC-EXs), accompanying with enhanced protective function on endothelial cells. Whether exercise can regulate the function of circulating EPC-EXs on protecting neurons against hypoxia/reoxygenation (H/R) injury remains unclear. Here, we isolated the circulating EPC-EXs from exercise mice (10 m/min) and investigated their role in neurons subjected to H/R. EPC-EXs were characterized by nanoparticle tracking and western blot analyses. Mouse neurons (N2a) cultured in hypoxia condition for 6 hr followed by 24 hr reoxygenation. The H/R-injured N2a cells were treated with EPC-EXs (4 x 10 7 EPC-EXs/ml) isolated from exercise (EPC-EXs E ) or sedentary mice (EPC-EXs s ). For mechanism study, N2a cells were transfected with miR-126 inhibitor or pre-treated with PI3k inhibitor (LY294002) for 2 hr. After 24 hr treatment, apoptosis and axon growth ability of N2a cells were determined. The growth factors (vascular endothelial growth factor/VEGF and brain derived neurotrophic factor/BDNF), miR-126 level and its downstream signal genes (PI3k and Akt) were measured. Our data showed that: 1) H/R insult induced decrease of VEGF and BDNF secretion as well as axon growth ability, increase of apoptosis of N2a cells; 2) EPC-EX S treatment raised miR-126 level and rescued N2a cells from H/R-induced injury, with better effect elicited by EPC-EXs E . 3) EPC-EXs E had better effect than EPC-EX S on activating the PI3k/Akt signal pathway, which was significantly reduced by miR-126 and PI3k inhibitors. Altogether, exercise boosts the effects of EPC-EXs on protecting neurons against H/R insult through activating the miR-126/PI3k/Akt signal pathway.

RETRACTED ARTICLE: Endothelial progenitor cell-derived exosomes, loaded with miR-126, promoted deep vein thrombosis resolution and recanalization

BackgroundDeep vein thrombosis (DVT) is caused by blood clotting in the deep veins. Thrombosis resolution and recanalization can be accelerated by endothelial progenitor cells. In this report, we investigated the effects of miR-126-loaded EPC-derived exosomes (miR-126-Exo) on EPCs function and venous thrombus resolution.MethodsIn vitro promotional effect of miR-126-Exo on the migration and tube incorporation ability of EPCs was investigated via transwell assay and tube formation assay. In addition, a mouse venous thrombosis model was constructed and treated with miR-126-Exo to clarify the therapeutic effect of miR-126-Exo by histological analysis. Lastly, this study predicted a target gene of miR-126 using target prediction algorithms and confirmed it by luciferase activity assay, RT-qPCR, and Western blot.ResultsTranswell assay and tube formation assay indicated that miR-126-Exo could enhance the migration and tube incorporation ability of EPCs. Moreover, in vivo study manifested enhanced thrombus organization and recanalization after miR-126-Exo treatment. Meanwhile, we identified that Protocadherin 7 as a target gene of miR-126.ConclusionsTo sum up, our results demonstrated that EPC-derived exosomes loaded with miR-126 significantly promoted thrombus resolution in an animal model of venous thrombosis, indicating exosomes as a promising potential vehicle carrying therapeutic molecules for DVT therapy.